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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. The study also confirmed liver as a target organ using metabolomics, which showed indications of a stress reaction in rats and changes in metabolites suggestive of a metabolic disorder. The metabolomics investigations did not find any other toxicologically relevant modes of action, and the observed metabolite changes were not associated with markers for mitochondrial dysfunction. The study concludes that integration of omics technologies, such as metabolomics, in regulatory toxicity studies is possible, confirms existing knowledge and adds additional information that can be used for mechanistic understanding of observed toxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Frank Faulhammer: is employed by BASF including stock ownership Bennard van Ravenzwaay: declares to have no known financial or personal relationships that could have appeared to influence the work reported in this paper A. Robert Schnatter: declares to have no known financial or personal relationships that could have appeared to influence the work reported in this paper Martijn Rooseboom: is employed by Shell including stock ownership Hennicke Kamp: is managing director of BASF Metabolome Solutions, a company that is offering metabolome analysis for various applications including toxicology research to external clients and BASF. He is employed by BASF including stock ownership Burkhard Flick: declares to have no known financial or personal relationships that could have appeared to influence the work reported in this paper Varun Giri: is employed by BASF including stock ownership Saskia Sperber: is employed by BASF including stock ownership Larry G Higgins and Michael G Penman: Penman Consulting provide consultancy services to the LOA REACH Consortium, and this article reflects the positions developed in the course of this work Neslihan Aygun Kocabas: is employed by TotalEnergies including stock ownership., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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. The MetAbolomics ring Trial for CHemical groupING (MATCHING), comprising six industrial, government and academic ring-trial partners, evaluated inter-laboratory reproducibility and worked towards best-practice. An independent team selected eight substances (WY-14643, 4-chloro-3-nitroaniline, 17α-methyl-testosterone, trenbolone, aniline, dichlorprop-p, 2-chloroaniline, fenofibrate); ring-trial partners were blinded to their identities and modes-of-action. Plasma samples were derived from 28-day rat tests (two doses per substance), aliquoted, and distributed to partners. Each partner applied their preferred liquid chromatography-mass spectrometry (LC-MS) metabolomics workflows to acquire, process, quality assess, statistically analyze and report their grouping results to the European Chemicals Agency, to ensure the blinding conditions of the ring trial. Five of six partners, whose metabolomics datasets passed quality control, correctly identified the grouping of eight test substances into three categories, for both male and female rats. Strikingly, this was achieved even though a range of metabolomics approaches were used. Through assessing intrastudy quality-control samples, the sixth partner observed high technical variation and was unable to group the substances. By comparing workflows, we conclude that some heterogeneity in metabolomics methods is not detrimental to consistent grouping, and that assessing data quality prior to grouping is essential. We recommend development of international guidance for quality-control acceptance criteria. This study demonstrates the reliability of metabolomics for chemical grouping and works towards best-practice., (© 2024. The Author(s).)

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. Here, 15 steam-cracked distillates, registered in the EU through the Lower Olefins Aromatics Reach Consortium (LOA), as well as six of the major substance constituents, were tested in a 14-day rat oral gavage study, in line with the fundamental elements of the OECD 407 guideline, in combination with plasma metabolomics. Beyond signs of clinical toxicity, reduced body weight (gain), and food consumption, pathological investigations demonstrated the liver, thyroid, kidneys (males only), and hematological system to be the target organs. These targets were confirmed by metabolome pattern recognition, with no additional targets being identified. While classical toxicological parameters did not allow for a clear distinction between the substances, univariate and multivariate statistical analysis of the respective metabolomes allowed for the identification of several subclusters of biologically most similar substances. These groups were partly associated with the dominant (> 50%) constituents of these UVCBs, i.e., indene and dicyclopentadiene. Despite minor differences in clustering results based on the two statistical analyses, a proposal can be made for the grouping of these UVCBs. Both analyses correctly clustered the chemically most similar compounds, increasing the confidence that this biological approach may provide a solution for the grouping of UVCBs., (© 2024. The Author(s).)

The current parameters for determining maternal toxicity (e.g. clinical signs, food consumption, body weight development) lack specificity and may underestimate the extent of effects of test compounds on the dams. Previous reports have highlighted the use of plasma metabolomics for an improved and mechanism-based identification of maternal toxicity. To establish metabolite profiles of healthy pregnancies and evaluate the influence of food consumption as a confounding factor, metabolite profiling of rat plasma was performed by gas- and liquid-chromatography-tandem mass spectrometry techniques. Metabolite changes in response to pregnancy, food consumption prior to blood sampling (non-fasting) as well as the interaction of both conditions were studied. In dams, both conditions, non-fasting and pregnancy, had a marked influence on the plasma metabolome and resulted in distinct individual patterns of changed metabolites. Non-fasting was characterized by increased plasma concentrations of amino acids and diet related compounds and lower levels of ketone bodies. The metabolic profile of pregnant rats was characterized by lower amino acids and glucose levels and higher concentrations of plasma fatty acids, triglycerides and hormones, capturing the normal biochemical changes undergone during pregnancy. The establishment of metabolic profiles of pregnant non-fasted rats serves as a baseline to create metabolic fingerprints for prenatal and maternal toxicity studies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)