The Science o f the Total Environment, 4 (1975) 1-52 © Elsevier Scientific Publishing Company, Amsterdam - Printed in Belgium

T H E C A R C I N C G E N I C I T Y OF D I E L D R I N * . P A R T I

SAMUEL S. EPSTEIN

Medical School, Case Western Reserve University, Cleveland, Ohio (U.S.A.) (Received November l l t h , 1974)

A. CARCINOGENICITY AS A PUBLIC HEALTH HAZARD

1. The scope o f human cancer There is now little doubt that many diseases hitherto regarded as spontaneous, particularly cancer, are caused by environmental pollutants. This realization is heightened by the exponential increase in human exposure to currently used and new synthetic chemicals--and their degradation products in air, water, and soil--which, in general, are inadequately characterized toxicologically and ecologically. More than 25% (51 million) of the 200 million people now living in the U.S.A. will develop some form of cancer, and approximately 20% of the American population dies of cancer 1' 2. Even with available methods of treatment, 34 million of these will die of cancer. In 1969, 323,000 people died of cancer in the U.S.A. In 1972, approximately 610,000 new cases of cancer were diagnosed and 1,000,000 were under treatment 3. Cancer is a leading cause of premature death (Table 1). TABLE 1 U.S. DEATHS F R O M VARIOUS CAUSES 2 Cancer deaths (1969) World War II battledeaths Auto accident deaths (1969) Vietnam war deaths (6 years) Korean war deaths (3 years) Polio deaths (1952--worst year)

323 000 292 000 59 600 41 000 34 000 3 300

The rate of recent increase of cancer deaths appears more rapid than the rate of increase in population and is even more rapid than the increase in the rate of death 2 ; this increase in new cancer cases is real and is oveI and above that due to increase in age alone. The economic impact of cancer is massive. The direct costs 4 of hospitalization and medical care for cancer in 1969 exceeded £500 million. The direct and indirect costs of cancer, including loss of earnings during illness and during the balance of normal life expectancy, were estimated at a total of £ 15 billion for 1971 a; on the basis o f analyses of selected cases, it appears that the total direct costs for a particular patient may range from £5,000 to over £20,000, * Based on Statement for Testimony at Cancellation Hearings on Aldrin/Dieldrin, Environmental Protection Agency and Environmental Defense Fund vs. Shell, March 1974.

2. Chemical carcinogens as a cause o f human cancer

Carcinogens are chemical, physical or biological agents, exposure to which, o f animals or humans, increases the probability of tumor (neoplasia) induction. This may manifest as follows: Increase in the tumor incidence in a given population, i.e., an increase in the number of tumor-bearing individuals. Increase in the number of tumors in each individual. Reduction in the latent period of tumor induction. Any combination of the above effects. Carcinogens may act at the site of initial contact, at the site of selective organ localization or accumulation, at the site of excretion or at the site of metabolism. Some carcinogens act at single sites only, others act at multiple sites. There is now growing recognition that the majority of human cancers are due to chemical carcinogens in the environment and that they are hence ultimately preventableS-8; there is also growing interest in the possible role of chemical carcinogens in activating oncogenic viruses. The World Health Organization has estimated that over 75% of human cancers are influenced by environmental factors ~. It has also been estimated that approximately 90% of human cancers are chemical in originS; other estimates indicate that between 60 to 80% of human cancers are environmental in origin. The basis for these estimates largely derives from epidemiological studies, in large community populations over extended periods, which have revealed wide geographic variations in the incidence of cancer of various organs 7' 9. In some of these studies, the role of specific environmental carcinogens has been implicated or identified. A much lesser degree of certainty on the causal relations between chemical agents, excluding cigarette smoking, drugs and "occupational airborne particles", was, however, recently expressed by a Presidentially-appointed panel, the great majority of whose members appeared to have expertise and qualifications in areas unrelated to chemical carcinogenesis 1°. Nevertheless, the panel, in apparent and unqualified contradiction to these views, stated in its general recommendations (p. 9) that: "Cancer incitements by so far unrecognized chemicals combine to form a threat to health, that may well be of at least the same general size as the three major threats just described (i.e., cigarette smoking, alcohol abuse and choice of dietary composition). These chemicals may be natural or synthetic". The best documented and significant data on carcinogenesis due to environmental chemicals are those on tobacco. It has been suspected for several decades that heavy tobacco smoking is directly and causally related to chronic lung disease, especially cancer. Over 29 retrospective epidemiologic studies on lung cancer, generally substantiating the causative role of smoking, have been published from 1939 to 196411. One of the most important of these studies demonstrated unambiguously that the incidence of lung cancer could be positively correlated with cigarette smoking 12. Mortality rates from lung cancer in both men and women have increased exponentially over the last few decades and have now reached epidemic proportions, particularly in the U.S. la.

There are also many studies which demonstrate the contributory role of urban air pollution in lung cancer; additionally numerous classes of chemical carcinogens have been identified in polluted urban air. These studies have shown that there is an excess of lung cancer deaths in smokers in polluted urban areas in contrast with those living in rural areas 14. There are marked regional differences in lung cancer mortality patterns in the U.S.A.; increased mortality is clearly related to increased urbanization and to increased levels of organic pollutants in the air. The higher lung cancer rates in urban areas cannot be fully explained by factors such as smoking or occupation. In a survey some 12 years ago, lung cancer rates in the U.S.A., standardized for smoking besides age, were found to be 39/100,000 in rural areas and 52/100,000 in cities with populations in excess of 50,000 (ref. 12); similar surveys in England also confirmed the importance of this urban factor, and stressed its interaction with smoking is. This urban excess, of 25% in U.S.A. mortality, is generally regarded as being due to air pollution. Confirmatory evidence is also afforded by several studies on immigrants who tend to retain the incidence pattern of lung cancer of their country of origin, even though they assume the smoking and other habits of their country of adoption. Similar striking regional variations in the occurrence of a wide range of other organ cancers are now well recognized 16-23. The high incidence of cancer of the oral cavity in Asia, representing some 35% of all Asiatic cancers, in contrast to 1% of all European cancers, is clearly related to the chewing of betel nuts and tobacco leaves. The high incidence of liver cancer in the Bantu and in Guam may well be due to dietary contamination with aflatoxin 24, a potent fungal carcinogen, and to eating Cycad plants, containing azoxyglucoside carcinogens, respectively1 v, is, 22-27. The high incidence of gastric cancer in Japan, Iceland, and Chile has been associated with high dietary intake of fish; suggestions have been made implicating nitrosamines, formed by reactions between secondary amines in fish and nitrite preservatives. The high incidence of cancer of the esophagus, in Zambians drinking Kachasu spirits, in the Calvados area of France and in other clearly defned geographic areas, may be related to contamination of alcoholic drinks or food with nitrosamines, some of which produce esophageal cancer in experimental animals 22' 23,2a. 29. More restricted data on chemical carcinogenesis in humans derive from followup studies on patients treated with carcinogenic drugs 3°-34. These include, immunosuppressive agents, used in transplant therapy; alkylating agents, used in treatment of cancer and to a lesser extent auto-immune diseases, radioactive phosphorus, used in treatment of polycythemia vera; estrogens, used for treatment of prostatic cancer and for hormone replacement therapy in women; and, diethyl stillbestrol, used for treatment of threatened abortions and as a "morning-after" pill. Data on occupational carcinogenesis extend back to the eighteenth century, with the discovery by Sir Percival Pott of a high incidence of scrotal cancers in young British chimney sweeps exposed to soot. A very wide range of occupational cancers has since been identified and studied in detail 35-38. These include bladder cancer in the aniline dye and rubber industry, induced by such chemicals as 2-naphthylamine, benzidine, 2-aminobiphen~l and 2-nitrobipheny139--4°; lung cancer in uranium miners

of Colorado, in coke oven workers, in nitrogen mustard factories in Japan and in U.S. workers even briefly exposed to bis(chloromethyl) ether41-*3; skin cancer in cutting and shale oil workers; nasal sinus cancer in wood workers; lung cancer and pleural mesotheliomas in insulation workers and in others, such as construction workers, exposed to asbestos44--*~; and, cancer of the pancreas and lymphomas in organic chemists 46.

3. Categorization of chemical carcinogens From toxicological standpoints, attempts have been made to differentiate "potent" from "weak" chemical carcinogens. Potent carcinogens, such as aflatoxins 24 and nitrosamines 23, produce a lelatively high incidence of cancers in conventional experiments with restricted numbers of test animals, even at the very low levels at which they have been found in foods, and elsewhere in the environment. Identification of such carcinogens has encouraged attempts to link their distribution, illustratively in foods, with local geographic patterns of cancer incidence. However, in the case of "weak carcinogens", such as atmospheric pollutants, their effects may easily escape detection by conventional biological tests. Additionally, as such weak carcinogens are unlikely to be clearly implicated epidemiologically, unless large populations with sharp differentials in exposure levels are followed up for prolonged periods of time, they are likely to pose greater hazards than more obvious and more easily recognizable potent carcinogens. From societal, legislative and regulatory standpoints, it is useful to differentiate two major categories of carcinogens on the basis of the voluntary or involuntary nature of human exposure to them. Firstly, there are carcinogens, such as cigarette smoke and certain drugs, to which individuals may voluntarily expose themselves, generally with some degree of cognizance as to the possible benefits, besides the potential hazards of such exposure. Secondly, there are the wider range of carcinogens, such as naturally-occurring or synthetic pollutants of air, water and food, such as Dieldrin, to which populations are involuntarily exposed and generally in the absence of information on such exposure. The 1958 Delaney Amendment to the Federal Food Drug and Cosmetic Act (P.L. 85-929) expressly proscribes the deliberate addition to food of additives which have been shown to be carcinogenic in man or experimental animals. No such protection appears to exist against widespread environmental contamination of food, air and water, following use of carcinogenic pesticides, such as Dieldrin; residues of carcinogenic pesticides in food are generally referred to as "accidental food additives", the word "accidental" qualifies their contamination of food and does not, of course, reflect the nature of their deliberate use or application. It should be emphasized that involuntary exposure to such carcinogens generally occurs in the absence of individual or collective information, of critical independent analysis of the benefits, alleged or real besides the potential hazards attendant on such exposure, and finally of the existence of similarly efficacious but less hazardous alternatives. Additional factors which properly influence the degree of concern on involuntary exposure to carcinogens include their persistence, environmental mobility and big-magnification or accumulation in the food chain.

REFERENCES 1 American Cancer Society, 1970, American Cancer Society, New York, 1974. 2 National Health Education Committee, Inc., New York, June 22, 1970, quoted in Report of the National Panel of Consultants on the Conquest of Cancer, for the Committee on Labor and Public Works, U.S. Senate, p. 31, November 1970. 3 National Cancer Program, The Strategic Plan, D.H.E.W. Publication No. N I H 74-569, January, 1973. 4 Report of the National Panel of Consultants on the Conquest of Cancer, prepared for the U.S. Senate Committee on Labor and Public Welfare, November, 1970. 5 S. S. Epstein, Nature, 228 (1970) 816. 6 World Health Organization, Technical Report No. 276, Prevention of Cancer, Report of WHO Expert Committee, 1964. 7 J. Higginson, Present Trends in Cancer Epidemiology, Prec. 8th Canadian Cancer Res. Conf., 1969. 8 E. Boyland, The Correlation of Experimental Carcinogenesis and Cancer in Man, Homburger & Karger (Eds.), in Experimental Tumor Research, 1964. 9 L. J. Dunham and J. C, Bailar, 3". Nat. Cancer Inst., 41 (1968) 155. 10 Chemicals and Health, Report of the Panel on Chemicals and Health of the President's Science Advisory Committee, September, 1973. 11 U.S. Dept. HEW, Smoking and Health, Report of the Advisory Committee to the Surgeon General of the PHS, PHS Publication 1103, Washington, D.C., 1964. 12 E. C. Hammond and D. Horn, 3".A.M.A., 166 0958) 1159 and 1294. 13 J. Clemmesen, Can. Med. Bull., 1 (1954) 37. 14 Particulate Polycyclic Organic Matter, National Academy of Sciences, Washington, D.C., 1972, p. 213. 15 P. Stocks, Brit. 3". Cancer, 14 (1960) 397. 16 W. C. Hueper, Ann. N. Y. Acad. Sci., 108 (1963) 963. 17 A. G. Oettle, J. Nat. Cancer Inst., 33 (1964) 383. 18 H. F. Kraybill and M. B. Shimkin, Adv. Cancer Res., 8 (1964) 191. 19 R. W. Miller, Yale Y. Biol. Med., 37 (1965) 487. 20 D. Schmal, Neoplasma, 15 (1968) 273. 21 J. Higginson, Present Trends in Cancer Epidemiology, Prec. 8th Candian Cancer Res. Conf., 1969. 22 J. Kmet and E. Mahboubi, Science, 175 (1972) 846. 23 W. Lijinsky and S. S. Epstein, Nature, 225 (1970) 21. 24 L. A. Goldblatt (Ed.), Aflatoxin. Scientific Background, Control and Implications, Academic Press, New York and London, 1969. 25 G. L. LaQuer, O. Mickelsen, M. Whiting and L. Kurland, J. Nat. Cancer Inst., 3 (1963) 919. 26 P. Keen and P. Martin, Trop. Geogr. Med., 23 (1971) 44. 27 Liver Cancer, I A R C - W H O Scientific Publication No. 1, 1971. 28 N. D. McGlashan, C. L. Waiters and A. E. M. McLean, Lancet, 2 (1968) 1017. 29 N. D. McGlashan, Gut, 10 (1969) 643. 30 L. S. Salyamon, Vopros. Onkologii, 9 (1963) 22, quoted in Federation Prec., 23, T.136. 31 Prec. Eur. Soc. for the Study of Drug Toxicity, Lausanne, January, 1964. 32 F. J. C. Roe, Clin. Pharm. Ther., 7 (1966) 77. 33 N. Napalkov, L. Shabad and R. Truhaut, Chemotherapia, 12 (1967) 47. 34 C. M. Bonser, Brit. Med. Y., 4 (1967) 129. 35 R. L. Eckhardt, Industrial Carcinogens, Grune and Stratton, New York, 1959. 36 W. C. Hueper, 3.. Occup. Med., 14 (1972) 150. 37 IARC, Monograph on the Evaluation of Carcinogenic Risk of Chemicals to Man, WHO, Geneva, 1972. 38 American Public Health Association Annual Meeting, Symposium on Occupational Carcinogenesis, November, 1972 39 K. F. Lampe, (Ed.) Bladder Cancer, Aesculapius, Birmingham, Ala., 1967. 40 W. C. Hueper, Occupational and Environmental Cancers in the Urinary System, New Haven, 1969. 41 W. C. Hueper, Occupational and Environmental Cancers of the Respiratory System, New York, Springer-Verlag, 1966. 42 S. Laskin, M. Kuschner, R. T, Drew, V. P. Cappiello and W. Nelson, Arch. Environ. Health, 23 (1971) 135.

43 J. W. Lloyd, .L Occup. Med., 13 (1971) 53. 44 Asbestos, The Need for and Feasibility of Air Pollution Controls, National Academy of Sciences, Washington, D.C., 1971. 45 I. Selikoff, E. C. Hammond and H. Seidman, Cancer Risk of Insulation Workers in the United States, presented at the Meeting of the Working Group to Assess Biological Effects of Asbestos,

Lyon, France, October 4, 1972. 46 F. P. Li, J. Fraumeni, N. Mantel and R. Miller, 3". Nat. Cancer Inst., 43 (1969) 1159.

B. PROBLEMS IN RECOGNITION OF CHEMICAL CARCINOGENS Current toxicological techniques are relatively insensitive and limited in their ability to detect carcinogens individually and in various combinations or mixtures realistically reflecting low or ambient levels and patterns of environmental exposure. Similarly, it is generally considered that epidemiological techniques are unlikely to detect weak carcinogens unless there are sharp differentials in exposure of the general population, as with cigarette smoking. For widely dispersed agents, including unintentional or accidental food additives, such as Dieldrin and DDT, to which the population-at-large is generally exposed, human experience is unlikely to provide any meaningful indication of safety or hazard.

1. Toxicological considerations a. Quantitative extrapolation from toxicological data Chemicals should be tested for carcinogenicity at higher levels than those of general human exposure 1. Irrespective of the route of administration, maximally tolerated doses (MTD) are recommended as the highest dose in dose-response studies for this purpose; it should be emphasized that life-long administration of these doses should not induce toxicity, as evidenced by weight loss and increased mortality. Testing at relatively high doses is now generally regarded as essential to the attempt to reduce the gross insensitivity imposed on tests by the small size of animal groups routinely tested, such as 50 or so rats or mice per dose level per chemical, compared with the millions of humans at presumptive risk 1. It should, however, be recognized that with doses in excess of the MTD, carcinogenic effects may be masked by premature death and by "competing risks" of toxicity. To illustrate the insensitivity of conventional animal test systems, assume that man is as sensitive to a particular carcinogen as the rat of mouse. Assume further that this particular agent has a risk that will produce cancer in one out of 10,000 humans exposed. Then the chances of detecting this in groups of 50 rats or mice, tested at ambient human exposure levels, are very low. Indeed, samples of 10,000 rats or mice would be required to yield one cancer, over and above any spontaneous occurrences: for statistical significance, perhaps 30,000 rodents would be needed 2' 3 Of course, in any particular instance, humans may be less or more sensitive than rodents to the chemical in question. There is consequently no valid basis for the a priori quantitative prediction of the relative sensitivities of test animals and man. Illustrative, for thalidomide the lowest effective human teratogenic dose is 0.5mg]kg/ day; the corresponding values for the mouse, rat, dog and hamster are 30, 50, 100, and 350 mg/kg/day, respectively4; thus, humans are 60 times more sensitive than mice, 100 times more sensitive than rats, 200 times more sensitive than dogs, and 700 times more sensitive than hamsters. Moreover, certain aromatic amines, such as 2-naphthylamine, are potent bladder carcinogens for man, monkeys and dogs, but not for rats, mice, guinea pigs, and rabbits s. Hence, it is not feasible to attempt to predict a finite safe level for carcinogens, such as 2-naphthylamine, from rodent data

or from derived mathematical models based on supposed safety factors. Such attempts would clearly expose humans to obvious potential carcinogenic hazards. Thus, threshold levels cannot be predicted for any individual, let alone for populations, comprised as they are of aggregates of genetically heterogeneous individuals with widely varying predispositions and susceptibilities. Apart from the gross insensitivity of animal test systems, and the impossibility of gauging human sensitivity from animal tests, ample data on interactions between carcinogens, and between carcinogens and a wide range of non-carcinogenic agents, further confirm that it is not possible to predict safe levels of carcinogens based on an arbitrary fraction of the lowest effective animal dose in a particular experimental situation. As H E W Secretai'y Flemming stated some 14 years agot: "Scientifically, there is no way to determine a safe level for a substance known to produce cancer in animals." Thus, the production of liver tumors in trout by feeding as little as 0.4 parts per billion of aflatoxin B 1 is sharply enhanced by addition of various non-carcinogenic oils to the diet 7. Similarly, the carcinogenic effects on mouse skin of low concentrations of benzo(a)pyrene and benz(a)anthracene are increased 1000-fold by the use of the non-carcinogenic n-dodecane as a solvent s. Intratracheal instillation of benzo(a)pyrene and ferric oxide in adult hamsters elicited a high incidence of lower respiratory tract tumors, only if the animals were pretreated at birth with a single low dose o f diethylnitrosamine 9. Thus, the quantitative response to a particular chemical carcinogen can be substantially influenced by a wide range of factors including, interaction with other carcinogens and non-carcinogens, and host determinants. Such considerations underlie the 1958 Delaney Amendment to the Federal Food, Drug and Cosmetic Act (P.L. 85-929), which imposes a zero tolerance for carcinogenic food additives. Although the apparent intent of Congress was clearly to protect the consumer, irrespective of the intent of the manufacturer, however, it appears that according to general legal interpretation, the Amendment applies ~nly to intentional food additives; unintentional carcinogenic food additives, such as residues of D D T and Dieldrin, are not apparently covered by its requirements. The Amendment states: "... no additive shall be deemed to be safe if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animals.." These conclusions were further emphasized, as follows, by an expert committee of the World Health Organizationt°: "... It is agreed that no assuredly safe level for carcinogens in human food can be determined from experimental findings at the present time." The scientific basis of the Delaney Amendment has been consistently endorsed by qualified independent expert committees, such as the 1966 Symposium of the International Union Against Cancer, and the 1970 Ad Hoc Committee Report to the

Surgeon General ~i which stated: "The principle of a zero tolerance for carcinogenic exposures should be retained in all areas of legislation presently covered by it and should be extended to cover exposures as well. Only... where contamination of an environmental source by a carcinogen has been proven to be unavoidable should exception he made (and then) only after the most extraordinary justification is presented... Periodic review.., should be made mandatory." No such instance of "extraordinary justification", necessitating open societal consideration of the benefit-risk calculus appears to have yet been presented or documented for any carcinogenic industrial chemical or consumer product, such as Dieldrin, whose use appears to result in widespread environmental contamination. The Scientific basis of the Delaney Amendment, and of the absence of threshold or "no-effect" levels for carcinogens, which has been recently further detailed TM 23, was overwhelmingly endorsed at a workshop on this subject convehed by the New York Academy o f Sciences, on January 15-16, 1973. It also must be emphasized that testing at high dosages does not produce "false positive" carcinogenic results. There is no basis for the contention that all chemicals are carcinogenic when tested at high doses. In fact, the experimental finding o f carcinogenicity in synthetic industrial chemicals, such as pesticides, is unusual. T o illustrate, in the recent National Cancer Institute-sponsored Bionetics study, about 140 pesticides and industrial chemicals were tested in mice of both sexes and two strains by continuous oral administration at M T D levels from the first weeks of life until sacrifice at 18 months; less than 10% of these agents were found to be carcinogenic 14. It is now dearly recognized that current toxicological techniques are insensitive and relatively limited in their ability to detect carcinogens, individually and in various combinations or mixtures realistically reflecting low or ambient levels and patterns of environmental exposure. While this is largely due to the relatively small numbers of animals tested compared with the massive human populations at presumptive risk, this also reflects the simplistic nature of toxicological approaches, which are generally based on the testing of single chemical agents in isolation from the multitude of other chemicals to which human populations are concurrently exposed. Thus, the potential for a wide range of interactions between two carcinogens, such as Dieldrin and D D T (see Section C.5), or between a carcinogen and a non-carcinogenic promoting agent, which may markedly enhance or synergize carcinogenicity, is not reflected in standard toxicological practice.

b. Qualitative extrapolation from toxicological data There is close parallelism between the pathological and biological characteristics of the very wide range of cancers seen in both rodents and humans. The use of rodent species, particularly the mouse and the rat, has been and still is standard practice in routine carcinogenicity testing in toxicological practice, in addition to'basic research. Considerations of convenience apart, the key role of the mouse, besides the rat, in carcinogenicity testing has been repeatedly and widely endorsed by a wide range of expert committees and authorities, as illustrated by the following statements:

"The rat and mouse have been shown to be susceptible to the carcinogenic action of a large variety of compounds and, indeed, most o f our knowledge of chemical carcinogenesis is based on the use of these species. The hamster and guinea pig are relatively resistant to the carcinogenic action of several compounds which produce tumors readily in the rat and mouse. On the other hand, no known carcinogens for the hamster and guinea pig are inactive in the rat and mouse .... The mouse has been the most widely used animal in cancer research, and many special strains have been developed that have known spontaneous incidences of different types of tumors." (ref. 15). "Both sexes of each of at least two species of animals should be used in the tests throughout their life span. In most cases these species would be rats and mice." (re/'. 10). "The mouse has come to be the classic animal for studies of carcinogenicity. Interest in genetics and the needs of investigators in carcinogenesis have led to the development of strains of inbred mice and rats which are uniform and show a standard response. They are the equivalent in biology of reagent chemicals in chemistry." (ref. 16). "The species most practical for testing are rats, mice, a n d - - a s more recently s h o w n - hamsters. Strains and colonies should be selected to provide adequate sensitivity to tumor induction, as revealed by positive control tests with known carcinogens. Their spontaneous tumor incidence should be recorded. Treatment should begin when the animals are young; the animals should be kept as free as possible from infectious diseases and parasites." (ref. 1.) A footnote to the above quotation states: "The use of non-rodent species.., has now been substantially dropped. A suitable, practical non-rodent species would be useful, but it is not available at this time. Carcinogenicity tests of food-borne pesticides require routine lifetime feeding of chemical compounds. While dogs have been employed for tests of carcinogenicity, with noteworthy success in selected cases (bladder carcinogenicity of aromatic amines), the requirement of lifetime feeding makes this species too expensive, in terms of time and funds, to be employed routinely." (ref.l .) "Rodent species (rats, mice and Syrian hamsters) should be the animals of choice for experiments of a screening nature or requiring large numbers of animals. Continued emphasis on rodent species is suggested since most comparative information has been derived from these species and their viral profiles, longevity, and suscept/bility to diseases have been well established over the years (Berenblum, 1969)." (re/'. 17.) " A t least two species are recommended for all carcinogenicity tests. It is suggested that two rodent species be used. There are many examples of species variation to carcinogens on record among rodents. The use of more than one species adds additional safeguards in consideration of extrapolation to man. The use of the dog for a two-year study is considered inadequate for the study of carcinogenicity, and it is felt unrealistic to extend this requirement routinely to the seven or more years needed to establish a negative finding. No other nonrodents have been sufficiently investigated with carcinogens to be recommended for routine tests at this time. Any new species or strains would have to be evaluated with a range of known carcinogens." (ref. 18.) " F r o m a practical point of view, the most useful species for cancer testing are the rat, mouse and hamster. The long latent period required for the production o f tumors with most carcinogens in the dog and primate reduces the usefulness o f this species . . . . Since it is the objective of some cancer studies to evaluate the potential carcinogenicity o f chemicals before they are introduced to the consumer, the choice of species for testing is usually not based on a methodical evaluation of the metabolism of the compound in m a n prior to the selection of an approved laboratory species. Rather, the rat, mouse or hamster is usually chosen; a selection that is based on the cost and availability of healthy animals and the investigator's experience and knowledge of the selected rodent species." (ref. 19.) It must be stressed that the general objective of requirements

for carcinogenicity

t e s t i n g i n a d d i t i o n a l s p e c i e s , b e s i d e s t h e m o u s e , s u c h a s t h e r a t a n d h a m s t e r , is t o provide a more extensive data base for attempts at the development

of protective

regulations against carcinogenic hazards to large and heterogeneous human 10

popula-

which has been vigorously presented at various scientific meetings, including at the Carcinogenesis panel of the Mrak Commission on Pesticides2° and one that has been since refuted by Shell data 21, has been that certain chemicals, such as Dieldrin, produced "compound-dependent tumors" in mice which were alleged to spontaneously regress following termination of administration of the chemical. The inference here was that such tumors could be induced by agents inducing microsomal enzyme synthesis in the liver and that the tumors were thus in the nature of physiological compensatory responses and not truly neoplastic. A further argument has been that the mouse and its liver is uniquely endowed in its response to certain carcinogens. The conclusion from this sequence of arguments would appear to be that carcinogenicity data derived from mice have limited or no relevance to man. Paradoxically, lack of carcinogenic effects in mice is generally preferred, in the absence of noted reservations, as evidence supporting claims for safety and petitions for registration. Such, presumably, would have been the case had the Tunstall tests, or other tests designed and submitted in support of tolerance petitions, demonstrated the noncarcinogenicity of Dieldrin in mice. The concept of "species-specific" mouse hepatocarcinogens has been recently examined, on the basis of a survey of the published literature, although no attempt was made to assess the adequacy of the reported tests, and unequivocally rejected by scientists on the staff of The International Agency for Research on Cancer 22; illustratively, Dieldrin is listed as being non-carcinogenic in the rat, in spite of evidence, which was, however, then largely unpublished, to the contrary (see Section D). Of 58 mouse liver carcinogens, identified in the literature, 40 induced additional tumors in other organs, of which only one, benz(a)anthracene was reportedly negative in inadequate rats and hamsters tests 23. None of the 18 carcinogens which only produced liver tumors in the mouse were shown to have been non-carcinogenic when adequately tested in rats and hamsters. It was thus clearly concluded that the induction of hepatomas in mice is as valid an indication of carcinogenicity as is the induction of tumors in any other site in the mouse or in any site in any other test species; hence, it follows that data on mouse carcinogenicity in general, and on liver cancers, in particular, may properly be extrapolated to humans. This conclusion was recently unequivocally endorsed by the Director of the Carcinogenesis Program of the Division of Cancer Cause and Prevention of the National Cancer Institute, who has major national responsibility for federally supported programs on testing for chemical carcinogens23. The following additional conclusions were made on the basis of this survey22: " A positive correlation appears to exist between t h e capacity o f a chemical t o i n d u c e liver t u m o r s in the m o u s e and its capacity to induce t u m o r s in a n y site in the rat o r t h e h a m ster. T h e strongest positive correlation was f o u n d w h e n the chemical given to t h e m o u s e d u r i n g adult life induces t u m o r s in the liver o f b o t h sexes, as well as t u m o r s at o t h e r sites. T h e induction o f liver t u m o r s in the m o u s e by a chemical does n o t imply that t h e l i v e r w o u l d be the target o r g a n in the rat or the hamster. " A m o n g the 58 chemicals considered, seven are recognized or s u s p e c t e d h u m a n carcinogens (BP, 4-aminobiphenyl, benzidine, a u r a m i n e , 2 - n a p h t h y l a m i n e , stilbestrol a n d aflatoxin). With the possible exception o f aflatoxin, there is n o evidence that t h e t a r g e t o r g a n

12

for man would be the liver. All were hepatocarcinogenic in the mouse and six were carcinogenic for the liver and/or other organs in the rat". N o a t t e m p t was m a d e in this survey to a t t e m p t differentiation b et w een h e p a t o m a s , as benign liver t u m o r s , a n d h e p a t o c e l l u l a r c a r c i n o m a s for the reason t h at ... "... it has been reportedly recognized that benign tumors may become malignant and probably no chemical exists which is capable of producing benign tumors only." (ref. 22.) The close biological r e l a t i o n o f m o u s e h e p a t o m a s a n d h u m a n liver cancers has b e e n recently stressed by r e c o g n i t i o n o f the value o f the alpha-feto p r o t e i n tests, first identified in studies o n m o u s e h e p a t o m a s , in the diagnosis o f h u m a n liver c a n c e r 24.

d. On the alleged distinction between tumorigens and carcinogens It is generally r e c o g n i z e d that a wide range o f apparently b e n i g n s p o n t a n e o u s h u m a n n e o p l a s m s a n d i n d u c e d a n i m a l n eo p l asm s m a y b e c o m e frankly m a l i g n a n t . In skin carcinogenesis studies with polycyclic h y d r o c a r b o n s , m o r p h o l o g i c a l l y b e n i g n p a p i l l o m a s first a p p e a r . S o m e o f these r e m a i n benign, o t h er regress, an d others u n d e r go m a l i g n a n t t r a n s f o r m a t i o n , as evidenced by m o r p h o l o g i c a l characteristics, invasiveness a n d metastasis. T h e invalidity o f recently alleged distinctions between t u m o r i g e n s a n d carcinogens has been r e p e a t e d l y and u n a m b i g u o u s l y e m p h a s i z e d by n u m e r o u s expert n a t i o n a l a n d i n t e r n a t i o n a l c o m m i t t e e s ; the terms t u m o r i g e n s a n d carcinogens thus h a v e s y n o n y m o u s i m p l ic a t io n s . T h e f o l l o w i n g q u o t a t i o n s are illustrative: "In the assessment of carcinogenic risk it is not considered relevant whether the tumor is benign or malignant since the conversion of the first to the second must be considered possible." (ref. 25.) "In the thinking of most experimentalists the induction of a benign tumor represents the production of neoplasia. Most would feel that this is an indication of carcinogenicity although it is usual to continue studies until morphologically malignant tumors have appeared. There are few studies on record in which only benign tumors are recorded (the neurofibromas induced by ergot appear to be such an instance). In the majority of experimental studies with epithelial tissues the induction of a benign tumor is merely a stage in the subsequent occurrence of a malignancy." (ref. 26.) "The response of test animals to carcinogens may take one of several forms: (1) an increased incidence of one or more of the tumor types noted in the controls; (2) the occurrence of tumors earlier than in the controls, without increased incidence; (3) the development of types of tumor not seen in the controls (this may or may not be associated with an overall increase in the number of tumors seen in the controls); and (4) a multiplicity of tumors in individual animals, the incidence in terms of tumor-bearing animals being the same. Furthermore, the tumors seen may be benign or malignant, or tumors of both categories may be present." (ref. 27.) "The Panel is unaware of the existence of any chemical which is capable of producing benign tumors only, wnich is to say, in the light of present knowledge, all tumorigens must be regarded as potential carcinogens: (a) No adequately tested chemical has been found to produce only benign neoplasms and, (b) a substantial percentage of benign-appearing tumors in mice has been demonstrated ultimately to eventuate in cancer." (ref. 1.) "In the first instance benign tumors may cause death in man and animals without even undergoing malignant transformation. The induction of a benign tumor is, itself, therefore, an indication of a serious adverse reaction. There can be no doubt from a survey of experimental studies that benign neoplasms are often precursors of malignancies. "Under these circumstances, it would be wise to take serious note of the occurrence 13

of benign neoplasms in experimental studies, although this alone is not sufficient for a conclusion of carcinogenesis. The occurrence of metastases provides an unequivocal demonstration of malignancy. There are, however, many tumors induced experimentally that are invasive and are classified as malignant, that metastasize only rarely during the average experiment. The absence of metastasis, in the view of most pathologists, does not rule out the diagnosis of malignancy. "Transplantation has been used by some investigators as additional proofof malignancy. This method is employed infrequently and has certain drawbacks. There are, for example, some tumors such as the mammary fibroadenoma of the rat that are benign in all respects and yet may he transplanted readily." (ref. 18.) The morphological resemblance between "benign hepatomas" and hyperplastic nodules 2s, has been used as the basis for arguments that agents inducing such effects in the mouse cannot be regarded as carcinogenic; in addition to the above-stated consensus on the invalidity of such distinctions between tumorigens and carcinogens, the argument has apparently ceased to be relevant with the recognition of pulmonary metastases resulting from a wide range of liver tumors induced in mice, illustratively by Dieldrin (see Section C).

2. General epidemioloyical considerations Even with well-planned and well-executed toxicologic testing, it is likely that unexpected carcinogenic effects from toxic chemicals will occur in humans, reflecting the inherent insensitivity and limitations of the test systems. Epidemiological surveys of human and animal populations may provide post hoc information on geographical or temporal clusters of unusual types or frequencies of carcinogenic effects after exposure to undetected or untested chemical agents in the environment. Additionally, inadequacies in surveillance systems, in the size of the population studied and in the duration of study may limit the sensitivity of epidemiological approaches even when temporal or geographical clusters of adverse effects have developed. Epidemiologic techniques serve to detect trends or fluctuations in mortality and morbidity of disease patterns. Provided that there are clear differentials in exposure of the general population levels to specific chemicals or agents, epidemiology may then correlate particular effects with particular agents. A classic example o f such a relationship is that between heavy cigarette smoking and lung cancer, although direct causality has only recently been belatedly recognized after decades o f human exposure. However, these relationships appear much more difficult to establish when exposure differentials are minimal, as with intentional or unintentional food additive residues or with feed additive residues consumed by the general population at not widely dissimilar levels. These considerations seem to apply with particular emphasis to Dieldrin, in view of its widespread environmental contamination in the U.S.A. (see Section F), resulting in its almost ubiquitous presence at relatively high levels in human adipose tissue 29. Epidemiologic studies may provide retrospective data relating to the apparent safety of chemicals in current use. However, human experience generally affords only limited indications of safety or hazard. The reasons for this include the long latent period, in some cases many decades, elapsing between exposure to carcinogenic 14

chemicals and the subsequent development of cancer; demonstration of causality may be obscured by mortality from competing risks during such latent periods. Illustratively, the latent period for induction of bladder cancer and of lung mesotheliomas following occupational exposure to aromatic amines and asbestos may average 18 and 30 years, respectively; contrastingly, latent periods of less than one decade have been noted for carcinogens such as cutting oils and for radiogenic leukaemia. Additional ditficulties include isolating the effects of any single chemical from the multitude of others to which populations are concurrently exposed and the lack of baseline data on regional and national incidences of cancer, besides other adverse human effects. These epidemiological limitations are compounded when the population-at-large is exposed to the carcinogen in question and when there are thus no sharp differentials in levels of human exposure. Even with tobacco, where such exposure differentials clearly exist in large population samples, ranging from heavy smokers to those who do not smoke at all or to those who are exposed by passive inhalation only, the epidemi~logical demonstration of causal relationships between smoking and lung cancer has been long delayed. It should be stressed that two of the most important major classes of carcinogenic agents, tobacco smoke and asbestos, whose effects are well documented in human populations, both produced multiple site tumors 31. Some problems in attempts to demonstrate the safety, in terms of non-carcinogenicity, of Dieldrin in varied human populations, of all ages and both sexes, are illustrated in a recent epidemiological study of occupational exposure of adult males to Dieldrin and related organochlorine pesticides 3°. This was based on a population 826 full-time male workers, including maintenance workers and cleaners, besides operators some of whom had additional exposure to unrelated pesticides, employed since 1954 at a Shell insecticide plant in Holland. Apparently, there was a high turnover rate in this plant, as the largest number of employees at any one time, 1962, was 230, and also "more or less frequent movement of workers between" units within the plant 3°. The approximate exposure periods of a specified number of 826 workers through 1970 were as follows: 277, less than one year; 316, 1-4 years; 198, 4-9 years; and 35, 10-13.25 years. The epidemiological aspects of this study have been reviewed independently by leading national experts who appear unanimous in their views that the numbers of workers at risk and duration of risk are quite inadequate for the development of valid inferences as to the non-carcinogenicity of Dieldrin31-33. The possibility that careful follow-up of this population over the next few decades could yield useful information on carcinogenicity was not, however, excluded. An additional complication in the future interpretation of such data is the fact that the problem of differential exposure is complicated since the population-at-large is exposed to Dieldrin as a general ubiquitous environmental contaminant in food, air and water (see Section F); Dieldrin workers would, of course, also be exposed to such incremental burdens in addition to their occupational exposures. In addition, since necessary safety precautions were apparently taken to minimize worker exposure, including the transfer of workers showing any acute toxic symptoms, this would tend to lessen the overall 15

exposure differential that one might expect between the workers and the general population. The following statements are illustrative of the views of the independent experts on this occupational study: " I t seems to me that the Shell group of workers and former workers is unlikely to yield much information on a possible cancer hazard for years to come at." " T h e assurance that the book gives as to the (carcinogenic) hazards for the general population is indeed small 32." " I cannot think of any environmental agent that has been implicated in h u m a n carcinogenesis by study of such a small sample over such a short period of time. Radiogenic leukemia, which has the shortest latent period for an environmentally induced cancer (to my knowledge), could be demonstrated in only one out of 60 survivors of Hiroshima who were within a thousand meters of the bomb within 12 years of exposure. The peak in occurrence was 5 years after exposure. Only 61 pesticide workers fell into the "extreme exposure" group, which had average exposure of 7.7-11.1 years. If one were looking for radiogenic leukemia in a sample of this size, he would expect one case." (ref. 33.)

Finally, in dismissing this worker observation study as "saying nothing" about carcinogenicity, Saffiotti testified in this Hearing that the study had also been reviewed by a committee of expert epidemiologists appointed by the International Agency for Research in Cancer of the World Health Organization which similarly concluded that the study "does not allow any conclusions on the existence of an excess risk of developing cancer" (ref. 23, Tt. 7539-40). REFERENCES

1 Report of the Advisory Panel of Carcinogenicity, Report of the Secretary's Commision on Pesticides and their Relationship to Environmental Health, U.S. Dept. HEW, Washington, D.C., December, 1969. 2 S. S. Epstein, et al., Science, 166 (1969) 1575. 3 S. S. Epstein, Nature, 228 (1970) 816. 4 H. Kalter, Teratology of the Central Nervous System, University of Chicago Press, 1968. 5 W. C. Hueper, Occupational and Environmental Cancers of the Urinary System, University Press, New Haven, 1969. 6 A. Flemming, Hearing on Color Additives, Committee on Interstate and Foreign Commerce, House of Representatives, 86th Congress, 2nd Session, 501 (1960). 7 R. O. Sinhuber, R. O. Wales, J. H. Ayres, J. L. Engebrecht and D. L. Amend, J. N A T . Cancer Inst., 41 (1968) 711. 8 E. Bingham and H. L. Falk, Arch. Environ. Health, 19 (1969) 779. 9 R. Montesano, U. Safliotti and P. Shubik, Inhalation Carcinogenesis, U.S. Atomic Energy Commission, April, 1970, p. 353. 10 Evaluation of The Carcinogenic Hazards of Food Additives, 5th Report of The Joint F A O / W H O Expert Committee on Food Additives, WHO Geneva, 1961, Tech. Rep. Ser. 220. 11 Ad Hoc Committee on the Evaluation of Low Levels of Environmental Chemical Carcinogens, Report to the Surgeon General, April 22, 1970. 12 U. Satliotti, Preventive Med., 2 (1973) 125. 13 S. S. Epstein, Preventive Med., 2 (1973) 140. 14 R. Innes et al., J. Nat. Cancer Inst., 42 (1969) 1101. 15 Problems in The Evaluation of Carcinogenic Hazard from Use of Food Additives, Food Protection Committee, Food and Nutrition Board, N A S - N R C , Publication 749, December 1959, Washington, D.C., 1960. 16 J. H. Weisburger and E. H. Weisburger, Chem. Eng. News, 54 (1966) 124. 17 Man's Health and the Environment--Some Research Needs, Report of The Task Force on Research Planning in Environmental Health Science, NIEHS, U.S. Dept. HEW, March, 1970, Ch. 7. 18 ToxicoL Appl. PharmacoL, 20 (1971) 49.

16

19 The Testing of Chemicals for Carcinogenicity, Mutagenicity and Teratogenicity, Health and Welfare Canada, September, 1973. 20 Memo from L. Golberg to the Carcinogenesis Panel of the Mrak Commission, Circulated by NCI to members of the Panel on Sept. 23, 1969, referring to a report by K . Weinbren (then a Shell consultant). 21 A. I. T. Walker, E. Thorpe and D. E. Stevenson, Fd. Cosmet. Toxicol., 11 (1973) 415. 22 L. Tomatis, C. Partensky and R. Montesano, Int. J. Cancer, 12 (1973) 1. 23 U. Saffiotti, Personal communication, and Exhibit 40, Testimony at Hearing o n Aldrin/Dieldrin. 24 E. Farber, Exhibit 45, Testimony and statement, 1/18/74, at Hearing on Aldrin/Dieldrin. 25 WHO Evaluation of the Carcinogenic Hazards of Food Additives, 5th R e p o r t , Tech. Rep. Ser. 220, Geneva, 1961. 26 WHO Prevention of Cancer, Tech. Report Ser. 276. Geneva, 1964. 27 WHO Principles of the Testing and Evaluation of Drugs for Carcinogenicity, Tech. Report Series 426, Geneva, 1969, 28 W. Butler, Liver Cancer, I.A.R.C. Lyon, 1971, pp. 30-41. 29 F. W. Kutz, Exhibit 47, Testimony at hearings on Aldrin/Dieldrin. 30 K. W. Jager, Aldrin, Dieldrin, Endrin and Telodrin, Elsevier, A m s t e r d a m / L o n d o n / N e w York, 1970. 31 H. Seidman, Exhibit 49, Testimony and statement at hearings on Aldrin/Dieldrin, Jan. 25, 1974. 32 N. Mantel, Memo to M. A. Schneiderman, Associate Director for Demography, The National Cancer Institute, Feb. 2, 1973. 33 R. W. Miller, Memo to M. A. Schneiderman, Feb. 8, 1973.

17

C. REVIEW OF CARCINOGENICITY TESTS IN MICE

1. FDA study 2: Davis and Fitzhuyh 1, 1962 This report is written in summary form. The Introduction section states, without reference, that... " A previous 2-year Aldrin and Dieldrin mouse feeding study raised the suspicion of tumorigenicity of these two pesticides; however, results o f the test were considered inconclusive because the majority of the animals were not available for pathological examination."

Groups of 215-218 young CaHeB/Fe mice (C3H mice), "approximately equally divided by sex", were started on a 2-year feeding test with Aldrin (10 ppm) and Dieldrin in their diet. It is stated that mice dying during experiments were autopsied, and that all 2-year survivors were sacrificed and autopsied. All results were reported for both sexes combined (Table 1). TABLE 1 RESULTS OF EXPERIMENTS WITH MICE (based on authors' Table 1)

Observation

Aldrin (10 ppm)

Dieldrin (10 ppm)

Control

Mice initially on experiment Average weeks on experiment Average weeks on experiment for mice with hepatomas Survivors at 18 months Survivors at 24 months Mice discarded at autopsy Nos. of mice with hepatomas: by gross pathology by microscopy

215 51.8

218 51.4

217 59.8

80 32 2 64

77 33 8 70

89 47 11 83

38 35

38 36

9

9

Overall mortality in the experiment was very high, particularly in Aldrin and Dieldrin groups where average survival (5,1.8 and 51.4 weeks, respectively) was approximately 2 months less than for controls. A "statistically significant" increase in the incidence of hepatomas in test groups was reported. The incidence of hepatomas, based on initial numbers of mice, in Aldrin groups was 35/215 (16%), in Dieldrin groups, 36/2,18 (17%), and in controls, 9/217 (4%). These values are probably underestimates because of very high mortality during the experiment, because data are reported for both sexes combined and also because large numbers of mice were "discarded at autopsy". The percentage of mice examined histologically appears to have been 70, 68 and 62% for Aldrin, Dieldrin and control groups, respectively; based on numbers of mice examined at autopsy, the incidence of hepatomas in these three groups is 23, 24 and 7%, respectively. Additionally, since no true survival data are given, the true incidence of hepatomas in relation to numbers of either sex at risk, may have been still higher. While the first 18

hepatoma occurred at 39 weeks in a mouse in the Aldrin group, it appears that the incidence of hepatomas was greater with longer surviving animals; the average survival for mice with hepatomas, 77-89 weeks, was longer than that for the total number of mice in the same group, 51,4-59.8 weeks. No metastases were noted in lung sections prepared from about half of the mice with hepatomas. Reference is made by the authors to a hepatoma transplantation study, but no data are cited. The histology of these hepatomas is neither adequately nor consistently described. While it is stated in the Results section that the hepatomas morphologically "ranged from very benign lesions to borderline carcinomas", in the Summary section they are, in apparent contradiction, characterized as "histologically benign liver tumors"; further, the Discussion section refers to the hepatomas as "morphologically benign", and states that Aldrin and Dieldrin must hence be regarded as "tumorigens" and not "carcinogens".

Conclusions This study, despite its inadequacies and the authors' qualifications of the data, presents strong evidence for the hepatocarcinogenicity of both Aldrin and Dieldrin in C3H mice. Independent re-evaluation of this study (see Section C.2) confirms these conclusions. 2. FDA study 3: Davis 2, 1965 This study was conducted in groups of 100 male and female C3H mice, and is presented in summary memorandum form (Table 2). TABLE 2 RESULTS OF E X P E R I M E N T S W I T H Call M I C E (based on a u t h o r ' s Table 1) Hy = Hyperplasia; H = "Benign h e p a t o m a ' ; H C = Hepatic carcinoma.

Dose (ppm)

0 Aldrin (10) Dieldrin (10)

Survivors at wks.

No. mice sacrificed with lesions

No. mice with tumor

52

78

104

Hy

H

HC

Benign

Malignant

188 152 169

150 121 117

64 31 39

48 72 71

27 65 69

4 3 5

30 61 71

21 9 9

Four to 12 mice from each group were lost or discarded; additionally, four males and females from Aldrin and Dieldrin groups and eight males from controls were used for liver tumor transplant studies; no results are reported by the author, although such data were apparently available 3. Decreased survival occurred particularly in test groups. No breakdown of data by sex and by time of death is given, and there is no indication as to the time of tumor detection or death in treated, in contrast with control groups. It is stated that: 19

"Incidence of both hepatic hyperplasia and o f benign hepatomas were approximately doubled in test groups. These pesticides had no significant effect on the incidence of malignant liver tumors. While there was a 2- to 3-fold increase in overall incidence o f benign tumors in all test groups, there were only approximately half as many mice with malignant tumors in each of the test groups as were noted in the control groups." (ref. 2.)

No indication is given that serial lung sections were performed, although metastases were restrictedly used, in addition to extra-hepatic invasion, as the major criteria in the diagnosis of malignant liver tumors. The liver pathology was ascribed to "hepatic stressor effects". It is finally concluded that: "We have now failed for the third time to prove that either Aldrin or Dieldrin are carcinogenic for inbred C3HeB/Fe/J mice when fed at near toxic levels for 2 years." (ref. 2.)

a. Re-evaluation o f both FDA tests I' 2 by Reuber 4 and others

This re-evaluation was based on histological material from both second I and third 2 FDA tests, using 10 ppm of Aldrin and Dieldrin; Dieldrin histology from the second test was not, however, available (Table 3). TABLE 3 HEPATOCARCINOGENIC EFFECTS IN MICE (based on Reuber's data) N H = No hyperplasia; H = hyperplasia; N = nodules; SC = small carcinomas ( < 5 ram) LC = large carcinomas; TC = total carcinomas.

Group

Nos. examined

Control, M Control, F Alddn, M Aldrin, F Dieldrin, M Dieldrin, F

73 53 91 85 71 71

Ave. survival weeks

89 93 86 80 91 81

% incidence o f liver lesions Nil

H

N

SC

LC

TC

40 72 1 1 0 0

12 11 3 6 3 4

18 13 13 8 10 8

!8 2 21 29 17 21

12 2 62 55 70 66

30 4 82 85 87 87

The following definitions4 should be noted: "Hyperplasia: Early changes that are dependent upon the continued feeding o f the chemical. If the chemical is discontinued early hyperplastic cells will not progress and become carcinomas." "Hyperplastic nodules: Nodules have reached the stage where they are no longer dependent upon the chemical. If the chemical is discontinued, nodules continue to progress and become carcinomas."

These data clearly indicate the malignancy of the hepatomas described in both FDA tests and demonstrate the hepatocarcinogenicity of Aldrin and Dieldrin in male and female C3H mice. Carcinomas in controls were generally small and single, in contrast with treated animals in which they were larger and sometimes multiple. The hepatocarcinogenic effects in males and females alone, as well as combined, were reported to be statistically highly significanP. Additionally, pulmonary metastases were found 20

in 4% of Aldrin-treated males and 5% of Dieldrin-treated females with liver carcinomas. Recognizing difficulties in differentiation between necrosis and post-mortem autolysis, however, hepatic vein thrombosis, causing massive liver necrosis and death, was diagnosed in approximately 5 % of treated mice: necrosis of this type is "unique" in Reuber's experience. Reuber also examined results of the transplant tests referred to in both FDA reports, which, however, cite no data. In these studies (Andervont Experiment No. 333), 24 hepatomas were transplanted into a total of 240 mice, each tumor generally being transplanted into 5 males and 5 femalesa. Reuber states that successful transplants occurred with 7/8 control tumors, 9/10 Aldrin tumors and 8/9 Dieldrin tumors. In Reuber's evaluation, the histology of the transplants was considered to resemble that of the original tumors. The degree of malignancy, as recognized histologically, correlated well with transplant behavior, as indicated by the fact that whereas highly malignant tumors produced successful transplants within 3 months, less malignant tumors took 6-9 months. It is stated that Popper, Farber, Firminger checked representative histology and concurred with the histological diagnoses of liver carcinomas in treated animals 4. b. Conclusions This FDA study, again despite its inadequacies and the authors' conclusions as to the "tumorigenicity" and not carcinogenicity of Dieldrin, confirms the results of the previous FDA study 1 on the hepatocarcinogenicity of Aldrin and Dieldrin in Call mice. These conclusions are further strengthened for both FDA studies, by subsequent independent histological re-evaluation4 and by transplantation studies a. 3. Song and Harville 5, 1964 Feeding tests for approximately one year were conducted on 120 CaHeB/Fe/J (Call) and CBA/J (CBA) mice of unspecified sex. The numbers of mice per group were controls, 10; Aldrin, 15 ppm, 55; Dieldrin, 15 ppm, 55. Two animals from each group were sacrificed "periodically". Liver cell necrosis was induced by Aldrin and Dieldrin in mice within 60 days. Liver "neoplasia" developed in four Call and three CBA mice, in unspecified groups, between 330 and 375 days. Conclusions Despite the author's implication of the hepatocarcinogenicity of Dieldrin in Call mice, this is totally unacceptable as a carcinogenicity test report because of the absence of minimal toxicological data. Furthermore, details of this work do not appear to have been subsequently published. 4. MacDonald et al., ~ 1972 This report was statedly prepared for publication on September, 27, 1972, but has apparently not been published yet.

21

Technical grade Dieldrin, dissolved in corn oil, was administered in the diet at two dose levels to weanling Swiss-Webster mice. The control and test groups consisted of 100 males and females each, the test thus being based on an initial total of 600 mice. The initial dosage in the two test groups was 1.5 and 5 ppm for the first two months; this was subsequently increased to 3 and 10 ppm, respectively. High "early mortality" from fighting occurred in males in all groups and was adjusted by subsequent enlarging of all male groups by approximately 30% of their original number. Intercurrent pathology, inflammatory lesions and amyloidosis, were noted and were presumably involved in the high mortality in all groups. The duration of this experiment is unclear. The authors' Table 1 and the Summary section refers to the study as "life-time"; the Results section, however, refers to the "termination 0f experiments", implying that the mice were all sacrificed at an unspecified time. No .data are given on the individual time of death of any animals. Only 71% (484/684) of the mice were examined histologically (Table 4). Marked decreases in the incidence of spontaneous mammary carcinomas were noted in treated groups--Dieldrin 10 ppm, 3/70; Dieldrin 3 ppm, 15/78; controls 11/71. No significant weight loss or changes in liver/body weight ratios occurred in mice in test groups. It is claimed that Dieldrin is not carcinogenic, although it induced a dose-related increase in "non-neoplastic lesions of the liver", such as "regenerative hypertrophy and nodulation or regenerative hyperplasia" (Table 4), which terms are, however, undefined; nodulation presumably implies focal nodular hyperplasia. The incidence of "nodulation", both sexes combined, in controls, 3 ppm and 10 ppm groups are 0, 2.5 and 48%, respectively. TABLE 4 S U M M A R Y A U T H O R S ' TABLES 1 AND 2

Observation

Dieldrin, 3 ppm

Dieldrin, 10 ppm

Control

M

F

M

M

F

100 20.7 78 0 2 2 (3)

130 100 13.3 21.8 91 70 0 0 62 51 32 (35) 44 (63)

125 12.7 93 0 0 0

100 18.8 71 2 0 0

1

Nos. mice/group 129 Mean survival, months 12 No. mice examined histologically 81 No. mice with hepatomas 0 No. mice with liver hypertrophy 8 No. mice with liver nodulation (%) 2 (2)

F

a. Re-evaluation o f the MacDonald et al. study 6 by Reuber 4 and others 7

Randomly selected slides from males and females in control, 3 and 10 ppm Dieldrin groups which had been examined, apparently separately by MacDonald and Blum and Anderson, the editor of a well-known textbook on human pathology, and reported on in the unpublished September 1972 paper of MacDonald, were reexamined by Anderson in September 1973 and examined for the first time by Reuber in March 19734, 7. 22

As indicated in Table 5, Anderson reversed his original diagnoses, presumably reflected in the unpublished MacDonald report co-authored by Anderson, from non-neoplastic to neoplastic lesions, including hepatocellular carcinoma, in approximately one-half of the only 14 liver sections he appears to have originally examined. It appears that the histological diagnoses in the MacDonald et al. report were largely made by MacDonald, and that the conclusions as to the non-carcinogenicity of Dieldrin in this report reflect his diagnoses4 which have since been challenged or reversed by several pathologists, including Anderson, one of MacDonald's co-authors. TABLE 5 RE-EVALUATION OF SOME STUDIES H a = hyperplasia; H y = h y p e r t r o p h y ; N = n o d u l a t i o n ; N N = no n e o p l a s i a ; A m = a m y l o i d ; H g -- h e m a n g i o m a ; H = h e p a t o m a ; H C - hepatocellular carcinoma.

Code No. (Sex)

MacDonald, 1971

Blum, 1971

Anderson, Sept. 1971

Anderson, Sept. 1973

Reuber, March 1973

Leukemic infiltration NN

--

NN

NN

NN

--

NN

Hg

Hg

Hg

Hg

Am

--

--

H H

-H

H --

Duct adenoma ? HC

---

--

Am Am H? HC HC Malignant Kupfer sarcoma t u m o r 7 - type NN NN N HC

Hy Am Hy Am Ha, Hy, N C l o u d y swelling

----

NN NN NN

Mild infiam. --

C l o u d y swelling

Ha, Ha, Ha, Ha,

Control 1, M 5, 6, 22, 11, 15,

M M M F F

17, F 24, F

D-3 ppm 3, M 7, M 13, 20, 9, 12,

M M F F

NN Hg

NN NN

NN Am N Am N Degen

NN Am HC Am HC NN

~

NN

NN

NN

N

--

NN

N N

Atrophic Cellular infiltr,

N, ? H Hy, N, ? H H, ? H C NC ? HC N, Hy, 7 H H, 7 H C Degen, N N

HC HC HC NC HC HC HC Am

~

NN

Duct Hy, N C

--

HC

HC

D-3 ppm 16, F

D-IO ppm 4, M 19, 23, 2, 8, 10, 14, 18,

M M F F F F F

21, F 25, F

Hy, Hy, Hy, Hy,

N

--

NN

Ha,

Hy, N Ha, Hy, N

-~

NN

Ha, Hy, N

~

Degen D u c t prolif. Hy, N, Duct adenorna Hy, N , 2 ° sarcoma

Degen

NN --

23

Of the livers in 10 mice on 10 ppm Dieldrin in which nodulation and non-neoplastic lesions were originally reported ~, Reuber diagnosed 7 instances of hepatocellular carcinoma; these diagnoses were all confirmed by Firminger and in the case of selected slides also by Popper and Farber 4. The results of this re-evaluated study thus confirm the hepatocarcinogenic effects of Dieldrin, although revised incidence figures cannot be given, as the re-evaluation was only based on a relatively small number of sections. b. Conclusions

This study, which is marred by high premature mortality in all groups, nevertheless demonstrates the hepatocarcinogenicity of Dieldrin in Swiss-Webster mice, thereby confirming similar t~ndings in two FDA studies on Call mice ~' 2. The authors' contrary conclusions, based on the alleged non-neoplastic nature of the Dieldrininduced liver lesions, has been refuted by independent re-evaluation of randomly selected histology4 and this refutation has apparently been endorsed by one of the co-authors of the original report 7. Detailed re-evaluation of the remainder of the histological material in this experiment appears to be indicated. 5. Tunstall study 1: Walker et al, s, 1973

This is the most extensive published report on carcinogenicity testing in mice, which is comprised by four related studies all using CF~ Carworth strain SPF mice fed Dieldrin (recrystallized and purity 99% HEOD) commencing at 4 weeks of age. "Study 1 '" Including the positive controls, this was based on a total of 1500 mice distributed by group and sex as follows: 0ppm, 300; 0.1 ppm, 125; 1 ppm, 125; 10 ppm, 200 (Table 6). The positive control and 10 ppm groups were palpated abdominally TABLE 6 RESULTS OF CARCINOGENICITY TESTS ON MICE (based on authors' Table 1) Dose (ppm)

0 0.1 1.0 10.0

No. animals

% with liver tumors A/B

M

F

M

F

288 124 111 176

297 90 87 148

20 (16/4) 12 (13/0) 26(22/4) 27 (23/4) 31(23/8) 37(31/6) 94 (37/57) 92 (37/55)

% with lung metastases

% with lung tumors Adeno-

Carct-

% with lymphoid tumors

% with other tumors

M

F

M

F

M

F

M

F

M

F

0.7 0.8 0.4 0.6

0 0 1.1 4.5

33 38 38 18

16 26 34 10

8 11 12 1

6 13 14 0

35 21 20 24

40 50 54 5

6 3 5 2

7 9 17 1

weekly, commencing at 16 weeks. Once masses were detected, animals were palpated twice weekly and animals were killed when masses became large, even if they were otherwise well. 24

"Cumulative percentage morbidity o f the various groups was calculated on the basis of the numbers of mice found dead, showing signs of ill.health and serit for autopsy, or sent ]br autopsy because o f the size o f the intra-abdominal mass" (my emphasis).

Half of all animals on 10 ppm had died or been killed by 15 months; the corresponding time for controls was 20-24 months. Palpable liver masses were identified in I0 ppm groups by 9 months, but were never detected in 0.1 or 1 ppm groups whose life spans were similar to those of controls. Surviving animals were sacrificed from 32-33 months. Histological examinations were based on 88% (1321/ 1500) of the total number of animals tested. No data are given on the time of death or sacrifice of animals with tumors. While overall life table data could be constructed from the authors' Figs. 1-5, which plot "cumulative morbidity" at various Dieldrin levels as a function of age, however, premature sacrifice of non-moribund animals, because of detection of large abdominal masses, made accurate determination of survival rates impossible, besides lowering the incidence of tumors, tumor size and metastases. It is not clear how the incidence of tumors in this and the other studies in the report were derived, as thes.-, were based on an undefined "number of animals" (authors' Tables 1--4); moreover, there appear to be discrepancies between numbers in the text and tables. Illustratively, 176 and 148 males, respectively, were dosed with 10 ppm, though the text indicates that these groups initially consisted of 200 animals each. Liver tumors were classified as Type A or B. Type A tumors were described as solid cords of closely packed parenchymal cells with morphology little different from the rest of the parenchyma, and which appear to grow by expansion and do not have a fibrous capsule; they can be single or multiple and can involve an entire lobe. These nodules did not show sufficient fibrosis to be regarded as hyperplastic or regenerative. "Since a clear-cut definition was not possible between a hyporplastic nodule and benign hepatoma on cell morphology or size of the growth only, these growths were classified a s benign tumors and were referred to as Type A tumors..."

Type B tumors were extremely rare in controls. In fact, n o n e of the 297 female controls in "Study 1 ", quite apart from the other 215 female controls (totalling 512 females) in "Studies 2.1, 2.2, 2.3 and 2.4", developed Type B tumors; similarly, of the 508 male controls in these studies, comprised by the 288 males in "Study 1" and the 220 controls in "Studies 2.1, 2.2, 2.3 and 2.4", an incidence of only 2.1% Type B tumors was noted. Focal necrosis and papilliform or adenoid areas were interspersed by irregular vascular channels. Abnormal mitotic activity and anaplasia were seen in varying degrees; type A tumors often co-existed. A high incidence of tumors occurred in 10 ppm groups, with a predominance of Type B lesions. Occurrence of tumors was dose-related, with effects detected at even the lowest dose tested, 0.1 ppm, at which the apparent "no effect" level was not reached. This classification into Type A and B tumors appears somewhat arbitary and possibly misleading. Type A appears to include hyperplastic nodules, as well as 25

histologically well-differentiated carcinomas, referred to by the authors as "benign hepatomas", which, as evidenced in the FDA studies, both metastasize and grow after transplantation. Categorization of well-differentiated carcinomas as Type A tumors thus decreases the true incidence of liver carcinomas, if the latter are defined in terms of Type B tumors, while inclusion of hyperplastic nodules appears to falsely elevate the incidence of Type A tumors. The resulting effect would thus probably induce an increase in the sum total of Type A and B tumors in controls, and thereby reduce the apparent difference in the incidence of total liver tumors between test and control groups. Thus, the Tunstall classification has two possible mutually conflicting effects. It is, however, clear that this possible conflict does not detract from the conclusive evidence in these experiments of the carcinogenicity of Dieldrin. The percentage of tumor-bearing animals with pulmonary metastases in this study, as well as in other studies in this report (Authors' Tables 1-4), is probably an under-estimate, as it is implied that these were looked for only when emboli were found at autopsy. "Detection of secondary tumor growth in the lung was difficult, as serial sections through a lung with small intravascular ©mboli commonly showed only a few positive slides" (ref. 8, p. 423).

Probably more importantly, the incidence of metastases was likely to have been markedly decreased by premature sacrifice. "Organochlorine" type liver changes were found to be progressive with exposure; however, centrilobular fatty change occurred only at later stages. Apart from the conclusive data on the hepatocarcinogenicity of Dieldrin in both male and female mice, probability tests on the incidence of non-hepatic tumors (Table 7) indicate that Dieldrin induced a highly significant incidence of pulmonary adenomas and carcinomas, and also lymphoid and "other" tumors in female m i c e g ; it must be stressed that such conclusions are based on large numbers of animals at risk. In spite of their own tabulated data, the authors anomalously conclude that: "... in the Dieldrin groups in Study 1 and the four experiments o f Study 2, no increase (of tumors) was found in other tissues (Tables 1-5)". (ref. 8, p. 427.)

These data clearly indicate the carcinogenic effects of Dieldrin in various target organs, besides the liver, in the mouse, and appear consistent with similar findings on multiple site tumors in FDA studies in the rat 1. "Study 2.1" This was a dose-response carcinogenicity test based on more test groups than in Study 1, but on a much smaller number of animals per group (Table 8). The lowest dose tested in this dose-response study was 1.25 ppm, although Study 1 clearly shows carcinogenic effects at the 0.1 ppm level, which is approximately 12 times lower than the lowest dose tested in this experiment. As can be seen (Table 8), the peak incidence of hepatomas in both males and females occurred at the 5 ppm level. "Competing risks" of toxicity and mortality appear to have been responsible for a reduced tumor incidence at higher dose levels. 26

a.

~dd

0

0

,-1

z

.1 0

0

~

~Sdd

:~

ddd

0

Z 0 u~ u~ 0

z ~~v. ~~ ~ =~ ~ 0 II II II U

.
5 0 ppm) ~. Liver cancers were found in 4/7 females and 1/11 males in the 100ppm Dieldrin group; this incidence is statistically significant (p = 0.0264), both sexes combined in relation to controls 7. It should be stressed that no hyperplastic nodules or carcinomas were found in the livers of control animals in this study 6. It appears that the latent period for induction of extra-hepatic tumors was comparatively longer than for hepatic tumors, as these were found at higher dosage levels where early death with acute hepatic and renal necrosis was observed 6. Thus, competing risks of acute toxicity may have decreased the incidence of extra-hepatic tumors at high dosage levels. Selected histological material was also examined by Firminger and Farber 6 who confirmed the above histological findings. b. Conclusions This F D A study, based on small groups of Osborne-Mendel rats, only twothirds of which were examined histologically, demonstrates dose-related decreased survival in groups fed more than I0 ppm of Aldrin and Dieldrin, and chronic nephritis, particularly in males in higher dosage Dieldrin groups. "Chlorinated insecticide" liver lesions were recognized even at the lowest doses tested, 0.5 ppm. No liver tumors were reported by the authors, who however, recognized a relatively high incidence of multiple site tumors at lower dosage levels of Aldrin and Dieldrin. Independent reevaluation 6 confirmed these multiple site tumors, whose incidence was statistically significant4; this is consistent with similar effects induced in mice in a subsequent study 5. Additionally, a statistically significant incidence of liver carcinomas was found at the 100 ppm Dieldrin dosage level7. It should be stressed that no liver carcinomas were found in controls ~, nor were they seen in another long-term F D A study with over 200 control Osborne-Mendel rats 8, nor in "thousands" of control Wistar, Buffalo or Sprague Dawley rats 9.

39

4. Song and Harville I°, 1964 Feeding tests were conducted on 60 Holtzman rats of unspecified sex for approximately 1 year. The number of rats per group was as follows: controls, 15; Aldrin, 250 ppm, 15; Dieldrin, 250ppm, 15; Dieldrin 15 ppm, increased by 10 ppm every 2 weeks until 385 ppm, 15. Two animals from each group were sacrificed "periodically ". Liver cell necrosis was induced by Aldrin and Dieldrin within 60 days. Nuclear hypertrophy and abnormal mitoses were noted by 90 days and were more m a r k e d in rats killed after 120 days. This report is totally unacceptable, even in abstract form, as a carcinogenicity test, particularly in view of its short duration and the absence of substantive data. Conclusions This is totally unacceptable, even in abstract form, as a carcinogenicity test, because of the absence of even minimal toxicological data. Furthermore, details of this work do not appear to have been subsequently published. 5. Deichman et al.1 i, 1967 This was designed to study synergistic interactions from feeding mixtures of carcinogenic pesticides, including Aldrin; the limited number of test groups, however, appears to preclude this objective. Aldrin, 5 ppm, was fed to 30 male and female Osborne-Mendel rats for 25 months at which time there was 65% survival. No significant effect on liver/body weight ratios was noted. It is stated that generally one tumor per rat was found and that these tumors were uncommon prior to 20 months (Table 4). TABLE 4 SUMMARY Group

Control Aldrin

OF AUTHORS'

TABLES 5 AND 6

No. tumors

No. rnal~gnant tumors

M

F

M

F

1 2

14 13

0 0

1 2

Conclusions Apart from the fact that only one dose level of Aldrin was tested in OsborneMendel rats, no life table data or data on mortality from organ specific t u m o r s are given. This is unacceptable as a carcinogenicity test.

6. Walker et al.l 2, 1969 Groups of 25 Carworth Farm strain " E " SPF male and female rats, were fed with Dieldrin at 0.1, 1 and 10 ppm for two years, with commencing exposure at five 40

weeks of age; control groups consisted of 45 animals (1 ppm = 0.0475 and 0.582 mg/ kg/day for seven-month-old male and female rats, respectively). Additional groups of 15 rats were serially sacrificed for haematological and biochemical investigations. Only 138 rats of an original total of 240 were examined histologically. TABLE 5 S U M M A R Y O F A U T H O R S ' T A B L E S 2, 4 A N D 6

Dose (ppm)

0

0.1 1 10

% mortality

Liver-body weight (mg/kg)

% tumor incidence

" O C " liver changes

Mean fat HEOD (ppm)

M

F

M

F

M

F

M

F

M

F

58 35 39 57

58 43 57 61

3.66 3.72 3.70 3.77

4.08 4.33 4.61 4.84

28 26 22 35

44 65 61 52

- -

- -

--1

--6

0.06 0.26 1.49 19.72

0.311 0.90 13.90 57.81

Irritability, tremors and occasional convulsions were noted after 2-3 months 10 ppm. No effect on body weight occurred at any dose level and mortality was similar in treated and control groups. Mortality was high in all groups (Table 5), so that only groups of 9 to 15 treated and 18 control rats survived to the end of the test. Increases in liver/body weight ratios were noted only in the 1 and 10 ppm female groups; however, the authors' Table 4 shows a dose-related increase at all doses in both sexes, though these were not all necessarily statistically significant. HEOD levels in fat and liver "plateaued" at 6 months; levels in females were between 2-10 times greater than in males. It is pointed out that these results are consistent with human studies, where an asymptotic relationship was claimed. Organochlorine (OC) liver changes were only seen in the 10 ppm group, three females of which had liver nodules. The "percentage tumor incidence" is reportedly similar in treated and control animals, as was that of chronic nephritis and myocardial fibrosis; however, this "incidence" is undefined. This could be based on the percentage of tumor-bearing rats or on the percentage of total tumors in individual rats; if the former, then this could underestimate tumor yields in test groups, as animals in these groups could have had multiple tumors. The tabulated data are, however, possibly suggestive of an enhanced extra-hepatic tumor incidence, particularly in the lower treatment female groups; this could be associated with their reportedly higher fat HEOD levels and also improved survival, and relative low toxicity, at these doses. Additionally, there appears to be an enhanced incidence of thyroid tumors in test and control groups after 2 years. The incidence of total tumors in females, at the 0.1 ppm and 1 ppm levels combined, approached statistical significance at the p = 0.058 level (ref. 7, Tr. 9429). Conclusions This carcinogenicity study in Carworth rats is inadequate, in its present form and in the absence of further analysis, particularly in view of the small number of

41

available animals at risk at two years, due to high premature mortality, and the small number of animals examined histologically. Additionally, no breakdown is given on tumor types or on their distribution and no data are given on the occurrence of tumors in relation to the number of animals surviving and at risk at any time. There has also been no independent re-evaluation of the complete histology of this study. Nevertheless, a near statistically significant incidence of total tumors in females was demonstrated at the lowest dose levels.

7. Deichman et al. la, 1970 Aldrin and Dieldrin at dietary concentrations o f 20, 30 and 50 p p m were each fed to groups of 50 male and 50 female Osborne-Mendel rats, with a composite control group of 100 males and 100 females, with commencing exposure at weanling age. Dosage for the first ten weeks was half the above final levels; experiments were terminated at 31 months. Dose-related acute toxicity, tremors and convulsions, were noted in a few rats in all test groups, particularly in females. It is stated that feeding Aldrin and Dieldrin at the above concentrations had no adverse effect on gain of body weight. However, mean survival was particularly reduced in females in higher dosage groups; Aldrin, 50 ppm, 13.0+2.5 months; Dieldrin, 50 ppm, 16.6+7.3; control, 19.5+4.1. Nevertheless, it is stated in the Discussion that: "... the reduced survival of the majority of these female rats does not present a too serious handicap in the acceptance of the results". TABLE 6 S U M M A R Y OF A U T H O R S ' TABLES 4 A N D 5

Female (ppm dosage groups)

No. of rats examined Male (ppm dosage groups) histologically~total 50 0 20 30

0

20

30

50

Aldrin Dieldrin

88/100 88/100

47/50 48/50

44/50 41/48

31/50 41/50

75/100 75/100

45/50 48/51

46/50 38/50

45/50 44/50

It is stated in the Experimental section: "Sections... of all livers, kidneys and lungs.., were stained.., and examined microscopically ".

This is also restated in the Discussion. However, this is clearly not the case, particularly for male controls and for some treated groups (Table 6). Marked intercurrent pathology, particularly in lungs, was found in all groups. A moderate, but not doserelated, increase in the incidence of centrilobular cloudy swelling and liver necrosis was noted in all Aldrin and Dieldrin male and female rats, but not in controls. A marked increase in chronic glomerulosclerosis was noted in Dieldrin-fed female rats. It is claimed that, in contrast to control animals, there was a reduced number of 42

benign and malignant tumors in Aldrin- and Dieldrin-fed rats, particularly in females, and that the latter was possibly due to increased microsomal enzyme activity, which increased excretion of polar steriods and hence protected against mammary cancers; a marked reduction in the incidence of lymphomas was also claimed for test animals. Although it is made clear that overall survival was reduced at higher dosages, particularly for Aldrin 50 ppm and Dieldrin 30 and 50 ppm females, no data are given on mortality rates by months to allow construction of a life table or to calculate the incidence of tumors in relation to numbers of animals at risk at any specified time. While the histological diagnoses of the tumors in each group are cited in the text, the animal age at which they were noted is not given, so that the incidence curves for individual tumors cannot be determined. Additionally, while feeding experiments were terminated at 31 months, when survivors were sacrificed, all controls were dead by 27 months! In fact, mean survival times in controls were 19.5-19.7 months only. Thus, high early mortality occurred in all groups and this may well have reduced overall tumor yields. a. Re-evaluation o f test o f Deichman et al. la by Reuber 6

Available histology in a limited sample, based on 36 males and 40 females in the:Dieldrin 30 ppm group, was re-examined 6. The results were at marked variance with the originally reported data (Table 7); however, there has been no independent re-evaluation of the remainder of the histological material on the basis of which it has been claimed that the tumor incidence is reduced in test animals in contrast with controls. It thus appears that the original authors under-estimated or under-reported the incidence of malignant tumors, at least in the specified test groups, by an approximately 3-fold factor. TABLE 7 R E S U L T S O F R E F S . 13 A N D 6

Total No. rats ( M and F) with malignant tumors D e i c h m a n et al., 1970 Reuber, 1973

10/79 = 12.6% 26/76 = 34.2%

The malignant tumors identified included one liver cancer, nine adenocarcinomas elsewhere, and 17 sarcomas6; benign tumors occurred in 15 rats. Additional unreported lesions included 23 rats with hyperplastic liver nodules, and acute renal necrosis in 22 females. The limited scope of this re-evaluation, apart from the fact that control histology was not available precluded conclusions on carcinogenicity. b. Conclusions

In this study on rats fed Aldrin and Dieldrin at relatively high levels only, the time of tumor occurrence is not given in relation to the numbers of animals at risk and there was reduced survival from intercurrent pathology and from toxicity 43

at higher dose levels. It is claimed that Aldrin and Dieldrin were not carcinogenic. Independent re-evaluation of a limited sample of histology in the 30 ppm Dieldrin group 6, indicates that the authors under-reported or under-estimated the incidence of induced malignant tumors by approximately 3-fold. REFERENCES 1 J. T. Treon and F. P. Cleveland, Agr. Food Chem., 3 (1955) 402. 2 F. P. Cleveland, Arch. Environ. Health, 13 (1966) 195. 3 0 . G. Fitzhugh, A. A. Nelson and M. L. Quaife, Fd. Cosmet. Toxicol., 2 (1964) 551. 4 T. Fears, Exhibit 52, Testimony at hearings on Aldrin/Di¢ldrin. 5 A. I. T. Walker, E. Thorpe and D. E. Stevenson, Fd. Cosmet. Toxicol., 11 (1973) 415. 6 M. Reuber, Exhibit 42, Testimony at Hearings on Aldrin/Dieldrin. 7 A. Gross, Personal Communication, February, 1974, and Testimony at Hearing on Aldrin/ Dieldrin. 8 0 . G. Fitzhugh, Toxicol. Appl. Pharmacol., 1 (1959) 315. 9 E. Farber, Testimony, at Hearing on Aldrin/Dieldrin, 18 Jan. 1974. 10 J. Song and W. E. HarviUe, Fed. Proc. (Abstr.), 23 (2) (1964) 336. 11 W. B. Deichman, M. Keplinger, F. Sala and E. Glass, Toxicol. AppL Pharmacol., 11 (1967) 88. 12 A. I. T. Walker, D. E. Stevenson, J. Robinson, E. Thorpe and M. Roberts, Toxicol. AppL Pharmacol., 15 (1969) 345. 13 W. B. Deichman, W. E. MacDonald, E. Blum, M. Bevilacqua, J. Radomski, M. Keplinger and M. Backus, Industr. Med., 39 (1970) 426.

44

E. REVIEW OF CARCINOGENICITY TESTS IN DOGS AND MONKEYS

1. Treon and Cleveland t, 1955 Groups of 2 male and 2 female beagles were fed on diets containing 1 and 3 ppm of Aldrin and Dieldrin and were sacrificed between 15 and 16 months; growth rates were similar to controls. Liver weights of test-fed animals were increased, even at 1 ppm. No "toxic changes" in tissues were noted microscopically at 1 ppm, apart from females on 1 p p m of Aldrin, in which vacuolation in distal renal tubule cells was noted. This is totally unacceptable as a carcinogenicity test. The number of dogs and duration of tests was totally inadequate for a carcinogenicity test. Additionally, there was high mortality from toxic effects, and the necropsy data were grossly inadequate; the testing should have been for periods of up to 10 years and should have extended down to doses where prolonged survival was possible. 2. Fitzhugh et al. 2, 1964 In a feeding study, conducted at the FDA about 1952, a total of 26 mongrel dogs, ranging in age from 5 months to 6 years, were dosed with Aldrin from 0.2 to 5 mg/kg/day, 6 days weekly up to 25 months. One male and one female were tested at each of 4 higher doses, and two males and two females at another lower dose. An apparent "'no-effect" level on both clinical and microscopic criteria, was seen in each of 2 dogs treated with Aldrin and Dieldrin at 0.2 mg/kg/day (-= 8 ppm diet); it is, however, pointed out that others ~ had found minimal effects at chronic dosages of 1 ppm. Doses >0.5 mg/kg induced gross toxic effects including marked weight loss and convulsions, and mortality was progressive with increasing dosage. With doses of 1 mg/kg/day, or greater, of Aldrin or Dieldrin, no survival was noted after 49 weeks of treatment. Fatty degenerative changes were seen in the liver and kidneys, and reduced erythropoietic activity was seen in bone marrow on dosages of 0.5 mg/kg/day of Dieldrin and 1.0 mg/kg/day of Aldrin. Diffuse severe hyperplasia of the liver was noted on re-examination of the histology of dogs given the 0.2 or 0.5 mg dose and which survived for 25 months 3. Conclusion This is totally unacceptable as a carcinogenicity test for reasons indicated in the previous dog study.

3. Walker et al. 4, 1969 Groups of 5 male and female dogs of unspecified breed were dosed by capsule with 0.005 mg/kg (------0.1 ppm) and 0.05 mg/kg and sacrificed after 2 years. No effects on weight, health, E E G , etc., were noted. An increase in plasma alkaline phosphatase activity, in the absence of other evidence of hepatic dysfunction, was noted at about 4 months at 0.05 mg/kg males and females. A dose-related increase in phosphatase activity was noted in both males and females at 2 years; illustratively, in females activity at 0, 0.005 and 0.05 mg/kg = 37, 57 and 122, respectively. There was no observed evidence o f gross or microscopic changes in any organs at 2 years. 45

Conclusion This study is totally unacceptable as a carcinogenicity test for the reasons set forth in the two previous dog studies 1' 2. 4. Zavon s, 1970 This report appeared as a preliminary draft dated 1970, which is still unpublished. Commencing 1973, groups of 4-year old Rhesus monkeys, totalling 31 animals, of which 30 were males, were fed on standard chow with Dieldrin for 5.5 to 6.0 years, as follows: controls, 6 monkeys (including the single female); 5 test groups of 5 mofikeys each at doses of 0.1, 0.1, 0.5, 0.1, 1.75 ppm. The highest dose group, 1.75 p p m , originally received 5.0 ppm for 4 months then, 2.5 ppm for approximately 2.5 months, and then 1.75 ppm subsequently. One monkey in the group then gradually had its intake increased to the original 5 ppm, on which it was maintained for 5 years. The author states that the minimum concentration fed, 0.01 ppm, was "... greater by at least one order of magnitude than the amount o f Dieldrin found in the human diet in the U.S.A."

Periodic fat and liver biopsies, serum HEOD determinations, serum enzyme liver function tests were taken; no data on these, however, were presented. Four monkeys "died prematurely", 2 of these had been on 5 ppm, the other 2 died of unspecified causes, and 27 animals were carried through to sacrifice. One monkey on 5 ppm had giant cell formation in the testes, otherwise no histological differences were found between treated or untreated animals in liver or other tissues. However, monkeys on 1.75 ppm (and 5 ppm)... "... demonstrate some adaptive change when measured by EM and this is confirmed on measurement of hepatic intracellular enzymes".

Conclusion This is clearly unacceptable as a carcinogenicity test. The test period of approximately 6 years is much too short, as the average lifespan of monkeys is 20 years. Additionally, the number of animals per dose group is small and testing is inadequate at higher dose levels. Finally, no detailed pathology data are cited. REFERENCES 1 J. T. T r e o n and F. P. Cleveland, Agr. Food Chem., 3 (1955) 402. 2 0 . G. Fitzhugh, A. A. Nelson and M. L. Quaife, Fd. Cosmet. Toxicol., 2 (1964) 551. 3 M. Reuber, Exhibit 42, Testimony at Hearings on Aldrin/Dieldrin. 4 A. I. T. Walker, D. E. Stevenson, J. Robinson, E. Thorpe and M. Roberts, Toxicol. Appl. PharmacoL, 15 (1969) 345. 5 M. R. Zavon, Preliminary Draft, The Effect of Long Continued Ingestion of Dieldrin on Rhesus Monkeys: A Six Year Study, unpublished, 1970.

46

F. GENERAL COMMENTS ON DIELDRIN AS AN ENVIRONMENTAL CARCINOGEN 1. Dieldrin as a carcinogen

Dieldrin is an unequivocal carcinogen, affecting a variety of target organs in both mice (see Section C) and rats (see Section D). It is carcinogenic at the lowest levels yet tested. Additionally, there is some evidence of interactive effects with another related carcinogen, DDT. 2. Environmental distribution

It appears that Dieldrin, as its complex, is now a widespread environmental contaminant of food, air and water; Dieldrin is stable, persistent, highly mobile, lipophilic and accumulates in the food chain t. Dieldrin tolerances in cattle meat fat, milk fat, and meat and meat by-products are being petitioned for at the levels 0.3, 0.2 and 0.1 ppm, respectively; on the basis of these petitions and existing tolerances on certain raw agricultural commodities the amount of Dieldrin in a standard diet has been calculated 2 to be 0.043 ppm. Thus, petitioned tolerances in some commodities exceed the lowest level, 0.1 ppm, at which Dieldrin has so far been tested and shown to be carcinogenic in conventional test systems3. While tolerances are generally expected to exceed mean residue levels, however, market basket surveys in 1973 indicated that residues in butter samples alone from diverse geographic areas ranged 4 from 0.014 to 0.056 ppm. Further evidence indicates that Dieldrin is present in mothers' milk in regions where sampling has been done in the U.S.A. 5. Positive values have ranged 5 from 1.2 to 21 ppb. Calculations based on mean sample values in a California study, 4.3 ppb, have shown that an infant can exceed the ADI solely on a diet of mothers' milk s. Excluding incremental burdens from air and water, apart from interactive or additive effects with other carcinogenic chlorinated hydrocarbon pesticides, such as DDT 3, these residues approach tolerance levels and the lowest level, 0.1 ppm, at which Dieldrin has so far been tested and shown to be significantly carcinogenic 3. Market basket surveys indicate that at least one-third of all children, age 0-5 years, besides segments of other age groups, exceed the ADI for Dieldrin6 ; it should be noted that ADI values were proposed prior to the definitive recognition of Dieldrin as a carcinogen. Dieldrin has been found in more than 85% of air samples monitored by EPA from 1970-19727; mean national values ranged from 1 to 2.8 ng/m 3, with calculated approximate daily respiratory intakes of 0.035 and 0.098/~g, respectively. Mean household dust levels of 2.9 ppm have been reported 6. This is approximately 30-fold higher than the lowest dose, 0.1 ppm, tested and shown to be carcinogenic. Dieldrin is reported to be distributed widely in natural fresh waters and in sediments of streams and lakes in the U.S.A.a; mean concentrations in surface waters were reported as ranging from 5 to 395 ng/1. Dieldrin has also been detected in various potable water samples, especially in rural areas, at concentrations ranging from 1 to 7 ng/1 (ref. 9) and 2 to 3 ng/l (ref. 8). Reflecting the environmental burden of Dieldrin, data from the National 47

Human Monitoring Program of EPA "indicate that Dieldrin residues have been found in virtually all of the [human] samples submitted" to the program 1°. The arithmetic mean of national human tissue levels (expressed on a % lipid basis) was 0.27 ppm in 1970 and 0.29 ppm in 1971. Maximum state, "design data", lipid extractable values ranged from 0.45 to 15.2 ppm in 1970 and from 0.50 to 2.91 in 1971 ~° It should be noted that feeding rodents with 1 ppm of Dieldrin results in tissue lipid levels 11 in the order of approximately 1.5 to 14 ppm. It should also be emphasized that Dieldrin is relatively stable in human tissues, with a reported half-life of 0.73 years12.

3. Involuntary nature of human exposure Continuing human exposure to Dieldrin, in food, besides in air and water, is involuntary and generally occurs in the absence of knowledge and consent, not only as to the degree of exposure and its attendant potential hazards to large populations, but also as to the nature of the benefits, alleged or real, to the user besides society-atlarge, apart from obvious benefits to the pesticide producer. Such considerations should be deafly contrasted with voluntary exposure to drugs, when the immediate benefits and risks are individualized. Considerations of the benefit-risk calculus clearly should be developed in open arenas in which the widest range of qualified viewpoints are properly represented. It seems reasonable that continued use o f Dieldrin, inter alia, should only be possibly justified if conscious societal decisions are made that the societal benefits of its use outweigh its potential public health hazards, and if the existence of alternativesa3, which are similarly efficacious but less hazardous, has been critically excluded. REFERENCES 1 C. F. Wurster, Environment, 13 (1971) 33. 2 A. Gross, Internal EPA Memorandum, Exhibit 50, Statement for Testimony at Hearing on Aldrin/Dieldrin. 3 A. I. T. Walker, E. Thorpe and P. E. Stevenson, Fd. Cosmet. Toxicol., 11 (1973) 415. 4 P. E. Corneliussen, Letter to J. A. Rogers, Office of Enforcement and General Counsel, EPA, 1 Nov. 1973. 5 B. W. De Lapp, EDF Exhibit 32, Statement for Testimony at Hearing on Aldrin/Dieldrin. 6 D. Bomkamp, Exhibit 38, Statement for Testimony at Hearing on Aldrin/Dieldrin. 7 M. A. Fisher, Exhibit 39, Atmospheric Hazards on Aldrin/Dieldrin 'Residues, Statement for Testimony at Hearing on Aldrin/Dieldrin. 8 J. J. Lichtenberg, Exhibit 7, Statement for Testimony at Hearing on Aldrin/Dieldrin. 9 R. L. Morris, Exhibit 8, Statement for Testimony at Hearing on Aidrin/Dieldrin. 10 F. W. Kutz, Exhibit 47, Statement for Testimony at Hearing on Aidrin/Dieldrin. 11 A. I. T. Walker, D. E. Stevenson, J. Robinson, E. Thorpe and M. Roberts, ToxicoL Appl. Pharmacol., 15 (1969) 345. 12 K. W. Jager, Aldrin, Dieldrin, Endrin and Telodrin, Elsevier, A m s t e r d a m / L o n d o n / N e w York, 1970. 13 Respondent's Brief Proposed Findings and Conclusions on Suspension, U.S.A., Environmental Protection Agency, Office of General Counsel, Fifra Pocket, Nos. 145 et al., September 16, 1974.

48

G. CONCLUSIONS 1. Carcinogenicity tests on Aldrin and Dieldrin in mice Table 1 presents in summary the salient data on the six carcinogenicity studies in mice which have been reviewed in Section C. These data unequivocally demonstrate the carcinogenicity of Dieldrin. Dieldrin has been shown to induce liver cancer in all these studies, excluding that of Song and HarviUe, 1964, which was unacceptable, involving three different strains of mice. Of additional interest is the fact that one of these studies (Walker et al., 1973) clearly demonstrates a dose-dependent induction of liver cancers, with the absence of an apparent "no-effect" level at even the lowest dose tested, 0.1 ppm, and also following only one to two months treatment at 10 ppm levels; the same study also clearly demonstrates the induction of a statistically significant incidence of extra-hepatic multiple site tumors, again at even the lowest dose tested, 0.1 ppm. Apart from histological criteria of malignancy of the liver cancers, which have been confirmed by independent re-evaluation, further evidence of their malignancy, has also been obtained by transplantation studies and by the development of pulmonary metastases. 2. Carcinoyenicity tests on Aldrin and Dieldrin in rats Table 2 presents in summary form the salient data in the seven carcinogenicity studies in rats which have been reviewed in Section D. As can be seen, the dosages tested were generally higher than for the mice tests and this resulted in poor survival and toxicity; higher dosages induced acute hepatic and renal necrosis. Data from one study (Fitzhugh et al., 1964), as confirmed by independent re-evaluation, clearly demonstrate the induction of a statistically significant incidence of multiple site tumors at the lowest dosages tested of Aldrin and Dieldrin, consistent with similar effects reported in mice (Walker et al., 1973). Also a statistically significant incidence of liver cancers, at 100 ppm of Dieldrin was demonstrated. Independent statistical evaluation of another study (Walker et al., 1969) indicates the induction of near significant increased incidence of total tumors in females at lower dose levels. The remaining studies are unacceptable or inadequate, at least in the absence of further analyses. 3. Carcinogenicity tests o.f Aldrin and Dieldrin in dogs and monkeys All four studies reported in Section D are unacceptable as carcinogenicity tests because of their very limited scope, the small number of animals tested over restricted dosage ranges and because of their brief duration of test, which was for a maximum of two years in the three dog studies and for six years in the monkey study. 4. Policy implications There is a growing and now general recognition that the majority of human cancers are due to environmental chemical carcinogens and that they are hence preventable. All chemicals, with the possible exception of trivalent arsenic, which are

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The carcinogenicity of dieldrin. Part I.

The Science o f the Total Environment, 4 (1975) 1-52 © Elsevier Scientific Publishing Company, Amsterdam - Printed in Belgium T H E C A R C I N C G E...
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