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. s RISK ASSESSMENT OF CHEMICAL CARCINOGENS: IS IT TIME FOR A CHANGE? By Robert J. Scheuplein, Ph.D. Director, Office of Toxicological Sciences Center for Food Safety and Applied Nutrition Food and Drug Administration Washington, D.C. 20204 Thank you, Carol, for the introduction and to ILSI for inviting me to, speak to you today. My subject is the Quantitative Risk Assessment of Carcinogens: Is it timE: for a change? Before I speculate about the direction in which risk assE>.s,sment might be headed or ought to be turned, I would like to give you some idea of where I think its come from, what science it rests on, what: it is and what it is not. This will involve a brief scientific history of cancer risk assessment with a few detours to places where it touches social policy or has been influ,enced by the Congress and the regulatory agencies. This interaction between science and regulation is important to understand, because a part of my thesis will be that despite well over 500 papers on cancer risk assessment, on the bioassay, on cancer thresholds and numerous related subjects, since 1961 (the date of Mantel and Bryan's paper) -- cancer risk assessment is still more of a regulatory tool than a scientific discipline and rests more on regulatory need than scientific plausibility. Presented at Brookings Institution, Washington, D.C.~June 17, 1991 ~

11 -2- Tt nonetheless involves several scientific matters and any good history should include them: (Slide 1) - Major Scientific_Issues in Cancer Risk Assessment. These issues are all involved in QRA and the final result - the deter- minai:ion of a safe level - depends on how these issues are resolved. And I`ll touch briefly on most of them. Righ- after World War I, scientists began to experiment with large colonies of rodents. Shortly thereafter, Yamagiwa and Ichikawa dis- covered that stdn cancers could be induced in mice by the repeated application of gas tars (ref.). The number of chemical agents tested grew steadily from year to year and it became difficult to analyze the available experimental results because of the variety of different methods adopted. By 1930, efforts began at standardizing these methods. (Slide 2) - Paper_TitlebyTwort and Twort. By 1939, methods were sufficiently well developed that lists of substances with relative potencies were published based on the ability of a c.ompound°s capacity to produce tumors in the shortest possible time. (Slide 3) - List-of Carcinogenic Comgounds Arranged in Descending Order of_Fotency - John_Iball_(1939). In this list by John Iball in 1939, the index of potency is the percentage of tumours A divided by the mean latent period B, recorded in the last column. . There were good social reasons for these academic efforts. It was becoming clear that environmental and occupational exposure to

N carcinogenic chemicals were contributing to the world-wide diversity in cancer incidence. Percival Pott had established the connection between soot and scrotal cancer in chimney sweepers several generations earlier. By the 20's it was clear that polycyclic hydrocarbons were the carcino- genic ingredients in soot, tar and oil. Some cancers on the abdomen could be attributed to carrying a basket of live coals beneath the clothes to keep warm in winter; some cancers in the buccal cavity could be attributed to chewing various mixtures of betel, tobacco and lime and some on the palate to smoking cigars. As Richard Doll (1977) has pointed out„ oncologists who worked chiefly in Europe and North America tended to regard these incidents as oddities and irrelevant to the production of ord:Cnary cancers. So it took a while for people to associate the major- ity of cancers with environmental factors, but soon the association became obvious. These concerns in the 30's and 40's motivated an effort to bring the kno,~nT occupational hazards under control, either by banning their produc- tion or controlling the manufacturing process to reduce exposure to employees. (Slide-4) - Occupational Cancers- Doll (1977). But these occupational hazards could not be responsible for the large observed incidence of cancer. Whole populations, however, had been exposed to lower levels of these same agents. These included polycyclic hydrocarbons, produced by the combustion of coal, wood and oil. It was known, for example, that residents of large towns in the U.K. may have been exposed - mainly through the combustion of domestic coal - to . something like 1/100 the amount of benzo(a)pyrene regularly inhaled by men working in the manufacture of coal gas and these men experienced only

r -4- an B0Z excess risk of lung cancer (Doll, et al, 1972). It was easy enough to dismiss the corresponding risks on the grounds that the doses were minute, but one did not then (or now) assume for cancer the exist:ence of a threshold. So some form of quantitative relationship between the dose and the resulting incidence was needed. But in the absence of such a relationship, decisions had to be made and FDA banned carcinogens from food during the years well prior to the enactment of the Delaney Clause. Arnold Lehman, the chief toxicologist at FDA in the 40's, stated in an article in 1949 that: "a finding that a substance caused cancer in animals was regarded as so °alarming' as to exclude it from consideration." In 1945, the FDA banned Butter Yellow; in 1950 Dulcin and P-4000, two artificial sweeteners; in 1950 also tonka beans and coumarin; and in 1959, aminotriazol on cranberries, all on the grounds they were carcinogens and had no place in foods. The reasons that scientists were unwilling to assume the existence of thresholds for carcinogens are interesting - primarily because they were large7'.y theoretical. Essentially the argument recast in modern terms went like this: Cancer is caused by agents known to be mutagenic suggesting that at least one crucial, rate limiting step is a somatic mutation. This focused attention on the nature of the genes that undergo mutat:ton and on the amount of chemical needed to affect that change. It was argued that only one molecule was necessary to produce a mutation in the DNA within the nucleus of a cell. This in turn could lead to a

r, -D- misc:oding sufficient to produce eventually a malignant cell. The cell then can reproduce itself in an irreversible and unregulated manner to yield a malignant tumor. But this is theory! What does the experimental evidence show? So far, for any carcinogenic or mutagenic response in any given situation, be it man, mouse, isolated organ or a Salmonella plate assay, there is a demo°:nstrable threshold or "no effect level." In thousands of studies with hundreds of thousands of animals, not a single carcinogen has been found that has not exhibited an experimental threshold. However, animal studies are insensitive and thresholds will vary from individual to individual. It is completely impractical to determine the level at which the most susceptible individual in the whole population might fail to respond. And worse yet, if a gargantuan animal study were done, assuming all the experimental difficulties involved in such a study could be overcome, people would point out that experimental animals are more inbred than people and the result would probably be discounted. The o,ne-molecule theory has to be argued at the theoretical level, so let's look at it. Nothing (in the one-molecule theory) is mentioned about the relationship between the intake dose and the final concen- tratiori of the chemical carcinogen in the nucleus of the cell where it interacts with DNA. Substances that are ingested have to be absorbed, distributed and metabolized usually before they can reach critical organs in chemically activated form. Then the activated molecules have to run a ; gauntlet of sequestration by other uninvolved macromolecules and overcome

r, -6- diffusional barriers before they can first enter a cell, and then later enter the nucleus of a cell. When the carcinogen is in the nucleus and poised to react with DNA, nothing is mentioned (in the one-molecule theory) about the constraints imposed on chemical reactions by the requirements of mass action or the need i:o acquire a transition state configuration or an activation energy prior to reaction, or even that the final adduct be somewhat stable so that 1Ct can last long enough for replication. And :ii` a reaction with DNA does occur, nothing is mentioned about the intron content of the DNA, that the amount of DNA in the nucleus that is not overtly expressed as protein may amount up to 90% of the total. These regions of DNA, which correspond mostly to the centromeric parts of the chromosomes almost certainly instruct no other process than their own repl:[c:ation. And finally nothing is said of the ability of the organism to accommodate to adverse effects - in this case by DNA repair mechanisms and enzyme induction. An example on this last:point was published in 1977 by Tony Pegg of the Hershey Medical Center. Dimei:hylnitrosamine (DMN) is a potent carcinogen in many species. It is well established that DitN exerts its carcinogenic effect after its metabclic conversion into a reactive methylating agent. The electrophile then methylates DNA nucleosides which are likely to miscode. One partic- ular adduct - 06 - methylguanine appears to be promutagenic or tumori- genic: in several studies and it has been identified as probably the adduc:t responsible for tumor induction in animals.

r, What Pegg did was to give DMN to rats and then analyze the livers for the 06-~adduct after 4 and 24 hours. His reE;ults are shown in the slide. (Slide 5). 06 _Methylguanine Levels in Rat Liver DNA At first glance, there appears to be a linear relationship between the administered dose and t:he formed adduct. But if the data are extended to lower doses, this is shown not to be true. At very low doses, 06-adducts levels are many times less than expected on the basis of linearity. Later studies showed that: a saturable enzymic repair system was responsible for the removal of the 06-adduct and that the repair system operated in both liver and kidney cells. These studies don't necessarily imply an absolute threshold for tumor induction for DMN because it was possible to detect some 06 - adduct in DNA 24 hours after a single dose and we don't know what level of 06- methy1guanine may be necessary to initiate tumor induction. But they do show t:hat liver and kidney can protect itself against a low-dose, car- cinogenic stimulus and that linear extrapolation is probably unjustified at low doses. These studies by Pegg are pertinent because they provide information well below the observable tumorigenicity range - over a 20,000 fold dose range - and measure the concentration of the specific adduc.t identified to be the one likely to initiate tumors. In summary, there are many reasons to contest the "one-molecule theory" and to anticipate, in conformity with the animal evidence, that signifi- cant concentrations of a carcinogen might be required to elicit cancer. But nevertheless, the "one-molecule" concept or its equivalent that exceed:Cngly small levels of carcinogens ingested daily for a lifetime !

-8- could be harmful, prevailed - in the 40's and 50's, and I suspect prevail today. Now the question of the existence of a threshold in an individual is a problem in biology - the question of determining the range of possible susceptibility in a large population that is assumed to have individuals capable of responding is a problem in the statistics of sampling to which we now turn. We have to skip over (in the interests of time) a rich part of the biological science that was developed in the 40's and 50's to arrive at our principal focus, the idea of using risk assessment for setting safe population exposure levels for carcinogens, This idea was first pub- lishe:d, so far as I am aware, by Mantel and Bryan in 1961. They showed that a negative animal study - particularly a small one with 100 animals or so, does not necessarily demonstrate that the treatment was safe. Studies of feasible size can be used to establish directly only risks of the order of 1/100 or higher. When a study with 100 animals is negative, we can only claim that we are reasonably sure (assurance levell of 99%) that the true risk is no greater than 4.5 percent. (Slide-6) - Interpretation of Negative Studies. Mantel and Brya:n then proposed to rely on the dose-response principle and extrapolate from this upper limit conservatively to human exposure levels. They examined the dose-responses of many chemicals and concluded that most chemical responses involving lethality decreased very rapidly, with dose-response slopes steeper than.10 or more probits per ten-fold . dose dilution. They noted probit slopes for the therapeutic effects of antibiotics of 2-3, and still lower probit slopes, the order of 2, in

-9- virus assay work. From this experience they felt that a probit slope of one per ten-fold dose dilution would likely be quite conservative for carcinogens. This would mean in the example above that the safe dose corrE:aponding to a risk of 1/100 million would be 1/8,300th of that which produced no tumors in the actual study. This 8,300 corresponds to a safety factor based on an upper confidence limit to a negative study. Later Carrol Weil would propose a factor of 5000 for known carcinogens, using a factor of 10 for animal. variation, a factor of 10 to translate animal results to human, a factor of 10 for cancer on the theory that it is less reversible than other toxic effects and a factor of 5 if the data used were a minimum effect level. (Weil 1972). The Delaney Clause had been passed by Congress a few years earlier as part of the Food Additives Amendment in 1958. In 1960,,there were hearings in the House to deal with some unfinished business regarding Color Additives. Cancer experts testified to the Committee,that there was a great deal of uncertainty about cancer induction at low doses. A report prepared by G. Burroughs Mider, then Associate Director for Research at NCI, was quoted by the Secretary of HEW at the hearings. It had an impact on the Committee. It stated: "No one at this time can tell how much or how little of a carcinogen would be required to produce cancer, or how long it would take the 0 c:a,ncer to develop."

-10- The Secretary also said, "'6,ihenever a sound scientific basis is developed for the establish- inent of tolerances for carcinogens, we will request the Congress to give us that authority:" and a].so, "'So long as the outstan,ding experts in the National Cancer Institute a.nd the Food and Drug Administration tell us that they do not know how to establish with any assurance at all a safe dose in man's food for a cancer-producing substance, the principal in the anticancer clause is sound." The Congressional response was, of course, predictable and the new Color Additives Amendments of 1960 contained its own Delaney anticancer clause. Congress concluded that there was too much uncertainty and it would require FDA to regulate on the side of caution by banning all animal carcinogens from the food supply. Of course, it was only 1960 - analyt- ical methods were typically capable of detecting a few parts per million at best:. There were relatively few carcinogens known and it was not apprer_j'.ated that traditional food and spices and ordinary cooking prac- tices would eventually be found to account for many if not most of them. The widespread contamination of food by low levels of environmental contaminants like dioxins, PCBs and aflatoxin had not yet been r discovered. Nor was the fact that the failure to specify "safe" levels would assure the triggering of the Delaney Clause on food and color