Table 8 Parameters and frequently employed assays for nanotoxicity assessment

Cytotoxicity

Apoptosis and necrosis

Annexin-V coupled with propidium iodide

CNTs are inappropriate for MTT evaluation because of their interference and possible false outcomes

Possibly, the concentration or dose employed for testing is influential towards the test results

Due to limitations of MTT assay, Flow cytometry and Annexin-V coupled with propidium iodide are the appropriate options

[262, 266]

Cell death determination

Flow cytometry

Cell proliferation

Colony-forming efficiency assay

Cell viability and altered metabolic activity

MTT/WST-1 assay + colorimetric assay using Alamar blue dye

Cell membrane integrity

LDH assay

Cell morphology assessment

Phase-contrast microscopy

Epithelial cell damage

Impedance based measurements

Inflammatory response

Inflammatory markers (cytokines, chemokines and other proteinaceous markers)

ELISA + cytokines determination in human peripheral blood mononuclear cells

Higher doses of NPs could cause cell lysis, which ultimately reduces the actual levels of inflammatory markers. Thus, the chances of false (underestimated) results are high

Metallic, silica NPs and CNTs show this type of interference

[265, 266, 295]

Genetic toxicity

Genetic material damage

Ames assay + mammalian cell micro-nuclear assay

Nanofibers induced genotoxicity is precipitable through mammalian assay but not with conventional Ames assay due to interference with results

[265, 271]

Oxidative stress

Evaluation of ROS

FRAS assay + nitric oxide evaluation + fluorescent or non-fluorescent markers i.e. dichloroflorescein

Interference of MWCNTs, TiO₂ NPs, Iron oxide NPs and graphene-based NPs with probe (fluorescent or non-fluorescent marking) method triggers the false results

[262]

Carcinogenicity

Carcinogenic potential

Colony transformation assay (CTA) + BALB/c 3T3 cell transformation assay + syrian hamster embryo assay

This technique can determine both genotoxic along non-genotoxic carcinogenicity

However, the “gold standard” method for NPs carcinogenicity evaluation is assessment in Laboratory animals (In vivo experimental model)

[272]