*3.4. Other Approaches*

Other approaches, which included *A. thaliana* transgenic lines for homologous recombination and transcriptional gene silencing, were adopted to assess the genotoxicity of ZnO NPs [48]. The results showed, at the level of roots, how exposure to ZnO NPs (0–20 mg L<sup>−</sup>1) resulted in an increase in homologous recombination (in particular the gene *atRad54-GFP-GUS* expression) and a reduction in transcriptional gene silencing in leaves (which contained the multicopy construct *P35S::GUS*), which can be ascribed to genotoxic effects triggered by ZnO NPs dissolution to free Zn ions. Methods described and relevant examples are reported in Table 1.

**Table 1.** Reference list of relevant experiments performed with different tools to identify ENM genotoxic effects in plants.


\*, treatment conditions information includes concentration, experimental setup, and time of exposure utilized. Reference list order in the table reflects the order of appearance in the text, depending on the type of analyses performed.

### **4. Conclusions**

In conclusion, in recent years, different techniques, previously exploited for animal cells, have been developed and applied to plants to assess the genotoxic effects related to ENM exposure. These approaches, considering their properties, and the relative pros and cons, which include high/low resolution vs. high/low target specificity, may be implemented for cross-validation of the results obtained. This may also include potential applications related to the utilization of novel methods of mutagenesis (e.g., CRISPR-*Cas9*) [49].

In this context, plants and microorganisms can be utilized as model organisms instead of animal models for Alternative Testing Strategies (ATS) to assess and characterize the risk, with particular regard to genotoxicity, related to ENMs exposure/effects [44,50]. Adoption of ATS for new organisms, endpoints, and span of variations in experimental scale and complexity have been increasingly functional in nanotoxicological literature through iterative processes able to combine results from physiological and molecular approaches [51]. Moreover, the monitoring of ENMs dispersal in the environment, especially at very early exposure stages and in realistic scenarios, can be further implemented [52] in accordance with the recently published EFSA guidance on risk assessment of the application of nanoscience and nanotechnologies in the food and feed chain, human and animal health, which considers in vitro/in vivo toxicological testing (e.g., in vitro degradation, toxicokinetics, genotoxicity, local and systemic toxicity), and the European Registration, Evaluation Authorization and Restriction of Chemicals (REACH) protocols for chemical safety assessment [1,53].

**Author Contributions:** Conceptualization, M.M., N.M. and L.P.; writing—original draft preparation, M.M., N.M. and L.P.; writing—review and editing, M.M., N.M. and L.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding authors.

**Acknowledgments:** All authors acknowledge the support of FIL ("Fondi Locali per la Ricerca").

**Conflicts of Interest:** Authors declare no competing financial interest.
