Symposium on the evaluation of the safety of food additives and chemical residues: III. The role of the chronic study in the laboratory animal for evaluation of safety

TOXICOLOGY

Symposium Chemical

AND

APPLIED

16, 507-512

PHARMACOLOGY

on the Evaluation Residues: III. The Laboratory Animal

(1970)

of the Safety Role of the for Evaluation

of Food Additives and Chronic Study in the of Safety’

PHILIPPE SHUBIK University

of Nebraska

College

of Medicine,

Omaha,

Nebraska

68105

I shall examine, from three standpoints, the necessity for performing chronic toxicity tests. First, examination will be made in the context of toxicity testing in animals and the question will be asked: How many effects are observed on a chronic basis that cannot be predicted from the observation of particular acute effects? Or phrased otherwise: How many chronic effects actually occur in the absence of acute manifestations ? In this framework many other detailed questions might be raised in an effort to define the best type of chronic test for various purposes. Second, chronic toxicity tests may be examined in the context of human disease and the question posed: How many diseases of man occur that are due to known toxic substances and that appear only after prolonged chronic exposure with no known premonitory signs? And as an addendum: How many diseases of man are unknown of origin but might be explained on such a basis? Third, the question may be put into the context of our technological civilization and discussed in theframeworkof the many new chemicals and other agents addedto our environment. Here the question might be posed in the form: What proportion of unknown substances are likely to have a toxic effect that can be detected only in long term studies ? In dealing with the first part of the first question: The most common reason for undertaking a chronic toxicity test is to detect possible carcinogenicity; at the present time it is assumed that the only unequivocal manifestation of carcinogenicity is the induction of a neoplasm. Indeed, in the terms of the interpretation of the Food Additives law, by the Food and Drug Administration, only the induction of a malignant neoplasm is used to classify an agent as a carcinogen. On many occasions hopes have been raised that an apparent carcinogen was really only inducing benign tumors and might be reprieved. This problem will be considered further in the context of chronic toxicity test procedures in general, but for the moment the important question of early manifestations will be considered. In some studies that I undertook about 10 years ago, with my colleague, Dr. Della Porta, we showed that a series of polycyclic aromatic hydrocarbon carcinogens that were considered to have little, if any, acute toxicity could induce a subacute toxicity picture reminiscent of the effects of X-ray-the progressive leukopenia with depletion of megakaryocytes to start with, then lymphocyte depletion and eventual death with many hemorrhages. Many other carcinogenic agents, including X-rays, certain alkylating agents, and urethan, had long been known to give rise to these “radiomimetic effects,” but these had not been observed in the instance of the polycyclic hydrocarbons. 1 Presented at the 8th Annual Meeting of the Society of Toxicology, Williamsburg, g-12,1969. 507

Virginia,

March

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The reason is that they had never been tested at sufficiently high dose levels (400 mg/kg) for the effect to be produced. Since that time, the curiously specific effect on the adrenal cortex by the carcinogenic hydrocarbon, 7,12-dimethylbenz(a)anthracene, has been studied exhaustively by Charles Huggins and others. The so-called “adrenal apoplexy” that occurs with large doses of this compound fed to the rat does not occur in the mouse, hamster, guinea pig, or rabbit (although in all these species similar neoplasms may be induced); this strange manifestation of acute toxicity peculiar to the rat may be due to a particular metabolite, although this is not sure. The metabolic disposition of carcinogenic chemicals is the crucial step in the initial determination of acute toxic manifestation. It is entirely possible that the “radiomimetic effects” of some carcinogenic compounds occur in relationship to the mysterious requirements of the induction of a neoplastic reaction; however, many carcinogens do not manifest this effect when given in large doses, but rather they either give rise to liver injury or are relatively nontoxic. However, in all these instances, it appears more than likely that the compounds in question are metabolized to “proximate carcinogens.” Transformation of the aromatic amine carcinogens to the N-hydroxymetabolites, so brilliantly discovered by the Millers, actually results in increase in quantity of metabolite excreted, with time. There have not yet been studies of metabolites of carcinogens for their acute effects for obvious reasons. Such studies will be performed in due course and may well assist us in the understanding of mechanisms of carcinogenesis. However, the knowledge that a compound may be metabolically converted to a more toxic substance over a period of time and that the relative amounts of different metabolites may also change over a period of time provides a solid basis for the requirement that only prolonged animal studies will provide the basis for negative findings. The viral oncologists currently use tissue culture systems to demonstrate “transformation” which is equated with oncogenicity. This test procedure, which involves the induction in a tissue culture system of manifestations, in the main, of loss of contact inhibition, is fraught with dangers. The same approach has been taken toward the investigation of chemical carcinogens. From a knowledge of the first principles of pathologic diagnosis, the use of tissue cultures is not likely to be able to provide an unequivocal basis for diagnosis of a neoplasm. It is entirely possible that some tissue culture procedure may well provide us with a screening method, and many of us are busy examining this possibility. At the present time, however, no characteristic findings exist that could be used to establish either a negative or a positive finding with confidence. The question whether or not any carcinogen can induce a tumor without having any initial manifestations has been asked often. In the system for studying the two stages of skin carcinogenesis using an initiating dose of a carcinogen followed by croton oil or some other promoting agent, no demonstrable changes are induced in the first phase. Thus, less than a microgram of a polycyclic hydrocarbon may be applied to skin, and such a tissue may be demonstrated to be “initiated” in the sense that it contains latent tumor cells and is functionally different as demonstrated by subsequent applications of the promotor. Light microscopy has revealed no changes in such “initiated” tissue. At one time there were reports that mitochondrial densities could be seen with the EM; but these changes were subsequently shown to be nonspecific. Of course, in the second stage,of the procedure, manifest and obvious changes do occur. These involve cell multiplication with an almost invariable overlay of some inflammatory changes;

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however, nothing which could be called specific to carcinogenesis is seen in static studies. On the other hand, the functional characteristics of such initiated tissues, as exemplified by a prolonged change in mitotic frequency, has been noted as a possibly specific concomitant of carcinogen treatment. These are a very few examples of the many studies that have been undertaken on the early changes accompanying treatment with carcinogenic agents. There are many other examples, but none as yet has revealed changes which can be called specific for carcinogenesis. We must know more of the intermediary metabolism of the range of carcinogens that we have at our disposal. It is perhaps too early to expect to be able to relate the intimate reactions of carcinogenic chemicals with cell constituents and biological effects. It is most unfortunate that more studies dealing with the metabolic characteristics of carcinogenic compounds have not been made. There have been few studies of metabolites of many of the new carcinogens (with notable exceptions in the instance of the aromatic amines). In any event, many more such studies, paralleled with additional studies of acute toxic effects, are required. The classic difficulty in the biochemical study of carcinogenesis is that a chemical may be administered to the animal and the effect observed as long as a year or more later. I would suggest that not enough attention has been paid to biochemical studies of the acute manifestations of some carcinogens, notably those inducing “radiomimetic effects.” These effects may not be associated with carcinogenesis, but it is more likely that they are. A toxic effect that is clearly defined and manifested in a period of days, rather than years, is, at least, amenable to study by modern biochemical methods in a way that will permit us to relate chemistry to biologic effect. In sum, it is my belief that our current lack of knowledge of the early changes induced by carcinogens is due to a lack of research in the area. The next problem is concerned with the need for chronic toxicity testing in the context of human disease. The most frequent causes of death in our population are diseases largely of unknown origin. That some of them might result from exposure to chemical factors in our environment is suspected by many investigators. It has been stated in recent years that up to 80% of human cancer might be due to environmental factors. There are few who would dispute the fact that the major cause of bronchiogenic carcinoma in man is cigarette smoking. We accept this piece of information easily these days without realizing how startling it is in terms of our philosophy of only a few years ago. I will be most surprised if other major cancers, such as carcinoma of the large bowel, are not explained in the reasonably near future by exposures to other factors. There is good reason to believe that much of the carcinoma of the liver among Africans might be related to atlatoxins or factors of this type. At the top of the list of causes of death are degenerative diseases of the cardiovascular system; these are of unknown origin in almost all instances. There are numerous suggestions made on their causation including diet, heredity, emotional status, and hormonal balance. So far, none of these possibilities has been confirmed. Unlike the carcinogenesis worker, the investigator in the field of arteriosclerosis has a paucity of good experimental models; arteriosclerosis is seen rarely in experimental animals and can be produced only with difficulty and only in a limited number of ways. From time to time, there is a remarkable overlap in the efforts of the cancer researcher and the heart investigator. In one series of studies that I came across while preparing some

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abstracts of the literature a few years ago, some arteriosclerosis students had induced the disease by feeding rats on a choline-deficient diet; reasonably similar diets had induced hepatomas in the hands of cancerologists. The determining factor in localizing the lesion appeared to be the source of research funds. This is a serious problem; I recollect having induced a series of interesting nephrosclerotic lesions in the rat in some experiments undertaken some years ago with Dr. Saffiotti. We had no time to study these renal lesions since we were only investigating skin carcinogenesis at the time and had no practical way of underwriting the additional investigation. My group is currently investigating a variety of drugs for possible carcinogenicity in a joint program with the National Cancer Institute. In these studies various drugs are being administered for a lifetime to several species. Although complete autopsies are done on all animals, in the instance of a tranquilizer no behavioral studies are performed and no detailed study of the central nervous system is done for the presence of demyelinating lesions. None of the various possible investigations are made for evaluation of alterations in physiological behavior. Even the blood pressure of the animals is not taken and the investigation of the state of vessels of the animals is cursory indeed. It is conceivable that clues might easily be obtained regarding the etiology of some of the mysterious degenerative diseases by being more complete in such animal toxicology studies. It is actually totally unrealistic to expect that such detailed studies can be accomplished because it is all we can do to complete proper carcinogenesis studies on a relatively few materials. Toxicologic studies have a way of never being absolutely completed; there is always more to learn from them and from continued studies. The liver damaging effects of aflatoxin were a very long way from the association of these products with the epidemic of hepatomas in the trout. The initial investigations of the occupational hazards from dimethylnitrosamine were remote indeed from the induction of various tumors of many different organ systems by the range of synthetic N-nitroso compounds. The late effects of many agents, both microbial and chemical, have been among the most difficult of all problems to elucidate in medicine, and experience tells us repeatedly not to make categorical assumptions, but rather to continuously use experimental data and clinical observations in a thorough and objective manner. In summary, I would suggest that many cancers, various degenerative diseases of the vascular system, the CNS, the eye and connective tissues among others, might be associated with chemical intoxication. Even where there are no specific leads, it is wise to attempt to be comprehensive in the animal toxicity study in the hope that such leads may emerge. With the almost complete understanding of microbiologically mediated diseases before us, the major killing diseases are still largely of unknown origin. Until the origin of such diseases is clarified, the assumption must be made that many will be related to the as yet unelucidated and often chronic effects of chemical and physical factors, again most often of extrinsic environmental origin. The last of the three questions which were proposed involves the consideration of the problem of chronic toxicity in the context of our modern environment. This is becoming such a hackneyed area that one hesitates to mention it in any discussion that makes an effort to be scientific. There is no doubt that “the problem” looms large and increases continuously. Simple observations that, for example, air pollution is undoubtedly an important causal factor in our commonest disabling disease, emphysema, makes one realize again the diversity of the ramifications of chronic toxicity studies. It is manifestly

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impossible to test everything in our environment, even for carcinogenicity, in an adequate manner. An immense problem in logistics is evident when consideration is made regarding the relationship between numbers of new chemicals coming into the environment and numbers of animals, and, above all, research workers needed for proper toxicologic evaluation. Clearly the decisions that have to be made involve considerations of a major economic and political nature. Many of the recommendations made by scientists on these problems must surely be recognized to be in the realm of theory for some years to come. However, an attempt will be made to answer the original question as it was posed. In the USPHS surveys of “Chemicals Tested for Carcinogenic Action,” it was found that when all compounds that had been tested in animals for more than 30 days were considered, one out of every four was carcinogenic (or perhaps, more correctly, tumorigenic). In these surveys this figure is perhaps a little biased since (a) many compounds were tested in a series of analogs synthesized for comparison with known carcinogens, and (b) a bias in favor of “compounds likely to be carcinogenic” appears among those compounds selected for test. In spite of this, subsequent figures with a little less bias, brought about by more chronic toxicity testing which was necessitated by the Delaney amendment, does seem to indicate that perhaps as many as one in six will be active. Thus a very appreciable number of compounds tested are active in animals when tested for carcinogenicity. There are no figures on other manifestations of toxicity. Carcinogenicity is the subject of legislation, and other manifestations of toxicity are not spelled out in the same way. At this point it is necessary to return to the first section of this presentation and further discuss the design and interpretation of chronic toxicity tests. There is little dissension from the view that special tests for carcinogens must be required for many environmental chemicals. Equally, there is little dissension from the view that the current test procedures involving, in the main, a chronic feeding study in rats is all right but that it could be a great deal better. It is my personal feeling that chronic toxicity testing, as currently performed, is providing us with a reasonably high degree of protection. Indeed, the record shows that the general population has already been protected against several potent carcinogens, such as, AAF, Aramite, and others. There has been no epidemic of cancer, except that of the lung, since the great changes in technology came to pass. 1 venture to believe that there might have been without these safeguards. Nevertheless, the laboratory procedures in common use require a great deal of improvement. Better animals and a higher degree of knowledge about species other than the rats and mice that have been used for so long are needed. Better standardization of the animal colonies and, above all, better-understood diets for the experimental animals are also greatly needed. For years, food additives have been tested in commecial diets which are absolute unknowns since they contain pesticide residues, processing contaminants, other food additives, and possibly, naturally occurring toxins. Such conditions complicate the immensely difficult task of interpreting the results which are obtained. Frequently it is not possible to distinguish between a compound which merely increases the incidence of a tumor that occurs commonly in control animals and one that results in the occurrence of a type of tumor not seen in the controls. The time of occurrence of the tumors is of importance but is difficult to assess. The levels of significance in the laboratory animals have to be established arbitrarily from knowledge of numbers of humans likely to be exposed.

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Transposing results obtained in 50 rats to 200,000,000 people is a very difficult problem, particularly in the instance of a disease in which a 1% incidence is very large indeed. Problem, after problem, arises from efforts to extrapolate from one species to another in the absence of much fundamental data on metabolism. Last, those investigators who deal with carcinogenesis testing in a practical manner are constantly faced with the special qualities imputed to carcinogenesis by legislation. The fact that any dose whatsoever of a carcinogen must be considered hazardous, placed this form of chronic toxicity into a unique class so that-dose-response, the keystone of logical toxicology, is removed. A revision of the approach in this regard is most important. It is much more important for the toxicologist to try to study all the manifestations of chronic toxicity. Arbitrary attempts by some investigators to reduce the length of tests required for purposes other than carcinogenicity are dangerous. No arbitrary times should be established. There is, just on the basis of simple logic, everything to be gained by knowing as much as possible about our environment, and as many tests lasting as long as possible and done as throughly as possible should be performed. This ideal obviously cannot be an overall goal. Some reasonable limits must be accepted. Much more individuality in choice of test, in interpretation of results, and in regulation, is needed in order that animal tests will reveal more about disease causation in the future than has been the case so far. It is tragic to think that the end point of many toxicity tests is merely acceptance or rejection of a product by a government agency. Every test is research and should be exploited to the full as such. A changed philosophy in this regard will undoubtedly result in the availability of many more trained people in the field and a continued upgrading of test procedures. I would venture to predict that such an approach would, in short order, result in a reasonable test for carcinogenicity-an ideal that few toxicologists feel is attainable in the face of present frustrations