Introduction to Experimental Toxicology

D. M. CONNING

Toxicology is defined, classically, as the study of the adverse effects of chemicals on living systems. Originally this meant the study of poisons and that meaning remains the most satisfactory of definitions, essentially because it embodies the concept of the effect being proportional to the administered dose. Although the popular concept of poison concerns poisoning to death, the true study of poisons embraces the induction of morbid changes that are not necessarily fatal.

In this sense, the study of toxicology came to be regarded as an extension of the study of pharmacology and it is still so regarded by those who take a pedantic view of the topic. Of necessity, a pharmacological assay must explore the optimum dose in relation to the maximum therapeutic effect, and thus the dosage at which the effect is counter-productive of the desired result. In many ways the association of pharmacology and toxicology has been beneficial to the latter as a burgeoning science because it instilled two basic constraints which reinforced the scientific nature of the study. First was the recognition that pharmacology involved the perturbation of physiological function. That is, it was realised that a variety of detectable changes were compatible with normal living, and pharmacology sought to enhance those aspects which would be beneficial in the presence of disease or inhibit others for the same purpose. Second, the pharmacological study was a study of a defined function such as heart function, nerve transmission, or renal reabsorption, and never involved a less specific or more abstruse objective such as that embodied in the question 'Do any effects occur?' In other words, the pharmacological approach demanded an investigation of the way a particular physiological function could be chemically modified.

In recent decades, the definition of toxicology has been expanded in a way which has taken it firmly and, it seems, irrevocably out of the field of pharmacology. The first and most fundamental change was to add another purpose to the study. Thus toxicology came to be defined as the study of the adverse effects of chemicals on living systems in order to predict chemical hazard to man. This had the effect of classifying toxicology as an ancillary to public or community health, and by extension to preventive medicine, always the poor brother of therapeutic medicine; and at the same time imposed impossible conditions on its practice as a science. Not only did the study of toxicology become a study of the effects of a chemical on any conceivable physiological function, defined or not, but under any conceivable circumstances because of the almost infinite variety of human activity and behaviour. Toxicology was expected thereby to predict the effect of a chemical in systems which themselves were not capable of being defined.

The problem was compounded by a further expansion of the definition to include 'chemicals or other agents' and the inclusion of 'man or his environment.' Thus toxicology is the study of the adverse effects of chemicals or other agents on living systems in order to predict hazard to man and his environment.

A number of very unsatisfactory consequences have resulted from these developments. The first was the birth of the concept of the 'no-effect level' and its embodiment in safety regulations. It is simply not possible at the present stage of development of toxicological knowledge to define with any precision the normal values for many biological activities and thus to define when abnormal values are detected. The best we can do is to define where the values in treated systems (e.g. the experimental rat) differ from those in untreated systems maintained in similar circumstances. We know only rarely whether any observed differences represent a toxic effect or an adaptive response. Sometimes we have great difficulty in determining if there are any differences at all, a problem which has given rise to a massive development in biometrics.

Our adherence to the 'no-effect level' has undermined our faith in epidemiology. Although the lack of epidemiological data and the relative insensitivity of epidemiological methods have themselves contributed to this outcome, it has seemed easier to put our faith in animal results which can be determined with some precision and therefore appear to be more easily judged. The result is that attempts to extrapolate the findings in animals, for example, to predictable effects in man have no basis in human experience and tend to assume that man's response will be the same as that of the animal.

Another consequence has been distortion of the economics of toxicological practice in that those who are involved with toxicological experiment spend so much of their time and resources generating data, there is very little available for scientific interpretation and further experiment. The construction and testing of hypotheses do not have a prominent role in toxicology.

All of this has come about for the best possible motives. The perception of the possible dangers consequent upon our chemical inventiveness has resulted in the appearance of potent forces to protect human communities against such consequences. The diligent pursuit of detail has extended very considerably the requisite observations before a 'no effect' dosage can be determined. It is a matter of profound regret that much of this invaluable data is not used to further our knowledge of biological function.

In this book we hope to lay the foundations on which future toxicologists can build the scientific practice of toxicology. Although we have acknowledged the demands of modern society and provided the basis for the career development of the toxicologist charged with the provision of data on which the acceptability of new chemical or physical agent can be defined, we hope this has been done in a way which does not stifle the needs of the enquiring mind. Despite the problems, toxicology still remains a science which promises real opportunities to unravel some of the fascinating problems of biology by identifying chemical and physical tools with which to probe living processes. In the end, it is this aspect of toxicology that will contribute most to our understanding of those features which determine the likelihood of human disease.

Effects of Physical Form, Route, and Species

A. B. WILSON

1 Introduction

When investigating the literature in reference texts such as Sax (1979), which summarise toxicological information on a wide range of compounds, there is the impression of simplicity and unequivocality . More detailed texts (Clayton and Clayton, 1981) review the toxicology of classes of compound, indicate gaps in knowledge, and variable, even contradictory results. Hence there is well founded dispute about the effects of low doses of heavy metals, whether or not certain chemicals are carcinogenic, and the mechanisms of action of many compounds. This is further explored in more general toxicology textbooks (Klaassen, Amdur, and Doull, 1986; Hayes, 1983; Lu, 1985) and in toxicology journals such as: Toxicology and Applied Pharmacology (Academic Press, Duluth), Fundamental and Applied Toxicology (Academic Press, Duluth), Food and Chemical Toxicology (Pergamon Journals Ltd., Exeter), Comments on Toxicology (Gordon and Breach Science Publishers, London), Archives of Toxicology (Springer-Verlag, Germany), Toxicology (Elsevier, Ireland), Regulatory Toxicology and Pharmacology (Academic Press, Duluth), and Human and Experimental Toxicology (Macmillan Press, Basingstoke).

This chapter discusses the major factors that are causes of variability in results — physical form, vehicles, routes of exposure, and animal species as encountered in studies designed to screen compounds for general toxic potential. Few papers are available on the variations that actually occur in laboratory animal toxicology but information is published by Gaines and Linder (1986) with regard to acute toxicology of pesticides, Haseman (1983) with regard to carcinogenicity investigations, Lu (1985) with regard to a selection of factors, and by Rao (1986), Gartner (1990), Wollink (1989), and Vogel (1993) with regard to factors affecting laboratory animals.

2 Nature of Toxicant

A Physical Form

It can be expected that the physical nature of a material and the route of exposure will alter toxicity. Experiments in animals have to take account of this and batches of compound representative of normal production are usually employed so that purity, particle size, and other factors will match the real situation.

In general, a reduction in particle size improves solubility, increases absorption, and is likely, therefore, to increase toxicity. The effects of particle size are probably greatest on inhalation toxicity and that is covered separately. Solids will, of course, encounter body fluids in the gastro-intestinal tract, lungs, eyes, and even the skin and the therefore the opportunity exists for the material to be dissolved and for absorption to be improved. Insoluble materials are often regarded as inert and non-toxic. There are exceptions : asbestos and coal dust induce toxic effects by virtue of their insolubility and non-absorption which lead to residence in tissues such as the lung.

The pharmacist, pharmacologist, and experimental toxicologist are more likely to be concerned with capsules (perhaps enteric coated to carry them through the stomach intact), modification or buffering of pH to alter dissociation, and selection of soluble salts (e.g. soluble aspirin) to reduce local effects. All these measures and many more can have potential profound effects upon the toxicity of a compound.

Liquids do not have variations in terms of particle size in quite the same manner as do solids. However, liquids can become droplets (aerosols) with varying dimensions, which may be of some relevance if these are immiscible and of substantial significance if exposure is by inhalation. Liquids may have significant vapour pressure so they can sometimes be considered in much the same way as gases.

Gases and vapours present their hazards by being readily inhaled or by penetrating the skin. They too can be dissolved in liquids or adsorbed onto solids; indeed some show a remarkable affinity for solid surfaces, a property exploited in the manufacture of plastic strips containing pesticides such as dichlorvos.

The physical form of a compound will alter the potential for the material to enter the body, the extent of likely absorption, and therefore the eventual toxicity and hazard.

B Vehicle and Concentration

It has already been mentioned that gases, liquids, or solids may be presented in a vehicle as mixtures, solutions, or suspensions. If the vehicle is well absorbed, there is a tendency for the solute to be more rapidly and completely absorbed and vice versu. Many complex factors are involved, including the permeability of the surface to vehicle and compound, the partition coefficients, the extent of ionisation, and the effect of the material on local blood flow.

The concentration of material in the vehicle can alter toxicity. Often the more dilute the solution, the more complete the absorption of the solute (presuming the vehicle itself is absorbed) though the rate of absorption may be slower if dilution is substantial, as happens with alcohol. A rapidly detoxified or excreted compound will show less toxicity if absorption is slow. If large volumes are administered, it is worth bearing in mind that the vehicle itself might contribute to the toxicity or variations between results, for example, the interaction of corn oil in gavage studies (Nutrition Foundation, 1983). More than one investigator has thought he was looking at a concentration effect when it was probably vehicle toxicity.

High concentrations are more likely to cause local reactions such as irritation, ulceration, altered blood flow, or necrosis, which will themselves affect absorption and systemic toxicity, most obviously in dermal studies but frequently by other routes. The local effects are, of course, expressions of toxicity in their own right.

The interactions between vehicle and compound are exploited in the art and science of formulating drugs. By skilful means the therapeutic action, pharmacokinetics, and toxicity can be balanced to favour the desirable over the adverse (Prescott and Nimmo, 1979; Prescott and Nimmo, 1985).

3 Routes of Exposure

A Frequently Encountered Routes

The most frequently encountered exposures to industrial chemicals are by the dermal and inhalation routes but these predominate in the place of work where much can be done to limit and control the actual risks. In fact, most of the experimental toxicology on laboratory animals is carried out via the oral route because:

(a) The oral route is the normal model of exposure (e.g. for food additives and for many pharmaceuticals).

(b) The widest exposure to herbicides and pesticides is likely to be as residues in crops or meat, which will be ingested.

(c) The oral route is simple to use and expedient. If reasonable evidence exists to show that differences in toxicity related to route are quantitative rather than qualitative then the bulk of the investigations will employ oral administration.

The route by which a chemical encounters the body can alter very substantially its effects and, in particular, the quantity required to cause that effect.

The physical presentation of a chemical and the route of exposure interact to influence greatly whether a chemical will be absorbed, its rate of absorption, tissue distribution, removal, and excretion. The integrity of the exposed surface, the residence time of the potential toxicant, and the metabolic activity of the surface are amongst the relevant factors. It may be reasonable to state that the route is likely to alter primarily the rate of absorption and the quantity absorbed (hence the dose required to cause toxicity) rather than the nature of the toxic effect. The exceptions that occur can generally be attributed to effects at the point of application or to rapid metabolism in the organs first encountered after absorption, e.g. by the liver after absorption through the gut.

With regard to route of exposure, coal dust on the skin does not present much of a hazard, but inhaled (as mentioned above) it can result in a severe and debilitating disease. However, the skin is not a universal barrier and many compounds, particularly liquids and gases, will penetrate quite readily. It must also be noted that the surface presented to a compound is particularly vulnerable to local effects as a result of high concentrations over a potentially small area, such as are likely to occur in mouse skin carcinogenicity studies (Wilson and Holland, 1982). One must therefore consider toxicity to the surface as well as through the surface. The local effects of an irritant or caustic material on the skin or gut might be quite dramatic but the systemic effects might only be marginal. Alternatively, compounds such as DDT are likely to have relatively insignificant local effects upon the skin but can result in very severe systemic toxicity and death if administered topically.

For completeness, it should also be remembered that toxicity to the skin or lungs does not necessarily depend upon the chemical being administered by that route — thallium does not have to be given topically to cause baldness nor does paraquat have to be inhaled to result in lung lesions.