Table 1.

Evaluation of suitability of cytotoxicity assays for SbD hazard testing.

Performance Criteria	Mitochondrial Activity (MTT, MTS, XTT, WST-1, Alamar Blue)	Cell Membrane Integrity (LDH)	Cell Membrane Integrity Staining (Trypan Blue, Propidium Iodide, Annexin V)	Lysosomal Integrity (Neutral Red Uptake)	Caspase 3/7 Assay
Simplicity and cost	Easy and cost-effective, commercial kits available.	Easy and cost-effective, commercial kits available.	Microscopic evaluation is time-consuming. Using flow cytometry increases time efficiency.	Easy and cost-effective, commercial kits available.	Easy and cost-effective, commercial kits available.
Predictivity (Sensitivity and Specificity)	Depends on the mechanism of toxicity of the particle, and the cell type used [110]. Macrophages seem more sensitive [114,117]. Assay better equipped to detect cytotoxicity of ion-shedding NMs [100]. Possibly suitable for making accurate rankings in toxicity [114].	Depends on the mechanism of toxicity of the particle, and the cell type used. LDH results have been shown to correlate with in vivo results for ion shedding NMs [112] as well as poorly soluble NMs [113].	Not assessed for NMs specifically.	Not assessed for NMs specifically	Not assessed for NMs specifically.
Robustness	For MTS assay, decent robustness but depending on cell type used [107], and only when interferences are correctly avoided [100,108]. More elaborate SOPs and harmonization between labs enhance assay robustness [52,102].	Similar robustness as MTS assay [108].	Not assessed for NMs specifically.	Not assessed for NMs specifically.	One study showed high inter-laboratory variability [100].
Compatibility	Many NMs interfere with the substrate, the product, or the optical readout. Can be overcome by washing cells before incubation with reagent, and centrifugation to get rid of NMs [100,108].	Many NMs interfere with the enzyme, the reagent, or the optical readout. Can be overcome via centrifugation. Washing not possible as LDH is measured in supernatant.	NMs may interfere with the dye.	NMs may interfere with the dye.	NMs may interfere with the dye or the readout.
Readiness	ISO protocol for MTS assay.	No NM-specific standardized protocol available.	No NM-specific standardized protocol available.	No NM-specific standardized protocol available.	No NM-specific standardized protocol available.