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.

Update of the DevTox data database for harmonized risk assessment and alternative methodologies in developmental toxicology: Report of the 9th Berlin Workshop on Developmental Toxicity.

Representatives of applied science (e.g. governmental organizations, academia, and industry) met to discuss the progress towards a harmonized human health risk assessment in developmental toxicology of plant protection products, biocidal products, and other environmental chemicals at the 9th Berlin Workshop on Developmental Toxicity held in September 2018. Within the focus of the scientific discussion were the future of in-vitro methods for developmental and reproductive toxicology, the potential relevance of alternative species in testing of developmental effects, and risk and hazard assessment of developmental and endocrine effects. Furthermore, the need for a harmonized terminology for classification of anomalies in laboratory animals in developmental toxicity studies aiming for human health risk assessment was determined. Here, the DevTox database was identified as an extremely valuable tool. Overall, the participants agreed that still one of the biggest challenges for testing developmental toxicity in the 21st century is the development of animal-free test strategies and alternatives to animal testing that could provide human-relevant information in a rapid, efficient, and mechanistically informative manner.

Report from the BfR expert hearing on practicability of hormonal measurements: recommendations for experimental design of toxicological studies with integrated hormonal end points.

This publication summarizes discussions that were held during an international expert hearing organized by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany, in October 2017. The expert hearing was dedicated to providing practical guidance for the measurement of circulating hormones in regulatory toxicology studies. Adequate measurements of circulating hormones have become more important given the regulatory requirement to assess the potential for endocrine disrupting properties for all substances covered by the plant protection products and biocidal products regulations in the European Union (EU). The main focus was the hypothalamus-pituitary-thyroid axis (HPT) and the hypothalamus-pituitary-gonadal axis (HPG). Insulin, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH) and vitamins A and D were also discussed. During the hearing, the experts agreed on specific recommendations for design, conduct and evaluation of acceptability of studies measuring thyroid hormones, thyroid stimulating hormone and reproductive hormones as well as provided some recommendations for insulin and IGF-1. Experts concluded that hormonal measurements as part of the test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) were necessary on the condition that quality criteria to guarantee reliability and reproducibility of measurements are adhered to. Inclusion of the female reproductive hormones in OECD TGs was not recommended unless the design of the study was modified to appropriately measure hormone concentrations. The current report aims at promoting standardization of the experimental designs of hormonal assays to allow their integration in OECD TGs and highlights research needs for better identification of endocrine disruptors using hormone measurements.

The mTORC1-autophagy pathway is a target for senescent cell elimination.

Cellular senescence has recently been established as a key driver of organismal ageing. The state of senescence is controlled by extensive rewiring of signalling pathways, at the heart of which lies the mammalian Target of Rapamycin Complex I (mTORC1). Here we discuss recent publications aiming to establish the mechanisms by which mTORC1 drives the senescence program. In particular, we highlight our data indicating that mTORC1 can be used as a target for senescence cell elimination in vitro. Suppression of mTORC1 is known to extend lifespan of yeast, worms, flies and some mouse models and our proof-of-concept experiments suggest that it can also act by reducing senescent cell load in vivo.

The bystander effect contributes to the accumulation of senescent cells in vivo.

Senescent cells accumulate with age in multiple tissues and may cause age-associated disease and functional decline. In vitro, senescent cells induce senescence in bystander cells. To see how important this bystander effect may be for accumulation of senescent cells in vivo, we xenotransplanted senescent cells into skeletal muscle and skin of immunocompromised NSG mice. 3 weeks after the last transplantation, mouse dermal fibroblasts and myofibres displayed multiple senescence markers in the vicinity of transplanted senescent cells, but not where non-senescent or no cells were injected. Adjacent to injected senescent cells, the magnitude of the bystander effect was similar to the increase in senescence markers in myofibres between 8 and 32 months of age. The age-associated increase of senescence markers in muscle correlated with fibre thinning, a widely used marker of muscle aging and sarcopenia. Senescent cell transplantation resulted in borderline induction of centrally nucleated fibres and no significant thinning, suggesting that myofibre aging might be a delayed consequence of senescence-like signalling. To assess the relative importance of the bystander effect versus cell-autonomous senescence, we compared senescent hepatocyte frequencies in livers of wild-type and NSG mice under ad libitum and dietary restricted feeding. This enabled us to approximate cell-autonomous and bystander-driven senescent cell accumulation as well as the impact of immunosurveillance separately. The results suggest a significant impact of the bystander effect for accumulation of senescent hepatocytes in liver and indicate that senostatic interventions like dietary restriction may act as senolytics in immunocompetent animals.

The senescent bystander effect is caused by ROS-activated NF-κB signalling.

Cell senescence is an important driver of the ageing process. The accumulation of senescent cells in tissues is accelerated by stress signals from senescent cells that induce DNA damage and ultimately senescence in bystander cells. We examine here the interplay of senescence-associated mitochondrial dysfunction (SAMD)-driven production of reactive oxygen species (ROS) and senescence-associated secretory phenotype (SASP) in causing the bystander effect. We show that in various modes of fibroblast senescence ROS are necessary and sufficient to activate the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which facilitates a large part of the SASP. This ROS-NF-κB axis causes the DNA damage response in bystander cells. Cytokines IL-6 and IL-8 are major components of the pro-inflammatory SASP in senescent fibroblasts. However, their activation in senescence is only partially controlled by NF-κB, and they are thus not strong candidates as intercellular mediators of the bystander effect as mediated by the ROS-NF-κB axis.

Persistent mTORC1 signaling in cell senescence results from defects in amino acid and growth factor sensing.

Mammalian target of rapamycin complex 1 (mTORC1) and cell senescence are intimately linked to each other and to organismal aging. Inhibition of mTORC1 is the best-known intervention to extend lifespan, and recent evidence suggests that clearance of senescent cells can also improve health and lifespan. Enhanced mTORC1 activity drives characteristic phenotypes of senescence, although the underlying mechanisms responsible for increased activity are not well understood. We have identified that in human fibroblasts rendered senescent by stress, replicative exhaustion, or oncogene activation, mTORC1 is constitutively active and resistant to serum and amino acid starvation. This is driven in part by depolarization of senescent cell plasma membrane, which leads to primary cilia defects and a resultant failure to inhibit growth factor signaling. Further, increased autophagy and high levels of intracellular amino acids may act to support mTORC1 activity in starvation conditions. Interventions to correct these phenotypes restore sensitivity to the mTORC1 signaling pathway and cause death, indicating that persistent signaling supports senescent cell survival.