Table 2:

in vitro toxicity and toxicokinetic testing

Toxicological and Toxicokinetic Responses	Assay	Assay Endpoints	Purpose	Assay outcomes	Availability/References
Developmental Toxicity	Zebrafish embryo assay	Developmental progression	Assess potential teratogenicity	Zebrafish point of departure (POD) and development information	Internal EPA assay doi:10.1016/j.reprotox.2011.10.018
Immunotoxicity	Bioseek Diversity Plus	Protein biomarkers across multiple primary cell types	Measure potential disease and immune responses	148 individual assays which report hitcall and AC50	Commercial doi:10.1177/1087057109345; doi:10.1038/nbt.2914
Developmental Neurotoxicity	Microelectrode array assay (rat primary neurons)	Neuronal electrical activity	Impacts on development of functional neural networks	17 different parameters measured to culminate in one in vitro POD	Internal EPA assay doi:10.1007/s00204-017-203505
Endocrine Disruption	ACEA real-time cell proliferation assay (T47D), plus selected endpoints from Attagene	Estrogen-dependent cell proliferation	Functional measurement of estrogen receptor activity	2 individual assays which report hitcall and AC50	Commercial doi:10.1021/tx400117y; doi:10.1016/j.tiv.2005.12.008
General Toxicity / Bioactivity	Attagene cis- and trans-Factorial assays	Nuclear receptor and other transcription factor activation in HepG2 cells	Activation of transcription factors involved in critical cell pathways and stress responses	81 individual assays which report hitcall and AC50	Commercial doi:10.1038/nmeh.1186; doi:10.1021/tx900325g
	High-throughput transcriptomic assay	Cellular mRNA, 2D HepaRG and U2OS cells	Measures changes in important biological pathways	1000s of genes which are used to derive an in vitro POD	Internal EPA assay with contract support doi:10.1371/journal.pone.0178302
	High-throughput phenotypic profiling	Nuclear, endoplasmic reticulum, nucleoli, golgi, plasma membrane, cytoskeleton, and mitochondria morphology, U2OS and MCF7 cells	Changes in cellular organelles and general morphology	Cell painting (CP) and cell viability (CV) PODs. 1300 features aggregated into 49 categories which are used to derive the CP POD.	Internal EPA assay doi:10.1177/2472555220928004; doi:10.1016/j.taap.2019.114876; doi:10.1038/nprot.2016.105
Intrinsic hepatic clearance	Hepatocyte stability assays	Time course metabolism of parent chemical in primary human hepatocytes	Measure metabolic breakdown by the liver	Clearance (Cl)	Commercial and Internal EPA and NTP assay doi:10.1124/dmd.30.8.892
Plasma protein binding	Ultra centrifugation assay	Fraction of chemical not bound to plasma protein	Measure amount of free chemical in the blood	Fraction unbound	Commercial and Internal EPA assay doi:10.1002/jps.21317