The short-term toxicity and metabolome of Benzene.

A 14-day rat study with plasma metabolomics was conducted to evaluate the toxicity of Benzene. Wistar rats were orally administered Benzene daily at doses of 0, 300 and 1000 mg/kg bw. The study identified liver and kidneys as target organs of Benzene toxicity and found reductions in total white blood cells, absolute lymphocyte and eosinophil cell counts, and increased relative monocyte counts suggesting bone marrow as a target organ.

Considerations for the development of guidance on dose level selection for developmental and reproductive toxicity studies.

In 2022, the European Chemicals Agency issued advice on the selection of high dose levels for developmental and reproductive toxicity (DART) studies indicating that the highest dose tested should aim to induce clear evidence of reproductive toxicity without excessive toxicity and severe suffering in parental animals. In addition, a recent publication advocated that a 10% decrease in body weight gain should be replaced with a 10% decrease in bodyweight as a criterion for dose adequacy. Experts from the European Centre for Ecotoxicology and Toxicology of Chemicals evaluated these recent developments and their potential impact on study outcomes and interpretation and identified that the advice was not aligned with OECD test guidelines or with humane endpoints guidance.

Demonstrating the reliability of in vivo metabolomics based chemical grouping: towards best practice.

While grouping/read-across is widely used to fill data gaps, chemical registration dossiers are often rejected due to weak category justifications based on structural similarity only. Metabolomics provides a route to robust chemical categories via evidence of shared molecular effects across source and target substances. To gain international acceptance, this approach must demonstrate high reliability, and best-practice guidance is required.

Utility of in vivo metabolomics to support read-across for UVCB substances under REACH.

Structure-based grouping of chemicals for targeted testing and read-across is an efficient way to reduce resources and animal usage. For substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), structure-based grouping is virtually impossible. Biology-based approaches such as metabolomics could provide a solution.

The short-term toxicity and metabolome of dicyclopentadiene.

Dicyclopentadiene (DCPD) was investigated in a 14-day oral rat toxicity study based on the OECD 407 guideline in combination with plasma metabolomics. Wistar rats received the compound daily via gavage at dose levels of 0, 50 and 150 mg/kg bw. The high dose induced transient clinical signs of toxicity and in males only reduced body weight gain.

Predicting acute paraquat toxicity using physiologically based kinetic modelling incorporating in vitro active renal excretion via the OCT2 transporter.

Including active renal excretion in physiologically based kinetic (PBK) models can improve their use in quantitative in vitro- in vivo extrapolation (QIVIVE) as a new approach methodology (NAM) for predicting the acute toxicity of organic cation transporter 2 (OCT2) substrates like paraquat (PQ). To realise this NAM, kinetic parameters Vmax and Km for in vitro OCT2 transport of PQ were obtained from the literature. Appropriate scaling factors were applied to translate the in vitro Vmax to an in vivo Vmax.

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology.

In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development.

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.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - Part IV: the ECETOC and CLE Proposal for a Thyroid Function-Related Neurodevelopmental Toxicity Testing and Assessment Scheme (Thyroid-NDT-TAS).

Following the European Commission Endocrine Disruptor Criteria, substances shall be considered as having endocrine disrupting properties if they (a) elicit adverse effects, (b) have endocrine activity, and (c) the two are linked by an endocrine mode-of-action (MoA) unless the MoA is not relevant for humans. A comprehensive, structured approach to assess whether substances meet the Endocrine Disruptor Criteria for the thyroid modality (EDC-T) is currently unavailable. Here, the European Centre for Ecotoxicology and Toxicology of Chemicals Thyroxine Task Force and CropLife Europe propose a Function-Related euroevelopmental oxicity esting and ssessment cheme (Thyroid-NDT-TAS).

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs).

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology.

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.

Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate.

In vitro models to measure effects on intestinal deconjugation and transport of mixtures of bile acids.

Bile acid metabolism and transport are critical to maintain bile acid homeostasis and host health. In this study, it was investigated if effects on intestinal bile acid deconjugation and transport can be quantified in vitro model systems using mixtures of bile acids instead of studying individual bile acids. To this end deconjugation of mixtures of selected bile acids in anaerobic rat or human fecal incubations and the effect of the antibiotic tobramycin on these reactions was studied.

Gut Microbiota as Well as Metabolomes of Wistar Rats Recover within Two Weeks after Doripenem Antibiotic Treatment.

An understanding of the changes in gut microbiome composition and its associated metabolic functions is important to assess the potential implications thereof on host health. Thus, to elucidate the connection between the gut microbiome and the fecal and plasma metabolomes, two poorly bioavailable carbapenem antibiotics (doripenem and meropenem), were administered in a 28-day oral study to male and female Wistar rats. Additionally, the recovery of the gut microbiome and metabolomes in doripenem-exposed rats were studied one and two weeks after antibiotic treatment (i.

Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects?

This review investigated which patterns of thyroid- and brain-related effects are seen in rats upon gestational/lactational exposure to 14 substances causing thyroid hormone imbalance by four different modes-of-action (inhibition of thyroid peroxidase, sodium-iodide symporter and deiodinase activities, enhancement of thyroid hormone clearance) or to dietary iodine deficiency. Brain-related parameters included motor activity, cognitive function, acoustic startle response, hearing function, periventricular heterotopia, electrophysiology and brain gene expression. Specific modes-of-action were not related to specific patterns of brain-related effects.

In vitro models to detect in vivo bile acid changes induced by antibiotics.

Bile acid homeostasis plays an important role in many biological activities through the bile-liver-gut axis. In this study, two in vitro models were applied to further elucidate the mode of action underlying reported in vivo bile acid changes induced by antibiotics (colistin sulfate, tobramycin, meropenem trihydrate, and doripenem hydrate). 16S rRNA analysis of rat fecal samples anaerobically incubated with these antibiotics showed that especially tobramycin induced changes in the gut microbiota.

Towards best use and regulatory acceptance of generic physiologically based kinetic (PBK) models for in vitro-to-in vivo extrapolation (IVIVE) in chemical risk assessment.

With an increasing need to incorporate new approach methodologies (NAMs) in chemical risk assessment and the concomitant need to phase out animal testing, the interpretation of in vitro assay readouts for quantitative hazard characterisation becomes more important. Physiologically based kinetic (PBK) models, which simulate the fate of chemicals in tissues of the body, play an essential role in extrapolating in vitro effect concentrations to in vivo bioequivalent exposures. As PBK-based testing approaches evolve, it will become essential to standardise PBK modelling approaches towards a consensus approach that can be used in quantitative in vitro-to-in vivo extrapolation (QIVIVE) studies for regulatory chemical risk assessment based on in vitro assays.

Investigating the gut microbiome and metabolome following treatment with artificial sweeteners acesulfame potassium and saccharin in young adult Wistar rats.

To elucidate if artificial sweeteners modify fecal bacterial composition and the fecal and plasma metabolomes, Wistar rats from both sexes were treated for 28 days with acesulfame potassium (40 and 120 mg/kg body weight) and saccharin (20 and 100 mg/kg body weight). Targeted MS-based metabolome profiling (plasma and feces) and fecal 16S gene sequencing were conducted. Both sweeteners exhibited only minor effects on the fecal metabolome and microbiota.

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.

Use of Physiologically Based Kinetic Modeling-Facilitated Reverse Dosimetry to Predict In Vivo Acute Toxicity of Tetrodotoxin in Rodents.

In this study, the ability of a new in vitro/in silico quantitative in vitro-in vivo extrapolation (QIVIVE) methodology was assessed to predict the in vivo neurotoxicity of tetrodotoxin (TTX) in rodents. In vitro concentration-response data of TTX obtained in a multielectrode array assay with primary rat neonatal cortical cells and in an effect study with mouse neuro-2a cells were quantitatively extrapolated into in vivo dose-response data, using newly developed physiologically based kinetic (PBK) models for TTX in rats and mice. Incorporating a kidney compartment accounting for active renal excretion in the PBK models proved to be essential for its performance.

A framework for chemical safety assessment incorporating new approach methodologies within REACH.

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach.

Predictive performance of next generation human physiologically based kinetic (PBK) models based on in vitro and in silico input data.

The goal of the present study was to assess the predictive performance of a generic human physiologically based kinetic (PBK) model based on in vitro and in silico input data and the effect of using different input approaches for chemical parameterization on those predictions. For this purpose, a dataset was created of 38,772 Cmax predictions for 44 compounds by applying different combinations of in vitro and in silico approaches for chemical parameterization, and these predicted Cmax values were compared to reported in vivo data. Best results were achieved when the hepatic clearance was parameterized based on in vitro (i.

Predictive Performance of Next Generation Physiologically Based Kinetic (PBK) Model Predictions in Rats Based on In Vitro and In Silico Input Data.

The goal of the present study was to assess the predictive performance of a minimal generic rat physiologically based kinetic (PBK) model based on in vitro and in silico input data to predict peak plasma concentrations (Cmax) upon single oral dosing. To this purpose, a dataset was generated of 3960 Cmax predictions for 44 compounds, applying different combinations of in vitro and in silico approaches for chemical parameterization, and comparison of the predictions to reported in vivo data. Best performance was obtained when (1) the hepatic clearance was parameterized based on in vitro measured intrinsic clearance values, (2) the method of Rodgers and Rowland for calculating partition coefficients, and (3) in silico calculated fraction unbound plasma and Papp values (the latter especially for very lipophilic compounds).

Ontogeny of renal, hepatic, and placental expression of ATP-binding cassette and solute carrier transporters in the rat and the rabbit.

Species differences in developmental toxicity can be due to varying expression of xenobiotic transporters. Hence, knowledge on the ontogeny of these transporters, especially in human, rat and rabbit, is pivotal. Two superfamilies of transporters, the ATP-binding cassette (ABC) and the solute carrier (SLC) transporters, are well known for their role in the absorption, distribution and/or elimination of xenobiotics and endogenous substances.