Federica Madia

The 10 key characteristics (KCs) of carcinogens form the basis of a framework to identify, organize, and evaluate mechanistic evidence relevant to carcinogenic hazard identification. The 10 KCs are related to mechanisms by which carcinogens cause cancer. The International Agency for Research on Cancer (IARC) Monographs programme has successfully applied the KCs framework for the mechanistic evaluation of different types of exposures, including chemicals, metals, and complex exposures, such as environmental, occupational, or dietary exposures. The use of this framework has significantly enhanced the identification and organization of relevant mechanistic data, minimized bias in evaluations, and enriched the knowledge base regarding the mechanisms of known and suspected carcinogens. We sought to report the main outcomes of an IARC Scientific Workshop convened by the IARC to establish appropriate, transparent, and uniform application of the KCs in future IARC Monographs evaluations. A group of experts from different disciplines reviewed the IARC Monographs experience with the KCs of carcinogens, discussing three main themes: a) the interpretation of end points forming the evidence base for the KCs, b) the incorporation of data from novel assays on the KCs, and c) the integration of the mechanistic evidence as part of cancer hazard identification. The workshop participants assessed the relevance and the informativeness of multiple KCs-associated end points for the evaluation of mechanistic evidence in studies of exposed humans and experimental systems. Consensus was reached on how to enhance the use of in silico, molecular, and cellular high-output and high-throughput data. In addition, approaches to integrate evidence across the KCs and opportunities to improve methodologies of mechanistic evaluation of cancer hazards were explored. The findings described herein and in a forthcoming IARC technical report will support future working groups of experts in reporting and interpreting results under the KCs framework within the IARC Monographs or in other contexts. https://doi.org/10.1289/EHP15389.

With recent rapid advancement of methodological tools, mechanistic understanding of biological processes leading to carcinogenesis is expanding. New approach methodologies such as transcriptomics can inform on non-genotoxic mechanisms of chemical carcinogens and can be developed for regulatory applications. The Organisation for the Economic Cooperation and Development (OECD) expert group developing an Integrated Approach to the Testing and Assessment (IATA) of Non-Genotoxic Carcinogens (NGTxC) is reviewing the possible assays to be integrated therein. In this context, we review the application of transcriptomics approaches suitable for pre-screening gene expression changes associated with phenotypic alterations that underlie the carcinogenic processes for subsequent prioritisation of downstream test methods appropriate to specific key events of non-genotoxic carcinogenesis. Using case studies, we evaluate the potential of gene expression analyses especially in relation to breast cancer, to identify the most relevant approaches that could be utilised as (pre-) screening tools, for example Gene Set Enrichment Analysis (GSEA). We also consider how to address the challenges to integrate gene panels and transcriptomic assays into the IATA, highlighting the pivotal omics markers identified for assay measurement in the IATA key events of inflammation, immune response, mitogenic signalling and cell injury.

In view of the need to enhance the assessment of consumer products called for in the EU Chemicals Strategy for Sustainability, we developed a methodology for evaluating hazard by combining information across different systemic toxicity endpoints and integrating the information with new approach methodologies. This integrates mechanistic information with a view to avoiding redundant in vivo studies, minimising reliance on apical endpoint tests and ultimately devising efficient testing strategies. Here, we present the application of our methodology to carcinogenicity assessment, mapping the available information from toxicity test methods across endpoints to the key characteristics of carcinogens. Test methods are deconstructed to allow the information they provide to be organised in a systematic way, enabling the description of the toxicity mechanisms leading to the adverse outcome. This integrated approach provides a flexible and resource-efficient means of fully exploiting test methods for which test guidelines are available to fulfil regulatory requirements for systemic toxicity assessment as well as identifying where new methods can be integrated.

Chemical substances are subjected to assessment of genotoxic and carcinogenic effects before being marketed to protect man and the environment from health risks. For agrochemicals, the long-term rodent carcinogenicity study is currently required from a regulatory perspective. Although it is the current mainstay for the detection of nongenotoxic carcinogens, carcinogenicity studies are shown to have prominent weaknesses and are subject to ethical and scientific debate. A transition toward a mechanism-based weight-of-evidence approach is considered a requirement to enhance the prediction of carcinogenic potential for environmental (agro)chemicals. The resulting approach should make optimal use of innovative (computational) tools and be less animal demanding. To identify the various mode of actions (MOAs) underlying the nongenotoxic carcinogenic potential of agrochemicals, we conducted an extensive analysis of 411 unique agrochemicals that have been evaluated for carcinogenicity by the United States Environmental Protection Agency (US EPA) and the European Chemicals Agency (ECHA). About one-third of these substances could be categorized as nongenotoxic carcinogens with an average of approximately two tumor types per substance, observed in a variety of organs. For two-third of the tumor cases, an underlying MOA (network) could be identified. This analysis demonstrates that a limited set of MOA (networks) is underlying nongenotoxic carcinogenicity of agrochemicals, illustrating that the transition toward a MOA-driven approach appears manageable. Ultimately the approach should cover relevant MOAs and its associated key events; this will also facilitate the evaluation of the human relevance. This manuscript describes the results of the analysis while identifying knowledge gaps and necessities to achieve a mechanism-based weight-of-evidence approach.

While regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests (which include mutagenicity assays). If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, but under most chemical regulations (except plant protection, biocides, pharmaceuticals), this is rare. The decision to conduct further testing based on genotoxicity test outcomes creates a regulatory gap for the identification of non-genotoxic carcinogens (NGTxC). With the objective of addressing this gap, in 2016, the Organization of Economic Cooperation and Development (OECD) established an expert group to develop an integrated approach to the testing and assessment (IATA) of NGTxC. Through that work, a definition of NGTxC in a regulatory context was agreed. Using the adverse outcome pathway (AOP) concept, various cancer models were developed, and overarching mechanisms and modes of action were identified. After further refining and structuring with respect to the common hallmarks of cancer and knowing that NGTxC act through a large variety of specific mechanisms, with cell proliferation commonly being a unifying element, it became evident that a panel of tests covering multiple biological traits will be needed to populate the IATA. Consequently, in addition to literature and database investigation, the OECD opened a call for relevant assays in 2018 to receive suggestions. Here, we report on the definition of NGTxC, on the development of the overarching NGTxC IATA, and on the development of ranking parameters to evaluate the assays. Ultimately the intent is to select the best scoring assays for integration in an NGTxC IATA to better identify carcinogens and reduce public health hazards.

To develop and evaluate scientifically robust and innovative approaches for the safety assessment of chemicals across multiple regulatory sectors, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) has started a project to explore how to better use the available information including that from existing animal studies. The aim is to minimize reliance on in vivo testing, to avoid redundancy and facilitate the integration of novel non-animal methods in the regulatory setting, with the ultimate goal of designing sustainable testing strategies. In this thought-starter paper, we present a number of examples to illustrate and trigger further discussions within the scientific and regulatory communities on ways to extrapolate useful information for predicting toxicity from one toxicity endpoint to another or across endpoints based on mechanistic information.