International Journal of Environmental Research and Public Health Review Nanotoxicology and Nanosafety: Safety-by-Design and Testing at a Glance Aleksandra Zieli ´nska 1,2 , Beatriz Costa 1, Maria V. Ferreira 1, Diogo Miguéis 1, Jéssica M. S. Louros 1, Alessandra Durazzo 3 , Massimo Lucarini 3, Piotr Eder 4 , Marco V. Chaud 5 , Margreet Morsink 6,7,8 , Niels Willemen 6,8, Patrícia Severino 6,9,10 , Antonello Santini 11,* and Eliana B. Souto 1,12,* 1 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal; [email protected] (A.Z.); [email protected] (B.C.); [email protected] (M.V.F.); [email protected] (D.M.); [email protected] (J.M.S.L.) 2 Institute of Human Genetics, Polish Academy of Sciences, Strzeszy´nska32, 60-479 Pozna´n,Poland 3 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy; [email protected] (A.D.); [email protected] (M.L.) 4 Department of Gastroenterology, Dietetics and Internal Diseases, Poznan University of Medical Sciences, Przybyszewskiego 49, 60-355 Pozna´n,Poland; [email protected] 5 Laboratory of Biomaterials and Nanotechnology, University of Sorocaba—UNISO, Sorocaba 18023-000, Brazil; [email protected] 6 Center for Biomedical Engineering, Department of Medicine, Brigham and Women& Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA; [email protected] (M.M.); [email protected] (N.W.); [email protected] (P.S.) 7 Translational Liver Research, Department of Medical Cell BioPhysics, Technical Medical Centre, Faculty of Science and Technology, University of Twente, 7522 NB Enschede, The Netherlands 8 Department of Developmental BioEngineering, Faculty of Science and Technology, Technical Medical Centre, University of Twente, 7522 NB Enschede, The Netherlands 9 Nanomedicine and Nanotechnology Laboratory (LNMed), Institute of Technology and Research (ITP), University of Tiradentes (Unit), Av. Murilo Dantas, 300, Aracaju 49010-390, Brazil 10 Tiradentes Institute, 150 Mt Vernon St, Dorchester, MA 02125, USA 11 Department of Pharmacy, University of Napoli Federico II, 80131 Napoli, Italy 12 CEB—Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal * Correspondence: [email protected] (A.S.); ebsouto@ff.uc.pt (E.B.S.); Tel.: +39-81-253-9317 (A.S.); +351-239-488-400 (E.B.S.) Received: 9 May 2020; Accepted: 23 June 2020; Published: 28 June 2020 Abstract: This review offers a systematic discussion about nanotoxicology and nanosafety associated with nanomaterials during manufacture and further biomedical applications. A detailed introduction on nanomaterials and their most frequently uses, followed by the critical risk aspects related to regulatory uses and commercialization, is provided. Moreover, the impact of nanotoxicology in research over the last decades is discussed, together with the currently available toxicological methods in cell cultures (in vitro) and in living organisms (in vivo). A special focus is given to inorganic nanoparticles such as titanium dioxide nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs). In vitro and in vivo case studies for the selected nanoparticles are discussed. The final part of this work describes the significance of nano-security for both risk assessment and environmental nanosafety. “Safety-by-Design” is defined as a starting point consisting on the implementation of the principles of drug discovery and development. The concept “Safety-by-Design” appears to be a way to “ensure safety”, but the Int. J. Environ. Res. Public Health 2020, 17, 4657; doi:10.3390/ijerph17134657 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020, 17, 4657 2 of 23 superficialityInt. J. Environ. Res. and Public the lackHealth of 2020 articulation, 17, x with which it is treated still raises many doubts. Although2 of the22 approach of “Safety-by-Design” to the principles of drug development has helped in the assessment of thecombination toxicity of of nanomaterials, scientific efforts acombination is constantly of urgent scientific to ensure efforts isthe constantly consistency urgent of methods to ensure and the consistencyprocesses. of This methods will andensure processes. that the This quality will ensure of nanomaterials that the quality is of controlled nanomaterials and istheir controlled safe anddevelopment their safe development is promoted. is promoted. Safety issues Safety issuesare considered are considered strategies strategies for fordiscovering discovering novel novel toxicological-relatedtoxicological-related mechanisms mechanisms still still needed needed to beto promoted.be promoted. Keywords:Keywords:nanotoxicology; nanotoxicology; nanosafety; nanosafety; nanomaterials; nanomaterial risk assessment;s; risk assessment; toxicity tests; toxicity nanoparticles; tests; humannanoparticles; health; Safety-by-Design; human health; Safe biologicalty-by-Design; systems biological systems 1. Introduction1. Introduction NanomaterialsNanomaterials (NMs) (NMs) are are natural natural or or manufactured manufactured materials materials basedbased onon nanosized particles particles in in a a disaggregateddisaggregated state state or inor thein the form form of anof aggregatean aggregat/agglomeratee/agglomerate [1]. [1]. The The number–size number–size distribution distribution of 50% of or more50% or of more the particles of the particles which have which one have or more one or external more external dimensions dimensions in the size in the range size comprised range comprised between 1 andbetween 100 nanometers 1 and 100 nanometers [2,3]. Due to[2,3]. the Due small to sizethe small of the size particles of the andparticles changes and inchanges their inner in their structure, inner NMsstructure, can have NMs diff canerent have properties different that properties stem from that a stem higher from surface a higher area surface to volume area to ratio volume [4]. Therefore, ratio [4]. theTherefore, physicochemical the physicochemical properties of NMs properties may di ffofer NM froms themay properties differ from of granularthe properties substances of granular or larger particlessubstances [5]. or larger particles [5]. ThereThere is ais huge a huge demand demand for for a definition a definition of of NMs NMs [6 ,[6,77]. On]. On the the other other hand, hand, the the life life cycle cycle assessment assessment of NMsof NMs has been has recognizedbeen recognized as a significant as a significant tool for tool regularly for regularly evaluating evaluating the potential the potential environmental environmental impacts of manufacturedimpacts of manufactured nanomaterial nanomaterial during their during complete their life complete cycles [ 8life], and cycles it is [8], schematically and it is schematically presented in Figurepresented1. in Figure 1. FigureFigure 1. 1.The The impactimpact ofof nanomaterial nanomaterial life life cycle cycle on on the the environment. environment. ForFor a long a long time, time, it hasit has been been assumed assumed that that NMs NMs have have a similar a similar toxicity toxicity to to materials materials of of larger larger size size [9 ]. However,[9]. However, studies havestudies shown have that shown nanosized that materialsnanosized exhibit materials different exhibit physicochemical different physicochemical properties from properties from those of the source material (thereby changing their reactivity in biological systems). It calls into the question whether applying conventional methodologies to evaluate the adverse effects of NMs is still valid [10]. According to “REACH” (Registration, Evaluation, Authorization and restriction of Chemical Substances), the safety assessment of NMs should follow the risk assessment methodology adopted for conventional chemicals, which is based on the following requirements [11]: (1) effects evaluation, (2) exposure assessment, and (3) risk characterization (Figure 2). Step 1 includes the evaluation of effects. The risk quotient is considered acceptable (higher than 1) when the Int. J. Environ. Res. Public Health 2020, 17, 4657 3 of 23 those of the source material (thereby changing their reactivity in biological systems). It calls into the question whether applying conventional methodologies to evaluate the adverse effects of NMs is still valid [10]. According to “REACH” (Registration, Evaluation, Authorization and restriction of Chemical Substances), the safety assessment of NMs should follow the risk assessment methodology adopted forInt. J. conventional Environ. Res. Public chemicals, Health 2020 which, 17, x is based on the following requirements [11]: (1) effects evaluation,3 of 22 (2) exposure assessment, and (3) risk characterization (Figure2). Step 1 includes the evaluation of e ffects. Theestimated risk quotient exposure is consideredvalue is lower acceptable than the (higherconcentrat thanion 1) of when the agent, the estimated in which no exposure adverse value effect is was lower thanobserved the concentration in the experimental of the agent, study in carried which noout adverse to evaluate effect the was point observed under in study—for the experimental example, study carriedinhalation out toxicity to evaluate or genotoxicity. the point under If it is study—for necessary example,to carry out inhalation in vitro toxicityand/or in or vivo genotoxicity. experiments If it is necessaryin order