Edinburgh Napier University

  The introduction of engineered nanomaterials (ENM) to new materials and products requires informed risk
assessment. The altered physico-chemical properties of ENM mean traditional assays to evaluate toxicity
must be modified or validated to ensure they are fit for purpose. Reproductive hazard testing is associated
with high animal burdens and monetary costs. Thus, in line with the 3Rs principle for Reduction,
Refinement and Replacement of animals in testing, alternative (non-animal) testing strategies are attractive options.
To explore whether in vitro testicular cell line studies could be indicative of findings in vivo, we exposed both cell lines and rats to a panel of highly relevant standardised ENM: copper oxide, two types of silver, silica and relevant ionic controls (provided by FP7 projects MARINA & SUN). To provide an indication of toxicity in vitro, the murine testicular steroidogenic Leydig cell line TM3 was exposed to ENM for 24 hours across a range of doses, and analysed using the WST-1 & Alamar Blue cytotoxicity assays to provide toxicity data inclusive of exposure time and dose factors.
In vitro outcomes were then compared to those of rats which had been treated in vivo either via traditional 28 day oral gavage (silver, silica) or an alternative 5 day short-term inhalation protocol (copper oxide). This was achieved by histological examination of testicular tissue, which included sterological analysis and quantitative evaluation of changes observed.
To date, results in vitro have proved indicative of adverse effects elicited by silver ENM exposure in vivo.
Initial examination of copper oxide and silica also show a correlation between findings in vitro and in vivo.
The results are being used to validate findings in vitro, and to guide intelligent testing strategy development for ENM-specific reproductive toxicity.