Neuroactivity screening of botanical extracts using microelectrode array (MEA) recordings

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

New Approach Methodologies for the Endocrine Activity Toolbox: Environmental Assessment for Fish and Amphibians

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.publishedVersio

Qualitative approach to comparative exposure in alternatives assessment

Most alternatives assessments (AAs) published to date are largely hazard-based rankings, thereby ignoring potential differences in human and/or ecosystem exposures; as such, they may not represent a fully informed consideration of the advantages and disadvantages of possible alternatives. Building on the 2014 US National Academy of Sciences recommendations to improve AA decisions by including comparative exposure assessment into AAs, the Health and Environmental Sciences Institute\u27s (HESI) Sustainable Chemical Alternatives Technical Committee, which comprises scientists from academia, industry, government, and nonprofit organizations, developed a qualitative comparative exposure approach. Conducting such a comparison can screen for alternatives that are expected to have a higher or different routes of human or environmental exposure potential, which together with consideration of the hazard assessment, could trigger a higher tiered, more quantitative exposure assessment on the alternatives being considered, minimizing the likelihood of regrettable substitution. This article outlines an approach for including chemical ingredient- and product-related exposure information in a qualitative comparison, including ingredient and product-related parameters. A classification approach was developed for ingredient and product parameters to support comparisons between alternatives as well as a methodology to address exposure parameter relevance and data quality. The ingredient parameters include a range of physicochemical properties that can impact routes and magnitude of exposure, whereas the product parameters include aspects such as product-specific exposure pathways, use information, accessibility, and disposal. Two case studies are used to demonstrate the application of the methodology. Key learnings and future research needs are summarized. Integr Environ Assess Manag 2018;00:000-000. (c) 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC

Investigating Alternatives to the fish early-life stage test: A strategy for discovering and annotating adverse outcome pathways for early fish development

The fish early-life stage (FELS) test (Organisation for Economic Co-operation and Development [OECD] test guideline 210) is the primary test used internationally to estimate chronic fish toxicity in support of ecological risk assessments and chemical management programs. As part of an ongoing effort to develop efficient and cost-effective alternatives to the FELS test, there is a need to identify and describe potential adverse outcome pathways (AOPs) relevant to FELS toxicity. To support this endeavor, the authors outline and illustrate an overall strategy for the discovery and annotation of FELS AOPs. Key events represented by major developmental landmarks were organized into a preliminary conceptual model of fish development. Using swim bladder inflation as an example, a weight-of-evidence–based approach was used to support linkage of key molecular initiating events to adverse phenotypic outcomes and reduced young-of-year survival. Based on an iterative approach, the feasibility of using key events as the foundation for expanding a network of plausible linkages and AOP knowledge was explored and, in the process, important knowledge gaps were identified.Given the scope and scale of the task, prioritization of AOP development was recommended and key research objectives were defined relative to factors such as current animal-use restrictions in the European Union and increased demands for fish toxicity data in chemical management programs globally. The example and strategy described are intended to guide collective efforts to define FELS-related AOPs and develop resource-efficient predictive assays that address the toxicological domain of the OECD 210 test.Keywords: Risk assessment, Aquatic toxicology, Adverse outcome pathways, Mode of action, Animal alternative, Fish early-life stage toxicity, Swim bladderKeywords: Risk assessment, Aquatic toxicology, Adverse outcome pathways, Mode of action, Animal alternative, Fish early-life stage toxicity, Swim bladde

Weight of evidence tools in the prediction of acute fish toxicity

Acute fish toxicity (AFT) is a key endpoint in nearly all regulatory implementations of environmental hazard assessments of chemicals globally. Although it is an early tier assay, the AFT assay is complex and uses many juvenile fish each year for the registration and assessment of chemicals. Thus, it is imperative to seek animal alternative approaches to replace or reduce animal use for environmental hazard assessments. A Bayesian Network (BN) model has been developed that brings together a suite of lines of evidence (LoEs) to produce a probabilistic estimate of AFT without the testing of additional juvenile fish. Lines of evidence include chemical descriptors, mode of action (MoA) assignment, knowledge of algal and daphnid acute toxicity, and animal alternative assays such as fish embryo tests and in vitro fish assays (e.g., gill cytotoxicity). The effort also includes retrieval, assessment, and curation of quality acute fish toxicity data because these act as the baseline of comparison with model outputs. An ideal outcome of this effort would be to have global applicability, acceptance and uptake, relevance to predominant fish species used in chemical assessments, be expandable to allow incorporation of future knowledge, and data to be publicly available. The BN model can be conceived as having incorporated principles of tiered assessment and whose outcomes will be directed by the available evidence in combination with prior information. We demonstrate that, as additional evidence is included in the prediction of a given chemical's ecotoxicity profile, both the accuracy and the precision of the predicted AFT can increase. Ultimately an improved environmental hazard assessment will be achieved.acceptedVersio

Weight of evidence tools in the prediction of acute fish toxicity

Acute fish toxicity (AFT) is a key endpoint in nearly all regulatory implementations of environmental hazard assessments of chemicals globally. Although it is an early tier assay, the AFT assay is complex and uses many juvenile fish each year for the registration and assessment of chemicals. Thus, it is imperative to seek animal alternative approaches to replace or reduce animal use for environmental hazard assessments. A Bayesian Network (BN) model has been developed that brings together a suite of lines of evidence (LoEs) to produce a probabilistic estimate of AFT without the testing of additional juvenile fish. Lines of evidence include chemical descriptors, mode of action (MoA) assignment, knowledge of algal and daphnid acute toxicity, and animal alternative assays such as fish embryo tests and in vitro fish assays (e.g., gill cytotoxicity). The effort also includes retrieval, assessment, and curation of quality acute fish toxicity data because these act as the baseline of comparison with model outputs. An ideal outcome of this effort would be to have global applicability, acceptance and uptake, relevance to predominant fish species used in chemical assessments, be expandable to allow incorporation of future knowledge, and data to be publicly available. The BN model can be conceived as having incorporated principles of tiered assessment and whose outcomes will be directed by the available evidence in combination with prior information. We demonstrate that, as additional evidence is included in the prediction of a given chemical's ecotoxicity profile, both the accuracy and the precision of the predicted AFT can increase. Ultimately an improved environmental hazard assessment will be achieved

Creating context for the use of DNA adduct data in cancer risk assessment: I. Data organization

The assessment of human cancer risk from chemical exposure requires the integration of diverse types of data. Such data involve effects at the cell and tissue levels. This report focuses on the specific utility of one type of data, namely DNA adducts. Emphasis is placed on the appreciation that such DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information. As emerging technologies provide even more sensitive quantitative measurements of DNA adducts, integration that establishes links between DNA adducts and accepted outcome measures becomes critical for risk assessment. The present report proposes an organizational approach for the assessment of DNA adduct data (e.g., type of adduct, frequency, persistence, type of repair process) in concert with other relevant data, such as dosimetry, toxicity, mutagenicity, genotoxicity, and tumor incidence, to inform characterization of the mode of action. DNA adducts are considered biomarkers of exposure, whereas gene mutations and chromosomal alterations are often biomarkers of early biological effects and also can be bioindicators of the carcinogenic process

It is time to develop ecological thresholds of toxicological concern to assist environmental hazard assessment

The Threshold of Toxicological Concern (TTC) concept is well established for assessing human safety of indirect food additives and has been reapplied for a variety of endpoints including carcinogenicity, teratogenicity, and reproductive toxicity. TTC approaches have benefits for screening-level risk assessments, including the potential for rapid decision-making, fully utilizing existing knowledge, reasonable conservativeness for chemicals used in lower volumes (Low Production Volume Chemicals (LPCVs – e.g. 1t/yr) would in principle always require specific information. TTC has found particular favor in the assessment of chemicals used in cosmetics and personal care products as well as other chemicals traditionally used in low volumes. Use of the TTC in environmental safety is just beginning and initial attempts are being published. Key questions focus on hazard extrapolation of diverse taxa across trophic levels, importance of mode of action, and whether safe concentrations for ecosystems estimated from acute or chronic toxicity data are equally useful and in what contexts. This paper will provide an overview of the theoretical basis for developing an eco-TTC with an initial exploration for chemical assessment and boundary conditions for use. An international collaboration under the ILSI Health and Environmental Sciences Institute has been established to address challenges related to developing and applying useful eco-TTC concepts.JRC.I.5-Systems Toxicolog