Predictive, integrative, and regulatory aspects of AI-driven computational toxicology - Highlights of the German Pharm-Tox Summit (GPTS) 2024.

The 9th German Pharm-Tox Summit (GPTS) and the 90th Annual Meeting of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT) took place in Munich from March 13-15, 2024. The event brought together over 700 participants from around the world to discuss cutting-edge developments in the fields of pharmacology and toxicology as well as scientific innovations and novel insights. A key focus of the conference was on the rapidly increasing role of computational toxicology, artificial intelligence (AI), and machine learning (ML) into the field, marking a shift away from traditional methods and allowing the reduction of animal testing as primary tool for toxicological risk assessment.

Limitations and Modifications of Skin Sensitization NAMs for Testing Inorganic Nanomaterials.

Since 2020, the REACh regulation requires toxicological data on nanoforms of materials, including the assessment of their skin-sensitizing properties. Small molecules' skin sensitization potential can be assessed by new approach methodologies (NAMs) addressing three key events (KE: protein interaction, activation of dendritic cells, and activation of keratinocytes) combined in a defined approach (DA) described in the OECD guideline 497. In the present study, the applicability of three NAMs (DPRA, LuSens, and h-CLAT) to nine materials (eight inorganic nanomaterials (NM) consisting of CeO, BaSO, TiO or SiO, and quartz) was evaluated.

Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms.

Background: Significant variations exist in the forms of ZnO, making it impossible to test all forms in in vivo inhalation studies. Hence, grouping and read-across is a common approach under REACH to evaluate the toxicological profile of familiar substances. The objective of this paper is to investigate the potential role of dissolution, size, or coating in grouping ZnO (nano)forms for the purpose of hazard assessment.

Avoiding a reproducibility crisis in regulatory toxicology-on the fundamental role of ring trials.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.

Molecular signatures of angiogenesis inhibitors: a single-embryo untargeted metabolomics approach in zebrafish.

Angiogenesis is a key process in embryonic development, a disruption of this process can lead to severe developmental defects, such as limb malformations. The identification of molecular perturbations representative of antiangiogenesis in zebrafish embryo (ZFE) may guide the assessment of developmental toxicity from an endpoint- to a mechanism-based approach, thereby improving the extrapolation of findings to humans. Thus, the aim of the study was to discover molecular changes characteristic of antiangiogenesis and developmental toxicity.

Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method.

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g.

A Novel and Fast Online-SPE-LC-MS/MS Method to Quantify Thyroid Hormone Metabolites in Rat Plasma.

Since the focus in regulatory toxicology has drifted toward the identification of endocrine disruptors, the improvement in determination of alterations in the thyroid hormone system has become more important. THs are involved in several molecular processes important for a proper pre- and postnatal development so that disturbances can inter alia lead to incorrect brain maturation and/or disturbed metabolic processes (thermogenesis or lipolysis). In this publication, a new automated online solid-phase extraction (SPE)-liquid chromatography (LC)-tandem mass spectrometry (MS/MS, xLC-MS/MS) is introduced which simultaneously analyzes total T4, T3, rT3, T2, and T1.

Three-tiered approach for standard information requirements for polymers requiring registration under REACH.

Polymers are a very large class of chemicals comprising often complex molecules with multiple functions used in everyday products. The EU Commission is seeking to develop environmental and human health standard information requirements (SIRs) for man-made polymers requiring registration (PRR) under a revised Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation. Conventional risk assessment approaches currently used for small molecules may not apply to most polymers.

Application of high throughput in vitro metabolomics for hepatotoxicity mode of action characterization and mechanistic-anchored point of departure derivation: a case study with nitrofurantoin.

Omics techniques have been increasingly recognized as promising tools for Next Generation Risk Assessment. Targeted metabolomics offer the advantage of providing readily interpretable mechanistic information about perturbed biological pathways. In this study, a high-throughput LC-MS/MS-based broad targeted metabolomics system was applied to study nitrofurantoin metabolic dynamics over time and concentration and to provide a mechanistic-anchored approach for point of departure (PoD) derivation.

Basic concepts of mixture toxicity and relevance for risk evaluation and regulation.

Exposure to multiple substances is a challenge for risk evaluation. Currently, there is an ongoing debate if generic "mixture assessment/allocation factors" (MAF) should be introduced to increase public health protection. Here, we explore concepts of mixture toxicity and the potential influence of mixture regulation concepts for human health protection.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil.

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity.

Transferability and reproducibility of exposed air-liquid interface co-culture lung models.

Background: The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes).

A high-throughput metabolomics in vitro platform for the characterization of hepatotoxicity.

Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here, we present the development of a 96-well plate LC-MS/MS-based targeted metabolomics screening platform for the classification of liver toxicity modes of action (MoAs) in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform.

Rapid equilibrium dialysis, ultrafiltration or ultracentrifugation? Evaluation of methods to quantify the unbound fraction of substances in plasma.

In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (f) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models.

Plant extracts, polymers and new approach methods: Practical experience with skin sensitization assessment.

Over the last decade, research into methodologies to identify skin sensitization hazards has led to the adoption of several non-animal methods as OECD test guidelines. However, predictive accuracy beyond the chemical domains of the individual validation studies remains largely untested. In the present study, skin sensitization test results from in vitro and in chemico methods for 12 plant extracts and 15 polymeric materials are reported and compared to available in vivo skin sensitization data.

Genotoxicity testing of nanomaterials.

Nanomaterials have outstanding and unprecedented advantageous material properties but may also cause adverse effects in humans upon exposure. Testing nanomaterials for genotoxic properties is challenging because traditional testing methods were designed for small, soluble molecules and may not be easily applicable without modifications. This review critically examines available genotoxicity tests for use with nanomaterials, including DNA damage tests such as the comet assay, gene mutation tests such as the mouse lymphoma and hprt assay, and chromosome mutation tests such as the micronucleus test and the chromosome aberration test.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12.

Toward Realistic Dosimetry : Determining Effective Concentrations of Test Substances in Cell Culture and Their Prediction by an Mass Balance Model.

Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the medium () can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log  -0.

Lessons Learned from the Grouping of Chemicals to Assess Risks to Human Health.

In analogy to the periodic system that groups elements by their similarity in structure and chemical properties, the hazard of chemicals can be assessed in groups having similar structures and similar toxicological properties. Here we review case studies of chemical grouping strategies that supported the assessment of hazard, exposure, and risk to human health. By the EU-REACH and the US-TSCA New Chemicals Program, structural similarity is commonly used as the basis for grouping, but that criterion is not always adequate and sufficient.

The Evolution of Regulatory Toxicology: Where is the Gardener?

There is a need for paradigm change in the methodology employed for toxicological testing and assessment. It could be said that this change is well on its way, through an evolutionary progress analogous to that of natural selection. Darwin's Theory of Evolution has defined the idea of evolution and descendancy since the last third of the 19th century.

Variation in dissolution behavior among different nanoforms and its implication for grouping approaches in inhalation toxicity.

Different nanoforms (NF) of the same substance each need to be registered under REACH, but similarities in physiological interaction -among them biodissolution- can justify read-across within a group of NFs, thereby reducing the need to perform animal studies. Here we focused on the endpoint of inhalation toxicity and explored how differences in physical parameters of 17 NFs of silica, and organic and inorganic pigments impact dissolution rates, half-times, and transformation under both pH 7.4 lung lining conditions and pH 4.

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

Background: The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases.