Editorial.

Changes to cosmetics regulations internationally have been instrumental in the move away from animal testing and toward the development and promotion of alternative methods. Brazil is one of the largest markets for personal care and cosmetics products in the world and on 1st March 2023, as Resolution No. 58/2023 came into force, Brazil joined over 40 countries moving towards a global ban on the testing of cosmetic ingredients on animals. It is encouraging to witness not only the ongoing progress in governmental policies in mandating alternatives to animals, but also the continual innovations in the associated science. Whilst, currently, there is significant focus on applications in cosmetics, success in this arena will undoubtedly drive forward the use and acceptance of alternatives in other areas. Further details on the Brazilian Resolution are given in the Spotlight on Three Rs Progress section of this issue. This section also includes summaries of different studies that are applying a diverse range of alternative methods, from bioprinting of reconstructed human epidermis for assessing skin irritation, to open-source software that can quantify animal behaviours. Studies on the effects of SARSCoV-2 on the developing fetal brain and a model for human ovarian follicles are also highlighted. The review article by da Silva and Valadares also focuses attention on SARS-CoV-2 and how the pandemic highlighted the need for improvements in non-animal based methods in preclinical research for respiratory diseases. Here, the authors provide a detailed review of key methods that can be applied in this area, including: monolayer cell cultures; air–liquid interface models; lung organoids; human precision-cut lung slices (PCLS); and lung-on-a-chip microfluidic systems. The advantages and limitations of each method are discussed, as well as the need for standardisation and quality control procedures to make these New Approach Methodologies (NAMs) more acceptable for regulatory purposes. The importance of selecting the most appropriate models and endpoints to measure, depending on the research question, is emphasised. Andersson et al. also propose an addition to what they describe as the “NAM-based predictive toolbox”. These authors report an in silicomethod that can be used to differentiate liquids with the potential for eliciting eye irritation, from those considered to be non-irritant. The authors describe a two-tier approach that identifies potential irritants by using predicted pKa values and also by using a model that is derived from Hansen Solubility Parameters. The model performed well, and in order to err on the side of caution, it has been optimised to give a low rate of false negative predictions. The last two articles in this issue both present results from assays for toxicity testing. Hautenen et al. present the results of a study to develop an assay to assess the induction of vasculature formation — a mechanism associated with non-genotoxic carcinogenicity. The coculturing of human umbilical vessel endothelial cells (HUVECs) with human adipose stromal cells (hASCs) has previously been shown to lead to more mature vessel formation, and this system was used as the basis for this study. The authors adapted their standardised vasculogenesis/angiogenesis model (VAM), previously used to detect inhibition of vascular formation, to instead measure induction of vasculature formation. Four new assay protocols were developed and compared, in order to determine the optimum conditions for an assay that can be used to assess the ability of chemicals to induce vasculature formation; this may be indicative of carcinogenesis. Kuruppuarachchi et al. used established assays, based on growth and the evaluation of mitotic indices and chromosomal aberration, for Allium cepa (onion) root tip, to assess potential cytotoxicity and genotoxicity of marine sponge extracts. For many years, extracts from marine sponges have been the subject of investigation due to their potential antibacterial, antiviral, antifungal, antimalarial or anticancer effects. The experiments reported here, showed that both cytotoxicity and genotoxicity were induced in the root tips by the extracts. Root growth was inhibited, mitodepressive effects were induced at all stages of cell division and chromosomal aberrations were also detected. These results indicate that the marine sponge investigated, Luffariella herdmani, may possess antiproliferative activity. This is worthy of further investigation, as there is increasing interest in the development of novel therapeutic agents derived from marine sponges. Finally, readers are directed to a range of information sources and freely available training materials that are signposted in the regular Resources Round-up section; we trust that you will find these useful.