Technical Report 130

Step IV: Evaluation and summary of the evidence for endocrine activity

(and non-endocrine activity, if available)

The purpose of Step IV of the ECETOC 7SI-ED is to determine whether a substance elicits endocrine activity in Level 2 and 3 assays. Evidence for endocrine activity may also be available from specific diagnostic endpoints included in some Level 4 and Level 5 studies or from assays and studies not mentioned in the OECD CF that provide comparable data (if justification of their comparability is provided). Further parameters from Level 4 and 5 studies (e.g. organ weight and histopathological parameters) may also be relevant to indicate the presence or absence of endocrine activity.

As compared to the corresponding step presented in Section V of ECHA and EFSA (2016), the focus of Step IV of the ECETOC 7SI-ED has been slightly modified to specify that the assays evaluated in this step allow identifying endocrine activity, and not endocrine MoA. MoAs extend to the manifestation of apical effects that are addressed in the studies evaluated in Step III, but not necessarily in the assays evaluated in Step IV. Additionally, Step IV has been expanded to cover not only the evaluation of in vitro and in vivo mechanistic evidence that is relevant to identify endocrine activity, but also available mechanistic evidence that is relevant to identify possible non-endocrine mechanisms that could explain the adverse effects identified in Step III. In Step V, such information may be relevant to identify the most plausible (endocrine or non-endocrine) MoA.

Level 2 assays are in vitro screening assays used for the identification of possible endocrine activity. Positive in vitro test results indicate the possibility of endocrine effects in vivo. However, in vitro assays do not include consideration of toxicokinetics (ADME). Therefore, their ability to predict effects in humans or in environmentally relevant species may be limited (OECD, 2012b). Consideration of toxicokinetics is particularly critical for the evaluation of taxonomic differences in effects. Current in vitro tests covered by the OECD CF are largely based on mammalian systems, but they are considered relevant for vertebrate wildlife species, because many of the assays are based on hormone receptors and molecular mechanisms that are highly conserved across vertebrates (OECD, 2012b). Mechanistic insight into endocrine activity is presently not available for all invertebrate groups (deFur et al., 1999; deFur, 2004; Weltje et al., 2013). Generally, an evaluation of the ecotoxicological relevance of endocrine activity identified in Level 2 for non-mammalian wildlife requires case-by-case expert judgement.

Level 3 assays are in vivo assays that allow screening for possible endocrine activity. While they may encompass apical endpoints, their study design as screening assays does not allow drawing conclusions on the presence of adverse effects that may be indicative of endocrine disrupting properties. The currently available Level 3 in vivo assays are designed to provide a yes/no (qualitative) answer about a substance’s ability to interact with the EATS pathways. These assays are designed to detect alterations in endocrine-sensitive tissues thereby providing alerts to substances with possible endocrine activity (Borgert et al., 2011, 2014; de Peyster and Mihaich, 2014). Therefore, they favour false positives rather than negatives and are in some cases highly sensitised models, e.g. neutered animals without a functional hypothalamic-pituitary-gonadal axis, which are therefore unable to compensate fully for endocrine perturbations.

In evaluating the consistency and coherence of types of activity amongst different assays, it should be taken into account that the routes of exposure applied in different assays may not be representative of normal conditions of use making direct extrapolation for relevance difficult (e.g. intraperitoneal exposure in a study when human exposure is dermal or oral).

OECD GD 150 provides guidance on the interpretation of Level 2 and 3 assays. Each Level 2 in vitro assay is primarily designed to detect a specific endocrine activity (e.g., oestrogen agonism, androgen antagonism, etc.), whilst some Level 3 in vivo ecotoxicological assays may provide information on more than one form of endocrine activity. For instance, the fish screening assays (OECD TG 230 and 229) can provide information on in vivo activity via the EAS pathways, whilst the amphibian metamorphosis assay (OECD TG 231) specifically detects in vivo activity via the T pathway.