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Assessment of Carcinogenicity: Generic Issues and Their Application to Diesel Exhaust DAVID G. KAUFMAN University of North Carolina Mechanisms of Carcinogenesis Relevant to Assessment of Mobile Source Emissions / 520 Experimental Models in Chemical Carcinogenesis / 520 Role of DNA Replication and Repair / 522 Genetic Effects of Carcinogen Damage to DNA / 522 Atypical Carcinogens / 524 Promotion, Cocarcinogenesis, and Enhancement / 525 Multistep Processes / 526 Variations in Susceptibility / 527 Metabolic Conversion and Carcinogen Activation / 530 Qualitative Assessments of Carcinogenicity / 530 Epidemiologic Evaluation / 530 Bioassays in Experimental Animals / 531 Short-Term Tests in Vivo and in Vitro / 533 Methods for Quantitative Extrapolations to Human Cancer Risk / 534 Estimation of Quantitative Risk in Laboratory Animals / 534 Extrapolations Among Species / 535 Extrapolations Among Routes of Administration or Exposure / 535 Extrapolation to Dose Levels of Human Exposure / 537 Experimental Evidence on Carcinogenicity of Diesel Exhaust / 539 Short-Term Tests of Activity of Diesel Emissions / 540 Data on Carcinogenic Activity of Diesel Exhaust Emissions / 541 Quantitative Assessment of the Cancer Risk of Diesel Exhaust in Humans / 543 Summary / 546 Summary of Research Recommendations: Priorities, Purposes, and Responsibilities / 547 Summary of Research Recommendations: A Research Plan / 549 Air Pollution, the Automobile, and Public Health. (it) 1988 by the Health Effects Institute. National Academy Press, Washington, D.C. 519

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520 Assessment of Carcinogenicity Despite our limited understanding of carci- nogenesis, practical concerns in the "real world" confront us with the need to assess the potential significance of diesel exhaust as a human carcinogen. Such an assessment requires progressing from fragmentary the- oretical insights into the process of carcino- genesis to estimates of the human risk posed by diesel exhaust. Confounding this effort is the fact that diesel exhaust is an imprecisely characterized and inconsis- tently constituted product composed of chemicals that may trigger carcinogenesis individually, cooperatively, or even se- quentially. Researchers are now confronting the dif- ficulties of understanding the etiology and pathogenesis of multifactorial, multistep disease processes, and they are just begin- ning to recognize general principles that may operate in most typical cases of cancer. There is awareness of the relationship be- tween the dose of carcinogens and the resulting tumor response, and recognition of the importance of the metabolism of a carcinogen into reactive intermediates that may cause damage. Cellular mechanisms such as DNA replication may provide op- portunities for carcinogens to transform genetic information, and targets in DNA may include specific genes or sites at which chromosomes are prone to breakage. En- hancing factors, such as promoters, may increase the likelihood of cancer develop- ment. Variations in human susceptibility to cancer make evaluation of the activity of specific carcinogens difficult, although it is clear that certain human tissues or certain individuals are more susceptible to cancer than others. Certain familial tendencies or acquired illnesses are also thought to pre- dispose people to cancer. In this chapter, the evidence on the car- cinogenicity of diesel engine exhausts and the methods used to make quantitative risk estimates from these data are evaluated. Specific evidence concerning carcinogen- esis of diesel exhaust in experimental sys- tems is reviewed, and relationships be . . , . . . tween t. his Information anc . reviews In other chapters are identified. Current knowledge as well as areas of ignorance influence efforts to estimate human risks by extrapolation from the experimental data on animals. A discussion of these issues serves as an outline for making such esti- mates in the future. Mechanisms of Carcinogenesis Relevant to Assessment of Mobile Source Emissions Chemical carcinogenesis is a very complex topic. Thus, this review is selective in its consideration of carcinogenesis, focusing on several general concepts rather than on specific details. The constructive role of studying cancer development in animal models is considered, and certain aspects of the general principles operating in most typical cases of carcinogenesis are exam- ined. The review also touches on unusual cases that appear not to fit the typical pattern of cancer development. It considers the evidence for and the problems associ- ated with evaluating a disease that develops as the result of a multistep process. Finally, the factors that define individual variations in susceptibility are discussed, and features of carcinogen metabolism and translocation are reviewed. Experimental Models in Chemical Carcinogenesis Experimental animal models have been employed to reproduce tumors of the his- tologic types and organs of origin that commonly occur in humans. Such models permit direct experimental study of factors that influence the development of the most common cancers in humans and the mech- anisms of action of particular carcinogens. Examples of valuable animal models and their applications are listed in table 1. Some unique insights have been derived from comparisons of the properties of animal tissues in which the tumor response is a good model for the human disease, to tissues of other species in which the re- sponse is very different from that of hu- mans. Studies of particular tissues have been facilitated by using organ cultures (Saff~otti

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David G. Kaufman 521 Table 1. Examples of Valuable Animal Models of Human Carcinogcncsis Rodent Species Type of Human Cancer Modeled References Application L)osc/ response Metabolic mechanisms Hormonal influences Dietary influences Hamster Rat Rat Mouse Lung Pancreatic Breast Colon Skin Saff~otti et al. (1968) Pour (1984) Scarpelli ct al. (1984) Muggins et al. (1961) Gullino et al. (1975) Ward et al. (1973) Rcddy et al. (1974) Berenblum and Shubik Promotion (1 947) and Harris 1979~. In this technique, pieces of intact tissue representative of the sam- pled organ are grown in culture. Many features of the tissue that exist in viva, including the interrelationship between the epithelial components and the supportive cells, are preserved. Such cultures can be used to assess morphological features, mac- romolecular synthesis, and responses to hormones as well as capacity to metabolize carcinogens and to repair DNA damage. Use of organ cultures has been a principal approach used for analysis of properties related to carcinogenesis in human tissues. Some of the attractive features of organ cultures for example, their maintenance of natural relationships between epithelial and supportive cells are related to some of their major shortcomings. In contrast to cell cultures, organ cultures cannot be propagated, and material from an individ- ual human subject is rapidly exhausted. Cell culture overcomes this limitation be- cause cells may be propagated in culture. However, the very process of propagation exerts a selective pressure, and the cell type that emerges may be unlike that predomi- nating in the intact tissue. Nevertheless, isolated cells have proven very useful in the study of common and unique features of carcinogenesis under far more controlled environmental conditions than is possible . . . In an intact an1ma. I. Although direct experimentation with the objective of inducing carcinogenesis is clearly unethical in humans, a broader, deeper information base is needed on the properties of human cells and tissues that relate to carcinogenesis. This goal has been approached by undertaking culture studies of human cells and tissues obtained at im- mediate autopsies or from surgical speci- mens (Harris and Trump 1983~. Studying the properties of human tissues in vitro allows examination of the human diversity in cancer development. For exam- ple, in vitro techniques can be used to explore the individual variability in metab- olizing carcinogens, repairing DNA dam- age, responding to various hormones, and perhaps even to determine the degree to which various nutrients serve as cofactors . . . In carcmogenesls. Although in vitro carcinogenesis with human cells in culture is rather new, trans- formation of normal cells to neoplastic ones has been accomplished with a number of cell types. Results from such studies permit the direct comparison of the stages in the presumed multistep process of carcinogen- esis in humans and in animals. For exam- ple, the apparently greater difficulty in transforming human cells than animal cells may parallel the comparative susceptibility to cancer of these various species. If the determinants of the various stages in carci- nogenesis are successfully characterized in human cells, it may be possible to develop improved methods for early detection of preneoplastic or early neoplastic lesions. Some human tissues have been main- tained as viable xenotransplants in nude mice (Valerio et al. 1981~. Such models are an ethically acceptable method for in viva study of the process of carcinogenesis in human tissue (Shimosato et al. 1980~. This

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522 Assessment of Carcinogenicity model may provide for direct comparisons of features of carcinogenesis between hu- mans and experimental animals that are commonly used in bioassays. Such infor- mation would clearly be valuable in de- termining the risks to humans of agents demonstrated as carcinogenic in animal bioassays. Role of DNA Replication and Repair It is a well-recognized clinical observation that cancer typically occurs in tissues that have a high rate of cell proliferation or in tissues in which cell proliferation occurs in response to injury. Conversely, cancer is extremely rare in adult tissues or cell types in which cell proliferation does not occur. It was the opinion of classical pathologists that chronic irritation or injury was the etiologic factor for the development of cancer. Subsequently, a variety of specific carcinogenic etiologic agents have been rec- ognized. Nonetheless, cell proliferation plays a significant role in the evolution of cancers (Grisham et al. 1983~. This is well illustrated in the case of liver cancers in- duced in rats by chemical carcinogens. Typical liver carcinogens at effectiv'e doses are also hepatotoxic, and they induce re- storative hyperplasia to replace cells lost as the result of the toxicity. The influence of cell proliferation as a contributing factor in the development of cancer presumably results from effects on the mitotic process and on DNA synthesis. Replicating DNA is vulnerable for a variety of reasons. First, replicating DNA is af- fected to a greater extent by chemical car- cinogens than is nonreplicating DNA (Cordeiro-Stone et al. 1982~. Second, rep- lication of DNA that contains carcinogen adducts may cause incorporation of incor- rect nucleotides at sites of altered or excised bases. Third, some carcinogens may mod- ify nucleotide precursors, and altered pre- cursors may be incorporated into DNA. Fourth, DNA replication itself occurs with a low, but nonzero, error rate. Situations that increase cell replication are likely to cause mutations strictly as the result of these errors. Mammalian cells have a number of mechanisms to repair DNA damage and to reduce the likelihood of errors during DNA replication. Treatments of cells or animals with chemical carcinogens or radi- ation cause the onset of DNA repair pro- cesses. In studies in which cell proliferation has been inhibited and DNA repair has been allowed to remove some or most carcinogen-induced DNA adducts, the transforming effects of the carcinogen dam- age have been reduced (Ikenaga and Kaku- naga 1977~. In contrast, in patients with defective DNA repair processes, such as the genetically determined syndrome known as xeroderma pigmentosum, increased inci- dences of tumors have been observed (Setlow 1978; Hanawalt and Sarasin 1986~. Thus, DNA repair processes appear to be protective against tumor development, whereas defects of DNA repair appear to be associated with increased risks of cancer. There appears to be a critical interrela- tionship between the repair and replication of DNA as factors in the etiology of cancer (Kakunaga 1975~. If DNA replication pro- ceeds within a damaged region prior to repair, there is a substantial risk of error- making during replication, which may cause a mutation to occur as the result of alteration of the base sequence of the com- plementary DNA strand. Of course, this does not occur if the repair of the damage precedes replication. Consequently, the re- lationship in time of the repair and replica- tion of DNA may be a major determinant of the potential for the occurrence of mu- tations and also oresumablv. of carcino- genes~s. , ~,, Genetic Effects of Carcinogen Damage to DNA Chemical carcinogens have been shown to produce a variety of types of DNA damage that can lead to genetic effects on cells (table 2) (Sarma et al. 1975; Drake and Baltz 1976; Singer and Grunberger 1983~. Point muta- tions and frameshift mutations can alter the regulatory or coding regions of genes. On a larger scale, carcinogens can directly af- fect chromatics and chromosomes (Evans 1983~. By still unknown mechanisms, car- cinogen damage can cause the exchange of

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David G. Kaufman 523 Table 2. Genetic EEects of Carcinogen Damage to DNA Point mutations transitions and transversions Frameshift mutations small deletions or additions Mutations at "hot spots" Chromosomal breakage at "fragile sites" Recombinations and rearrangements Sister chromatic exchanges Translocations of portions of chromosomes Gene amplification Aneuploidy DNA segments between sister chromatics, and chromosomal breakage that leads to large deletions or transposition of chromo- somal segments to other chromosomes. Pre- sumably, such damage may lead to failures of mitotic division with unequal distribution of chromosomes between daughter cells, result- ing in abnormal DNA content. DNA dam- age is also thought to be one mechanism for the amplification of segments of DNA. The significance of many or all of these forms of damage to DNA does not concern the chemical composition of this molecule but rather its content of genetic informa- tion. Valuable insights about these genetic effects, particularly with regard to onco- genes, have arisen from recent studies in viral carcinogenesis and molecular biology. Investigations of the mechanism of cell transformation by oncogenic retroviruses have shown that their transforming genes, designated as oncogenes, are derived from the coding regions of cellular precursor genes known as proto-oncogenes (Bishop 1983~. Proto-oncogenes are believed to play an important, though as yet unknown, role in normal cellular function or differen- tiation because they are highly conserved in widely divergent species from yeast to humans. Recent studies have shown that proto-oncogenes can acquire transforming activity as the result of genetic alterations that affect their DNA sequence or place them under abnormal genetic regulation by chromosomal rearrangements, insertion of promoters, or gene amplification (Wein- berg 1985; Barbacid 1986~. The number of known retroviral oncogenes is quite lim- ited about two dozen. Even when the proto-oncogenes from which they are de- rived and the closely related cellular genes (for example, N-myc and N-ras) are added to the sum, the total of retrovirus-related oncogenes is still small. Although further studies of human and animal tumors have identified additional genes with transform- ing activity, it is not yet possible to esti- mate the number of cellular genes that have transforming activity induced by genetic alteration. It is well known that mutations occur at exceptionally high rates at specific sites in DNA of viruses and other prokaryotic or- ganisms. This nonhomogeneous effect is recognized for spontaneous mutations as well as mutations induced by radiation or chemicals. The location of these so-called "hot spots" relates to the specific form of radiation or chemical carcinogen that in- duces the mutations. DNA sequence as well as the structural features of DNA, including bending and association with proteins, appears to influence the spectrum of hot spots. Clearly the DNA sequence in higher organisms such as mammals is far less completely defined, and the means for cataloging the spectra of hot spots in DNA of these organisms are very limited. None- theless, some evidence suggests that there are sites selectively affected by carcinogens where mutations occur at high frequency. Fragile sites are locations in chromo- somes that are particularly prone to break- age. When cell growth conditions are al- tered, such as through deprivation of thymidine and folic acid, chromosomes have been found to break consistently at the same sites. These sites are closely related to sites where chromosomal rearrangements occur in human cancers (Yunis and Soreng 1984), suggesting that structural peculiari- ties that make these sites prone to breakage may be important factors in the develop- ment of cancers. Another notable point is the chromosomal location of these fragile sites relative to several of the known proto- oncogenes. Although the power of the scientific methods used to compare the locations of these sites is not great, the apparent statistical relationship within the experimental error of the methods suggests that some very important feature of cancer development is related to the structure of DNA at these sites.

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524 Assessment of Carcinogenicity Techniques for identifying subregions (bands) within chromosomes now allow abnormal chromosomes in cancer cells to be examined with far greater resolution and specificity than previously possible. Sur- veys of the chromosomal banding patterns of a wide spectrum of cancer cells have shown some consistent patterns of chro- mosomal abnormalities for many different types of cancer (Sandberg 1983; Mitelman 1986~. For some cancers- for example, Burkitt's lymphoma there is a very high degree of consistency in the type of alter- ation observed. Most Burkitt's lymphomas show balanced translocations of portions of specific chromosomes. In other cases, such as the development of the Philadelphia chromosome (loss of a portion of the long arms of chromosome 22) in chronic my- elogenous leukemia, the appearance of the chromosomal abnormality accompanies the chronic phase of the disease. Another very common feature of cancer cells is the development of aneuploidy, with cells having more or less than the normal diploid number of chromosomes or an abnormal DNA content. In fact, the large size and the hyperchromaticity char- acteristic of cancer cell nuclei are largely due to the increased DNA content of typ- ical aneuploid cells. Aneuploidy is pre- sumed to develop as a consequence of unequal mitotic divisions during the evolu- tion of cancer cells. The presence of abnor- mal mitotic figures is one feature of cancers used to arrive at a pathological diagnosis. One of the consequences of the abnormal chromosomal content of cancer cells is that particular genes are present in low or high copy number. One can speculate how the loss of a normal inhibiting function can occur with the loss of a chromosome in hypodiploid cancer cells. Hyperdiploid cells can greatly overexpress particular gene products, or they may generate insufficient inhibitory activity to balance the high copy number of some cancer-related gene. Atypical Carcinogens A number of substances very different from the typical chemical carcinogens have been shown to be carcinogenic in humans and in experimental animals. With atypical carcin- ogens, carcinogenesis can be induced by physical agents and chemicals that do not directly alter DNA. The differences be- tween these atypical carcinogens and the common carcinogens challenge the classical concepts of carcinogenesis and demand the development of theories of carcinogenesis that can include their mode of action. For some time, asbestos has been recog- nized as carcinogenic, first in humans (Doll 1955) and later confirmed in experimental animals (Wagner et al. 1973~. When di- rectly instilled into the pleural cavity of experimental animals it has been shown to produce tumors like those that follow as- bestos exposure in humans (Wagner et al. 1973~. The critical property of asbestos best associated with carcinogenicity is the phys- ical dimensions of fibers (Stanton and Wrench 1972) rather than the chemical composition of the asbestos or the sub- stances adsorbed on it. This was confirmed by showing that glass fibers, prepared in length and width comparable to asbestos fibers, were also carcinogenic. The cellular response to asbestos fibers and other foreign bodies involves the for- eign-body inflammatory reaction wherein the fibers are surrounded by macrophages and fibroblasts (Brand et al. 1975~. Current hypotheses suggest that the inflammatory cells or epithelial cells produce reactive forms of oxygen molecules which may affect the DNA of the epithelial cells, and this damage is fundamental to the carcino- genic process. Others suggest that asbestos acts by affecting chromosomal segregation during mitosis. On a practical level, asbes- tos is relevant to the topic of mobile source emissions. It is known that asbestos expo- sure is associated with mesothelial cancer in humans. However, in individuals in whom asbestos exposure is combined with ciga- rette smoking, the risk of cancer is greatly increased, and the leading type of cancer is bronchogenic carcinoma (Selikoff et al. 1968~. It is conceivable that individuals who have been exposed to asbestos will represent a group at increased risk from the combined effects of asbestos and mobile . . source em1sslons. A number of studies have shown that

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David G. Kaufman 525 unusual substances, functioning as atypical carcinogens, can produce cancers in exper- imental animals. Plastic films have been shown to produce tumors when implanted into animals. However, when the films were sufficiently fenestrated, or when they were ground to a powder, the material was not carcinogenic. Several metals, in the forms of ores, refinery process by-prod- ucts, and ions and salts, have been shown to be carcinogenic in humans or experimental animals (International Agency for Research on Cancer 1980~. Examples include various forms of arsenic, chrome, and nickel. A number of reports in recent years have noted that chemicals, including therapeutic agents that cause proliferation of peroxi- somes, are carcinogenic (Ready et al. 1980~. Unlike chemicals such as phenobar- bital, these agents appear to function as complete carcinogens rather than just as promoters. Investigations of examples of this class of chemicals have shown that they do not form adducts with DNA. Several other chemicals and drugs are carcinogenic in animals or humans, but are not known to interact with or form adducts with DNA. Among these are agents that affect enzymes involved in the metabolism of DNA precursors or that more directly affect DNA precursor pools. These prop- erties make some of these chemicals effec- tive therapeutic agents for treating cancer. The action of some of these agents is be- lieved to be a consequence of imbalances in DNA precursor pools, which also cause mutations. Promotion, Cocarcinogenesis, and Enhancement . Exposure to carcinogens is not the only determinant of cancer development. Other substances or other processes can influence the risk for cancer development, particu- larly when they complement exposures to carcinogens or act on animals that have a high spontaneous tumor incidence. These factors must be recognized when the obser- vational data derived from carcinogenicity tests in animals are being interpreted mech- anistically. The terms "enhancers" and "enhancement" describe effects that include those typically classified as promoters or cocarcinogens but without attribution of a mechanism of action. Promotion is defined operationally, based on classical experiments in which tumors were induced in mouse skin by a two-step treatment protocol (Berenblum 1975~. The first treatment involved the application of a subcarcinogenic dose of a strong carcinogen to the mouse skin, fol- lowed by a prolonged series of applications of a noncarcinogenic agent. The combined treatments produced a tumor response, whereas the same dose of the carcinogen or the second agent, which has come to be known as a promoter when used alone, was ineffective or vastly weaker. The two steps of the treatment process were designated as . . . . . . nltlatlon anc . promotion, ant . have come to be interpreted as separate events or pro- cesses in the evolution of cancers. In contrast to the separate application of . . . . initiator anc promoter, cocarclnogens are agents that enhance the development of cancers when administered concurrently with a carcinogen. Cocarcinogens act through a variety of mechanisms. They may modify the metabolism of carcinogens to yield a greater quantity or proportion of ultimate carcinogenic metabolites. They may act by causing cell or tissue toxicity with accompanying accelerated cell prolif- eration; this in turn may increase the risk of malignant transformation. They may also act by interfering with normal defense mechanisms that function to counteract the detrimental effects of carcinogens. Enhancing or inhibiting effects from ex- posure to a wide variety of substances (for example, certain constituents of mobile source emissions), not just exposure to car- cinogens (such as the possibly carcinogenic constituents of such mobile source emis- sions), determine the tumor response. Our understanding of these effects and the inter- actions between substances is very limited. More-specific enhancing effects, in some cases affecting individual tissues, may come from exposures to noncarcinogens that ex- ert a promoting or cocarcinogenic effect. Individual genetics, prior or concurrent medical conditions, and diet all may con- tribute to an individual's specific risk from

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526 Assessment of Carcinogenicity a given level of exposure. Such enhancers presumably increase the effects of other carcinogens. It is particularly important to ascertain whether mobile source emissions contain constituents with enhancing activ- ~ty. Mobile source emissions may represent a serious public health problem if they en- hance carcinogenesis initiated by other exposures. This is an important general problem that requires further attention. Different methods of bioassay from those used to detect carcinogens will have to be developed to determine whether these emissions have enhancing activity. Such methods are needed to explore the possibil- ities that enhancing activities are specific in augmenting the activity of particular types of carcinogens or that their activity differs according to tissue sites at which they act. It is clear that standard carcinogenicity bioassavs are not cure tests for either can cer-~nit~at~ng activity or activity as a com- plete carcinogen. They are phenomeno- logic studies that associate excess cancers with particular treatments, but they do not indicate the mechanism by which the can- cers are produced. Particularly in the case of tumors in tissues with a high spontane- ous tumor incidence in untreated animals, increases in the incidence of tumors may reflect a toxic or promoting activity of the tested chemical. If this effect is not the result of toxicity associated with high exposure levels, then the result may be a demonstration of pro- motion activity. Such a conclusion might distinguish these compounds from stan- dard carcinogens, but it does not indicate that they are without risk. In view of the hazard posed by chemicals of this type, it is important to develop methods to demon- strate how they cause tumors. Since some of the constituents of diesel exhaust may also have this kind of activity, it would be useful to have the means of identifying and quantitating these chemicals. Promoting or enhancing activity may involve a number of organs and tissues other than the skin. For this reason it will be necessary to evaluate the differences in enhancing effects in different tissues. For example, 12-0-tetradecanoylphorbol-13 acetate (TPA) is a good promoter for mouse skin, but is not a good promoter for rat liver; conversely, phenobarbital is very effective in rat liver, but not in skin. The possibility also exists that enhancing activ- ity relates to the type of initiator. The type of initiator may determine which tissues or organs will be sensitized to promoter or enhancer effects. It is entirely conceivable that the broad spectrum of chemicals in diesel exhaust contains enhancing agents with dis- tinctive patterns of organ selectivity. To test these hypotheses it will be neces- sary to examine the enhancing activity of materials such as diesel exhaust following an initiating treatment with any of a variety of carcinogens with a range of organ spec- ificities. In this manner it may be possible to develop a standard panel of animal test models that would have the ability to detect and quantify promoters and enhancers that are active in any of a number of tissues. Recommendation 1. . . . . The role of pro- moters and enhancers In human carcinogen- esis should be determined. Multistep Processes On the basis of a variety of clinical and experimental evidence, the development of cancer is believed to be a multistep process (Armitage 1985~. Clinical experience has shown that the incidence of most cancers rises with age and most are seen to pass through premalignant stages prior to the development of clinically overt cancer (Doll 1971~. The most thoroughly studied case is that of the multistep evolution of squamous cell carcinoma of the uterine cervix. The validity of a multistep interpre- tation is attested to by the fact that clinical intervention at an early stage vastly reduces the incidence of the overt, invasive tumors of this type. A similar sequence of prema- lignant lesions of the bronchial epithelium precedes invasive lung cancer (Auerbach et al. 1961~. One study followed uranium miners with repetitive sputum cytologies for many years (Schreiber et al. 1974~. Progressive cnanges In Cyrologlc nnalngs proceeded from squamous metaplasia through various stages of dysplasia, in situ . ~. . . . . .. ..

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David G. Kaufman 527 . . . . . carcinoma, and Invasive carcinoma over the course of several years. Subsequently, comparable sequences of epithelial lesions have been found to precede overt cancers in a number of sites (Farber 1984~. Initiation and promotion in the mouse skin bioassay is an example of carcinogen- esis as a two-step process (Berenblum 1975~. Comparatively recent studies have shown that the process of promotion itself can be divided into stages (Slaga et al. 1980~. In the case of the evolution of tu- mors of rat liver, cancer is believed to be the end result of a process in which foci or areas of enzyme-altered hepatocytes and neoplastic nodules precede malignant tu- mors (Farber 1980~. In the respiratory tract of hamsters, carcinogen treatment has been found to cause a progressive sequence of histologic alterations that culminate in invasive, malignant tumors (Saff~otti and Kaufman 1975~. These lesions demonstrate a spectrum of morphological changes very close in appearance to the lesions of the respiratory tract seen in humans. In fact, the evolving lesions shed cells analogous to those observed in the cytology preparations from the uranium miners cited above (Schreiber et al. 1974~. Methods to study the transformation of cells by chemical carcinogens in tissue cul- ture have been available for about two decades. These studies first were successful in rodent embryo and fibroblast cells. More recently, similar results have been achieved using rodent epithelial cells, and in the past few years human cells have also been trans- formed with chemical carcinogens. A num- ber of morphological, biological and phe- notypic changes have often been observed in these in vitro transformation systems as the cells progress from the original cell population to demonstrably malignant cells. In some cases, for example in studies using Syrian hamster embryo cells, a spe- cific sequence of changes in the culture has been linked to malignancy (Barrett and Ts'o 1978; Smith and Sager 1982~. With cultured rat tracheal epithelial cells, a se- quence of progressive changes in the bio- logical behavior of carcinogen-treated cells has been observed (Nettesheim and Barrett 1984~. In this system, the cultured cells can be evaluated for their relationship to the morphological alterations observed in vivo by allowing them to repopulate a trans- planted rat trachea which had been deepi- thelialized (Klein-Szanto et al. 1982~. Other evidence of the multistep nature of trans- formation found in this system is a two . . . . . . . step transformation envoy Ding an ~n~t~at~ng carcinogen treatment followed by in vitro promotion with TPA (Steele et al. 1980~. Studies of the transformation of human cells in vitro have also shown that several distinct alterations occur consistently and in a generally similar order (Kakunaga et al. 1983). Variations in Susceptibility Rates of development of spontaneous be- nign and malignant tumors vary in animals of different species and strains (Grasso and Hardy 1974~. Some animals are highly re- sistant to tumor development, and even after a long life few will die with tumors. In contrast, some species and strains of ani- mals have a very high incidence of cancers, in some cases 100 percent. In these species and strains, the type and quantity of these background tumors are characteristic of the animal and are presumed not to be the result of unusual exposures to environmen- tal factors. Among the animals typically chosen for carcinogenesis bioassays, mice have an exceptionally high incidence of liver tumors, particularly in males. In fe- male rats, mammary tumors are very com- mon. Treatment of these animals with chemical carcinogens results in tumors at various locations and of types that depend on the activity and dose of the carcinogen as well as the route of administration and other factors. Commonly, these treatments also affect the incidence and multiplicity of the tumors characteristic of the untreated animals, indicative of the sensitivity of these tissues to transformation. The human population, in comparison, appears to have a relatively low back- ground level of cancer, as determined from cancer incidence data for certain low-risk groups in underdeveloped nations or in specific populations such as Mormons or Seventh Day Adventists in the United

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528 Assessment of Carcinogenicity Table 3. Factors Affecting Human Susceptibility to Carcinogenesis E. xposures to carcinogens Diet composition and nutritional status Personal habits including cigarette smoking and alcohol consumption Determinants of geographic variations in cancer development Genetic diseases or heterozygous carrier states Acquired illnesses and infections Unknown factors determining familial . . . prec .lsposltlons Variations in metabolic activation or inactivation of carcinogens States in which there are religious restric- tions on smoking or certain dietary prac- tices. Despite the low overall cancer rates in these groups, certain cancers are seen and these may represent the background tu- mors of humans. These include leukemias and lymphomas, soft-tissue sarcomas, skin tumors, and a low rate of tumors of several epithelial tissues. Above this background, several factors appear to affect the suscep- tibility of humans to the development of cancers (table 3~. The incidence in humans of tumors of various organs differs by country and even by population group within countries. In the United States, cancer of the lung is the most common significant cancer in males and females, whereas in Egypt and Japan, cancers of the urinary bladder and stomach, respectively, are the most common. Within the United States, there appear to be geo- graphic differences in incidence of tumors of various organs (Pickle et al. 1987~. Clearly, a large proportion of these tumors are induced rather than spontaneous and are ~ . . . . 01 environmental orlgln. In contrast to the general population, there are individuals who are genetically predisposed to the development of cancers. Examples of genetic diseases associated with a high incidence of cancer include xeroderma pigmentosum, ataxia telangiec- tasia, familial retinoblastoma, Fanconi's anemia, Gardner's syndrome, familial polyposis coli, and many others. Studies have shown that for the recessively inher- ited genetic disease ataxia telangiectasia, close relatives who do not have the disease, but are heterozygous carriers, also have an elevated cancer risk (Swift et al. 1976~. In fact, the heterozygous carriers of the ataxia . . . te anglectasla trait may represent up to a few percent of the human population. At present, the biological basis for these ge- netic diseases and their link to cancer are unknown. However, it is known that there are defects of DNA repair functions, pre- sumably different defects, for xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia. Familial retinoblastoma has been shown to be consistent with a deletion or mutation of chromosome 13. Familial polyposis cold and Gardner's syn- drome are associated with abnormalities of cellular growth control. These observations provide clues to pos- sible steps in the presumed multistep pro- cess of malignant transformation. To the extent that carrier states for these diseases are common in the population, these ge- netic traits may be factors that influence individual risk for developing cancer (Swift et al. 19764. It is likely that other genetically determined factors may influence cancer risks even if they do not yield recognized genetic diseases. For example, there may be genetically determined influences on the rate or route of metabolism of chemicals. The racial differences in alcohol metabo- lism illustrate that such differences occur in the human population. Individual varia- tions in other factors, such as those affecting responses to injury, may also influence cancer risk. Knowledge about such factors is limited at present but may be an impor- tant and useful area for continued research. There are a variety of illnesses and infec- tions that predispose people to the devel- opment of cancer. For examples, hepatitis B and schistosomiasis of the bladder are important factors in the causation of liver and bladder cancers, respectively. Certain lymphomas are associated with infectious diseases (for example, human T-cell lym- photrophic virus types I and III, or Epstein- Barr virus), and colon cancers are associ- ated with ulcerative colitis. These diseases and conditions cause a high level of cell proliferation in specific target cell popula- tions which may predispose to cancer de- velopment in the affected tissue.

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David G. Kaufman 529 Some tumors of the lung are associated with scars of the parenchyma. There has also been speculation that other lung con- ditions predispose people to lung cancer development (Kuschner 1985~. Although it is likely that these conditions predispose people to lung cancer because of increased cell proliferation rates, it is also possible that these conditions affect the capacity of the lung to clear exogenous materials, in- cluding potential environmental carcino- gens. Omitting the known genetic diseases that predispose to cancer development, and in the absence of acquired diseases that are associated with cancer, there are still a number of families with an unusually high incidence of cancer. In most of these fami- lies there is variable penetrance of tumor risk with less than 100 percent incidence of cancer in these populations. In some cases there are distinct patterns of tumor devel- opment with particular organs affected to unusual extents and with different tumors predominating in males and females. It is unclear whether these occurrences are pri- marily the result of unrecognized genetic diseases or a heterozygous carrier state for a recessive genetic disease. Alternatively, these families may develop these cancers because of elusive environmental factors passed socially from generation to genera- tion, such as diet or personal habits. Clinical and experimental evidence sug- gest that there are important differences in susceptibility to cancer among individuals in the human population. This could be a very significant factor in efforts to control specific types of cancer, including any re- lated to exposure to diesel exhausts. The population of susceptible individuals may account for a disproportionate share of particular types of cancer. It may be pos- sible to significantly change the overall in- cidence of specific types of cancer by identifying susceptible individuals and con- centrating cancer prevention activities on this population. It might be possible to identify individuals for whom specific types of carcinogens or diesel exhaust rep- resent a particular hazard and protect them from such hazards. We have limited knowledge of biological and enzymatic factors that determine these states of unusual susceptibility. More data are needed about the range of variation of metabolic processes, DNA repair pro- cesses, constitutive and induced cell prolif- eration rates, and responsiveness to hor- mones in tissues from human subjects. These data are needed for each tissue in which cancer is common, and this informa- tion should be obtained where possible to determine the variability according to age, gender, genetic background, and personal factors such as diet, therapeutic drug use, and personal habits (for example, cigarette smoking). Accomplishing these goals will require development of methods to obtain human tissues in an acceptable manner. Furthermore, human subjects will have to be chosen scientifically so that they are representative of the population as a whole or the subpopulations that appear to be at unusual risk. If these studies are successful, the next step will be development of meth- ods to test these characteristics in samples of tissue that can be obtained from normal individuals with little or no risk. On the basis of epidemiologic observa- tions that there is a range of responses within populations apparently exposed to the same levels of a carcinogenic substance, it is conceivable that there are individual factors that are major determinants of risk. Identifying the portion of the population at exceptional risk and concentrating protec- tive efforts on that population might have a major impact on the overall cancer inci- dence at particular tissue-specific sites. Such an approach has proven notably effec- tive in reducing myocardial infarction rates in individuals with genetic abnormalities of low-density lipoprotein metabolism and in individuals with acquired coronary artery disease. Developing methods to determine the elements of individual risk will require great attention. The development of appro- priate and acceptable methods for obtain- ing cells from various body sites with little or no risk should be included in this method. Methods to test various cellular characteristics that have been associated with the development of cancer will also have to be devised. Such factors as the

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544 Assessment of Carcinogenicity culations for other compounds and the approach used to make this assessment was an extrapolation by comparisons and anal- ogy (Albert et al. 1983; Lewtas et al. 1983~. Results of skin carcinogenesis and skin ini- tiation in SENCAR mice were compared for diesel exhaust extracts and extracts from gasoline engine exhaust, cigarette smoke condensate, roofing tar vapors, and coke oven emissions. Similarly, results of short-term assays for genotoxicity were compared for these same compounds. From these data the comparative potency of diesel exhaust was estimated on the basis of the most active diesel extract. To relate these values to estimates of risk for the human population, estimates of human lung cancer risk were made for coke oven emissions, roofing tar, and cigarette smoke condensate. For each of these compounds there were epidemiologic data relating ex- posure to human cancer and experimental data in test systems identical to that for diesel exhausts. The risk per unit quantity for diesel exhausts was extrapolated by determining the human risk on the basis of a unit quantity of organic extractable ma- terial. The estimated unit risk obtained for human lung cancer was 0.02-0.60 x 10-4 (lung cancers)/,ug exhaust particulates/m3 of air. To understand this estimate, it is impor- tant to recognize the inherent assumptions of the method (Lewtas et al. 1983~. The method assumes that the relative potency ~ . Or carcinogens in one carclnogenesls assay is directly proportional to that in another bioassay. Further, this comparability is as- sumed to apply across biological systems and species. This assumes that the bioavail- ability of the active compounds at the tar- get tissue is proportional even when ex- trapolations are made between species and between routes of exposure. As Cuddihy and McClellan (1983) note, the estimates derived by this method suggest that ex- tracts of diesel exhaust particles are not "orders of magnitude more potent than other emissions." This risk assessment has a number of limitations. These studies and extrapola- tions are not based on whole, fresh ex- hausts; the exhausts are not acting on a . population exposed to a myriad of other carcinogens and active compounds unre- lated to diesel exhaust; and the exhausts are acting on a homogeneous population where genetic factors, prior illness, and personal habits do not influence the suscep- tibility to these insults. Also, this assess- ment offers an estimate of risk strictly for lung cancer, although cancers in other sites might also be affected. The risk estimate is also provided as specific risk per unit of exposure. This specific risk is not very dissimilar to those for the other materials to which it was compared experimentally. Thus, the comparison with the specific risk for gasoline engines is somewhat mislead- ing when one considers the fact that diesel engines may generate one to two orders of magnitude more particles than a gasoline engine with a catalytic converter. More recently, the comparative potency approach has been used to assess the human cancer risk associated with diesel exhaust in a more comprehensive analysis that in- cludes estimates of population exposures (Cuddihy et al. 1984; McClellan 1986~. Those analyses used previously reported estimates of specific risk of lung cancer development (lung cancers/,ug diesel par- ticulates/m3 of air/year) (Albert et al. 1983; Cuddihy et al. 1983; Harris 1983~. Expo- sure estimates were based on environmen- tal concentrations in various locations and distribution of the population according to concentration levels and assuming a 20 percent proportion of diesel-powered light- duty vehicles. Analyses were also based on the estimates of risk from epidemiologic studies. From these data, calculated values for excess lung cancer deaths per year ranged from 100 to 3,500, a range attribut- able to an increase to a 20 percent light- duty diesel-powered fleet. As with the ear- lier estimate noted above, there are numerous potential sources of error in these calculated risk values. Despite these limitations the risk estimate offers a starting point for determining the overall potential for changes in the rate of cancer deaths as a result of increasing the use of diesel engines in the U.S. transportation fleet. Too little generic information exists on the carcinogenicity of the gaseous and par

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David G. Kaufman 545 ticulate emissions of mobile sources. Stud- ies have been performed on representative emissions generated by particular sources operating under specific conditions. Scien- tifically, it is not clear to what extent such results apply to different engines operating with different fuel or other different condi- tions. Further, it is not clear how these . . . results relate to the same engine operating under other conditions or even to the same engine operating under presumably identi- cal conditions at a different time. Differ- ences between individual engines or oper- ating conditions can result in the generation of emissions with quantitative and even qualitative differences in the products formed. These differences in turn can be the major determinant of the activity of the emission in carcinogenicity tests. This sit- uation is quite unlike the testing of a pure chemical, in which case there is a reason- able assurance of repeatability upon retest- ing. Given that there is no standard mixture for mobile source emissions, the question arises whether these mixtures can be eval- uated on the basis of quantity and activity of certain "sentinel" constituents. If these most active components could be moni- tored and minimized, then optimum en- gines and operating conditions might be selected. Although this idea has merit as a comparative measure, the assessment of the actual quantitative risk at any level of these sentinel compounds may be difficult to determine. A further complication is the fact that little is known about the possibil- ity of interactions between carcinogenic compounds. It is unlikely that the risk associated with the mixture of sentinel compounds is simply the sum of the effects of the individual compounds. This uncer- tainty is further amplified when the numer- ous other constituents of emissions are con- sidered as influencing the activities of the sentinel compounds. Further bioassays are needed to provide a sufficient body of information about the carcinogenicity of diesel exhausts in exper- imental animals. In view of the variability of diesel engine exhausts due to engine design, conditions of operation, and the fuel used, it is necessary to perform more studies to evaluate the influence of these variables on tumor responses. Exposure to diesel alone should be complemented by studies in which diesel exhaust exposures follow initiating carcinogen treatment in each of several organs or tissues. Exposures should not be limited to a particular frac- tion of diesel exhaust condensate or to the particulate material, since the complete ex- hausts may have additive or inhibitory ef- fects that would otherwise not be detected. ~ Recommendation 6. Additional stud- ies should be performed on the carcinoge- nicity of diesel exhaust. Assessment of the carcinogenicity of mixtures poses two conflicting problems. The first concerns the nature of mixtures and the fact that each mixture represents a unique case. The second concerns attempts to evaluate mixtures by dividing them into their constituents. In such cases it is difficult to determine how to reconstitute the effects of the individual constituents into the ef- fects of the total mixture. Mixtures such as the diverse combustion products in mobile source emissions can be administered to experimental animals and tested for carcinogenicity. However, these mixtures are not intentionally formulated with precise analytical procedures. They are the products of a process or source that may not have exceptional reproducibility. Thus, the emissions from two different diesel engines may have quantitative differ- ences in the products of combustion. Even the same engine, operating under slightly different, or even unmodified conditions, may yield mixtures of products with some quantitative differences. Despite the slight quantitative differences in the various com- bustion products, it is likely that the mix- tures will have similar qualitative effects: they will prove to be carcinogenic or they will not. The magnitude of the carcino- genic response may be affected by the ac- tual composition of the mixtures. Under any condition of operation or engine design, diesel exhaust is a mixture of many chemicals. The composition of this mixture is highly variable and depends on such factors as the engine, fuel, and oper

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546 Assessment of Carcinogenicity ating conditions. It is conceivable that the Summary interaction of the components of this mix ture is the determinant of overall carcino genicity of the complete mixture. There fore, the only currently valid method to determine the carcinogenic activity of each form of diesel exhaust is by a separate animal bioassay. However, animal bioassay is not practi cal for evaluating modifications of diesel engine design or other aspects of their operation as they affect carcinogenicity. It would be valuable to have some method of estimating changes in carcinogenic activity based on knowledge about the changes in composition of diesel exhaust. This would require a better understanding of the in teractions of components of complex mixtures in causing cancer. To learn how constituents of mixtures interact in carcin ogenesis, it will be necessary to determine how carcinogenicity changes with the vari ation of the concentration of individual components. Choices of chemicals to study would presumably be based on the activ ity of the isolated compound or its rela tive abundance in the diesel exhaust mix ture. In addition, constituents that have demonstrated or are suspected of enhanc ing (or inhibiting, for that matter) the action of carcinogens (for example, pro moters or enhancers) will also need to be studied. This problem will require additional study to evaluate the effects of variation in the composition of diesel exhaust on the tumorigenicity of other carcinogens; this serves as a model of the multiple complex exposures associated with human environ ments and lifestyles. Optimally it would be possible to achieve a reasonable estimate for the complex exhaust mixture that is based on measurements of the concentrations of a certain small number of index compounds. This hypothesis and experimental approach should be tested and validated. If it is found to predict certain levels of carcinogenic activity, the predictions should be tested bv performing animal bioassays. , Recommendation 7. Methods should be developed for assessing the carcinoge . . . . nlclty ot mixtures. This chapter reviews information on mech- anisms of carcinogenesis and considers fac- tors that influence the rate of tumor forma- tion. It also considers the criteria for identifying a chemical qualitatively as a carcinogen, and methods that have been used to extrapolate from these data to quantitative estimates of cancer risk in hu- mans. Finally, data have been reviewed regarding the qualitative assessment of die- sel exhaust as a carcinogen and the extrap- olations made using these data to estimate human cancer risk from diesel exhaust ex- posure. The review of current knowledge about . . . . carclnogenes1s points out great advances that have been made in our understanding of cancer and also reflects the vast remain- ing ignorance. Cancer development has come to be recognized as a slowly pro- gressing, multifactorial, multistep process. Cell proliferation is known to have one or several roles in the process, and abnor- malities of the control of this process are fundamental features of cancer cells. Fac- tors such as chronic injury or toxicity (for example, toxicity that is produced by high, but nonlethal doses of administered drugs) can result in elevated rates of cell proliferation with the attendant increase in cancer risk. Some chemicals are known to act as carcinogens by direct effects on DNA; in some cases, specific mutations induced by chemicals have been identified. Other genes whose effects are recognized in their absence or altered state in certain genetic diseases predisposing to cancer have been localized cytogenetically, and efforts are in progress to isolate the genes. Studies of atypical carcinogens that do not have a direct mutagenic effect have suggested alternate pathways or separate steps in the pathway to the development of cancer. Similarly, studies of the role of promoters in carcinogenesis have pointed out the multistep nature of the process, and the potential influence of factors that may act by selecting cells with abnormal properties. The human population is diverse in its genetic back- ground and its exposures to harmful

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David G. Kaufman 547 materials. Many chemicals require enzy- matic activation to become reactive ulti- mate carcinogenic metabolites; these meta- bolic processes may be included among the factors influenced by genetics and environ- mental exposures, that determine the indi- vidual variations in susceptibility to cancer. The list of issues about the process of carcinogenesis considered in this section was necessarily incomplete, limited by space constraints rather than the exhaustion of important facts. The section on qualitative evaluations of carcinogenicity and quantitative estimates of cancer risks in humans considered the criteria for designating a chemical as a carcinogen and how these data are quanti- fied and extrapolated to estimates of human cancer risk. The section shows that there are reasonably well-defined criteria for judging whether a chemical is a carcinogen. That is not to say that this evaluation is not without problems. Weak carcinogenic re- sponses may be difficult to distinguish from background levels of tumors. Increased rates of tumors may be observed as the result of exposure to promoters of carcino- genesis; on the basis of a positive tumor response, the promoter is classified for- mally as a carcinogen. However, the tumor response produced by promoters may be critically dependent on dose and may even have a threshold level for activity. Conse- quently, promoters may become classified as carcinogens although their mode of ac- tion may be quite different from that of strong mutagenic carcinogens. The review of methods for quantitative risk assessment includes discussions of the several factors that must be considered in extrapolating estimates of human cancer risk. The discus- sion of extrapolation to humans from the species used in the carcinogenicity tests includes consideration of differences be . . . . . tween species in t :le sensitivity to tumor formation in particular organs. Also noted are the considerations that must be given to account for differences in dose and in the route of exposure to a compound when extrapolating from animal tests to estimates of human risk. The review of data specifically concerned with diesel engine exhaust emissions dem onstrates that these exhausts have biologi- cal activity. Short-term tests have shown that diesel engine exhausts are mutagenic and can cause chromosomal damage. The activity in these studies was influenced by the source of the emissions tested, for ex- ample, the type of engine used. A variety of studies have evaluated the activity of diesel engine exhaust as a complete carcinogen and as an initiator or promoter of carcino- genesis. Some of the studies failed to pro- duce positive results or were equivocal. Positive results, however, have been found in inhalation studies and in mouse skin painting and lung adenoma formation as- says. The results of these studies have been used with current, though admittedly im- perfect, risk extrapolation methods, and values for projected human cancers have been calculated. The risk for diesel engine exhaust was calculated to be comparable to the approximate range found for other car- cinogenic human exposures such as coke oven emissions and roofing tar. Within the limitations of these estimates, diesel engine exhausts do not appear to be notably more active than these other materials. Review of the epidemiologic studies of the risk of diesel engine exhausts shows that exposure to these exhausts does not cause a strong effect like cigarette smoking. How- ever, because of the limitations of the stud- ies, it is difficult to conclude conversely that the carcinogenic activity is negligible or absent. Summary of Research Recommendations: Priorities, Purposes, and Responsibilities Many factors must be considered in devel- oping a research plan that sets priorities for the pursuit of the various recommended research goals. For example, these prior- ities might be selected on the basis of the unique mission of the Health Effects Insti- tute, or they might be viewed on the basis of the more general need for furthering our knowledge about how to make quantitative risk assessments. From a practical point of view, it might be preferable to place the

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548 Assessment of Carcinogenicity highest priority on goals that will require the longest time to accomplish or that are not getting adequate attention and support from other sources. It is also reasonable to place highest priority on goals that might significantly affect the cancer risks that might be attributable to diesel engine ex- haust, even without accomplishing all of the proposed research goals. If accomplishment of the unique mission of the Health Effects Institute is the per- spective from which priorities are deter- mined, then highest priority must be given to performing additional studies on the carcinogenicity of diesel exhaust (Recom- mendation 6) and developing methods for . . . . . assessing t he carclnogenlclty of mixtures (Recommendation 7~. It is unlikely that other sources or organizations will place comparable emphasis on the direct study of diesel exhaust as a carcinogen. The issue of the carcinogenicity of mixtures is a more general problem, but it is essential for the evaluation of diesel exhaust, although only a secondary concern in the evaluation of many other materials. If the view is taken that adequate assess- ment of the hazards of diesel exhaust will not be possible without obtaining more knowledge about how to make quantita- tive risk assessments in general, then prior- ities might be set somewhat differently. In this case, the highest priority might be placed on evaluating the role of toxicity in carcinogenesis (Recommendation 3) and gathering critical data for quantitative as- sessments (Recommendation 4~. By learn- ing how to make quantitative risk assess- ments that account for effects of toxicity, and which involve extrapolations to low doses, among routes of administration, anc among species, In a manner more firmly founded on scientific knowledge, better estimates of human cancer risks in general will become possible, and this will benefit the assessments of diesel ex- haust. Another basis for setting priorities might be consideration of practical issues. For example, priorities could be set so that the research goals all might be accomplished in the shortest time. From this perspective, highest priority might be placed on goals that require the longest time to accomplish. Thus, priority might be given to evaluating the role of toxicity in carcinogenesis (Rec- ommendation 3) Lathering critical data for . . ~ ~ 7 quantitative assessments (Recommenda- tion 4), and developing methods for assess- ing the carcinogenicity of mixtures (Rec- ommendation 7~. Each of these is a complex problem that will require the per- formance of long-term studies to accom- plish, and may require several such studies in sequence. By beginning these studies at the earliest time and phasing in other goals later, it might be possible to have the more complete body of knowledge with which to make scientific risk assessments at the . . . earliest time. Another perspective is to place the high- est priority on goals that are not receiv- ing adequate attention and support from other sources. It could be argued that many or most of the Research Recommen- dations are not being pursued with the vigor that might be desired. The conclu- sion from this, however, is that all of the Recommendations should be given a high priority. This point of view may be accu- rate but it does not contribute to a practical plan. Another view might be predicated on the idea that early availability of certain critical knowledge might make it possible to affect the cancer risks from diesel exhaust signif- icantly even before all of the needed infor- mation for scientific risk assessments has been obtained. A possible scenerio that might fit this perspective would place the highest priority on developing methods to identify individuals at high risk (Recom- mendation 2) and developing acceptable methods for dosimetry in humans (Recom- mendation 5~. For example, if one could identify the individuals who were at high risk for the development of cancer if they are excessively exposed to diesel engine exhaust, then it would be possible to focus preventive health measures on this group. If it were possible to carry out dosimetry on exposed individuals, then preventive measures might be developed that would reduce exposure and risk.

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David G. Kaufman 549 Summary of Research Recommendations: A Research Plan From the preceding discussion, it is clear that there are many ways to assign priorities for the pursuit of the various Research Recommendations. The following plan considers these different perspectives in defining a preferred set of priorities. HIGH PRIORITY No other organization will commit comparable attention or resources to the study of diesel engine exhaust, and therefore this must be done by the Health Effects Institute. One would hope that research on the scientific problems in making critical extrapolations in quantitative risk assessment and in validating the process would be widely supported and actively pursued. Unfortunately, this need has been clear for some time, yet there has been less progress in solving this problem than might have been expected. Accom- plishment of the following two goals will provide the most urgently needed information to perform better assessments of the human risks resulting from diesel exhausts. Recommendation 4 Critical data should be gathered for quantitative assessments. Recommendation 6 Additional studies should be performed on the carcinogenicity of diesel exhaust. MEDIUM PRIORITY Development of methods for human dosimetry may benefit from investigator-initiated basic research and even from the re search of commercial enterprises. Therefore, the pursuit of these goals may be given somewhat lower priority. A similar lower priority may be given to the evaluation of the carcinogenicity of mixtures. This problem is not unique to the assessment of diesel exhausts and knowledge may be gained from studies supported by other regulatory programs. Recommendations Acceptable methods should be developed for dosimetry in humans. Recommendation 7 Methods should be developed for assessing the carcinogenicity of mixtures. LOW PRIORITY The remaining recommendations are important but are generic goals that would improve our general ability to make risk assess ments. These issues touch on basic research that is being pursued in investigator-initiated studies. Investigations of this type may also be pursued by other agencies that are required to make risk assessments. Thus, although these are important goals, they may deserve lower priority in this program.

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550 Assessment of Carcinogenicity Recommendation 1 The role of promoters and enhancers in human carcinogenesis should be determined. Recommendation 2 Methods should be developed to identify individuals at high risk. Recommendation 3 The role of toxicity in carcinogenesis should be evaluated. Acknowledgments The author thanks Dianne Shaw for excel- lent technical editing and Brigitte Cooke for skillful secretarial assistance. References diesel and related environmental emissions: in vitro mutagenesis and oncogenic transformation, Envi- ron. Int. 5:403-409. Claxton, L. D. 1981. Mutagenic and carcinogenic potency of diesel and related environmental emis- sions: Salmonella bioassay, Environ. Int. 5:389-391. Cordeiro-Stone, M., Topal, M. D., and Kaufman, D. G. 1982. DNA in proximity to the site of replication is more alkylated than other nuclear DNA in S Phase 10T1/2 cells treated with N- methyl-N-nitrosourea, Carcinogenesis 3:1119-1127. Cuddihy, R. G., and McClellan, R. O. 1983. Evalu- ating lung cancer risks from exposures to diesel engine exhaust, Risk Anal. 3:119-123. Cuddihy, R. G., Griffith, W. C., and McClellan, R. O. 1984. Health risks from light-duty diesel vehicles, Environ. Sci. Technol . 18:14A-21 A. Curren. R. D.. Kouri. R. E.. Kim C. M.. and Albert, R. E., Lewtas, J., Nesnow, S., Thorslund, T. W., and Anderson, E. 1983. Comparative po- tency method for cancer risk assessment: applica- tion to diesel particulate emissions, Risk Anal. 3:101-117. Armitage, P. 1985. Multistage models of carcinogen- esis, Environ. Health Perspect. 63:195-201. Auerbach, O., Stout, A. P., Hammond, E. C., and Garfinkel, L. 1961. Changes in bronchial epithelium in relation to cigarette smoking and in relation to lung cancer, N. Engl. J. Med. 265:253-267. Barbacid, M. 1986. Oncogenes and human cancer: cause or consequence? Carcinogenesis 7:1037-1042. Barrett, J. C., and Tsto, P. O. P. 1978. Evidence for the progressive nature of neoplastic transformation in vitro, Proc. Natl. Acad. Sci. USA 75:3297-3301. Berenblum, I. 1975. Sequential aspects of chemical carcinogenesis: skin. In: Cancer: A Comprehensive Treatise (F. F. Becker, ed.), pp. 323-344, Plenum Press, New York. Berenblum, I., and Shubik, P. 1947. A new, quanti- tative, approach to the study of the stages of chem- ical carcinogenesis in the mouse's skin, Br. J. Cancer 1 :383-391. Bishop, J. M. 1983. Cellular oncogenes and retrovi- ruses, Ann. Rev. Biochem. 52:301-354. Brand, K. G., Buoen, L. C., Johnson, K. H., and Brand, I. 1975. Etiological factors, stages, and the role of the foreign body in foreign body tumorigen- esis: a review, Cancer Res. 35:279-286. Cairns, T. 1980. The EDo~ study: introduction, ob- jectives, and experimental design, J. Environ. Pathol. Toxicol. 3:1-7. Casto, B. C., Hatch, G. G., and Huang, S. L. 1981. Mutagenic and carcinogenic potency of extracts of Correspondence should be addressed to David G. Kaufman, Department of Pathology, School of Med icine, University of North Carolina, Brinkhous-Bul litt Building, 228H, Chapel Hill, NC 27514. , ~7 - - 7 Schechtman, L. M. 1981. Mutagenic and carcino- genic potency of extracts from diesel related envi- ronmental emissions: simultaneous morphological transformation and mutagenesis in BALB/c 3T3 cells, Environ. Int. 5:411-415. Doll, R. 1955. Mortality from lung cancer in asbestos workers, Br. J. Ind. 12:81-86. Doll, R. 1971. The age distribution of cancer: impli- cations for models of carcinogenesis, J. Roy. Soc. Med. 134:133-166. Drake, J. W., and Baltz, R. H. 1976. The biochemis- try of mutagenesis, Ann. Rev. Biochem. 45:11-37. Evans, H. J. 1983. Effects on chromosomes of carci- nogenic rays and chemicals, In: Chromosome Muta- tions and Neoplasia a German, ed.), pp. 253-279, A. R. Liss, New York. Farber, E. 1980. The sequential analysis of liver cancer induction, Biochim. Biophys. Acta 605:149-166. Farber, E. 1984. Chemical carcinogenesis: a current biological perspective, Carcinogenesis 5:1-5. Gaylor, D. W. 1980. The EDo, study: summary and conclusions, J. Environ. Pathol. Toxicol. 3:179-183. Grasso, P., and Hardy, J. 1974. Strain differences in natural incidence and response to carcinogens, In: Mouse Hepatic Neoplasia (W. H. Butler and P. M. Newberne, eds.), pp. 111-132, Elsevier Press, Amsterdam. Grisham, J. W., KauLmann, W. K., and Kaufman, D. G. 1983. The cell cycle and chemical carcinogen- esis, Sure. Synth. Pathol. Res. 1:49-66. Gullino, P. M., Pettigrew, H. M., and Grantham, F. H. 1975. N-Nitrosomethylurea as mammary gland carcinogen in rats, J. Nat. Cancer Inst. 54:401-414. Hall, N. E., and Wynder, E. L. 1984. Diesel exhaust

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