Resource Summary:Scientific confidence frameworks (SCFs) are alternatives to traditional validation for new approach methodologies (NAMs). The SCFs adapted by the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) and the American Chemistry Council (ACC) both address inference performance—the ability of NAMs to predict or inform the biological effect of interest. Inference performance is a distinct evaluation procedure in ACC’s SCF but is blended into several steps of ICCVAM’s SCF. Here, we first reproduce the previously derived human relevant potency threshold (HRPT) for the estrogen receptor alpha (ERα) agonism of Borgert et al. (2018) using guideline and guideline-like studies; we found that a HRPT of 1 to 10–1 positively and consistently predicted clinical endometrial and endocervical effects. We next mapped inference performance to ICCVAM’s SCF and found that it can be used as an effective initial screen prior to performing more detailed characterizations in their SCF. We first conclude that a HRPT for ERα agonism of 10–2 to 10–4 is a health-protective NAM based on an established mode of action that could potentially be used in early screening, much like the threshold of toxicological concern. We then conclude that inference performance is a core requirement for SCFs.

Resource Summary:IARC has begun using ToxCast/Tox21 data in efforts to represent key characteristics of carcinogens to organize and weigh mechanistic evidence in cancer hazard determinations and this implicit inference approach also is being considered by USEPA. To determine how well ToxCast/Tox21 data can explicitly predict cancer hazard, this approach was evaluated with statistical analyses and machine learning prediction algorithms. Substances USEPA previously classified as having cancer hazard potential were designated as positives and substances not posing a carcinogenic hazard were designated as negatives. Then ToxCast/Tox21 data were analyzed both with and without adjusting for the cytotoxicity burst effect commonly observed in such assays. Using the same assignments as IARC of ToxCast/Tox21 assays to the seven key characteristics of carcinogens, the ability to predict cancer hazard for each key characteristic, alone or in combination, was found to be no better than chance. Hence, we have little scientific confidence in IARC's inference models derived from current ToxCast/Tox21 assays for key characteristics to predict cancer. This finding supports the need for a more rigorous mode-of-action pathway-based framework to organize, evaluate, and integrate mechanistic evidence with animal toxicity, epidemiological investigations, and knowledge of exposure and dosimetry to evaluate potential carcinogenic hazards and risks to humans.

Resource Summary:The endocrine system operates via ligand-receptor interactions, where ligand potency (affinity × efficacy) and concentration dictate physiological effects, governed by mass action laws relating affinity to receptor occupancy. Previously, we established a Human-Relevant Potency Threshold (HRPT) for ERα agonists at 1/10,000 the potency of 17β-estradiol (E2), as weak ligands (e.g., from botanicals) lack clinically observable estrogenic effects. Here, we hypothesize the HRPT stems from competition with the endogenous metabolic milieu (hormone precursors, metabolites). Fractional ERα occupancy calculations, incorporating normal circulating E2 and other endogenous ligands, show that exogenous ligands with potencies greater than 1/1,000 relative to 17β-estradiol may compete effectively against individual or mixed milieu components; those with relative potencies less than 1/1,000 cannot. Thus, the HRPT proposed originally (1/10,000 of E2) is conservative, arguing against estrogenic disruption potential. For potencies above 1/1,000 of E2, disruption is equivocal, requiring corroboration. Critically, the endogenous milieu drives the HRPT for both agonists and antagonists, providing a kinetics-based mechanistic rationale using established metabolic properties.

Resource Summary:The International Agency for Research on Cancer's (IARC) key characteristics of carcinogens (KCCs) have sparked debate, offering a framework to organize data for simplifying cancer hazard identification and risk assessment. However, guidance for integrating KCC evidence into decisions is limited and inconsistent; examples of KCC use show failure to predict classifications and can lead to mislabeling compared to IARC and U.S. EPA outcomes. Modern understanding of chemical carcinogenesis—encompassing multifaceted molecular, cellular, and dose-dependent processes—far surpasses knowledge from decades ago, demanding application in risk assessments to safeguard public health while enabling chemical benefits. Scientific progress in cancer and risk evaluation trends toward greater complexity, emphasizing hypothesis testing and causal analysis; simplistic approaches like the Delaney Clause or standalone KCCs hinder this. Becker et al. advocate returning to hypothesis generation and testing via four archetypal chemical carcinogenesis modes of action (MOA), exemplified by an advanced causal evidence scoring system that compares support for each MOA, identifies the probable operative one, and advances it with toxicity values for risk assessment.

Resource Summary:The Consensus Key Characteristics Approach (CKCA) applied to carcinogens or endocrine disruptors is either: 1. Unscientific: as it cannot be falsified (all endogenous and biologically relevant chemicals are carcinogens and endocrine disruptors). OR 2. False: CKCA rewards confirmation bias. Evidence of meeting 1 single criterion is sufficient to be labeled a Carcinogen or Endocrine Disruptor. Proponents specifically state the following question should be answered: “Does exposure to the agent induce end points associated with one or more specific key characteristic properties of carcinogens?

Resource Summary:Regulatory WoE assessments are clear that GLY does not show EATS modalities. • The methodological deficiencies of KCs explain the incorrect classification of glyphosate’s potential as an ED using this novel approach compared to the application of wellestablished WoE approaches developed by regulatory agencies over the past decade. • ED KCs like other KCs have not been validated against negative controls and the GLY data is an ideal example of a negative control and demonstrates how the KC approach lacks a means to get to a negative conclusion about a chemical’s ED properties.

Resource Summary: For more than a decade, weight of evidence (WoE) evaluations have been the standard method for determining whether a chemical meets the definition of an endocrine disrupting chemical (EDC). WoE methods consider all data pertinent to satisfying the EDC definition and evaluating those data with respect to relevance, reliability, strength, and coherence with established endocrine physiology and pharmacology. A new approach for identifying EDC hazards has been proposed that organizes and evaluates data according to ten so-called “Key Characteristics (KCs) of EDCs”.