Octahydro-tetramethyl-naphthalenyl-ethanone (OTNE) is a synthetic fragrance ingredient. OTNE was evaluated in repeated-dose toxicological studies. Target organs via oral and dermal routes were the liver and skin/liver, respectively. Effects were observed on the thyroid and thyroid hormones, suggesting hypothalamic-pituitary-thyroid axis perturbation. We investigated the molecular initiating event(s) (MIEs), key events (KEs), and adverse outcomes of OTNE-induced thyroid perturbation within an adverse outcome pathway (AOP). Data were generated using new approach methodologies (NAMs) on human, mouse, and/or rat receptors exploring MIEs using in vitro receptor ligand-binding assays for androstane receptor variant 3 (CAR), farnesoid X receptor (FXR), liver X receptor alpha (LXRα), peroxisome proliferator-activated receptors alpha, delta, and gamma (PPARα, δ, and γ), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AhR). These data inform an AOP network where CAR, FXR, and PXR activation serve as MIEs with thyroid perturbation occurring as secondary effects. These data represent a robust evaluation using NAMs for mapping OTNE-induced thyroid effects and identifying activation of receptor-ligand binding as MIEs in lieu of additional in vivo experimentation. These data indicate the observed thyroid effects are secondary to liver effects and the thyroid effects, therefore, should not be the basis for assessing potential OTNE-induced human health hazards. •OTNE induced dose-dependent agonist increases on human/mouse CAR variant 3 and human/mouse/rat FXR and PXR.•OTNE did not exhibit agonist or antagonist activity on human/mouse/rat LXRα and PPARα, γ, or δ, or human/rat AhR.•OTNE caused statistically significant increases in liver weight following repeated oral and dermal administration in rodents.•OTNE caused statistically significant increases in rat thyroid weights after repeated oral administration in an EOGRT study.•In vitro studies assessing MIEs within an AOP suggests OTNE produces thyroid effects in rodent studies via secondary effects on the liver.