Technical Report 130

Step V: Integration of the evidence and evaluation of biological plausibility that adverse effect and endocrine activity are linked by specific endocrine MoA

The purpose of Step V of the ECETOC 7SI-ED is to determine if adverse effects identified in Step III and endocrine activity identified in Step IV are linked by a specific endocrine MoA. The ECHA and EFSA (2016) outline calls for a reasonable degree of proof that adverse effects identified in Step III and the endocrine activity identified in Step IV are linked by a specific endocrine MoA.

As compared to the corresponding step presented in Section V of ECHA and EFSA (2016), the title of Step V of the ECETOC 7SI-ED has been reformulated to highlight that the adverse effects identified in Step III and the endocrine activity identified in Step IV are (or are not) linked by a specific endocrine MoA. Therefore, evidence supporting different endocrine MoAs needs to be evaluated separately in Step V. In accordance with ECHA and EFSA (2016), the biological plausibility for this link is established, i.e. the extent of evidence to support that a specific substance operates via a specific endocrine MoA (Commission, 2016). In the following, specific criteria are presented for the assessment of the extent of evidence for a biologically plausible link.

The ECETOC 7SI-ED uses aspects of the modified Bradford Hill Considerations presented in the updated WHO/IPCS MoA/species concordance framework (Meek et al., 2014a, b) to determine if there is reasonable degree of proof for a biologically plausible link as defined in ECHA and EFSA (2016). The WoE evaluation conducted in Step V (just as in Steps III and IV) should never be based upon a mere weighing up of positive versus negative outcomes.

Biological concordance: There is evidence of an adverse effect(s) that may be indicative of endocrine disrupting properties (Step III) and evidence for endocrine activity (Step IV), and the endocrine activity is known to be associated with the endocrine MoA that is relevant for the adverse effect(s). The pattern of adverse effects identified in Step III and the evidence for endocrine activity identified in Step IV should be indicative of the same (hypothesised) endocrine MoA.

Dose/concentration-response concordance: The endocrine activity (key event(s)) observed in the Level 2 and 3 assays is observed at similar or lower doses/concentrations than the adverse effects that are indicative of endocrine disrupting properties seen in the Level 4 and 5 apical studies. The principles used within the in vitro-in vivo extrapolation model presented by Wetmore et al. (2012) are recommended to address the extrapolation of in vitro to in vivo data.

Plausible key events of the endocrine MoA and temporal association: Plausible key events of the endocrine MoA that are affected by the substance have been identified. If applicable, depending on the design of the available assays and studies, temporal association of the key events and adverse effects can be determined. For instance, if adverse effects only occur in juvenile stages, whereas endocrine activity is only observed in the adult, the link between the two is not biologically plausible.

Essentiality of key events: To establish a MoA, the essentiality of the key events can be demonstrated by showing that the downstream events are absent or reversed if the key event is stopped or prevented. To demonstrate this conclusively, relevant inhibitors, antagonists or agonists can be used.

AOPs, i.e. the evaluation and integration of many different types of chemical and biological information following an MoA-based approach to understanding adverse effects (OECD, 2012d; Munn and Goumenou, 2013), have the potential to aid in the determination of the biological concordance of an endocrine MoA for adverse effects observed in in vivo (eco)toxicological studies. As such, they can help identify if an observed adverse effect can be plausibly linked to an endocrine mechanism. While the term AOP originated in ecotoxicology (Ankley et al., 2010), the AOP approach is equally applicable in toxicology, just as the WHO/IPCS MoA concept that was originally developed for toxicology is also applicable in ecotoxicology (Munn and Goumenou, 2013). The ECETOC Guidance on assessment and application of AOPs relevant to the endocrine system (ECETOC, 2016) presents a detailed scheme of additional considerations for assessing the biological plausibility of a link between adverse effect and endocrine MoA.