Table 3.

Validated or suggested test approaches.

Method	Principle	Effects Analyzed	Status	Reference
OECD standardized test guidelines for evaluating EDs	Repeated-dose 28-day/90-day study	Body and organ weight, (histo)pathology, clinical chemistry	Harmonized test guidelines approved for regulatory use	[190,191]
In vivo endpoints (to characterize metabolic phenotype)	Glucose and insulin tolerance test (GTT, ITT)	Blood glucose levels are measured upon administration of glucose/insulin	Additional techniques might be added as new endpoints	[199,200]
	Non-targeted metabolomics	Non-targeted liquid chromatography/mass spectrometry (LC/MS)		[196,197,205,206]
	Targeted metabolomics	Triglyceride measurement by gas chromatography
In silico approach	Computerized models (e.g., (Q)SAR) predicting physicochemical, biological, and environmental fate properties based on chemical structure	Interaction of a chemical with a defined biological target (modeling of molecular docking simulations to receptors)	Use for identification of MIEs of AOPs	[178]
Grouping of substances and read-across	Use of relevant information from tested substances to predict the properties of target substances	Alternative approach for filling data gaps	In registrations submitted under the REACH regulation	[204]
In vitro toolbox	AOP-based in vitro assays measuring MIEs or KEs	Combinations of NR activation, gene and protein expression, lipid accumulation, mitochondrial respiration/dysfunction, formation of fatty liver cells	Use for AOPs	[118,178,202]
Transcriptomic signatures	In vitro model	Gene expression markers for accumulation of triglycerides		[203]