- Developing an internationally acceptable in vitro testing batteries new approach methodologies and integrated approaches to testing and assessment. The test methods will be subject to testing of a sufficiently large set of reference compounds, including prioritised PARC compounds, to allow the evaluation of reliability and relevance considering sensitivity and specificity of the various systems as well as their complementarity detecting different modes of action involved on non-genotoxic carcinogenicity.
- The innovative methods developed therein will allow for the first time a rapid mechanistic non-genotoxic carcinogens hazard analysis of a high number of substances.
- The utility and applicability of a shift towards a more quantitative approach for assessment of non-genotoxic carcinogens toxicity hazard will be explored.
Key messages
- The battery of genotoxicity in vitro assays is sensitive for the detection of genotoxic carcinogens, but the multifactorial nature of the carcinogenesis process greatly hinders the detection of non-genotoxic carcinogens.
- The overarching aim of this project is to improve human carcinogenic evaluation in vitro based on reliable and relevant New Approach Methodologies (NAMs ↗), ultimately enabling a better prediction of human risk in line with regulatory requirements.
Overview
This project aims to advance the identification of non-genotoxic carcinogens ↗ (NGTXCs) by applying innovative, human-relevant NAMs to a range of organ and cellular model systems, including liver, breast, colon, and adipocytes. Techniques such as transcriptomic, high-content analysis, cell painting, epigenetics, and high-throughput-compatible reporter systems will be used to investigate key mechanisms of carcinogenesis, including oxidative stress, epithelial-mesenchymal transition, inflammation, changes in metabolism, epigenetic marks or nuclear receptors signaling and cell proliferation.
In addition, in silico tools will be optimised to support the identification of substances acting through non-genotoxic carcinogens, addressing the needs for the development of an Integrated Approach to Testing and Assessment (IATA ↗) for NGTXCs. This work is carried out in collaboration with IATA and aligns with ongoing EU and OECD activities.
The project will contribute to a better understanding of the links between specific substances and Adverse Outcome Pathways (AOPs ↗) that lead to cancer. It will also evaluate methods of increasing biological complexity, from traditional 2D cell cultures to 3D spheroids and zebrafish models to integrate physiologically relevant toxicological information.
By comparing the different assays, the project will define the applicability domain and limitations of each method. This will ultimately inform chemical risk assessment and regulatory decisions, aligning with international efforts on NGTXCs and supporting the development of more predictive, ethical and efficient safety assessment strategies.
Achievements & Results
All partners finalized the testing of the first set of reference substances (at least 4 positives and 4 negatives) and at least two (up to 10 substances) for each of the 5 different selected mode-of actions: proliferation, oxidative stress, nuclear receptor activation, inflammation and changes in cellular morphology indicative for cell migration as well as cytotoxicity that might be indicative for regenerative proliferation. The data was reported in an already agreed common data template for each chemical.
The analysis of tissue, model and substance specific effects related to distinct aspects of carcinogenesis is ongoing, including a comparison of the lowest concentration needed to detect a significant and/or potentially biological relevant effect in the various test systems. This will facilitate the (further) development of prediction models for each assay to especially define thresholds that allow identification of physiological/ biological and regulatory relevant activities and to develop a complementary and predictive test battery.
However, some more complex methods are still in a status of development and optimization, in particular different 3D liver organoids that include liver cells, macrophages and stellate cells and a 3D mammary duct model.