Ement, Reduction and Refinement (i.e., 3 Rs1) of animal testing. In addition, Attain (2020g) and the Cosmetic Items Regulation (2020e) have either contributed to the implementation from the 3Rs by, respectively referring to and encouraging the useThree Rs and 3Rs are used interchangeably.Vol.:(0123456789)Archives of Toxicology (2021) 95:1867of options to animal testing, or banning animal testing altogether. Along this line, for some specific toxicological endpoints (e.g., skin H2 Receptor manufacturer corrosion and irritation, critical eye damage and irritation, skin sensitisation, and mutagenicity and genotoxicity), the possible hazard of chemical substances is typically evaluated working with non-animal approaches. Nevertheless, for other endpoints, including acute systemic toxicity, repeated dose toxicity and reproductive and developmental toxicity, the regulatory specifications, and hence chemical security evaluation, still heavily relies on the use of animals. Understanding present regulatory needs particular for the assessment of chemical and cosmetic ingredient effects on human well being is crucial to recognize doable understanding gaps, and evaluate how alternative (non-animal) techniques might be integrated in present regulatory practice. This is in line with recent initiatives taken by the International Cooperation on Alternative Test Approaches (ICATM) (https://ec.europa.eu/jrc/en/eurl/ecvam/alter native- metho ds- toxic ity- testi ng/ advis ory- bodies/ icatm), whose members consist of EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) in the European Commission’s Joint Study Centre (JRC), ICCVAM (the US Interagency Coordinating Committee on the Validation of Alternative Approaches) at the National Institute of Environmental Overall health Sciences, JaCVAM (Japanese Center for the Validation of Option Strategies) at the National Institute of Wellness Sciences, Well being Canada, and KoCVAM (South Korean Center for the Validation of Alternative Strategies) in the National Institute of Environmental Well being Sciences, with ad hoc participation from governmental institutions from Brazil, Singapore, China and Taiwan. Using the aim of comparing requirements in different jurisdictions and coordinating the promotion and in the end the implementation of non-animal approaches worldwide, a summary of regulatory needs for skin sensitisation testing across the countries represented by the ICATM partners was published (Daniel et al. 2018), with each other using a proposal of sensible strategies to promote the adoption and regulatory use of defined approaches (DAs)two for the assessment of skin sensitisation (Casati et al. 2018). Here we summarise current EU regulatory requirements for the human wellness assessment of chemical substances beneath Attain plus the Cosmetic Solutions Regulation, thinking of the following toxicological endpoints: skin corrosion and irritation, serious eye damage/eye irritation, photo-induced toxicity, mutagenicity/genotoxicity, acute toxicity, skin sensitisation, repeated dose toxicity, carcinogenicity, reproductive anddevelopmental toxicity, also as absorption, distribution, metabolism and excretion (ADME) and toxicokinetics (TK), and determine the principle challenges in existing regulatory testing practice. We widen the discussion on the D1 Receptor Species availability and advancement of new technologies and in vitro (non-animal) models, highlighting how new frameworks and initiatives undertaken in the European and international level could help to market the 3Rs and implement twent.