# Characterization of Nanoparticle Batch-To-Batch Variability
## 4 Discussion
### ROS Generation  
The radical formation potential of NMs is an important intrinsic parameter for predicting the formation of ROS in cells and organisms. Together with cellular ROS production (e.g., activation of NADPH oxidases) this is considered as a key event in inducing oxidative stress and adverse effects such as oxidative damage of proteins, lipids or DNA. Different detection methods of the radical formation potential have been developed. They are summarized in a recent review by [PERSON] et al. [52] and strengths and weaknesses of the singe assays were discussed. The DCF assay was evaluated as suitable in principle, however, other methods seem to be more sensitive in a cell-free system. The authors proposed to use multiple methods to identify potential artefacts in individual assays. They furthermore found that some types of NMs caused oxidative stress in cells while producing low amounts of radicals in a cell-free assay. Other NMs triggering marked cell-free radical formation induced only minimal oxidative stress in cells [53]. Therefore, a correlation between radical formation potential of NMs and toxicity is difficult and should be studied for each type of NM. In the OECD WPMN testing program, a suite of techniques was evaluated to determine physico-chemical properties of NMs [14; 15]. With respect to radical formation, methods different to the DCF assay were assessed for silica, ceria, zinc oxide and titania. Electron spin resonance seemed to be most appropriate to detect radical formation by the respective NMs. Here we used the DCF assay which also indicates reactivity of these NMs but also of PS-NH\({}_{2}\). Figure 7 shows a comparison of the ability to induce DCF fluorescence of all tested NMs which indicates their potential to generate ROS. Some NMs such as ZnO and PS-NH\({}_{2}\) moderately induced DCF fluorescence but showed no difference between the batches. The other NMs showed a moderate (silica-flame, titania) to high (silica-Stober, ceria) B2B variability when assessing the formation of ROS in a cell-free assay. As ROS production is one of the central mechanisms to explain and predict toxicity of NMs, in the future there is certainly a need to investigate and understand the basis for this B2B variability. Correlation between impurity level and ROS generation was found in some cases but not in general. Note that in the case of carbon NMs, the correlation between toxicity and impurity level is well recognized [54; 55]. For silica, either produced by flame or colloidal synthesis, outliers could be identified with strong potential to produce ROS. Although the reason for this different behaviour could not be resolved,also in previous studies with colloidal silica a high B2B variability of the surface chemistry, which is critically linked to ROS production, could be observed [56]. Therefore, future studies should correlate differences in ROS formation as measured here in cell-free assays with toxicity for example, in cells to investigate the relevance of reactivity as proposed by the OECD WPMN for safety assessment in case by case studies. It is likely that other parameters such as size distribution, crystallinity and surface properties also play a role in ROS generation. Concerning the surface properties, the surface charge does not seem to be significantly affected by the preparation method, as the zeta potential varies only with the pH of the solutions. Interestingly, clustering of the batches produced by the same synthesis conditions revealed in the case of ceria a strong correlation of lower DCF oxidation with the presence of the reducing agent HMT. Overall, in the case of ROS formation there seems to be a higher B2B variability between batches produced by different synthesis conditions compared to batches produced in the same way, although other parameters such as size and the zeta potential are fairly similar.