Table 4 Toxicological endpoints used and/or recommended in in vitro and in vivo iENM toxicity investigations, and studies of the gut microbiome

 Nanoparticle ion release and accumulation location

Fluorescent labeling of ions [156], ICP-MS

[63, 129]

 Cell proliferation

Cell count using hemocytometer

[128]

 Cellular energetics

WST-1, WST-8, live/dead kit, CellTiter-Glo, XTT, MTS, MTT, NRU, Prestoblue assay

[46, 69, 127]

 ROS generation

Electron paramagnetic resonance, total glutathione content, DCFH-DA assay

[63, 64, 68, 127]

 Cell membrane damage

LDH and trypan blue assay

[59]

 Apoptosis initiation

Annexin V-FITC, monodansylcadaverine staining

[59, 64]

 Necrosis

Sytox red and propidium iodide (PI) staining

[59, 63]

 Pro-inflammatory and inflammatory cytokine release

ELISA, Wester blotting

[129]

 DNA damage

Fpg-modified comet assay

[62, 68]

 Brush border morphology

Immunocytochemistry, electron microscopy (TEM and SEM)

[66]

 Barrier integrity

Trans-epithelial electrical resistance measurement

[46, 128]

 Barrier permeability

Dextran-FITC and Lucifer yellow transport

[46, 128]

 Gene expression

qRT-PCR

[46, 64, 66, 68]

 Coefficients of organs

Ratio of tissue (wet weight) to body weight

[21, 85]

 Changes in tissues

Histopathological evaluation

[48, 123, 149]

 Testicular toxicity

Sperm count, motility and % abnormal sperms

[65]

 Tissue accumulation

ICMP-MS or ICP-AES

[100]

 Reductive stress

GSH/GSSG ratio in plasma

[96]

 Tissue function

Blood biochemical and hematological analysis

[123, 149]

 Inflammatory cells quantification in blood and the GI segment of interest

Flow cytometry, imaging flow cytometry^a

[48, 50]

 Apoptosis in the GI segment of interest

TUNEL assay

[81, 125]

 Cytokine release in blood and the GI segment of interest

ELISA (IL-1β, IL-2, IL-6, TNF-α, IFN-γ, IL-8, IL-10, IL-17 and GM-CSF), Western blotting

[48, 49]

 Tumor progression biomarkers in colon tissue

Immunohistochemistry (COX2, β-catenin and Ki67), ELISA^a, Western blotting^a

[49]

 Intestinal permeability

⁵¹Cr-EDTA radioactivity

[50]

 Aberrant crypts formation in the GI tract

Bird’s procedure [122]

[50]

 Local tissue concentration

Micro X-ray fluorescence, NanoSIMS imaging

[46, 50]

 Gut microbiome composition

16S rRNA pyrosequencing, Shotgun metagenomic sequencing^a, Microbial transcriptomics^a

[124]

 Gas production

Gas chromatography

[45]

 Fatty acid production

Fatty acid methyl ester analysis

[45]

 Microbiome diversity

16S rRNA 454 pyrosequencing, Shotgun metagenomic sequencing^a, Microbial transcriptomics^a

[45, 124]