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The International Agency for Research on Cancer (IARC) has recently proposed employing “ten key characteristics of human carcinogens” (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

It is time to say goodbye to the standard two-year rodent bioassay. While a few, primarily genotoxic, compounds which are clearly associated with human cancer test positive in the bioassay, there is no science-based, sound foundation for presuming it provides either a valid broad (across different chemicals) capability for discerning potential human carcinogens or a valid starting point for making human risk assessment decisions. The two basic assumptions underlying the bioassay are: (1) rodent carcinogens are human carcinogens; and (2) results obtained at high doses are indicative of results that will occur at lower, environmentally relevant, doses. Both of these assumptions are not correct. Furthermore, a reevaluation of National Toxicology Program bioassay data has revealed that if the dose group size were increased from 50 to 200 rodents per group the number of bioassays deemed to be positive would increase from approximately 50% to very close to 100%. Thus, under the extreme conditions of the bioassay (e.g., high doses, lifetime exposure and, at times, a non-physiological route of administration) virtually all chemicals tested could be made into rodent carcinogens. In recent years there have been a number of proposals to move away from the standard bioassay. In particular, a recently formulated decision tree (Cohen, 2017), which places an emphasis on dose–response relationships and invites the use of MOA information, provides a sound basis for moving on from the bioassay and towards a rational approach to both identify chemicals which appear to have the potential to cause cancer in humans and take dose–response relationships into consideration in order to place the extent, if any, of the risk they might pose into proper perspective.

The rapidly evolving field of epigenetic regulation of gene expression is having an impact across the spectrum of biomedical research. Toxicologists have embraced this area as evidenced by their increasing focus on discerning potential epigenetic mechanisms underlying mechanisms by which chemical and physical agents might cause toxicity. It is not surprising that an interest in epigenetic mechanisms of toxicity would lead to a desire to incorporate an epigenetic component into safety assessment. However, premature movement in this direction carries the risk of imposing more confusion than light. This commentary provides an overview of epigenetics, with an emphasis on how the various epigenetic parameters are integrated, as a basis for understanding the complexity behind the desire to include epigenetic evaluations in safety evaluations. Basically, we have much more to learn before turning the goal into a reality. However, considerable progress has been made with regard to using epigenetic profiles as signatures of xenobiotic exposure.

The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.

It is time to say goodbye to the standard two-year rodent bioassay. While a few, primarily genotoxic, compounds which are clearly associated with human cancer test positive in the bioassay, there is no science-based, sound foundation for presuming it provides either a valid broad (across different chemicals) capability for discerning potential human carcinogens or a valid starting point for making human risk assessment decisions. The two basic assumptions underlying the bioassay are: (1) rodent carcinogens are human carcinogens; and (2) results obtained at high doses are indicative of results that will occur at lower, environmentally relevant, doses. Both of these assumptions are not correct. Furthermore, a reevaluation of National Toxicology Program bioassay data has revealed that if the dose group size were increased from 50 to 200 rodents per group the number of bioassays deemed to be positive would increase from approximately 50% to very close to 100%. Thus, under the extreme conditions of the bioassay (

The International Life Sciences Institute, Health and Environmental Sciences Institute sponsored a workshop entitled "State of the Science: Evaluating Epigenetic Changes," hosted by the National Institute of Environmental Health Sciences, Research Triangle Park, NC, 28-30 October 2009. The goal was to evaluate and enhance the scientific knowledge base regarding epigenetics and its role in disease, including potential relationships between epigenetic changes and transgenerational effects. A distinguishing aspect of the workshop was the highly interactive discussion session on the final morning. Meeting participants formed breakout groups (with representation from academia, industry, and government in each group) and were tasked with integrating their previous knowledge of epigenetics with what was learned during the workshop. The participants addressed the issue of what needs to be known prior to thinking about incorporating an epigenetic evaluation into safety assessment. To this end, the breakout groups were asked to address the following questions: (1) What model systems might be employed to evaluate the ability of a chemical to produce an epigenetic change (affecting the F1 and/ or F3 generation); (2) What end points/targets might be evaluated; (3) What techniques might be employed; and (4) Regulatory Perspective: When is it appropriate to incorporate "new" science, in this case epigenetics, into the regulatory process? What does one need to know, what are the pitfalls and how might these be overcome/avoided? The basis of this paper is a synopsis of these discussions. The workshop highlighted the fact that the field of epigenetics is evolving at a very rapid pace and indicated that a great deal needs to be learned prior to being able to rationally incorporate an epigenetic evaluation into safety assessment. The value of the workshop is that it called attention to key data/ knowledge gaps that should serve to focus attention on the areas where research and new thinking are needed to better understand epigenetics and its relationship to safety assessment.

Our overall goal is to elucidate progressive changes, in expression and methylation status, of genes which play key roles in phenobarbital (PB)–induced liver tumorigenesis, with an emphasis on their potential to affect signaling through critical pathways involved in the regulation of cell growth and differentiation. PB-elicited unique expression changes of genes, including some of those identified previously as exhibiting regions of altered DNA methylation, were discerned in precancerous liver tissue and/or individual liver tumors from susceptible constitutive active/androstane receptor (CAR) wild-type (WT) compared with resistant CAR knockout (KO) mice. Many of these function in crucial cancer-related processes, for example, angiogenesis, apoptosis, cell cycle, DNA methylation, Hedgehog signaling, invasion/metastasis, Notch signaling, and Wnt signaling. Furthermore, a subset of the uniquely altered genes contained CAR response elements (CAREs). This included Gadd45b, a coactivator of CAR and inhibitor of apoptosis, and two DNA methyltransferases (Dnmt1, Dnmt3a). The presence of CAREs in Dnmts suggests a potential direct link between PB and altered DNA methylation. The current data are juxtaposed with the effects of PB on DNA methylation and gene expression which occurred uniquely in liver tumor-prone B6C3F1 mice, as compared with the resistant C57BL/6, following 2 or 4 weeks of treatment. Collectively, these data reveal a comprehensive view of PB-elicited molecular alterations (i.e., changes in gene expression and DNA methylation) that can facilitate hepatocarcinogenesis. Notably, candidate genes for initial “fingerprints” of early and late stages of PB-induced tumorigenesis are proposed.

At 2 and 4 weeks following treatment with phenobarbital (PB), the classical nongenotoxic rodent liver carcinogen, we elucidated unique gene expression changes (both induction and repression) in liver tumor-susceptible B6C3F1 mice, as compared with the relatively resistant C57BL/6. Based on their cancer-related roles, we believe that altered expression of at least some of these genes might underlie PB-induced liver tumorigenesis. Putative constitutive active/androstane (CAR) response elements (CAREs), a subset of PB response elements, were present within multiple genes whose expression was uniquely altered in the B6C3F1 mice, suggesting a role for CAR in their regulation. Additionally, three DNA methyltransferase genes (Dnmt1, Dnmt3a, and Dnmt3b) were repressed uniquely in the tumor-prone B6C3F1 mice, and all possess putative CAREs, providing a potential direct link between PB and expression of key genes that regulate DNA methylation status. Previously, we demonstrated that PB-elicited unique regions of altered methylation (RAMs) in B6C3F1 mice, as compared with the relatively resistant C57BL/6, at 2 and 4 weeks, and annotation of the regions harboring these changes revealed 51 genes. This is extended by the current study, which employed RNA isolated from the same liver tissue used in the earlier investigations. Genes elucidated from both the methylation and expression analyses are involved in identical processes/pathways (e.g., cell cycle, apoptosis, angiogenesis, epithelial-mesenchymal cell transition, invasion/metastasis, and mitogen-activated protein kinase, transforming growth factor-beta, and Wnt signaling). Therefore, these changes might represent very early events that directly contribute to PB-induced tumorigenesis. It is instructive to consider the possibility that, in a hypothesis-driven fashion, these genes are initial candidates that could be utilized to develop a biomarker “fingerprint” of early exposure to PB and PB-like compounds.

The constitutive active/androstane receptor (CAR) mediates responses to the nongenotoxic rodent liver tumor promoter phenobarbital (PB), including certain gene expression changes, hepatomegaly, and tumor formation. Aberrant DNA methylation represents epigenetic events that can play multiple roles in tumorigenesis. Previously, 146 unique PB-induced regions of altered DNA methylation (RAMs) were observed in liver tumor-susceptible CAR wild-type (WT) mice (in 23 weeks, precancerous tissue, and 32 weeks, tumor tissue), as compared to the resistant knockout (KO). We believe that at least some of these might be key for tumorigenesis. In the current study, cloning and annotation of a subset (82%) of the unique RAMs revealed 47 genes exhibiting altered methylation; 17 are already implicated in cancer or related processes and, thus, we have identified 30 "new" candidate genes that might be involved in carcinogenesis due to an epigenetic alteration. These may contribute to tumor development through their involvement in angiogenesis, apoptosis, epithelial-mesenchymal cell transition, growth/survival, and invasion/migration/metastasis. We have also, previously, discerned unique PB-elicited RAMs in liver tumor-prone B6C3F1 mice, as compared to the relatively resistant C57BL/6 strain, at 2 or 4 weeks, and identified 51 genes exhibiting altered methylation. Importantly, 11 of these genes were identified from identical, unique RAMs discerned in both the sensitive B6C3F1 and CAR WT mice, thus representing an initial, potential candidate "fingerprint" which might serve as a biomarker for PB-induced tumorigenesis. These two studies reveal "new" genes whose epigenetic statuses changed uniquely in liver tumor-susceptible mice (B6C3F1 and CAR WT), as compared to their resistant counterparts (C57BL/6 and CAR KO, respectively), within a continuum of PB-induced tumorigenesis.

Aberrant DNA methylation plays important roles in tumorigenesis, and the nongenotoxic rodent tumor promoter phenobarbital (PB) alters methylation patterns to a greater extent in liver tumor susceptible as compared to resistant mice (Watson and Goodman, 2002). Unique hepatic regions of altered DNA methylation (RAMs) were identified in sensitive B6C3F1, as compared to resistant C57BL/6, mice at 2 or 4 weeks of PB treatment using a novel approach involving methylation-sensitive restriction digestion, arbitrarily primed PCR, and capillary electrophoresis (Bachman et al., 2006b). PCR products representing 90 of 170 (53%) total unique B6C3F1 RAMs at 2 or 4 weeks were cloned and subjected to BLAST-like alignment tool searches that resulted in 51 gene matches; some of these have documented oncogenic or tumor suppressor roles. Importantly, uniquely hypomethylated genes play roles in angiogenesis (e.g., chymase 1, tyrosine kinase nonreceptor 2, and possibly ephrin B2 and triple functional domain, PTPRF interacting) and invasion and metastasis, including those involved in the epithelial-mesenchymal transition (transcription factor 4, transforming growth factor beta receptor II, and ral guanine nucleotide dissociation stimulator). Common cellular targets and regulators of the genes representing unique B6C3F1 RAMs were uncovered, indicating that they might act in concert to more efficiently promote tumorigenesis. Genes not previously associated with mouse liver tumorigenesis exhibited altered methylation at these very early times following PB treatment. We hypothesize that at least some of the unique PB-induced B6C3F1 RAMs represent key events facilitating transformation, which is consistent with a causative role of altered DNA methylation during early stages of tumorigenesis.

To predict important strategic issues in product safety during the next 10 years, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute initiated a mapping exercise to evaluate which issues are likely to be of societal, scientific, and regulatory importance to regulatory authorities, the HESI membership, and the scientific community at large. Scientists representing government, academia, and industry participated in the exercise. Societal issues identified include sensitive populations, alternative therapies, public education on the precautionary principle, obesity, and aging world populations. Scientific issues identified include cancer testing, children's health, mixtures and co-exposures, sensitive populations, idiosyncratic reactions, "omics" or bioinformatics, and environmental toxicology. Regulatory issues identified include national and regional legislation on chemical safety, exposure inputs, new technologies, transitioning new science into regulations and guidelines, conservative default factors, data quality, and sensitive populations. Because some issues were identified as important in all three areas (e.g. sensitive populations), a comprehensive approach to assessment and management is needed to ensure consideration of societal, scientific, and regulatory implications. The resulting HESI Combined Challenges Map is not intended to offer a universal description of challenges in safety assessment, nor is it intended to address, advocate, or manage the prioritized issues. Rather, the map focuses on and predicts issues likely to be central to the strategic agendas of individual companies and regulatory authorities in the developed world. Many of these issues will become increasingly important in the future in rapidly developing economies, such as India and China. The scientific mapping exercise has particular value to the toxicology community because it represents the contributions of key scientists from around the world from government, academia, and industry.

A public appeal has been advanced by a large group of scientists, concerned that science has been misused in attempting to quantify and regulate unmeasurable hazards and risks.1 The appeal recalls that science is unable to evaluate hazards that cannot be measured, and that science in such cases should not be invoked to justify risk assessments in health, safety and environmental regulations. The appeal also notes that most national and international statutes delineating the discretion of regulators are ambiguous about what rules of evidence ought to apply. Those statutes should be revised to ensure that the evidence for regulatory action is grounded on the standards of the scientific method, whenever feasible. When independent scientific evidence is not possible, policies and regulations should be informed by publicly debated trade-offs between socially desirable uses and social perceptions of affordable precaution. This article explores the premises, implications and actions supporting the appeal and its objectives.

The HESI-led RISK21 effort has developed a framework supporting the use of twenty-first century technology in obtaining and using information for chemical risk assessment. This framework represents a problem formulation-based, exposure-driven, tiered data acquisition approach that leads to an informed decision on human health safety to be made when sufficient evidence is available. It provides a transparent and consistent approach to evaluate information in order to maximize the ability of assessments to inform decisions and to optimize the use of resources. To demonstrate the application of the framework’s roadmap and matrix, this case study evaluates a large number of chemicals that could be present in drinking water. The focus is to prioritize which of these should be considered for human health risk as individual contaminants. The example evaluates 20 potential drinking water contaminants, using the tiered RISK21 approach in combination with graphical representation of information at each step, using the RISK21 matrix. Utilizing the framework, 11 of the 20 chemicals were assigned low priority based on available exposure data alone, which demonstrated that exposure was extremely low. The remaining nine chemicals were further evaluated, using refined estimates of toxicity based on readily available data, with three deemed high priority for further evaluation. In the present case study, it was determined that the greatest value of additional information would be from improved exposure models and not from additional hazard characterization.

Large granular lymphocyte leukemia (LGLL) is a common fatal disease in aging F344 rats. The current understanding of rat LGLL and a search for mechanistic data/correlations to human leukemia were examined with the goal of improving evaluation of the LGLL endpoint in cancer bioassays as it relates to human cancer risk assessments. The exact cell of origin of the F344 rat LGLL is not fully resolved, although natural killer (NK) cell characteristics were demonstrated in most, if not all cases. Similarities between rat LGLL and a rare human NK-LGLL exist, invalidating claims of no human counterpart, although the underlying etiopathogenesis may be different. There is insufficient data to establish a mode of action of chemical-induced rat LGLL. Evaluation of the National Toxicology Program database revealed only 34 substances (out of over 500 studied) that were possibly associated with increased incidences of LGLL. Of these, only five produced definitive LGLL effects in both sexes; the remaining 29 produced single sex responses and/or only "equivocal" associations with LGLL. Trends of increasing background/variability in LGLL incidence and its modulation by extraneous factors (e.g., corn oil gavage) are key confounders in interpretation. Given that LGLL is a common tumor in control F344 rats, interpretations of bioassays can be improved by increasing the statistical stringency (e.g., p

Altered DNA methylation, an epigenetic mechanism, likely contributes to tumorigenesis, with an inverse relationship existing between methylation in a promoter region and transcription. Using the SENCAR two-stage mouse skin tumorigenesis model, altered methylation was characterized in precancerous tissue and in tumor tissue. Mouse skin was initiated with 7,12-dimethylbenz[a]anthracene and promoted three times a week with 3, 9, 18, or 27 mg cigarette smoke condensate (CSC) for 4, 8, or 29 weeks; tumors were collected at 29 weeks. In addition, reversibility of changes in methylation was assessed following cessation of the promoting stimulus. DNA was isolated, and GC-rich methylation was assessed quantitatively via methylation-sensitive restriction digestion, arbitrarily primed PCR, and electrophoretic separation of PCR products. Analysis focused on regions of altered methylation (RAMs), which persisted from 4 to 8 weeks and from 8 weeks to tumor tissue. Persistent RAMs (i.e., seen in precancerous tissue and carried forward to tumors) are likely to play a key role in tumorigenesis. Twenty-two CpG sites in the upstream region of the Ha-ras promoter were unmethylated in control skin, 27 mg CSC, and tumor tissue. At two CpG sites closer to the transcriptional start site the incidence of hypomethylation increased with the dose of CSC. Hypomethylation was detected in all tumor samples. Expression of Ha-ras increased with 18 and 27 mg CSC promotion and more so in tumor tissue. These data support our hypothesis that tumor promotion involves instability of the epigenome, providing an environment where changes in the methylation status of specific regions of the genome accumulate progressively and contribute to the clonal expansion of initiated cells that leads to tumor formation.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.

DNA methylation is an epigenetic mechanism regulating transcription, which when disrupted, can alter gene expression and contribute to carcinogenesis. Diethanolamine (DEA), a non-genotoxic alkanolamine, produces liver tumors in mice. Studies suggest DEA inhibits choline uptake and causes biochemical changes consistent with choline deficiency (CD). Rodents fed methyl-deficient diets exhibit altered methylation of hepatic DNA and an increase in liver tumors, e.g., CD causes liver tumors in B6C3F1 mice. We hypothesize that DEA-induced CD leads to altered methylation patterns which facilitates tumorigenesis. B6C3F1 hepatocytes in primary culture were grown in the presence of either 4.5 mM DEA, 3 mM Phenobarbital (PB), or CD media for 48 h. These concentrations induced comparable increases in DNA synthesis. PB, a nongenotoxic rodent liver carcinogen known to alter methylation in mouse liver, was included as a positive control. Global, average, DNA methylation status was not affected. The methylation status of GC-rich regions of DNA, which are often associated with promoter regions, were assessed via methylation-sensitive restriction digestion and arbitrarily primed PCR with capillary electrophoretic separation and detection of PCR products. DEA, PB, and CD treatments resulted in 54, 63, and 54 regions of altered methylation (RAMs), respectively, and the majority were hypomethylations. A high proportion of RAMs (72%) were identical when DEA was compared to CD. Similarly, 70% were identical between PB and CD. Altered patterns of methylation in GC-rich regions induced by DEA and PB resemble that of CD and indicate that altered DNA methylation is an epigenetic mechanism involved in the facilitation of mouse liver tumorigenesis.

The current status of the GRAS evaluation program of flavoring substances operated by the Expert Panel of FEMA is discussed. The Panel maintains a rigorous rotating 10-year program of continuous review of scientific data related to the safety evaluation of flavoring substances. The Panel concluded a comprehensive review of the GRAS (GRASa) status of flavors in 1985 and began a second comprehensive review of the same substances and any recently GRAS materials in 1994. This second re-evaluation program of chemical groups of flavor ingredients, recognized as the GRAS reaffirmation (GRASr) program, is scheduled to be completed in 2005. The evaluation criteria used by the Panel during the GRASr program reflects the significant impact of advances in biochemistry, molecular biology and toxicology that have allowed for a more complete understanding of the molecular events associated with toxicity. The interpretation of novel data on the relationship of dose to metabolic fate, formation of protein and DNA adducts, enzyme induction, and the cascade of cellular events leading to toxicity provides a more comprehensive basis upon which to evaluate the safety of the intake of flavor ingredients under conditions of intended use. The interpretation of genotoxicity data is evaluated in the context of other data such as in vivo animal metabolism and lifetime animal feeding studies that are more closely related to actual human experience. Data are not viewed in isolation, but comprise one component that is factored into the Panel's overall safety assessment. The convergence of different methodologies that assess intake of flavoring substances provides a greater degree of confidence in the estimated intake of flavor ingredients. When these intakes are compared to dose levels that in some cases result in related chemical and biological effects and the subsequent toxicity, it is clear that exposure to these substances through flavor use presents no significant human health risk.

This publication is the eighth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of benzyl derivatives as flavoring ingredients is evaluated. The group of benzyl derivatives was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals, their low level of flavor use, the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential. This evidence of safety is supported by the fact that the intake of benzyl derivatives as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

This publication is the ninth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of phenethyl alcohol, aldehyde, acid, and related acetals and esters as flavoring ingredients is evaluated. The group of phenethylalcohol, aldehyde, acid, and related acetals and esters was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food, their rapid absorption, metabolic detoxication, and excretion in humans and other animals, their low level of flavor use, the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential. This evidence of safety is supported by the fact that the intake of phenethyl alcohol, aldehyde, acid, and related acetals and esters as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

It is necessary to determine whether chemicals or drugs have the potential to pose a threat to human health. Research conducted over the last two decades has led to the paradigm that chemicals can cause cancer either by damaging DNA or by altering cellular growth, probably via receptor-mediated changes in gene expression. However, recent evidence suggests that gene expression can be altered markedly via several diverse epigenetic mechanisms that can lead to permanent or reversible changes in cellular behavior. Key molecular events underlying these mechanisms include the alteration of DNA methylation and chromatin, and changes in the function of cell surface molecules. Thus, for example, DNA methyltransferase enzymes together with chromatin-associated proteins such as histone modifying enzymes and remodelling factors can modify the genetic code and contribute to the establishment and maintenance of altered epigenetic states. This is relevant to many types of toxicity including but not limited to cancer. In this paper, we describe the potential for interplay between genetic alteration and epigenetic changes in cell growth regulation and discuss the implications for drug discovery and safety assessment.

DNA methylation is an epigenetic mechanism regulating patterns of gene expression. Our goal was to see if the assessment of DNA methylation might be a useful tool, when used in conjunction with initial, basic in vitro tests, to provide a more informative preliminary appraisal of the toxic potential of chemicals to prioritize them for further evaluation. We sought to give better indications of a compound's toxic potential and its possible mechanism of action at an earlier time and, thereby, contribute to a rational approach of an overall reduction in testing by making improved early decisions. Global and GC-rich patterns of DNA methylation were evaluated along with more traditional cytolethality measurements, e.g., cytolethality and genotoxicity assessments, on rat hepatoma (H4IIE) cells. The relative toxic potential of model compounds camptothecin, 5-fluorouracil, rotenone, and staurosporine was estimated by employing DNA methylation assessments combined with our cytolethality data plus genotoxicity information gleaned from the literature. The overall contribution of the methylation assessment was threefold; it (1) strengthened a ranking based on genotoxicity; (2) provided an indication that a compound might be more potentially problematic than what cytolethality and genotoxicity assessments alone would indicate; and (3) suggested that compounds, particularly nongenotoxins, that are more potent regarding their ability to alter methylation, especially at noncytolethal concentrations, may be more potentially toxic. Altered methylation per se is not proof of toxicity; this needs to be viewed as a component of an evaluation.

The Alternatives to Carcinogenicity Testing Committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) conducted a large-scale, multinational collaborative research program to evaluate several genetically modified mouse assays for assessing the human carcinogenic potential of compounds. The data from this testing program have made an important contribution to the general understanding of how these models can be best applied in hazard identification; however, questions still exist regarding methodology and data interpretation. To address these issues, ILSI HESI hosted a February 2003 workshop on the Utility of Transgenic Assays for Risk Assessment. The purpose of this workshop was to reach an understanding of how data from genetically modified mouse models are viewed by different regulatory bodies in the pharmaceutical sector and, based on this understanding, to identify areas in which more experimental work may be needed to increase the utility of data derived from these assays. In the course of discussions, various data gaps related to model selection and protocol issues were identified. Based on the outcome of the workshop, various studies are proposed to provide data to improve the utility of currently available assays for cancer hazard identification and risk assessment purposes.

Promoter-region DNA methylation inhibits transcription. A two-stage SENCAR (sensitive to mouse carcinogenesis) mouse skin carcinogenicity model was used to examine gene-specific changes in methylation during skin tumor promotion. Analysis was performed on 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin promoted with 9, 18, 27, or 36 mg cigarette smoke condensate (CSC) for 9 wk, or 27 mg CSC for 9 wk and sacrificed 6 wk afterwards (recovery group). Additionally, tumors arising following promotion with 27 mg CSC for 29 wk were assessed. Gene array analysis identified differentially expressed genes. Expression of HoxA5, a tumor suppressor gene, was decreased following 9 wk of treatment with 27 mg CSC, and returned to control levels during recovery. HoxA5 promoter methylation was measured with the enzymatic regional methylation assay (ERMA). DNA was bisulfite-modified, PCR-amplified with primers containing dam sites, incubated with [14C-methyl] S-adenosyl-l-methionine (SAM) and dam methyltransferase for DNA quantification, then incubated with [3H-methyl] SAM and SssI methylase to quantify methylation status. Higher 3H/14C ratios indicate increased methylation. The 3H/14C ratios of animals promoted with 27 or 36 mg CSC (48.2 ± 6.9 and 24.2 ± 6.1, respectively) were higher than the control or recovery group ratios (12.3 ± 0.1 and 12.6 ± 0.3, respectively); sequence analysis supported these findings. Increased methylation of p16 or O6 methylguanine methyltranferase (MGMT) was detected in 4/8 (50%) of the tumor samples from mice promoted with 27 mg CSC. These data suggest that increased DNA methylation contributes to the downregulation of HoxA5, and combined with hypermethylation of p16 or MGMT, this might facilitate the clonal expansion of increasingly aberrant cells during promotion. © 2004 Wiley-Liss, Inc.

published

It is necessary to determine whether chemicals or drugs have the potential to pose a threat to human health. Chemicals that can damage DNA are detected in short-term assays, but the detection of nongenotoxic carcinogens relies upon bioassays in laboratory animals. However, there are marked differences between rodents and humans in response to nongenotoxic carcinogens, which makes the relevance of rodent data to human risk assessment questionable. Here, we address the background issues concerning rodent nongenotoxic carcinogenesis and then focus upon peroxisome proliferators, chloroform, and dioxins as examples of toxicants that cause rodent-specific oxidative stress, cell proliferation, and the suppression of apoptosis. In the case of peroxisome proliferators and dioxins, this response is receptor-mediated. The evidence presented suggests that, at least for some toxicants, the molecular mechanisms of the rodent carcinogenic responses do not operate in humans; this is discussed in the context of human risk assessment. Finally, consideration is given to incorporating mechanism-based information into risk assessment for regulatory purposes.

DNA methylation is an important epigenetic mechanism involved in transcriptional control and altered patterns of methylation may lead to the aberrant gene expression contributing to carcinogenesis. Three groups of mice were used in the current study: the relatively liver-tumor-sensitive C3H/He strain and B6C3F1 stock (C57BL/6xC3H/He), as well as the relatively resistant C57BL/6 strain. For a 2-week period, animals from each group were given drinking water containing a tumor-promoting dose of phenobarbital (PB), a nongenotoxic rodent carcinogen. Methylation-sensitive restriction digests using HpaII or MspI were followed by PCR amplification using an arbitrary primer or primer pair, binding preferentially to guanine and cytysine (GC)-rich regions of DNA, including CpG islands. This procedure allows for assessment of methylation at the internal and/or external cytosine of the 5'-CCGG-3' sites recognized by MspI and HpaII. Results with the single primer indicated marked differences in PB-induced hypermethylation at external and internal cytosines of 5'-CCGG-3' sites: C3H/He >> B6C3F1 > C57BL/6. Results with the arbitrary primer pair indicated PB-induced hypermethylation at the external cytosine of 5'-CCGG-3' site: B6C3F1 > C3H/He, and a low level of hypomethylation at internal and external cytosine sites in C57BL/6. Thus, there was a clear indication of more methylation changes in GC-rich regions of DNA, primarily hypermethylation, in the tumor-sensitive groups of mice in response to PB treatment. Therefore, this study supports our hypothesis that the capacity to maintain normal methylation patterns is related inversely to tumor susceptibility.

A wide variety of chemical and physical agents have the potential to produce adverse effects by causing heritable changes to the genome, resulting in heritable alterations in phenotype. These are often assumed to be a consequence of mutation. However, mutagenesis is not the only mechanism underlying heritable alterations to the genome. It is important to understand that there may also be an epigenetic basis for this. DNA methylation is the epigenetic mechanism that this review focuses upon. We indicate how altered methylation may play a key role in a variety of chemical-induced toxicities, including, but not limited to, carcinogenesis, and we point out how an assessment of methylation status can provide important information as a component of an overall safety assessment.

This review focuses on the role that DNA methylation plays in the regulation of normal and aberrant gene expression and on how, in a hypothesis-driven fashion, altered DNA methylation may be viewed as a secondary mechanism involved in carcinogenesis. Research aimed at discerning the mechanisms by which chemicals can transform normal cells into frank carcinomas has both theoretical and practical implications. Through an increased understanding of the mechanisms by which chemicals affect the carcinogenic process, we learn more about basic biology while, at the same time, providing the type of information required to make more rational safety assessment decisions concerning their actual potential to cause cancer under particular conditions of exposure. One key question is: does the mechanism of action of the chemical in question involve a secondary mechanism and, if so, what dose may be below its threshold?

This is the fifth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of pyrazine derivatives as flavoring ingredients is evaluated.

Octamethylcyclotetrasiloxane (D4) has been described as a phenobarbital-like inducer of hepatic enzymes. Phenobarbital (PB) and phenobarbital-like chemicals induce transient hepatic and thyroid hyperplasia and sustained hypertrophy in rats and mice. The extent to which these processes are involved with D4-induced hepatomegaly is not known. The present study has evaluated the effects of repeated inhalation exposure to D4 vapors on hepatic and thyroid cell proliferation and hypertrophy with respect to time and exposure concentration. Female Fischer 344 rats were exposed via whole body inhalation to 0 ppm D4, 700 ppm D4 vapors (6 h/day; 5 days/week), or 0.05% PB in drinking water over a 4-week period. Incorporation of 5'-bromo-2-deoxyuridine (BrdU) and the abundance of proliferating cell nuclear antigen were used as indicators of cell proliferation. Designated animals from each treatment group were euthanized on study days 6, 13, and 27. The effect of D4 exposure concentration on hepatic cell proliferation was evaluated at 0, 7, 30, 70, 150, 300, or 700 ppm. Liver-to-body weight ratios in animals exposed to 700 ppm D4 were increased 18, 20, and 22% over controls while PB-treated animals showed increases of 33, 27, and 27% over controls on days 6, 13, and 27 respectively. Hepatic incorporation of BrdU following exposure to D4 was highest on day 6 (labeling index = 15-22%) and was at or below control values by day 27. This pattern of transient hyperplasia was observed in all hepatic lobes examined and was similar to the pattern observed following treatment with PB.

This perspective is based upon the data presented at the International Life Sciences Institute (ILSI), Health and Environmental Sciences Institute Workshop on the Evaluation of Alternative Methods for Carcinogenicity Testing (ILSI Workshop). It is important to understand that all models discussed at the Workshop have limitations and that they are not designed to be employed as stand-alone assays. Although they may have other, appropriate applications, I do not recommend use of the SHE cell assay and the Tg.AC model for the regulatory purposes of a safety assessment. In my view, the neonatal mouse, p53 +/- , XPA -/- , XPA-/- and p53 +/-, and the rasH2 models can, as a component of an overall assessment, provide information on potential carcinogenicity of a chemical that is appropriate for consideration in a regulatory context. Generally, these models exhibit the ability to detect genotoxic compounds. In most cases these compounds would be detected in a standard battery of genotoxicity tests and, therefore, quite often the use of an alternative is not necessary. Actually, I believe that a bioassay in rats will suffi ce most of the time, that is, in my view, a routine bioassay in mice is not necessary. Specifi c circumstances where data obtained from one of the “recommended” alternative models might be helpful are discussed. With regard to lessons for the future, there is a particular need for models that are responsive to chemicals that exhibit a nongenotoxic mode of action. Additionally, new models will continue to be developed and their half-life will likely be substantially shorter than the time required for traditional validation. The development of enhanced paradigms for validation should be a priority so that improved safety assessment decisions can be made more quickly. However, while evaluating and validating such models, it is important to consider the fundamental issues, for example, rational dose selection, evaluation of mode of action in the context of dose-response relationships including the existence of thresholds and secondary mechanisms, and species-to-species extrapolation. The alternatives to carcinogenicity testing project was a very major undertaking. In addition to the valuable information provided, it serves to illustrate the value of cooperation between academia, government, and industry. Furthermore, the involvement of the International Life Sciences Institute as the overall organizing, facilitating umbrella was crucial for the success of the project.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

DNA methylation (DNA-5-methylcytosine [MeC]) plays a key role in regulation of gene expression. Highly methylated genes tend to be silenced whereas hypomethylated genes have an increased potential for expression. One way in which methylation may lead to inhibition of transcription is by permitting the binding of methylated DNA-binding proteins, which then block access of transcription factors to DNA. A particular methylated DNA-binding protein, MDBP-1, has been identified in nuclear extracts prepared from various human and rat tissues, and binds in a sequence-specific manner to DNA containing methylated cytosines (P. C. Supakar et al., 1988, Nucleic Acids Res. 16, 8029-8044). In the current study, an electrophoretic mobility shift assay (EMSA) was employed to assess the presence of MDBP-1 in protein isolated from mouse liver and to examine characteristics of its binding to DNA. The ligand used in our EMSA was a 32P-end-labeled, double-stranded, symmetrically methylated oligonucleotide containing the protein's binding site, 5'-CTAGATMGT-CAMGGMGAT-3' (M denotes 5-methyl-cytosine). Utilizing protein extracted from B6C3F1 mouse liver, we observed a complex that is competed by non-labeled, double-stranded, fully methylated ligand but not by an excess of a 26-mer, double-stranded oligonucleotide containing the binding site for AP-1. No complex was formed when a nonmethylated, double-stranded ligand, or our single-stranded ligands (methylated or unmethylated) were used as EMSA ligands. Complex formation was observed utilizing double-stranded, hemimethylated ligands; however, the affinity of MDBP-1 for these was one-tenth the affinity of MDBP-1 for fully methylated, double-stranded ligand. Additionally, protein isolated from C57BL/6 and C3H/He mouse liver was found to bind specifically to fully methylated ligand and hemimethylated ligands for MDBP-1, with similar characteristics as the binding of B6C3F1 mouse liver protein. These results indicate that MDBP-1 is present in mouse liver, and that the degree of methylation determines the strength of binding of MDBP-1 to DNA.

Dose influences mechanism; and over a wide range of doses, one can envision that mechanism will change with changing dose. This basic concept in toxicology is juxtaposed with the biologic importance of maintaining normal DNA methylation status to provide the focus of this paper. The idea that altered DNA methylation plays a variety of roles in carcinogenesis is compatible with three key features of this multistage process: clonal selection of abnormal cells in a progressive fashion, the reversibility of tumor promotion, and the multiplicity of tumor phenotypes. A relatively low capacity to maintain normal methylation status appears to explain, in part, the high propensity of the B6C3F1 mouse to develop liver tumors. This observation supports the view that a mouse liver tumor response is not an appropriate end point for human risk assessment. Additionally, it is suggested that altered DNA methylation can be viewed as a secondary mechanism underlying carcinogenesis. The knowledge that a chemical is acting by a mode of action involving a secondary mechanism can be used to support a safety factor or multiplicity of exposure approach to risk assessment.

Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and β-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesis.

The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA) has assessed the safety of furfural for its continued use as a flavour ingredient. The safety assessment takes into account the current scientific information on exposure, metabolism, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. Furfural was reaffirmed as GRAS (GRASr) as a flavour ingredient under conditions of intended use based on: (1) its mode of metabolic detoxication in humans; (2) its low level of flavour use compared with higher intake levels as a naturally occurring component of food; (3) the safety factor calculated from results of subchronic and chronic studies, (4) the lack of reactivity with DNA; and (5) the conclusion that the only statistically significant finding in the 2-year NTP bioassays, an increased incidence of hepatocellular adenomas and carcinomas in the high-dose group of male mice, was secondary to pronounced hepatotoxicity. Taken together, these data do not indicate any risk to human health under conditions of use as a flavour ingredient. This evidence of safety is supported by the occurrence of furfural as a natural component of traditional foods, at concentrations in the diet resulting in a 'natural intake' that is at least 100 times higher than the intake of furfural from use as a flavour ingredient.

Modifications to DNA-5-methylcytosine (5MeC) content (i.e., alterations in the level of 5MeC) constitute epigenetic events. In general, hypomethylation of a gene is necessary but not sufficient for expression, while methylated genes typically are quiescent. Ha-ras is an oncogene commonly implicated in murine liver tumorigenesis, often, though not always, involving mutation. A PCR-based approach using pre-PCR digestion with methylation-sensitive enzymes was employed to determine the 5MeC content of the 5' flanking region of this gene in (i) B6C3F 1 and C57BU6 mouse liver from young animals (4 months old) and (ii) B6C3F, mouse liver from aged animals (24 months old). Two segments of the 5' flanking region of Ha-ras were examined. We demonstrate the presence of 5MeC in a portion of the 5' flanking region of Ha-ras that does not share characteristics of a CpG island, while a region that shares CpG island characteristics is primarily unmethylated. Differences in methylation status in these areas of Ha-ras were not observed between B6C3F 1 and C57BU6 mouse livers. Increases in methylation status were observed with ageing in B6C3F, mouse liver. These data provide a role for methylation in regulating Ha-ras expression in mouse liver. Ha-ras in human liver has been reported to be unmethylated. There are substantial sequence differences in a key region of the 5' flanking region of Ha-ras in mice as compared to humans. These differences in DNA methylation and sequence may, in part, provide a basis for the frequent involvement of Ha-ras in mouse liver tumors and its virtual lack of involvement in human tumors.

A workshop entitled “Implementation of EPA Revised Cancer Assessment Guidelines: Incorporation of Mechanistic and Pharmacokinetic Data” was held in Anaheim, California, in 1996 at the 35th Annual Meeting of the Society of Toxicology (SOT). This workshop was jointly sponsored by the Carcinogenesis, Risk Assessment, and Veterinary Specialty Sections of the SOT. The thrust of the workshop was to discuss the scientific basis for the revisions to the EPA Guidelines for cancer assessment and EPA's plans for their implementation. This is the first revision to the original EPA guidelines which have been in use by EPA since 1986. The principal revisions are intended to provide a framework for an increased ability to incorporate biological data into the risk assessment process. Two cases were presented, for chloroform and trichloroethylene, that demonstrated the use of the revised guidelines for specific cancer risk assessments. Using these new guidelines, nonlinearmargin of exposureanalyses were proposed for these chemicals instead of thelinearized multistage modelpreviously used by the EPA as the default method. The workshop participants generally applauded the planned revisions to the EPA guidelines. For the most part, they considered that the revised guidelines represented a positive step which should allow for and encourage the use of biological information in the conduct of cancer risk assessments. Several participants cautioned however that the major problem with cancer risk assessments would continue to be the inadequacy of available data on which to conduct more scientific risk assessments.

The effects of different liver tumor-promoting treatments (i.e., a choline-devoid, methionine-deficient (CMD) diet, phenobarbital (PB), or both) on Ha-ras and raf methylation status and expression were determined in mouse strains with different susceptibilities to liver tumor formation: the relatively sensitive B6C3F1 and the relatively resistant C57BL/6. Additionally, B6C3F1 mouse liver tumors, spontaneous or PB induced, were assessed for alterations in global DNA methylation status and expression of Ha-ras and raf. The CMD diet led to hypomethylation of Ha-ras and raf after 12 wk of administration in B6C3F1 and C57BL/6 mice. At this early phase of tumor promotion, the frequency of increased expression of both Ha-ras and raf mRNAs was higher in the B6C3F1 but not the C57BL/6 mice. This is a mechanism that may, in part, underlie the heightened sensitivity of the B6C3F1 mouse to liver tumorigenesis. Subpopulations of B6C3F1 mouse liver tumors displayed altered global methylation status, with both hypomethylation and hypermethylation evident. Carcinomas were significantly more hypomethylated than adenomas. The level of raf mRNA was not changed in spontaneous or PB-induced B6C3F1 mouse liver tumors. Increased expression of Ha-ras was evident in some spontaneous B6C3F1 liver tumors and in most of the PB-induced liver tumors. These experiments support the concept that altered DNA methylation plays a key role in tumorigenesis and indicate that the high propensity of the B6C3F1 mice to liver tumorigenesis may be due, in part, to a decreased ability to maintain normal methylation status.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

A workshop entitled "NTP Studies: Principles of Dose Selection and Applications to Mechanistic Based Risk Assessment" was held at the 34th Annual Meeting of the Society of Toxicology in Baltimore, Maryland. The purpose of the workshop was to provide an overview of factors currently considered important in the selection of doses for NTP studies, to describe some of the confounding factors that can result from the indiscriminate use of bioassay data in quantitative risk assessment, and to suggest ways in which information from mechanistic studies or studies of biomarkers of exposure or effect might be used to better advantage in risk assessment.

The molecular events during nongenotoxic carcinogenesis and their temporal order are poorly understood but thought to include long-lasting perturbations of gene expression. Here, we have investigated the temporal sequence of molecular and pathological perturbations at early stages of phenobarbital (PB) mediated liver tumor promotion in vivo. Molecular profiling (mRNA, microRNA [miRNA], DNA methylation, and proteins) of mouse liver during 13 weeks of PB treatment revealed progressive increases in hepatic expression of long noncoding RNAs and miRNAs originating from the Dlk1-Dio3 imprinted gene cluster, a locus that has recently been associated with stem cell pluripotency in mice and various neoplasms in humans. PB induction of the Dlk1-Dio3 cluster noncoding RNA (ncRNA) Meg3 was localized to glutamine synthetase-positive hypertrophic perivenous hepatocytes, suggesting a role for β-catenin signaling in the dysregulation of Dlk1-Dio3 ncRNAs. The carcinogenic relevance of Dlk1-Dio3 locus ncRNA induction was further supported by in vivo genetic dependence on constitutive androstane receptor and β-catenin pathways. Our data identify Dlk1-Dio3 ncRNAs as novel candidate early biomarkers for mouse liver tumor promotion and provide new opportunities for assessing the carcinogenic potential of novel compounds.

Propiconazole (PPZ) is a conazole fungicide that is not mutagenic, clastogenic, or DNA damaging in standard in vitro and in vivo genetic toxicity tests for gene mutations, chromosome aberrations, DNA damage, and cell transformation. However, it was demonstrated to be a male mouse liver carcinogen when administered in food for 24 months only at a concentration of 2,500 ppm that exceeded the maximum tolerated dose based on increased mortality, decreased body weight gain, and the presence of liver necrosis. PPZ was subsequently tested for mutagenicity in the Big Blue® transgenic mouse assay at the 2,500 ppm dose, and the result was reported as positive by Ross et al. ([2009]: Mutagenesis 24:149-152). Subsets of the mutants from the control and PPZ-exposed groups were sequenced to determine the mutation spectra and a multivariate clustering analysis method purportedly substantiated the increase in mutant frequency with PPZ (Ross and Leavitt. [2010]: Mutagenesis 25:231-234). However, as reported here, the results of the analysis of the mutation spectra using a conventional method indicated no treatment-related differences in the spectra. In this article, we re-examine the Big Blue® mouse findings with PPZ and conclude that the compound does not act as a mutagen in vivo. © Environ. Mol. Mutagen. 2012. Published 2011 Wiley Periodicals, Inc.