Botanicals have long been used to promote health and treat diseases, but the safety of many currently marketed botanicals has not been adequately evaluated. Given the chemical complexity of botanicals, which often contain numerous unknown constituents, and their widespread use, comprehensive toxicity assessments are needed. The Botanical Safety Consortium was established to address this challenge. This international group of experts in toxicology, chemistry, bioinformatics, and pharmacognosy is developing a toolkit of assays to generate reliable toxicological profiles for botanicals. Genotoxicity assessment is especially critical, because, unlike other toxicities, genotoxicity is not adequately identified by adverse event and history-of-use reports, and genotoxicity is directly linked to health consequences such as cancer and birth defects. The Consortium's Genotoxicity Technical Working Group is exploring a genotoxicity testing strategy based on the use of in silico modeling and the bacterial reverse mutation and in vitro micronucleus assays and including several options for additional tests to further characterize genotoxicity and mode of action when indicated. The effectiveness of this testing strategy is being evaluated using 13 well-characterized botanicals with existing toxicological data as case studies. A brief overview of each of these 13 botanicals is provided. The final strategy for developing comprehensive genotoxicity profiles of botanicals will incorporate published genotoxicity data, chemical composition information, in silico and in vitro test data, and human exposure data, reducing the need for animal testing.

The amphibian metamorphosis assay (AMA) is an in vivo screen to assess potential interactions of chemicals with the amphibian thyroid system. Tadpoles are exposed for 21-days, then assessed for development and growth after 7 days and at test termination. This paper presents data from studies performed to satisfy test orders from the US EPA's Endocrine Disruptor Screening Program. Data Evaluation Records were used to collate the control variability and performance of biological endpoints in AMAs conducted in different laboratories, then supplemented with recent studies. We examine the statistical power of AMA endpoint analysis and assess whether historical control data (HCD) can assist evidence-based interpretation of the endpoints, with 52 studies from 7 different laboratories. HCD can be used to understand assay performance post validation. The analysis identifies some need for flexibility in the interpretation of the Test Guidelines' performance criteria, including latitude with analytical variability and statistical analysis of late-stage animals. Additionally, more guidance is suggested for feed regiments and the selection criteria for batches of animals to initiate the assay. Potential Guideline refinements that improve interpretation of the data and have potential to reduce the number of vertebrate animals used in the conduct of AMAs are identified and discussed.

Botanicals are widely consumed as dietary supplements, medicines, and consumer products. Their chemical complexity and variability present challenges in safety assessment, including potential neurotoxicity. The Botanical Safety Consortium (BSC)'s Neurotoxicity Working Group is exploring new approach methodologies (NAMs) for the screening of botanical extracts for neurotoxic effects.Areas coveredThis paper outlines the selection of NAMs, including in vitro assays using primary rat cortical neurons, zebrafish embryos, and Caenorhabditis elegans. These assays aim to assess neurotoxic endpoints such as neuronal activity and behavioral responses. Microelectrode array recordings of rat cortical neurons provide insights into the impact of botanical extracts on neuronal function, while the zebrafish embryos and C. elegans assays evaluate neurobehavioral responses. The paper also provides an account of the selection of botanical case studies based on expert judgment and existing neuroactivity/toxicity information. The proposed battery of assays will be tested with these case studies to evaluate their utility for neurotoxicity screening.Expert opinionThe complexity of botanicals necessitates the use of multiple NAMs for effective neurotoxicity screening. This paper discusses the evaluation of methodologies to develop a robust framework for evaluating botanical safety, including complex neuronal models and key neurodevelopmental process assays. It aims to establish a comprehensive screening framework.

Toxicity testing of botanicals is challenging because of their chemical complexity and variability. Since botanicals may affect many different modes of action involved in neuronal function, we used microelectrode array (MEA) recordings of primary rat cortical cultures to screen 16 different botanical extracts for their effects on cell viability and neuronal network function in vitro. Our results demonstrate that extract materials (50 μg/mL) derived from goldenseal, milk thistle, tripterygium, and yohimbe decrease mitochondrial activity following 7 days exposure, indicative of cytotoxicity. Importantly, most botanical extracts alter neuronal network function following acute exposure. Extract materials (50 μg/mL) derived from aristolochia, ephedra, green tea, milk thistle, tripterygium, and usnea inhibit neuronal activity. Extracts of kava, kratom and yohimbe are particularly potent and induce a profound inhibition of neuronal activity at the low dose of 5 μg/mL. Extracts of blue cohosh, goldenseal and oleander cause intensification of the bursts. Aconite extract (5 μg/mL) evokes a clear hyperexcitation with a marked increase in the number of spikes and (network) bursts. The distinct activity patterns suggest that botanical extracts have diverse modes of action. Our combined data also highlight the applicability of MEA recordings for hazard identification and potency ranking of botanicals.

Botanical supplements and herbal products are widely used by consumers for various purported health benefits, and their popularity is increasing. Some of these natural products can have adverse effects on liver function and/or interact with prescription and over-the-counter (OTC) medications. Ensuring the safety of these readily available products is a crucial public health concern; however, not all regulatory authorities require premarket safety review and/or testing. To address and discuss these and other emerging needs related to botanical safety, a symposium was held at the Society of Toxicology Annual Meeting in Salt Lake City (UT) on March 11, 2024. The symposium addressed the latest research on botanical-induced liver toxicity and botanical-drug interactions, including new approach methods to screen for toxicity, challenges in assessing the safety of botanicals, and relating human adverse events to specific products. The presentations and robust panel discussion between the speakers and audience highlighted the need for further research and collaboration to improve the safety of botanical supplements and herbal products, with the ultimate goal of protecting consumer health. Although utility of many of the modern tools presented in the symposium requires further study, the synergistic efforts of diverse experts hold promise for effective prediction and evaluation of botanical-induced hepatotoxicity and botanical-drug interaction potential.

The Ames test is required by regulatory agencies worldwide for assessing the mutagenic and carcinogenic potential of chemical compounds. This test uses several strains of bacteria to evaluate mutation induction: positive results in the assay are predictive of rodent carcinogenicity. As an initial step to understanding how well the assay may detect mutagens present as constituents of complex mixtures such as botanical extracts, a cross-sector working group examined the within-laboratory reproducibility of the Ames test using the extensive, publicly available National Toxicology Program (NTP) Ames test database comprising more than 3000 distinct test articles, most of which are individual chemicals. This study focused primarily on NTP tests conducted using the standard Organization for Economic Co-operation and Development Test Guideline 471 preincubation test protocol with 10% rat liver S9 for metabolic activation, although 30% rat S9 and 10 and 30% hamster liver S9 were also evaluated. The reproducibility of initial negative responses in all strains with and without 10% S9, was quite high, ranging from 95% to 99% with few exceptions. The within-laboratory reproducibility of initial positive responses for strains TA98 and TA100 with and without 10% rat liver S9 was >= 90%. Similar results were seen with hamster S9. As expected, the reproducibility of initial equivocal responses was lower, <50%. These results will provide context for determining the optimal design of recommended test protocols for use in screening both individual chemicals and complex mixtures, including botanicals.

Botanicals contain complex mixtures of chemicals most of which lack pharmacokinetic data in humans. Since physicochemical and pharmacokinetic properties dictate the in vivo exposure of botanical constituents, these parameters greatly impact the pharmacological and toxicological effects of botanicals in consumer products. This study sought to use computational (i.e., in silico) models, including quantitative structure-activity relationships (QSAR) and physiologically based pharmacokinetic (PBPK) modeling, to predict properties of botanical constituents. One hundred and three major constituents (e.g., withanolides, mitragynine, and yohimbine) in 13 botanicals (e.g., ashwagandha, kratom, and yohimbe) were investigated. The predicted properties included biopharmaceutical classification system (BCS) classes based on aqueous solubility and permeability, oral absorption, liver microsomal clearance, oral bioavailability, and others. Over half of these constituents fell into BCS classes I and II at dose levels no greater than 100 mg per day, indicating high permeability and absorption (%Fa > 75%) in the gastrointestinal tract. However, some constituents such as glycosides in ashwagandha and Asian ginseng showed low bioavailability after oral administration due to poor absorption (BCS classes III and IV, %Fa < 40%). These in silico results fill data gaps for botanical constituents and could guide future safety studies. For example, the predicted human plasma concentrations may help select concentrations for in vitro toxicity testing. Additionally, the in silico data could be used in tiered or batteries of assays to assess the safety of botanical products. For example, highly absorbed botanical constituents indicate potential high exposure in the body, which could lead to toxic effects.

Botanical supplements, herbal remedies, and plant-derived products are used globally. However, botanical dietary supplements are rarely subjected to robust safety testing unless there are adverse reports in post-market surveillance. Botanicals are complex and difficult to assess using current frameworks designed for single constituent substances (e.g. small molecules or discrete chemicals), making safety assessments costly and time-consuming. The liver is a primary organ of concern for potential botanical-induced hepatotoxicity and botanical-drug interactions as it plays a crucial role in xenobiotic metabolism. The NIH-funded Drug Induced Liver Injury Network noted that the number of botanical-induced liver injuries in 2017 nearly tripled from those observed in 2004-2005. New approach methodologies (NAMs) can aid in the rapid and cost-effective assessment of botanical supplements for potential hepatotoxicity. The Hepatotoxicity Working Group within the Botanical Safety Consortium is working to develop a screening strategy that can help reliably identify potential hepatotoxic botanicals and inform mechanisms of toxicity. This manuscript outlines the Hepatotoxicity Working Group's strategy and describes the assays selected and the rationale for the selection of botanicals used in case studies. The selected NAMs evaluated as a part of this effort are intended to be incorporated into a larger battery of assays to evaluate multiple endpoints related to botanical safety. This work will contribute to a botanical safety toolkit, providing researchers with tools to better understand hepatotoxicity associated with botanicals, prioritize and plan future testing as needed, and gain a deeper insight into the botanicals being tested.

Drug-induced kidney injury (DIKI) refers to kidney damage resulting from the administration of medications. The aim of this project was to identify reliable urinary microRNA (miRNAs) biomarkers that can be used as potential predictors of DIKI before disease diagnosis. This study quantified a panel of six miRNAs (miRs-210-3p, 423-5p, 143-3p, 130b-3p, 486-5p, 193a-3p) across multiple time points using urinary samples from a previous investigation evaluating effects of a nephrotoxicant in cynomolgus monkeys. Exosome-associated miRNA exhibited distinctive trends when compared to miRNAs quantified in whole urine, which may reflect a different urinary excretion mechanism of miRNAs than those released passively into the urine. Although further research and mechanistic studies are required to elucidate how these miRNAs regulate signaling in disease pathways, we present, for the first time, data that several miRNAs displayed strong correlations with histopathology scores, thus indicating their potential use as biomarkers to predict the development of DIKI in preclinical studies and clinical trials. Also, these findings can potentially be translated into other non-clinical species or human for the detection of DIKI.

Increases in botanical use, encompassing herbal medicines and dietary supplements, have underlined a critical need for an advancement in safety assessment methodologies. However, botanicals present unique challenges for safety assessment due to their complex and variable composition arising from diverse growing conditions, processing methods, and plant varieties. Historically, botanicals have been largely evaluated based on their history of use information, based primarily on traditional use or dietary history. However, this presumption lacks comprehensive toxicological evaluation, demanding innovative and consistent assessment strategies. To address these challenges, the Botanical Safety Consortium (BSC) was formed as an international, cross-sector forum of experts to identify fit-for purpose assays that can be used to evaluate botanical safety. This global effort aims to assess botanical safety assessment methodologies, merging traditional knowledge with modern in vitro and in silico assays. The ultimate goal is to champion the development of toxicity tools for botanicals. This manuscript highlights: 1) BSC's strategy for botanical selection, sourcing, and preparation of extracts to be used in in vitro assays, and 2) the approach utilized to characterize botanical extracts, using green tea and Asian ginseng as examples, to build confidence for use in biological assays.

The COVID-19 pandemic sparked the development of novel anti-viral drugs that have shown to be effective in reducing both fatality and hospitalization rates in patients with elevated risk for COVID-19 related morbidity or mortality. Currently, nirmatrelvir/ritonavir (Paxlovid™) fixed-dose combination is recommended by the World Health Organization for treatment of COVID-19. The ritonavir component is an inhibitor of cytochrome P450 (CYP) 3A, which is used in this combination to achieve needed therapeutic concentrations of nirmatrelvir. Because of the critical pharmacokinetic effect of this mechanism of action for Paxlovid™, co-administration with needed medications that inhibit or induce CYP3A is contraindicated, reflecting concern for interactions with the potential to alter the efficacy or safety of co-administered drugs that are also metabolized by CYP3A. Some herbal medicines are known to interact with drug metabolizing enzymes and transporters, including but not limited to inhibition or induction of CYP3A and P-glycoprotein. As access to these COVID-19 medications has increased in low- and middle-income countries (LMICs), understanding the potential for herb-drug interactions within these regions is important. Many studies have evaluated the utility of herbal medicines for COVID-19 treatments, yet information on potential herb-drug interactions involving Paxlovid™, specifically with herbal medicines commonly used in LMICs, is lacking. This review presents data on regionally-relevant herbal medicine use (particularly those promoted as treatments for COVID-19) and mechanism of action data on herbal medicines to highlight the potential for herbal medicine interaction Herb-drug interaction mediated by ritonavir-boosted antiviral protease inhibitors This work highlights potential areas for future experimental studies and data collection, identifies herbal medicines for inclusion in future listings of regionally diverse potential HDIs and underscores areas for LMIC-focused provider-patient communication. This overview is presented to support governments and health protection entities as they prepare for an increase of availability and use of Paxlovid™.

Interest in botanicals, particularly as dietary supplement ingredients, is growing steadily. This growth, and the marketing of new ingredients and combination products as botanical dietary supplements, underscores the public health need for a better understanding of potential toxicities associated with use of these products. This article and accompanying template outline the resources to collect literature and relevant information to support the design of botanical toxicity studies. These resources provide critical information related to botanical identification, characterization, pre-clinical and clinical data, including adverse effects and interactions with pharmaceuticals. Toxicologists using these resources should collaborate with pharmacognosists and/or analytical chemists to enhance knowledge of the botanical material being tested. Overall, this guide and resource list is meant to help locate relevant information that can be leveraged to inform on decisions related to toxicity testing of botanicals, including the design of higher quality toxicological studies.

Zusammenfassung Die Verwendung von pflanzlichen Heilmitteln und Nahrungsergänzungsmitteln ist weit verbreitet. In vielen Ländern der Welt weisen die Verkaufszahlen auf eine steigende Beliebtheit dieser Produkte hin. Daher ist die Gewährleistung der Sicherheit von pflanzlichen Produkten eine Priorität der öffentlichen Gesundheit. Dieser Artikel beschreibt die Aufgaben und Ziele des Botanical Safety Consortiums (BSC) – einer öffentlich-privaten Partnerschaft. Diese Partnerschaft ist das Ergebnis einer Absichtserklärung zwischen der Gesundheitsbehörde der USA (FDA), dem National Institute of Environmental Health Sciences (NIEHS, eine Unterabteilung des Ministeriums für Gesundheitspflege und Soziale Dienste der Vereinigten Staaten) und einer gemeinnützigen Organisation, dem Health and Environmental Sciences Institute (HESI). Das BSC dient als globales Forum für Wissenschaftler aus Regierungen, Hochschulen, Gesundheitsfürsorgegruppen, Industrie und gemeinnützigen Organisationen, um gemeinsam an der Anpassung und Integration neuer Ansatzmethoden (NAM) für routinemäßige Sicherheitsbewertungen von Pflanzenstoffen zu arbeiten. Die Ziele des BSC sind: 1) eine weltweite Zusammenarbeit, um existierende Sicherheitstests besser zu nutzen; 2) geeignete Methoden der chemischen Charakterisierung von Pflanzenstoffen festzusetzen; 3) pragmatische, zweckmäßige NAM zu identifizieren, um die Sicherheit von pflanzlichen Inhaltsstoffen und Fertigpräparaten zu bewerten; 4) eine Bewertung dieser Methoden mittels Vergleich mit derzeit verfügbaren Informationen zur Sicherheit von ausgewählten Pflanzenstoffen; 5) und die Integration dieser NAM in ein System, das die Bewertung von Pflanzenstoffen erleichtern kann. Der Schwerpunkt des BSC liegt zunächst auf Phytopharmaka und Nahrungsergänzungsmitteln, welche oral verabreicht werden, jedoch kann dieser Ansatz in zukünftigen Arbeitsphasen erweitert werden. Dieser Artikel beinhaltet einen Überblick über die Struktur, Ziele und Strategien dieser Initiative und erläutert die ersten Ziele, nämlich die Auswahl der Test-Pflanzenstoffe, basierend auf publizierten toxikologischen Daten.

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource‐intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an “animal‐free” testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross‐sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical‐induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight‐of‐evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit‐for‐purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757–777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Targeted protein degraders (TPDs) are emerging classes of therapeutics that have the potential to address difficult drug targets in new ways. In 2021, experts convened to discuss challenges in TPD safety evaluation, and supported supplementing individual efforts to address these challenges with collective development of novel decision frameworks, tools and a shared evidence base. Targeted protein degraders (TPDs) are emerging classes of therapeutics that have the potential to address difficult drug targets in new ways. In 2021, experts convened to discuss challenges in TPD safety evaluation, and supported supplementing individual efforts to address these challenges with collective development of novel decision frameworks, tools and a shared evidence base.

There is growing recognition across broad sectors of the scientific community that use of genomic biomarkers has the potential to reduce the need for conventional rodent carcinogenicity studies of industrial chemicals, agrochemicals, and pharmaceuticals through a weight-of-evidence approach. These biomarkers fall into 2 major categories: (1) sets of gene transcripts that can identify distinct tumorigenic mechanisms of action; and (2) cancer driver gene mutations indicative of rapidly expanding growth-advantaged clonal cell populations. This call-to-action article describes a collaborative approach launched to develop and qualify biomarker gene expression panels that measure widely accepted molecular pathways linked to tumorigenesis and their activation levels to predict tumorigenic doses of chemicals from short-term exposures. Growing evidence suggests that application of such biomarker panels in short-term exposure rodent studies can identify both tumorigenic hazard and tumorigenic activation levels for chemical-induced carcinogenicity. In the future, this approach will be expanded to include methodologies examining mutations in key cancer driver gene mutation hotspots as biomarkers of both genotoxic and nongenotoxic chemical tumor risk. Analytical, technical, and biological validation studies of these complementary genomic tools are being undertaken by multisector and multidisciplinary collaborative teams within the Health and Environmental Sciences Institute. Success from these efforts will facilitate the transition from current heavy reliance on conventional 2-year rodent carcinogenicity studies to more rapid animal- and resource-sparing approaches for mechanism-based carcinogenicity evaluation supporting internal and regulatory decision-making.

Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). “Extraction-less” or “direct” real time–reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.