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C.A. Shacklady
Institute for Animal Nutrition Research (ILOB) Wageningen, Netherlands
Toxicological evaluations present, in general, three problems The first is inherent in the science itself; the others relate to the nature of the material to be evaluated and the circumstances in which its use is contemplated.
By definition, the inherent problem will exist regardless of the type of product or its intended use, and can be stated in very simple terms. Nothing is totally innocuous, so every toxicological evaluation resolves itself into the assessment of an acceptable level of risk because it cannot be based upon absolute criteria.
The problem associated with the nature of the material derives from the fact that the accepted classical evaluation procedures were not developed with bioconversion products in mind. With the exception of biogas, these fall into the category of materials now known, generically, as single-cell protein (SCP), comprising yeasts from fermentation, algae from photosynthesis, and fungi and bacteria from fermentation, with or without photosynthetic intervention. Experimental procedures designed for quantitatively minor additives and pharmacologically active agents must be modified to take account of the practical limitations imposed when evaluating products intended to form a significant proportion of the dietary intake.
The third type of problem - the one concerned with the circumstances in which a material may be used - can be subdivided into two parts. In broad terms, these could be regarded as the practical, and the ethical or philosophical considerations about the use of the product. The former would take into account such factors as whether the material would be used on a regular or intermittent basis and the probable level of intake in either event
The ethical consideration is, in fact, related to the inherent problem of toxicological evaluation in that the circumstances in which a product may be used will have a major bearing on the degree of risk that could be regarded as acceptable. As has already been said, absolute safety does not exist; consequently, in any given situation, the question arises as to the balance between the risk attendant upon the use of a product and the benefit that would accrue. Clearly, this ratio will vary according to circumstances; what might be acceptable in one case could be quite unacceptable in another.
Having stated the problems, it becomes necessary to define them in more depth to see if the potential for solving them exists within the framework of the general programme under consideration.
In the present state of knowledge, it is improbable that any solution to the fundamental shortcomings of the science itself will emerge in the foreseeable future.
The need to determine the acceptability of single-cell proteins as food or feed ingredients required some adjustments to be made in existing toxicological procedures. These had been developed for materials that, although frequently highly active biologically, formed a quantitatively insignificant proportion of the dietary intake. The procedures allowed the construction of response curves to a wide range of dose levels in experimental animals and, in many cases, required the administration of the test material by more than one route. SCPs, on the other hand, were intended to make a significant quantitative contribution to the nutrient intake and would be ingested, thus imposing constraints that did not exist in the case of drugs or similar additives.
Nonetheless, a practical scheme that took these limitations into account, but retained the principles of the classical approach, was developed for yeasts grown on hydrocarbons and was adopted in 1964 by the Central Institute for Nutrition Research (CIVO) in Zeist, Netherlands. Concurrently with this, the Institute for Research in Animal Nutrition (ILOB) in Wageningen, Netherlands, was carrying out the nutritional evaluation of the same materials in farm animals. These studies examined the short-, medium-, and long-term effects of an SCP on growth and reproduction in experimental animals and have been reported in some detail by Engel (1) and de Groot and co-workers (2 - 5). In the early 1970s, the then Protein-Calorie Advisory Group (PAG) of FAD/WHO/ UNICEF published Guideline Nos. 6 and 7 (6,7) dealing with the pre-clinical and clinical evaluation of novel protein sources, and these were followed by Guideline No. 15 (8). The first two pertained primarily to the use of SCP by man, whereas No. 15 was directed towards use for animals. These guidelines have formed the basis for those subsequently adopted by various national regulatory authorities, and do not differ in principle from the CIVO/ILOB approach
The programmes recommended in these guidelines have proved satisfactory in evaluating the toxicological and nutritional characteristics of SCP. It might be assumed, therefore, that no major problem would be encountered in applying them to any bioconversion products. In practice, this is not so. The reason is that these evaluations are both extensive in scope and expensive in implementation Neither the facilities nor the financial support to conduct them are likely to be available in the areas where the need is greatest. Experience to date suggests that, in these circumstances, the toxicological evaluation of products derived from bioconversion on the farm or in rural communities will almost certainly be neglected. The situation is also aggravated by the fact that the nature and quality of products from organic residues may vary according to the composition of the residues themselves. In contrast to this, SCP produced by industry is generally, and justifiably, assumed to be acceptably constant in composition and to conform with that previously tested.
It is not the intention here to discuss the possible effects of production variables upon the toxicological and nutritional characteristics of bioconversion products other than to draw attention to them as factors that should not be ignored. From what has been said, however, it may be gathered that it would be irresponsible to use these materials as food or feed ingredients without attempting to assess their toxicological and nutritional acceptability. At the same time, it would be unrealistic to expect that, in the circumstances in which they will be produced, they would, or could, be subjected to the exhaustive evaluation applied to SCP produced on a large scale by industrial plants.
This introduces the third type of problem, which, in essence, is whether or not this apparent impasse may be avoided. Bear in mind that no absolute determination of product safety can be made because absolute safety does not exist. The question is, therefore, can a practical method for adequately evaluating toxicity of small-scale rural bioconversion products be designed?
It is suggested that this can be done by a modification of what may be regarded as the classical procedure outlined in the PAG Guidelines, taking advantage of very recent developments reported since they were published.
The modification lies in the area of the long-term feeding studies that have been a feature of the classical approach and that, because of their complexity and duration, have effectively eliminated themselves from consideration in rural projects. These long-term studies for orally administered materials required them to be given at more than one dosage level for the full lifespan of two species of test animal. Administration commenced with the pregnant female because of the possibility of transfer of toxic materials to the fetus, and was continued until only 20 per cent of animals in the subsequent litters were still alive. It is evident that this procedure cannot be applied to the target species for which the product is intended, whether this be man or farm animals. In the case of man, ethical considerations alone would prohibit it, and in farm animals the extended lifespan and the impracticability of having treatment groups large enough to give statistically significant results would preclude such studies. Even using the short-lived laboratory animals, in particular the rat, and evaluation of this nature would require several hundred animals and could take up to four years to complete.
United States Food and Drug Administration regulations, which are probably as stringent as any, propose as acceptable a one in one-million level of risk and consider this to be ". . . of insignificant public health concern" (9). Here may be seen the application of the concept of risk/benefit ratio and the introduction of the acceptability of a risk rather than a declaration of absolute safety. The concept cannot be criticized; the acceptable level of risk is a matter of judgement, and there is nothing to suggest that this has not been the subject of deep deliberation and extensive discussion by those competent to judge At the same time, it must be remembered that the judgement is made in the context of conditions - social, political, economic, and industrial - prevailing in the United States of America.
What is now suggested is that a programme be designed based essentially on short-term studies, that is, chemical analyses of feeding studies in laboratory and/or target species of farm animal, with in vitro or in vivo mutagenicity studies as indicators of possible long-term hazard. In parenthesis it might be added that a consideration of these could be incorporated into a review of PAG Guideline 15.
A considerable amount of information may be obtained in a relatively short time by exhaustive chemical analysis of a bioconversion product. The nature of the substrate and the process conditions may indicate areas of potential danger. Thus, algae grown on waste water, or organisms grown on waste liquor from the paper industry may contain undesirably high levels of toxic elements such as lead, cadmium, etc. If crop residues are used as a substrate, it may be possible to determine whether or not any significant amount of pesticide or herbicide has been carried over into the biomass. The presence of unusual components, or unusually high levels of more common components, should be noted as a possible hazard.
Results obtained by chemical analysis of the biomass should be regarded as indicative rather than predictive of potentially adverse effects. For example, a high concentration of an undesirable element in the biomass does not necessarily mean that it would be transferred to an animal eating that biomass. It might be unavailable biologically and excreted rather than stored. This can, and should, be determined in animal feeding studies.
Short term screening studies on the target species of animal are described in the paper by van Weerden in these proceedings, so little will be said about them here except to add that it would always be desirable to supplement these by a well designed but simple 90-day study in rats. Histopathological examination of the more important organs and tissues will give a very good indication of the likelihood of there being any chronic toxic, but not carcinogenic, effect. Feeding studies with target species of animals can also provide useful information in this respect and, in particular, chemical analysis of the edible portions should determine whether or not undesirable or toxic components have accumulated to an extent likely to present a hazard to the consumer.
There remains the question of potential carcinogenicity. The standard method in current use, as has already been noted, is lifespan feeding studies in laboratory animals. A major disadvantage of this type of evaluation lies in the length of time and the facilities required to carry out a study that will give valid results. It was this that encouraged the search for more rapid alternatives that would require fewer facilities. Tests now exist that do not require extensive animal housing and maintenance facilities, and that can be completed in a few days or weeks.
All of these tests are based on the mutagenic or nonmutagenic activity of the material under examination, involving the use of certain bacteria and mammalian cells in vitro or in vivo. A recent report of the Committee of the European Environment Mutagen Society (10) contains the following passage: "Carcinogenic substances have, in most cases, produced positive results in screening programmes for mutagens in contrast to non-carcinogens. The theoretical basis for this correlation has yet to be fully established although it seems likely from available evidence that DNA damage is intimately involved in both processes. Screening tests for mutagens thus serve a useful additional function in identifying potential carcinogens."
There appears to be no single test that will detect every type of mutation, and it is necessary to employ a battery of tests to cover the whole spectrum. Probably the best known and most widely used single test at present is that developed by Ames and his coworkers (11), in which S. typhimurium mutants have been selected for sensitivity and selectivity in their ability to revert from requiring histidine to histidine independence in the presence of a wide variety of mutagens. While this method is not well suited to the screening of proteinaceous materials as such, it has been used with success on extracts from such materials by Pamukcu et al. (12).
Renner and Münzner (13) have described the use of various mammalian test systems in the mutagenic evaluation of bacterial protein preparations, and there seems to be no reason why these systems could not be used for other proteins.
Although a considerable degree of expertise is needed in the conduct of these tests, as well as experience in their interpretation, they are now being performed on a routine basis in a number of institutes. Compared to lifespan feeding studies with laboratory animals, they are very rapid and inexpensive. Moreover, they do not require equipment other than that generally found in a moderately well-equipped microbiological laboratory. They appear to be well-suited to the facilites available in the biological departments of universities and research institutes in areas where rural development is receiving most attention.
The present view is that there is a correlation of more than 90 per cent between the mutagenic and carcinogenic activity of the several hundred chemical compounds so far examined. The suitability of various tests for predicting carcinogenicity has been surveyed in a paper by McCann et al. (14). In a recent paper Parke (15), in considering carcinogenicity, mutagenicity, and reproductive effects, refers to a new screening technique of McMahon et al. (16) for mutagenicity, and warns that results of in vitro mutagenicity studies must be interpreted with care before they can be extrapolated to in vivo carcinogenesis.
Although the mutagenicity studies mentioned were carried out on bacteria or in mammalian cell systems, these systems were in laboratory, not farm, animals. There does not appear to be any reason why, with the exception of the dominant lethal test for which sequential matings are necessary, the effect on at least some of the mammalian cell systems could not be examined in the target species of farm animal, possibly combining these studies with short term feeding studies, but to my knowledge. no reports of this nature have been published.
A further advantage of these mutagenicity tests is that, because of their short duration, they could be used to monitor the effect of process changes in the course of development of a production system. This would be out of the question in the case of lifespan studies.
In summary, therefore, it is suggested that the toxicological evaluation of products of bioconversion in rural areas requires a different approach from that taken by large industries. The main divergence from the procedure used in the latter is the substitution of short-term mutagenicity studies for lifespan studies as indicators of potential carcinogenicity. The 90-day rat feeding studies and relatively short-term experiments on target species of farm animals remain common to both situations. It is unrealistic to imagine that many of the products arising from small-scale projects would be evaluated in a manner identical to that adopted by industry. So far, there is no indication that this has been done, even when biomass preparations have been available.
The alternatives are either those suggested in this paper or virtually no testing at all - certainly no tests that would indicate chronic toxic or carcinogenic effects.
Circumstances do alter cases When circumstances require food supplies to be increased as a matter of necessity, the situation is different from that in which additions to the food available are novelties, not necessities It has been estimated that malnutrition in the developing countries is responsible for between six and twelve million deaths annually. In these circumstances, it is suggested that the long-term risk to benefit ratio consequent upon the adoption of the very recent abbreviated testing procedures described here would be acceptable and, certainly, infinitely preferable to the alternative of no tests to indicate such risk.
The problem is not the same in all countries, hence the solution is not necessarily the same. That is the premise upon which the suggestions outlined here are based.
The non-gaseous products from bioconversion of organic residues fall into the category of materials known generically as single-cell proteins (SCP). Methods for evaluating the potential toxicity of these materials in food or feed preparations have been developed and put into practice in recent years. In common with procedures for the similar evaluation of food additives and pharmacologically active substances required by most regulatory authorities, these methods are extensive, expensive, and time-consuming
It is doubtful whether either the facilities or the funds to carry out such procedures would be deployed for rural community projects in non-industrial countries There is, consequently, the danger that little or no long-term toxicological evaluation will be performed in these cases.
The suggestion is made that, when a new source of food is just a novelty, the situation is quite different from that prevailing when it is a necessity. A different problem may justify a different solution. To determine an acceptable level of safety in these cases for SCP produced by bioconversion processes, it is recommended that the long-term feeding studies with laboratory animals be replaced by the recently developed tests for mutagenicity, using bacterial or mammalian cell systems. These are rapid and relatively inexpensive; they do not require elaborate equipment, and give a high degree of correlation with the results obtained in studies of in vivo carcinogenicity.*
1. C. Engel, "Safety Evaluation of Yeast Grown on Hydrocarbons," in H. Gounelle de Pontanel (ed.), Proteins from Hydrocarbons, pp. 53 - 81, Academic Press, New York, 1972.
2. A.P. de Groot, M.P. Til, and V.J. Feron, "Safety Evaluation of Yeast Grown on Hydrocarbons. I. One-year Feeding Study in Rats with Yeast Grown on Gas Oil," Food Cosmet. Toxicol. 8:267 (1970).
3. A.P. de Groot, M.P. Til, and V,J. Feron, "Safety Evaluation of Yeast Grown on Hydrocarbons. II. One-Year Feeding Study in Rats with Yeast Grown on Pure e-Paraffins," Food Cosmet. Toxicol, 8:499 (1970).
4. A.P. de Groot, M.P. Til, and V.J. Feron, "Safety Evaluation of Yeast Grown on Hydrocarbons, lilt Two-Year Feeding and Multigeneration Study in Rats with Yeast Grown on Gas Oil," Food Cosmet. Toxicol. 9: 787 11971).
5. A.P. de Groot, H.C. Dreef-van der Meulen, M.P. Til, and V.J. Feron, "Safety Evaluation of Yeast Grown on Hydrocarbons. IV. Two-Year Feeding and Multigeneration Study in Rats with Yeast Grown on Pure e-Paraffins," Food Cosmet. Toxicol. 13:619 (1975).
6. Protein Advisory Group, Guideline No. 6: Preclinical Testing, 1972, United Nations, New York,
7. Protein Advisory Group, Guideline No.7: Human Testing of Supplementary Food Mixtures, 1972 United Nations, New York.
8. Protein-Calorie Advisory Group, Guideline No. 15: Evaluating Novel Proteins for Anima/Feeding, 1974. United Nations, New York.
9. FDA Criteria and Procedures for Evaluating Assays for Carcinogenic Residues. Federal Register 42. 10412, U.S. Food and Drug Administration, Washington, D.C., 1977.
10. European Environment Mutagen Society, "Mutagenicity Screening: General Principles and Minimal Criteria, "Biol. Zbl. 97: 217 (1978)
11. B.N. Ames, F.D, Leed, and W.E, Durston, ''An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens," Proc. Natl. Acad. Sci. (U.S.) 70. 782 (1973).
12. A.M. Pamukcu, E. Erturk, S. Yalciner, U. Milli, and G.T. Bryan, "Carcinogenic and Mutagenic Activities of Milk from Cows Fed Bracken Fern (Pteridium aquilinum)," Cancer Res. 38 (6): 1556 (1978).
13. H.W. Renner and R. Munzner, "Mutagenic Evaluation of Single Cell Protein with Various Mammalian Test Systems," Toxicology 10: 141 (1978).
14. J. McCann, E. Choi, E. Yamasaki, and B.N. Ames, "Detection of Carcinogens as Mutagens in the Salmonella Microsome Test Assay of 300 Chemicals," Proc, Natl. Acad. Sci. (U.S.) 72: 5135 (19751.
15. D.V. Parke, "Importance of Biochemical Parameters in Toxicological Evaluation of Food Additives and Contaminants," Proceedings of the 3rd World Congress on Animal Feeding (Madrid) 7:523 (1978).
16. R.E. McMahon, J.C. Cline, and C.Z. Thompson, "Assay of 855 Test Chemicals in Ten Tester Strains Using a New Modification of the Ames Test for Bacterial Mutagens," Cancer Res. 39 13): 682 (1979).