Integrated Approach for Testing and Assessment for Developmental Neurotoxicity (DNT) to Prioritize Aromatic Organophosphorus Flame Retardants.

Organophosphorus flame retardants (OPFRs) are abundant and persistent in the environment but have limited toxicity information. Their similarity in structure to organophosphate pesticides presents great concern for developmental neurotoxicity (DNT). However, current in vivo testing is not suitable to provide DNT information on the amount of OPFRs that lack data. Over the past decade, an in vitro battery was developed to enhance DNT assessment, consisting of assays that evaluate cellular processes in neurodevelopment and function. In this study, behavioral data of small model organisms were also included. To assess if these assays provide sufficient mechanistic coverage to prioritize chemicals for further testing and/or identify hazards, an integrated approach to testing and assessment (IATA) was developed with additional information from the Integrated Chemical Environment (ICE) and the literature. Human biomonitoring and exposure data were identified and physiologically-based toxicokinetic models were applied to relate in vitro toxicity data to human exposure based on maximum plasma concentration. Eight OPFRs were evaluated, including aromatic OPFRs (triphenyl phosphate (TPHP), isopropylated phenyl phosphate (IPP), 2-ethylhexyl diphenyl phosphate (EHDP), tricresyl phosphate (TMPP), isodecyl diphenyl phosphate (IDDP), tert-butylphenyl diphenyl phosphate (BPDP)) and halogenated FRs ((Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), tris(2-chloroethyl) phosphate (TCEP)). Two representative brominated flame retardants (BFRs) (2,2'4,4'-tetrabromodiphenyl ether (BDE-47) and 3,3',5,5'-tetrabromobisphenol A (TBBPA)) with known DNT potential were selected for toxicity benchmarking. Data from the DNT battery indicate that the aromatic OPFRs have activity at similar concentrations as the BFRs and should therefore be evaluated further. However, these assays provide limited information on the mechanism of the compounds. By integrating information from ICE and the literature, endocrine disruption was identified as a potential mechanism. This IATA case study indicates that human exposure to some OPFRs could lead to a plasma concentration similar to those exerting in vitro activities, indicating potential concern for human health.

Reproductive and developmental toxicity following exposure to organophosphate ester flame retardants and plasticizers, triphenyl phosphate and isopropylated phenyl phosphate, in Sprague Dawley rats.

Two organophosphate esters used as flame retardants and plasticizers, triphenyl phosphate (TPHP) and isopropylated phenyl phosphate (IPP), have been detected in environmental samples around the world. Human exposure primarily occurs via oral ingestion with reported higher concentrations in children. Currently, there are no data to evaluate potential risk from exposure to either TPHP or IPP during fetal development. These short-term perinatal studies in rats provide preliminary toxicity data for TPHP and IPP, including information on transfer to fetus/offspring and across the pup blood-brain barrier. In separate experiments, TPHP or IPP were administered via dosed feed at concentrations 0, 1000, 3000, 10 000, 15 000, or 30 000 ppm to time-mated Hsd:Sprague Dawley SD rats from gestation day (GD) 6 through postnatal day (PND) 28; offspring were provided dosed feed at the same concentration as their dam (PND 28-PND 56). TPHP- and IPP-related toxicity resulted in removal of both 30 000 ppm groups on GD 12 and 15 000 ppm IPP group after parturition. Body weight and organ weights were impacted with exposure in remaining dams. Reproductive performance was perturbed at ≥10 000 ppm TPHP and all IPP exposure groups. In offspring, both TPHP- and IPP-related toxicity was noted in pups at ≥10 000 ppm as well as reduction in bodyweights, delays in pubertal endpoints, and/or reduced cholinesterase enzyme activity starting at 1000 ppm TPHP or IPP. Preliminary internal dose assessment indicated gestational and lactational transfer following exposure to TPHP or IPP. These findings demonstrate that offspring development is sensitive to 1000 ppm TPHP or IPP exposure.

Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches.

Neurotoxicology is the study of adverse effects on the structure or function of the developing or mature adult nervous system following exposure to chemical, biological, or physical agents. The development of more informative alternative methods to assess developmental (DNT) and adult (NT) neurotoxicity induced by xenobiotics is critically needed. The use of such alternative methods including in silico approaches that predict DNT or NT from chemical structure (e.g., statistical-based and expert rule-based systems) is ideally based on a comprehensive understanding of the relevant biological mechanisms. This paper discusses known mechanisms alongside the current state of the art in DNT/NT testing. In silico approaches available today that support the assessment of neurotoxicity based on knowledge of chemical structure are reviewed, and a conceptual framework for the integration of in silico methods with experimental information is presented. Establishing this framework is essential for the development of protocols, namely standardized approaches, to ensure that assessments of NT and DNT based on chemical structures are generated in a transparent, consistent, and defendable manner.

Exploring the Influence of Experimental Design on Toxicity Outcomes in Zebrafish Embryo Tests.

Compound toxicity data obtained from independent zebrafish laboratories can vary vastly, complicating the use of zebrafish screening for regulatory decisions. Differences in the assay protocol parameters are the primary source of variability. We investigated this issue by utilizing data from the NTP DNT-DIVER database (https://doi.org/10.22427/NTP-DATA-002-00062-0001-0000-1, last accessed June 2, 2022), which consists of data from zebrafish developmental toxicity (devtox) and locomotor response (designated as "neurotox") screens from 3 independent laboratories, using the same set of 87 compounds. The data were analyzed using the benchmark concentration (BMC) modeling approach, which estimates the concentration of interest based on a predetermined response threshold. We compared the BMC results from 3 laboratories (A, B, C) in 3 toxicity outcome categories: mortality, cumulative devtox, and neurotox, in terms of activity calls and potency values. We found that for devtox screening, laboratories with similar/same protocol parameters (B vs C) had an active call concordance as high as 86% with negligible potency difference. For neurotox screening, active call concordances between paired laboratories are lower than devtox screening (highest 68%). When protocols with different protocol parameters were compared, the concordance dropped, and the potency shift was on average about 3.8-fold for the cumulative devtox outcome and 5.8-fold for the neurotox outcome. The potential contributing protocol parameters for potency shift are listed or ranked. This study provides a quantitative assessment of the source of variability in zebrafish screening protocols and sets the groundwork for the ongoing Systematic Evaluation of the Application of Zebrafish in Toxicology effort at the National Toxicology Program.

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery.

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell-based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 µM (5 FRs), 1<10 µM (7 FRs) to the >10 µM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell-based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment.

Beyond Cholinesterase Inhibition: Developmental Neurotoxicity of Organophosphate Ester Flame Retardants and Plasticizers.

BACKGROUND: To date, the toxicity of organophosphate esters has primarily been studied regarding their use as pesticides and their effects on the neurotransmitter acetylcholinesterase (AChE). Currently, flame retardants and plasticizers are the two largest market segments for organophosphate esters and they are found in a wide variety of products, including electronics, building materials, vehicles, furniture, car seats, plastics, and textiles. As a result, organophosphate esters and their metabolites are routinely found in human urine, blood, placental tissue, and breast milk across the globe. It has been asserted that their neurological effects are minimal given that they do not act on AChE in precisely the same way as organophosphate ester pesticides. OBJECTIVES: This commentary describes research on the non-AChE neurodevelopmental toxicity of organophosphate esters used as flame retardants and plasticizers (OPEs). Studies in humans, mammalian, nonmammalian, and in vitro models are presented, and relevant neurodevelopmental pathways, including adverse outcome pathways, are described. By highlighting this scientific evidence, we hope to elevate the level of concern for widespread human exposure to these OPEs and to provide recommendations for how to better protect public health. DISCUSSION: Collectively, the findings presented demonstrate that OPEs can alter neurodevelopmental processes by interfering with noncholinergic pathways at environmentally relevant doses. Application of a pathways framework indicates several specific mechanisms of action, including perturbation of glutamate and gamma-aminobutyric acid and disruption of the endocrine system. The effects may have implications for the development of cognitive and social skills in children. Our conclusion is that concern is warranted for the developmental neurotoxicity of OPE exposure. We thus describe important considerations for reducing harm and to provide recommendations for government and industry decision makers. https://doi.org/10.1289/EHP9285.

Cobalt-induced oxidative stress contributes to alveolar/bronchiolar carcinogenesis in B6C3F1/N mice.

Rodent alveolar/bronchiolar carcinomas (ABC) that arise either spontaneously or due to chemical exposure are similar to a subtype of lung adenocarcinomas in humans. B6C3F1/N mice and F344/NTac rats exposed to cobalt metal dust (CMD) by inhalation developed ABCs in a dose dependent manner. In CMD-exposed mice, the incidence of Kras mutations in ABCs was 67% with 80% of those being G to T transversions on codon 12 suggesting a role of oxidative stress in the pathogenesis. In vitro studies, such as DMPO (5,5-dimethyl-1-pyrroline N-oxide) immune-spin trapping assay, and dihydroethidium (DHE) fluorescence assay on A549 and BEAS-2B cells demonstrated increased oxidative stress due to cobalt exposure. In addition, significantly increased 8-oxo-dG adducts were demonstrated by immunohistochemistry in lungs from mice exposed to CMD for 90 days. Furthermore, transcriptomic analysis on ABCs arising spontaneously or due to chronic CMD-exposure demonstrated significant alterations in canonical pathways related to MAPK signaling (IL-8, ErbB, Integrin, and PAK pathway) and oxidative stress (PI3K/AKT and Melatonin pathway) in ABCs from CMD-exposed mice. Oxidative stress can stimulate PI3K/AKT and MAPK signaling pathways. Nox4 was significantly upregulated only in CMD-exposed ABCs and NOX4 activation of PI3K/AKT can lead to increased ROS levels in human cancer cells. The gene encoding Ereg was markedly up-regulated in CMD-exposed mice. Oncogenic KRAS mutations have been shown to induce EREG overexpression. Collectively, all these data suggest that oxidative stress plays a significant role in CMD-induced pulmonary carcinogenesis in rodents and these findings may also be relevant in the context of human lung cancers.

Evaluation of chemical compounds that inhibit neurite outgrowth using GFP-labeled iPSC-derived human neurons.

Due to the increasing number of drugs and untested environmental compounds introduced into commercial use, there is recognition for a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. One process that has been implicated in neurodevelopment is neurite outgrowth; the disruption of which can result in adverse outcomes that persist later in life. Here, we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content, high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. The assay was optimized for use in a 1536-well plate format. Then, we used this assay to screen a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 h and then treated with compounds at 11 concentrations ranging from 1.56 nM to 92 µM for 24 and 48 h. Effects of compounds on neurite outgrowth were evaluated by quantifying total neurite length, number of segments, and maximum neurite length per cell. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2',4,4'-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization.

Organophosphorus flame retardants are developmental neurotoxicants in a rat primary brainsphere in vitro model.

Due to regulatory bans and voluntary substitutions, halogenated polybrominated diphenyl ether (PBDE) flame retardants (FR) are increasingly substituted by mainly organophosphorus FR (OPFR). Leveraging a 3D rat primary neural organotypic in vitro model (rat brainsphere), we compare developmental neurotoxic effects of BDE-47-the most abundant PBDE congener-with four OPFR (isopropylated phenyl phosphate-IPP, triphenyl phosphate-TPHP, isodecyl diphenyl phosphate-IDDP, and tricresyl phosphate (also known as trimethyl phenyl phosphate)-TMPP). Employing mass spectroscopy-based metabolomics and transcriptomics, we observe at similar human-relevant non-cytotoxic concentrations (0.1-5 µM) stronger developmental neurotoxic effects by OPFR. This includes toxicity to neurons in the low µM range; all FR decrease the neurotransmitters glutamate and GABA (except BDE-47 and TPHP). Furthermore, n-acetyl aspartate (NAA), considered a neurologic diagnostic molecule, was decreased by all OPFR. At similar concentrations, the FR currently in use decreased plasma membrane dopamine active transporter expression, while BDE-47 did not. Several findings suggest astrogliosis induced by the OPFR, but not BDE-47. At the 5 µM concentrations, the OPFR more than BDE-47 interfered with myelination. An increase of cytokine gene and receptor expressions suggests that exposure to OPFR may induce an inflammatory response. Pathway/category overrepresentation shows disruption in 1) transmission of action potentials, cell-cell signaling, synaptic transmission, receptor signaling, (2) immune response, inflammation, defense response, (3) cell cycle and (4) lipids metabolism and transportation. Taken together, this appears to be a case of regretful substitution with substances not less developmentally neurotoxic in a primary rat 3D model.

Qualitative and Quantitative Neuropathology Approaches Using Magnetic Resonance Microscopy (Diffusion Tensor Imaging) and Stereology in a Hexachlorophene Model of Myelinopathy in Sprague-Dawley Rats.

It is well established that hexachlorophene, which is used as an antibacterial agent, causes intramyelinic edema in humans and animal models. The hexachlorophene myelinopathy model, in which male Sprague-Dawley rats received 25 to 30 mg/kg hexachlorophene by gavage for up to 5 days, provided an opportunity to compare traditional neuropathology evaluations with magnetic resonance microscopy (MRM) findings. In addition, stereology assessments of 3 neuroanatomical sites were compared to quantitative measurements of similar structures by MRM. There were positive correlations between hematoxylin and eosin and luxol fast blue stains and MRM for identifying intramyelinic edema in the cingulum of corpus callosum, optic chiasm, anterior commissure (aca), lateral olfactory tracts, pyramidal tracts (py), and white matter tracts in the cerebellum. Stereology assessments were focused on the aca, longitudinal fasciculus of the pons, and py and demonstrated differences between control and treated rats, as was observed using MRM. The added value of MRM assessments was the ability to acquire qualitative 3-dimensional (3-D) images and obtain quantitative measurements of intramyelinic edema in 26 neuroanatomical sites in the intact brain. Also, diffusion tensor imaging (fractional anisotropy [FA]) indicated that there were changes in the cytoarchitecture of the white matter as detected by decreases in the FA in the treated compared to the control rats. This study demonstrates creative strategies that are possible using qualitative and quantitative assessments of potential white matter neurotoxicants in nonclinical toxicity studies. Our results lead us to the conclusion that volumetric analysis by MRM and stereology adds significant value to the standard 2-D microscopic evaluations.

Multigenerational reproductive assessment of 4-methylimidazole administered in the diet to Hsd:Sprague Dawley SD rats.

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

Corrigendum to A high-throughput approach to identify specific neurotoxicants / developmental toxicants in human neuronal cell function assays.

In this manuscript, which appeared in ALTEX 35 , 235-253 ( doi:10.14573/altex.1712182 ), the Acknowledgements should read: This work was supported by the Land BW, the Doerenkamp-Zbinden Foundation, the DFG (RTG1331, KoRS-CB), the BMBF (NeuriTox), and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

Screening for neurotoxic potential of 15 flame retardants using freshwater planarians.

Asexual freshwater planarians are an attractive invertebrate model for high-throughput neurotoxicity screening, because they possess multiple quantifiable behaviors to assess distinct neuronal functions. Planarians uniquely allow direct comparisons between developing and adult animals to distinguish developmentally selective effects from general neurotoxicity. In this study, we used our automated planarian screening platform to compare the neurotoxicity of 15 flame retardants (FRs), consisting of representative phased-out brominated (BFRs) and replacement organophosphorus FRs (OPFRs). OPFRs have emerged as a proposed safer alternative to BFRs; however, limited information is available on their health effects. We found 11 of the 15 FRs (3/6 BFRs, 7/8 OPFRs, and Firemaster 550) caused adverse effects in both adult and developing planarians with similar nominal lowest-effect-levels for BFRs and OPFRs. This suggests that replacement OPFRs are comparably neurotoxic to the phased-out compounds. BFRs were primarily systemically toxic, whereas OPFRs, except Tris(2-chloroethyl) phosphate, shared a behavioral phenotype in response to noxious heat at sublethal concentrations, indicating specific neurotoxic effects. We found this behavioral phenotype was correlated with cholinesterase inhibition, thus linking behavioral outcomes to molecular targets. By directly comparing effects on adult and developing planarians, we further found that one BFR (3,3',5,5'-Tetrabromobisphenol A) caused a developmental selective defect. Together, these results demonstrate that our planarian screening platform yields high content data from various behavioral and morphological endpoints, allowing us to distinguish selective neurotoxic effects and effects specific to the developing nervous system. Ten of these 11 bioactive FRs were previously found to be bioactive in other models, including cell culture and alternative animal models (nematodes and zebrafish). This level of concordance across different platforms emphasizes the urgent need for further evaluation of OPFRs in mammalian systems.

Organophosphate Ester Flame Retardants: Are They a Regrettable Substitution for Polybrominated Diphenyl Ethers?

As the use of polybrominated diphenyl ethers (PBDEs), and the entire class of organohalogen flame retardants, is declining, the use of organophosphate esters flame retardants (OPFRs) is increasing. In this paper, we ask whether OPFRs are a better choice than PBDEs. To address this question, we compared OPFRs with PBDEs for a wide range of properties. OPFRs exposure is ubiquitous in people and in outdoor and indoor environments, and are now often found at higher levels compared to PBDE peak exposure levels. Furthermore, data from toxicity testing, epidemiological studies, and risk assessments all suggest that there are health concerns at current exposure levels for both halogenated and non-halogenated OPFRs. Obtaining the scientific evidence needed for regulation of OPFRs can take many years. Given the large number of OPFRs in use, manufacturers can move towards healthier and safer products by developing innovative ways to reduce fire hazard for electronics enclosures, upholstered furniture, building materials and other consumer products without adding flame retardant chemicals.

Application of Benchmark Concentration (BMC) Analysis on Zebrafish Data: A New Perspective for Quantifying Toxicity in Alternative Animal Models.

Over the past decade, the zebrafish is increasingly being used as a model to screen for chemical-mediated toxicities including developmental toxicity (DT) and neurotoxicity (NT). One of the major challenges is lack of harmonization in data analysis approaches, thereby posing difficulty in comparing findings across laboratories. To address this, we sought to establish a unified data analysis strategy for both DT and NT data, by adopting the benchmark concentration (BMC) analysis. There are two critical aspects in the BMC analysis: having a toxicity endpoint amenable for BMC and selecting a proper benchmark response (BMR) for the endpoint. For the former, in addition to the typical endpoints in NT assay (eg, hyper/hypo- response quantified by distance moved), we also used endpoints that assess the differences in movement patterns between chemical-treated embryos and control embryos. For the latter, we standardized the selection of BMR, which is analogous to minimum activity threshold, based on intrinsic response variations in the endpoint. When comparing our BMC results with a traditionally used LOAEL method (lowest-observed-adverse-effect level), we found high active compound concordance (100% for DT vs 74% for NT); generally, the BMC was more sensitive than LOAEL (no. of BMC more sensitive/no. of concordant active compounds, 43/50 for DT vs 16/26 for NT). Using the BMC with standardized toxicity endpoints and an appropriate BMR, we may now have a unified data-analysis approach to comparing results across different zebrafish datasets, for a better understanding of strengths and challenges when using the zebrafish as a screening tool.

International Regulatory and Scientific Effort for Improved Developmental Neurotoxicity Testing.

The Organisation for Economic Co-Operation and Development (OECD) coordinates international efforts to enhance developmental neurotoxicity (DNT) testing. In most regulatory sectors, including the ones dealing with pesticides and industrial chemicals registration, historical use of the in vivo DNT test guideline has been limited. Current challenges include a lack of DNT data and mechanistic information for thousands of chemicals, and difficulty in interpreting results. A series of workshops in the last decade has paved the way for a consensus among stakeholders that there is need for a DNT testing battery that relies on in vitro endpoints (proliferation, differentiation, synaptogenesis, etc.) and is complemented by alternative species (eg, zebrafish) assays. Preferably, a battery of in vitro and alternative assays should be anchored toward mechanistic relevance for applying an integrated approach for testing and assessment (IATA) framework. Specific activities have been initiated to facilitate this OECD project: the collation of available DNT in vitro methods and their scoring for readiness; the selection of these methods to form a DNT testing battery; the generation of a reference set of chemicals that will be tested using the battery; the case studies exemplifying how DNT in vitro data can be interpreted; and the development of an OECD guidance document. This manuscript highlights these international efforts and activities.

Functional and Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural 3D Cultures.

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.

Screening for Developmental Neurotoxicity at the National Toxicology Program: The Future Is Here.

The National Toxicology Program (NTP) receives requests to evaluate chemicals with potential to cause adverse health effects, including developmental neurotoxicity (DNT). Some recent requests have included classes of chemicals such as flame retardants, polycyclic aromatic compounds, perfluoroalkyl substances, and bisphenol A analogs with approximately 20-50 compounds per class, many of which include commercial mixtures. However, all the compounds within a class cannot be tested using traditional DNT animal testing guideline studies due to resource and time limitations. Hence, a rapid and biologically relevant screening approach is required to prioritize compounds for further in vivo testing. Because neurodevelopment is a complex process involving multiple distinct cellular processes, one assay will unlikely address the complexity. Hence, the NTP sought to characterize a battery of in vitro and alternative animal assays to quantify chemical effects on a variety of neurodevelopmental processes. A culmination of this effort resulted in a NTP-hosted collaborative project with approximately 40 participants spanning across domains of academia, industry, government, and regulatory agencies; collaborators presented data on cell-based assays and alternative animal models that was generated using a targeted set of compounds provided by the NTP. The NTP analyzed the assay results using benchmark concentration (BMC) modeling to be able to compare results across the divergent assays. The results were shared with the contributing researchers on a private web application during the workshop, and are now publicly available. This article highlights the overview and goals of the project, and describes the NTP's approach in creating the chemical library, development of NTPs data analysis strategy, and the structure of the web application. Finally, we discuss key issues with emphasis on the utility of this approach, and knowledge gaps that need to be addressed for its use in regulatory decision making.

Detection and Prioritization of Developmentally Neurotoxic and/or Neurotoxic Compounds Using Zebrafish.

The standard methods for toxicity testing using rodent models cannot keep pace with the increasing number of chemicals in our environment due to time and resource limitations. Hence, there is an unmet need for fast, sensitive, and cost-effective alternate models to reliably predict toxicity. As part of Tox21 Phase III's effort, a 90-compound library was created and made available to researchers to screen for neurotoxicants using novel technology and models. The chemical library was evaluated in zebrafish in a dose-range finding test for embryo-toxicity (ie, mortality or morphological alterations induced by each chemical). In addition, embryos exposed to the lowest effect level and nonobservable effect level were used to measure the internal concentration of the chemicals within the embryos by bioanalysis. Finally, considering the lowest effect level as the highest testing concentration, a functional assay was performed based on locomotor activity alteration in response to light-dark changes. The quality control chemicals included in the library, ie, negative controls and replicated chemicals, indicate that the assays performed were reliable. The use of analytical chemistry pointed out the importance of measuring chemical concentration inside embryos, and in particular, in the case of negative chemicals to avoid false negative classification. Overall, the proposed approach presented a good sensitivity and supports the inclusion of zebrafish assays as a reliable, relevant, and efficient screening tool to identify, prioritize, and evaluate chemical toxicity.

Toxicity profiling of flame retardants in zebrafish embryos using a battery of assays for developmental toxicity, neurotoxicity, cardiotoxicity and hepatotoxicity toward human relevance.

Following the voluntary phase-out of brominated flame retardants (BFRs) due to their environmental persistence and toxicity, the organophosphorus flame retardants (OPFRs) are emerging replacements. However, there is limited information on the potential human health effects of the OPFRs. Zebrafish embryos are a viable vertebrate model organism with many advantages for high throughput testing toward human hazard assessment. We utilized zebrafish embryos to assess developmental toxicity, neurotoxicity, cardiotoxicity and hepatotoxicity, of eight replacement OPFRs: (triphenyl phosphate [TPHP], isopropylated phenyl phosphate [IPP], 2-ethylhexyl diphenyl phosphate [EHDP], tert-butylated phenyl diphenyl phosphate [BPDP], trimethyl phenyl phosphate [TMPP], isodecyl diphenyl phosphate [IDDP], tris(1,3-dichloroisopropyl) phosphate [TDCIPP], and tris(2-chloroethyl) phosphate [TCEP]) and two BFRs (3,3',5,5'- tetrabromobisphenol A [TBBPA] and 2,2'4,4'-brominated diphenyl ether [BDE-47]). To determine potential effects on teratogenicity, embryos were exposed to flame retardants (FRs) at 4 h post fertilization (hpf) to 4 days post fertilization (dpf) and morphological alterations and corresponding survival were evaluated at 2 and 4 dpf. Internal concentrations were measured in larvae used in this assay by liquid chromatography-mass spectrometry. Locomotor activity was assessed in larvae treated for 48 h (from 3 dpf to 5 dpf), followed by hepatotoxicity evaluation. Finally, alterations in heart rate and rhythmicity were assessed to determine cardiotoxicity in 48 hpf embryos exposed to compounds for 3 h. Results suggest that several OPFRs (BPDP, EHDP; IPP, TMPP; TPHP and TDCIPP) produced adverse effects in multiple target organs at concentrations comparable to the two BFRs. As these OPFRs have the capacity to disrupt an integrated vertebrate model, they potentially have the capacity to affect mammalian biology. Then, we compared the lowest effective levels (LEL) in zebrafish with estimated or measured human plasma concentrations using biomonitoring data (human plasma, breast milk, handwipe samples and house dust) and a high throughput toxicokinetic (HTTK) model. Results indicate that for some compounds, the nominal LELs were within the range of human exposures, while internal LELs in zebrafish are above internal exposures in humans. These findings demonstrate the value of the zebrafish model as a relevant screening tool and support the need for further hazard characterization of the OPFRs.