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Mechanisms of Mutagenicity and Carcinogenicity
Genetic Toxicology Association Spring Meeting
Thursday, May 17, 2006
Clayton Hall Conference Center, University of Delaware

Analyses of the Performance of InVitro Genotoxicity Tests to Predict Rodent Carcinogenicity

Marilyn Aardema (Procter & Gamble)

In the 1980-90's the NTP evaluated the performance of in vitro genotoxicity tests for predicting rodent carcinogenicity. Since these early analyses were on limited numbers of chemicals, we undertook an extensive evaluation of the battery of the 3 most commonly used in vitro genotoxicity tests - Ames + mouse lymphoma assay (MLA) + in vitro micronucleus (MN) or chromosomal aberrations (CA) test - for its ability to discriminate rodent carcinogens and non-carcinogens, from a database of over 700 chemicals compiled from the CPDB ("Gold"), NTP, IARC and other publications. Of the 554 carcinogens, 93% gave positive results in at least 1 of the 3 tests. Unfortunately, 75-95% of non-carcinogens also gave positive results in at least one test in the battery. This extremely low specificity highlights the importance of understanding the mechanism by which genotoxicity is induced in order to determine whether a genotoxic effect is relevant for the whole animal or human, which is the subject of this meeting. This low specificity also highlights the importance of considering whether it is time for new approaches for predicting carcinogenicity.

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Role of Topoisomerase Inhibition in Clastogenicity of Non-Structurally Alerting Drugs and Chemica

Ron Snyder (Shering Plough Research Institute)

We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. This presentation will review the application of a V79 cell-based model and a 3-D DNA docking model for predicting non-covalent chemical/DNA interactions. Results obtained from these approaches suggest that very widely structurally-diverse molecules may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (45%) clastogenic drugs were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction. Furthermore, data will be presented suggesting that genotoxicity arising from DNA intercalators most likely is a consequence of inhibition of DNA topoisomerases.

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Point Mutation as a Mechanism of Antimicrobial Resistance: A Focus on Fluorquinolones

Steve Yan (US FDA)

Antimicrobial resistance has become an increasing medical and public health concern. Bacteria can develop resistance to virtually every available antimicrobial drug. There are three basic routes that microorganisms may use to develop resistance to an antimicrobial drug: 1) inactivation of the drug; 2) reduction in the drug permeability through cell membranes; and 3) modification of drug targets. Point mutation of relevant genes may be implicated in each of these approaches; therefore, they may contribute, as a mechanism, to resistance development. For example, point mutations in TEM-1 beta-lactamase greatly increase its enzymatic activity and spectrum, which is one of the mechanisms of resistance for the extended spectrum beta-lactam drugs in some of enteric bacteria. Modification through point mutations in the 23S rRNA has also been found to be responsible for resistance to macrolides. Point mutation as a mechanism of resistance may be best presented in the case of fluoroquinolone resistance. Fluoroquinolones act upon inhibition of DNA topoisomerases II (gyrase) and IV, both of which are essential during bacterial DNA replication. Point mutations in the quinolone resistance-determining region of the genes correlate well with reduced susceptibility in a variety of bacterial species. In the presentation, the role of point mutations is explored through a case report of multiple fluoroquinolone-resistant Streptococcus pyogenes and through follow-up results. The possible relationship between fluoroquinolone exposure and point mutation selection is also discussed. Improving rapid molecular detection methods to identify key point mutations in resistant clinical isolates will benefit patient care and public health.

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Framework for Assessing Mode of Action and Human Relevance of Animal Tumors

Vicki Dellarco (US EPA)

A carcinogenic mode of action (MOA) is defined as a biologically plausible sequence of key events that are obligatory and quantifiable steps leading to the development of tumors. Several years ago EPA and the International Programme of Chemical Safety (IPCS) published similar analytical frameworks (in 1999 and 2001, respectively) for evaluating a chemical’s proposed cancer MOA. The IPCS and EPA Framework put forth the concept that it is sometimes possible to establish a series of key events that are along the causal path for an induced tumor by using criteria based on those described by Bradford Hill for epidemiologic investigations (i.e., taking account of factors such as dose-response and temporal concordance, biological plausibility, coherence and consistency). In 2003, the International Life Sciences Institute Risk Science Institute (ILSI RSI) published a Human Relevance Framework, which extended the EPA and IPCS cancer MOA framework to provide additional guidance on how to evaluate the evidence that the same mode of action will occur in humans. The ILSI/RSI HRF is essentially a two-step process: (1) evaluation of the weight of evidence for the proposed MOA in the test species (typically rodents), and once an MOA is established, qualitative and quantitative comparisons of each key event are made between the experimental animal and humans to enable a conclusion as to the likely relevance of the animal MOA for human risk. Recently, IPCS updated its 2001 MOA framework based on experience gained and considered the 2003 ILSI/RSI human cancer relevance framework to produce a unified Human Cancer Relevance Framework (IPCS HRF). This HRF provides a rigorous and transparent approach for determining the sufficiency of evidence and the relevance of an animal MOA for humans. The concept of mode of action (MOA) has permitted information on critical precursor events to be incorporated in the assessment of potential risk and has lead to a better scientific basis for characterizing human relevance and the dose response of animal tumor responses. A case study on organic arsenic will be presented to illustrate the usefulness of the Human Relevance Framework.

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An Evaluation of the Mode of Action Framework for Mutagenic Carcinogens Case Study: Cyclophosphamide

Nancy McCarrol (US EPA)

In response to the 2005 revised U.S EPA Cancer Guidelines, a Risk Assessment Forum's Technical Panel devised a strategy in which genetic toxicology data combined with other information are assessed to determine whether a carcinogen operates through a mutagenic mode of action (MOA). This information is necessary for EPA to decide whether the age-dependent adjustment factors (ADAFs) should be applied to the cancer risk assessment. The decision tree, developed as part of this approach, outlines the critical steps for analysis of a mutagenic MOA (e.g., data analysis, determination of mutagenicity in animals and in humans). Agents satisfying these criteria proceed through the Agency’s framework analysis for MOAs. Cyclophosphamide (CP), an antineoplastic agent, which is carcinogenic in animals and humans and mutagenic in vitro and in vivo, was selected as a case study. There were consistent positive results for mutagenic activity in numerous in vitro assays. Similarly, there was evidence of mutagenicity in animals (mice, rats, and hamsters) and in humans (adult and pediatric patients, hospital workers, and workers in industrial settings). CP is being processed through the framework analysis and it has been found that the key steps leading to tumor formation may be: metabolism to the main alkylating metabolites, DNA damage, cytotoxicity and/or induction of multiple adverse genetic events, cell proliferation, and bladder tumors. Genetic changes in rats (SCE induction in bone marrow at 0.62 mg/kg) can commence within 30 minutes and in cancer patients (chromosome aberrations and SCEs at 35 mg/kg) are seen by 1 hour, well within the timeframe and tumorigenic dose range for early events. Supporting evidence is also found for cytotoxicity and cell proliferation, indicating that mutagenicity and cytotoxicity leading to a proliferative response occur early (48 hours) in the process of tumor induction. No convincing data were found that an alternative MOA other than mutagenicity may be operative. CP was designated as carcinogenic by IARC in 1981 and there is convincing evidence that it operates through a mutagenic MOA. Other evidence also shows chromosome breakage in pediatric noncancer patients (2-17 years) and micronucleated polychromatic erythrocytes in mouse fetal blood. If the weight of the evidence supports a mutagenic MOA, the Cancer Guidelines recommend a linear extrapolation for the risk assessment; additionally, the ADAFs would be applied. Note: This case study does not represent a regulatory decision by the EPA. It attempts to demonstrate a proposed process by which chemicals can be evaluated.

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Low-Dose Extrapolation in Various Fields of Toxicology

Ajit Thakur (Covance)

Low-dose extrapolation of effects of chemicals from animal experiments goes under several different names in toxicological literature. For example, probable carcinogenic responses in life-time mouse and rat studies may be fitted with empirical mathematical models to extrapolate to extremely small dose levels for estimating acceptable doses that do not exceed more than one-in-a-million cancer risk in human. In the usual bioassay terminology, this is called carcinogenic risk assessment. There are many noncarcinogenic responses in toxicological battery, such as teratology, reproductive toxicology, etc. that may require similar low-dose extrapolation exercises. Depending on the discipline, such exercises may be called bench mark dose (BMD) computation, ED01 estimation, virtual safe dose (VSD) computation, etc. Once again, these exercises are accomplished using similar empirical models as in cancer risk assessment with transformation of experimental data in different domains. Additionally, non-cancer models involving either linear or nonlinear functions of various types are also used for these circumstances. Irrespective of the model or models used, the mathematical techniques used all have certain characteristics that may not be ideal for this purpose. Almost invariably, the computation involves linear, or occasionally nonlinear, numerical techniques. All these extrapolation techniques may have high levels of uncertainty and numerical instability. Moreover, some of these mathematical functions may not necessarily be appropriate for extrapolation beyond or within data range. We will briefly discuss some of the above issues from several areas of toxicology to elicit the above points with real examples from several areas of toxicology.

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The Role of Pharmacology and Off-Target Pharmacology in the High Frequenqy of Aneuploidy Findings with Inhibitors of the Protein Kinase Gene Family

Maik Schuler (Pfizer)

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Carcinogenic Impurities in Color Additives

Arthur Lipman (FDA/CFSAN)

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Issues Related to Genotoxic Impurities in Drug Products

Tim McGovern (FDA/CDER)

Genotoxic impurities in drug substances and products can have an impact on pharmaceutical drug development and product approval due to their potential for being carcinogenic to humans. This presentation will provide a regulatory perspective related to genotoxic impurities. In general, a suspected or known genotoxic impurity should either be removed or reduced to a level that conveys no significant increase in cancer risk. The determination of a safe level could be based upon various parameters including, but not necessarily limited to, characterization of the genotoxic profile, structural activity relationships (SARs), risk assessment based on relevant carcinogenicity data, threshold approaches based on appropriate databases, and the proposed use of the drug. An overall safety assessment should consider the relative benefit to be derived from a given drug product versus the risk conveyed by the presence of a genotoxic impurity.

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