Strategies for toxicity testing of food chemicals and components

Fd Chem. Toxic. Vol. 28, No. 1I, pp. 735-738, 1990 Printed in Great Britain

0278-6915/90 $3.00+ 0.00 Pergamon Press plc

STRATEGIES FOR TOXICITY TESTING OF FOOD CHEMICALS A N D COMPONENTS D. M. CONNING The British Nutrition Foundation, 15 Belgrave Square, London SWIX 8PS, UK

Introduction The now traditional approach to food chemical toxicology is based on three major assumptions. First, all 'natural' food and food processed by methods developed before the emergence of synthetic organic chemistry as a separately recognizable human activity (around 1850) are deemed safe. Thus, food processes that depend on fermentation or acid production by bacteria for example, using time-hallowed procedures, are not subject to critical evaluation. Secondly, a synthetic chemical or one extracted and purified according to modern methods may be safely consumed provided that the daily dose does not exceed one-hundredth of the highest dose found to induce no detectable biological effects in any laboratory animal. Thirdly, given that food is consumed from birth until death, and toxicity has to be related to a possible lifetime exposure, the lifespan of a laboratory animal may be assumed to equate with the lifespan of man. Over the years these assumptions have been eroded remarkably little, though there have been changes. In studies of carcinogenesis, for example, the no-effect level is not usually employed but replaced by a negative response from the maximum dosage that does not cause premature death. The no-effect level itself is often taken as the no-adverse-effect level (dosage), though rarely is the term 'adverse' defined because of the difficulty in establishing the boundaries of normal values in biological systems. Frequently, the significance of an effect on the diagnosis of premature death relies on mathematical rather than biological analysis. Toxicologists have long complained of the lack of scientific rationale inherent in these assumptions and have been frustrated at the willingness of the regulatory authorities to consider alternative approaches only if the traditional data are also provided. The frustration arises because it is now very expensive in terms of both money and time to provide this basic data given the range of biological assays and the depth of analysis demanded by the conventional approach. Few manufacturers are willing to meet the additional costs. Despite the extraordinary amounts of information generated, two fundamental questions remain unanswered in all respects: (1) what is the biological basis of the dose-response relationship? (2) Is the lifespan of, for example, the rat equivalent to that of man? The first question is important because the lack of such an understanding does not permit the assump-

tion that man will be less than 100 times more sensitive than the rat to a given toxic effect. This is certainly not true in respect of immunotoxicity. On the other hand, human DNA repair mechanisms appear to be much more resistant to toxic interference and man may be much less sensitive to reparable defects (Frosina and Abbondandolo, 1985). The second question is assuming increasing importance as we recognize the interdependence of degenerative disease, ageing, heritable characteristics and dietary influences. Is it conceivable that ageing is an inverse function of DNA repair? If so, chronic toxicity might be dependent on the ability of a chemical to which low level exposure occurs for prolonged periods, to erode the capability of DNA repair. Similarly, there may be nutritional influences, though as yet the putative mechanisms are confined to the simple notion of nutrient overload. Two other developments should be considered as part of the background to further thinking on toxicity testing. In recent years the movement against the use of animals in research has achieved substantial publicity and, as always, this has had an effect on our political masters whatever the public may think. There has been a tendency, therefore, to consider reducing the use of animals whatever the biological necessities dictate. This in itself has led to the second development, namely demands for an increase in the use of human tissues and human volunteers in safety evaluation. In addition, many toxicologists believe that interspecies extrapolation is on such shaky ground, at present, that human data are indispensable. Against this conceptual background, then, what is the strategy for the future development of toxicity testing of compounds administered at low dosages? First, three basic premises must be accepted: (1) some studies with animals are necessary simply because we are not yet in a position to predict the complete range of biochemical reactions to which a given chemical will be subjected, (2) The range of metabolic transformations that occurs in the laboratory animal will serve as a qualitative model for man but might not serve as a quantitative model. That is, a fairly complete range of metabolic changes can be detected in a number of species but usually one predominates, and that one may be species-specific. It follows that some studies in man or with human tissues will be necessary before a prediction of human toxicity can be made where metabolic transformation is an essential prerequisite for the production of a toxic effect. (3) Further research is required before a new system of risk assessment can be established. Such research

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must be organized against specific agreed objectives and conducted in several laboratories for which some co-ordinating mechanism is required.

Strategy for toxicity testing of food chemicals Three phases of analysis are required.

Phase I studies (1) All studies would be preceded by structureactivity analyses in an attempt to obtain guidance on, for example, target organ(s) or principal metabolic pathways. It is now possible for experienced biochemists to predict probable metabolic pathways from the molecular configuration of a compound and in some instances this approach can be formalized. The prediction cannot, however, be certain. It is unlikely that much progress will be made until more is known about the pure chemistry of biological reactions (Gangolli and Phillips, 1988). Allied to these analyses would be some priority setting related to how far the test compound is likely to be alien to biological systems; and to the scale of production and consumption. (2) An elaborate 90-day feeding study in an outbred strain of rat utilizing enough dose levels to establish a dose-response relationship. Lower dose levels should, where possible, be related to the expected concentration of the compound in use. The higher dose levels should be designed to induce effects. During the course of this study the following studies should be undertaken. (a) Biochemical. (i) Absorption/excretion studies; (ii) tissue distribution; (iii) principal metabolites: (iv) accumulation and adaptation. The aim of these studies is: to equate metabolic disposition to emergent toxicology; to define the limits of normal metabolism and determine the relationship of dose response to abnormal transformations; to determine whether enzyme induction influences the toxicological potential; and to better characterize target tissue responses. (b) Clinical pharmacology. Functional studies of: (i) cardiovascular system; (ii) central nervous and locomotor systems; (iii) gastro-intestinal tract; (iv) genito-urinary system; and (v) pulmonary system. The aim of these studies is to identify functional impairment not associated with morbid histological change. (c) Pathology. To identify target tissue responses, to relate the dose-response relationship where possible to severity of tissue damage and to provide clues to organelle toxicity. The latter would require selected ultramicroscopic studies. The pathologist needs to be well trained in experimental pathology designed to investigate the biochemical mechanisms involved in toxic phenomena. It will not always be enough merely to classify lesions according to conventional pathology. (3) Genotoxicity screening procedures These studies should be designed to detect whether the test substance can interact with and damage DNA and also to determine whether such damage can be repaired in mammalian systems. It will be important to determine whether residual DNA adducts are

formed. Many schemes (batteries) have been devised, none entirely satisfactory because of the lack of human data. This position might be improved if more analyses based on molecular biology could be undertaken but it is unlikely that such progress will be made until the discipline itself is more advanced. The information acquired from phase 1 studies would indicate: (1) overt or functional toxicity in a dose range related to use: (2) the role of the primary compound or a metabolite in relation to specific tissue toxicity: (3) subcellular organelles implicated in a toxic event: (4) possible excretion products that might be used to define that a toxic process had occurred and therefore serve as markers of such an event in humans; (5) whether irreversible interaction with DNA could occur. The relevance of findings to the concept of nongenotoxic carcinogenicity would be judged by the type of tissue damage and reaction that occurred. For example, a chronic inflammatory response to recurrent tissue necrosis or a persistent reactive hyperplasia with or without tissue damage would raise suspicion. This body of information might be enough in itself to determine whether further development of the compound was warranted. If, for example, toxicity was associated with a particular metabolic route it would be necessary to ascertain whether that route occurred in man and to what extent. If irreversible interaction with DNA was demonstrated, or there was interference with DNA repair, it might be deemed prudent to abandon the compound. These decisions could only be taken in the light of other information such as the economic impact of the compound, but for food use the balance would weigh heavily against further development. If, however, no adverse effects were detected, or it could be argued that the findings were not relevant to man, the investigation should proceed to phase 2.

Phase 2 studies These would involve primarily: (1) Embryotoxicity and teratology. Although animal models exist and are extensively used for such studies there is continuing uncertainty as to their validity for man (Lansdown, 1983). If it can be agreed that such studies are valid, they can be used as definitive indicators. If not, a debate on the ethical issue of using human embryos should be generated. A great deal of work is required to elucidate the relationship between specific and probably focal embryotoxicity and consequent teratology defined here as abnormal development compatible with life. (2) Adverse effects on lymphocyte function as the basis of an immunotoxic event. As yet it is doubtful whether the technology of immunotoxicity testing can define a suitable method to determine immune function (Parish, 1981). Additional studies in phase 2 could involve human organ or cell cultures to elucidate whether the primary events observed at phase 1 do occur and if so by the same processes in human tissues. The determination of reproductive potential is not measured directly in this scheme. Sperm function is determined as part of the genito-urinary screens and ovarian toxicity during pathological analysis, If a

Testing food chemicals and components teratological study is deemed necessary, this can be used to determine fertility. Three-generation reproduction studies as currently practised are cumbersome and of little relevance to the study of food additives or contaminants.

Phase 3 studies In the event that studies in phases 1 and 2 do not indicate that serious and irreversible toxicity occurs it would be necessary to proceed to volunteer studies in man. These would be akin to the clinical trials of therapeutic agents but would employ very small dosages and be designed to determine the principal metabolites excreted. The rationale is to identify the primary metabolic pathway in man in comparison with the laboratory species in which toxicity studies are undertaken. In due course, this phase could involve in vitro studies with human cell or organ cultures. Given the outbred nature of man it may prove difficult to generate a reliable databank, and considerable effort will be required to classify the reactivity of cell lines according to toxic responses. Conclusions The information generated by the studies outlined would allow the following conclusions: (1) a toxicological profile in the laboratory animal including the dose response, the role of metabolism and the primary cell dysfunction. (2) The likelihood of irreversible interaction with DNA by the compound or a metabolite and the consequences of that in terms of DNA repair. (3) A crude measure of embryotoxicity, teratology and immunotoxicity. (4) Whether the experimental model is likely to be appropriate for predicting human effects and whether the dosages employed are pertinent. Clearly, further research is required before these suggestions can be considered seriously for regulatory purposes. The priority objectives are: (1) an effective procedure for detecting human embryotoxicity and human teratology; (2) an effective procedure for detecting human immunotoxicity; and (3) the development of human cell cultures and exploration of the influence of heritable characteristics on toxic responses in human tissues. Strategies for the testing of new foods Increasingly, food scientists can be expected to develop materials that fulfil all the desirable characteristics of a nutrient with few or none of the undesirable. We should recognize that 'desirability' is often dictated by fashion rather than science but we may consistently expect there to be a quest for satisfying foods with reduced energy delivery. This will be achieved either by increased content of zero-energy components or by resistance to absorption from the gut. Three types of novel foods can be identified, therefore: (1) non-absorbed materials; (2) diluted materials; and (3) straight replacements. Examples would be olestra (Bernhardt, 1988), mycoprotein (by virtue of its increased fibre content compared with meat) (Edelman et al., 1983) and any type

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of bacterial biomass, respectively. Where one nutrient is made to mimic another (e.g. simplesse) it seems very unlikely that a toxicological problem could exist. It is unlikely that such foods will completely replace the nutrient they mimic but it is possible that they will achieve levels of incorporation up to approximately 15% of the normal equivalent nutrient content. For example, the use of olestra initially will be to replace 5% of energy from fat. A person utilizing 2000 kcal/day and getting, say, 40% of that energy from fat eats 89 g fat/day. To reduce this by 5% energy means eating about 11 g olestra/day (5/40 x 89), that is about 12% of total fat. The average consumption is likely to be around 7 g/day. The toxicological examination of such products must be considered in three phases.

Nutritional evaluation The principle here is that the substitute nutrient must fulfil the nutritional properties, both positive and negative, claimed for it, in a normal animal and in man. This means that paired feeding studies are required in which the normal nutrient component is replaced at different levels of incorporation, related to the intended use, for sufficient periods of time to detect significant changes in growth rates. It will of course be easier to conduct such studies with synthetic diets where the concentrations of purified nutrients can be more easily manipulated. The aim is to detect variations in the efficiency of food utilization. A novel nutrient that adversely affected food utilization, outwith the variability seen in paired feed studies, would be suspect. The levels of incorporation in the diet might include one that could induce nutrient toxicity (e.g. excess protein), though in practice this ought not to be necessary. Such studies should be repeated with materials that have been subject to the kind of processing they are likely to receive in practice to ensure that heating or freezing, or the production of fine emulsions utilizing emulsifiers and stabilizers, do not adversely affect the nutrient properties. Toxicological studies These would vary with the type of material under examination. (a) Non-absorbed materials. The objectives should be: (i) to ensure absorption does not occur; (ii) to ensure there are no adverse effects in the gut itself; (iii) to ensure that the material does not interfere with the absorption of other nutrients: and (iv) to determine any interactions with gut flora. (b) Energy-diluted materials. The toxicology here depends on the nature of the diluting non-absorbed or energy-free material. Where this too is a normal constituent of the diet (e.g. fibre) specific toxicity testing does not seem warranted. (c) Replacement materials. These rarely consist of the pure nutrient but come with cell-wall materials and nucleic acids. The latter are more of a theoretical problem than real in that the concentrations are easily reduced by hydrolysis or spray-drying procedures, which would be a normal part of the manufacturing process. The presence of unusual components such as lipopolysaccharides or branched-

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chain fatty acids should be excluded as part of the analytical procedures for specification purposes. The toxicological assessment of these materials must depend initially on a long-term animal study in which the material is incorporated into the diet at the intended level of use with one or two multiples of that level. With most materials it is likely that the nutritional and toxicological evaluation can be combined, but with additional ancillary studies indicated in section (a). The efficiency of food utilization can itself be an indicator of toxic effect but the main purpose will be to determine the relative incidence of chronic disease including turnouts. The selection of control materials is an important consideration. These should resemble as closely as possible the nutrient that the novel material is intended to replace. For example, the main objective of the use of olestra would be to replace saturated fatty acids so that the control material would be a triglyceride with predominantly saturated fatty acid composition. Similarly, with protein replacements, the amino-acid profile should be matched.

of a defective nutrient. This would be more important, obviously, with materials designed not to be absorbed but might also be necessary where aminoacid profiles are changed. Overall, it is unlikely that the use of alternative nutrients is likely to raise serious toxicological problems and it would be a mistake to exaggerate this aspect of the safety assessments provided the novel material is sufficiently well characterized chemically to exclude the presence of unidentified compounds. Safety evaluation or risk assessment is the essence of applied science. Hitherto the role of a study of toxicology based on mechanisms has been devalued because of the length of time required. If, however, such studies can be constrained by the objectives defined here, it is possible that toxicological assays could be better related to the mechanisms of action without requiring 20 years of basic research. It is, of course, axiomatic that an understanding of mechanisms is a prerequisite for interspecies extrapolation.

Human studies

Bernhardt C. A. (1988) Olestra -a non-caloric fat replacement. Fd Technol. hm, pp. 176 178. Ede[man J., Fewell A. and Solomons G. L. (1983) Mycoprotein a new food. Nutr. Ahstr. Rec. 53, 471 480. Frosina G. and Abbondandolo A. (1985) The current evidence for an adductive response to alkylating agents in mammalian cells. Mutation Res. 154, 85- 100. Gangolli S. D. and Phillips J. C. (1988) The metabolism and disposition of xenobiotics. In Experimental Toxicology. Edited by D. Anderson and D. M. Conning. pp. 130 179. Royal Society of Chemistry, London. Lansdown A. B. G. (1983) Teratogenicity and reduced fertility resulting from factors present in food. In Toxic Hazards m Food. Edited by D. M. Conning and A. B. G. Lansdown. pp. 73 121. Croom Helm, London. Parish W. E. (1981) Immunological tests to predict toxicological hazards in man. In Testing[or Toxicity. Edited by J. W. Garrod. Taylor & Francis, London.

Three types of studies are envisaged. (1) Balance studies that determine the relative turnovers of the nutrient of primary importance such as fat or nitrogen. Such studies should also look at the status of micronutrients and minerals that might be adversely affected, such as fat soluble vitamins and calcium when examining fat replacements. A positive result would not necessarily be construed as adverse but might indicate the need for supplementation. (2) Clinical studies on patients with, for example, a compromised gut or other disease process (e.g. defective hepatic or renal function) that could render the individual more vulnerable to adverse affects. (3) Total energy balance studies to determine whether the individual compensates for the presence

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