2 Humanising Nanotoxicology Testing: Moving Towards Animal-free Approaches for Hazard Assessment of Nanomaterials

Abstract The vast array of nanomaterials (NMs) under development raises ethical concerns over the reliance and extent of animal usage in the hazard assessment of substances. Grouping which allows read-across of data from NMs predicted to pose similar hazard could provide justification for waiving of in vivo tests. The GRACIOUS Framework has defined Integrated Approaches to Testing and Assessment (IATAs) to guide the generation of data to support grouping. Each IATA is underpinned by a tiered testing strategy promoting the use of acellular and in vitro assays, (Tier 1-2), which are predictive of in vivo mechanisms of toxicity. Tier 3 in vivo studies are only required to confirm grouping or fill specific gaps. Adopting the IATA approach allows the effective triage of vast number of NMs prioritized for in vivo hazard assessment, thus reducing overall animal testing. However, many of the methods recommended at Tier 1-2 are reliant on the use of animal-derived materials and reagents. The information gathered at early Tiers therefore cannot be considered animal-free unless adaptions are made to standard in vitro protocols to replace all animal-derived reagents. The objective of this study was to review the GRACIOUS Inhalation IATA and identify where animal-free options could be selected when designing experimental hazard studies. The replacement of animal-derived reagents with human alternatives was trialed in multiple Tier 1 assays using a CuO NM as a case study material. This proof-of-concept study informs the suitability of animal-free methods for NM hazard assessment and demonstrates only minor adaptions may be required.