A method to assess the relevance of nanomaterial dissolution during reactivity testing.

The reactivity of particle surfaces can be used as a criterion to group nanoforms (NFs)
based on similar potential hazard. Since NFs may partially or completely dissolve over the duration
of the assays, with the ions themselves inducing a response, reactivity assays commonly measure the
additive reactivity of the particles and ions combined. Here, we determine the concentration of ions
released over the course of particle testing, and determine the relative contributions of the released
ions to the total reactivity measured. We differentiate three classes of reactivity, defined as being (A)
dominated by particles, (B) additive of particles and ions, or (C) dominated by ions. We provide
examples for each class by analyzing the NF reactivity of Fe2O3, ZnO, CuO, Ag using the ferric
reduction ability of serum (FRAS) assay. Furthermore, another two reactivity tests were performed:
Dichlorodihydrofluorescin diacetate (DCFH2-DA) assay and electron paramagnetic resonance (EPR)
spectroscopy. We compare assays and demonstrate that the dose-response may be almost entirely
assigned to ions in one assay (CuO in DCFH2-DA), but to particles in others (CuO in EPR and FRAS).
When considering this data, we conclude that one cannot specify the contribution of ions to NF
toxicity for a certain NF, but only for a certain NF in a specific assay, medium and dose. The extent of
dissolution depends on the buffer used, particle concentration applied, and duration of exposure.
This culminates in the DCFH2-DA, EPR, FRAS assays being performed under different ion-to-particle
ratios, and differing in their sensitivity towards reactions induced by either ions or particles. If applied
for grouping, read-across, or other concepts based on the similarity of partially soluble NFs, results on
reactivity should only be compared if measured by the same assay, incubation time, and dose range