Determining nanoform similarity via assessment of surface reactivity by abiotic and in vitro assays.

Grouping of substances is a method used to streamline hazard and risk assessment. Assessment of similarity provides the scientific evidence needed for formation of groups. This work reports on justification of grouping of nanoforms (NFs) via similarity of their surface reactivity. Four reactivity assays were used for concentration dependent detection of reactive oxygen species (ROS) generated by NFs: abiotic assays FRAS, EPR and DCFH2-DA, as well as the in vitro assay of NRF2/ARE responsive luciferase reporter activation in the HEK293 cell line. Representative materials (CuO, Mn2O3, BaSO4, CeO2 and ZnO) and three case studies of each several NFs of iron oxides, Diketopyrrolopyrroles (DPP)-based organic pigments and silicas were assessed. A novel similarity assessment algorithm was applied to quantify similarities between pairs of NFs, in a four-step workflow on concentration-response curves, individual concentration and response ranges, and finally the representative materials. We found this algorithm to be applicable to all abiotic and in vitro assays that were tested. Justification of grouping must include the increased potency of smaller particles via the scaling of effects with specific surface, and hence quantitative similarity analysis was performed on concentration-response in mass-metrics. CuO and BaSO4 were the most and least reactive representative materials respectively, and all assays found BaSO4/CuO not similar, as confirmed by their different NOAECs of in vivo studies. However, similarity outcomes from different reactivity assays were not always in agreement, highlighting the need to generate data by one assay for the representative materials and the candidate group of NFs. Despite low similarity scores in vitro some pairs of case study NFs can be accepted as sufficiently similar because the in vivo NOAECs are similar, highlighting the conservative assessment by the abiotic assays.

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 (DCFH2­DA) assay and electron paramagnetic resonance (EPR) spectroscopy. We compare assays and demonstrate that the dose­response may be almost entirely assigned to ions in one assay (CuO in DCFH2­DA), 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 DCFH2­DA, EPR, FRAS assays being performed under different ion­to­particle ratios, and differing in their sensitivity towards reactions induced by either ions or particles. If applied for grouping, read­across, 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.