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Toxicology beyond the LD-50 test

- Prof Bjorn Ekwall, MD,
Assistant Professor, Dept pf Toxicology, University of Uppsala
Speech delivered at the International Scientific Congress, London, April 1991

My present lecture will consist of four parts.

Firstly, I want to emphasise the importance of toxicity testing in our modern society.

Secondly, I am going to criticise animal toxicity tests. The shortcomings of these tests will be scrutinised from a scientific standpoint. The LD50 test – that is, a measure of the dose of a chemical which kills 50 per cent of a population of rodents – will be taken as an example of a relatively ineffective and cruel toxicity test. At the same time, I shall point out that sole reliance on such a test for risk assessment is decreasing, and in many areas of toxicity testing this test is in fact already obsolete.

Thirdly, a more refined animal-based approach to toxicity testing will be described which is today typical in most sectors of the pharmaceutical and chemical industry.

Fourthly, the basis for non-animal toxicity testing in the near future is described. This is cell toxicology, with its possibilities of using batteries of cell culture and other test-tube tests as models of human reaction to the toxicity of chemicals.

I shall start from the beginning and remind you that we do not live in the stone, bronze or iron ages any longer – we now live in the chemical age. At the same time we live in the plastics age, the communications age, the information-explosion age and so on. The chemical age denotes the fact that man is surrounded by four million chemicals, which penetrate every segment of his surroundings. Most of these chemicals have only recently been synthesised, and new ones are pouring out of industry every day, to the extent of hundreds per month. And most of them are not released in distinct and easily controlled doses, as are (or should be) drugs and pesticides. No, the vast majority of them occur unexpectedly and diffusely, in food, clothing, furniture, cars, electronic items et cetera –that is when they are not environmental pollutants, scattered abroad without any control whatsoever.

The nastiest fact, however, is that most of these chemicals have been poorly tested for the various effects of their toxicity on man, such as acute toxicity, chronic toxicity, local irritancy to the eyes or skin, foetal toxicity, allergic sensitisation potential and so forth. And many of the chemicals have not been tested at all, in spite of the production and dispersion of hundreds of tons of them. This is the so-called “mountain” of untested or poorly tested chemicals feared by many government agencies today.

Many laymen and, indeed, many professional toxicologists believe that this state of affairs is somehow checked by the medical profession: if some of untested chemicals really are dangerous, physicians will surely detect this in a typical or unusual illness. I think that all the physicians in this auditorium would agree that this “clinical” safety-net is an illusion. A human being can only respond to injury to cells and organs in a relatively stereotyped way and limited to few illnesses, such as cancers, various degrees of inflammation and so on, connected with each organ. Nearly always, then, only components of the so-called general panorama of illnesses will result from chemical toxicity (for example, heart, liver and kidney problems, headache, cough, vertigo, sleepiness) – that is, diagnoses are made without obvious reason. It is very seldom that a new syndrome, specific to the offending agent only, will result from toxicity. Consequently, there is no clinical safety-net for untested chemicals as long as a percentage of the population is ill from unknown causes.

Systematic toxicity testing of chemicals, including human risk assessment based on the tests, is only about 50 years old. Up to now, most of this testing has been done by using animal subjects. At least 25 per cent of all animals used in biomedical and other research are destined for toxicity tests. Groups of five to ten animals receive a dose of the chemical by one method of administration or another, making up several groups, plus control animals, per experiment. Symptoms of injury, illness and/or death are then recorded after certain intervals of time. Often an autopsy is performed, including histopathological studies. The LD50 test, taken as an example here, often has an observation period of two weeks. The LD50 test is the first to be performed on an unknown chemical, followed by a series of other tests on local irritancy, subchronic and chronic toxicity, teratogenicity, phototoxicity, sensitisation, fertility and short- or long-term carcinogenicity. The most common animal toxicity test is the LD50 assay or small modifications of that test.

A basic animal test, such as the LD50 test, has several shortcomings:
Firstly, the idea behind the test – that is, the use of an animal as a representative of the human body and mind for testing purposes – is not correct. We have a species gap between the various animals due to differences in bodily functions, making their responses to chemicals differ from one to another. The toxic dose depends on the reaction of the most sensitive target in the body. The target is always the molecules constituting parts of cells, intercellular materials, extra-cellular transmitters or hormones. But often the effect will also depend on various toxico-kinetic factors, such as absorption of the chemical in the intestine or skin, metabolism of the chemical in the liver, distribution of the chemical to bodily compartments, storage of the chemical and finally excretion of it through kidneys, intestines, lungs or skin. A variation in only one of these factors between the test animal and the human will make the toxic dose administered to the animal inapplicable to or unpredictable for man. Thus, animal experiments are a gamble. Additional tests in other animal species will not improve the odds – often, confusion is increased, at the expense of further suffering and costs.
A second flaw in animal testing is that primary toxic events, such as chemical interference with molecules in cells, cell organelles and extracellular receptors, are not measured. Instead, a host of secondary effects from the original insult obscures the picture – that is, the symptoms of poisoning such as fall in blood pressure, confusion, convulsions and so on. Often these secondary effects, which have no quantitative relation to the original insult, are the basis for the measurement of toxicity. Animal experiments simply have a very low resolving power in interpreting toxicity. Routine autopsy may compensate for 5this to a degree but may also, in most cases, overlook the functional effects of injury. The main problem with the measurement of secondary phenomena is that they make test results still more difficult to extrapolate to man.

Why have such blunt and crude investigative tools been used in the past and are still being used? The obvious answer seems to be that nobody has yet come up with better ideas. The real answer is probably that until recent times nobody has bothered to try to come up with better ideas. Twenty or thirty years ago, when industry and regulatory agencies had not yet been influenced by the modern consciousness of risks in this chemical age, or by a deeper concern about opinions on animal experiments, these scientifically crude tests were considered good enough.

However, for decades now, modern chemical and pharmaceutical industrial and regulatory governmental authorities have not relied solely on animal tests to measure toxic doses, body counts and toxic symptoms. Nowadays, the routine toxicity testing of pharmaceuticals, pesticides, industrial chemicals, cosmetics et cetera is much more refined. As the first step towards such a composite toxicology study of a compound, accumulated historical data on the toxicity of chemical analogues are used, together with simple physico-chemical data, on the investigated compound in order to predict human toxicity. This is done by structure-activity analysis with the help of computer programs. Guided by such studies, qualified toxicity tests on relatively few animals are performed. These include toxico-kinetic tests (blood levels and metabolites of the compound) and tests of subtle clinical toxicity (blood and urine analysis, including enzymology and cell counts). A more refined system of histopathology is applied and related to the other measurements to find out the cause of primary toxic events. Another characteristic feature of modern toxicology tests is the tiered-test approach, in which subsequent tests are individually guided and motivated by earlier tests. This is very different from the old-time approach of performing the standard animal tests in parallel, just to gain time. Instead, the modern approach gains understanding. Often the primary toxic mechanism is appreciated, which enhances the effectiveness of human risk prediction enormously. These routines are indeed a step forward beyond the LD50 test, although still based on animal testing.

One important problem with the modern, refined use of animals in toxicological studies is that these studies are very expensive. Thus, economical reasons are now a limiting factor in extending such scientifically improved testing – that is, replacing crude animal tests such as the LD50 or Draize tests in the mass testing of food additives, industrial chemicals, household products and so forth. The problem is similar to one in the technical developments in medicine where economic reasons prevent the unlimited use of transplantations, heart surgery et cetera.

To sum up: refined composite animal toxicity experiments have improved the test situation, but they have not solved the important problems in that field. Thus, toxicology still relies on unscientific animal experiments.

In the last ten years, a new method or subdiscipline has evolved in toxicology, namely, cell toxicology. This method may also be called test-tube toxicology. Cell toxicology is cell biology and molecular biology applied to toxicity studies. One main incentive in this development was the discovery and promotion of bacterial mutagenicity and carcinogenicity tests by Dr Bruce Ames in the early 70s. These mechanistically based test-tube tests have for 15 years been used routinely as adjuncts to animal carinogenicity tests. Additionally, cultures of specific cells from diverse organs have been used for 20 years as adjuncts to animal experiments in the study of toxic mechanisms. As a third development, ten years ago Christian, Waters, Zucco, Paganuzzi-Stammati, Walum, Nardone, Halle et al began to realise that cell cultures could also be used in toxicity testing.

To understand the almost revolutionary effect of our ideas on how to use cell cultures as replacements for animals in toxicity testing (and the almost counter-revolutionary reactions against these ideas), you must be acquainted with the thinking of the toxicologist of some years ago. His discipline came from pharmacology, which had been much influenced by the basic science of physiology. Thus, most toxic effects of chemicals – apart from the development of cancer and the toxicity of anti-cancer drugs – were thought to be the results of interactions between those chemicals and the regulatory mechanisms of the body, especially the receptor-mediated regulatory mechanisms. Furthermore, if toxicity in a few cases was attributed to direct cell injury, the organ-specific functions of cells were thought to be the target of this toxicity. Any target-organ toxicity was thus thought to be interference with typical cells (liver cells in liver injury, brain cells in brain injury and so on). Hence, the use of cells in the testing of general toxicity was, according to this thinking, impossible, because: 1) few toxic effects result in cell injury; and 2) if cellular toxic effects were to be tested, a very large number of organ-specific cells (liver, kidney, heart, brain, lung, thyroid, et cetera), must be used in a battery to cover the toxic insults to man, and this would be costly and unpractical. Mechanistically based cell tests of general toxicity, analogous with the Ames test, would also be impossible, since general toxicity is known by the toxicologist to operate via many different mechanisms.

To summarise results from my own and other cell toxicologists’ research: it seems to be possible to construct batteries of test-tube tests on various types of general toxicity (LD50, Draize eye and skin tests and so forth). The core of such batteries would be cell-line toxicity tests measuring basal cytotoxicity. Other more costly, animal-dependent, primary cultures of some key organ-specific cells may be added to the batter, to cover common types of specific organ toxicity. Test-tube tests with important macro-molecules in the body such as proteins and phospholipids, as well as similar tests with important transmitters, may also be added to the battery to cover non-cellular toxicity. Such a battery would have a good chance of predicting toxic blood and tissue levels of unknown compounds. These results would probably at times be falsely negative – when a rare toxic effect not covered by the battery occurs – but would certainly not produce false positive results. A new toxicology will emerge, performing risk assessment based on comparisons of blood and tissue concentrations from typical exposure in man, and resulting from the above-described battery.

To be able also to predict toxic dosage for humans, and to predict the possible toxicity of chemicals which are metabolised by the liver into more toxic compounds, the battery must also include toxicokinetic tests, and this is under way, although the necessary in-vitro methods have not yet been fully developed. Human liver cells and slices are already routinely used by some pharmaceutical companies as an adjunct in predicting the metabolism of drugs in man. Absorption tests (intestine, skin et cetera) are under investigation. Distribution, storage and excretion tests have not yet been developed.

To summarise: the concept of animal-free toxicity testing has recently evolved, aspects of which are now being realised in practical terms by cell toxicologists. Batteries of cellular and other test-tube tests of toxicity and toxicokinetic events are taking shape. These batteries will prove a great deal more scientific and more effective than animal tests. The reasons for this assertion are as follows. 1) Each component of a battery is calibrated directly to human conditions for better prediction. 2) the batteries focus on testing the primary cellular events. 3) High test sensitivity is ensured by the high number of cells involved and by the sensitive toxicity criteria which it is possible to apply to cell cultures. 4) Mechanistic understanding is very easy to achieve by further studies on the cellular level.

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