Treatment of a variety of highly tumorigenic mouse lines in vitro with chemical mutagens, such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-Nnitrosoguanidine (MNNG), can result in extraordinarily high frequencies (sometimes in excess of 90%) of strongly immunogenic clones unable to grow progressively in normal syngeneic hosts. These clones will, however, grow in immunosuppressed hosts and gradually regain tumorigenic ability in normal mice i f maintained in long-term (several months-I year) culture, i.e., they are often phenotypically unstable. These features-phenotypic drift and high frequency-make it unlikely that point mutations are the underlying mechanism involved in the generation of the variants. Results presented here demonstrate that these observations can be reproduced on the same tumor lines using 5-azacytidine-an analogue of cytidine which can be incorporated into DNA causing subsequent extensive hypomethylation of cytosine residues in the absence of any significant mutagenic effects. Furthermore, 5-azacytidine treatment of a nonmetastatic mouse mammary tumor led to the emergence of a small number of heritable but unstable tumor clones capable of spontaneous metastatic spread. Because it is known that DNA hypomethylation can lead to transcriptional activation of normally silent genes, that altered methylation patterns can be somatically replicated with a high but not perfect fidelity, and that mutagens can cause DNA hypomethylation, we propose that DNA hypomethylation followed by de novo methylation represents a plausible mechanism to account not only for the induction of the nontumorigenic variants but for a number of aspects of tumor progression and tumor heterogeneity, as well. In particular, we refer to heritable phenotypic alterations in tumor cell populations which occur at very high frequency but which are not necessarily stable over very long periods of time.Perhaps the most insidious characteristic of cancers is their relentless capacity for cellular diversification. Even if they originate from the transformation of a single cell-and most evidence indicates that this is so (Fialkow, 1979)-a tumor cell population nonetheless will be phenotypically and genotypically heterogeneous by the time it is clinically detectable. Because this phenotypic diversity encompasses the development of resistance to virtually all commonly employed anticancer therapeutic agents, therapy of cancer over the long term will inevitably lead to unsuccessful results. Moreover, there will also be an inexorable tendency for tumors to become progressively *To whom reprint requestsicorrespondence should be addressed. 1984 ALAN R. LISS, INC

Formaldehyde has been assessed as a Priority Substance under the Canadian Environmental Protection Act. Probabilistic estimates of exposure of the general population in Canada to formaldehyde in ambient and indoor air are presented. Critical health effects include sensory irritation and the potential to induce tumors in the upper respiratory tract (the nasal region in rodents and potentially the lungs of humans). The majority of the general population is exposed to airborne concentrations of formaldehyde less than those typically associated with sensory irritation (i.e., 0.1 mg/m3). Based primarily upon data derived from laboratory studies, the inhalation of formaldehyde under conditions that induce cytotoxicity and sustained regenerative proliferation within the respiratory tract is considered to present a carcinogenic hazard to humans. At airborne levels for which the prevalence of sensory irritation is minimal (i.e., 0.1 mg/m3), risks of respiratory-tract cancers for the general population estimated on the basis of a biologically motivated case-specific model are exceedingly low. This biologically motivated case-specific model incorporates two-stage clonal expansion and is supported by dosimetry calculations from computational fluid dynamics analyses of formaldehyde flux in various regions of the nose and single-path modeling for the lower respiratory tract. The degree of confidence in the underlying database and uncertainties in estimates of exposure and in characterization of hazard and dose response are delineated.

For more than three decades chronic studies in rodents have been the benchmark for assessing the potential long-term toxicity, and particularly the carcinogenicity, of chemicals. With doses typically administered for about 2 years (18 months to lifetime), the rodent bioassay has been an integral component of testing protocols for food additives, pesticides, pharmaceuticals, industrial chemicals, and all manner of byproducts and environmental contaminants. Over time, the data from these studies have been used to address an increasing diversity of questions related to the assessment of human health risks, adding complexity to study design and interpretation. An earlier ILSI RSI working group developed a set of principles for the selection of doses for chronic rodent studies (ILSI, 1997). The present report builds on that work, examining some of the issues that arise and offering new perspectives and approaches for putting the principles into practice. Dose selection is considered both from the prospective viewpoint of the choosing of dose levels for a study and from the retrospective interpretation of study results in light of the doses used. A main theme of this report is that the purposes and objectives of chronic rodent studies vary and should be clearly defined in advance. Dose placement, then, should be optimized to achieve study objectives. For practical reasons, most chronic studies today must be designed to address multiple objectives, often requiring trade-offs and innovative approaches in study design. A systematic approach to dose selection should begin with recognition that the design of chronic studies occurs in the context of a careful assessment of the accumulated scientific information on the test substance, the relevant risk management questions, priorities and mandates, and the practical limitations and constraints on available resources. A stepwise process is described. The aim is to increase insofar as possible the utility of an expensive and time-consuming experiment. The kinds of data that are most commonly needed for dose selection and for understanding the dose-related results of chronic rodent studies, particularly carcinogenicity studies, are discussed as "design/interpretation factors." They comprise both the inherent characteristics of the test substance and indicators of biological damage, perturbation or stress among the experimental animals. They may be primary toxicity endpoints, predictors or indicators of appropriate dose selection, or indicators of conditions to be avoided in dose selection. The application and interpretation of design/interpretation factors is conditioned by the study objectives-what is considered desirable will depend on the strategy for choice of doses that is being followed. The challenge is to select doses that accommodate all of the issues raised by the relevant design/interpretation factors. Three case studies are presented here that illustrate the interplay between study objectives and the design and selection of doses for chronic rodent studies. These exa...

Treatment of normally tumorigenic murine tumor cell lines in vitro with chemical mutagens followed by cloning of the surviving cells, results in the selection, at extraordinarily high frequencies (anywhere from <1% to >90%), of clones unable to grow progressively in normal syngeneic mice (1-7). Such clones, which are phenotypically stable in culture over a period of several weeks or months, have been designated by Boon and his colleagues (1-6) as "tum-" (nontumorigenic in normal hosts). ~ They have been derived using such standard mutagens as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethyl methanesulfonate (EMS), from at least eight different murine tumors of viral, chemical, or spontaneous origin (1-7). Because the turn-clones will grow readily in highly immunosuppressed recipients, e.g., X-irradiated or nude mice (1-8), the turn-phenotype appears to have an underlying immunological basis, a view confirmed by detailed in vitro studies of T cell-mediated cytotoxicity (CMC).These studies have also shown that each turn-clone derived from a particular mutagenized parent line possesses an individual tumor-specific antigen distinct from the new antigen found on any other turn-clone, i.e., there is a startling degree of tumor antigen polymorphism (1-7). Of considerable interest is that this holds true even for totally non-immunogenic tumors of spontaneous origin (5-7) and as such, has very broad and important implications for the field of tumor immunology, especially in relation to the controversies surrounding the antigenic and immunologic status of neoplastic cells (9-11). Nevertheless, very little is known about how mutagens can cause such drastic and heritable changes in the behavioral properties of tumors in vivo, and at such high frequencies.

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