Table 2.

Key hallmarks of NGTxC and representative international standardized tests, that can address these hallmarks

Block	Key hallmarks /characteristics (Mechanism/ mode of action)	IATA modules	Readiness level/currently used in hazard assessmenta	Selected examples of representative standardized (or commonly used) assays
1a	Receptor binding and activation also as part of hormone-mediated processes	MIE
	Testing for receptor binding		A: Adopted as OECD, EU and US EPA TG	Oestrogen Receptor (ER) Binding Assay OECD TG 493 US EPA 890.1250
	Testing for receptor agonism/antagonism		A: Adopted as OECD, EU and US EPA TG	Oestrogen receptor (ER) transactivation assays OECD TG 455 US EPA 890.1300
			A: Adopted as OECD TG	Androgen receptor transactivation assay OECD TG 458
			B: Undergoing ISO validation 2019-current	Aryl hydrocarbon receptor (AhR) transactivation assay, US EPA METHOD 4435 (2008); JIS K 0463 (2009)(Aarts et al. 1995; Garrison et al. 1996; Han et al. 2004)
	Alteration of hormone synthesis		A: Adopted as OECD and US EPA TG	Steroidogenesis H295R Assay OECD TG 456 US EPA 890.1550
			A: Adopted as US EPA OCSPP HTGb	Aromatase US EPA 890.1200
1b	CYP P450 induction	MIE	B: On OECD TGP workplan: validated, draft OECD TG under consideration	HepRG® CYP induction test method (Bernasconi et al. 2019)
2	Cell proliferation	(Sustained) proliferation, mitogenic signalling	No in vitro TGs, no in vitro assays on the OECD TGP workplan	Repeated Dose 90-Day Oral Toxicity Study in Rodents OECD TG 408, US EPA 870.3100
			A: Adopted as OECD and US EPA TG In vivo
			Test methods currently used to contribute to regulatory WoE assessment	Cell proliferation in vivo (Ki-67) (Jouinot and Bertherat 2018) (Wood et al. 2015) Hepatic DNA synthesis and cell proliferation (US FDA 2018; Wood et al. 2015) Bromodeoxyuridine (BrdU) assays in vivo
3	Cell transformation	Change in morphology	A: Adopted as OECD and US EPA TG	TG 408 US EPA 870.3100 90 day in vivo histopathological features
			A: ICH guideline	ICH S6 PART 1(ICH 2011a, b; Wang et al. 2010)
			B: Validated assays not adopted as OECD TG; OECD GD (OECD 2015, 2017b)	Bhas 42 Cell Transformation Assay (OECD 2017b)
				Syrian Hamster Embryo (SHE) Cell Transformation Assay(OECD 2015)
				BALB/c 3T3 Transformation Assay
4	Gap junction intercellular communication	Change in morphology, mitogenic signalling	No TGs, no assays on the OECD TGP workplan
5	Indicators of oxidative stress	Inflammation, immune response, Cell injury	B: Assays in (pre) validation and on the OECD TGP workplan	Reactive Oxygen Species (ROS) generation assay (phototoxicity)
				Toxtracker (Hendriks et al. 2012, 2016)
			C: Test methods currently used to contribute to regulatory WoE assessment	HepG2 peroxide formation
				Malondialdehyde (MDA)—Thiobarbituric Acid Reactive Substances (TBARs) ex vivo assays
				in vivo assays, ICH (ICH 2005a, b)
6	Immunosuppression/ evasion	Immune response	A: ICH S8 (ICH 2005a, b) and OECD TGs (TG 443)	NK cell/Host resistance and others
			A: ICH S8(ICH 2005a, b)	TDAR- immunosuppression tier 1 screening
			A: TG 443(OECD 2018c) US EPA, OPPTSc 870.7800 Immunotoxicity. (1996) US EPA, OCSPP Test Guideline No. 870. 7800). (2013)
7	Gene expression and cell signalling pathways	Inflammation, immune response, mitogenic signalling	B: Assays in (pre) validation and on the OECD TGP workplan	Toxtracker (Henriks et al. 2012, 2016)
8	Increased resistance to apoptotic cell death	Cell injury	A: OECD TG 408	Histopathology from TG 408 90 days studies (standard picnotic nuclei)
				Histopathology from single or repeated dose studies (eg TG 408 90 day studies): Use of special stains on ex vivo Tissue slices Hematoxilin/Eosin staining Fluoro-jade staining
				Caspase 3/7 activation in vivo
				DNA fragmentation
9	Pathogenic angiogenesis and neoangiogenesis	Change in morphology	No TGs, no assays on the OECD TGP workplan
10	Genetic Instability (Disturbed DNA repair, quick establishment of mutations = “mutator phenotype”)	Change in morphology Cell injury	No TGs, no assays on the OECD TGP workplan
11	Cellular senescence/ telomerase	Change in morphology, Cell injury	No TGs, no assays on the OECD TGP workplan
12	Metastasis (migration, intra- and extravasation), survival outside of original tissue	Tumour	No TGs, no assays on the OECD TGP workplan
13	Epigenetic mechanisms and associated genetic instability	All	No TGs, no assays on the OECD TGP workplan	(Greally and Jacobs 2013) reviews how TGs could be augmented

^aTG readiness levels A, B, C are described in the methodology section

^bOCSPP HTG: US EPA Office of Chemical Safety and Pollution Prevention Harmonized Test Guidelines

^cOPPTS: US EPA Office of Prevention, Pesticides and Toxic Substances