In vitro Toxicity Testing in the Twenty-First Century

The National Research Council (NRC) article “Toxicity Testing in the 21st Century: A vision and A Strategy” (National Research Council, 2007) was written to bring attention to the application of scientific advances for use in toxicity tests so that chemicals can be tested in a more time and cost efficient manner while providing a more relevant and mechanistic insight into the toxic potential of a compound. Development of tools for in vitro toxicity testing constitutes an important activity of this vision and contributes to the provision of test systems as well as data that are essential for the development of computer modeling tools for, e.g., system biology, physiologically based modeling. This article intends to highlight some of the issues that have to be addressed in order to make in vitro toxicity testing a reality in the twenty-first century

Applying the adverse outcome pathway (AOP) for food sensitization to support in vitro testing strategies

Background Before introducing proteins from new or alternative dietary sources into the market, a compressive risk assessment including food allergic sensitization should be carried out in order to ensure their safety. We have recently proposed the adverse outcome pathway (AOP) concept to structure the current mechanistic understanding of the molecular and cellular pathways evidenced to drive IgE-mediated food allergies. This AOP framework offers the biological context to collect and structure existing in vitro methods and to identify missing assays to evaluate sensitizing potential of food proteins. Scope and approach In this review, we provide a state-of-the-art overview of available in vitro approaches for assessing the sensitizing potential of food proteins, including their strengths and limitations. These approaches are structured by their potential to evaluate the molecular initiating and key events driving food sensitization. Key findings and conclusions The application of the AOP framework offers the opportunity to anchor existing testing methods to specific building blocks of the AOP for food sensitization. In general, in vitro methods evaluating mechanisms involved in the innate immune response are easier to address than assays addressing the adaptive immune response due to the low precursor frequency of allergen-specific T and B cells. Novel ex vivo culture strategies may have the potential to become useful tools for investigating the sensitizing potential of food proteins. When applied in the context of an integrated testing strategy, the described approaches may reduce, if not replace, current animal testing approaches

Prevalence and significance of sexually transmitted diseases among Ethiopian women attending antenatal clinics in Addis Ababa

Abstract: To determine the prevalence of sexually transmitted diseases (STDs) and the risk for (i) the mother regarding pregnancy wastage and puerperal sepsis and (ii) the child with regard to congenital and neonatal infection, 342 routine antenatal clinic (ANC) at tenders were investigated. The prevalence of antibodies showing exposure to specific STD pathogens in pregnant women attending ANC was: syphilis (TPHA) 27%, (VDR:) 28%, gonorrhoea 43%, genital chlamydiae 54%, HBV 37%, HSV-2 35 %, H ducreyi 10%. High titre seropositivity suggestive of active infection was: gonorrhoea 10%, genital chlamydiae 31 %, HSV2 19%; with HBV SAg 5% -all of which are likely to be transmitted to the foetus in utero or during delivery. Only 10% of ANC at tenders had no serological evidence of any STD: 72% had serological evidence for two or more STDs. Among conditions requiring treatment vaginitis was the most important, 20% having a severe trichomonal infection. Despite the frequency of this condition it was noted that few women (4%) complained of vaginal discharge. Thus women attending the ANC revealed a high prevalence of STD. Consequently the foetus and neonate are put at risk because of intrauterine or intrapartum transmission of infection. The high prevalence among ANC at tenders also reflects the relative prevalence of STDs in the community. Measures such as screening at ANC and information and education regarding prevention are required to reduce STDs in pregnant women and their sexual partners. Prophylaxis for the neonate can be considered until this goal is achieved. [Ethiop. J. Health Dev. 1995;9(1):31-40

A sociological and serological study of at tenders of family planning clinics in Addis Ababa

Abstract: A study of 542 women attending family planning clinics (FPC) and 1568 women attending obstetric and gynaecologic clinics in Addis Ababa showed utilisation of FPC was highest in those with a family income of 100-500 EB per month (36%), in women who were: Tigrawi (33%) or Amara (31 %), aged 20-34 years (30%), age 16 or older at first marriage/coitus (28%), parity of 2... 2 children (35%), > 5 lifetime husbands/sexual partners (39%), or were bargirls (73%) or prostitutes (43%). FPC attendance was lowest among the nulliparous (2.3%), women from rural areas (10%), the Guragie (10%) and Oromo women (19%), Moslem women 14(%), those of subsistence income ( < 10EB per month) (14%). The seroprevalence rates indicative of exposure to STD pathogens were high as was the prevalence of essentially asymptomatic pelvic inflammatory disease (PID). Only 4% of FPC at tenders had no serological evidence of STD: 64% had 3 or more different STD. Specific present or active STD infection prevalence for syphilis (VDRL) 28%, Neisseria gonorrhoea 31 %, genital chlamydia 46% and HSV-2 21% was higher in FPC at tenders than among women attending other clinics. Clinical evidence of PID was also more common in the FPC at tenders (54%), 37% having evidence of salpingitis. Thus FPCs provide a useful setting for screening women particularly at risk. Because of lack of symptoms, these women are unlikely to attend either an STDs clinic or a hospital for routine check up, and as such are not treated and represent a population from which STDs can spread into the population. Measures to screen, treat and educate FPC at tenders, their partners and their clients, are recommended in an attempt to Control STDs and ultimately HIV in the community. [Ethiop. J. Hea/th Dev. 1995;9(1):19-30

An Adverse Outcome Pathway for Sensitization of the Respiratory Tract by Low-Molecular-Weight Chemicals: Building Evidence to Support the Utility of In Vitro and In Silico Methods in a Regulatory Context

Sensitization of the respiratory tract is an important occupational health challenge, and understanding the mechanistic basis of this effect is necessary to support the development of toxicological tools to detect chemicals that may cause it. Here we use the adverse outcome pathway (AOP) framework to organize information that may better inform our understanding of sensitization of the respiratory tract, building on a previously published skin sensitization AOP, relying on literature evidence linked to low-molecular-weight organic chemicals and excluding other known respiratory sensitizers acting via different molecular initiating events. The established key events (KEs) are as follows: (1) covalent binding of chemicals to proteins, (2) activation of cellular danger signals (inflammatory cytokines and chemokines and cytoprotective gene pathways), (3) dendritic cell activation and migration, (4) activation, proliferation, and polarization of T cells, and (5) sensitization of the respiratory tract. These events mirror the skin sensitization AOP but with specific differences. For example, there is some evidence that respiratory sensitizers bind preferentially to lysine moieties, whereas skin sensitizers bind to both cysteine and lysine. Furthermore, exposure to respiratory sensitizers seems to result in cell behavior for KEs 2 and 3, as well as the effector T cell response, in general skewing toward cytokine secretions predominantly associated with T helper 2 (Th2) response. Knowledge gaps include the lack of understanding of which KE(s) drive the Th2 polarization. The construction of this AOP may provide insight into predictive tests that would in combination support the discrimination of respiratory-sensitizing from non- and skin-sensitizing chemicals, a clear regulatory need

Developing a framework for assessing respiratory sensitization: A workshop report

Respiratory tract sensitization can have significant acute and chronic health implications. While induction of respiratory sensitization is widely recognized for some chemicals, validated standard methods or frameworks for identifying and characterizing the hazard are not available. A workshop on assessment of respiratory sensitization was held to discuss the current state of science for identification and characterization of respiratory sensitizer hazard, identify information facilitating development of validated standard methods and frameworks, and consider the regulatory and practical risk management needs. Participants agreed on a predominant Th2 immunological mechanism and several steps in respiratory sensitization. Some overlapping cellular events in respiratory and skin sensitization are well understood, but full mechanism(s) remain unavailable. Progress on non-animal approaches to skin sensitization testing, ranging from in vitro systems, –omics, in silico profiling, and structural profiling were acknowledged. Addressing both induction and elicitation phases remains challenging. Participants identified lack of a unifying dose metric as increasing the difficulty of interpreting dosimetry across exposures. A number of research needs were identified, including an agreed list of respiratory sensitizers and other asthmagens, distinguishing between adverse effects from immune-mediated versus non immunological mechanisms. A number of themes emerged from the discussion regarding future testing strategies, particularly the need for a tiered framework respiratory sensitizer assessment. These workshop present a basis for moving towards a weight-of-evidence assessment

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

Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins

[Background] The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP).[Main body] The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell–cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential.[Conclusion] The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.Peer reviewe