Chemical carcinogen safety testing: OECD expert group international consensus on the development of an integrated approach for the testing and assessment of chemical non-genotoxic carcinogens

Abstract

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.

Keywords: Cancer hallmarks; Cancer microenvironment; Cancer model; Cancer prevention; Hazard assessment; IATA; Integrated approaches to testing and assessment; Non-genotoxic carcinogenicity.

Fig. 1

Steps undertaken in the development of the NGTxC IATA and assay evaluations

Fig. 2

Simplified comminality models of the natural history of cancer for colon, breast and gall bladder exemplifying common key events. Simplified pathways for modelling the natural history of cancer to exemplify critical common stages of key events for use as a basis to derive an overarching commonality IATA. Examples given for colon, breast and gall bladder cancers, with colour and shape coded boxes and text to draw out the commonalities (Reference examples include and are not limited to: (Arpino et al. ; Espinoza et al. ; Giuliano et al. ; Kanthan et al. ; Sakamaki et al. ; Sun et al. ; Tariq and Ghias ; Villanueva ; Yu and Schwabe 2017)

Fig. 3

A general integrated approach for the testing and assessment of non -genotoxic carcinogens. The first step of a cancer endpoint hazard assessment is to conduct mutagenicity and genotoxicity testing (top far left hand thick green framed box), for which there are already well established in vitro and in vivo testing paradigms in regulatory toxicology. Considerations in relation to metabolism, exposure and quantitative in vitro and in vivo extrapolations (QIVIVE) would contribute to the overall risk assessment, as indicated on the far left. Sustained exposure is a critical consideration throughout all the modules of the IATA, as this is likely to trigger subsequent modules. Substances that are negative for mutagenicity and genotoxicity would enter the NGTxC IATA, particularly screening, for the cascade of downstream key events for which there are several suitable assays including some validated assays and TGs. Each module sits within a box frame that will be populated by relevant assays, including epigenetic and cofactor assay components, as many of the modules may be subjected to epigenetic deregulation known to be influential in modulating the specific hallmark module. Bound by broken lines on both the left and right-hand sides, central to the IATA, are six pivotal modules, of which four are not consequent or sequential to each other and can lead to (sustained) proliferation. These are as follows: inflammation (including assays that address the hallmark blocks covering oxidative stress and gene and cell signalling); immune response (again including assays for oxidative stress, but also immune evasion assay models, as they mark the passage /turning point from the body’s immune defence to the immune evasion by the tumour); mitotic signalling (including assays addressing the gap junction hallmark); and finally cell injury (including assays addressing the hallmarks of genetic instability, gap junction, oxidative stress and senescence and telomerase). The fifth module is (sustained) proliferation, and here the essential assay hallmark to be addressed is cell proliferation, triggering investigations on gene and cell signalling and resistance to apoptotic cell death. The sixth module, a change in morphology (dysplastic change), represents the point at which adaptive (sustained) proliferation, -hyperplasia becomes mal-adaptive. The change in morphology module also includes early key events of cell transdifferentiation (at the cellular level that is conversion of one differentiated cell type into another cell type), such as changes in the organization of the cytoskeleton, acquisition of different morphology) and progression to mal-adaptive/irreversible modifications, specifically pathogenic angiogenesis (in contrast to neoangiogenesis which could be adaptive modifications), genetic instability, and then senescence and telomerase activation. The seventh and final module is the tumour stage that is addressed by the metastasis cancer hallmark

Fig. 4

Conceptual overview of the adaptive versus mal-adaptive critical data gaps for adverse outcome recognition in NGTxC. From adaptive to mal-adaptive disease progression: key data gaps in the testing and assessment of non-genotoxic carcinogenicity (adapted from Paparella et al. 2016). There are numerous in vitro assays to address the early key events from receptor binding and transactivation, gene transcription, metabolism and cell proliferation (indicated by the green circle on the left of the figure). Assays are also available for cell transformation, both for early (initiation) and later (promotion) phases (broken red line elipse). A change in morphology represents the point at which adaptive (sustained) proliferation and hyperplasia/dysplasia become mal-adaptive, and this is the key data gap to make the in vivo evidence-based step from hyperplasia to tumour formation (solid red lined elipse). This tipping point is histopathologically characterized with cellular and/or structural atypia. This change is often observed as abnormal nuclear division and disorganized cell proliferation with loss of cell polarity; therefore, in vitro assays that can be used to explore and test these aspects are of high priority