TABLE 4.

Advantages and limitations of animal models and NAMs

	Advantages	Limitations
Animal models	Effect on entire body assessed (i.e., histology, clinical chemistry)	Translation issue; does not identify all adverse events seen in humans nor drugs that prove to be nonefficacious in humans
	Good safety record for phase I clinical trials	Optimal test species and strain not always clear
	Many disease models exist for efficacy assessment	Some models do not replicate human diseases accurately, and this can lead to clinical failures
	Identify mechanistic issues	Cost and throughput an issue for classic toxicology studies, to model efficacy in disease models, and when a fast assessment is needed (e.g., prepared food on the market potentially showing unexpected adverse events)
	Can study developmental stages although may not mirror human totally	Limited genetic variability in inbred strains and genetic drift in animal colonies
	Enables studies of medical devices	Irreproducibility is sometimes an issue
NAMs	May address the 3Rs	Animals may need to be euthanized to provide cells for in vitro systems or other NAMs
In vitro assays	Use of human cells may provide better prediction of human responses	Translation issue; does not identify all types of injury within a tissue, adaptive responses, or interactions among body systems
	May enable precision medicine by studying donors with unique characteristics	May reflect the response of an individual donor versus population; must investigate how many donors required; in vivo human studies do not predict all other humans
	Can control the test environment (e.g., dose of drug, duration of exposure)	May be difficult to keep cells differentiated, particularly if trying to mimic an in vivo chronic study
	Can be easier to study mechanistic questions of toxicity and efficacy	Fresh human cells may be difficult to obtain, particularly for complex platforms with multiple cell types (e.g., liver)
	Faster and can be less expensive than in vivo studies	Cost and throughput may depend on the question being asked; complex NAMs are expensive, usually just address one organ/tissue type, and are often low throughput
	May enable toxicity and efficacy testing in disease models	May be difficult to replicate disease models in vitro
	A relatively small amount of test materials is needed	Difficult to replicate responses that involve multiple cell types (e.g., immune cells and liver cells)
		At this time, cannot study all organs/tissues in the body
		Irreproducibility is an issue
		Do not replicate complexity of human system
		Difficult to replicate pregnancy and developmental stages
		Domain of applicability might be limited
		Physical/chemical properties of substances might not be compatible with assay
		Not able to replicate complex human/animal traits, like behavior
	Can identify a bioactivity point of departure	Unclear how many cell types are needed to provide sensitivity/confidence that toxicity has been adequately evaluated
		Limited assessment over time versus in vivo studies (e.g., disease progression)
In silico assays	Might avoid the need for any new biologic testing	Critical that models are trained and tested accurately
	Fast and expensive once models are built	May be difficult to obtain sufficient data
	Flexible in terms of what models can be built (e.g., disease, normal)	May be limited to chemical structure space
		Cannot always predict metabolic breakdown of compounds
		At this time, models do not exist for all organs and tissues
		Do not replicate complexity of human systems