Table A.12.

Empirical support for the KERs; WoE analysis

Empirical support for KERs	Defining question: Does the empirical evidence support that a change in the KEup leads to an appropriate change in the KEdown? Does KEup occur at lower doses and earlier time points than KEdown and is the incidence of KEup higher than that for KEdown? Are inconsistencies in empirical support cross taxa, species and stressors that don't align with expected pattern of hypothesised AOP?	High (Strong)	Moderate	Low (weak)
		Multiple studies showing dependent change in both exposure to a wide range of specific stressors (extensive evidence for temporal, dose–response and incidence concordance) and no or few critical data gaps or conflicting data	Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc	Limited or no studies reporting dependent change in both events following exposure to a specific stressor (i.e. endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don't align with expected pattern for hypothesised AOP
MIE => KE1 Binding of inhibitor to NADH‐ubiquinone oxidoreductase leads to partial or total inhibition of complex I	Strong	Rationale: The inhibition of complex I is well documented in a variety of studies using isolated mitochondria or cells as well as in in vivo experiments and in human post mortem PD brains. In many experiments using different inhibitors i.e. rotenone and MPTP, the observed relationship between the two events was temporal, response and dose concordant (Grivennikova et al., 1997; Barrientos and Moraes, 1999; Okun et al., 1999; Betarbet et al., 2000, 2006; Koopman et al., 2007; Choi et al., 2008)
KE1 => KE2 Inhibition of complex I leads to mitochondrial dysfunction	Strong	Rationale: There is a large amount of studies showing that the inhibition of CI inhibition results in mitochondrial dysfunctions in a response and dose dependent manner (Barriento and Moraes, 1999)
KE2 => KE3 Mitochondrial dysfunction results in impaired proteostasis	Strong	Rationale: Based on the existing in vitro and in vivo data it is suggested that mitochondrial dysfunction impairs protein homeostasis (impairment of the UPS and ALP system) through oxidative and nitrosative stress resulting in accumulation of misfolded proteins (including α‐synuclein), disruption of microtubule assembly and damaged intracellular transport of proteins and cell organelles. A number of studies performed with chemical stressors showed evidence of temporal, response and dose concordance (Chou et al., 2010; Betarbet et al., 2000, 2006; Fornai et al., 2005)
KE2 => KE4 Mitochondrial dysfunction directly leads to degeneration of DA neurons of nigrostriatal pathway	Strong	Rationale: Multiple in vitro studies indicate dose and response–response concordance. As most of the studies were conducted in vitro, the temporal concordance is difficult to establish; however, can be expected based on the well know temporal sequence of the two KEs. (Swedlow et al., 1996; Jha et al., 2000; Sherer et al., 2003, 2007; Chinta et al., 2006; Marella et al., 2008; Hajieva et al., 2009; Du et al., 2010; Jana et al., 2011; Wen et al., 2011; Choi et al., 2014; Park et al., 2014)
KE3 => KE4 Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway	Strong	Rationale: The empirical support linking impaired proteostasis with degeneration of DA neurons of the nigrostriatal pathway is strong and comes from in‐vivo and in‐vitro studies performed with different stressor (i.e. Rotenone, MPTP or proteasome inhibitors) and post‐mortem human evidences in PD patients supporting a causative link between the two key events. Temporal, effect and dose concordance was established in a number of experiments (Betabret et al., 2000, 2006; Fornai et al., 2003, 2005)
KE4 <=> KE5 Neuroinflammation directly leads to degeneration of DA neurons of the nigrostriatal pathway	Moderate	Rationale: multiple in vivo and in vitro experiments support the link between neuroinflammation and degeneration of DA neurons in the nigrostriatal pathway as well as vice versa. The observation of concomitant presence of glial and astrocytic cells and degenerated/degenerating DA neurons is also reported in many studies with a good temporal and response concordance
KE4 => AO Degeneration of DA neurons of nigrostriatal pathway leads to parkinsonian motor symptoms	Strong	Rationale: The experimental support linking the degeneration of DA neurons of nigrostriatal pathways with the manifestation of motor symptoms of PD comes from human in vivo observations as well as from monkey, mice and rat in vivo models exposed to an experimental toxin i.e. rotenone and MPTP. Observations in human allow defining correlation between the levels of striatal DA with the onset of motor dysfunction (Bernheimer et al., 1973; Lloyd et al., 1975; Hornykiewicz et al., 1986). Temporal, effect and dose concordance comes from studies performed in multiple animal species following administration of rotenone and MPTP (Lloyd et al., 1975; Bezard et al., 2001; Petroske et al., 2001; Alvarez‐Fischer et al., 2008; Cannon et al., 2009; Blesa et al., 2012)