*4.2. In Vitro Techniques*

As stated by (Drasler et al., 2017, [199]), assays could be carried out using eternal cell lines or primary cultures. As stated by these authors, cell lines are preferred as they provide increased stability as well as homogeneity, which favors the consistency in test results, particularly in preliminary examinations. For more precise examinations, the same investigators indorse the usage of three-dimensional co-cultures for better understanding the action mechanisms of nano-sized materials on tissues. In order to assess nanomaterial toxicity, the utilization of epithelial cell lines (lung, skin, etc.) is typically designated because the aforementioned cells demonstrate characteristics of actual barriers against destructive agents and are consequently the foremost to suffer the effect of these compounds (Rothen-Rutishauser et al., 2012, [201]). Conversely, certain strains might not be receptive to the impacts of nanomaterials and here the primary cultures might be better designated.

In the case of in vitro comet assay using the mammalian cell culture, Collins and his team members (Collins et al., 2017, [202]) put forward certain recommendations: (i) Utilize non-cytotoxic concentrations; (ii) select the type of cell as per the exposure scenario; (iii) identify both long (24 h) and short (2–3 h) tests for obtaining a clear knowledge of the action mode of the nanomaterial; and (iv) identify if the genotoxic destruction demonstrated is a consequence of the direct effect with DNA or because of DNA oxidation. As stated by (Catalán et al., 2017, [203]), the limitations as well as relevance of mutagenicity/genotoxicity assays must be considered while selecting the most suitable monitoring technique. As per the aforementioned study, examinations considered in the assessment must be based on three classes: (1) DNA damage, (2) chromosomal destruction, and (3) gene mutation. According to OECD guidelines (OECD, 2014, [204]), for selecting a test and evaluating the genotoxicity of a nanoform, the solubility, absorption, exposure, metabolites, as well as other derivatives must be taken into account, along with possible side effects.

### **5. Environmental Risk Assessment—Case Studies**

In a study performed by (Voelker et al., 2015, [205]), a standard environmental risk assessment of silver nanomaterials applied in textiles has been carried out. Environmental exposure scenarios were developed for three distinct categories of textiles equipped with silver nanomaterials. Based on these scenarios, the predicted environmental concentrations were deduced for sewage treatment plants and for the environmental compartments such as surface water, sediment, and soil. The information on

ecotoxicology were obtained from di fferent analysis on earthworms, chironomids, macrophytes, duckweed, fish, daphnids, algae, cyanobacteria, activated sludge, terrestrial plants, and soil microorganisms. Emission information on silver nanomaterials NM-300K from textiles were obtained from washing experiments. The environmental risk assessment performed was based on the specifications defined in the European Chemicals Agency (ECHA) guidance on information requirements as well as chemical safety assessment. Depending on the selected scenarios as well as preconditions, no environmental hazard of the silver nanomaterials NM-300K discharged from textiles was noticed. Under conservative assumptions, a risk quotient for surface water pointed out that the marine compartment might be influenced by a higher emission of silver nanomaterials to the surroundings because of the higher sensitivity of marine life to silver. Depending on the e ffective retention of silver nanomaterials in sewage sludge and the continuing application of sewage sludge on farm land it is suggested to introduce a threshold for total silver concentration in sewage sludge. With regards to the potential risk mitigation measures, it is highlighted that one should directly introduce silver nanomaterials into the textile fiber due to the fact that this would lessen the discharge of silver nanomaterials extremely in the course of washing. If this is not conceivable because of technical restrictions or some other reason, then the inclusion of a threshold level controlling the discharge of silver nanomaterials from textiles is recommended. It should be noted that the aforementioned specific study is a case study that is only valid for examined silver nanomaterial NM-300K and its possible application in textiles.

The study conducted by (Yasin et al., 2019, [206]) elaborated on certain points for technical textile waste. Initially, the Life Cycle Assessment method for end-of-life is feasible if the waste treatment depends on the technical textile functionality instead of common textile waste. Secondly, this end-of-life study confirmed that the Life cycle assessment results of any technical textile product at its disposal are also case dependent and must not be considered the same as collective textile waste, in spite of environmental correspondence being considered or not. Figure 11 demonstrates the life cycle of a textile product system as well as its environmental interventions at di fferent phases. The life cycle assessment "gate-to-grave" method was used for studying two technical textiles with di fferent functionalities however with the same weight, one is a silver nanoparticle-treated polyester and the other one is flame retardant-treated wool. They were examined for having an improved understanding of environmental parity, particularly in their usage phase as well as at the "end-of-life" phase. Ten-midpoint categories were employed for analyzing the environmental impacts at the time of the use phase and end-of-life phase of both technical textiles. With regard to the technical textile curtain recycling, both use functionality substances, flame retardant (for its flame retardancy), and silver nanoparticles (for its antimicrobial properties). Their life-cycle impact perspectives can be di fferent with their functionality lost in the application phase, for example, substantial loss of silver nanoparticles as the time of laundering, as compared to the well-bond flame retardants to the fibers. This will increase the environmental cost of one technical textile (silver nanoparticle treated) in use phase imposing severe wastewater treatment. In the same way, the behavior of other functionality substance on technical textile (flame retardant treated) needs di fferent considerations for either end-of-life, incineration, or landfill. Thus, the overall results indicated that in the use phase, the life cycle impact of technical textiles is upfront and changes with the variation in number of washes, the types of applied attributional substances, as well as the rate of release. At the "end-of-life" phase, it has been noted that there is no relationship between the two types of technical textiles with respect to environmental impacts.

**Figure 11.** Life cycle of a textile product system as well as its environmental interventions at different phases. Reproduced from (Yasin et al., 2019, [206]).
