Search

Abstract Scientific, financial, and ethical drivers have led to unprecedented interest in implementing human-relevant, mechanistic in vitro and in silico testing approaches. Further, as non-animal approaches are being developed and validated, researchers are interested in strategies that can immediately reduce the use of animals in toxicology testing. Here, we aim to outline a testing strategy for assessing genotoxicity beginning with standard in vitro methods, such as the bacterial reverse mutation test and the in vitro micronucleus test, followed by a second tier of in vitro assays including those using advanced 3D tissue models. Where regulatory agencies require in vivo testing, one demonstrated strategy is to combine genotoxicity studies traditionally conducted separately into a single test or to integrate genotoxicity studies into other toxicity studies. Standard setting organisations and regulatory agencies have encouraged such strategies, and examples of their use can be found in the scientific literature. Employing approaches outlined here will reduce animal use as well as study time and costs.

Scientists are using in vitro methods to answer important research questions and implementing strategies to maximize the reliability and human relevance of these methods. One strategy is to replace the use of fetal bovine serum (FBS)-an undefined and variable mixture of biomolecules-in cell culture media with chemically defined or xeno-free medium. In this study, A549 cells, a human lung alveolar-like cell line commonly used in respiratory research, were transitioned from a culture medium containing FBS to media without FBS. A successful transition was determined based on analysis of cell morphology and functionality. Following transition to commercially available CnT-Prime Airway (CELLnTEC) or X-VIVO™ 10 (Lonza) medium, the cells were characterized by microscopic evaluation and calculation of doubling time. Their genotype, morphology, and functionality were assessed by monitoring the expression of gene markers for lung cell types, surfactant production, cytokine release, the presence of multilamellar bodies, and cell viability following sodium dodecyl sulphate exposure. Our results showed that A549 cells successfully transitioned to FBS-free media under submerged and air-liquid-interface conditions. Cells grown in X-VIVO™ 10 medium mimicked cellular characteristics of FBS-supplemented media while those grown in CnT-Prime Airway medium demonstrated characteristics possibly more reflective of normal human alveolar epithelial cells.

Many companies and global regulatory programs have expressed the intent to move away from in vivo animal testing to new approach methods (NAMs) as part of product safety assessments. NAMs, which include non-animal approaches for testing and assessment – from computer-based modeling to in chemico or in vitro models – allow faster data gener­ation with potentially greater relevance to humans while avoiding animal use. To monitor progress implementing NAMs, each organization first must define what is in scope, starting with the definition of “animal” (e.g., mammals, vertebrates) and applicable studies (e.g., animals used for “in-house” experiments, at contract research organizations, as part of envi­ronmental monitoring). Next, organizations must establish baseline animal use, including defined rules for inclusion/ exclusion of animals that ensure consistency in future assessments. Lastly, organizations must establish metrics for animal savings based on the utility of NAM data. This paper presents one approach to establish “animal use” metrics in a toxi­cology program at The Dow Chemical Company. The premise of our program is that most NAM information has value for animal savings, but the value depends on how data are used (e.g., research and development, screening, or regulatory requirements) and the level of certainty for internal decision-making. This manuscript provides metrics on the impact of NAMs, allowing a quantitative assessment of animal use numbers over time, accountability for resources spent on NAM development, and identification of areas where NAM development is still needed. This approach can be refined for use at other organizations.