Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Mixture Risk Assessment of Complex Real-Life Mixtures-The PANORAMIX Project.

Humans are involuntarily exposed to hundreds of chemicals that either contaminate our environment and food or are added intentionally to our daily products. These complex mixtures of chemicals may pose a risk to human health. One of the goals of the European Union's Green Deal and zero-pollution ambition for a toxic-free environment is to tackle the existent gaps in chemical mixture risk assessment by providing scientific grounds that support the implementation of adequate regulatory measures within the EU. We suggest dealing with this challenge by: (1) characterising 'real-life' chemical mixtures and determining to what extent they are transferred from the environment to humans via food and water, and from the mother to the foetus; (2) establishing a high-throughput whole-mixture-based in vitro strategy for screening of real-life complex mixtures of organic chemicals extracted from humans using integrated chemical profiling (suspect screening) together with effect-directed analysis; (3) evaluating which human blood levels of chemical mixtures might be of concern for children's development; and (4) developing a web-based, ready-to-use interface that integrates hazard and exposure data to enable component-based mixture risk estimation. These concepts form the basis of the Green Deal project PANORAMIX, whose ultimate goal is to progress mixture risk assessment of chemicals.

Non-Newly Generated, "Immature" Neurons in the Sheep Brain Are Not Restricted to Cerebral Cortex.

A newly proposed form of brain structural plasticity consists of non-newly generated, "immature" neurons of the adult cerebral cortex. Similar to newly generated neurons, these cells express the cytoskeletal protein Doublecortin (DCX), yet they are generated prenatally and then remain in a state of immaturity for long periods. In rodents, the immature neurons are restricted to the paleocortex, whereas in other mammals, they are also found in neocortex. Here, we analyzed the DCX-expressing cells in the whole sheep brain of both sexes to search for an indicator of structural plasticity at a cellular level in a relatively large-brained, long-living mammal. Brains from adult and newborn sheep (injected with BrdU and analyzed at different survival times) were processed for DCX, cell proliferation markers (Ki-67, BrdU), pallial/subpallial developmental origin (Tbr1, Sp8), and neuronal/glial antigens for phenotype characterization. We found immature-like neurons in the whole sheep cortex and in large populations of DCX-expressing cells within the external capsule and the surrounding gray matter (claustrum and amygdala). BrdU and Ki-67 detection at neonatal and adult ages showed that all of these DCX+ cells were generated during embryogenesis, not after birth. These results show that the adult sheep, unlike rodents, is largely endowed with non-newly generated neurons retaining immature features, suggesting that such plasticity might be particularly important in large-brained, long-living mammals.SIGNIFICANCE STATEMENT Brain plasticity is important in adaptation and brain repair. Structural changes span from synaptic plasticity to adult neurogenesis, the latter being highly reduced in large-brained, long-living mammals (e.g., humans). The cerebral cortex contains "immature" neurons, which are generated prenatally and then remain in an undifferentiated state for long periods, being detectable with markers of immaturity. We studied the distribution and developmental origin of these cells in the whole brain of sheep, relatively large-brained, long-living mammals. In addition to the expected cortical location, we also found populations of non-newly generated neurons in several subcortical regions (external capsule, claustrum, and amygdala). These results suggests that non-neurogenic, parenchymal structural plasticity might be more important in large mammals with respect to adult neurogenesis.