Proliferative Hepatocellular Lesions of the Rat: Review and Future Use in Risk Assessment Prepared for the Risk Assessment Forum U.S. Environmental Protection Agency Washington, DC February 1986 Esther Rinde, Ph.D. Richard Hill, M.D., Ph.D. )ffice of Pesticides and Toxic Substances Authors Arthur Chiu, M.D., Ph.D. Bernard Haberman, D.V.M., M.S. Office of Research and Development |arry Anderson, Ph.D. (ODW)* lary Argus, Ph.D. (OPTS) \i\n Baumel, Ph.D. (OPTS)* Judith Bellin, Ph.D. (OSWER) Iteven Bayard, Ph.D. (ORD) Jhao Chen, Ph.D. (ORD) Irthur Chiu, M.D., Ph.D. (ORD) Technical Panel David Cleverly, M.S. (OAR) Bernard Haberman, D.V.M., M.S. (ORD)* Richard Hill, M.D., Ph.D. (OPTS) Louis Kasza, Ph.D., D.V.M. (OPTS) Paul Milvy, Ph.D. (OPPE) Esther Rinde, Ph.D. (OPTS) Jackson Schad, M.S. (OSWER) *Technical Panel Co-Chairman Risk Assessment Forum Staff Dorothy Patton, Ph.D., J.D., Executive Director (Acting) Alan Ehrlich, Ph.D., Executive Secretary U.S. ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, DC 20460 ------- IN MEMORY OF DR. BERNARD HABERMAN ------- Disclaimer ------- ------- Contents Page List of Figures List of Tables I. Introduction .................................... 1 A. Background .................................... ........... ^ B. Purpose and Scope .......................... ............. 1 II. Chemical Hepatocarcinogenesis, A Model ..................... 3 III. Characteristics of Proliferative Hepatocellular Lesions ........ 5 A. Morphological Classification ....................... ........... .5 B. Background Incidence of Lesions in Untreated Animals. '.'.'.'.'.'.'.'.'. 7 C. Comparative Morphology .............................. ..... 7 IV. Relationship Between Certain Hepatocellular Proliferative Lesions and Hepatocellular Carcinoma ........................... 9 A. Regression of Early Lesions .................... ............... B 1 . Foci ............................................ ......... g 2. Neoplastic Nodules ........................... ........... 10 B. Progression .................................. ............. 1 1 1 . Foci ........................................ ........... 12 2. Neoplastic Nodules ................................. ..... 12 V. Current Governmental Use of Data on Rat Hepatocellular Lesions ... 1 4 VI. Future Use of Rat Hepatocellular Lesions by EPA ................ 15 A. Rationale for Positions ....................... " ' ] ] .......... 15 B. EPA Positions ....................... ............... ........ 16 References ...................................... 1 o ------- List of Figures Page ... 4 Number 1. Hypothetical scheme of neoplastic development in rat liver.. 2. Terminology of rat liver lesions 5 ------- List of Tables Number Page 1. Proliferative Liver Lesions: Background Incidence in 2-Year-Old Rats 8 2. Evidence for Progressions of Hepatocellular Lesions 11 ------- ------- I. Introduction A. Background When liver carcinogens are administered to rats, there is an increase in the frequency of certain morphological lesions and often a chronology in their formation. Generally foci of cellular alteration are first to appear, then nepplastic nodules, and finally carcinomas are noted in the livers of the rats This time sequence suggests an interpretation of the carcinogenic process in which foci are a precursor stage in the development of neoplastic nod- ules, which in turn are precursors of carcinomas. However, under certain circumstances, if carcinogen administration ceases, many foci and neoplas- tic nodules disappear, and the formation of carcinomas occurs at a low frequency. Thus, an alternative interpretation is that foci and neoplastic nodules may be independent of carcinoma development. Because of these general findings, it is difficult to evaluate the signifi- cance of increases in lesions in long-term tests in rats. Since there is no certain scientific knowledge or consensus about the biological significance of liver nodules, the EPA needs a policy for the interpretation of rat carcino- gen bioassays in which liver nodules are an end point. Differences as to the way rat hepatocellular lesions might be used in risk assessment arose within the Agency. In July 1984, the Office of Toxic Sub- stances submitted to the EPA Risk Assessment Forum a proposal to inves- tigate the nature and use of neoplastic nodules of the rat liver. Issues were formulated that dealt with the histological classification of hepatocellular proliferative lesions and the way these lesions would be used for hazard assessment and dose-response assessment. It was agreed that the EPA should have a clear position on these issues. B. Purpose and Scope The classification of certain experimentally induced proliferative hepato- cellular lesions in the rat liver, other than carcinoma, has been the subject of great controversy. This paper addresses three questions that are crucial in the evaluation of such rat tumor data and in the assessment of carcino- genic risk for humans. One question is whether to consider these lesions hyperplastic" or "neoplastic" in nature, the latter classification being in- dicative of tumor formation.1 A second question is to interpret the finding that in some experimental systems, certain nonmalignant proliferative le- sions may regress following removal of an inciting, carcinogenic stimulus with only a small proportion of the lesions persisting. Finally, it is important to know whether any of these lesions may progress to carcinoma. This paper begins with a composite view of chemically induced hepato- carcmogenesis. Proliferative hepatocellular lesions are described histo- iDorland's Illustrated Medical Dictionary (1974) defines the term neoplasm as "... a mass of new nh^? 7 ' Per»'tf an, 9r°WS indePendent|y °f "s surrounding structures and which has no physologic use." Neoplasm has also been defined as ". . . an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease" (Stedman's Medical Dictionary, 1982). Dorland's defines hyper- plasia as ... abnormal multiplication or increase in the number of normal cells in normal arrangement m tissue." Stedman's definition of hyperplasia is ". . . increase in the nCmS™or cells in a tissue or organ, excluding tumor formatioq, where-by the bulk of the organ is in- crsssGu. 1 ------- pathologically, and comparisons among species are made. Discussions are developed on the regression of proliferative lesions following removal of a carcinogenic stimulus, as well as their potential for progression to car- cinoma. Current uses of rat liver proliferative lesions in risk assessments by governmental agencies are reviewed, and recommendations are made for the future use of these lesions by EPA in cancer risk assessment. ------- II. Chemical Hepatocarcinogenesis, A Model This section presents concepts generally accepted to represent the natu- ral history of chemical hepatocarcinogenesis, and provides a background for the larger discussion of neoplastic nodules and foci of cellular alteration in evaluating chemical carcinogenesis. Only key elements are discussed.2 Hepatocellular carcinoma, like other tumors, is generally recognized as the last stage in a multistage process, in which several stages can be de- scribed in terms of the atypical phenotypic expression(s) of cells. The con- cept of a stepwise development in the carcinogenic process is mainly based on morphological observations, correlated with cellular behavior. However, the precise developmental stages in carcinogenesis have not been unequiv- ocally demonstrated, but are mainly inferred by post-hoc reconstruction. The first step in hepatocarcinogenesis (usually termed initiation) is thought to be an irreversible alteration of one or more hepatocytes, either as a "spontaneous" event or induced by contact with an active chemical substance. The initial lesion becomes fixed following one or more cycles of cell proliferation, such that there appears to be lasting memory of its occur- rence in all following cell generations. In some systems initiation appears to be manifested by a resistance of the initiated cell to the growth-inhibiting effect of carcinogens on normal cells; thus the cell can proliferate into a focus, the first observable morphologic hepatocellular lesion (Scherer and Emmelot, 1976; Farber, 1980; Pitot and Sirica, 1980; Williams, 1982). Fol- lowing initiation, the progression of the initiated cell to cancer can be greatly accelerated by the administration of promoting agents (Berenblum, 1975; Pitot and Sirica, 1980). Foci are microscopic lesions that can be distinguished from surrounding normal liver tissue by a number of phenotypic markers. For example, rou- tine staining with hematoxylin and eosin differentiates foci into several types (e.g., clear cells, eosinophilic foci, and basophilic foci). Foci can also differ in their histochemical properties, such as alterations in enzyme activ- ities (e.g., deficiencies in glucose-6-phosphatase and beta-glucuronidase; increases in gamma-glutamyl transpeptidase), and reduced iron accumula- tion. While these are general characteristics, individual lesions may differ in their phenotypic marker expression. Foci do not show any disruption of normal hepatic lobular architecture, although some types of foci show ele- vated rates of cell division as compared with the surrounding "normal" parenchyma (Squire and Levitt, 1975; Farber, 1980; Williams, 1980; Bannasch et al., 1980; Pitot and Sirica, 1980; Emmelot and Scherer, 1980; Stewart, 1980; Bannasch et al., 1982). Neoplastic nodules usually appear later in time than foci, but before the advent of carcinoma; there may be overlap, however. Nodules are com- posed of cells that are similar to those of foci (e.g., show similar phenotypic 2Various authors have used a plethora of terms over the years to describe the various hepatocel- lular proliferative lesions and associated phenomena. Sometimes they have meant to indicate specific types of lesions or cellular processes. However, in order to keep this review simple in concept, we use the term "focus" (foci) for any of the various literature references that describe small, focal proliferative areas or areas of cellular alteration. We use the term "neoplastic nod- ules" to refer to any "nodular" proliferative lesions, whether the authors called them hyperplas- tic, neoplastic, or any other comparable terms. Lastly, processes leading to morphological rever- sal of proliferative lesions back to normal-appearing morphology (termed variably such things as phenotypic maturation, remodeling, reversion, re-differentiation) are called regression. ------- alterations following routine and special histochemical staining) and show elevated rates of cell division. Nodules, however, are larger in size and, unlike foci, compress the surrounding parenchyma, from which they are sharply demarcated (Squire and Levitt, 1975; Stewart, 1980; Williams, 1980; Farber, 1980). Still later following initiation and the development of foci and nodules, hepatocellular carcinomas are seen to develop. Carcinogens usually induce multiple foci or nodules and generally fewer carcinomas. Although the morphology and timing of the lesions suggests a progres- sion from focus to nodule to carcinoma, the case has certainly not been proven. Farber (1984) states that nodules are one identified source, while Williams (1981) suggests that foci do not necessarily give rise to neoplastic nodules, but may be direct precursors of carcinoma. Yet, unidentified le- sions that are antecedents of carcinoma may also exist. A hypothetical projection consistent with these observations is shown in Figure 1. Progression of lesions to carcinoma is only part of the scheme. As will be discussed later in this paper, under certain experimental conditions it ap- pears that some foci and neoplastic nodules may regress following removal of a chemical stimulus. Figure 1. Hypothetical scheme of neoplastic development in rat liver. Normal Hepatocyte I Initiated Cell . Focus Neoplastic Nodule Carcinoma ------- III. Characteristics of Proliferative Hepatocellular Lesions A. Morphological Classification In recognition of the diversity of terms used to describe proliferative lesions of the rat liver, a conference was held in 1974 to develop a consen- sus among researchers in the field (Squire and Levitt, 1975). Many of the previous terms that had existed in the literature up to that time, such as nodular hyperplasia, hyperplastic nodule, hepatoma, and hepatic cell ade- noma, were replaced by other terms. Three hepatocellular proliferative di- agnoses were identified: foci (or areas) of cellular alteration, neoplastic nodule, and hepatocellular carcinoma (see Figure 2). Figure 2, Terminology of rat liver lesions. Pre-1975 Since 1975 1985(NTP) Preneoplastic Hyperplastic Foci \ Basophilic Hyperplasia Enzyme-Deficient Islands Hyperplastic Nodule Nodular Hyperplasia Neoplastic Benign Malignant Hepatoma Hepatic Cell Adenoma Hepatoma Hepatic Cell Carcinoma Trabecular Carcinoma Neoplastic Nodule \ V Hepatocellular Carcinoma Hepatocellular Carcinoma SOURCES: Squire and Levitt, 1975; Stewart et al., 1980; Maronpot et al., 1985. Foci were defined as small lesions of less than one liver lobule in size, whereas areas of alteration were the size of a lobule or greater. The primary distinguishing features involve the staining and texture of the cytoplasm, leading to the use of descriptive terms such as, clear cell, eosinophilic (or ground glass), and basophilic to describe the differences. Foci did not show any disruption of the normal hepatic architecture. Because of the structural similarities with neoplastic nodules, most participants agreed that foci and areas of alteration may be part of a spectrum of lesions capable of progress- ing to neoplastic nodules (Squire and Levitt, 1975). ------- Neoplastic nodules were defined as generally spherical lesions, several hepatic lobules in size, which show architectural distortion and sharp de- marcation from the surrounding liver over at least a portion of its periphery. Neoplastic nodules also display compression of surrounding liver par- enchyma. It was recognized that nodules occur at a low incidence in control animals, could be induced by administration of carcinogenic substances, and "at the least, indicate an increased probability for the development of hepatocellular carcinoma" (Squire and Levitt, 1975). A majority of persons thought the previously used term, hyperplastic nodule, was a misnomer, and preferred the designation neoplastic nodule. Some question still ex- isted about the nature of neoplastic nodules, however, since the conferees concluded at that time that benign hepatocellular tumors (that is, those without malignant behavior potential) were not recognized in rats and, therefore, the term adenoma was not recommended. The hepatocellular carcinoma designation was applied to lesions with characteristic histological and cytological features. They are larger and more irregular than neoplastic nodules and may involve major portions of the liver lobes. At the periphery they compress or extend into the surround- ing parenchyma (Squire and Levitt, 1975). A second group of experts was assembled by the National Academy of Sciences (NAS) (Stewart et al., 1980) to consider the nomenclature of rat liver proliferative lesions. This group affirmed and extended the classifica- tion suggested by the previous group (Squire and Levitt, 1975). Hepatocel- lular lesions were divided into focus and primary neoplasms (i.e., neoplastic nodule and various subtypes of carcinoma). The NAS group recognized that although foci occur in aging control rats, an increase in their incidence following chemical administration raises the suspicion that the substance may be carcinogenic. The neoplastic nodule was interpreted as a manifestation of the carcinogenic process, with the focus representing an earlier stage. The nature and significance of neoplastic nodules was captured in the report by the following: Before and to some extent after this workshop, some pathologists termed this lesion a "hyperplastic nodule." Some who use this term regarded, and continue to regard, the lesion so designated as a stage of the neoplastic process; others consider it to be unrelated to neoplasia. Still others have called the neoplastic nodule a hepatoma or adenoma, thus implying that the lesion is a benign neoplasm. The consensus of those attending the workshop, however, was that neoplastic nodules, as described, do progress. End-stage benign neoplasms of hepatic cell origin have not been identified with certainty in the rat liver. From these considerations, the use of the term "hyperplastic nodule" as a synonym for neoplastic nodule is to be discouraged. (Stewart et al., 1980). The Squire and Levitt and NAS papers developed a new classification scheme for hepatocellular proliferative lesions, but the exact position and significance of the neoplastic nodule was not totally clarified. The nodule was seen not as a hyperplastic lesion but as somewhat neoplastic in nature, yet still not an end-stage benign tumor. Bannasch (1976) proposed that neoplastic nodules seem to include a mixture of forms, some precancerous, some cancerous, and various inter- mediate cells. Dissatisfaction with the term neoplastic nodule is reflected in the discus- sion of a recent international conference organized by the Society of Toxico- logic Pathologists (Symposium Panel Discussion, 1982). An informal con- ------- sensus of the conference participants (determined by a show of hands) recommended that the term neoplastic nodule be abandoned. Most recently, after discussion with an outside group of experts in the field, the National Toxicology Program (NTP) has instituted a change in the classification of rat liver proliferative lesions in the chronic toxicity program (Maronpot et al., 1986; Maronpot, personal communication, 1985). Basi- cally, the NTP has retained the terms focus and hepatocellular carcinoma as used before, but has replaced neoplastic nodule with two terms: hyper- plasia and hepatocellular adenoma (see Figure 2). Hyperplasia is character- ized by mild compression of the surrounding parenchyma that is not noted in the focus of cellular alteration. Hyperplasia is viewed as the result of cell injury with accompanying regeneration. In contrast, hepatocellular ade- noma shows clear-cut demarcation from surrounding normal tissue with compression and loss of the normal lobular architecture. The new NTP classification scheme is similar to that for some other ep- ithelial organs: cellular reactions include non-neoplastic hyperplasia as well as benign and malignant neoplastic lesions. The choice of terms should help to alleviate some of the problems associated with the term neoplastic nodule. The effect that the change in nomenclature will have on the propor- tion of neoplastic nodules that will now be called hyperplasia must await its use in a number of chronic studies. Although the NTP is applying the new terminology to the Fischer 344 rat, the new nomenclature could be extended to other strains. Finally, it is important to see how the proposed classifica- tion will hold up in experimental studies concerned with hepatocarcinogen- esis in which a host of biological indicators are used to assess the nature of the various proliferative lesions. B. Background Incidence of Lesions in Untreated Animals A few authors have reported the incidence of hepatocellular proliferative lesions in control animals in various strains of rats. A representative set of values is tabulated in this report (Table 1). Over a lifetime, it appears that the bulk of untreated animals develop foci, whereas many fewer develop neo- plastic nodules or carcinomas. For instance, for Fischer 344 rats, it seems that over 75% of control animals develop foci, whereas usually less than 5% and less than 1% of animals develop neoplastic nodules and liver-cell can- cers, respectively (Goodman et al., 1979; Haseman et al., 1984; Popp et al., 1985). C. Comparative Morphology Proliferative liver lesions have been identified in a number of different species. Foci showing alteration in routine or special (e.g., enzymatic) histo- logical staining have been identified in the mouse, rat, golden hamster, monkey, and human (Ward, 1984; Stenback et al., 1986). The same is true for "benign" nodules and carcinomas. Both Ward (1984) and Popper et al. (1977) point out the similarity in proliferative lesions (i.e., foci, nodules [adenomas], and carcinomas) among species, although Ward claims that "dysplastic" foci are more common in the monkey and human than in the mouse,, and Popper et al. indicate that human lesions show a much more pronounced fibrotic reaction than those in rodents. This latter difference is probably a reflection of the fact that normal rat liver contains only one-third the hydroxyprolihe (a measure of collagen, a necessary component in fibro- sis) of normal human liver. ------- Table 1. Proliferative Liver Lesions: Background Incidence in 2-Year-Old Rats Animals with (%) Strain Wistar Sherman COBS CD (Charles River) Fischer 344a Fischer 344b Fischer 344 (early NCI experi- ence) Fischer 344 (recent NTP ex- perience) Sprague- Dawley0 (Dow Chemical) Wistar (Col- worth strain) Number Neoplastic Sex examined Foci nodules M F M F M M F M F M F M F M F 7 100 173 16 20 15 20 50 9 100 85 78 79 87 1794 1754 2306 2356 171 172 45 45 — — — — 11 4.6 1.3 2.7 3.4 3.0 3.5 7.0 13 11 Carcinomas Reference — — — — 1.2 0 0.39 0.39 0.8 0.2 2.9 1.7 16 29 Ogawa et al.. 1981 Kimbrough et al., 7975 Ulland et al., 1977 Ogawa et al., 1981 Popp et al., 1985 Goodman et al., 1979 Haseman et al., 1984 Adapted from Thorpe et al., 1982 Adapted from Thorpe et al., 1982 "72 weeks old. b111 weeks old; total from three laboratories. Tote/ from two studies. ------- IV. Relationship Between Certain Hepatocellular Proliferative Lesions and Hepatocellular Carcinoma Administration of hepatocarcinogenic substances to rats is associated with two different phenomena. One is the induction of proliferative hepato- cellular lesions, foci, and neoplastic nodules, and usually later, hepatocellu- lar carcinomas. The other is reversion of early lesions back to "normal" morphology, which occurs under some experimental conditions, if the car- cinogenic stimulus is removed early in the process. This section summa- rizes some of the evidence dealing with the potential of various early prolif- erative lesions either to progress to carcinoma or regress to normal appearance. A. Regression of Early Lesions Early proliferative lesions induced by hepatocarcinogens often change following discontinuation of administration of the carcinogen. The mor- phology of foci and nodules becomes more like that of normal adult liver. Many of the biochemical changes noted in these lesions reverse (e.g., glucose-6-phosphatase activity is regained, gamma-glutamyl transpepti- dase activity declines). Due to this regression, the area becomes grossly indistinguishable from the surrounding liver (Kitagawa and Pitot, 1975- Farber, 1980). 1. Foci Goldfarb and Zak (1961) reported regression of foci 4 weeks after with- drawal of 3'-methyl butter yellow, fed for 26 days. (When the same carcino- gen was fed for 40 days, neoplastic nodules and hepatocellular carcinomas were found.) Kitagawa (1976) demonstrated after feeding N-2-fluorenylacetamide (2-AAF) that a majority of "hyperplastic" areas (it is assumed that the author was referring in part to foci) underwent regression. On the other hand, in a small group of foci, enzyme deficiency persisted during the observation period. The author speculated that these areas appeared to be more impor- tant than the ones that had regressed, in relation to the later development of carcinoma. Williams and Watanabe (1978) reported that over 95% of altered foci, induced by 2-AAF, regressed within 6 months after withdrawal of 2-AAF (but that a small fraction progressed to neoplastic nodules and carcinoma). Also after promotion with phenobarbital (following 2-AAF withdrawal) "over 90% of livers developed carcinoma by 6 months." Regression of foci has also been reported with N-nitrosomorpholine (NNM) (Moore et al., 1983). There have also been reports in which foci did not regress. For instance, daily oral doses of NNM for 7 weeks at doses of 80-200 mg/L in drinking water resulted in increases in the number of foci which persisted for periods up to 50 weeks after cessation of dosing (Moore et al., 1982). Irreversible foci were also induced with a single dose administration of diethylnitrosamine (DEN) followed by partial hepatectomy (Scherer and Emmelot, 1975) or followed by feeding of phenobarbital (Pitot et al., 1978). ------- 2. Neoplastic Nodules Sprague-Dawley rats fed 0.064% methyl butter yellow for 26 days or 40 days and then put on a standard lab diet showed different carcinogenic responses (Goldfarb and Zak, 1961). Neoplastic nodules formed in animals treated for 40 days, but most underwent regression upon cessation of treat- ment; many animals had developed hepatocellular carcinomas by the end of the study. However, few nodules or carcinomas were noted after 26 days of treatment. This compound induced hepatotoxic effects in addition to parenchymal cell changes (e.g., lesions of bile ducts and cirrhosis). Confirm- ing studies showing regression of nodules initially produced by methyl butter yellow were reported by Goldfarb (1973). Teebor and Becker (1971) reported heterogeneity among neoplastic nod- ules. They found that when Sprague-Dawley rats were fed 0.06% 2-AAF for 3 monthly cycles (3 weeks on treatment; 1 week on basal diet) and then returned to control diet, many nodules were produced which then re- gressed. Only a low incidence of hepatocellular carcinomas developed over time. However, in another group of rats that had been fed for only 1 addi- tional monthly cycle, nodules persisted, and there was a high incidence of animals with hepatocellular carcinomas. Kitagawa (1976) found that many neoplastic nodules induced by feeding 0.03% 2-AAF to Sprague-Dawley or Donryu rats for 12 weeks returned to a more normal phenotype upon withdrawal of the agent. In contrast to the above experiments, Hirota and Williams (1979) failed to find regression of neoplastic nodules in F344 rats fed 0.021% 2-AAF for 3,4, or 5 cycles (4 weeks on treatment; 1 week on basal diet) and then returned to control diet, although some nodules did show changes in enzyme stain- ing that are often seen in nodules that regress. Moore et al. (1982) also failed to find regression of neoplastic nodules and foci in male Sprague-Dawley rats that had been given NNM in drinking water (80-200 mg/L) for 1 to 20 weeks and then sacrificed 8 weeks after removal of the chemical. Some animals given NNM for 7 weeks were not sacrificed until 50 weeks post-treatment; again, there was no indication of regression. Unlike these findings, when the carcinogen is given for a limited period of time and then stopped, when the same laboratory (Moore et al., 1983) evaluated NNM in a short-term exposure system,3 regression of nod- ules and foci was observed. In a series of experiments using a related model,4 Farber and co-workers have clearly indicated that nodules synchronously appear in great numbers following treatment, only to regress following termination of 2-AAF (Solt et al., 1977; Ogawa et al., 1979b; Enomoto and Farber, 1982; Farber, 1982; Tatematsu et al., 1983). These results have been confirmed by Van der Heijden and Dormans (1981). During regression, staining characteristics in most nodules return to normal and the characteristic morphology of the lesions gradually disappears, with the cells in the area again appearing "normal." Tritiated thymidine labeling has demonstrated that regresssion to normalcy in this system is not attended with profound cellular loss; instead, existing cells in nodules return to normal phenotypes (Kitagawa, 1976; Enomoto and Farber, 1982). SPartial hepatectomy, 3 days NNM (400 mg/L in drinking water), 2 weeks of 2-AAF (0.2 mg/kg in feed) with a single dose of carbon tetrachloride (1 mL/kg) midway during 2-AAF exposure. 4A single dose of N-nitrosodiethylamine followed 2 weeks later by feeding 2-AAF for 1 week and partial hepatectomy. 10 ------- Thus, it appears that both foci and neoplastic nodules can regress under at least two circumstances following removal of a carcinogenic stimulus. In "stop" experiments, where a carcinogen is given for a short period and then stopped, both foci and nodules have reverted to normal morphology. In addition, foci and nodules have also regressed in short-term experiments, when a carcinogen is given for a period of time following administration of another carcinogen to initiate the process and a stimulus is given to cause liver regeneration. Regression has not, however, been noted by every inves- tigator, nor have all experimental systems been investigated. Even in those cases in which regression has been demonstrated, some proliferative lesions persist. Farber has stated that despite a high rate of regression of nodules in his experimental model, 70% of animals develop hepatocellular carcinomas by 9 months following cessation of treatment (Ogawa et al., 1979a; Farber, 1982). For those proliferative lesions that regress, the question remains whether they retain "memory" of the initial lesion. Farber (1982) hypothesized that these hepatocytes revert back to an initiated cell; however, evidence for this position is still lacking. B. Progression The general acceptance of the developmental sequence: focus to neo- plastic nodule (NN) to hepatocellular carcinoma (HCC) (i.e., progression) is based mainly on indirect evidence. Table 2 presents a summary of some of this evidence in simplified form. Although there has been much discussion of candidates for a phenotypic marker of cells committed to develop carcinoma, none as yet has proven to be a reliable indicator of the origin of hepatocellular carcinoma cells. Thus, Table 2. Evidence for Progressions of Hepatocellular Lesions Foci NN HCC Incidence increased by carcino- gen administration + Sequence in the development of lesions ^t Proliferative lesion + Number of lesions per liver + + + Morphology: compression of adjacent tissue — Markers, Enzyme-staining abnormalities + Decreased iron accumulation + Site of hepatocellular carcinoma formation ? Transplantability -/? + 2nd + + + +/++? + 3rd +/_ +/_ + -f n/a 11 ------- most of the conclusions in the literature on this aspect of hepatocarcinogen- esis are based on inference, rather than direct evidence. Some of these are presented. 1. Foci It has been claimed that some carcinomas arise within foci (Schulte- Hermann et al., 1982). However, the authors cautioned that there was no proof that all foci progressed to malignancy. Cells of certain carcinogen-initiated foci cells are able to proliferate (in- crease in number) in the absence of the carcinogen for considerable periods of time (Scherer and Emmelot, 1975). Bannasch et al. (1982) observed that "foci became larger and acquired phenotypic markers closerto neoplasia, independent of further action of the carcinogen." Although foci undergo regression, it has been suggested that these re- verted cells may retain some permanent carcinogen-induced alteration which could make them susceptible to progression if exposed to additional carcinogens (Williams and Watanabe, 1978). These authors describe an experiment where PB was given as a promotor after withdrawal of AAF, which resulted in increased persistence of foci, and up to 90% of livers so treated developed carcinomas by 6 months. Scherer and Emmelot (1975) suggested there is a precursor-product rela- tionship between foci and hepatocellular carcinoma, which they claim to have demonstrated by a quantitative technique. (See Emmelot and Scherer, 1980 for a detailed discussion.) It has also been claimed that only a small fraction of foci may develop into nodules or carcinomas: about 1 in 1000 following administration of AAF (Watanabe and Williams, 1978) and about 1 in 11,000 after partial hepatec- tomy and dosing with methyl(acetoxy-methyl)nitrosamine (Kaufman et al., 1985). 2. Neoplastic Nodules Cancer has been found to arise in nodules (Goldfarb, 1973; Williams, 1976 [observed cancer arising both inside and outside nodules]; Solt et al., 1977; Popper et al., 1977; Farber, 1982). Some nodules have been found to be a site for cancer development with five different carcinogens in different mod- els (Farber et al., 1984); however, cancer cannot be said to arise exclusively inside nodules. A minority of nodules do not undergo regression following carcinogen removal, but persist (Ogawa et al., 1979a; Van der Heijden and Dormans, 1981; Enomoto and Farber, 1982; Moore et al., 1983; Tatematsu et al., 1983; Farber, 1984). These may play a role in the development of carcinoma. Autonomy Neoplastic nodules sometimes develop long after removal of a carcino- gen and are progressively growing entities: a carcinogen can be withdrawn for 8 to 12 weeks, and liver cancer will still appear weeks or months later (Epstein etal., 1967). Several researchers have published experimental re- sults that clearly demonstrate this concept of autonomous growth for neo- plastic nodules. For many weeks after chemical withdrawal, neoplastic nod- ules continue to enlarge or invariably increase in number (Goldfarb, 1973; Hirota and Williams, 1979). In fact, Hirota and Williams (1979) presented data from studies in F344 rats fed N-2-fluorenylacetamide for a limited number of cycles, which clearly showed that nodules were persistent after carcinogen withdrawal and had progressive growth ability (in situ), a char- acteristic of neoplasms. Similar conclusions regarding persistence and au- 12 ------- tonomy were also reported for various experimental protocols (Scherer and Emmelot, 1975; Emmelot and Scherer, 1980). Transplantation Since on transplantation, hepatocellular carcinomas display continued growth, invasiveness, and metastasis, several investigators have studied the behavior of "putative preneoplastic lesions" following transplantation. The results are not consistent. Lee et al. (1983) described the transplantation of carcinogen-altered hep- atocytes and their subsequent development into metastasizing cancer. Dis- sociated cells from livers bearing chemically induced nodules were im- planted into spleens of syngenic rats. Cells from "early nodules,"5 grew slowly in the spleen, but their evolution into cancer was not observed in studies lasting up to 2 years. Cells from "persistent nodules,"6 on the other hand, generated hepatocellular carcinoma in the spleen within 18 months (Finklestein et al., 1983; Farber, 1984). In another transplantation model, hepatocytes from nodules showed pro- gressive invasion into chick embryo heart fragments in the same way as did those from hepatocellular carcinoma. Control hepatocytes showed no inva- sion (Wanson et al., 1981). In still another series of studies summarized by Williams (1980), earlier attempts at transplantation of neoplastic nodules were unsuccessful. Nod- ule transplants placed in the mammary fat pads of syngenic rats were found to persist for months, but did not display "continued growth, invasiveness, or metastasis," although cell division was noted. Ward et al. (1975) claimed that two well-delineated nodules, induced by dietary aflatoxin B-i and transplanted into syngenic recipients, grew slowly. In a later study, Ward and Lynch (1984) were unsuccessful in transplanting a hepatocellular "adenoma" and a hepatocellular carcinoma. In summary, there is some information that indicates that foci and nod- ules may continue to grow and possibly progress toward carcinoma. The case is stronger for the progression of some nodules to carcinoma than it is for foci, but in neither case is the evidence conclusive. It is also recognized that under certain experimental conditions many foci and nodules regress after the administration of a hepatocarcinogen has been discontinued. Thus, even if some foci or nodules do progress, all such lesions are not necessarily obligate precursors of carcinoma. One can conclude that foci and nodules are part of the carcinogenic process. It is generally recognized that foci are hyperplastic lesions, but it is still difficult to discern whether rat liver nodules are hyperplastic or neoplas- tic lesions. A position consistent with the data is that at least some nodules may be neoplastic (e.g., those that persist after carcinogen removal) and have the potential to progress to carcinoma. 5Those nodules induced in the short-term test system employed by Farber and co-workers; see footnote 4. 6The small percentage of nodules that do not regress following removal of a carcinogenic stim- ulus. 13 ------- V. Current Governmental Use of Data on Rat Hepatocellular Lesions Several governmental agencies have adopted procedures for the use of proliferative lesions in experimental animals in the evaluation of chemicals for carcinogenicity. The National Toxicology Program (NTP) commonly pools the incidence of certain benign and their corresponding malignant neoplasms in evaluating the carcinogenic hazard of chemicals in long-term animal tests (McConnell et al., 1986); this practice has been endorsed by an advisory group to the NTP (1984). For rat hepatocellular lesions, separate incidence figures are generated for animals bearing neoplastic nodules, hepatocellular car- cinomas, and for the combination of the two. Carcinogenicity determina- tions are based on treatment versus control comparisons and dose-trend relationships for all these groupings. Neoplastic nodules have been inter- preted by NTP as if they were benign neoplasms (Maronpot and Boorman, 1982); chemically related increases in any of the three groupings constitute "clear" evidence of carcinogenicity. The NTP also collects information on the frequency of different types of foci of cellular alteration among animals in their long-term studies. Recently, NTP has proposed to change the classification of rat hepatocel- lular proliferative lesions in their chronic toxicity testing program (Maronpot et al., 1986). The major difference in their new classification scheme is replacement of the term neoplastic nodule with two terms: hyperplasia and hepatocellular adenoma. At the end of a 2-year trial period, NTP will evalu- ate the adequacy of this new nomenclature. The Office of Science and Technology Policy (1985) report and the EPA Proposed Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1984) sup- port the pooling of benign and malignant tumors of the same histological type when scientifically defensible. The EPA Guidelines go on to state that the contribution of benign tumors to the total extrapolated risk should be expressed when pooling has been used. In qualitative hazard assessment, the Guidelines accord less weight to cases where the only neoplastic re- sponse attributed to chemical exposure is the production of benign tumors. Such data constitute limited evidence of animal carcinogenicity, and such chemicals are generally put into Group C (Possible Human Carcinogen). The EPA Proposed Guidelines also state that decisions as to whether to proceed with quantitative risk assessment on Group C substances would be made on a case-by-case basis. The Food and Drug Administration, Occupational Safety and Health Ad- ministration, and Consumer Product Safety Commission have used rat neo- plastic nodules in the assessment of carcinogenicity of chemical sub- stances, but no specific policies have been established. Hepatocellular carcinomas and nodules are used in a weight-of-evidence determination as to whether a substance may pose a carcinogenic hazard. All agencies use foci in their hazard assessments to help in the interpretation of hepatocellu- lar neoplastic responses. 14 ------- VI. Future Use of Rat Proliferative Hepatocellular Lesions by EPA A. Rationale for Positions Although the disagreements seem to have narrowed in recent years, there are still differences of opinion within the scientific community on the biological significance of certain parenchyma! cell proliferative lesions in rat liver. Several factors have influenced this situation, including questions about nomenclature and possible progression and regression of these le- sions under certain circumstances. As the National Academy of Sciences (NRC, 1983) pointed out, even in the absence of scientific consensus, Fed- eral agencies have the responsibility for developing inference guidelines (i.e., science policiesj which lay out the positions to be taken on specific issues together with their rationale. From the accompanying review it seems apparent that scientists have described a number of proliferative lesions of rat hepatocytes which are in some way part of the carcinogenic process. Morphological entities similar to those found in the rat have been described in the livers of other rodents, monkeys, and humans. Foci appear to be some type of preneoplastic lesion, but controversy still surrounds the term neoplastic nodule. Some consider the nodule to be a hyperplastic lesion while others interpret it to be a neoplastic entity (a benign or possibly a malignant lesion), and the term may include entities from all such morphological designations. Foci are commonly found in the livers of control rats at the end of long- term studies; neoplastic nodules and hepatocellular carcinomas are less common. Administration of carcinogens, but not of noncarcinogens, in- creases the incidence of these three lesions. These lesions are also in- creased in animals initially given a carcinogen and then a promoter. There seems to be agreement that an increase in foci and nodules in treated animals is some indication that the liver is at increased risk of cancer forma- tion. The appearance of morphological entities generally follows a time- dependent pattern following administration of a hepatocarcinogen: foci form first, followed by nodules, and still later, by carcinomas. Although there is some evidence supporting the notion of progression from a latent-initiated cell to focus to nodule and then to carcinoma, that relationship cannot be said to be established. Carcinoma sometimes seems to arise from a nodule; a focus or some yet unidentified precursor or a combination of the ab'ove may be other sources. Regression experiments serve two roles: they help to elucidate steps in the carcinogenic process, and they may give insights into phenomena that may occur under conditions of limited or intermittent exposure to carcino- genic substances. Human exposures to environmental toxicants may be limited or intermittent and, therefore, regression experiments may be rele- vant to risk evaluation. It also appears that under certain experimental conditions, early prolifer- ative lesions (i.e., some foci and nodules) that have been induced by hepa- tocarcinogens will largely regress following removal of the carcinogenic stimulus. Whether these lesions return to a latent-initiated state or back to a totally normal cell, or whether the cells may be lost or destroyed under some circumstances, is not known. To the extent that proliferative lesions 15 ------- regress, however, there may be some modulation of the potential hazard for cancer development. Even in those cases in which the bulk of the foci and nodules have been shown to regress following carcinogen removal, some lesions persist. The persistent lesions are often associated with carcinoma formation at a later time. It also seems possible that some of the persistent lesions may actual ly progress to carcinoma. Federal guidance for carcinogen risk assessments, as developed in the Office of Science and Technology Policy (1985) document, recommends that the incidence of animals bearing malignant and corresponding benign lesions should be combined when scientifically defensible. It also points out the importance of reviewing the incidence of preneoplastic lesions in target organs as being an adjunct to the assessment of a chemical's carcinogenic- ity. EPA's Proposed Guidelines for Carcinogen Risk Assessment (1984) af- firm these positions, but state that an increase in benign tumors alone is accorded less weight in assessing carcinogenic hazards than an increase in malignant tumors or a combination of malignant and corresponding benign tumors. Increases in benign tumors only are given a weight-of-evidence classification of Group C (Possible Human Carcinogen). Decisions as to whether to proceed with a quantitative risk assessment are made on a case-by-case basis. Thus, in regard to rat liver proliferative lesions, it appears that Federal agencies are using foci and neoplastic nodules along with hepatocellular carcinomas in risk assessments on chemicals, and it seems appropriate to continue such practices. Accordingly, the Agency provides the following guidance for interpreting liver toxicity data in rats. B. EPA Positions In formulating a policy position on the interpretation of neoplastic nod- ules, one needs to be cognizant of the existing scientific evidence as well as the inconsistencies in the data, the absence of information in certain areas, and the lack of a consensus in the scientific community. Morphologically described proliferative lesions of rat parenchymal cells will be used in carcinogen risk assessments in conjunction with the EPA Proposed Guidelines for Carcinogen Risk Assessment (1984) as follows: 1. Foci of cellular alteration are to some extent indicative of an ongoing carcinogenic process and should be used in risk assessments. Accordingly, EPA adopts the following position: An analysis of the incidence of foci of cellular alteration among treat- ment groups can contribute to the interpretation of a given rat toxicity study and, thus, becomes part of the hazard identification step of the carcinogenic risk assessment. Dose-response and time-to-occurrence parameters are useful in this qualitative analysis. 2. Neoplastic nodules are increased in animals receiving carcinogens, and that occurrence is almost always associated with an increase in hepato- cellular carcinoma. However, it appears that there is not a one-to-one rela- tionship between nodules and carcinomas. Although some nodules may have "malignant potential," others may only be "hyperplastic" lesions. Still other nodules'may regress following cessation of carcinogenic administra- tion. Therefore, the exact contribution of neoplastic nodules to the overall incidence of hepatocellular tumors in the rat is unclear at this time. EPA adopts the following position until better means of articulating uncertainties are developed. Determination of carcinogenic hazard will be based upon consider- ation of the incidence of hepatocellular carcinoma alone, neoplastic 16 ------- nodule alone, and a combination of carcinoma and nodule. The range of responses could vary from cases where there are very significant increases in the incidence of carcinomas backed up by increases in nodules, through situations where only the combined frequency of animals with carcinomas or nodules is significant, to cases where in- creases are limited to nodules alone. (i) At one end, responses showing an overwhelming increase in car- cinomas alone will be interpreted as providing sufficient evidence of animal carcinogen/city when other criteria for the sufficient category are met. In the absence of human evidence and supporting information, such evidence will generally be given a weight-of-evidence designation of Probable Human Carcinogen (Group B2), and quantitative risk as- sessment is appropriate. (ii) At the other end, where increases in lesions and statistical signif- icance are restricted to neoplastic nodules alone, such data will be interpreted as only limited evidence of animal carcinogenioity. When neither human evidence nor supporting information accompany the increase in nodules, the weight-of-evidence designation is Possible Hu- man Carcinogen (Group C), but quantitative assessment is not war- ranted. (Hi) Situations within the two extremes will be evaluated on a case- by-case basis. In determining carcinogenic hazard, different factors should be considered in deciding whether there is sufficient or limited evidence of animal carcinogenicity. Some of these factors include the proportion of proliferative lesions that are malignant; the magnitude of increase in incidence of nodules and carcinomas; considerations of dose response and time-to-tumor; and the nature and the outcome of short-term tests and other support- ing information. In deciding whether or not to proceed with quantita- tion, consideration will be given to the overall weight of evidence. In cases where quantitation is performed, methods of expressing the pos- sible uncertainty in the estimated risks should be explored (e.g., ex- pressing risk as a range between that determined from extrapolation of carcinoma incidence alone and that from a combination of carcinomas and nodules). 3. Hepatocellular adenoma will constitute a diagnostic classification in some rat studies reviewed by the Agency in the future. The designation adenoma includes a yet unknown proportion of lesions previously called neoplastic nodules. The Agency concludes that: The EPA current position on benign tumors will apply to the designa- tion, hepatocellular adenoma. The pooled incidence of hepatocellular adenomas and carcinomas will be used in making qualitative judg- ments as to whether a rat study demonstrates evidence of a carcino- genic hazard. Pooled incidence will also be used for dose-response assessments. 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