An evaluation of the mode of action framework for mutagenic carcinogens case study: Cyclophosphamide.

In response to the 2005 revised US Environmental Protection Agency (EPA) Cancer Guidelines, a Risk Assessment Forum's Technical Panel has 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 age-dependent adjustment factors (ADAFs) should be applied to the cancer risk assessment. A decision tree has been developed as a part of this approach and outlines the critical steps for analyzing a compound for carcinogenicity through a mutagenic MOA (e.g., data analysis, determination of mutagenicity in animals and in humans). Agents, showing mutagenicity in animals and humans, 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 to illustrate how the framework analysis would be applied to prove that a carcinogen operates through a mutagenic MOA. Consistent positive results have been seen for mutagenic activity in numerous in vitro assays, in animals (mice, rats, and hamsters) and in humans. Accordingly, CP was processed through the framework analysis and key steps leading to tumor formation were identified as follows: metabolism of the parent compound to alkylating metabolites, DNA damage followed by induction of multiple adverse genetic events, cell proliferation, and bladder tumors. Genetic changes in rats (sister chromatid exchanges at 0.62 mg/kg) can commence within 30 min and in cancer patients, chromosome aberrations at 35 mg/kg are seen by 1 hr, well within the timeframe and tumorigenic dose range for early events. Supporting evidence is also found for cell proliferation, indicating that mutagenicity, associated with cytotoxicity, leads to a proliferative response, which occurs early (48 hr) in the process of tumor induction. Overall, the weight of evidence evaluation supports CP acting through a mutagenic MOA. In addition, no data were found that an alternative MOA might be operative. Therefore, the cancer guidelines recommend a linear extrapolation for the risk assessment. Additionally, data exist showing that CP induces mutagenicity in fetal blood and in the peripheral blood of pediatric patients; thus, the ADAFs would be applied.

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim.

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data. Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.