Table 3.

Summary of assays used for in vitro toxicological and biological assessment.

	Endpoint	Techniques	Biological System	Test matrix	References
Classical regulatory toxicology	Cytotoxicity
	Induction of cell death	Neutral red uptake (NRU) assay	Mouse fibroblast cells (Balb/c 3T3)	TPM/ACM	[29]
	Chromosome damage
	Formation of micronuclei due to chromosomal loss/damage induced by clastogens or aneugens	In vitro micronucleus (IVMn) assay	Chinese hamster V79 cells	TPM/ACM	[31]
	Mutation
	Induction of mutations in mammalian cells by the test substance	Mouse lymphoma assay (MLA)	Mouse lymphoma L5178Y tkþ/e cells	TPM/ACM	[29]
	Induction of reverse mutations in bacteria	Ames test	Salmonella typhimurium (strains TA98, TA100, TA1535, TA1537 and TA102)	WA/TPM/ACM	[29], [12]
	Cell transformation (tumour promotion)
	Assessment of tumour-promoting potential by an established rodent cell-based cell transformation assay	Bhas 42 cell transformation assay (promotion protocol)	Bhas 42 mouse embryo fibroblast cells	TPM/ACM	[32]; [33]
Contemporary high content screening	Cell health and oxidative stress
	Endpoints assessed: cell count, nuclear size, DNA structure, mitochondrial mass, mitochondrial membrane potential, oxidative stress, glutathione content and cellular ATP	Image analysis based HCS analysis of fluorescently stained cellular and molecular targets, using Cellomics Arrayscan VTI platform.	Normal human bronchial epithelial (NHBE) cells	TPM/ACM	[21]
	DNA damage and stress kinase
	p-H2AX (DNA damage), and phospho-cJun (stress kinase)	As above	NHBE cells	TPM/ACM	[21]
Lung cell toxicity assays	Oxidative stress
	Transcriptional activation of antioxidant response element (ARE)	H292-ARE-Luc2P reporter gene assay	H292-ARE-Luc2P reporter cells	TPM	[21]
	Cytotoxicity
	Evaluation of concentration of the test substance to cause cell death	Neutral red uptake (NRU) assay	NCI-H292 human bronchial epithelial cells	WA	[34]
CVD	Endothelial wound repair
	Inhibition of wound repair in injured cell monolayers	Scratch wound assay	Human umbilical vein endothelial cells (HUVEC)	AqE	[35]

Abbreviations: AqE, aqueous extract; ARE, antioxidant response element; DSB, double-strand break; EC₅₀, smoke concentration that kills 50 % of cells; TPA, TPM, total particulate matter; WA, whole aerosol/smoke.