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This study uses biogas, an environmentally friendly renewable energy resource, to operate the prototype of a micro-gas turbine (MGT) system called the Proto X-3 Bioenergy Micro-gas Turbine, designed for green building application. The biogas is produced by an anaerobic digester. The aim of this research is to simulate slurry flow in an anaerobic digester as the basis for developing a biogas digester that will produce biogas to meet the requirements of the Proto X-3 Bioenergy Micro-gas Turbine. The digester is a rectangular type with 3.4 m3 capacity. The flow calculations and simulations were done using Computational Fluid Dynamics (CFD) methods in two-dimensional, body-fitted coordinate mesh. The simulations were conducted with various baffle clearances for the digester: 50 mm, 100 mm, and 150 mm. The CFD simulations showed that the recirculation phenomena was found in all flows but that the 50-mm baffle clearance model had the largest recirculation, and it would lead to better mixing of the slurry.

This paper presents a numerical analysis of gas flow in the annular combustion chamber of a Proto X-3 Bioenergy micro gas turbine for green building applications. The computational fluids dynamics (CFD) simulation was conducted in two dimensions, turbulent flow and gas phase combustion, with the goal of comparing the effects of different models in real conditions. Two different turbulence models, standard (STD) k-? and renormalization group (RNG) k-?, were applied for simulations. The fuel used was biogas produced from animal waste. Fuel consumption was assumed to be 100 kJ/s for simulations. The results of the simulations were analyzed and compared for reference. The temperature and the mass fraction of CH4, H2, O2, and CO2 distributions gave almost the same results for both models; therefore, both models (STD k-? and RNG k-?) could be used to represent the combustion process phenomenon without many significant differences.

Read-Across (RAx) serves as a strategy to fill a data gap in the toxicological profile of a substance (target) using existing information on similar source substances. The principle is applied also to a category of substances for which similarity may follow a regular trend. Demonstration of similarity is not trivial and requires the analysis of different steps, starting from the precise analytical characterization of both target and source substances and including the analysis of the impact that each minor difference can have on the final outcome. Application of QSARs and performing new experimental tests within the new approach methodologies (NAMs) is necessary to increase confidence in the final prediction and reduce the uncertainty., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This work aims at studying why azodicarbonamide (ADCA), a formally apolar compound with good hydrogen bond (HB) acceptors, is soluble only in polar aprotic solvents like dimethyl sulfoxide (DMSO) but not in water. Solubility measurements, as well as quantum mechanical and classical molecular dynamics simulations, were employed to tackle the problem. We found that in the liquid phase a polar conformer of ADCA ( µ = 8.7 D), unreported to date, is favoured under the enthalpic drive provided by a highly polar solvent. At the same time, the very high hydrogen bond propensity of water with itself prevents this solvent from providing an effective hydrogen bond-mediated solvation. Solvents bearing good HB acceptors, while lacking strong HB donors, contribute to further stabilizing solute-solvent adducts through weak and fluxional HBs that involve the amide groups of ADCA. Implications for the solubility of ADCA down to µ M concentrations were evaluated, also with the aid of classical simulations of solution nanodroplets., Competing Interests: We declare we have no competing interests., (© 2024 The Authors.)

The EU's chemicals regulation, REACH, requires that most chemicals in the EU be evaluated for human health and ecosystem risks, with a mandate to minimize use of animal tests for these evaluations. The REACH process has been ongoing since about 2008, but a calculation of the resulting animal use is not publicly available. For this reason, we have undertaken a count of animals used for REACH. With EU legislators set to consider REACH revisions that could expand animal testing, we are releasing results for test categories counted to date: reproductive toxicity tests, developmental toxicity tests, and repeat-ed-dose toxicity tests for human health. The total animal count as of December 2022 for these categories is about 2.9 million. Additional tests involving about 1.3 million animals are currently required by a final proposal authorization or compliance check but not yet completed. The total, 4.2 million, for just these three test categories exceeds the original European Com-mission forecast of 2.6 million for all REACH tests. The difference is primarily because the European Commission estimate excluded offspring, which are most of the animals used for REACH. Other reasons for the difference are extra animals included in tests to ensure sufficient survive to meet the minimum test requirement; dose range-finding tests; extra test animal groups, e.g., for recovery analysis; and a high rejection rate of read-across studies. Given higher than forecast animal use, the upcoming debate on proposed REACH revisions is an opportunity to refocus on reducing animal numbers in keeping with the REACH mandate.

The EU's REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) Regulation requires animal testing only as a last resort. However, our study (Knight et al., 2023) in this issue reveals that approximately 2.9 million animals have been used for REACH testing for reproductive toxicity, developmental toxicity, and repeated-dose toxicity alone as of December 2022. Currently, additional tests requiring about 1.3 million more animals are in the works. As compliance checks continue, more animal tests are anticipated. According to the European Chemicals Agency (ECHA), 75% of read-across methods have been rejected during compliance checks. Here, we estimate that 0.6 to 3.2 million animals have been used for other endpoints, likely at the lower end of this range. The ongoing discussion about the grouping of 4,500 regis-tered petrochemicals can still have a major impact on these numbers. The 2022 amendment of REACH is estimated to add 3.6 to 7.0 million animals. This information comes as the European Parliament is set to consider changes to REACH that could further increase animal testing. Two proposals currently under discussion would likely necessitate new animal testing: extending the requirement for a chemical safety assessment (CSA) to Annex VII substances could add 1.6 to 2.6 million animals, and the registration of polymers adds a challenge comparable to the petrochemical discussion. These findings high-light the importance of understanding the current state of REACH animal testing for the upcoming debate on REACH revisions as an opportunity to focus on reducing animal use.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA 's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

EU cosmetic ingredients are governed by two regulations that conflict. Regulation EC 1223/2009, the Cosmetic Regulation, bans in vivo (animal) testing for cosmetic product safety assessments, including both final products and ingredients. At the same time, the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation can impose in vivo testing of those same ingredients under its chemical testing requirements. Here, we examined REACH dossiers for chemicals for which the only reported use is cosmetics to determine the extent of new in vivo testing caused by REACH. We found the REACH database has 3,206 chemical dossiers with cosmetics as a reported use. Of these, 419 report cosmetics as the only use, and 63 of these have in vivo tests completed after the Cosmetic Regulation ban on in vivo testing. Registrants largely used alternative, non-animal methods to evaluate ingredients for REACH, but some still conducted new in vivo tests to comply with REACH requirements for toxicity data and worker safety assessments. In some cases, ECHA, the agency that evaluates REACH dossiers, rejected registrants’ alternative methods as insufficient and required new in vivo tests. As ECHA continues to evaluate dossiers, more requests for in vivo tests are likely. REACH tests on cosmetic ingredients appear only as “industrial chemicals legislation” tests in EU reports. Given the importance to consumers and the cosmetic industry of having cosmetics free of animal testing, the public should be made aware of REACH testing until the conflict between the regulations is resolved.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

This study aims to evaluate the risk assessment approach of the REACH legislation in industrial chemical departments with a focus on the use of three models to calculate exposures, and discuss those factors that can determine a bias between the estimated exposure (and therefore the expected risk) in the extended safety data sheets (e-SDS) and the expected exposure for the actual scenario. To purse this goal, the exposure estimates and risk characterization ratios (RCRs) of registered exposure scenarios (ES; "communicated exposure" and "communicated RCR") were compared with the exposure estimates and the corresponding RCRs calculated for the actual, observed ES, using recommended tools for the evaluation of exposure assessment and in particular the following tools: (i) the European Centre for Ecotoxicology and Toxicology of Chemicals Targeted Risk Assessment v.3.1 (ECETOC TRA), (ii) STOFFENMANAGER ® v.8.0 and (iii) the Advanced REACH Tool (ART). We evaluated 49 scenarios in three companies handling chemicals. Risk characterization ratios (RCRs) were calculated by dividing estimated exposures by derived no-effect levels (DNELs). Although the calculated exposure and RCRs generally were lower than communicated, the correlation between communicated and calculated exposures and RCRs was generally poor, indicating that the generic registered scenarios do not reflect actual working, exposure and risk conditions. Further, some observed scenarios resulted in calculated exposure values and RCR higher than those communicated through chemicals' e-SDSs; thus 'false safe' scenarios (calculated RCRs > 1) were also observed. Overall, the obtained evidences contribute to doubt about whether the risk assessment should be performed using generic (communicated by suppliers) ES with insufficient detail of the specific scenario at all companies. Contrariwise, evidences suggested that it would be safer for downstream users to perform scenario-specific evaluations, by means of proper scaling approach, to achieve more representative estimates of chemical risk.

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission’s H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

Seven years after the last release, the European Commission has again collated and released data on laboratory animal use. The new report is the first to correspond to the requirements of the new Directive 2010/63/EU. Beside minor problems in reporting, the new reporting format is a major step forward, with additional new categories like severity allowing insight into animal use related questions that goes far beyond the previous reports. An in-depth analysis confirms a slight decrease in animal use from 2015 to 2017, but also compared to the 2005, 2008 and 2011 reports, though the new reporting scheme makes this comparison difficult. Notable success is evident for replacing rabbit pyrogen testing but, in general, the implementation of accepted alternative methods lags behind expec-tations. Beside the roughly 10 million animals per year covered in the report, about 8 million animals were identified that fall under the Directive but are not included in this number. Their omission downplays the impact of REACH on animal use. The report, second to none in its detail internationally, represents an important instrument for benchmarking and strategi-cally focusing activities in the 3Rs.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

For a long time, the discussion about animal testing vs its alternatives centered on animal welfare. This was a static warfare, or at least a gridlock, where life scientists had to take a position and make their value choices and hardly anyone changed sides. Technical advances have changed the frontline somewhat, with in vitro and in silico methods gaining more ground. Only more recently has the economic view begun to have an impact: Many animal tests are simply too costly, take too long, and give misleading results. As an extension and update to previous articles in this series written a decade ago, we reanalyze the economic landscape of especially regulatory use of animal testing and this time also consider respective alternative tests. Despite some ambiguity and data gaps, which we have filled with crude estimates, a picture emerges of globally regulated industries that are subject to stark geographic and sectorial differences in regulation, which determine their corresponding animal use. Both animal testing and its alternatives are industries in their own right, offering remarkable business opportunities for biotech and IT companies as well as contract research organizations. In light of recent revelations as to the reproducibility and relevance issues of many animal tests, the economic consequences of incorrect results and the reasons for still maintaining often outdated animal test approaches are discussed.

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes. General discussions raised the duality related to the difficulties in implementing GCCP in an academic innovative research framework on one hand, and on the other hand, the need for such GCCP principles in order to ensure reproducibility and robustness of in vitro test methods for toxicity testing. Indeed, if good cell culture principles are critical to take into consideration for all uses of in vitro test methods for toxicity testing, the level of application of such principles may depend on the stage of development of the test method as well as on the applications of the test methods, i.e., academic innovative research vs. regulatory standardized test method., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The European Chemicals Agency (ECHA) warehouses the largest public dataset of in vivo and in vitro toxicity tests. In December 2014 this data was converted into a structured, machine readable and searchable database using linguistic search engines. It contains data for 9,801 unique substances, 3,609 unique study descriptions and 816,048 study documents.This allows exploring toxicological data on a scale far larger than previously available. Substance similarity analysis was used to determine clustering of substances for hazards by mapping to PubChem. Similarity was measured using PubChem 2D conformational substructure fingerprints, which were compared via the Tanimoto metric. Following K-Core filtration, the Blondel et al.(2008) module recognition algorithm was used to identify chemical modules showing clusters of substances in use within the chemical universe. Global Harmonized System of Classification and Labelling provides a valuable information source for hazard analysis. The most prevalent hazards are H317 "May cause an allergic skin reaction" with 20% and H318 "Causes serious eye damage" with 17% positive substances. Such prevalences obtained for all hazards here are key for the design of integrated testing strategies. The data allowed estimation of animal use. ECHA cover about 20% of substances in the high-throughput biological assay database Tox21 (1,737 substances) and have a 917 substance overlap with the Comparative Toxicogenomics Database (~7% of CTD). The biological data available in these datasets combined with ECHA in vivo endpoints have enormous modeling potential. A case is made that REACH should systematically open regulatory data for research purposes.

The public data on skin sensitization from REACH registrations already included 19,111 studies on skin sensitization in December 2014, making it the largest repository of such data so far (1,470 substances with mouse LLNA, 2,787 with GPMT, 762 with both in vivo and in vitro and 139 with only in vitro data). 21% were classified as sensitizers. The extracted skin sensitization data was analyzed to identify relationships in skin sensitization guidelines, visualize structural relationships of sensitizers, and build models to predict sensitization. A chemical with molecular weight > 500 Da is generally considered non-sensitizing owing to low bioavailability, but 49 sensitizing chemicals with a molecular weight > 500 Da were found. A chemical similarity map was produced using PubChem's 2D Tanimoto similarity metric and Gephi force layout visualization. Nine clusters of chemicals were identified by Blondel's module recognition algorithm revealing wide module-dependent variation. Approximately 31% of mapped chemicals are Michaell's acceptors but alone this does not imply skin sensitization. A simple sensitization model using molecular weight and five ToxTree structural alerts showed a balanced accuracy of 65.8% (specificity 80.4%, sensitivity 51.4%), demonstrating that structural alerts have information value. A simple variant of k-nearest neighbors outperformed the ToxTree approach even at 75% similarity threshold (82% balanced accuracy at 0.95 threshold). At higher thresholds, the balanced accuracy increased. Lower similarity thresholds decrease sensitivity faster than specificity. This analysis scopes the landscape of chemical skin sensitization, demonstrating the value of large public datasets for health hazard prediction.

Public data from ECHA online dossiers on 9,801 substances encompassing 326,749 experimental key studies and additional information on classification and labeling were made computable. Eye irritation hazard, for which the rabbit Draize eye test still represents the reference method, was analyzed. Dossiers contained 9,782 Draize eye studies on 3,420 unique substances, indicating frequent retesting of substances. This allowed assessment of the test's reproducibility test based on all substances tested more than once. There was a 10% chance of a non-irritant evaluation given after a prior severe-irritant result as given by UN GHS classification criteria. The most reproducible outcomes were the results negative (94% reproducible) and severe eye irritant (73% reproducible). To evaluate whether other GHS categorizations predict eye irritation we built a dataset of 5,629 substances (1,931 'irritant' and 3,698 'non-irritant'). The two best decision trees with up to three other GHS classifications resulted in balanced accuracies of 68% and 73%, i.e., in the rank order of the Draize rabbit eye test itself, but both use inhalation toxicity data ("May cause respiratory irritation"), which is not typically available. Next, a dataset of 929 substances with at least one Draize study was mapped to PubChem to compute chemical similarity using 2D conformational fingerprints and Tanimoto similarity. Using a minimum similarity of 0.7 and simple classification by the closest chemical neighbor resulted in balanced accuracy from 73% over 737 substances to 100% at a threshold of 0.975 over 41 substances. This represents a strong support of read-across and (Q)SAR approaches in this area.

The European Chemicals Agency, ECHA, made available a total of 13,832 oral toxicity studies for 8,568 substances up to December 2014. 75% of studies were from the retired OECD Test Guideline 401 (11% TG 420, 11% TG 423 and 1.5% TG 425). Concordance across guidelines, evaluated by comparing LD50 values ≥ 2000 or < 2000 mg/kg body weight from chemicals tested multiple times between different guidelines, was at least 75% and for their own repetition more than 90%. In 2009, Bulgheroni et al. created a simple model for predicting acute oral toxicity using no observed adverse effect levels (NOAEL) from 28-day repeated dose toxicity studies in rats. This was reproduced here for 1,625 substances. In 2014, Taylor et al. suggested no added value of the 90-day repeated dose oral toxicity test given the availability of a low 28-day study with some constraints. We confirm that the 28-day NOAEL is predictive (albeit imperfectly) of 90-day NOAELs, however, the suggested constraints did not affect predictivity. 1,059 substances with acute oral toxicity data (268 positives, 791 negatives, all Klimisch score 1) were used for modeling: The Chemical Development Kit was used to generate 27 molecular descriptors and a similarity-informed multilayer perceptron showing 71% sensitivity and 72% specificity. Additionally, the k-nearest neighbors (KNN) algorithm indicated that similarity-based approaches alone may be poor predictors of acute oral toxicity, but can be used to inform the multilayer perceptron model, where this was the feature with highest information value.