Table 2.

Key questions and example secondary questions by pillar

Pillar	Key/primary question	Example secondary questions	Example assessment/tests/strategies
Bioavailability	Does the substance access biological systems?	Is this substance bio-persistent or bio-accumulative? Does the substance produce a persistent metabolite? How much of the substance is bioavailable in various tissues, including locally to epithelial surfaces? Does the substance enter circulation? Can the substance enter target cells/tissues (eg, cross the blood-brain barrier)?	Physicochemical properties (eg, molecular weight, solubility, pKa, logP) ADME
Bioactivity	Does the substance induce a host response?	Is the effect measurable? In what tissues/cells does the bioactivity occur? At what dose does the bioactivity occur? Is the host response specific or nonspecific? Are there consequences of this response in other organ systems?	Receptor screens QSAR and/or read-across approaches Cytotoxicity Key characteristics-based assessments
Adversity	Does the substances have the potential to cause harm?	How does the bioactivity relate to known mechanisms or modes of action of toxicity? Is the effect reversible? Is there an adaptive response after the first dose/exposure? Are defined key characteristics of toxicity observed?	Functional agonist or antagonist activity Cell/tissue health Organ system health AOP-based assessments
Susceptibility	Is there differential sensitivity to potential harm within a population?	Is there an increased risk for adversity relative to the general population that is due to intrinsic factors such as Particular life stages (ie, developing fetus, puberty, etc.)? Certain genotypes/genetic polymorphisms?	DART and DNT testing Genetically diverse cell-based testing systems Integration with other relevant hazard information