The production and use of engineered nanomaterials (NMs) is constantly expanding; however, there are still many uncertainties surrounding the potential risks posed by NMs to human health. Exposure of humans is expected in occupational, consumer and environmental settings via inhalation, ingestion, injection and dermal routes. The biological effects elicited by NMs therefore need to be considered following different exposure routes. Assessment of NM toxicity is required at the exposure site (i.e. lungs, skin, gut), but also at secondary sites (e.g. liver) due to the capacity of NMs to translocate from their portal of entry into the body. There is a relatively large amount of information relating to the pulmonary response following NM exposure, when compared to other routes of exposure and target sites. Due to ethical and financial constraints it is not possible to test all NMs in vivo using all possible routes of exposure. It is therefore important to develop reliable alternatives to animal testing. Many in vitro models use a single cell type exposed to a NM in culture medium with serum, but questions have been raised regarding the suitability of such models.