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Current guidelines for toxicity testing: Their strengths and weaknesses
Vol. 28, No. 11, pp. 789-790, 1990 Printed in Great Britain.All rights reserved
0278-6915/90$3.00+ 0.00 Copyright ~', 1990PergamonPress plc
Fd Chem. Toxic.
C U R R E N T GUIDELINES FOR TOXICITY TESTING: THEIR STRENGTHS A N D WEAKNESSES R. KROES National Institute of Public Health and Environmental Protection, Antonie van Leeuwenhoeklaan 9, Postbus l, NL 3720 BA Bilthoven, The Netherlands
Other papers in these proceedings illustrate some of the difficulties involved in achieving adequate health risk assessments for the wide diversity of foods that are now available to the average consumer. In particular, the case studies demonstrate that for some dietary components it is physically impossible to comply with the existing guidelines, which are based on traditional toxicity testing. Furthermore, and of equal conceptual importance, if existing guidelines were to be used to set safe limits for some essential nutrients, for example vitamin A, selenium, iron etc., their toxicity is such that they would only be permitted at levels well below those required to maintain normal health. Current practice thus tacitly demonstrates that the benefit side of the risk/benefit equation has to be given due weight in the final evaluation of food safety, and the Working Group at the workshop had no difficulty in accepting the concept that different margins of safety could be, and indeed would have to be, established for different foods and food components. It is clear that lower safety margins are acceptable for essential nutrients than would be permitted for additives, processing aids and contaminants. For the last three categories, where the test compounds are present in small quantities (less than 1% of the food) the validity and importance of the existing OECD (1982) guidelines for toxicity testing was recognized and acknowledged by the Working Group. However, significant problems now arise in deciding how to assess the safety of new foods, nutrtent replacers and bulk food products, in such a way that the safety of the consumer is not compromised but at the same time, the potential for innovation and introduction of variety into the diet is not stifled. It was recognized by the Working Group that the OECD guidelines were developed a decade ago and had stood well the test of time. However, they now appear to be too rigid in terms of the number of animals involved, parameters used and endpoints studied, to be easily adaptable to meet new challenges in food safety. On the other hand it was acknowledged that the OECD guidelines had provided a basis for proper international comparison and acceptance of results so obtained, thus reducing the expenditure of animals, manpower and facilities. Any change in these guidelines thus needs to be scientificallyjustified and undertaken only with great care. A number of principles emerged from the discussion that the Working Group felt should be incorporated into any new strategies that may evolve from the existing guidelines.
It was recommended that foods and food ingredients should be tested at dose levels comparable with probable human exposure levels and one or two multiples beyond that. The practice of using the maximum tolerated dose level of a substance in order to maximize the chances of observing toxic damage may be satisfactory for pharmaceuticals and dietary micro-ingredients (though not always even for these categories). When applied to major dietary components, however, it can introduce confounding factors such as energy imbalance and/or disturbance in normal nutrient balance to the extent that any physiological or biochemical disturbances observed cannot be meaningfully interpreted. For many new foods and nutrient replacers it was agreed that physiological, biochemical and functional endpoints will need to be measured to ensure safety; nutritional disturbance rather than cytotoxicity may well be the determining factor for questioning the safety or acceptability of a dietary component. The effect of a dietary component on such parameters as the absorption of other nutrients, energy balance, the digestibility of other foods and the competence of metabolic synthetic and excretory pathways may be far more relevant to the assessment of the 'safety' of a new food than knowing its LDs0, even if that could be determined. Equally well, the validity of applying safety factors to a determined no-observable-effect level depends on which effects are observed. There was a consensus in the Working Group that nutritional and toxicological evaluation of foods should not be undertaken as isolated exercises but should be integrated into a co-ordinated evaluation protocol, and that the doses, endpoints and systems studied should be tailored to the overall aims of the study. These might differ according to the material being tested; thus the parameters used to assess the safety of bread made with a new bioengineered yeast mxght be quite different from those used to assess the health risk of a non-nutrient fat replacer. However, a basic assurance of absence of toxicity would always be required as well as an indication of the type of nutritional side effects, if any, that might be expected at high consumption levels (such as alterations in the absorption or utilization of other dietary components, and any disturbances in normal physiology). The absence of systemic toxicity including the absence of chronic disease should also be demonstrated. It was suggested that in any new guidelines, the importance of relevant controls with respect to energy density and nutrient availability should be
789
790
R. KROES
stressed. Also the Working Party emphasized that food safety assessment must include human trials and not be confined to animal models, which, in general, are inadequate for the detection of allergenicity and behavioural disturbances. Where the margins of safety appear to be small in terms of anticipated human exposure in relation to estimated lowest toxic effect levels, as they are, for example, for both naturally occurring and synthetic retinoids, the importance was emphasized of conducting well designed mechanistic studies to understand the biochemical basis of the toxic response. The potential for using cultured human as well as animal cells and tissues for in vitro mechanistic studies was highlighted. There is an opportunity here to develop guidelines and criteria for such mechanistic studies, which would cover the conditions under which in vitro studies should be done, the precautions to be taken, the ethical and scientific conditions to be fulfilled and the questions that such studies need to answer if they are to facilitate safety assurance. The intrinsic chemical properties of a substance and the initial observations made when it is first fed to an animal are of prime importance in determining the extent and type of toxicity testing that will be necessary. The Working Group was of the opinion that a case-by-case approach would be unavoidable if appropriate testing and evaluation was to be achieved, although elements of a decisiontree approach might be incorporated in the revised guidelines. The degree of ~risk' that is acceptable for a therapeutic drug designed to ameliorate other pathology, is conceptually quite different from that which is appropriate for food. Food, after all, provides not only the basic essential nutrients for normal healthy life but is also the source of much sensory satisfaction. A decision to revise the current guidelines would provide an excellent opportunity to re-evaluate the endpoints used to assess toxicity in the context of relevance to probable/possible effects of dietary change on human populations bearing in mind the
quality of life as well as basic safety and nutrition. Consumers are very aware of what they perceive as diet-related adverse effects such as food allergies, behavioural disturbances and long-term health effects such as obesity, cardiovascular disease, etc. Improved methods of assessing the long-term effects of human dietary components are still needed, but where information and endpoints are available they should be incorporated into revised guidelines. It was suggested that emphasis should be placed on improved methods for detecting any potential genotoxicity, neurotoxicity and immunotoxicity of dietary components. A retrospective analysis of adequately performed toxicity studies and how effective they had been as predictors of human response might help to rank the endpoints currently used in terms of more, less or no relevance to the human condition. Guidelines on detailed protocols for animal studies could have the additional bonus of reducing the numbers of animals required, if they were systematically designed to obtain more information from each animal. Thus, the inclusion in long-term toxicity studies of satellite groups for interim kills with careful immediate postmortem examination, should give information about developing target-organ toxicity and enable proper and appropriate examination and evaluation of animals surviving to the end of the long-term study. This approach would reduce the number of parameters that had to be studied and focus the attention of the investigator on those phenomena that appeared to be important to safety assurance. At present, all too often a study has to be repeated because a toxic effect emerges during the final post mortems that was not anticipated and that was not properly controlled for. A careful reassessment of endpoints in the context of different, specific toxic effects that are relevant to man would be a valuable basis for any such revision. It seems likely that although some currently used measurements, for example extensive haematology and urine analyses, may be dropped, others such as indicators of immunological and endocrinological function could be included with advantage.
0278-6915/90$3.00+ 0.00 Copyright ~', 1990PergamonPress plc
Fd Chem. Toxic.
C U R R E N T GUIDELINES FOR TOXICITY TESTING: THEIR STRENGTHS A N D WEAKNESSES R. KROES National Institute of Public Health and Environmental Protection, Antonie van Leeuwenhoeklaan 9, Postbus l, NL 3720 BA Bilthoven, The Netherlands
Other papers in these proceedings illustrate some of the difficulties involved in achieving adequate health risk assessments for the wide diversity of foods that are now available to the average consumer. In particular, the case studies demonstrate that for some dietary components it is physically impossible to comply with the existing guidelines, which are based on traditional toxicity testing. Furthermore, and of equal conceptual importance, if existing guidelines were to be used to set safe limits for some essential nutrients, for example vitamin A, selenium, iron etc., their toxicity is such that they would only be permitted at levels well below those required to maintain normal health. Current practice thus tacitly demonstrates that the benefit side of the risk/benefit equation has to be given due weight in the final evaluation of food safety, and the Working Group at the workshop had no difficulty in accepting the concept that different margins of safety could be, and indeed would have to be, established for different foods and food components. It is clear that lower safety margins are acceptable for essential nutrients than would be permitted for additives, processing aids and contaminants. For the last three categories, where the test compounds are present in small quantities (less than 1% of the food) the validity and importance of the existing OECD (1982) guidelines for toxicity testing was recognized and acknowledged by the Working Group. However, significant problems now arise in deciding how to assess the safety of new foods, nutrtent replacers and bulk food products, in such a way that the safety of the consumer is not compromised but at the same time, the potential for innovation and introduction of variety into the diet is not stifled. It was recognized by the Working Group that the OECD guidelines were developed a decade ago and had stood well the test of time. However, they now appear to be too rigid in terms of the number of animals involved, parameters used and endpoints studied, to be easily adaptable to meet new challenges in food safety. On the other hand it was acknowledged that the OECD guidelines had provided a basis for proper international comparison and acceptance of results so obtained, thus reducing the expenditure of animals, manpower and facilities. Any change in these guidelines thus needs to be scientificallyjustified and undertaken only with great care. A number of principles emerged from the discussion that the Working Group felt should be incorporated into any new strategies that may evolve from the existing guidelines.
It was recommended that foods and food ingredients should be tested at dose levels comparable with probable human exposure levels and one or two multiples beyond that. The practice of using the maximum tolerated dose level of a substance in order to maximize the chances of observing toxic damage may be satisfactory for pharmaceuticals and dietary micro-ingredients (though not always even for these categories). When applied to major dietary components, however, it can introduce confounding factors such as energy imbalance and/or disturbance in normal nutrient balance to the extent that any physiological or biochemical disturbances observed cannot be meaningfully interpreted. For many new foods and nutrient replacers it was agreed that physiological, biochemical and functional endpoints will need to be measured to ensure safety; nutritional disturbance rather than cytotoxicity may well be the determining factor for questioning the safety or acceptability of a dietary component. The effect of a dietary component on such parameters as the absorption of other nutrients, energy balance, the digestibility of other foods and the competence of metabolic synthetic and excretory pathways may be far more relevant to the assessment of the 'safety' of a new food than knowing its LDs0, even if that could be determined. Equally well, the validity of applying safety factors to a determined no-observable-effect level depends on which effects are observed. There was a consensus in the Working Group that nutritional and toxicological evaluation of foods should not be undertaken as isolated exercises but should be integrated into a co-ordinated evaluation protocol, and that the doses, endpoints and systems studied should be tailored to the overall aims of the study. These might differ according to the material being tested; thus the parameters used to assess the safety of bread made with a new bioengineered yeast mxght be quite different from those used to assess the health risk of a non-nutrient fat replacer. However, a basic assurance of absence of toxicity would always be required as well as an indication of the type of nutritional side effects, if any, that might be expected at high consumption levels (such as alterations in the absorption or utilization of other dietary components, and any disturbances in normal physiology). The absence of systemic toxicity including the absence of chronic disease should also be demonstrated. It was suggested that in any new guidelines, the importance of relevant controls with respect to energy density and nutrient availability should be
789
790
R. KROES
stressed. Also the Working Party emphasized that food safety assessment must include human trials and not be confined to animal models, which, in general, are inadequate for the detection of allergenicity and behavioural disturbances. Where the margins of safety appear to be small in terms of anticipated human exposure in relation to estimated lowest toxic effect levels, as they are, for example, for both naturally occurring and synthetic retinoids, the importance was emphasized of conducting well designed mechanistic studies to understand the biochemical basis of the toxic response. The potential for using cultured human as well as animal cells and tissues for in vitro mechanistic studies was highlighted. There is an opportunity here to develop guidelines and criteria for such mechanistic studies, which would cover the conditions under which in vitro studies should be done, the precautions to be taken, the ethical and scientific conditions to be fulfilled and the questions that such studies need to answer if they are to facilitate safety assurance. The intrinsic chemical properties of a substance and the initial observations made when it is first fed to an animal are of prime importance in determining the extent and type of toxicity testing that will be necessary. The Working Group was of the opinion that a case-by-case approach would be unavoidable if appropriate testing and evaluation was to be achieved, although elements of a decisiontree approach might be incorporated in the revised guidelines. The degree of ~risk' that is acceptable for a therapeutic drug designed to ameliorate other pathology, is conceptually quite different from that which is appropriate for food. Food, after all, provides not only the basic essential nutrients for normal healthy life but is also the source of much sensory satisfaction. A decision to revise the current guidelines would provide an excellent opportunity to re-evaluate the endpoints used to assess toxicity in the context of relevance to probable/possible effects of dietary change on human populations bearing in mind the
quality of life as well as basic safety and nutrition. Consumers are very aware of what they perceive as diet-related adverse effects such as food allergies, behavioural disturbances and long-term health effects such as obesity, cardiovascular disease, etc. Improved methods of assessing the long-term effects of human dietary components are still needed, but where information and endpoints are available they should be incorporated into revised guidelines. It was suggested that emphasis should be placed on improved methods for detecting any potential genotoxicity, neurotoxicity and immunotoxicity of dietary components. A retrospective analysis of adequately performed toxicity studies and how effective they had been as predictors of human response might help to rank the endpoints currently used in terms of more, less or no relevance to the human condition. Guidelines on detailed protocols for animal studies could have the additional bonus of reducing the numbers of animals required, if they were systematically designed to obtain more information from each animal. Thus, the inclusion in long-term toxicity studies of satellite groups for interim kills with careful immediate postmortem examination, should give information about developing target-organ toxicity and enable proper and appropriate examination and evaluation of animals surviving to the end of the long-term study. This approach would reduce the number of parameters that had to be studied and focus the attention of the investigator on those phenomena that appeared to be important to safety assurance. At present, all too often a study has to be repeated because a toxic effect emerges during the final post mortems that was not anticipated and that was not properly controlled for. A careful reassessment of endpoints in the context of different, specific toxic effects that are relevant to man would be a valuable basis for any such revision. It seems likely that although some currently used measurements, for example extensive haematology and urine analyses, may be dropped, others such as indicators of immunological and endocrinological function could be included with advantage.