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A critical study of methods of assessment of effects of low doses Prof. Etienne Fournier

A critical study of methods of assessment of the effects of low doses P. Etienne Fournier (1993) This paper sets out to be a consideration of the positions taken by experimental toxicologists and regulatory bodies for more than 40 years and on their necessary confrontation with the facts from human observation conducted by clinical physicians and, preferably, by clinical toxicologists. One objective is apparent, in any case part of all the legislation - that of suppressing - in theory completely (objective 0), in practice in such a way as to become indiscernible, and at worst to reduce substantially - ailments connected with the absorption of chemical products however absorbed and the clinical course of cancer. Let us admit that in the usual constitution of discussion panels, clinical toxicologists (representing internal medicine or occupational medicine) although the only qualified observers, are practically excluded from the final report in favour of experimental toxicologists or analysts. This is not a paradox, since each of their contributions stresses actual facts including a strong probability of correlation between a known exposure and the too premature, too frequent and excessively atypical incidence of certain cancers. From these comes the set of agreed procedures which are transcribed and quantified to achieve national and international regulation. Calculations in this matter are those of epidemiologists and biostatisticians and evaluation of doses those of analysts: A first logical, quasi-mathematical, relationship will be established: For one exposure to x, ppm in the air or for oral absorption of x2 mglKg/day, n cancers appear (in the context of the study: target population, - exposure time, time before appearance, & -).

A second relationship, no less classical, normally follows. It is defined in accordance with methods which avoid the essential bias of the number of these cancers observed in a reference population suffering no exposure to the target chemical hazard. In general, it results in a mortality coefficient implying excess mortality for a defined exposure. From the moment when the set of interpretations begins, the most frequent being a major increase through a purely formal movement to sufficiently large numbers: For example, if X has observed two fatal cancers in the target population and only one in the control population, Y can say that the SMR ratio is 200%. Whilst recognizing immediately that this simple outline is not only unacceptable but is far from representing the reality of the cancerous condition. a) Most common hormone-sensitive cancers currently treated are cured or benefit from a prolonged remission. Indeed the morbidity of cancer is exceptionally well-documented. b) The relative importance of cancers subject to hormonal influence has not ceased to grow and this group does not always have an obvious connection with toxic impregnation, with the exception of thyroid cancers, although an associated effect should be observed. c) Conversely, cancers appear in subjects treated and cured by the use of radiation or drugs primarily acting on DNA. d) Other pathological phenomena certainly recognized apart from the transmission of transplacental products, individual predispositions in individuals who are carriers of inherited cancers, identify genetic criteria in families where the preponderance of cancer amongst the causes of morbidity is important. This notion is particularly useful in the study of u~i a childhood cancers. ~ ~ ~ ~ 2

This is found in an exaggerated manner in subjects who are carriers of inherited abnormalities relating to the DNA and its repair, and who present a greater prevalence of cancers of the skin or blood (leukaemias and lymphomas). There are too few such families to identify from them response criteria to chemical products. And caffeine, the classical inhibitor of DNA repair, has no demonstrated role in experimental carcinogenesis. e) The most common cancers of chemical origin are cancers of the lung due to chronic addiction to smoking with constant exposure to several grams of carcinogenic substances over the bronchial mucous membrane, photosensitive skin cancers susceptible to activation by chemical products and of the bladder after excessively prolonged impregnation (aromatic amines). In all three cases cellular exposure is massive and prolonged. Asbestos cancers in the form of mesothelioma are comparable with them because of a considerable accumulation of asbestos fibrilla irreversibly accumulated in the serous membrane. In fact, human cancers due to chemical products (the "may cause human cancer" category) appear after long periods of close and significant contact between a cellular type and the product itself or its metabolites. The "one hit - one cancer" hypothesis should therefore be questioned. This slogan is suspect because it is a slogan. It cannot simply be accepted. Clinicians have never taken steps to observe a cancer occurring after one single minimal contact, which certainly does not mean that this method of occurrence cannot be suggested as one possible hypothesis. Each individual is free to express his views. But this first attitude, an extreme one, is also one which prevents all subsequent discussion, since no individual has been totally protected from the sun or fumes. In advance we are all cancerous - which will perhaps be confirmed but in different ways. 3

A better quantitative approach to the initial mutation phenomenon might be assessment involving tests on procarocytes of the minimum concentration effective. In the usual literature, the biologist looks for an obvious effect which he calls positive and which he contrasts with doubtful or negative results. It would be interesting to test the molecules by specifying the threshold-concentration from observation of a rise in mutations compared with the spontaneous mutations of the original preparation. Even if we do not know the cause of spontaneous mutations we may assume that they relate to a random process on the scale of a micro-organism which becomes a measurable constant for the population, and the deviation from the constant may be a good experimental index for the effect of low dose- concentrations (less than 1-9M). The same reasoning is proposed for organs and their cellular population. Proposed extrapolations a) anirnal references Since no cancer due to a chemical product can have been observed in man in the purest imaginable environmental conditions, the logical stance would be to take experimental data supported by control animals reared under rigorous conditions: water, food, air, accommodation and free of viral immunological reactions. Even if all is not yet perfect in the field of good laboratory practice, experimenters are nearing perfection. They also note.that the spontaneous mortality, apparently inescapable, of such animals is largely of cancerous origin, and that the date of appearance of cancers depends on the species and the breed. N.B. 1 Epidemiologists, for their part, give us to understand that the prevalence of human cancers is a function of age:lcA5, but this proposition has only modest consequences if the average lifespan varies little from one population to another. Thus the variation from 70 to 75 years (considerable average variation) only increases the probability caused by 41%. 4 2501171199

N.B.2 An extrapolation by linear or even semi-logarithmic function towards doses - or concentrations - considerably lower than those for which cancers have been observed in man or animals, leads to non observable rates of effect still comparable with the initial doses, generally very high (n mg/Kg/day). Over some forty years an abstract approach has developed based on hypotheses which at first were the interpretation of extremely simplistic elementary principles but which have evolved through the introduction of the biological knowledge accumulated during recent years and the biology of DNA. Let us briefly recall them: First hypothesis: only one particular shock - production of a single radical OH* - causes DNA to explode (cellular death) or deforms it sufficiently for the cell to become uncontrollable (one hit one cancer). Apart from its fundamental drawbacks, the hypothesis ill applies to the absorption of chemical substances or to the effect of their metabolites. Avogadro's constant 6.02 1023 implies that the nanogram supports an average of 3 10 12 reactive poles. This is considerably more than can be supported by an organism if each cell absorbing a single reactive molecule were to become cancerous. Second hypothesis: It refers to the most generally accepted knowledge of cancerisation, the current theory 'making to succeed' an initial stage which remains latent in successive phases of advancement. If the same molecule is initiator and promoter, the hypothesis of a multiple stage reaction is acceptable. Unfortunately our knowledge about promoters is still very hazy compared with what we know about initiators and complete carcinogens. If we admit that a very large number of molecules such as some phenols are promoters and that the human being always carries them, we are brought back to the previous stage. 5

For initiators the current theory would be that of incomplete repairs leaving adduct-mutations in place, becoming more and more numerous. For promoters a consensus without formal reason agrees somewhat shamefacedly to consider that they only act above a certain threshold. Third hypothesis: This results from knowledge of anticancerous genes - emerogenes. These can as well be stimulated by both chemical products as pro-oncogenes. Similarly, damaged DNA excision-repair phenomena unite to prevent the appearance of immortal cells with carcinogenic potential. The theory seeks a differential function between the initiator effect and the repairer effect. Fourth hypothesis: Coming finally to the in situ control of formed cancer and its own evolution by metastases, attacks and phases of stabilization. The simple theory holds that once formed, the initiated and promoted cell divides in an inescapable way. In this case, whatever the duration of a pathological division, the carrier of the cancerous mass should die within a few days or months, which is effectively observed in acute forms. The actual phenomenon becomes at least doubly random - uncertainty about the progress and uncertainty about regression - in so far as we are incapable of gauging the different factors and measuring the various cytokines which regulate the complete process. Mathematical analysis of sequential and contradictory cellular phenomena calls on models of physio-pathological regulation. In respect of mathematical carcinogenesis, we are unfortunately at the point where the ancient Egyptian surveyors of a random expanse - the silt of the Nile - were, before fundamental data about plane geometry. But additional data is gradually appearing. Evaluation of resistance to a cancer has barely begun. For we already know that not all asbestos workers die of mesothelioma even if exposed to the maximum amount of dust. 25aiiil20i 6

On a simpler mode, not all the bacteria of the Ames system mutate when they divide in a milieu containing a reference carcinogen, but it is clear that the random nature of the mutation is located at a level other than that of the non exposed population. The deregulation is explained by a coefficient of mutagenesis: It is in homologous terms that the coefficients of morbidity (rarely recognized) and mortality (which are only valid for cancers which are often fatal) appear. Uncertainty increases in the proximity of the coefficient 1 in as much as the first serious observation was that of the "healthy worker effect" which brings the coefficient to 0.8 during adult life. Hence the extraordinary confusion of demands for nil risk, faced by biologists and doctors who admit that this notion is strictly speaking unrealistic (Krewoki et al 1984), which is not to say that to propose and tolerate an added acceptable risk (between 10-5 and 10-8) makes more sense: 10'6 x 70 years: about 6 hours in terms of life expectancy. The biologist accustomed to margins of error otherwise large has a poor grasp of the practical value of attitudes, which may be compared to a proposition of Pure Behavioral Act: Art. 1 "the designated population should live without sin". Should the risk of sinning be agreed to be 10-5 or 10-8? Should attempts at evaluating a carcinogenic effect be similarly rejected wholesale? The answer is certainly no, provided the limits of models are known. * N.B. I would especially like to thank Professors A.J. Valleron and G. Thomas, who described to me the methods used and the limits of use. a) The model with threshold (tolerance model) assumes that a subject exposed to a dose (cumulative) of a carcinogen will develop a cancerous 2501171202 7

tumour if the dose exceeds a threshold called a tolerance. Various approaches are suggested (see Appendix). These models are only valid for binary situations excluding all interference from other factors, eliminating the time factor to the advantage of the single cumulative dose. Elemental toxicology, throughout life, permits these calculations. These models are however little used, for it is rare that human observations concern more than three situations: A lot, a little, or no chemical product. Experiments on animals rarely involve more than three to four doses: one close to the maximum dose more or less well tolerated (in the general sense) by the animal; another fairly low dose is selected in the reasonable expectation that nothing will be observed; and one or two intermediate doses which are the only ones genuinely compatible with a sub-normal life expectancy. In these conditions it is difficult to draw up a graph with a single point - or two - and the regulations most frequently allow for the lowest dose which showed no effect (NOEL). Models allowing for the effect of time Time is a fundamental variable of carcinogenesis but its introduction necessitates a biological unity such as average life span or the extreme life expectancy of the species or ethnic group, or that of the appearance of perceptible phenomena of which cancers form a part. There is no consensus about the mechanism of the increase in the prevalence of cancer according to age (accumulation of errors, progressive chromosomal abnormality, perigenic abnormality of the histones, epigenetic abnormalities of cellular regulators (hormones, adenylcyclases, calcic mediators etc.), but an experimental gain is confirmed by monitoring animals throughout their lives. Little by little the notion is taking hold that in certain mammals the prevalence of cancerous mortality becomes preponderant in excess of 70%. 2501171203 g

In man the situation is evolving in the same direction although the part played by degenerative causes with cellular death remains high. If the average life span reaches 80 years cancerous morbidity should become considerable. N.B. This discussion is different from that about the sensitivity of elderly subjects to exposure to carcinogens. Models using time refer to empirical models called log-linear, of the type T (probability - distribution according to observation) T=exp( B1 Z + yW ) p. vector p. vector actual random variable of parameters of functions of a single dose or of regression (Lox) y (t:d) = yo W exp (6'Z(t))) and models based on biological hypotheses : multi-hits, multi-stage. These already old models like those of Fisher and Holloman (1951) have had the merit of taking parallel events into account (more than six cells transformed together - abandoned- ) or more DNA disorders (6-7 successive mutations on the same cell). The latter argument was essential to explain why the incidence of many human cancers would grow with age to the power of 5 or 6. We acknowledge that currently the appearance of a cancer supposes at least two, and probably fewer than seven predisposing factors affecting one cell. The model derived from the work of Moolgavkar, Venzon and Knudson ( 1981) results in an outline consisting of normal and intermediate cells and those proliferating out of control, capable of reproducing themselves as they are, of leading to the later stages, or dying. Recent models associated with validated experimental or epidemiological data, studies of absorption or metabolisms, encompass usable results for 9