Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.

Computational toxicology models have been successfully implemented to prioritize and screen chemicals. There are numerous in silico (quantitative) structure-activity relationship ([Q]SAR) models for the prediction of a range of human-relevant toxicological endpoints, but for a given endpoint and chemical, not all predictions are identical due to differences in their training sets, algorithms, and methodology. This poses an issue for high-throughput screening of a large chemical inventory as it necessitates several models to cover diverse chemistries but will then generate data conflicts. To address this challenge, we developed a consensus modeling strategy to combine predictions obtained from different existing in silico (Q)SAR models into a single predictive value while also expanding chemical space coverage. This study developed consensus models for nine toxicological endpoints relating to estrogen receptor (ER) and androgen receptor (AR) interactions (i.e., binding, agonism, and antagonism) and genotoxicity (i.e., bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus). Consensus models were created by combining different (Q)SAR models using various weighting schemes. As a multi-objective optimization problem, there is no single best consensus model, and therefore, Pareto fronts were determined for each endpoint to identify the consensus models that optimize the multiple-criterion decisions simultaneously. Accordingly, this work presents sets of solutions for each endpoint that contain the optimal combination, regardless of the trade-off, with the results demonstrating that the consensus models improved both the predictive power and chemical space coverage. These solutions were further analyzed to find trends between the best consensus models and their components. Here, we demonstrate the development of a flexible and adaptable approach for in silico consensus modeling and its application across nine toxicological endpoints related to ER activity, AR activity, and genotoxicity. These consensus models are developed to be integrated into a larger multi-tier NAM-based framework to prioritize chemicals for further investigation and support the transition to a non-animal approach to risk assessment in Canada.

Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

β-Myrcene is a flavoring substance that occurs naturally in a large variety of foods. At the request of the European Food Safety Authority (EFSA) for additional toxicological data on β-myrcene, groups of Sprague Dawley rats (10/sex/group) were administered diets containing 0, 700, 2100, or 4200 ppm of β-myrcene designed to provide nominal doses of 0, 50, 150, or 300 mg/kg bw/day in a 90-day GLP-compliant study. Based on body weights, feed consumption, and substance stability data, final estimated daily intakes of β-myrcene were calculated to be 20.4, 58.8, and 115.2 mg/kg bw for males and 24.2, 70.0, and 135.9 mg/kg bw for females. No effects on clinical observations, hematology and clinical chemistry parameters, organ weights, or macroscopic and histopathological examinations were found attributable to ingestion of β-myrcene. The oral no-observed-adverse-effect level (NOAEL) for rats of both sexes was the highest dose tested. Based on feed consumption and test substance stability in the diet, the NOAEL was calculated to be 115 and 136 mg/kg bw/day for males and females, respectively.

The Organization for Economic Cooperation and Development (OECD) recently revised the test guidelines (TGs) for genetic toxicology. This article describes the main issues addressed during the revision process, and the new and consistent recommendations made in the revised TGs for: (1) demonstration of laboratory proficiency; (2) generation and use of robust historical control data; (3) improvement of the statistical power of the tests; (4) selection of top concentration for in vitro assays; (5) consistent data interpretation and determination of whether the result is clearly positive, clearly negative or needs closer consideration; and, (6) consideration of 3R\'s for in vivo assay design. The revision process resulted in improved consistency among OECD TGs (including the newly developed ones) and more comprehensive recommendations for the conduct and the interpretation of the assays. Altogether, the recommendations made during the revision process should improve the efficiency, by which the data are generated, and the quality and reliability of test results. Environ. Mol. Mutagen. 58:284-295, 2017. © 2017 Wiley Periodicals, Inc.