Table A.20.

Empirical support for the KERs; WoE analysis

3 Empirical support for KERs	Defining question	High (strong)	Moderate	Low (weak)
	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?	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 to KE1	Moderate	With the chemical tool paraquat, studies are mainly conducted at fixed doses and dose relationships studies are very limited for the O2 production, which is relevant for the intra MIE dose relationship (Cantu et al., 2011; Mitra et al. 2011; Dranka et al., 2012; Huang et al., 2012; Rodriguez‐Rocha et al., 2013; De Oliveira et al., 2016). However, intra KE1 dose relationship is observable for ROS production/lipid peroxidation using the same stressor compound (McCormack et al., 2005; Mitra et al., 2011; Lopert et al., 2012; de Oliveira et al., 2016). In‐vitro, high concentrations of PQ showing activation of the MIE are showing the most pronounced ROS production indicating that a concordance in dose and response relationship exists between the MIE and KE1 and cell death (Chau et al., 2009; Lopert et al., 2012; Rodriguez‐Rocha et al. 2013; de Oliveira et al., 2016). Temporal relationship between MIE and KE1 is indistinguishable due to the fast conversion of O2 to H2O2 and other ROS species (Cohen and Doherty, 1987). However, when considering cell death as the observational end point, a dose response and time concordance exists. PQ (0.1–1 mM) induces O2°− and H2O2 production within minutes in isolated mitochondria and mitochondrial brain fraction (Castello et al., 2007; Cochemé and Murphy, 2008), while in cells this process is detectable after 4–6 h from the exposure (Cantu et al., 2011; Dranka et al. 2012; Huang et al., 2012; Rodriguez‐Rocha et‐al., 2013). At these time points no death is generally detected. In‐vivo, there is limited evidence of intra MIE dose relationship with paraquat and temporal concordance cannot be defined as the experiments are conducted at single time point descriptive assessment (Mitra et al., 2011). However, circumstantial evidences are supported by the knowledge on the chronic and progressive nature of parkinsonian syndromes
KE1 to KE2 Mitochondrial dysfunction (ROS production) results in impaired proteostasis	Low	Evidence is provided that exposure to PQ and deficiency of DJ‐1 might cooperatively induce mitochondrial dysfunction resulting in ATP depletion and contribute to proteasome dysfunction in mouse brain (Yang et al., 2007). Moreover, exacerbation of Paraquat effect on the autophagic degradation pathway is observed in an in vitro system with silenced DJ‐1 (González‐Polo et al., 2009). In C57BL/6J mice 10 mg/kg i.p. for 1–5 doses, increased level of lipid peroxides in ventral midbrain was associated impaired proteostasis (Prasad et al., 2007) Temporal and dose concordance cannot be elaborated from in vivo studies as they are conducted at the same dose and observational time‐point. However, in vitro studies are indicative of a temporal and concentration concordance, evidencing concentration‐and/or time‐dependent effects on mitochondrial and proteasome functions (Ding and Keller, 2001; Yang and Tiffany‐Castiglioni, 2007)
KE2 to KE3 Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway	Moderate	The empirical support linking impaired proteostasis with degeneration of DA neurons of the nigrostriatal pathway comes from post‐mortem human evidences in PD patients supporting a causative link between the two key events.. With paraquat, a response concordance was observed in multiple in vivo studies (Manning‐Bog, 2003; Fernagut, 2007; Mitra, 2011).Temporal and dose concordance cannot be elaborated from these studies as they are conducted at the same dose and observational time‐point. Some inconsistencies were observed, i.e. partial effect on proteasomal inhibition which is likely due to compensatory effects and/or lower toxicity of PQ when compared to other chemical stressor (e.g. rotenone, MPTP) In vivo studies with Paraquat are showing a more relevant effect on the ALP and α‐synuclein overexpression with a less evident effect on proteasome inhibition. A dose and temporal concordance was more consistently observed in in vitro studies (Gonzalez‐Polo, 2009; Chinta, 2010)
KE3 <=> KE4 Neuroinflammation	Moderate	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 (Cicchetti et al., 2005; Wu et al., 2005; Purisai et al., 2007; Mitra et al., 2011; Mangano et al., 2012). A similar relationship was observed with compounds like rotenone and MPTP
KE3 to AO Degeneration of DA neurons of nigrostriatal pathway leads to parkinsonian motor symptoms	Moderate to low	PQ is reported to induce motor deficits and loss of nigral dopaminergic neurons in a dose‐(Brooks et al., 1999) and age (Thiruchelvam et al., 2003) dependent manner. The concomitant observation of dopaminergic neuronal loss and parkinsonian motor symptoms has been confirmed by other authors (Cicchetti et al., 2005; Prasad et al., 2009; Mitra et al., 2011). However, at similar doses and experimental design a number of inconsistencies or lack of reproducibility were noted and described in the uncertainties (Smyne et al., 2016). In human (and animal models using rotenone and MPTP), motor symptoms are expected to be clinically visible when striatal dopamine levels drop of approximately 80%, corresponding to a DA neuronal loss of approximately 60% (Bernheimer et al., 1973; Lloyd et al., 1975; Hornykiewicz et al., 1986; Jellinger et al., 2009). This threshold of pathological changes was only achieved when paraquat was administered directly in the SN and the link between neuronal loss and clinical symptoms was empirically consolidated by the following treatment with apomorphine or the concomitant treatment with the MAOB inhibitors (Liou, 1996, 2001). When different routes of administration were applied, neuronal loss was below this pathological threshold, not consistently related to drop in striatal DA and motor symptoms were only occasionally observed (Smyne et al., 2016). Some methodological biases can explain the inconsistency in the lack of reproducibility observed for some experiments conducted in‐vivo with the chemical stressor PQ (Smyne et al., 2016); however, when considering the multiple factors associated with host susceptibility and the complexity of the local disposition of PQ i.e. concentration and activation of PQ in the SNpc and striatum, the biological variability can account for the different outcomes observed in the studies