The Cell Transformation Assay: A Historical Assessment of Current Knowledge of Applications in an Integrated Approach to Testing and Assessment for Non-Genotoxic Carcinogens

Abstract

The history of the development of the cell transformation assays (CTAs) is described, providing an overview of in vitro cell transformation from its origin to the new transcriptomic-based CTAs. Application of this knowledge is utilized to address how the different types of CTAs, variously addressing initiation and promotion, can be included on a mechanistic basis within the integrated approach to testing and assessment (IATA) for non-genotoxic carcinogens. Building upon assay assessments targeting the key events in the IATA, we identify how the different CTA models can appropriately fit, following preceding steps in the IATA. The preceding steps are the prescreening transcriptomic approaches, and assessment within the earlier key events of inflammation, immune disruption, mitotic signaling and cell injury. The CTA models address the later key events of (sustained) proliferation and change in morphology leading to tumor formation. The complementary key biomarkers with respect to the precursor key events and respective CTAs are mapped, providing a structured mechanistic approach to represent the complexity of the (non-genotoxic) carcinogenesis process, and specifically their capacity to identify non-genotoxic carcinogenic chemicals in a human relevant IATA.

In vitro evaluation of the carcinogenic potential of perfluorinated chemicals

“…This procedure also applies to BALB/c 3T3 cells that undergo apoptosis in the absence of serum. However, the sourcing of FCS from animal-derived material raises ethical concerns and poses challenges related to variability, batch-to-batch consistency, and the potential for contamination with adventitious agents (Verma et al, 2020;Lang et al, 2018;Colacci et al, 2023).…”

“…Efforts to develop non-animal-derived alternatives for FCS are ongoing and motivated by concerns regarding animal welfare and the need for more standardized and defined culture conditions. Although some progress has been made in the development of serum-free or defined media formulations that utilize recombinant growth factors and synthetic substitutes, the complete replacement of FCS remains a challenging task (Lang et al, 2018;Subbiahanadar Chelladurai et al, 2021;Jacobs et al, 2023;Colacci et al, 2023). Despite advancements in cell culture technology, non-animal-derived alternatives often fail to fully replicate the growth-promoting properties of FCS, resulting in suboptimal cell growth, reduced cell viability, and altered cellular behavior (Colacci et al, 2023).…”

“…Indeed, to support the testing strategy for carcinogenesis and fill the critical gaps in the use of CTA, as a stand-alone test, CTA can be coupled with a transcriptomics approach, using the so-called transformics assay, allowing the evaluation of whole-genome transcriptional changes at different times along the process leading to foci formation (Mascolo et al, 2018;Pillo et al, 2022;Colacci et al, 2023). The use of this integrated approach would provide information of the key events at the molecular and cellular level leading to oncotransformation, allowing a better understanding of the MoA of these chemicals, and allowing the identification of an effective threshold.…”

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens—a PARC project

“…UKHSA: Augmentation of the CYP induction test method for use in the NGTxC IATA and validation management for the cell transformation Assay using transcriptomics: the transformics assay UKHSA will provide input on the further development of the HepaRG CYP induction test method (Bernasconi et al, 2019) (Jacobs et al, 2022), to support the NGTxC IATA (Jacobs et al, 2020) and will be implicated for the (pre)validation of promising test methods that address the outstanding needs as identified in the work of the OECD NGTxC IATA expert group, specifically here in relation to cell transformation (Colacci et al, 2023) and gap junction (Sovadinova et al, 2021) in the first instance. A regulatory framework for the IATA for NGTxC developed with ECHA will also be provided (Louekari et al paper in prep), this will assist in supporting the regulatory targeted assay development.…”

“…In 2020, an OECD expert group developed an IATA for NGTxCs and published a consensus paper describing the overarching IATA, with the molecular initiating events (MIE) of cellular metabolism and receptor interactions, followed by the early key events (KEs) of inflammation and immune dysfunction, mitotic signaling, and cell injury, leading to (sustained) proliferation, morphological transformation and tumor formation ( Jacobs et al, 2020 ). Several assay evaluations and reviews have been conducted and published in relation to epigenetics ( Desaulniers et al, 2021 ), cell transformation ( Colacci et al, 2023 ), gap junction ( Sovadinova et al, 2021 ), gene signaling ( Oku et al, 2022 ), cell proliferation and oxidative stress ( Veltman et al, 2023 ). These publications provide details on the next steps needed to facilitate developments that will have regulatory relevance for the safety assessment of NGTxCs.…”