Nanomaterials are increasingly being added to food handling and packaging materials, or directly, to human food and animal feed. To ensure the safety of such engineered nanomaterials (ENMs), in May 2011, the European Food Safety Authority (EFSA) published a guidance document on Risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain. It states that risk assessment should be performed by following a step-wise procedure. Whenever human or animal exposure to nanomaterials is expected, the general hazard characterisation scheme requests information from in vitro genotoxicity, toxicokinetic and repeated dose 90-day oral toxicity studies in rodents. Numerous prevailing uncertainties with regard to nanomaterial characterisation and their hazard and risk assessment are addressed in the guidance document. This article discusses the impact of these knowledge gaps on meeting the goal of ensuring human safety. The EFSA's guidance on the risk assessment of ENMs in food and animal feed is taken as an example for discussion, from the point of view of animal welfare, on what level of uncertainty should be considered acceptable for human safety assessment of products with non-medical applications, and whether animal testing should be considered ethically acceptable for such products.