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JMIR Research Protocols - Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments
<!doctype html><html data-n-head-ssr lang="en" data-n-head="%7B%22lang%22:%7B%22ssr%22:%22en%22%7D%7D"><head ><meta data-n-head="ssr" charset="utf-8"><meta data-n-head="ssr" name="viewport" content="width=device-width, initial-scale=1"><meta data-n-head="ssr" name="msapplication-TileColor" content="#247CB3"><meta data-n-head="ssr" name="msapplication-TileImage" content="https://asset.jmir.pub/assets/static/images/mstile-144x144.png"><meta data-n-head="ssr" name="description" content="Background: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study. Objective: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays. Methods: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures. Results: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity. Conclusions: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals. "><meta data-n-head="ssr" name="keywords" content="general population; workers; blood-brain barrier; organic solvent exposure; neurotoxicity; liver toxicity; human cell cultures"><meta data-n-head="ssr" name="DC.Title" content="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments"><meta data-n-head="ssr" name="DC.Subject" content="general population; workers; blood-brain barrier; organic solvent exposure; neurotoxicity; liver toxicity; human cell cultures"><meta data-n-head="ssr" name="DC.Description" content="Background: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study. Objective: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays. Methods: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures. Results: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity. Conclusions: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals. "><meta data-n-head="ssr" name="DC.Publisher" content="JMIR Research Protocols"><meta data-n-head="ssr" name="DC.Publisher.Address" content="JMIR Publications // 130 Queens Quay East, Unit 1100 // Toronto, ON, M5A 0P6"><meta data-n-head="ssr" name="DC.Date" scheme="ISO8601" content="2024-01-18"><meta data-n-head="ssr" name="DC.Type" content="Text.Serial.Journal"><meta data-n-head="ssr" name="DC.Format" scheme="IMT" content="text/xml"><meta data-n-head="ssr" name="DC.Identifier" content="doi:10.2196/50300"><meta data-n-head="ssr" name="DC.Language" scheme="ISO639-1" content="EN"><meta data-n-head="ssr" name="DC.Relation" content="World"><meta data-n-head="ssr" name="DC.Source" content="JMIR Res Protoc 2024;13:e50300 https://www.researchprotocols.org/2024/1/e50300"><meta data-n-head="ssr" name="DC.Rights" content="Unless stated otherwise, all articles are open-access distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work ("first published in JMIR Research Protocols...") is properly cited with original URL and bibliographic citation information. The complete bibliographic information, a link to the original publication on http://www.researchprotocols.org/, as well as this copyright and license information must be included."><meta data-n-head="ssr" property="og:title" content="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments"><meta data-n-head="ssr" property="og:type" content="article"><meta data-n-head="ssr" property="og:url" content="https://www.researchprotocols.org/2024/1/e50300"><meta data-n-head="ssr" property="og:image" content="https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png"><meta data-n-head="ssr" property="og:site_name" content="JMIR Research Protocols"><meta data-n-head="ssr" name="twitter:card" content="summary_large_image"><meta data-n-head="ssr" name="twitter:site" content="@jmirpub"><meta data-n-head="ssr" name="twitter:title" content="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments"><meta data-n-head="ssr" name="twitter:description" content="Background: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study. Objective: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays. Methods: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures. Results: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity. Conclusions: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals. "><meta data-n-head="ssr" name="twitter:image" content="https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png"><meta data-n-head="ssr" name="citation_title" content="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments"><meta data-n-head="ssr" name="citation_journal_title" content="JMIR Research Protocols"><meta data-n-head="ssr" name="citation_publisher" content="JMIR Publications Inc., Toronto, Canada"><meta data-n-head="ssr" name="citation_doi" content="10.2196/50300"><meta data-n-head="ssr" name="citation_issue" content="1"><meta data-n-head="ssr" name="citation_volume" content="13"><meta data-n-head="ssr" name="citation_firstpage" content="e50300"><meta data-n-head="ssr" name="citation_date" content="2024-01-18"><meta data-n-head="ssr" name="citation_abstract_html_url" content="https://www.researchprotocols.org/2024/1/e50300"><meta data-n-head="ssr" name="citation_abstract_pdf_url" content="https://www.researchprotocols.org/2024/1/e50300/PDF"><meta data-n-head="ssr" name="DC.Creator" content="Nancy B"><meta data-n-head="ssr" name="DC.Contributor" content="Nancy B Hopf"><meta data-n-head="ssr" name="DC.Contributor" content="Laura Suter-Dick"><meta data-n-head="ssr" name="DC.Contributor" content="Jörg Huwyler"><meta data-n-head="ssr" name="DC.Contributor" content="Myriam Borgatta"><meta data-n-head="ssr" name="DC.Contributor" content="Lucie Hegg"><meta data-n-head="ssr" name="DC.Contributor" content="David Pamies"><meta data-n-head="ssr" name="DC.Contributor" content="Hélène Paschoud"><meta data-n-head="ssr" name="DC.Contributor" content="Ramya Deepthi Puligilla"><meta data-n-head="ssr" name="DC.Contributor" content="Elena Reale"><meta data-n-head="ssr" name="DC.Contributor" content="Sophie Werner"><meta data-n-head="ssr" name="DC.Contributor" content="Marie-Gabrielle Zurich"><meta data-n-head="ssr" name="citation_authors" content="Nancy B Hopf"><meta data-n-head="ssr" name="citation_authors" content="Laura Suter-Dick"><meta data-n-head="ssr" name="citation_authors" content="Jörg Huwyler"><meta data-n-head="ssr" name="citation_authors" content="Myriam Borgatta"><meta data-n-head="ssr" name="citation_authors" content="Lucie Hegg"><meta data-n-head="ssr" name="citation_authors" content="David Pamies"><meta data-n-head="ssr" name="citation_authors" content="Hélène Paschoud"><meta data-n-head="ssr" name="citation_authors" content="Ramya Deepthi Puligilla"><meta data-n-head="ssr" name="citation_authors" content="Elena Reale"><meta data-n-head="ssr" name="citation_authors" content="Sophie Werner"><meta data-n-head="ssr" name="citation_authors" content="Marie-Gabrielle Zurich"><title>JMIR Research Protocols - Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure 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in the following <span class="collection__span">e-collection/theme issue:</span></h2> <a href="/themes/99" data-test="article-collection" aria-label="311 articles belongs to RCTs - Protocols/Proposals (non-eHealth) e-collection/theme issue" class="collection__link"> RCTs - Protocols/Proposals (non-eHealth) (311) </a><a href="/themes/102" data-test="article-collection" aria-label="186 articles belongs to Neurology and Neurosciences e-collection/theme issue" class="collection__link"> Neurology and Neurosciences (186) </a></div></div> <div class="row"><div class="main col-lg-9 mb-1"><!----> <div data-test="details" class="details"><div><p id="main-content" tabindex="0"> Published on <time datetime="18.01.2024">18.01.2024 </time> in <span data-test="issue-info"><a href="/2024/1" class="nuxt-link-active"> Vol 13<!----> (2024)<!----></a></span></p> <!----></div> <div class="preprints-version"><span aria-hidden="true" class="icon fas fa-thumbtack"></span> <div><span class="ml-2"> Preprints (earlier versions) of this paper are available at <a data-test="preprint-link" aria-label="'Preprints (earlier versions) of this paper are available at preprints.jmir.org/preprint/'50300" href="https://preprints.jmir.org/preprint/50300" target="_blank">https://preprints.jmir.org/preprint/50300</a>, first published <time datetime="June 26, 2023">June 26, 2023</time>. </span></div></div></div> <div class="info mt-3"><div class="info__article-img"><div data-v-10f10a3e><img data-srcset="https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png 480w,https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png 960w,https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png 1920w,https://asset.jmir.pub/assets/472e3c942ef8c40f33ac7fef996e9769.png 2500w" alt="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments" title="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments" aria-label="Article Thumbnail Image" src="https://asset.jmir.pub/placeholder.svg" data-v-10f10a3e></div> <div data-test="article-img-info" class="info__article-img-info"><span aria-hidden="true" class="icon fas fa-search-plus"></span></div></div> <div class="info__title-authors"><h1 tabindex="0" aria-label="Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments" class="h3 mb-0 mt-0">Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments</h1> <h2 class="info__hidden-title"> Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments </h2> <div class="mt-3"><p tabindex="0" class="authors-for-screen-reader"> Authors of this article: </p> <span data-test="authors-info" class="info__authors"><a href="/search?term=Nancy%20B%20Hopf&type=author&precise=true" aria-label="Nancy B Hopf. Search more articles by this author."> Nancy B Hopf<sup>1, 2</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-4490-6109"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Laura%20Suter-Dick&type=author&precise=true" aria-label="Laura Suter-Dick. Search more articles by this author."> Laura Suter-Dick<sup>2, 3</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-1449-3913"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=J%C3%B6rg%20Huwyler&type=author&precise=true" aria-label="Jörg Huwyler. Search more articles by this author."> Jörg Huwyler<sup>2, 4</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0003-1748-5676"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Myriam%20Borgatta&type=author&precise=true" aria-label="Myriam Borgatta. Search more articles by this author."> Myriam Borgatta<sup>1, 2</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-6326-0614"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Lucie%20Hegg&type=author&precise=true" aria-label="Lucie Hegg. Search more articles by this author."> Lucie Hegg<sup>1, 2</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0009-0002-3105-9577"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=David%20Pamies&type=author&precise=true" aria-label="David Pamies. Search more articles by this author."> David Pamies<sup>2, 5</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-1224-573X"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=H%C3%A9l%C3%A8ne%20Paschoud&type=author&precise=true" aria-label="Hélène Paschoud. Search more articles by this author."> Hélène Paschoud<sup>1, 2</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0001-5429-5148"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Ramya%20Deepthi%20Puligilla&type=author&precise=true" aria-label="Ramya Deepthi Puligilla. 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Search more articles by this author."> Elena Reale<sup>1, 2</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-0853-0693"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Sophie%20Werner&type=author&precise=true" aria-label="Sophie Werner. Search more articles by this author."> Sophie Werner<sup>2, 3, 4</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0009-0007-6531-2610"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <span style="margin-left: -2px;"> ; </span></span><span data-test="authors-info" class="info__authors"><a href="/search?term=Marie-Gabrielle%20Zurich&type=author&precise=true" aria-label="Marie-Gabrielle Zurich. Search more articles by this author."> Marie-Gabrielle Zurich<sup>2, 5</sup> <!----></a> <span><a aria-label="Visit this author on ORCID website" data-test="orcid-link" target="_blank" href="https://orcid.org/0000-0002-3168-5968"><img src="https://asset.jmir.pub/assets/static/images/Orcid-ID-Logo-Colour.png" alt="Author Orcid Image" aria-label="Author Orcid Image" class="info__orcid-img"></a></span> <!----></span></div> <!----></div></div> <div role="tablist" aria-label="Article" class="tabs"><a href="/2024/1/e50300/" aria-current="page" role="tab" aria-label="Article" data-test="tabs" class="nuxt-link-exact-active nuxt-link-active active"> Article </a><a href="/2024/1/e50300/authors" role="tab" aria-label="Authors" data-test="tabs"> Authors </a><a href="/2024/1/e50300/citations" role="tab" aria-label="Cited by " data-test="tabs"> Cited by </a><a href="/2024/1/e50300/tweetations" role="tab" aria-label="Tweetations (3)" data-test="tabs"> Tweetations (3) </a><a href="/2024/1/e50300/metrics" role="tab" aria-label="Metrics" data-test="tabs"> Metrics </a></div> <div class="container"><div class="row"><div class="col-lg-3 mb-5 sidebar-sections"><div class="sidebar-nav"><div class="sidebar-nav-sticky"><ul></ul></div></div></div> <div data-test="keyword-links" class="col-lg-9 article"><main id="wrapper" class="wrapper ArticleMain clearfix"><section class="inner-wrapper clearfix"><section class="main-article-content clearfix"><article class="ajax-article-content"><h4 class="h4-original-paper"><span class="typcn typcn-document-text"></span>Protocol</h4><div class="authors-container"><div class="authors clearfix"></div></div><div class="authors-container"><div class="authors clearfix"></div></div><div class="authors-container"><div class="authors clearfix"><ul class="clearfix"><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Nancy%20B+Hopf" class="btn-view-author-options">Nancy B Hopf<sup><small>1,</small></sup><sup><small>2</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-4490-6109" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Laura+Suter-Dick" class="btn-view-author-options">Laura Suter-Dick<sup><small>2,</small></sup><sup><small>3</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-1449-3913" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Jörg+Huwyler" class="btn-view-author-options">Jörg Huwyler<sup><small>2,</small></sup><sup><small>4</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0003-1748-5676" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Myriam+Borgatta" class="btn-view-author-options">Myriam Borgatta<sup><small>1,</small></sup><sup><small>2</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-6326-0614" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Lucie+Hegg" class="btn-view-author-options">Lucie Hegg<sup><small>1,</small></sup><sup><small>2</small></sup>, MSc</a><a class="author-orcid" href="https://orcid.org/0009-0002-3105-9577" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=David+Pamies" class="btn-view-author-options">David Pamies<sup><small>2,</small></sup><sup><small>5</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-1224-573X" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Hélène+Paschoud" class="btn-view-author-options">Hélène Paschoud<sup><small>1,</small></sup><sup><small>2</small></sup>, MSc</a><a class="author-orcid" href="https://orcid.org/0000-0001-5429-5148" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Ramya%20Deepthi+Puligilla" class="btn-view-author-options">Ramya Deepthi Puligilla<sup><small>2,</small></sup><sup><small>4</small></sup>, MSc</a><a class="author-orcid" href="https://orcid.org/0000-0002-0767-9402" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Elena+Reale" class="btn-view-author-options">Elena Reale<sup><small>1,</small></sup><sup><small>2</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-0853-0693" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Sophie+Werner" class="btn-view-author-options">Sophie Werner<sup><small>2,</small></sup><sup><small>3,</small></sup><sup><small>4</small></sup>, MSc</a><a class="author-orcid" href="https://orcid.org/0009-0007-6531-2610" target="_blank" title="ORCID"> </a>; </li><li><a href="/search/searchResult?field%5B%5D=author&criteria%5B%5D=Marie-Gabrielle+Zurich" class="btn-view-author-options">Marie-Gabrielle Zurich<sup><small>2,</small></sup><sup><small>5</small></sup>, PhD</a><a class="author-orcid" href="https://orcid.org/0000-0002-3168-5968" target="_blank" title="ORCID"> </a></li></ul><div class="author-affiliation-details"><p><sup>1</sup>Center for Primary Care and Public Health (Unisanté), University of Lausanne, Lausanne, Switzerland</p><p><sup>2</sup>Swiss Centre for Applied Human Toxicology (SCAHT), Basel, Switzerland</p><p><sup>3</sup>School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland</p><p><sup>4</sup>Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland</p><p><sup>5</sup>Department of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland</p></div></div><div class="corresponding-author-and-affiliations clearfix"><div class="corresponding-author-details"><h3>Corresponding Author:</h3><p>Marie-Gabrielle Zurich, PhD</p><p></p><p>Department of Biomedical Sciences</p><p>University of Lausanne</p><p>Bugnon 7</p><p>Lausanne, 1005</p><p>Switzerland</p><p>Phone: 41 21 692 5542</p><p>Email: <a href="mailto:mzurich@unil.ch">mzurich@unil.ch</a></p><br></div></div></div><section class="article-content clearfix"><article class="abstract"><h3 id="Abstract" class="navigation-heading" data-label="Abstract">Abstract</h3><p><span class="abstract-sub-heading">Background: </span>Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study.<br></p><p><span class="abstract-sub-heading">Objective: </span>This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays.<br></p><p><span class="abstract-sub-heading">Methods: </span>The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures.<br></p><p><span class="abstract-sub-heading">Results: </span>The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity.<br></p><p><span class="abstract-sub-heading">Conclusions: </span>This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals.<br></p><p><span class="abstract-sub-heading">International Registered Report Identifier (IRRID): </span>DERR1-10.2196/50300<br></p><strong class="h4-article-volume-issue">JMIR Res Protoc 2024;13:e50300</strong><br><br><span class="article-doi"><a href="https://doi.org/10.2196/50300">doi:10.2196/50300</a></span><br><br><h3 class="h3-main-heading" id="Keywords">Keywords</h3><div class="keywords"><span><a href="/search?type=keyword&term=organic%20solvent%20exposure&precise=true">organic solvent exposure</a>; </span><span><a href="/search?type=keyword&term=workers&precise=true">workers</a>; </span><span><a href="/search?type=keyword&term=general%20population&precise=true">general population</a>; </span><span><a href="/search?type=keyword&term=neurotoxicity&precise=true">neurotoxicity</a>; </span><span><a href="/search?type=keyword&term=blood-brain%20barrier&precise=true">blood-brain barrier</a>; </span><span><a href="/search?type=keyword&term=liver%20toxicity&precise=true">liver toxicity</a>; </span><span><a href="/search?type=keyword&term=human%20cell%20cultures&precise=true">human cell cultures</a> </span></div><div id="trendmd-suggestions"></div></article><br><article class="main-article clearfix"><br><h3 class="navigation-heading h3-main-heading" id="Introduction" data-label="Introduction">Introduction</h3><p class="abstract-paragraph">Environmental and occupational exposure to chemicals may contribute to the development of several neurological diseases [<span class="footers"><a class="citation-link" href="#ref1" rel="footnote">1</a></span>,<span class="footers"><a class="citation-link" href="#ref2" rel="footnote">2</a></span>]. In particular, organic solvents used in industries such as car repair, painting, furniture manufacturing, printing, and cleaning have been associated with several central nervous system (CNS) conditions. These include mild to severe toxic encephalopathy [<span class="footers"><a class="citation-link" href="#ref3" rel="footnote">3</a></span>]; deficits in cognitive function [<span class="footers"><a class="citation-link" href="#ref4" rel="footnote">4</a></span>-<span class="footers"><a class="citation-link" href="#ref7" rel="footnote">7</a></span>]; and, in some cases, neurodegenerative diseases [<span class="footers"><a class="citation-link" href="#ref8" rel="footnote">8</a></span>,<span class="footers"><a class="citation-link" href="#ref9" rel="footnote">9</a></span>]. The diffuse neuropathological effects of acute solvent intoxication reflect neurophysiological abnormalities involving multiple brain regions. With increasingly intense or prolonged exposure, the severity of acute impairment may progress along the spectrum of delirium. Chronic high-level exposure may lead to global cognitive impairment including deficits in memory, attention, energy, and personality, which are well-described forms of dementia [<span class="footers"><a class="citation-link" href="#ref10" rel="footnote">10</a></span>-<span class="footers"><a class="citation-link" href="#ref13" rel="footnote">13</a></span>]. Much of the initial work on organic solvent toxicity originated in Scandinavia, where a neurobehavioral syndrome in painters leading to their early retirement was first described [<span class="footers"><a class="citation-link" href="#ref14" rel="footnote">14</a></span>]. However, although the neurotoxicity of solvents such as toluene, trichloroethylene, and n-hexane has been recognized, the neurotoxicity of common solvents currently on the market has not been evaluated. Notably, neurotoxicity testing is only required if the chemical is deemed a pesticide; otherwise, all other chemicals are evaluated on a case-by-case basis. The only exception is if the compound structure is suspected to have nervous system targets and no data are available for read-across or when effects on the nervous system are found in single-dose (Organisation for Economic Co-operation and Development [OECD] Test Guidelines [TGs] 402, 403, 420, 423, or 425) or repeated-dose toxicity studies (TG 407 or 408). Because the nervous system effect endpoints considered as triggers (ie, modifications of wet brain weight or basic histopathology, or both) are quite insensitive, high amounts of potentially neurotoxic compounds are available on the market. The European Union Classification, Labelling, and Packaging Regulation does not include a classification for neurotoxicity. Exposure to organic solvents, especially among workers with higher exposure than the general population, can produce neurotoxic effects, depending on the internal dose. Therefore, to efficiently protect the population from possible solvent toxicity, it is important to determine the air concentrations at which neurotoxicity does not occur. To this end, we propose a strategy using a combination of in vivo (zebrafish embryo), in vitro, and in silico tools coupled with controlled in vivo human exposure experiments to assess the neurotoxicity of solvents (<span class="footers"><a class="citation-link" href="#figure1" rel="footnote">Figure 1</a></span>).</p><figure><a name="figure1">‎</a><a class="fancybox" title="Figure 1. Overview of project rationale, organization, and outcomes. The project will develop a strategy based on various models to assess the neurotoxicity of solvents. Scientific and regulatory outcomes are foreseen. AOP: adverse outcome pathway; BBB: blood-brain barrier; DNT: developmental neurotoxicity; KE: key event; NT: neurotoxicity; OECD: Organisation for Economic Co-operation and Development; PBTK: physiologically based toxicokinetic; TG: Test Guideline; WP: work package." href="https://asset.jmir.pub/assets/aa69821f56726f0f092800dbbc197d31.png" id="figure1"><img class="figure-image" src="https://asset.jmir.pub/assets/aa69821f56726f0f092800dbbc197d31.png"></a><figcaption><span class="typcn typcn-image"></span><b>Figure 1. </b> Overview of project rationale, organization, and outcomes. The project will develop a strategy based on various models to assess the neurotoxicity of solvents. Scientific and regulatory outcomes are foreseen. AOP: adverse outcome pathway; BBB: blood-brain barrier; DNT: developmental neurotoxicity; KE: key event; NT: neurotoxicity; OECD: Organisation for Economic Co-operation and Development; PBTK: physiologically based toxicokinetic; TG: Test Guideline; WP: work package. </figcaption></figure><p class="abstract-paragraph">The recognized method for the evaluation of the neurotoxic potential of chemicals, the OECD TG 424 (neurotoxicity in rodents), uses complex in vivo tests in rodents, which are laborious, expensive, difficult to apply in a standardized manner, and ethically debatable. Regulators from different agencies worldwide as well as the scientific community are becoming increasingly aware of the limitations of the current toxicity testing paradigm. Animal-based high-dose testing in typically 1 stand-alone guideline test is not always relevant for human exposure scenarios [<span class="footers"><a class="citation-link" href="#ref15" rel="footnote">15</a></span>]. One of the most challenging aspects of this animal-centric approach is the impossibility of coping with the thousands of chemicals for which data are still lacking. Conducting animal tests is time consuming and expensive. Therefore, they cannot be carried out routinely because of the sheer number of chemicals that are currently on the market and those anticipated to enter it in the coming years [<span class="footers"><a class="citation-link" href="#ref16" rel="footnote">16</a></span>]. In addition, there are shortcomings regarding interspecies concordance between different mammalian or rodent species as well as with respect to extrapolation from experimental animals to humans. These ambiguities in results or poor reproducibility performance call into question the relevance of such test methods for human risk assessment [<span class="footers"><a class="citation-link" href="#ref16" rel="footnote">16</a></span>-<span class="footers"><a class="citation-link" href="#ref21" rel="footnote">21</a></span>]. All this prompts a move away from animal testing toward a combination of in vitro and in silico approaches that address functional mechanistic endpoints [<span class="footers"><a class="citation-link" href="#ref15" rel="footnote">15</a></span>,<span class="footers"><a class="citation-link" href="#ref16" rel="footnote">16</a></span>,<span class="footers"><a class="citation-link" href="#ref22" rel="footnote">22</a></span>].</p><p class="abstract-paragraph">Numerous in vitro models have been proposed for the evaluation of neurotoxicity in the last decades. Monolayer cultures of a single brain cell type are far from representing the human brain in terms of architecture and functionality. Given the sophistication of brain cell-to-cell interactions, some complexity is required to recapitulate human-relevant cellular processes and functions in vitro. However, a good balance must be found between this complexity and the simplicity needed to have robust and reproducible systems that can be applied for chemical screening in a high-throughput manner [<span class="footers"><a class="citation-link" href="#ref23" rel="footnote">23</a></span>]. The 3D hiPSC–derived brain test system (BrainSpheres) we previously developed [<span class="footers"><a class="citation-link" href="#ref24" rel="footnote">24</a></span>] will be used in this study, as it fulfills these requirements.</p><p class="abstract-paragraph">The blood-brain barrier (BBB) protects the brain parenchymal cells from the deleterious effects of xenobiotics. However, some chemicals are able to cross or impair the BBB [<span class="footers"><a class="citation-link" href="#ref25" rel="footnote">25</a></span>]. The transient or permanent opening of the BBB provides xenobiotics, plasma proteins, and immunoregulatory mediators access to the CNS, where they can induce toxic effects. Therefore, we will implement a predictive model to assess the impact of solvents on BBB based on in vivo (zebrafish embryo), in vitro (human brain microvascular endothelial cells [hCMEC/D3]), and in silico models [<span class="footers"><a class="citation-link" href="#ref26" rel="footnote">26</a></span>] to assist in the interpretation of the results obtained in in vitro neurotoxicity testing.</p><p class="abstract-paragraph">Glycol ethers will be used as a case study to evaluate the feasibility of our protocol. Glycol ethers form a wide family of a few dozen solvents with different physicochemical properties making them versatile and usable in a variety of industrial applications ranging from pharmaceuticals and microelectronics to domestic cleaning, personal care, and printing. The 2 main groups of glycol ethers are the E series (ie, ethylene glycol ethers [EGEs]) and the P series (ie, propylene glycol ethers [PGEs]). EGEs and PGEs show differences in their toxicological properties regarding teratogenicity, hemolysis, and testicular atrophy [<span class="footers"><a class="citation-link" href="#ref23" rel="footnote">23</a></span>], apparently resulting from their distinct production of metabolites [<span class="footers"><a class="citation-link" href="#ref24" rel="footnote">24</a></span>,<span class="footers"><a class="citation-link" href="#ref25" rel="footnote">25</a></span>]. EGEs have a primary alcohol group and are oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to form the toxic alkoxyacetic acid. Therefore, PGEs are progressively introduced as a less toxic replacement of EGEs. PGEs are sold as a mixture of 2 isomers, with the bulk having a secondary alcohol (a) group and generally <5% of primary alcohol (b) groups [<span class="footers"><a class="citation-link" href="#ref22" rel="footnote">22</a></span>]. The b-isomer is oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to form the toxic alkoxypropionic acid in the body. However, the actual toxicity of PGEs is poorly characterized. Therefore, it is essential for our strategy to study not only the parent compound but also the metabolites. Metabolically competent liver cell models will be used to screen for liver toxicity of solvents and for the production of potential metabolites.</p><p class="abstract-paragraph">Exposure to solvents in human volunteers is fundamental when quantifying possible risks from chemicals because toxicological effects are related to internal exposure, that is, the concentration of a chemical inside the body and its biotransformation. Therefore, in vivo human volunteer studies are necessary to quantify absorption, distribution, metabolism, and excretion (ADME) kinetics in humans under controlled exposure conditions, which are necessary to understand the relationship between solvent concentrations in the air and biological samples, such as blood, urine, or exhaled air. These experiments provide important data on the total absorbed dose after inhaled solvent concentration, absorption rate (ie, from the site of absorption into the bloodstream), biotransformation rate (ie, metabolization of the parent chemical with production of metabolites), and elimination rate (ie, excretion of the parent chemical and metabolites from the body) [<span class="footers"><a class="citation-link" href="#ref27" rel="footnote">27</a></span>]. These toxicokinetic parameters are representative of the target organ or of the tissue concentrations that may trigger an effect and are, therefore, relevant for understanding chemical risks in humans.</p><p class="abstract-paragraph">Toxicokinetic models quantitatively describe the body’s ADME of a chemical or substance (different terms for this concept are preferred in different fields, including “toxicokinetics,” “pharmacokinetics,” and “biokinetics”) [<span class="footers"><a class="citation-link" href="#ref28" rel="footnote">28</a></span>]. Furthermore, using a toxicokinetic model in reverse dosimetry, we can predict the solvent air concentrations leading to brain concentrations below the levels found to produce neurotoxic effects in vitro in the BrainSpheres. The toxicokinetic model will incorporate metabolism parameters derived from the in vitro liver system and passage through or toxicity to BBB. Once calibrated, the toxicokinetic model can be used to simulate chronic exposure scenarios to predict cumulative brain concentrations and used in reverse dosimetry to predict air concentrations that will not likely result in brain concentrations associated with toxicity.</p><br><h3 class="navigation-heading h3-main-heading" id="Methods" data-label="Methods">Methods</h3><h4>Ethical Considerations</h4><p class="abstract-paragraph">Ethics committee approval was obtained from Swiss ethics (Commission cantonale d’éthique de la recherche sur l’être humain) in 2022 for this nonclinical human study (2022-01567). Healthy women and men were recruited as participants in our study. Each participant signed a written informed consent form before inclusion in the study. The participants will be reimbursed for their time and inconvenience according to the Swiss guidelines.</p><h4>Global Strategy</h4><p class="abstract-paragraph">The choice of solvents to be included in the study will be based on the amount annually placed on the European market and the number of products registered containing known glycol ethers. The selected organic solvents will be applied to various in vitro models to determine their neurotoxicity. They must be amphiphilic to be solubilized in the cell culture media. We established the following solvent selection criteria: (1) used or produced >1 metric ton per year, (2) incorporated in numerous industrial and commercial products, and (3) water solubility. This selection process involves consulting government databases and contacting different industry sectors. We will start the project concomitantly by testing 2 solvents of the P series, propylene glycol methyl ether (PGME), for which we have already developed a toxicokinetic model, and propylene glycol butyl ether. We will test 1 additional solvent from the E series with the in vitro test system, namely, ethylene glycol methyl ether (EGME), which has been banned for use in cosmetic products in Europe [<span class="footers"><a class="citation-link" href="#ref29" rel="footnote">29</a></span>].</p><p class="abstract-paragraph">The study is organized into 5 work packages (WPs). The information workflow between the WPs is shown in <span class="footers"><a class="citation-link" href="#figure2" rel="footnote">Figure 2</a></span>. All results collected from the abovementioned systems will contribute to refining the toxicokinetic model we previously developed for PGME [<span class="footers"><a class="citation-link" href="#ref30" rel="footnote">30</a></span>]. The toxicokinetic parameters of the solvent and the metabolites will then be characterized in human volunteers after exposure to PGE vapors under controlled conditions. These results will be used to calibrate and expand our toxicokinetic model [<span class="footers"><a class="citation-link" href="#ref30" rel="footnote">30</a></span>]. The toxicokinetic model will be constructed to predict brain concentrations of selected solvents and, consequently, will include a brain compartment to predict the target organ solvent and metabolite concentrations. BBB parameters such as barrier transport, transport of the solvent once in the brain, and solvent-brain binding will be incorporated. Solvent neurotoxicity may depend on the metabolic modifications of the substances; therefore, we will incorporate the parameters for the parent compound and the metabolites found in the in vitro liver system. The data necessary to build the model will be retrieved from peer-reviewed scientific literature for tissue:blood partition coefficients (PCs) [<span class="footers"><a class="citation-link" href="#ref31" rel="footnote">31</a></span>] following the fit-for-purpose dose-response analysis approach. The toxicokinetic model should be able to predict human brain concentration for each of the tested solvents after inhalation exposure, given the air concentration of vapors and duration of exposure. The simulated human brain concentrations will then be compared with the no observed adverse effect concentrations (NOAECs) obtained from the neurotoxicity in vitro system (BrainSpheres). In addition, the toxicokinetic model will be used to predict solvent air concentrations that are unlikely to lead to brain concentration equal to or superior to the brain NOAECs using reverse dosimetry. The specific aims of each WP are summarized in <span class="footers"><a class="citation-link" href="#table1" rel="footnote">Table 1</a></span>.</p><figure><a name="figure2">‎</a><a class="fancybox" title="Figure 2. Information workflow between the work package (WP). BBB: blood-brain barrier; IVIVE: in vitro-in vivo extrapolation; NOAEC: no observed adverse effect concentration; PBTK: physiologically based toxicokinetic." href="https://asset.jmir.pub/assets/d2e4708e87d2c800445c8e84997b83ee.png" id="figure2"><img class="figure-image" src="https://asset.jmir.pub/assets/d2e4708e87d2c800445c8e84997b83ee.png"></a><figcaption><span class="typcn typcn-image"></span><b>Figure 2. </b> Information workflow between the work package (WP). BBB: blood-brain barrier; IVIVE: in vitro-in vivo extrapolation; NOAEC: no observed adverse effect concentration; PBTK: physiologically based toxicokinetic. </figcaption></figure><div class="figure-table"><figcaption><span class="typcn typcn-clipboard"></span><b>Table 1. </b>Specific aims of the work packages (WPs).</figcaption><table width="1000" cellpadding="5" cellspacing="0" border="1" rules="groups" frame="hsides"><col width="70" span="1"><col width="270" span="1"><col width="660" span="1"><thead><tr valign="top"><td rowspan="1" colspan="1">WP</td><td rowspan="1" colspan="1">Name</td><td rowspan="1" colspan="1">Specific aims</td></tr></thead><tbody><tr valign="top"><td rowspan="1" colspan="1">WP1</td><td rowspan="1" colspan="1">In vitro neurotoxicity testing</td><td rowspan="1" colspan="1"><ol type="1"><li>Determine NOAEC<sup>a</sup> for neurotoxicity of each solvent<br></li><li>Determine the in vitro distribution kinetics of solvents<br></li><li>Identify toxicity pathways and KEs<sup>b</sup> for solvent neurotoxicity<br></li></ol></td></tr><tr valign="top"><td rowspan="1" colspan="1">WP2</td><td rowspan="1" colspan="1">In vivo or in vitro or in silico BBB<sup>c</sup> functionality testing</td><td rowspan="1" colspan="1"><ol type="1"><li>Evaluate the suitability of the zebrafish embryo model to study BBB integrity and functionality<br></li><li>Determine the impact of solvents on BBB integrity and transport in zebrafish and hCMEC/D3<sup>d</sup><br></li><li>Determine the solvents permeability coefficient (Pe)<br></li><li>Provide quantitative data on BBB permeability and tissue distribution of solvents based on computational modeling<br></li></ol></td></tr><tr valign="top"><td rowspan="1" colspan="1">WP3</td><td rowspan="1" colspan="1">In vitro hepatic metabolism and clearance</td><td rowspan="1" colspan="1"><ol type="1"><li>Elucidate hepatic metabolism<br></li><li>Calculate substrate-enzymatic parameters (Vmax<sup>e</sup> and Km<sup>f</sup>)<br></li><li>Detect and identify possible metabolites produced by the liver<br></li></ol></td></tr><tr valign="top"><td rowspan="1" colspan="1">WP4</td><td rowspan="1" colspan="1">In vivo volunteer exposure</td><td rowspan="1" colspan="1"><ol type="1"><li>Characterize human blood absorption and urinary elimination kinetics for parent glycol ether as well as the metabolites identified in WP3<br></li><li>Find neurotoxic and vascular injury effect biomarkers for solvent exposure<br></li></ol></td></tr><tr valign="top"><td rowspan="1" colspan="1">WP5</td><td rowspan="1" colspan="1">In silico PBTK<sup>g</sup> modeling</td><td rowspan="1" colspan="1"><ol type="1"><li>Establish and calibrate the PBTK model for various organic solvents<br></li><li>Use reverse dosimetry to determine air concentrations below human brain toxicity concentrations<br></li></ol></td></tr></tbody></table><fn id="table1fn1"><p><sup>a</sup>NOAEC: no observed adverse effect concentration.</p></fn><fn id="table1fn2"><p><sup>b</sup>KE: key event.</p></fn><fn id="table1fn3"><p><sup>c</sup>BBB: blood-brain barrier.</p></fn><fn id="table1fn4"><p><sup>d</sup>hCMEC/D3: human brain microvascular endothelial cells.</p></fn><fn id="table1fn5"><p><sup>e</sup>V<sub>max</sub>: maximum velocity.</p></fn><fn id="table1fn6"><p><sup>f</sup>Km: Michaelis constant.</p></fn><fn id="table1fn7"><p><sup>g</sup>PBTK: physiologically based toxicokinetic.</p></fn></div><h4>WP1: In Vitro Neurotoxicity Testing</h4><p class="abstract-paragraph">Testing strategies are needed to evaluate the neurotoxicity of chemicals in a more cost-effective, efficient, and ethical manner. Participating in an international effort, we developed a 3D human-induced pluripotent stem cells (hiPSC)–derived brain model containing several subtypes of neurons, astrocytes, and oligodendrocytes [<span class="footers"><a class="citation-link" href="#ref24" rel="footnote">24</a></span>]. This system allows the cells to reach a high level of differentiation and cellular maturation, exemplified by the presence of functional synapses and compact myelin. The presence of myelin is important for this project because solvents more easily target lipid-rich structures [<span class="footers"><a class="citation-link" href="#ref32" rel="footnote">32</a></span>]. This 3D human brain model has already proven its usefulness for neurotoxicity testing [<span class="footers"><a class="citation-link" href="#ref33" rel="footnote">33</a></span>-<span class="footers"><a class="citation-link" href="#ref36" rel="footnote">36</a></span>]. We hypothesized that glycol ethers are neurotoxic. Therefore, we propose to take advantage of our hiPSC-derived BrainSpheres model to study the neurotoxicity induced by uncharacterized glycol ethers present on the market, which will be compared with the neurotoxicity of well-characterized solvents known to induce human encephalopathy.</p><p class="abstract-paragraph">Solvents are data-poor substances. It was originally hypothesized that they exert their toxic effects largely through nonspecific physicochemical effects that modulate membrane fluidity and perturb the hydrophobic force regulating macromolecular interactions [<span class="footers"><a class="citation-link" href="#ref37" rel="footnote">37</a></span>]. However, recent evidence supports the view that solvents interact with lipophilic areas on protein receptors [<span class="footers"><a class="citation-link" href="#ref38" rel="footnote">38</a></span>,<span class="footers"><a class="citation-link" href="#ref39" rel="footnote">39</a></span>]. They have also been shown to induce lipid peroxidation, leading to mitochondrial dysfunction, failure of electron transport, and energy production [<span class="footers"><a class="citation-link" href="#ref40" rel="footnote">40</a></span>,<span class="footers"><a class="citation-link" href="#ref41" rel="footnote">41</a></span>]. In this study, omics (eg, proteomics, metabolomics, and lipidomics) technology will be used to decipher the mechanisms of glycol ether neurotoxicity and to identify potential biomarkers of toxic effects.</p><p class="abstract-paragraph">Primary 3D hiPSC-derived brain cell cultures will be prepared and maintained as previously described [<span class="footers"><a class="citation-link" href="#ref24" rel="footnote">24</a></span>]. This model contains neurons that form synapses, astrocytes, and oligodendrocytes myelinating the axons (<span class="footers"><a class="citation-link" href="#figure3" rel="footnote">Figure 3</a></span>). Cytotoxicity will be determined by a resazurin assay after repeated exposure (7 d) to the selected glycol ethers (parent compounds and metabolites). Gene expression for cell type–specific genes, markers of synapses and myelin, and markers of cell stress will be quantified by quantitative reverse transcription polymerase chain reaction at concentrations of solvents under half-maximal effective concentration (EC50) for cytotoxicity. Immunostaining will be performed to assess the effects of solvents on synapses, myelin, and astrocyte reaction, and immunofluorescence will be quantified. NOAECs (<span class="footers"><a class="citation-link" href="#figure2" rel="footnote">Figure 2</a></span>) will be determined for all tested endpoints, as previously shown for gene expression [<span class="footers"><a class="citation-link" href="#ref42" rel="footnote">42</a></span>]. Brain cell cultures will also be exposed to the metabolites of PGME, propylene glycol butyl ether, and EGME and to the metabolites of the newly selected uncharacterized solvents, potentially produced by liver metabolism (WP3).</p><figure><a name="figure3">‎</a><a class="fancybox" title="Figure 3. Brain model used in work package (WP) 1. Immunostainings of human induced pluripotent stem cell–derived 3D BrainSpheres after 8 weeks of differentiation, showing the presence of proteins specific for neurons (neurofilament heavy polypeptide [NF200]), synapses (postsynaptic density-95 protein [PSD95] and synaptophysin [SYP]), astrocytes (glial fibrillary acidic protein [GFAP]) and oligodendrocyte (proteolipid protein 1 [PLP1]). Scale bars: 40 µm." href="https://asset.jmir.pub/assets/285cd4f3d30da35eb2f91825020c0bbf.png" id="figure3"><img class="figure-image" src="https://asset.jmir.pub/assets/285cd4f3d30da35eb2f91825020c0bbf.png"></a><figcaption><span class="typcn typcn-image"></span><b>Figure 3. </b> Brain model used in work package (WP) 1. Immunostainings of human induced pluripotent stem cell–derived 3D BrainSpheres after 8 weeks of differentiation, showing the presence of proteins specific for neurons (neurofilament heavy polypeptide [NF200]), synapses (postsynaptic density-95 protein [PSD95] and synaptophysin [SYP]), astrocytes (glial fibrillary acidic protein [GFAP]) and oligodendrocyte (proteolipid protein 1 [PLP1]). Scale bars: 40 µm. </figcaption></figure><p class="abstract-paragraph">To establish the in vitro distribution kinetics of selected solvents necessary for toxicokinetic modeling, 3D brain cell cultures and medium will be collected 3, 6, 24, and 48 hours after the first exposure and after the last exposure of the repeated treatment. The solvent and its main metabolites (if relevant) will be quantified to establish a time course of disappearance from the medium and appearance in the cells as well as to assess the potential accumulation for the entire period of exposure. The fraction bound to culture plates’ plastic will be quantified after desorption. In silico modeling of glycol ethers in vitro distribution kinetics will then be developed. This model will be able to predict the change in cell-associated concentrations of solvents in BrainSpheres with time, as previously shown for amiodarone [<span class="footers"><a class="citation-link" href="#ref43" rel="footnote">43</a></span>].</p><h4>WP2: In Vivo, In Vitro, and In Silico BBB Functionality Testing</h4><p class="abstract-paragraph">We previously established the zebrafish as a predictive vertebrate screening model to study the systemic circulation and tissue distribution of particulate drug carriers [<span class="footers"><a class="citation-link" href="#ref44" rel="footnote">44</a></span>,<span class="footers"><a class="citation-link" href="#ref45" rel="footnote">45</a></span>]. At 72 hours postfertilization, zebrafish embryos have a functional CNS and, presumably, a fully functional BBB. Anatomical structures such as the vascular endothelium can be visualized using transgenic fish lines expressing fluorescent proteins (<span class="footers"><a class="citation-link" href="#figure4" rel="footnote">Figure 4</a></span>). Defined exposure of the zebrafish can be achieved by the simple addition of solvents to the fish incubation medium within a closed container. Other advantages of the model include the possibility of studying BBB functionality under physiological conditions in vivo and the high throughput. A well-known in vitro model for the human brain endothelium, hCMEC/D3 cell line (<span class="footers"><a class="citation-link" href="#figure4" rel="footnote">Figure 4</a></span>) showing the formation of tight junctions and the expression of most transporters and receptors of the in vivo BBB [<span class="footers"><a class="citation-link" href="#ref46" rel="footnote">46</a></span>], cultured in a transwell system, will also be used. Furthermore, extrapolation of in silico, in vitro, and zebrafish data to higher vertebrates seems feasible [<span class="footers"><a class="citation-link" href="#ref47" rel="footnote">47</a></span>].</p><figure><a name="figure4">‎</a><a class="fancybox" title="Figure 4. Blood-brain barrier (BBB) models used in work package (WP) 2: zebrafish larvae (ZFL) and human brain microvasculatur endothelial cells (hCMEC/D3). ZFL (2 top panels): tracer permeability across BBB. Dorsal view of the midbrain region of the zebrafish lines Tg (kdrl:enhanced green fluorescent protein [eGFP]), which expresses eGFP (green signal) in the endothelial cell membranes. ZFL were injected with the tracer 1 kDa maleimide (red signal). Scale bars: 50 µm. hCMEC/D3 cells (lowest panel): actin filament stained with fluorescein isothiocyanate phalloidin. Scale bars: 100 µm." href="https://asset.jmir.pub/assets/d06b22bb63eb0b19db2296d78cf9072c.png" id="figure4"><img class="figure-image" src="https://asset.jmir.pub/assets/d06b22bb63eb0b19db2296d78cf9072c.png"></a><figcaption><span class="typcn typcn-image"></span><b>Figure 4. </b> Blood-brain barrier (BBB) models used in work package (WP) 2: zebrafish larvae (ZFL) and human brain microvasculatur endothelial cells (hCMEC/D3). ZFL (2 top panels): tracer permeability across BBB. Dorsal view of the midbrain region of the zebrafish lines Tg (kdrl:enhanced green fluorescent protein [eGFP]), which expresses eGFP (green signal) in the endothelial cell membranes. ZFL were injected with the tracer 1 kDa maleimide (red signal). Scale bars: 50 µm. hCMEC/D3 cells (lowest panel): actin filament stained with fluorescein isothiocyanate phalloidin. Scale bars: 100 µm. </figcaption></figure><p class="abstract-paragraph">Zebrafish larvae are frequently used in developmental biology or toxicological studies. However, in this study, we will use zebrafish larvae exclusively to study BBB integrity and functionality [<span class="footers"><a class="citation-link" href="#ref48" rel="footnote">48</a></span>]. Fluorescently labeled reference compounds (<span class="footers"><a class="citation-link" href="#figure4" rel="footnote">Figure 4</a></span>) will be intravenously injected into the Duct of Cuvier, as markers of paracellular permeability (eg, fluorescein isothiocyanate dextran 70 or fluorescently labeled liposomes), substrates of drug export transporters (eg, rhodamine-123 as P-glycoprotein substrate), or nutrient transporters (eg, fluorescently labeled transferrin as a marker for receptor-mediated transcytosis). PGME will be the first reference compound to be tested because of its high water miscibility. To precisely assess exposure, analytical methods (gas chromatography-tandem mass spectrometry [GC-MS/MS]) will be used to determine the concentrations of solvents and their metabolites in zebrafish medium, in the headspace of closed incubation vessels and tissue samples (ie, zebrafish homogenates). Circulation, tissue distribution, and brain uptake of the reference compounds will be monitored by confocal laser scanning microscopy (live imaging of anesthetized fish embryos for up to 24 hours). The concentration-dependent toxicity of solvents or their metabolites will be monitored based on the viability and malformations of embryos. The integrity of vasculature will be visualized in transgenic zebrafish kdrl: enhanced green fluorescent protein embryos. The metabolic capacity of the zebrafish will be determined by quantifying potential metabolites (determined in WP3; <span class="footers"><a class="citation-link" href="#figure2" rel="footnote">Figure 2</a></span>) in zebrafish tissue homogenates. The concentration-dependent toxicity to BBB and the coefficient of permeation of solvents will additionally be evaluated in the hCMEC/D3 cell line cultured in a transwell system.</p><p class="abstract-paragraph">Finally, quantitative estimates of passive cellular uptake and BBB permeability of solvents and their metabolites will be provided based on computational modeling using physicochemical molecular descriptors according to the methods we previously established [<span class="footers"><a class="citation-link" href="#ref26" rel="footnote">26</a></span>,<span class="footers"><a class="citation-link" href="#ref49" rel="footnote">49</a></span>]. These methods provide very high throughput, allowing the screening of web-based chemical libraries.</p><h4>WP3: In Vitro Hepatic Metabolism and Clearance</h4><p class="abstract-paragraph">Because the liver is the main organ responsible for metabolism and a large contributor to compound clearance, we will implement a system suitable for predicting the hepatic metabolism of solvents. In recent years, 3D liver cell models have been proposed as an alternative to less physiological 2D cell monolayers, and their applications have progressed substantially [<span class="footers"><a class="citation-link" href="#ref50" rel="footnote">50</a></span>]. They are widely used for the assessment of hepatotoxicity [<span class="footers"><a class="citation-link" href="#ref51" rel="footnote">51</a></span>-<span class="footers"><a class="citation-link" href="#ref55" rel="footnote">55</a></span>]. An advantage of spheroids is that they overcome the limitation of rapid decline of drug-metabolizing enzyme activities in primary human hepatocyte suspension culture and cell lysates [<span class="footers"><a class="citation-link" href="#ref56" rel="footnote">56</a></span>], such as microsomes and liver S9 fractions. In this study, we will use 3D liver cultures of the well-characterized human HepaRG cell line (<span class="footers"><a class="citation-link" href="#figure5" rel="footnote">Figure 5</a></span>), which represent a promising model to evaluate hepatotoxicity and hepatic metabolism [<span class="footers"><a class="citation-link" href="#ref57" rel="footnote">57</a></span>].</p><figure><a name="figure5">‎</a><a class="fancybox" title="Figure 5. Liver model used in work package (WP) 3. Bright field and immunostainings of liver 3D HepaRG cultures showing the presence of albumin and cytochrome P450 3A4 (CYP3A4). Bars: 100 µm." href="https://asset.jmir.pub/assets/6204f2bfbb95dd4f300acdb86c9b47a3.png" id="figure5"><img class="figure-image" src="https://asset.jmir.pub/assets/6204f2bfbb95dd4f300acdb86c9b47a3.png"></a><figcaption><span class="typcn typcn-image"></span><b>Figure 5. </b> Liver model used in work package (WP) 3. Bright field and immunostainings of liver 3D HepaRG cultures showing the presence of albumin and cytochrome P450 3A4 (CYP3A4). Bars: 100 µm. </figcaption></figure><p class="abstract-paragraph">Determining the appropriate experimental test system (eg, cell plate, incubation time, and exposure concentration) will be an essential part of the development of the 3D model. Moreover, analytical methods (GC-MS/MS and liquid chromatography-tandem mass spectrometry) to detect and quantify the solvents and the metabolites formed must be developed to calculate hepatic metabolism and clearance. Then, proof of metabolic competence and maintenance of the 3D HepaRG cells will be carried out by assessing the metabolism of known P450 substrates. The presence and secretion of albumin as a specific hepatocyte marker will be assessed using immunostaining and enzyme-linked immunosorbent assay. Solvent- and metabolite-induced cytotoxicity will be assessed after 48 hours and 7 days (repeated) of exposure to determine the nontoxic concentration range for subsequent experiments. The metabolic abilities of the 3D HepaRG model and 3D primary human hepatocytes will be compared. Furthermore, the clearance data obtained from the 3D HepaRG model will be compared with the short-term clearance measured in the human liver cell lysate (S9 fractions). In addition, Michaelis-Menten-Kinetic parameters (V<sub>max</sub> and Km) for the formation of the metabolites will be derived using the S9 fractions. These data will be used to build a physiologically based toxicokinetic (PBTK) model (<span class="footers"><a class="citation-link" href="#figure2" rel="footnote">Figure 2</a></span>).</p><h4>WP4: In Vivo Volunteer Exposure</h4><p class="abstract-paragraph">Human biomonitoring refers to monitoring exposure-related health risks by analyzing biological samples, usually blood and urine samples [<span class="footers"><a class="citation-link" href="#ref58" rel="footnote">58</a></span>]. The biomonitoring limit values (BMLVs) are set to protect human populations against the potential toxic effects of chemical substances. These limit values account for all routes through which a chemical can enter the body. These are most often the inhalation and skin routes in occupational and environmental settings. Kinetic studies that provide absorption, biotransformation, and elimination rates as well as the absorption and elimination half-lives of the parent compound and its metabolites are necessary to set BMLVs. The apparent urinary elimination half-lives of the parent compound and its metabolites will later be used to develop a biomonitoring method. Sample collection time is crucial and is determined by the apparent elimination half-life of the chemical. Blood concentrations will be used to calibrate the air:blood PC for the toxicokinetic models.</p><p class="abstract-paragraph">We will recruit 4 participants for 2 of the selected solvents. All participants must meet the following criteria: they should be healthy individuals who do not smoke or use contraceptive hormones, do not consume alcohol, be aged between 18 and 65 years, have normal red blood cells and hemoglobin concentrations, maintain a BMI between 18 and 25, and should not be working with glycol ethers. Pregnant and breastfeeding women will be excluded from this study. Participants will be recruited using flyers and announcements distributed at the teaching hospital, university websites, and bulletin boards. All participants will sign a written informed consent form before being included in the study.</p><p class="abstract-paragraph">The participants will be exposed to a single glycol ether for 4 hours under controlled conditions in an exposure chamber (12 m<sup>3</sup>). PGE concentrations will be set at or below the Swiss occupational exposure level (OEL) if one exists. In the absence of an OEL, we will rely on existing OELs for other propylene glycols. The parent compound (free and conjugated) and the oxidative metabolites (free and conjugated) of the selected glycol ethers will be monitored in blood, urine, and exhaled air samples. These are noninvasive methods used for human participants, and the results will be used in WP5 to estimate brain concentrations. All compounds will be quantified using capillary gas (parent compound in blood, urine, and exhaled air) or liquid (metabolites in blood and urine) chromatograms with tandem mass spectroscopy detection.</p><h4>WP5: In Silico PBTK Modeling</h4><p class="abstract-paragraph">PBTK models can be used to estimate human brain concentrations. The risk of neurotoxic effects can be estimated by comparing the predicted solvent-brain concentrations with the NOAEC obtained from the in vitro models. Mathematical models such as PBTK models can be used to predict the ADME of a chemical and its metabolites. In these PBTK models, the body is represented by 1 or more compartments. Each compartment represents 1 or more tissues that are kinetically homogeneous, that is, that have similar perfusion rates and an assumed similar substance solubility. PBTK models are described by a set of parameters that define the compartments and a set of mass balance differential equations for each compartment.</p><p class="abstract-paragraph">A previously developed toxicokinetic model for PGME with metabolism that is assumed to follow Michaelis-Menten kinetics calibrated for different age groups serves as the basis for our development [<span class="footers"><a class="citation-link" href="#ref30" rel="footnote">30</a></span>,<span class="footers"><a class="citation-link" href="#ref31" rel="footnote">31</a></span>,<span class="footers"><a class="citation-link" href="#ref59" rel="footnote">59</a></span>]. We aim to modify this previously developed toxicokinetic model to include a separate compartment for the brain using BBB flux rates obtained from in vivo, in vitro, and in silico models (WP2). In addition, we will implement PC obtained from empirical human experiments (WP4) and metabolic parameters assessed in a hepatocyte assay (WP3). The toxicokinetic models will be able to simulate not only acute but also chronic exposures; therefore, both short-term and long-term exposures can be explored in silico. We will develop the toxicokinetic model into a physiologically based pharmacokinetic model based on the existing inhalation-only toxicokinetic model originally developed for PGME [<span class="footers"><a class="citation-link" href="#ref40" rel="footnote">40</a></span>] and build it in the Berkeley Madonna software or equivalent. We will model the brain as a single compartment with direct contact with the blood flow and where organic solvent uptake will be assumed to be diffusion limited, which is in line with other physiologically based pharmacokinetic models [<span class="footers"><a class="citation-link" href="#ref60" rel="footnote">60</a></span>]. Values for physiological parameters (volume of vascular brain, as fraction of brain volume [FVvb], volume of extravascular brain, as fraction of brain volume [FVevb], volume fraction of brain tissue [FVB; as percent of body weight], BBB surface [Sh] in cm<sup>2</sup>, fraction of cardiac output in brain at rest [BFbrainrest]/cardiac output in brain at light work [BFbrain]) required to build the TK model are from the scientific literature. Depending on the substance, values of chemical-specific parameters such as the pulmonary retention (Rpulm), central:air PC (Pca), blood:air PC (Pba), and brain tissue:vascular brain PC (Pevb_vb) are either taken from the literature or estimated in silico. Since the partitioning of organic compounds between human tissue homogenate and blood is a function of water and lipid content of tissues and the n-octanol:water PC (Kow), PCs are estimated in silico based on LogKow. Kinetic coefficients needed for each organic solvent included in this study will be found in WP3 for liver metabolism (Michaelis-Menten parameters [V<sub>max</sub> and Km]), WP2 for BBB uptake (BBB permeability-surface area product [PS]). The fraction unbound in blood (Fu_blood) will be estimated based on the fraction unbound in plasma (Fu_plasma) and the blood-to-plasma ratio (Rb), and the fraction unbound in brain (Fu_brain) will be considered when modeling each solvent as only the free fraction is able to distribute to different tissues and is biologically active. The model will be calibrated by comparing the predicted and actual urinary organic solvent concentrations obtained from the controlled human experiments (WP4). Both the free and total organic solvent concentrations (free+conjugated) will be obtained for calibration.</p><br><h3 class="navigation-heading h3-main-heading" id="Results" data-label="Results">Results</h3><p class="abstract-paragraph">With this project, we expect to provide a strategy to rank uncharacterized solvents and their potential liver-formed metabolites, according to their potential neurotoxicity, and in comparison with the banned EGME. More importantly, a series of PBTK simulations will be conducted to predict occupational exposure, assuming 8 hours of exposure per day, 5 days per week, physical activity for 12 hours per day, and rest for the remaining 12 hours. We will use the PBTK model in reverse dosimetry to estimate air concentrations that do not produce brain concentrations determined as neurotoxic in the hiPSC-derived 3D brain model. We will recommend that authorities setting occupational exposure and public health limits consult these values. Keeping the exposure below the brain effect level should ultimately increase the protection of exposed workers and the general population with domestic exposures.</p><p class="abstract-paragraph">We also anticipate gaining insights into the mechanisms of action of solvents of the glycol ether family. We will elucidate the possible toxic endpoints in the brain, liver, and zebrafish models. Furthermore, we will be able to establish how toxicity is related to the compounds’ lipophilicity and metabolites.</p><br><h3 class="navigation-heading h3-main-heading" id="Discussion" data-label="Discussion">Discussion</h3><p class="abstract-paragraph">Overall, our strategy combining multiple, fit-for-purpose 3D advanced cell culture systems; zebrafish larvae; biomarker analysis; human ADME experiments; and in silico prediction is expected to contribute to the improvement of human risk assessment. Although we identified some risks we could encounter during the project, we are confident that our already determined mitigation measures will be able to overcome potential pitfalls.</p><p class="abstract-paragraph">Determination of the passage of solvent through the BBB may be challenging; hence, we are applying 3 different complementary methods: in vivo zebrafish larvae, in vitro human cells (hCMEC/D3), and in silico models. We are also considering and assessing the effects of the hepatic metabolites of the solvents on human BBB cells. With this experimental strategy, issues regarding the potential direct effect of solvents on cell membranes, the relatively low miscibility of solvents with water, and the physiological differences between zebrafish and humans (eg, metabolism and route of expected) should be overcome.</p><p class="abstract-paragraph">We have extensive experience in recruiting human volunteers for controlled human exposure sessions in the exposure chamber. Sometimes, recruitment takes longer than anticipated, and if that is the case, we will extend the timeline to not compromise the size of the study. New analytical chemical methods will need to be determined, which is time consuming. However, we will use a laboratory with extensive experience in analyzing PGME in urine and blood samples. This will also have to be accommodated with a delay in the timeline.</p><p class="abstract-paragraph">Future developments, not included in this study, are a strategy extended to include developmental neurotoxicity by determining other endpoints, such as proliferation and neurite outgrowth, after exposure of BrainSpheres to solvents at earlier developmental stages and by adding an in vitro test system to take into account the passage of solvents through the placental barrier [<span class="footers"><a class="citation-link" href="#ref61" rel="footnote">61</a></span>]. We might also consider combining zebrafish embryo behavioral assays (eg, spontaneous tail coiling) with the BrainSpheres model as readouts for developmental neurotoxicity. Finally, the PBTK model could be adapted to determine solvent air concentrations that are unlikely to cause neurotoxic effects in fetuses or pregnant women.</p></article><p><h4 class="h4-border-top">Acknowledgments</h4></p><p class="abstract-paragraph">This work was partially funded by the Swiss Centre for Human Applied Toxicology (SCAHT), Basel, Switzerland.</p><h4>Data Availability</h4><p class="abstract-paragraph">Data will be available from the corresponding author on reasonable request.</p><h4 class="h4-border-top">Authors' Contributions</h4><p><p class="abstract-paragraph">NBH, LSD, JH, and MGZ conceptualized the paper. NBH, LSD, JH, and MGZ wrote the original draft. All authors reviewed and edited the paper. LH, DP, HP, RDP, and SW visualized the paper. NBH, LSD, JH, and MGZ administered the project. NBH, LSD, JH, and MGZ acquired the funding.</p></p><h4 class="h4-border-top">Conflicts of Interest</h4><p><p class="abstract-paragraph">None declared.</p></p><div class="footnotes"><h4 id="References" class="h4-border-top navigation-heading" data-label="References">References</h4><ol><li><span id="ref1">Brown RC, Lockwood AH, Sonawane BR. Neurodegenerative diseases: an overview of environmental risk factors. Environ Health Perspect. Sep 2005;113(9):1250-1256. [<a href="https://ehp.niehs.nih.gov/doi/10.1289/ehp.7567?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed" target="_blank">FREE Full text</a>] [<a target="_blank" href="https://dx.doi.org/10.1289/ehp.7567">CrossRef</a>] [<a href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16140637&dopt=Abstract" target="_blank">Medline</a>]</span></li><li><span id="ref2">Pearce N, Kromhout H. Neurodegenerative disease: the next occupational disease epidemic? Occup Environ Med. 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Jul 27, 2021;12(8):884. [<a href="https://www.mdpi.com/resolver?pii=mi12080884" target="_blank">FREE Full text</a>] [<a target="_blank" href="https://dx.doi.org/10.3390/mi12080884">CrossRef</a>] [<a href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=34442506&dopt=Abstract" target="_blank">Medline</a>]</span></li></ol></div><br><hr><a name="Abbreviations">‎</a><h4 class="navigation-heading" id="Abbreviations" data-label="Abbreviations">Abbreviations</h4><table width="80%" border="0" align="center"><tr><td><b>ADME:</b> absorption, distribution, metabolism, and excretion</td></tr><tr><td><b>BBB:</b> blood-brain barrier</td></tr><tr><td><b>BFbrain:</b> cardiac output in brain at light work</td></tr><tr><td><b>BFbrainrest:</b> cardiac output in brain at rest</td></tr><tr><td><b>BMLV:</b> biomonitoring limit value</td></tr><tr><td><b>CNS:</b> central nervous system</td></tr><tr><td><b>EGE:</b> ethylene glycol ether</td></tr><tr><td><b>EGME:</b> ethylene glycol methyl ether</td></tr><tr><td><b>Fu_blood:</b> fraction unbound in blood</td></tr><tr><td><b>Fu_brain:</b> fraction unbound in brain</td></tr><tr><td><b>Fu_plasma:</b> fraction unbound in plasma</td></tr><tr><td><b>FVB:</b> volume fraction of brain tissue</td></tr><tr><td><b>FVevb:</b> volume of extravascular brain, as fraction of brain volume</td></tr><tr><td><b>FVvb:</b> volume of vascular brain, as fraction of brain volume</td></tr><tr><td><b>GC-MS/MS:</b> gas chromatography-tandem mass spectrometry</td></tr><tr><td><b>hCMEC/D3:</b> human brain microvascular endothelial cells</td></tr><tr><td><b>hiPSC:</b> human induced pluripotent stem cell</td></tr><tr><td><b>NOAEC:</b> no observed adverse effect concentration</td></tr><tr><td><b>OECD:</b> Organisation for Economic Co-operation and Development</td></tr><tr><td><b>OEL:</b> occupational exposure level</td></tr><tr><td><b>Pba:</b> blood:air partition coefficient</td></tr><tr><td><b>PBTK:</b> physiologically based toxicokinetic</td></tr><tr><td><b>PC:</b> partition coefficient</td></tr><tr><td><b>Pca:</b> central:air partition coefficient</td></tr><tr><td><b>Pevb_vb:</b> brain tissue:vascular brain partition coefficient</td></tr><tr><td><b>PGE:</b> propylene glycol ether</td></tr><tr><td><b>PGME:</b> propylene glycol methyl ether</td></tr><tr><td><b>PS:</b> blood-brain barrier permeability-surface area product</td></tr><tr><td><b>Rb:</b> blood-to-plasma ratio</td></tr><tr><td><b>Rpulm:</b> pulmonary retention</td></tr><tr><td><b>Sh:</b> blood-brain barrier surface</td></tr><tr><td><b>TG:</b> Test Guideline</td></tr><tr><td><b>Vmax:</b> maximum velocity</td></tr><tr><td><b>WP:</b> work package</td></tr></table><br><hr><p style="font-style: italic">Edited by A Mavragani; submitted 26.06.23; peer-reviewed by T Dong, J Parkin Kullmann; comments to author 18.09.23; revised version received 10.10.23; accepted 13.10.23; published 18.01.24.</p><a href="https://support.jmir.org/hc/en-us/articles/115002955531" id="Copyright" target="_blank" class="navigation-heading h4 d-block" aria-label="Copyright - what is a Creative Commons License?" data-label="Copyright">Copyright <span class="fas fa-question-circle"></span></a><p class="article-copyright">©Nancy B Hopf, Laura Suter-Dick, Jörg Huwyler, Myriam Borgatta, Lucie Hegg, David Pamies, Hélène Paschoud, Ramya Deepthi Puligilla, Elena Reale, Sophie Werner, Marie-Gabrielle Zurich. 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title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Laura+Suter-Dick\" class=\"btn-view-author-options\"\u003ELaura Suter-Dick\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E3\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-1449-3913\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Jörg+Huwyler\" class=\"btn-view-author-options\"\u003EJörg Huwyler\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E4\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0003-1748-5676\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Myriam+Borgatta\" class=\"btn-view-author-options\"\u003EMyriam Borgatta\u003Csup\u003E\u003Csmall\u003E1,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E2\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-6326-0614\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Lucie+Hegg\" class=\"btn-view-author-options\"\u003ELucie Hegg\u003Csup\u003E\u003Csmall\u003E1,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E2\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, MSc\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0009-0002-3105-9577\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=David+Pamies\" class=\"btn-view-author-options\"\u003EDavid Pamies\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E5\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-1224-573X\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Hélène+Paschoud\" class=\"btn-view-author-options\"\u003EHélène Paschoud\u003Csup\u003E\u003Csmall\u003E1,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E2\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, MSc\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0001-5429-5148\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Ramya%20Deepthi+Puligilla\" class=\"btn-view-author-options\"\u003ERamya Deepthi Puligilla\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E4\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, MSc\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-0767-9402\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Elena+Reale\" class=\"btn-view-author-options\"\u003EElena Reale\u003Csup\u003E\u003Csmall\u003E1,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E2\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-0853-0693\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Sophie+Werner\" class=\"btn-view-author-options\"\u003ESophie Werner\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E3,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E4\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, MSc\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0009-0007-6531-2610\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E; \u003C\u002Fli\u003E\u003Cli\u003E\u003Ca href=\"\u002Fsearch\u002FsearchResult?field%5B%5D=author&criteria%5B%5D=Marie-Gabrielle+Zurich\" class=\"btn-view-author-options\"\u003EMarie-Gabrielle Zurich\u003Csup\u003E\u003Csmall\u003E2,\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E\u003Csup\u003E\u003Csmall\u003E5\u003C\u002Fsmall\u003E\u003C\u002Fsup\u003E, PhD\u003C\u002Fa\u003E\u003Ca class=\"author-orcid\" href=\"https:\u002F\u002Forcid.org\u002F0000-0002-3168-5968\" target=\"_blank\" title=\"ORCID\"\u003E \u003C\u002Fa\u003E\u003C\u002Fli\u003E\u003C\u002Ful\u003E\u003Cdiv class=\"author-affiliation-details\"\u003E\u003Cp\u003E\u003Csup\u003E1\u003C\u002Fsup\u003ECenter for Primary Care and Public Health (Unisanté), University of Lausanne, Lausanne, Switzerland\u003C\u002Fp\u003E\u003Cp\u003E\u003Csup\u003E2\u003C\u002Fsup\u003ESwiss Centre for Applied Human Toxicology (SCAHT), Basel, Switzerland\u003C\u002Fp\u003E\u003Cp\u003E\u003Csup\u003E3\u003C\u002Fsup\u003ESchool of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland\u003C\u002Fp\u003E\u003Cp\u003E\u003Csup\u003E4\u003C\u002Fsup\u003EDivision of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland\u003C\u002Fp\u003E\u003Cp\u003E\u003Csup\u003E5\u003C\u002Fsup\u003EDepartment of Biomedical Sciences, University of Lausanne, Lausanne, Switzerland\u003C\u002Fp\u003E\u003C\u002Fdiv\u003E\u003C\u002Fdiv\u003E\u003Cdiv class=\"corresponding-author-and-affiliations clearfix\"\u003E\u003Cdiv class=\"corresponding-author-details\"\u003E\u003Ch3\u003ECorresponding Author:\u003C\u002Fh3\u003E\u003Cp\u003EMarie-Gabrielle Zurich, PhD\u003C\u002Fp\u003E\u003Cp\u003E\u003C\u002Fp\u003E\u003Cp\u003EDepartment of Biomedical Sciences\u003C\u002Fp\u003E\u003Cp\u003EUniversity of Lausanne\u003C\u002Fp\u003E\u003Cp\u003EBugnon 7\u003C\u002Fp\u003E\u003Cp\u003ELausanne, 1005\u003C\u002Fp\u003E\u003Cp\u003ESwitzerland\u003C\u002Fp\u003E\u003Cp\u003EPhone: 41 21 692 5542\u003C\u002Fp\u003E\u003Cp\u003EEmail: \u003Ca href=\"mailto:mzurich@unil.ch\"\u003Emzurich@unil.ch\u003C\u002Fa\u003E\u003C\u002Fp\u003E\u003Cbr\u003E\u003C\u002Fdiv\u003E\u003C\u002Fdiv\u003E\u003C\u002Fdiv\u003E\u003Csection class=\"article-content clearfix\"\u003E\u003Carticle class=\"abstract\"\u003E\u003Ch3 id=\"Abstract\" class=\"navigation-heading\" data-label=\"Abstract\"\u003EAbstract\u003C\u002Fh3\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EBackground: \u003C\u002Fspan\u003EChemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study.\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EObjective: \u003C\u002Fspan\u003EThis study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays.\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EMethods: \u003C\u002Fspan\u003EThe proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures.\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EResults: \u003C\u002Fspan\u003EThe Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity.\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EConclusions: \u003C\u002Fspan\u003EThis study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals.\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cp\u003E\u003Cspan class=\"abstract-sub-heading\"\u003EInternational Registered Report Identifier (IRRID): \u003C\u002Fspan\u003EDERR1-10.2196\u002F50300\u003Cbr\u003E\u003C\u002Fp\u003E\u003Cstrong class=\"h4-article-volume-issue\"\u003EJMIR Res Protoc 2024;13:e50300\u003C\u002Fstrong\u003E\u003Cbr\u003E\u003Cbr\u003E\u003Cspan class=\"article-doi\"\u003E\u003Ca href=\"https:\u002F\u002Fdoi.org\u002F10.2196\u002F50300\"\u003Edoi:10.2196\u002F50300\u003C\u002Fa\u003E\u003C\u002Fspan\u003E\u003Cbr\u003E\u003Cbr\u003E\u003Ch3 class=\"h3-main-heading\" id=\"Keywords\"\u003EKeywords\u003C\u002Fh3\u003E\u003Cdiv class=\"keywords\"\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=organic%20solvent%20exposure&precise=true\"\u003Eorganic solvent exposure\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=workers&precise=true\"\u003Eworkers\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=general%20population&precise=true\"\u003Egeneral population\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=neurotoxicity&precise=true\"\u003Eneurotoxicity\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=blood-brain%20barrier&precise=true\"\u003Eblood-brain barrier\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=liver%20toxicity&precise=true\"\u003Eliver toxicity\u003C\u002Fa\u003E; \u003C\u002Fspan\u003E\u003Cspan\u003E\u003Ca href=\"\u002Fsearch?type=keyword&term=human%20cell%20cultures&precise=true\"\u003Ehuman cell cultures\u003C\u002Fa\u003E \u003C\u002Fspan\u003E\u003C\u002Fdiv\u003E\u003Cdiv id=\"trendmd-suggestions\"\u003E\u003C\u002Fdiv\u003E\u003C\u002Farticle\u003E\u003Cbr\u003E\u003Carticle class=\"main-article clearfix\"\u003E\u003Cbr\u003E\u003Ch3 class=\"navigation-heading h3-main-heading\" id=\"Introduction\" data-label=\"Introduction\"\u003EIntroduction\u003C\u002Fh3\u003E\u003Cp class=\"abstract-paragraph\"\u003EEnvironmental and occupational exposure to chemicals may contribute to the development of several neurological diseases [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref1\" rel=\"footnote\"\u003E1\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref2\" rel=\"footnote\"\u003E2\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. In particular, organic solvents used in industries such as car repair, painting, furniture manufacturing, printing, and cleaning have been associated with several central nervous system (CNS) conditions. These include mild to severe toxic encephalopathy [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref3\" rel=\"footnote\"\u003E3\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]; deficits in cognitive function [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref4\" rel=\"footnote\"\u003E4\u003C\u002Fa\u003E\u003C\u002Fspan\u003E-\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref7\" rel=\"footnote\"\u003E7\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]; and, in some cases, neurodegenerative diseases [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref8\" rel=\"footnote\"\u003E8\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref9\" rel=\"footnote\"\u003E9\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The diffuse neuropathological effects of acute solvent intoxication reflect neurophysiological abnormalities involving multiple brain regions. With increasingly intense or prolonged exposure, the severity of acute impairment may progress along the spectrum of delirium. Chronic high-level exposure may lead to global cognitive impairment including deficits in memory, attention, energy, and personality, which are well-described forms of dementia [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref10\" rel=\"footnote\"\u003E10\u003C\u002Fa\u003E\u003C\u002Fspan\u003E-\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref13\" rel=\"footnote\"\u003E13\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. Much of the initial work on organic solvent toxicity originated in Scandinavia, where a neurobehavioral syndrome in painters leading to their early retirement was first described [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref14\" rel=\"footnote\"\u003E14\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. However, although the neurotoxicity of solvents such as toluene, trichloroethylene, and n-hexane has been recognized, the neurotoxicity of common solvents currently on the market has not been evaluated. Notably, neurotoxicity testing is only required if the chemical is deemed a pesticide; otherwise, all other chemicals are evaluated on a case-by-case basis. The only exception is if the compound structure is suspected to have nervous system targets and no data are available for read-across or when effects on the nervous system are found in single-dose (Organisation for Economic Co-operation and Development [OECD] Test Guidelines [TGs] 402, 403, 420, 423, or 425) or repeated-dose toxicity studies (TG 407 or 408). Because the nervous system effect endpoints considered as triggers (ie, modifications of wet brain weight or basic histopathology, or both) are quite insensitive, high amounts of potentially neurotoxic compounds are available on the market. The European Union Classification, Labelling, and Packaging Regulation does not include a classification for neurotoxicity. Exposure to organic solvents, especially among workers with higher exposure than the general population, can produce neurotoxic effects, depending on the internal dose. Therefore, to efficiently protect the population from possible solvent toxicity, it is important to determine the air concentrations at which neurotoxicity does not occur. To this end, we propose a strategy using a combination of in vivo (zebrafish embryo), in vitro, and in silico tools coupled with controlled in vivo human exposure experiments to assess the neurotoxicity of solvents (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure1\" rel=\"footnote\"\u003EFigure 1\u003C\u002Fa\u003E\u003C\u002Fspan\u003E).\u003C\u002Fp\u003E\u003Cfigure\u003E\u003Ca name=\"figure1\"\u003E‎\u003C\u002Fa\u003E\u003Ca class=\"fancybox\" title=\"Figure 1. Overview of project rationale, organization, and outcomes. The project will develop a strategy based on various models to assess the neurotoxicity of solvents. Scientific and regulatory outcomes are foreseen. AOP: adverse outcome pathway; BBB: blood-brain barrier; DNT: developmental neurotoxicity; KE: key event; NT: neurotoxicity; OECD: Organisation for Economic Co-operation and Development; PBTK: physiologically based toxicokinetic; TG: Test Guideline; WP: work package.\" href=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Faa69821f56726f0f092800dbbc197d31.png\" id=\"figure1\"\u003E\u003Cimg class=\"figure-image\" src=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Faa69821f56726f0f092800dbbc197d31.png\"\u003E\u003C\u002Fa\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-image\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003EFigure 1. \u003C\u002Fb\u003E Overview of project rationale, organization, and outcomes. The project will develop a strategy based on various models to assess the neurotoxicity of solvents. Scientific and regulatory outcomes are foreseen. AOP: adverse outcome pathway; BBB: blood-brain barrier; DNT: developmental neurotoxicity; KE: key event; NT: neurotoxicity; OECD: Organisation for Economic Co-operation and Development; PBTK: physiologically based toxicokinetic; TG: Test Guideline; WP: work package. \u003C\u002Ffigcaption\u003E\u003C\u002Ffigure\u003E\u003Cp class=\"abstract-paragraph\"\u003EThe recognized method for the evaluation of the neurotoxic potential of chemicals, the OECD TG 424 (neurotoxicity in rodents), uses complex in vivo tests in rodents, which are laborious, expensive, difficult to apply in a standardized manner, and ethically debatable. Regulators from different agencies worldwide as well as the scientific community are becoming increasingly aware of the limitations of the current toxicity testing paradigm. Animal-based high-dose testing in typically 1 stand-alone guideline test is not always relevant for human exposure scenarios [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref15\" rel=\"footnote\"\u003E15\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. One of the most challenging aspects of this animal-centric approach is the impossibility of coping with the thousands of chemicals for which data are still lacking. Conducting animal tests is time consuming and expensive. Therefore, they cannot be carried out routinely because of the sheer number of chemicals that are currently on the market and those anticipated to enter it in the coming years [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref16\" rel=\"footnote\"\u003E16\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. In addition, there are shortcomings regarding interspecies concordance between different mammalian or rodent species as well as with respect to extrapolation from experimental animals to humans. These ambiguities in results or poor reproducibility performance call into question the relevance of such test methods for human risk assessment [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref16\" rel=\"footnote\"\u003E16\u003C\u002Fa\u003E\u003C\u002Fspan\u003E-\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref21\" rel=\"footnote\"\u003E21\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. All this prompts a move away from animal testing toward a combination of in vitro and in silico approaches that address functional mechanistic endpoints [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref15\" rel=\"footnote\"\u003E15\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref16\" rel=\"footnote\"\u003E16\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref22\" rel=\"footnote\"\u003E22\u003C\u002Fa\u003E\u003C\u002Fspan\u003E].\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003ENumerous in vitro models have been proposed for the evaluation of neurotoxicity in the last decades. Monolayer cultures of a single brain cell type are far from representing the human brain in terms of architecture and functionality. Given the sophistication of brain cell-to-cell interactions, some complexity is required to recapitulate human-relevant cellular processes and functions in vitro. However, a good balance must be found between this complexity and the simplicity needed to have robust and reproducible systems that can be applied for chemical screening in a high-throughput manner [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref23\" rel=\"footnote\"\u003E23\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The 3D hiPSC–derived brain test system (BrainSpheres) we previously developed [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref24\" rel=\"footnote\"\u003E24\u003C\u002Fa\u003E\u003C\u002Fspan\u003E] will be used in this study, as it fulfills these requirements.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EThe blood-brain barrier (BBB) protects the brain parenchymal cells from the deleterious effects of xenobiotics. However, some chemicals are able to cross or impair the BBB [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref25\" rel=\"footnote\"\u003E25\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The transient or permanent opening of the BBB provides xenobiotics, plasma proteins, and immunoregulatory mediators access to the CNS, where they can induce toxic effects. Therefore, we will implement a predictive model to assess the impact of solvents on BBB based on in vivo (zebrafish embryo), in vitro (human brain microvascular endothelial cells [hCMEC\u002FD3]), and in silico models [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref26\" rel=\"footnote\"\u003E26\u003C\u002Fa\u003E\u003C\u002Fspan\u003E] to assist in the interpretation of the results obtained in in vitro neurotoxicity testing.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EGlycol ethers will be used as a case study to evaluate the feasibility of our protocol. Glycol ethers form a wide family of a few dozen solvents with different physicochemical properties making them versatile and usable in a variety of industrial applications ranging from pharmaceuticals and microelectronics to domestic cleaning, personal care, and printing. The 2 main groups of glycol ethers are the E series (ie, ethylene glycol ethers [EGEs]) and the P series (ie, propylene glycol ethers [PGEs]). EGEs and PGEs show differences in their toxicological properties regarding teratogenicity, hemolysis, and testicular atrophy [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref23\" rel=\"footnote\"\u003E23\u003C\u002Fa\u003E\u003C\u002Fspan\u003E], apparently resulting from their distinct production of metabolites [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref24\" rel=\"footnote\"\u003E24\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref25\" rel=\"footnote\"\u003E25\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. EGEs have a primary alcohol group and are oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to form the toxic alkoxyacetic acid. Therefore, PGEs are progressively introduced as a less toxic replacement of EGEs. PGEs are sold as a mixture of 2 isomers, with the bulk having a secondary alcohol (a) group and generally <5% of primary alcohol (b) groups [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref22\" rel=\"footnote\"\u003E22\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The b-isomer is oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to form the toxic alkoxypropionic acid in the body. However, the actual toxicity of PGEs is poorly characterized. Therefore, it is essential for our strategy to study not only the parent compound but also the metabolites. Metabolically competent liver cell models will be used to screen for liver toxicity of solvents and for the production of potential metabolites.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EExposure to solvents in human volunteers is fundamental when quantifying possible risks from chemicals because toxicological effects are related to internal exposure, that is, the concentration of a chemical inside the body and its biotransformation. Therefore, in vivo human volunteer studies are necessary to quantify absorption, distribution, metabolism, and excretion (ADME) kinetics in humans under controlled exposure conditions, which are necessary to understand the relationship between solvent concentrations in the air and biological samples, such as blood, urine, or exhaled air. These experiments provide important data on the total absorbed dose after inhaled solvent concentration, absorption rate (ie, from the site of absorption into the bloodstream), biotransformation rate (ie, metabolization of the parent chemical with production of metabolites), and elimination rate (ie, excretion of the parent chemical and metabolites from the body) [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref27\" rel=\"footnote\"\u003E27\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. These toxicokinetic parameters are representative of the target organ or of the tissue concentrations that may trigger an effect and are, therefore, relevant for understanding chemical risks in humans.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EToxicokinetic models quantitatively describe the body’s ADME of a chemical or substance (different terms for this concept are preferred in different fields, including “toxicokinetics,” “pharmacokinetics,” and “biokinetics”) [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref28\" rel=\"footnote\"\u003E28\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. Furthermore, using a toxicokinetic model in reverse dosimetry, we can predict the solvent air concentrations leading to brain concentrations below the levels found to produce neurotoxic effects in vitro in the BrainSpheres. The toxicokinetic model will incorporate metabolism parameters derived from the in vitro liver system and passage through or toxicity to BBB. Once calibrated, the toxicokinetic model can be used to simulate chronic exposure scenarios to predict cumulative brain concentrations and used in reverse dosimetry to predict air concentrations that will not likely result in brain concentrations associated with toxicity.\u003C\u002Fp\u003E\u003Cbr\u003E\u003Ch3 class=\"navigation-heading h3-main-heading\" id=\"Methods\" data-label=\"Methods\"\u003EMethods\u003C\u002Fh3\u003E\u003Ch4\u003EEthical Considerations\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EEthics committee approval was obtained from Swiss ethics (Commission cantonale d’éthique de la recherche sur l’être humain) in 2022 for this nonclinical human study (2022-01567). Healthy women and men were recruited as participants in our study. Each participant signed a written informed consent form before inclusion in the study. The participants will be reimbursed for their time and inconvenience according to the Swiss guidelines.\u003C\u002Fp\u003E\u003Ch4\u003EGlobal Strategy\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EThe choice of solvents to be included in the study will be based on the amount annually placed on the European market and the number of products registered containing known glycol ethers. The selected organic solvents will be applied to various in vitro models to determine their neurotoxicity. They must be amphiphilic to be solubilized in the cell culture media. We established the following solvent selection criteria: (1) used or produced >1 metric ton per year, (2) incorporated in numerous industrial and commercial products, and (3) water solubility. This selection process involves consulting government databases and contacting different industry sectors. We will start the project concomitantly by testing 2 solvents of the P series, propylene glycol methyl ether (PGME), for which we have already developed a toxicokinetic model, and propylene glycol butyl ether. We will test 1 additional solvent from the E series with the in vitro test system, namely, ethylene glycol methyl ether (EGME), which has been banned for use in cosmetic products in Europe [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref29\" rel=\"footnote\"\u003E29\u003C\u002Fa\u003E\u003C\u002Fspan\u003E].\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EThe study is organized into 5 work packages (WPs). The information workflow between the WPs is shown in \u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure2\" rel=\"footnote\"\u003EFigure 2\u003C\u002Fa\u003E\u003C\u002Fspan\u003E. All results collected from the abovementioned systems will contribute to refining the toxicokinetic model we previously developed for PGME [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref30\" rel=\"footnote\"\u003E30\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The toxicokinetic parameters of the solvent and the metabolites will then be characterized in human volunteers after exposure to PGE vapors under controlled conditions. These results will be used to calibrate and expand our toxicokinetic model [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref30\" rel=\"footnote\"\u003E30\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The toxicokinetic model will be constructed to predict brain concentrations of selected solvents and, consequently, will include a brain compartment to predict the target organ solvent and metabolite concentrations. BBB parameters such as barrier transport, transport of the solvent once in the brain, and solvent-brain binding will be incorporated. Solvent neurotoxicity may depend on the metabolic modifications of the substances; therefore, we will incorporate the parameters for the parent compound and the metabolites found in the in vitro liver system. The data necessary to build the model will be retrieved from peer-reviewed scientific literature for tissue:blood partition coefficients (PCs) [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref31\" rel=\"footnote\"\u003E31\u003C\u002Fa\u003E\u003C\u002Fspan\u003E] following the fit-for-purpose dose-response analysis approach. The toxicokinetic model should be able to predict human brain concentration for each of the tested solvents after inhalation exposure, given the air concentration of vapors and duration of exposure. The simulated human brain concentrations will then be compared with the no observed adverse effect concentrations (NOAECs) obtained from the neurotoxicity in vitro system (BrainSpheres). In addition, the toxicokinetic model will be used to predict solvent air concentrations that are unlikely to lead to brain concentration equal to or superior to the brain NOAECs using reverse dosimetry. The specific aims of each WP are summarized in \u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#table1\" rel=\"footnote\"\u003ETable 1\u003C\u002Fa\u003E\u003C\u002Fspan\u003E.\u003C\u002Fp\u003E\u003Cfigure\u003E\u003Ca name=\"figure2\"\u003E‎\u003C\u002Fa\u003E\u003Ca class=\"fancybox\" title=\"Figure 2. Information workflow between the work package (WP). BBB: blood-brain barrier; IVIVE: in vitro-in vivo extrapolation; NOAEC: no observed adverse effect concentration; PBTK: physiologically based toxicokinetic.\" href=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Fd2e4708e87d2c800445c8e84997b83ee.png\" id=\"figure2\"\u003E\u003Cimg class=\"figure-image\" src=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Fd2e4708e87d2c800445c8e84997b83ee.png\"\u003E\u003C\u002Fa\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-image\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003EFigure 2. \u003C\u002Fb\u003E Information workflow between the work package (WP). BBB: blood-brain barrier; IVIVE: in vitro-in vivo extrapolation; NOAEC: no observed adverse effect concentration; PBTK: physiologically based toxicokinetic. \u003C\u002Ffigcaption\u003E\u003C\u002Ffigure\u003E\u003Cdiv class=\"figure-table\"\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-clipboard\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003ETable 1. \u003C\u002Fb\u003ESpecific aims of the work packages (WPs).\u003C\u002Ffigcaption\u003E\u003Ctable width=\"1000\" cellpadding=\"5\" cellspacing=\"0\" border=\"1\" rules=\"groups\" frame=\"hsides\"\u003E\u003Ccol width=\"70\" span=\"1\"\u003E\u003Ccol width=\"270\" span=\"1\"\u003E\u003Ccol width=\"660\" span=\"1\"\u003E\u003Cthead\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EName\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003ESpecific aims\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003C\u002Fthead\u003E\u003Ctbody\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP1\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EIn vitro neurotoxicity testing\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003E\u003Col type=\"1\"\u003E\u003Cli\u003EDetermine NOAEC\u003Csup\u003Ea\u003C\u002Fsup\u003E for neurotoxicity of each solvent\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EDetermine the in vitro distribution kinetics of solvents\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EIdentify toxicity pathways and KEs\u003Csup\u003Eb\u003C\u002Fsup\u003E for solvent neurotoxicity\u003Cbr\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP2\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EIn vivo or in vitro or in silico BBB\u003Csup\u003Ec\u003C\u002Fsup\u003E functionality testing\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003E\u003Col type=\"1\"\u003E\u003Cli\u003EEvaluate the suitability of the zebrafish embryo model to study BBB integrity and functionality\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EDetermine the impact of solvents on BBB integrity and transport in zebrafish and hCMEC\u002FD3\u003Csup\u003Ed\u003C\u002Fsup\u003E\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EDetermine the solvents permeability coefficient (Pe)\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EProvide quantitative data on BBB permeability and tissue distribution of solvents based on computational modeling\u003Cbr\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP3\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EIn vitro hepatic metabolism and clearance\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003E\u003Col type=\"1\"\u003E\u003Cli\u003EElucidate hepatic metabolism\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003ECalculate substrate-enzymatic parameters (Vmax\u003Csup\u003Ee\u003C\u002Fsup\u003E and Km\u003Csup\u003Ef\u003C\u002Fsup\u003E)\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EDetect and identify possible metabolites produced by the liver\u003Cbr\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP4\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EIn vivo volunteer exposure\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003E\u003Col type=\"1\"\u003E\u003Cli\u003ECharacterize human blood absorption and urinary elimination kinetics for parent glycol ether as well as the metabolites identified in WP3\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EFind neurotoxic and vascular injury effect biomarkers for solvent exposure\u003Cbr\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr valign=\"top\"\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EWP5\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003EIn silico PBTK\u003Csup\u003Eg\u003C\u002Fsup\u003E modeling\u003C\u002Ftd\u003E\u003Ctd rowspan=\"1\" colspan=\"1\"\u003E\u003Col type=\"1\"\u003E\u003Cli\u003EEstablish and calibrate the PBTK model for various organic solvents\u003Cbr\u003E\u003C\u002Fli\u003E\u003Cli\u003EUse reverse dosimetry to determine air concentrations below human brain toxicity concentrations\u003Cbr\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003C\u002Ftbody\u003E\u003C\u002Ftable\u003E\u003Cfn id=\"table1fn1\"\u003E\u003Cp\u003E\u003Csup\u003Ea\u003C\u002Fsup\u003ENOAEC: no observed adverse effect concentration.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn2\"\u003E\u003Cp\u003E\u003Csup\u003Eb\u003C\u002Fsup\u003EKE: key event.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn3\"\u003E\u003Cp\u003E\u003Csup\u003Ec\u003C\u002Fsup\u003EBBB: blood-brain barrier.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn4\"\u003E\u003Cp\u003E\u003Csup\u003Ed\u003C\u002Fsup\u003EhCMEC\u002FD3: human brain microvascular endothelial cells.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn5\"\u003E\u003Cp\u003E\u003Csup\u003Ee\u003C\u002Fsup\u003EV\u003Csub\u003Emax\u003C\u002Fsub\u003E: maximum velocity.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn6\"\u003E\u003Cp\u003E\u003Csup\u003Ef\u003C\u002Fsup\u003EKm: Michaelis constant.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003Cfn id=\"table1fn7\"\u003E\u003Cp\u003E\u003Csup\u003Eg\u003C\u002Fsup\u003EPBTK: physiologically based toxicokinetic.\u003C\u002Fp\u003E\u003C\u002Ffn\u003E\u003C\u002Fdiv\u003E\u003Ch4\u003EWP1: In Vitro Neurotoxicity Testing\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003ETesting strategies are needed to evaluate the neurotoxicity of chemicals in a more cost-effective, efficient, and ethical manner. Participating in an international effort, we developed a 3D human-induced pluripotent stem cells (hiPSC)–derived brain model containing several subtypes of neurons, astrocytes, and oligodendrocytes [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref24\" rel=\"footnote\"\u003E24\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. This system allows the cells to reach a high level of differentiation and cellular maturation, exemplified by the presence of functional synapses and compact myelin. The presence of myelin is important for this project because solvents more easily target lipid-rich structures [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref32\" rel=\"footnote\"\u003E32\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. This 3D human brain model has already proven its usefulness for neurotoxicity testing [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref33\" rel=\"footnote\"\u003E33\u003C\u002Fa\u003E\u003C\u002Fspan\u003E-\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref36\" rel=\"footnote\"\u003E36\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. We hypothesized that glycol ethers are neurotoxic. Therefore, we propose to take advantage of our hiPSC-derived BrainSpheres model to study the neurotoxicity induced by uncharacterized glycol ethers present on the market, which will be compared with the neurotoxicity of well-characterized solvents known to induce human encephalopathy.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003ESolvents are data-poor substances. It was originally hypothesized that they exert their toxic effects largely through nonspecific physicochemical effects that modulate membrane fluidity and perturb the hydrophobic force regulating macromolecular interactions [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref37\" rel=\"footnote\"\u003E37\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. However, recent evidence supports the view that solvents interact with lipophilic areas on protein receptors [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref38\" rel=\"footnote\"\u003E38\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref39\" rel=\"footnote\"\u003E39\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. They have also been shown to induce lipid peroxidation, leading to mitochondrial dysfunction, failure of electron transport, and energy production [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref40\" rel=\"footnote\"\u003E40\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref41\" rel=\"footnote\"\u003E41\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. In this study, omics (eg, proteomics, metabolomics, and lipidomics) technology will be used to decipher the mechanisms of glycol ether neurotoxicity and to identify potential biomarkers of toxic effects.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EPrimary 3D hiPSC-derived brain cell cultures will be prepared and maintained as previously described [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref24\" rel=\"footnote\"\u003E24\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. This model contains neurons that form synapses, astrocytes, and oligodendrocytes myelinating the axons (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure3\" rel=\"footnote\"\u003EFigure 3\u003C\u002Fa\u003E\u003C\u002Fspan\u003E). Cytotoxicity will be determined by a resazurin assay after repeated exposure (7 d) to the selected glycol ethers (parent compounds and metabolites). Gene expression for cell type–specific genes, markers of synapses and myelin, and markers of cell stress will be quantified by quantitative reverse transcription polymerase chain reaction at concentrations of solvents under half-maximal effective concentration (EC50) for cytotoxicity. Immunostaining will be performed to assess the effects of solvents on synapses, myelin, and astrocyte reaction, and immunofluorescence will be quantified. NOAECs (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure2\" rel=\"footnote\"\u003EFigure 2\u003C\u002Fa\u003E\u003C\u002Fspan\u003E) will be determined for all tested endpoints, as previously shown for gene expression [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref42\" rel=\"footnote\"\u003E42\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. Brain cell cultures will also be exposed to the metabolites of PGME, propylene glycol butyl ether, and EGME and to the metabolites of the newly selected uncharacterized solvents, potentially produced by liver metabolism (WP3).\u003C\u002Fp\u003E\u003Cfigure\u003E\u003Ca name=\"figure3\"\u003E‎\u003C\u002Fa\u003E\u003Ca class=\"fancybox\" title=\"Figure 3. Brain model used in work package (WP) 1. Immunostainings of human induced pluripotent stem cell–derived 3D BrainSpheres after 8 weeks of differentiation, showing the presence of proteins specific for neurons (neurofilament heavy polypeptide [NF200]), synapses (postsynaptic density-95 protein [PSD95] and synaptophysin [SYP]), astrocytes (glial fibrillary acidic protein [GFAP]) and oligodendrocyte (proteolipid protein 1 [PLP1]). Scale bars: 40 µm.\" href=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002F285cd4f3d30da35eb2f91825020c0bbf.png\" id=\"figure3\"\u003E\u003Cimg class=\"figure-image\" src=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002F285cd4f3d30da35eb2f91825020c0bbf.png\"\u003E\u003C\u002Fa\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-image\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003EFigure 3. \u003C\u002Fb\u003E Brain model used in work package (WP) 1. Immunostainings of human induced pluripotent stem cell–derived 3D BrainSpheres after 8 weeks of differentiation, showing the presence of proteins specific for neurons (neurofilament heavy polypeptide [NF200]), synapses (postsynaptic density-95 protein [PSD95] and synaptophysin [SYP]), astrocytes (glial fibrillary acidic protein [GFAP]) and oligodendrocyte (proteolipid protein 1 [PLP1]). Scale bars: 40 µm. \u003C\u002Ffigcaption\u003E\u003C\u002Ffigure\u003E\u003Cp class=\"abstract-paragraph\"\u003ETo establish the in vitro distribution kinetics of selected solvents necessary for toxicokinetic modeling, 3D brain cell cultures and medium will be collected 3, 6, 24, and 48 hours after the first exposure and after the last exposure of the repeated treatment. The solvent and its main metabolites (if relevant) will be quantified to establish a time course of disappearance from the medium and appearance in the cells as well as to assess the potential accumulation for the entire period of exposure. The fraction bound to culture plates’ plastic will be quantified after desorption. In silico modeling of glycol ethers in vitro distribution kinetics will then be developed. This model will be able to predict the change in cell-associated concentrations of solvents in BrainSpheres with time, as previously shown for amiodarone [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref43\" rel=\"footnote\"\u003E43\u003C\u002Fa\u003E\u003C\u002Fspan\u003E].\u003C\u002Fp\u003E\u003Ch4\u003EWP2: In Vivo, In Vitro, and In Silico BBB Functionality Testing\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EWe previously established the zebrafish as a predictive vertebrate screening model to study the systemic circulation and tissue distribution of particulate drug carriers [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref44\" rel=\"footnote\"\u003E44\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref45\" rel=\"footnote\"\u003E45\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. At 72 hours postfertilization, zebrafish embryos have a functional CNS and, presumably, a fully functional BBB. Anatomical structures such as the vascular endothelium can be visualized using transgenic fish lines expressing fluorescent proteins (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure4\" rel=\"footnote\"\u003EFigure 4\u003C\u002Fa\u003E\u003C\u002Fspan\u003E). Defined exposure of the zebrafish can be achieved by the simple addition of solvents to the fish incubation medium within a closed container. Other advantages of the model include the possibility of studying BBB functionality under physiological conditions in vivo and the high throughput. A well-known in vitro model for the human brain endothelium, hCMEC\u002FD3 cell line (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure4\" rel=\"footnote\"\u003EFigure 4\u003C\u002Fa\u003E\u003C\u002Fspan\u003E) showing the formation of tight junctions and the expression of most transporters and receptors of the in vivo BBB [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref46\" rel=\"footnote\"\u003E46\u003C\u002Fa\u003E\u003C\u002Fspan\u003E], cultured in a transwell system, will also be used. Furthermore, extrapolation of in silico, in vitro, and zebrafish data to higher vertebrates seems feasible [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref47\" rel=\"footnote\"\u003E47\u003C\u002Fa\u003E\u003C\u002Fspan\u003E].\u003C\u002Fp\u003E\u003Cfigure\u003E\u003Ca name=\"figure4\"\u003E‎\u003C\u002Fa\u003E\u003Ca class=\"fancybox\" title=\"Figure 4. Blood-brain barrier (BBB) models used in work package (WP) 2: zebrafish larvae (ZFL) and human brain microvasculatur endothelial cells (hCMEC\u002FD3). ZFL (2 top panels): tracer permeability across BBB. Dorsal view of the midbrain region of the zebrafish lines Tg (kdrl:enhanced green fluorescent protein [eGFP]), which expresses eGFP (green signal) in the endothelial cell membranes. ZFL were injected with the tracer 1 kDa maleimide (red signal). Scale bars: 50 µm. hCMEC\u002FD3 cells (lowest panel): actin filament stained with fluorescein isothiocyanate phalloidin. Scale bars: 100 µm.\" href=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Fd06b22bb63eb0b19db2296d78cf9072c.png\" id=\"figure4\"\u003E\u003Cimg class=\"figure-image\" src=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002Fd06b22bb63eb0b19db2296d78cf9072c.png\"\u003E\u003C\u002Fa\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-image\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003EFigure 4. \u003C\u002Fb\u003E Blood-brain barrier (BBB) models used in work package (WP) 2: zebrafish larvae (ZFL) and human brain microvasculatur endothelial cells (hCMEC\u002FD3). ZFL (2 top panels): tracer permeability across BBB. Dorsal view of the midbrain region of the zebrafish lines Tg (kdrl:enhanced green fluorescent protein [eGFP]), which expresses eGFP (green signal) in the endothelial cell membranes. ZFL were injected with the tracer 1 kDa maleimide (red signal). Scale bars: 50 µm. hCMEC\u002FD3 cells (lowest panel): actin filament stained with fluorescein isothiocyanate phalloidin. Scale bars: 100 µm. \u003C\u002Ffigcaption\u003E\u003C\u002Ffigure\u003E\u003Cp class=\"abstract-paragraph\"\u003EZebrafish larvae are frequently used in developmental biology or toxicological studies. However, in this study, we will use zebrafish larvae exclusively to study BBB integrity and functionality [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref48\" rel=\"footnote\"\u003E48\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. Fluorescently labeled reference compounds (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure4\" rel=\"footnote\"\u003EFigure 4\u003C\u002Fa\u003E\u003C\u002Fspan\u003E) will be intravenously injected into the Duct of Cuvier, as markers of paracellular permeability (eg, fluorescein isothiocyanate dextran 70 or fluorescently labeled liposomes), substrates of drug export transporters (eg, rhodamine-123 as P-glycoprotein substrate), or nutrient transporters (eg, fluorescently labeled transferrin as a marker for receptor-mediated transcytosis). PGME will be the first reference compound to be tested because of its high water miscibility. To precisely assess exposure, analytical methods (gas chromatography-tandem mass spectrometry [GC-MS\u002FMS]) will be used to determine the concentrations of solvents and their metabolites in zebrafish medium, in the headspace of closed incubation vessels and tissue samples (ie, zebrafish homogenates). Circulation, tissue distribution, and brain uptake of the reference compounds will be monitored by confocal laser scanning microscopy (live imaging of anesthetized fish embryos for up to 24 hours). The concentration-dependent toxicity of solvents or their metabolites will be monitored based on the viability and malformations of embryos. The integrity of vasculature will be visualized in transgenic zebrafish kdrl: enhanced green fluorescent protein embryos. The metabolic capacity of the zebrafish will be determined by quantifying potential metabolites (determined in WP3; \u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure2\" rel=\"footnote\"\u003EFigure 2\u003C\u002Fa\u003E\u003C\u002Fspan\u003E) in zebrafish tissue homogenates. The concentration-dependent toxicity to BBB and the coefficient of permeation of solvents will additionally be evaluated in the hCMEC\u002FD3 cell line cultured in a transwell system.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EFinally, quantitative estimates of passive cellular uptake and BBB permeability of solvents and their metabolites will be provided based on computational modeling using physicochemical molecular descriptors according to the methods we previously established [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref26\" rel=\"footnote\"\u003E26\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref49\" rel=\"footnote\"\u003E49\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. These methods provide very high throughput, allowing the screening of web-based chemical libraries.\u003C\u002Fp\u003E\u003Ch4\u003EWP3: In Vitro Hepatic Metabolism and Clearance\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EBecause the liver is the main organ responsible for metabolism and a large contributor to compound clearance, we will implement a system suitable for predicting the hepatic metabolism of solvents. In recent years, 3D liver cell models have been proposed as an alternative to less physiological 2D cell monolayers, and their applications have progressed substantially [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref50\" rel=\"footnote\"\u003E50\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. They are widely used for the assessment of hepatotoxicity [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref51\" rel=\"footnote\"\u003E51\u003C\u002Fa\u003E\u003C\u002Fspan\u003E-\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref55\" rel=\"footnote\"\u003E55\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. An advantage of spheroids is that they overcome the limitation of rapid decline of drug-metabolizing enzyme activities in primary human hepatocyte suspension culture and cell lysates [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref56\" rel=\"footnote\"\u003E56\u003C\u002Fa\u003E\u003C\u002Fspan\u003E], such as microsomes and liver S9 fractions. In this study, we will use 3D liver cultures of the well-characterized human HepaRG cell line (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure5\" rel=\"footnote\"\u003EFigure 5\u003C\u002Fa\u003E\u003C\u002Fspan\u003E), which represent a promising model to evaluate hepatotoxicity and hepatic metabolism [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref57\" rel=\"footnote\"\u003E57\u003C\u002Fa\u003E\u003C\u002Fspan\u003E].\u003C\u002Fp\u003E\u003Cfigure\u003E\u003Ca name=\"figure5\"\u003E‎\u003C\u002Fa\u003E\u003Ca class=\"fancybox\" title=\"Figure 5. Liver model used in work package (WP) 3. Bright field and immunostainings of liver 3D HepaRG cultures showing the presence of albumin and cytochrome P450 3A4 (CYP3A4). Bars: 100 µm.\" href=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002F6204f2bfbb95dd4f300acdb86c9b47a3.png\" id=\"figure5\"\u003E\u003Cimg class=\"figure-image\" src=\"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002F6204f2bfbb95dd4f300acdb86c9b47a3.png\"\u003E\u003C\u002Fa\u003E\u003Cfigcaption\u003E\u003Cspan class=\"typcn typcn-image\"\u003E\u003C\u002Fspan\u003E\u003Cb\u003EFigure 5. \u003C\u002Fb\u003E Liver model used in work package (WP) 3. Bright field and immunostainings of liver 3D HepaRG cultures showing the presence of albumin and cytochrome P450 3A4 (CYP3A4). Bars: 100 µm. \u003C\u002Ffigcaption\u003E\u003C\u002Ffigure\u003E\u003Cp class=\"abstract-paragraph\"\u003EDetermining the appropriate experimental test system (eg, cell plate, incubation time, and exposure concentration) will be an essential part of the development of the 3D model. Moreover, analytical methods (GC-MS\u002FMS and liquid chromatography-tandem mass spectrometry) to detect and quantify the solvents and the metabolites formed must be developed to calculate hepatic metabolism and clearance. Then, proof of metabolic competence and maintenance of the 3D HepaRG cells will be carried out by assessing the metabolism of known P450 substrates. The presence and secretion of albumin as a specific hepatocyte marker will be assessed using immunostaining and enzyme-linked immunosorbent assay. Solvent- and metabolite-induced cytotoxicity will be assessed after 48 hours and 7 days (repeated) of exposure to determine the nontoxic concentration range for subsequent experiments. The metabolic abilities of the 3D HepaRG model and 3D primary human hepatocytes will be compared. Furthermore, the clearance data obtained from the 3D HepaRG model will be compared with the short-term clearance measured in the human liver cell lysate (S9 fractions). In addition, Michaelis-Menten-Kinetic parameters (V\u003Csub\u003Emax\u003C\u002Fsub\u003E and Km) for the formation of the metabolites will be derived using the S9 fractions. These data will be used to build a physiologically based toxicokinetic (PBTK) model (\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#figure2\" rel=\"footnote\"\u003EFigure 2\u003C\u002Fa\u003E\u003C\u002Fspan\u003E).\u003C\u002Fp\u003E\u003Ch4\u003EWP4: In Vivo Volunteer Exposure\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EHuman biomonitoring refers to monitoring exposure-related health risks by analyzing biological samples, usually blood and urine samples [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref58\" rel=\"footnote\"\u003E58\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. The biomonitoring limit values (BMLVs) are set to protect human populations against the potential toxic effects of chemical substances. These limit values account for all routes through which a chemical can enter the body. These are most often the inhalation and skin routes in occupational and environmental settings. Kinetic studies that provide absorption, biotransformation, and elimination rates as well as the absorption and elimination half-lives of the parent compound and its metabolites are necessary to set BMLVs. The apparent urinary elimination half-lives of the parent compound and its metabolites will later be used to develop a biomonitoring method. Sample collection time is crucial and is determined by the apparent elimination half-life of the chemical. Blood concentrations will be used to calibrate the air:blood PC for the toxicokinetic models.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EWe will recruit 4 participants for 2 of the selected solvents. All participants must meet the following criteria: they should be healthy individuals who do not smoke or use contraceptive hormones, do not consume alcohol, be aged between 18 and 65 years, have normal red blood cells and hemoglobin concentrations, maintain a BMI between 18 and 25, and should not be working with glycol ethers. Pregnant and breastfeeding women will be excluded from this study. Participants will be recruited using flyers and announcements distributed at the teaching hospital, university websites, and bulletin boards. All participants will sign a written informed consent form before being included in the study.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EThe participants will be exposed to a single glycol ether for 4 hours under controlled conditions in an exposure chamber (12 m\u003Csup\u003E3\u003C\u002Fsup\u003E). PGE concentrations will be set at or below the Swiss occupational exposure level (OEL) if one exists. In the absence of an OEL, we will rely on existing OELs for other propylene glycols. The parent compound (free and conjugated) and the oxidative metabolites (free and conjugated) of the selected glycol ethers will be monitored in blood, urine, and exhaled air samples. These are noninvasive methods used for human participants, and the results will be used in WP5 to estimate brain concentrations. All compounds will be quantified using capillary gas (parent compound in blood, urine, and exhaled air) or liquid (metabolites in blood and urine) chromatograms with tandem mass spectroscopy detection.\u003C\u002Fp\u003E\u003Ch4\u003EWP5: In Silico PBTK Modeling\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EPBTK models can be used to estimate human brain concentrations. The risk of neurotoxic effects can be estimated by comparing the predicted solvent-brain concentrations with the NOAEC obtained from the in vitro models. Mathematical models such as PBTK models can be used to predict the ADME of a chemical and its metabolites. In these PBTK models, the body is represented by 1 or more compartments. Each compartment represents 1 or more tissues that are kinetically homogeneous, that is, that have similar perfusion rates and an assumed similar substance solubility. PBTK models are described by a set of parameters that define the compartments and a set of mass balance differential equations for each compartment.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EA previously developed toxicokinetic model for PGME with metabolism that is assumed to follow Michaelis-Menten kinetics calibrated for different age groups serves as the basis for our development [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref30\" rel=\"footnote\"\u003E30\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref31\" rel=\"footnote\"\u003E31\u003C\u002Fa\u003E\u003C\u002Fspan\u003E,\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref59\" rel=\"footnote\"\u003E59\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. We aim to modify this previously developed toxicokinetic model to include a separate compartment for the brain using BBB flux rates obtained from in vivo, in vitro, and in silico models (WP2). In addition, we will implement PC obtained from empirical human experiments (WP4) and metabolic parameters assessed in a hepatocyte assay (WP3). The toxicokinetic models will be able to simulate not only acute but also chronic exposures; therefore, both short-term and long-term exposures can be explored in silico. We will develop the toxicokinetic model into a physiologically based pharmacokinetic model based on the existing inhalation-only toxicokinetic model originally developed for PGME [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref40\" rel=\"footnote\"\u003E40\u003C\u002Fa\u003E\u003C\u002Fspan\u003E] and build it in the Berkeley Madonna software or equivalent. We will model the brain as a single compartment with direct contact with the blood flow and where organic solvent uptake will be assumed to be diffusion limited, which is in line with other physiologically based pharmacokinetic models [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref60\" rel=\"footnote\"\u003E60\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. Values for physiological parameters (volume of vascular brain, as fraction of brain volume [FVvb], volume of extravascular brain, as fraction of brain volume [FVevb], volume fraction of brain tissue [FVB; as percent of body weight], BBB surface [Sh] in cm\u003Csup\u003E2\u003C\u002Fsup\u003E, fraction of cardiac output in brain at rest [BFbrainrest]\u002Fcardiac output in brain at light work [BFbrain]) required to build the TK model are from the scientific literature. Depending on the substance, values of chemical-specific parameters such as the pulmonary retention (Rpulm), central:air PC (Pca), blood:air PC (Pba), and brain tissue:vascular brain PC (Pevb_vb) are either taken from the literature or estimated in silico. Since the partitioning of organic compounds between human tissue homogenate and blood is a function of water and lipid content of tissues and the n-octanol:water PC (Kow), PCs are estimated in silico based on LogKow. Kinetic coefficients needed for each organic solvent included in this study will be found in WP3 for liver metabolism (Michaelis-Menten parameters [V\u003Csub\u003Emax\u003C\u002Fsub\u003E and Km]), WP2 for BBB uptake (BBB permeability-surface area product [PS]). The fraction unbound in blood (Fu_blood) will be estimated based on the fraction unbound in plasma (Fu_plasma) and the blood-to-plasma ratio (Rb), and the fraction unbound in brain (Fu_brain) will be considered when modeling each solvent as only the free fraction is able to distribute to different tissues and is biologically active. The model will be calibrated by comparing the predicted and actual urinary organic solvent concentrations obtained from the controlled human experiments (WP4). Both the free and total organic solvent concentrations (free+conjugated) will be obtained for calibration.\u003C\u002Fp\u003E\u003Cbr\u003E\u003Ch3 class=\"navigation-heading h3-main-heading\" id=\"Results\" data-label=\"Results\"\u003EResults\u003C\u002Fh3\u003E\u003Cp class=\"abstract-paragraph\"\u003EWith this project, we expect to provide a strategy to rank uncharacterized solvents and their potential liver-formed metabolites, according to their potential neurotoxicity, and in comparison with the banned EGME. More importantly, a series of PBTK simulations will be conducted to predict occupational exposure, assuming 8 hours of exposure per day, 5 days per week, physical activity for 12 hours per day, and rest for the remaining 12 hours. We will use the PBTK model in reverse dosimetry to estimate air concentrations that do not produce brain concentrations determined as neurotoxic in the hiPSC-derived 3D brain model. We will recommend that authorities setting occupational exposure and public health limits consult these values. Keeping the exposure below the brain effect level should ultimately increase the protection of exposed workers and the general population with domestic exposures.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EWe also anticipate gaining insights into the mechanisms of action of solvents of the glycol ether family. We will elucidate the possible toxic endpoints in the brain, liver, and zebrafish models. Furthermore, we will be able to establish how toxicity is related to the compounds’ lipophilicity and metabolites.\u003C\u002Fp\u003E\u003Cbr\u003E\u003Ch3 class=\"navigation-heading h3-main-heading\" id=\"Discussion\" data-label=\"Discussion\"\u003EDiscussion\u003C\u002Fh3\u003E\u003Cp class=\"abstract-paragraph\"\u003EOverall, our strategy combining multiple, fit-for-purpose 3D advanced cell culture systems; zebrafish larvae; biomarker analysis; human ADME experiments; and in silico prediction is expected to contribute to the improvement of human risk assessment. Although we identified some risks we could encounter during the project, we are confident that our already determined mitigation measures will be able to overcome potential pitfalls.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EDetermination of the passage of solvent through the BBB may be challenging; hence, we are applying 3 different complementary methods: in vivo zebrafish larvae, in vitro human cells (hCMEC\u002FD3), and in silico models. We are also considering and assessing the effects of the hepatic metabolites of the solvents on human BBB cells. With this experimental strategy, issues regarding the potential direct effect of solvents on cell membranes, the relatively low miscibility of solvents with water, and the physiological differences between zebrafish and humans (eg, metabolism and route of expected) should be overcome.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EWe have extensive experience in recruiting human volunteers for controlled human exposure sessions in the exposure chamber. Sometimes, recruitment takes longer than anticipated, and if that is the case, we will extend the timeline to not compromise the size of the study. New analytical chemical methods will need to be determined, which is time consuming. However, we will use a laboratory with extensive experience in analyzing PGME in urine and blood samples. This will also have to be accommodated with a delay in the timeline.\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EFuture developments, not included in this study, are a strategy extended to include developmental neurotoxicity by determining other endpoints, such as proliferation and neurite outgrowth, after exposure of BrainSpheres to solvents at earlier developmental stages and by adding an in vitro test system to take into account the passage of solvents through the placental barrier [\u003Cspan class=\"footers\"\u003E\u003Ca class=\"citation-link\" href=\"#ref61\" rel=\"footnote\"\u003E61\u003C\u002Fa\u003E\u003C\u002Fspan\u003E]. We might also consider combining zebrafish embryo behavioral assays (eg, spontaneous tail coiling) with the BrainSpheres model as readouts for developmental neurotoxicity. Finally, the PBTK model could be adapted to determine solvent air concentrations that are unlikely to cause neurotoxic effects in fetuses or pregnant women.\u003C\u002Fp\u003E\u003C\u002Farticle\u003E\u003Cp\u003E\u003Ch4 class=\"h4-border-top\"\u003EAcknowledgments\u003C\u002Fh4\u003E\u003C\u002Fp\u003E\u003Cp class=\"abstract-paragraph\"\u003EThis work was partially funded by the Swiss Centre for Human Applied Toxicology (SCAHT), Basel, Switzerland.\u003C\u002Fp\u003E\u003Ch4\u003EData Availability\u003C\u002Fh4\u003E\u003Cp class=\"abstract-paragraph\"\u003EData will be available from the corresponding author on reasonable request.\u003C\u002Fp\u003E\u003Ch4 class=\"h4-border-top\"\u003EAuthors' Contributions\u003C\u002Fh4\u003E\u003Cp\u003E\u003Cp class=\"abstract-paragraph\"\u003ENBH, LSD, JH, and MGZ conceptualized the paper. NBH, LSD, JH, and MGZ wrote the original draft. All authors reviewed and edited the paper. LH, DP, HP, RDP, and SW visualized the paper. NBH, LSD, JH, and MGZ administered the project. NBH, LSD, JH, and MGZ acquired the funding.\u003C\u002Fp\u003E\u003C\u002Fp\u003E\u003Ch4 class=\"h4-border-top\"\u003EConflicts of Interest\u003C\u002Fh4\u003E\u003Cp\u003E\u003Cp class=\"abstract-paragraph\"\u003ENone declared.\u003C\u002Fp\u003E\u003C\u002Fp\u003E\u003Cdiv class=\"footnotes\"\u003E\u003Ch4 id=\"References\" class=\"h4-border-top navigation-heading\" data-label=\"References\"\u003EReferences\u003C\u002Fh4\u003E\u003Col\u003E\u003Cli\u003E\u003Cspan id=\"ref1\"\u003EBrown RC, Lockwood AH, Sonawane BR. Neurodegenerative diseases: an overview of environmental risk factors. Environ Health Perspect. Sep 2005;113(9):1250-1256. 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[\u003Ca href=\"https:\u002F\u002Fwww.mdpi.com\u002Fresolver?pii=mi12080884\" target=\"_blank\"\u003EFREE Full text\u003C\u002Fa\u003E] [\u003Ca target=\"_blank\" href=\"https:\u002F\u002Fdx.doi.org\u002F10.3390\u002Fmi12080884\"\u003ECrossRef\u003C\u002Fa\u003E] [\u003Ca href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fentrez\u002Fquery.fcgi?cmd=Retrieve&db=PubMed&list_uids=34442506&dopt=Abstract\" target=\"_blank\"\u003EMedline\u003C\u002Fa\u003E]\u003C\u002Fspan\u003E\u003C\u002Fli\u003E\u003C\u002Fol\u003E\u003C\u002Fdiv\u003E\u003Cbr\u003E\u003Chr\u003E\u003Ca name=\"Abbreviations\"\u003E‎\u003C\u002Fa\u003E\u003Ch4 class=\"navigation-heading\" id=\"Abbreviations\" data-label=\"Abbreviations\"\u003EAbbreviations\u003C\u002Fh4\u003E\u003Ctable width=\"80%\" border=\"0\" align=\"center\"\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EADME:\u003C\u002Fb\u003E absorption, distribution, metabolism, and excretion\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EBBB:\u003C\u002Fb\u003E blood-brain barrier\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EBFbrain:\u003C\u002Fb\u003E cardiac output in brain at light work\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EBFbrainrest:\u003C\u002Fb\u003E cardiac output in brain at rest\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EBMLV:\u003C\u002Fb\u003E biomonitoring limit value\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ECNS:\u003C\u002Fb\u003E central nervous system\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EEGE:\u003C\u002Fb\u003E ethylene glycol ether\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EEGME:\u003C\u002Fb\u003E ethylene glycol methyl ether\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFu_blood:\u003C\u002Fb\u003E fraction unbound in blood\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFu_brain:\u003C\u002Fb\u003E fraction unbound in brain\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFu_plasma:\u003C\u002Fb\u003E fraction unbound in plasma\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFVB:\u003C\u002Fb\u003E volume fraction of brain tissue\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFVevb:\u003C\u002Fb\u003E volume of extravascular brain, as fraction of brain volume\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EFVvb:\u003C\u002Fb\u003E volume of vascular brain, as fraction of brain volume\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EGC-MS\u002FMS:\u003C\u002Fb\u003E gas chromatography-tandem mass spectrometry\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EhCMEC\u002FD3:\u003C\u002Fb\u003E human brain microvascular endothelial cells\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EhiPSC:\u003C\u002Fb\u003E human induced pluripotent stem cell\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ENOAEC:\u003C\u002Fb\u003E no observed adverse effect concentration\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EOECD:\u003C\u002Fb\u003E Organisation for Economic Co-operation and Development\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EOEL:\u003C\u002Fb\u003E occupational exposure level\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPba:\u003C\u002Fb\u003E blood:air partition coefficient\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPBTK:\u003C\u002Fb\u003E physiologically based toxicokinetic\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPC:\u003C\u002Fb\u003E partition coefficient\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPca:\u003C\u002Fb\u003E central:air partition coefficient\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPevb_vb:\u003C\u002Fb\u003E brain tissue:vascular brain partition coefficient\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPGE:\u003C\u002Fb\u003E propylene glycol ether\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPGME:\u003C\u002Fb\u003E propylene glycol methyl ether\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EPS:\u003C\u002Fb\u003E blood-brain barrier permeability-surface area product\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ERb:\u003C\u002Fb\u003E blood-to-plasma ratio\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ERpulm:\u003C\u002Fb\u003E pulmonary retention\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ESh:\u003C\u002Fb\u003E blood-brain barrier surface\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003ETG:\u003C\u002Fb\u003E Test Guideline\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EVmax:\u003C\u002Fb\u003E maximum velocity\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003Ctr\u003E\u003Ctd\u003E\u003Cb\u003EWP:\u003C\u002Fb\u003E work package\u003C\u002Ftd\u003E\u003C\u002Ftr\u003E\u003C\u002Ftable\u003E\u003Cbr\u003E\u003Chr\u003E\u003Cp style=\"font-style: italic\"\u003EEdited by A Mavragani; submitted 26.06.23; peer-reviewed by T Dong, J Parkin Kullmann; comments to author 18.09.23; revised version received 10.10.23; accepted 13.10.23; published 18.01.24.\u003C\u002Fp\u003E\u003Ca href=\"https:\u002F\u002Fsupport.jmir.org\u002Fhc\u002Fen-us\u002Farticles\u002F115002955531\" id=\"Copyright\" target=\"_blank\" class=\"navigation-heading h4 d-block\" aria-label=\"Copyright - what is a Creative Commons License?\" data-label=\"Copyright\"\u003ECopyright \u003Cspan class=\"fas fa-question-circle\"\u003E\u003C\u002Fspan\u003E\u003C\u002Fa\u003E\u003Cp class=\"article-copyright\"\u003E©Nancy B Hopf, Laura Suter-Dick, Jörg Huwyler, Myriam Borgatta, Lucie Hegg, David Pamies, Hélène Paschoud, Ramya Deepthi Puligilla, Elena Reale, Sophie Werner, Marie-Gabrielle Zurich. Originally published in JMIR Research Protocols (https:\u002F\u002Fwww.researchprotocols.org), 18.01.2024.\u003C\u002Fp\u003E\u003Csmall class=\"article-license\"\u003E\u003Cp class=\"abstract-paragraph\"\u003EThis is an open-access article distributed under the terms of the Creative Commons Attribution License (https:\u002F\u002Fcreativecommons.org\u002Flicenses\u002Fby\u002F4.0\u002F), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https:\u002F\u002Fwww.researchprotocols.org, as well as this copyright and license information must be included.\u003C\u002Fp\u003E\u003C\u002Fsmall\u003E\u003Cbr\u003E\u003C\u002Fsection\u003E\u003C\u002Farticle\u003E\u003C\u002Fsection\u003E\u003C\u002Fsection\u003E\u003C\u002Fmain\u003E\n"}],fetch:{},error:a,state:{host:a,environment:e,journalPath:B,keys:{},domains:{},screensize:"desktop",accessibility:{filter:"none","font-weight":"inherit","font-size":.625,"text-align":"initial"},announcements:{data:[{announcement_id:489,title:"JMIR Research Protocols Receives a Journal Impact Factor of 1.4",description_short:"\u003Cp\u003EJMIR Publications is proud to announce that \u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E has received a Journal Impact Factor (JIF) of 1.4 as published in the 2024 Journal Citation Report (JCR) from Clarivate.\u003C\u002Fp\u003E",date_posted:"2024-06-27T09:53:01.000Z",journal_id:f},{announcement_id:469,title:"JMIR Research Protocols Accepted for MEDLINE Indexing",description_short:"\u003Cp\u003EJRP has been accepted for MEDLINE indexing.\u003C\u002Fp\u003E",date_posted:"2024-06-13T16:33:35.000Z",journal_id:f},{announcement_id:380,title:"JMIR Research Protocols Receives Inaugural Impact Factor of 1.7",description_short:"\u003Cp class=\"p1\" style=\"margin: 0px; font-variant-numeric: normal; font-variant-east-asian: normal; font-variant-alternates: normal; font-kerning: auto; font-optical-sizing: auto; font-feature-settings: normal; font-variation-settings: normal; font-stretch: normal; font-size: 14.7px; line-height: normal; color: rgb(0, 0, 0);\"\u003E\u003Cspan style=\"font-family: Arial, Helvetica, sans-serif;\"\u003EJMIR Publications is pleased to announce that \u003Cem style=\"\"\u003EJMIR Research Protocols,\u003C\u002Fem\u003E indexed in the Emerging Sources Citation \u003Cspan style=\"font-size: 14.7px;\"\u003EIndex (ESCI), has been given an inaugural Journal Impact Factor (JIF) of 1.7 from the 2023 Journal Citation Reports™ (JCR).\u003C\u002Fspan\u003E\u003C\u002Fspan\u003E\u003C\u002Fp\u003E",date_posted:"2023-06-28T15:55:24.000Z",journal_id:f},{announcement_id:346,title:"JMIR Research Protocols Expected to Receive Impact Factor in 2023",description_short:"\u003Cp\u003EThe JMIR Research Protocols has been indexed by Clarivate since 2015 and will receive its' first Impact Factor in 2023\u003C\u002Fp\u003E",date_posted:"2022-07-29T15:31:52.000Z",journal_id:f},{announcement_id:250,title:"JMIR Research Protocols Receives Prestigious DOAJ Seal",description_short:"\u003Cp\u003EJMIR Publications is happy to announce that JMIR Research Protocols has been awarded the prestigious Directory of Open Access Journals (DOAJ) Seal. The DOAJ Seal is awarded to journals that demonstrate best practice in open access publishing. Only 10% of the 15,000 peer-reviewed journals indexed in DOAJ have been awarded this Seal. \u003C\u002Fp\u003E",date_posted:"2021-04-14T15:54:59.000Z",journal_id:f},{announcement_id:195,title:"JMIR Research Protocols Now Indexed in Scopus",description_short:"\u003Cp\u003E(Toronto, Oct 9, 2019) We are happy to announce that \u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E, which published its first article in 2012, has been accepted for inclusion in Elsevier’s Scopus.\u003C\u002Fp\u003E\r\n\r\n\u003Cp\u003EJMIR Publications is pleased to have the following journals indexed\u002Faccepted in Scopus:\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003E\u003Cem\u003EJournal of Medical Internet Research\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR mHealth & uHealth \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Research Protocols \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Human Factors \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Mental Health \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Serious Games \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Medical Informatics \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Formative Research \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Diabetes \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Cancer \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Pediatrics and Parenting \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Medical Education \u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Aging\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Rehabilitation and Assistive Technologies\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Cardio\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJMIR Public Health and Surveillance\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003Cli\u003E\u003Cem\u003EJournal of Participatory Medicine\u003C\u002Fem\u003E\u003C\u002Fli\u003E\u003C\u002Ful\u003E\u003Cp\u003EAdditional journals may currently be under evaluation.\u003C\u002Fp\u003E\r\n\r\n\u003Cp\u003EScopus is used by more than 5,000 institutions worldwide and JMIR Publications is glad to be a part of it.\u003C\u002Fp\u003E\u003Cp\u003EKnowledge base article: \u003Ca href=\"https:\u002F\u002Fsupport.jmir.org\u002Fhc\u002Fen-us\u002Farticles\u002F360007270972-Which-JMIR-journals-are-indexed-in-Scopus-\"\u003EWhich JMIR journals are indexed in Scopus?\u003C\u002Fa\u003E\u003C\u002Fp\u003E",date_posted:"2019-10-09T16:34:46.000Z",journal_id:f},{announcement_id:126,title:"JMIR Research Protocols is open for nominations for Section Editors and an Editor-in-Chief",description_short:"\u003Cp\u003EAre you interested in research methods? Are you a specialist in a specific research area or clinical discipline? Do you have a special interest in research ethics and integrity of research, and do you see the importance of prospectively publishing research protocols? Do you see the educational value of publishing research proposals, ideas, and formative research?\u003C\u002Fp\u003E\r\n\r\n\u003Cp\u003EWe are currently seeking academics to apply as \u003Cstrong\u003Esection editors\u003C\u002Fstrong\u003E for Editorial Board positions for JMIR Res Protoc, including the \u003Cstrong\u003EEditor-in-Chief \u003C\u002Fstrong\u003Eposition. There will be remuneration in form of a credit system, rewarding actions such as taking on papers as submission editor. EB members will also be able to use a small discount for their own papers, or papers they invite from other authors. \u003C\u002Fp\u003E\r\n\u003Cp\u003EPrequisites include a scholarly track-record, demonstrated by being a first author on peer-reviewed publications and having served as peer-reviewer (preferably this should include JMIR journals. Applicants can self-assign themselves to papers to be peer-reviewed at \u003Ca href=\"http:\u002F\u002Fpreprints.jmir.org\u002F\"\u003EJMIR Preprints\u003C\u002Fa\u003E).\u003C\u002Fp\u003E\r\n\u003Cp\u003EPlease apply at \u003Ca href=\"http:\u002F\u002Ftinyurl.com\u002Fjmir-eb-appl\" title=\"application form for EB\" target=\"_blank\"\u003Ehttp:\u002F\u002Ftinyurl.com\u002Fjmir-eb-appl\u003C\u002Fa\u003E\u003C\u002Fp\u003E",date_posted:"2016-02-05T14:46:00.000Z",journal_id:f},{announcement_id:118,title:"Looking for ideas, innovators, designs, a solution? JMIR Challenges connects solution seekers with Digital Health innovators, epatients and experts",description_short:"\u003Cp\u003E(7 Dec 2015, Toronto) JMIR Publications is proud to announce the creation of a new platform and journal for\u003Cstrong\u003E innovators and organizations seeking solutions and trying to stimulate innovation:\u003C\u002Fstrong\u003E JMIR Challenges.\u003C\u002Fp\u003E\r\n\u003Cp\u003EThe platform is now open for submissions from solution seekers or \"sponsors\" of a challenge or an award\u002Fprize.\u003C\u002Fp\u003E\r\n\u003Cp\u003EDo you have a problem or question for the worlds' leading health innovators? For example, are you looking for software wireframes, blueprints, behavior change architectures, workflows, ideas or feedback from experts in the field, or do you need a piece of software development or software architecture outsourced? Or do you have an open dataset or big data resource and looking for experts to analyze the data? Do you want to sponsor a monetary or non-monetary prize\u002Faward for compelling solutions for a pressing public health problem? Is your research or development stuck because you need input from a broader community? JMIR has a network of over 60.000 potential problem-solvers and idea generators: eHealth researchers and health experts, e-patients, leaders and innovators, including the top scientists in the fields of informatics, behavioral sciences, mental health, serious games, mHealth, ubiquitous computing, human factors, bioinformatics and biotechnology (\u003Ca href=\"..\u002F..\u002F..\u002F..\u002F..\u002Fuser\u002Fprofile\"\u003Esign up here\u003C\u002Fa\u003E). JMIR Challenges is a new platform connecting \u003Cem\u003E\"solution-seekers\"\u003C\u002Fem\u003E (\u003Cstrong\u003Esponsors\u003C\u002Fstrong\u003E) (companies, or other researchers) with \u003Cem\u003E\"solution-providers\"\u003C\u002Fem\u003E (\u003Cstrong\u003Eentrants\u003C\u002Fstrong\u003E) (innovators, researchers, developers in the ehealth space).\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003Cstrong\u003ESolution seekers\u003C\u002Fstrong\u003E submit a \u003Cstrong\u003Ecompetition document\u003C\u002Fstrong\u003E (\u003Ca href=\"http:\u002F\u002Fjmir.org\u002Fojs\u002Fpublic\u002Fjournals\u002F1\u002Fchallenges-template.docx\"\u003Etemplate here\u003C\u002Fa\u003E) specifying what they are looking for, the prize\u002Faward, and the detailed rules of the competition including evaluation process and criteria. After an internal review, JMIR Challenges will publish the competition document (the solution seeker can remain anonymous, if requested).\u003C\u002Fp\u003E\r\n\u003Cp\u003EFor a template for a competition document and submission guidelines see\u003Cstrong\u003E \u003Ca href=\"..\u002F..\u002F..\u002F..\u002F..\u002Fabout\u002Fsubmissions#authorGuidelines\"\u003EAuthor Guidelines\u003C\u002Fa\u003E.\u003C\u002Fstrong\u003E\u003C\u002Fp\u003E\r\n\u003Cp\u003EThere are no costs for the first challenges hosted on \u003Ca href=\"http:\u002F\u002Fchallenges.jmir.org\u002F\"\u003EJMIR Challenges\u003C\u002Fa\u003E.\u003C\u002Fp\u003E",date_posted:"2015-12-21T10:24:58.000Z",journal_id:f},{announcement_id:105,title:"JMIR Res Protoc among journals selected for Web of Science (New Edition)",description_short:"\u003Cp\u003E(June 2015) Thomson Reuters, producer of the Journal Citation Reports and Web of Science and other database products, is creating a new edition of Web of Science (\u003Cem\u003EEmerging Sources Citation Index\u003C\u002Fem\u003E, ESCI); and we are proud to report that JMIR journals have been selected for the content expansion. \u003C\u002Fp\u003E\r\n\u003Cp\u003EThe new Thomson Reuters Web of Science edition ESCI, which launches later in 2015, will include influential journals covering a variety of disciplines. \"The journals selected have been identified as important to key opinion leaders, funders, and evaluators worldwide.\", says a Thomson Reuters communication about the database. \"We are proud that the Thomson Reuters team recognizes the influence of the JMIR journals\", commented Gunther Eysenbach, publisher at JMIR Publications.\u003C\u002Fp\u003E\r\n\u003Cp\u003EThe following journals are confirmed to be part of the initial ESCI release, with more JMIR journals to be added:\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003EINTERACTIVE JOURNAL OF MEDICAL RESEARCH, \u003Ca href=\"http:\u002F\u002Fwww.i-jmr.org\"\u003Ehttp:\u002F\u002Fwww.i-jmr.org\u003C\u002Fa\u003E, ISSN 1929073X\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003EJMIR MHEALTH AND UHEALTH, \u003Ca href=\"http:\u002F\u002Fmhealth.jmir.org\u002F\"\u003Ehttp:\u002F\u002Fmhealth.jmir.org\u002F\u003C\u002Fa\u003E, ISSN 22915222\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003EJMIR MEDICAL INFORMATICS, \u003Ca href=\"http:\u002F\u002Fmedinform.jmir.org\u002F\"\u003Ehttp:\u002F\u002Fmedinform.jmir.org\u002F\u003C\u002Fa\u003E, ISSN 22919694\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003EJMIR RESEARCH PROTOCOLS, \u003Ca href=\"http:\u002F\u002Fwww.researchprotocols.org\u002F\"\u003Ehttp:\u002F\u002Fwww.researchprotocols.org\u002F\u003C\u002Fa\u003E, ISSN 19290748\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cul\u003E\r\n\u003Cli\u003EJMIR SERIOUS GAMES, \u003Ca href=\"http:\u002F\u002Fgames.jmir.org\u002F\"\u003Ehttp:\u002F\u002Fgames.jmir.org\u002F\u003C\u002Fa\u003E, ISSN 22919279\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E\u003C\u002Fp\u003E\r\n\u003Cp\u003EJMIR Publications is working on getting its newer journals such as \u003Ca href=\"http:\u002F\u002Fmentalhealth.jmir.org\" target=\"_blank\"\u003EJMIR Mental Health\u003C\u002Fa\u003E into the collection as well. \u003Ca href=\"http:\u002F\u002Fjmirpublications.com\u002F\" target=\"_blank\"\u003EJMIR Publications is now publishing over a dozen journals\u003C\u002Fa\u003E with topics covering innovation in health and technology.\u003C\u002Fp\u003E",date_posted:"2015-07-01T09:09:55.000Z",journal_id:f},{announcement_id:97,title:"New JMIR journals - no submission or publication fees!",description_short:"\u003Cp\u003E\u003Cem\u003E(Update Jun 2nd, 2015: This is an older posting, and some of the journals below including those marked with * now have Article Processing Fees. Please make sure to check the \u003Ca href=\"http:\u002F\u002Fwww.jmir.org\u002Fabout\u002FeditorialPolicies#custom8\"\u003EFee Schedule\u003C\u002Fa\u003E). \u003C\u002Fem\u003E\u003C\u002Fp\u003E\r\n\u003Cp\u003E\u003Cem\u003E\u003C\u002Fem\u003EWe are pleased to announce our forthcoming new journals, all of which have currently no submission or publication fees, and all of which focus on emerging technologies and patient-centered innovations in specific areas, going beyond Internet\u002Fwebbased interventions: * JMIR Cancer (http:\u002F\u002Fcancer.jmir.org) * JMIR Medical Education (http:\u002F\u002Fmededu.jmir.org) * JMIR Public Health and Surveillance (http:\u002F\u002Fpublichealth.jmir.org) We welcome submissions for the inaugural issues of these journals. The following journals have already published articles and are still free of charge to publish in (no submission or publication fees): * JMIR Human Factors (http:\u002F\u002Fhumanfactors.jmir.org) * JMIR Rehabilitation and Assistive Technologies (http:\u002F\u002Frehab.jmir.org) * JMIR Mental Health (http:\u002F\u002Fmentalhealth.jmir.org) To submit to these journals, simply append \u002Fauthor to the URLs above, or submit to the main JMIR journal and use the dropdown-box in step 1 to change the journal name. All journals offer careful copyediting and typesetting of manuscripts, and submission to PubMed and PubMed Central (being new journals it may however take a few month until they appear in PubMed). We are also happy to announce that JMIR Medical Informatics and JMIR Serious Games are now indexed in PubMed.\u003C\u002Fp\u003E",date_posted:"2015-02-19T12:00:43.000Z",journal_id:f}],pagination:{from:b,to:r,total:n,perPage:r,firstPage:b,lastPage:b}},article:{data:{article_id:50300,published_at:"2024-01-18T08:30:04.000Z",submitted_at:ag,section_id:ah,journal_id:f,year:ai,issue:aj,volume:n,identifier:"50300",url:"\u002F2024\u002F1\u002Fe50300",pdf_url:"https:\u002F\u002Fwww.researchprotocols.org\u002F2024\u002F1\u002Fe50300\u002FPDF",html_url:"https:\u002F\u002Fwww.researchprotocols.org\u002F2024\u002F1\u002Fe50300",xml_url:"https:\u002F\u002Fwww.researchprotocols.org\u002F2024\u002F1\u002Fe50300\u002FXML",title:"Novel Strategy to Assess the Neurotoxicity of Organic Solvents Such as Glycol Ethers: Protocol for Combining In Vitro and In Silico Methods With Human-Controlled Exposure Experiments",public_id:"JMIR Res Protoc 2024;13:e50300",thumbnail:"https:\u002F\u002Fasset.jmir.pub\u002Fassets\u002F472e3c942ef8c40f33ac7fef996e9769.png",doi:"10.2196\u002F50300",pmid:38236630,pmcid:"10835597",issue_title:"Jan-Dec",pages:[],transfer:a,authors:[{first_name:"Nancy B",last_name:"Hopf",degrees:h,deceased:a,orcid:"0000-0002-4490-6109",equal_contrib:g,matchedAffiliations:[b,c]},{first_name:"Laura",last_name:"Suter-Dick",degrees:h,deceased:a,orcid:"0000-0002-1449-3913",equal_contrib:g,matchedAffiliations:[c,j]},{first_name:"Jörg",last_name:"Huwyler",degrees:h,deceased:a,orcid:"0000-0003-1748-5676",equal_contrib:g,matchedAffiliations:[c,k]},{first_name:"Myriam",last_name:"Borgatta",degrees:h,deceased:a,orcid:"0000-0002-6326-0614",equal_contrib:g,matchedAffiliations:[b,c]},{first_name:"Lucie",last_name:"Hegg",degrees:s,deceased:a,orcid:"0009-0002-3105-9577",equal_contrib:g,matchedAffiliations:[b,c]},{first_name:"David",last_name:"Pamies",degrees:h,deceased:a,orcid:"0000-0002-1224-573X",equal_contrib:g,matchedAffiliations:[c,f]},{first_name:"Hélène",last_name:"Paschoud",degrees:s,deceased:a,orcid:"0000-0001-5429-5148",equal_contrib:g,matchedAffiliations:[b,c]},{first_name:"Ramya Deepthi",last_name:"Puligilla",degrees:s,deceased:a,orcid:"0000-0002-0767-9402",equal_contrib:g,matchedAffiliations:[c,k]},{first_name:"Elena",last_name:"Reale",degrees:h,deceased:a,orcid:"0000-0002-0853-0693",equal_contrib:g,matchedAffiliations:[b,c]},{first_name:"Sophie",last_name:"Werner",degrees:s,deceased:a,orcid:"0009-0007-6531-2610",equal_contrib:g,matchedAffiliations:[c,j,k]},{first_name:ak,last_name:al,degrees:h,deceased:a,orcid:"0000-0002-3168-5968",equal_contrib:g,matchedAffiliations:[c,f]}],affiliations:[{aff_id:11139430,author_id:am,phone:a,fax:d,corresp_aff:o,aff_type:a,seq:b,article_id:a,institution_line_1:"Center for Primary Care and Public Health (Unisanté)",institution_line_2:C,institution_line_3:d,address_line_1:a,address_line_2:a,city:D,prov_state:a,postal_code:a,country:l},{aff_id:11139434,author_id:am,phone:a,fax:d,corresp_aff:o,aff_type:a,seq:c,article_id:a,institution_line_1:"Swiss Centre for Applied Human Toxicology (SCAHT)",institution_line_2:d,institution_line_3:d,address_line_1:"Missionsstrasse 64",address_line_2:d,city:an,prov_state:a,postal_code:"4055",country:l},{aff_id:11139420,author_id:360226,phone:a,fax:d,corresp_aff:o,aff_type:a,seq:b,article_id:a,institution_line_1:"School of Life Sciences",institution_line_2:"University of Applied Sciences and Arts Northwestern Switzerland",institution_line_3:d,address_line_1:a,address_line_2:a,city:"Muttenz",prov_state:a,postal_code:a,country:l},{aff_id:11139424,author_id:360227,phone:a,fax:d,corresp_aff:o,aff_type:a,seq:b,article_id:a,institution_line_1:"Division of Pharmaceutical Technology",institution_line_2:"Department of Pharmaceutical Sciences",institution_line_3:"University of Basel",address_line_1:a,address_line_2:a,city:an,prov_state:a,postal_code:a,country:l},{aff_id:11139433,author_id:360230,phone:a,fax:d,corresp_aff:o,aff_type:a,seq:b,article_id:a,institution_line_1:ao,institution_line_2:C,institution_line_3:d,address_line_1:ap,address_line_2:d,city:D,prov_state:a,postal_code:aq,country:l}],primaryAuthor:{first_name:ak,last_name:al,email:"mzurich@unil.ch",degrees:h,primaryAffiliation:{fax:d,phone:"41 21 692 5542",country:l,postal_code:aq,prov_state:a,city:D,address_line_1:ap,address_line_2:d,institution_line_1:ao,institution_line_2:C,institution_line_3:d}},abstract:"Background: Chemicals are not required to be tested systematically for their neurotoxic potency, although they may contribute to the development of several neurological diseases. The absence of systematic testing may be partially explained by the current Organisation for Economic Co-operation and Development (OECD) Test Guidelines, which rely on animal experiments that are expensive, laborious, and ethically debatable. Therefore, it is important to understand the risks to exposed workers and the general population exposed to domestic products. In this study, we propose a strategy to test the neurotoxicity of solvents using the commonly used glycol ethers as a case study.\nObjective: This study aims to provide a strategy that can be used by regulatory agencies and industries to rank solvents according to their neurotoxicity and demonstrate the use of toxicokinetic modeling to predict air concentrations of solvents that are below the no observed adverse effect concentrations (NOAECs) for human neurotoxicity determined in in vitro assays.\nMethods: The proposed strategy focuses on a complex 3D in vitro brain model (BrainSpheres) derived from human-induced pluripotent stem cells (hiPSCs). This model is accompanied by in vivo, in vitro, and in silico models for the blood-brain barrier (BBB) and in vitro models for liver metabolism. The data are integrated into a toxicokinetic model. Internal concentrations predicted using this toxicokinetic model are compared with the results from in vivo human-controlled exposure experiments for model validation. The toxicokinetic model is then used in reverse dosimetry to predict air concentrations, leading to brain concentrations lower than the NOAECs determined in the hiPSC-derived 3D brain model. These predictions will contribute to the protection of exposed workers and the general population with domestic exposures.\nResults: The Swiss Centre for Applied Human Toxicology funded the project, commencing in January 2021. The Human Ethics Committee approval was obtained on November 16, 2022. Zebrafish experiments and in vitro methods started in February 2021, whereas recruitment of human volunteers started in 2022 after the COVID-19 pandemic–related restrictions were lifted. We anticipate that we will be able to provide a neurotoxicity testing strategy by 2026 and predicted air concentrations for 6 commonly used propylene glycol ethers based on toxicokinetic models incorporating liver metabolism, BBB leakage parameters, and brain toxicity.\nConclusions: This study will be of great interest to regulatory agencies and chemical industries needing and seeking novel solutions to develop human chemical risk assessments. It will contribute to protecting human health from the deleterious effects of environmental chemicals.\n",keywords:"general population; workers; blood-brain barrier; organic solvent exposure; neurotoxicity; liver toxicity; human cell cultures",date_submitted:ag,title_html:a,sections:[{title:"RCTs - Protocols\u002FProposals (non-eHealth)",section_id:ah,journal_id:f,colour:E,count:311},{title:"Neurology and Neurosciences",section_id:102,journal_id:j,colour:ar,count:186}],preprint:i,articleKD:as,isOldOjphiMigrated:as}},articles:{recent:[],openReview:[]},articleTypes:{},authentication:{data:a,jwt:a},countries:{data:[]},departments:{data:[]},help:{data:{}},journal:{data:{journal_id:f,title:F,tag:at,description:au,path:B,slug:av,seq:c,enabled:b,environment:e,url:aw,batch:b,year:t,colour:E,impact:G,order:u,published:ax,transfers:a,cite_score:ay,settings:{aboutJournal:"\u003Cp class=\"p1\"\u003E\u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E (JRP, ISSN 1929-0748) is a unique journal indexed in \u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F?term=JMIR+Res+Protoc%5Bjour%5D\"\u003E\u003Cspan class=\"s1\"\u003EPubMed,\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fpmc\u002Fjournals\u002F2066\u002F\"\u003E\u003Cspan class=\"s1\"\u003EPubMed Central (PMC),\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fnlmcatalog\u002F?term=jmir+research+protocols\"\u003EMEDLINE,\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fv2.sherpa.ac.uk\u002Fid\u002Fpublication\u002F32254\"\u003E\u003Cspan class=\"s1\"\u003ESherpa Romeo,\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fdoaj.org\u002Ftoc\u002F1929-0748?source=%7B%22query%22%3A%7B%22bool%22%3A%7B%22must%22%3A%5B%7B%22terms%22%3A%7B%22index.issn.exact%22%3A%5B%221929-0748%22%5D%7D%7D%5D%7D%7D%2C%22size%22%3A100%2C%22sort%22%3A%5B%7B%22created_date%22%3A%7B%22order%22%3A%22desc%22%7D%7D%5D%2C%22_source%22%3A%7B%7D%2C%22track_total_hits%22%3Atrue%7D\"\u003E\u003Cspan class=\"s1\"\u003EDOAJ,\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fwww.scopus.com\u002Fsourceid\u002F21100967335\"\u003E\u003Cspan class=\"s1\"\u003EScopus,\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fmjl.clarivate.com\u002Fjournal-profile\"\u003E\u003Cspan class=\"s1\"\u003EWeb of Science(WoS)\u002FESCI\u002FSCIE,\u003C\u002Fspan\u003E\u003C\u002Fa\u003E and EBSCO, publishing peer-reviewed, openly accessible research ideas and grant proposals, and study and trial protocols (also referred to as Registered Report Stage 1 papers). \u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003E\u003Cspan\u003EIn 2024, \u003C\u002Fspan\u003E\u003Cem\u003EJMIR Research Protocols \u003C\u002Fem\u003E\u003Cspan\u003Ereceived a \u003C\u002Fspan\u003E\u003Ca href=\"https:\u002F\u002Fjmir.org\u002Fannouncements\u002F476\"\u003EJournal Impact Factor™ of 1.4\u003C\u002Fa\u003E\u003Cspan\u003E (5-Year Journal Impact Factor™: 1.5) according to the latest release of the Journal Citation Reports™ from Clarivate, 2024. \u003C\u002Fspan\u003E\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003EWith a \u003Ca href=\"https:\u002F\u002Fwww.scopus.com\u002Fsourceid\u002F21100967335\"\u003ECiteScore of 2.4,\u003C\u002Fa\u003E \u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E ranks in the 66th percentile (#211 of 636) as a Q2 journal in the field of General Medicine.\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003EIt should be stressed however that most authors do not publish their protocols for \"impact\" or citations, rather to document their ideas to how to design experiments, to document their successful grant proposals, or to publish (and maybe brag a little about) their already funded protocols (which do not require additional peer-review). We offer this platform for scientists to publish peer-reviewed protocols for a very low APF, and unfunded protocols for a reasonable fee that includes peer-review. \u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003EWhile the original focus was on eHealth studies, \u003Cem\u003EJRP\u003C\u002Fem\u003E now publishes protocols and grant proposals \u003Cstrong\u003Ein all areas of medicine,\u003C\u002Fstrong\u003E and their peer-review reports, if available (preliminary results from pilot studies, early results, and formative research should now be published in \u003Cem\u003E\u003Ca href=\"http:\u002F\u002Fformative.jmir.org\u002F\"\u003E\u003Cspan class=\"s1\"\u003EJMIR Formative Research\u003C\u002Fspan\u003E\u003C\u002Fa\u003E\u003C\u002Fem\u003E).\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E is fully open access, with full-text articles deposited in PubMed Central.\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003E\u003Cstrong\u003EWhy should I publish my protocol?\u003C\u002Fstrong\u003E \u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E publishes research protocols, grant proposals, pilot\u002Ffeasibility studies and early reports of ongoing and planned work that encourages collaboration and early feedback, and \u003Cstrong\u003Ereduces duplication of effort.\u003C\u002Fstrong\u003E\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E will be a valuable \u003Cstrong\u003Eeducational resource\u003C\u002Fstrong\u003E for researchers who want to learn about current research methodologies and how to write a winning grant proposal.\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E creates an \u003Cstrong\u003Eearly scientific record\u003C\u002Fstrong\u003E for researchers who have developed novel methodologies, software, innovations or elaborate protocols.\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP \u003C\u002Fem\u003Eprovides a \"dry-run\" for peer-review of the final results paper, and \u003Cstrong\u003Eallows feedback\u002Fcritique of the methods\u003C\u002Fstrong\u003E, often while they still can be fixed.\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E \u003Cstrong\u003Eenhances rigor\u003C\u002Fstrong\u003E and demonstrates to reviewers of subsequent results papers that authors followed and adhered to carefully developed and described a-priori methods, rather than fishing for P-values (HARKing).\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E \u003Cstrong\u003Efacilitates and guarantees subsequent publication of results\u003C\u002Fstrong\u003E demonstrating that the methodology has already been reviewed, and reduces the effort of writing up the results, as the protocol can be easily referenced.\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E is compatible with the concept of \"Registered Reports\" and since May 2018, published protocols receive an \u003Cstrong\u003EInternational Registered Report Identifier\u003C\u002Fstrong\u003E (\u003Ca href=\"https:\u002F\u002Fjmir.zendesk.com\u002Fhc\u002Fen-us\u002Farticles\u002F360003797672\"\u003E\u003Cspan class=\"s1\"\u003EWhat is a Registered Report Identifier?\u003C\u002Fspan\u003E\u003C\u002Fa\u003E) and acceptance of the subsequent results paper is \"in principle\" guaranteed in any JMIR journal and partner journals - see \u003Ca href=\"https:\u002F\u002Fjmir.zendesk.com\u002Fhc\u002Fen-us\u002Farticles\u002F360003450852\"\u003E\u003Cspan class=\"s1\"\u003EWhat is a Registered Report?\u003C\u002Fspan\u003E\u003C\u002Fa\u003E. We assign an \u003Ca href=\"https:\u002F\u002Firridregistry.org\u002F\" target=\"_blank\"\u003EIRRID (International Registered Report Identifier)\u003C\u002Fa\u003E to each published protocol, faciliating the linking between protocol and final study, and also indicating that results papers of studies are also \u003Cstrong\u003E\"in principle accepted\"\u003C\u002Fstrong\u003E for subsequent publication in other JMIR journals (or other members of the \u003Ca href=\"https:\u002F\u002Firridregistry.org\u002F\"\u003EIRRID Registry Network\u003C\u002Fa\u003E) as long as authors adhere to their original protocol - regardless of study results (even if they are negative), reducing publication bias in medicine.\u003C\u002Fli\u003E\r\n\u003Cli class=\"p1\"\u003EAuthors publishing their protocols in \u003Cem\u003EJRP\u003C\u002Fem\u003E will receive a 20% discount on the article processing fee if they publish their results in another journal of the \u003Ca href=\"https:\u002F\u002Fjmir.zendesk.com\u002Fhc\u002Fen-us\u002Farticles\u002F115001442707-Which-journal-titles-is-JMIR-Publications-currently-publishing-\"\u003E\u003Cspan class=\"s1\"\u003E\u003Cem\u003EJMIR\u003C\u002Fem\u003E journal family\u003C\u002Fspan\u003E\u003C\u002Fa\u003E (for example, \u003Cem\u003E\u003Ca href=\"http:\u002F\u002Fwww.jmir.org\u002F\"\u003E\u003Cspan class=\"s1\"\u003EJMIR\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003C\u002Fem\u003Efor e-health studies,\u003Cem\u003E \u003Ca href=\"http:\u002F\u002Fwww.i-jmr.org\u002F\"\u003E\u003Cspan class=\"s1\"\u003Ei-JMR\u003C\u002Fspan\u003E\u003C\u002Fa\u003E \u003C\u002Fem\u003Efor others).\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp class=\"p1\"\u003ENeed more reasons? Read the Knowledge Base article on \"\u003Ca href=\"https:\u002F\u002Fjmir.zendesk.com\u002Fhc\u002Fen-us\u002Farticles\u002F115002860428\"\u003E\u003Cspan class=\"s1\"\u003EWhy should I publish my protocol\u002Fgrant proposal\u003C\u002Fspan\u003E\u003C\u002Fa\u003E\"!\u003C\u002Fp\u003E\r\n\u003Cdiv\u003E \u003C\u002Fdiv\u003E",announcementLink:"https:\u002F\u002Fjmirpublications.com\u002Fannouncements\u002F475",copyrightNotice:"Unless stated otherwise, all articles are open-access distributed under the terms of the Creative Commons Attribution License (http:\u002F\u002Fcreativecommons.org\u002Flicenses\u002Fby\u002F2.0\u002F), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work (\"first published in JMIR Research Protocols...\") is properly cited with original URL and bibliographic citation information. The complete bibliographic information, a link to the original publication on http:\u002F\u002Fwww.researchprotocols.org\u002F, as well as this copyright and license information must be included.",focusScopeDesc:"\u003Cp\u003E\u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E (\u003Cem\u003EJRP\u003C\u002Fem\u003E, ISSN 1929-0748,\u003Cem\u003E \u003C\u002Fem\u003E\u003Ca href=\"https:\u002F\u002Fjmir.org\u002Fannouncements\u002F476\"\u003EImpact Factor: 1.4\u003C\u002Fa\u003E\u003Cem\u003E)\u003C\u002Fem\u003E publishes peer-reviewed, openly accessible research ideas and grant proposals, study and trial protocols, reports of ongoing research, current methods and approaches (Preliminary results from pilot studies, early results, and formative research should now be published in \u003Ca href=\"http:\u002F\u002Fformative.jmir.org\" target=\"_blank\" rel=\"noopener\"\u003EJMIR Formative Research\u003C\u002Fa\u003E). While the original focus was on the design of medical and health-related research and technology innovations, \u003Cem\u003EJMIR Research Protocols\u003C\u002Fem\u003E publishes research protocols, proposals, and methods \u003Cem\u003Ein all areas of medical and health research\u003C\u002Fem\u003E.\u003C\u002Fp\u003E\r\n\u003Cul\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E publishes protocols and grant proposals in all areas of medicine (and their peer-review reports, if available) including wet lab methods\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP \u003C\u002Fem\u003Eis fully open access, with full-text articles deposited in PubMed Central\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP \u003C\u002Fem\u003Epublishes research protocols, grant proposals, and early reports of ongoing and planned work that encourages collaboration and early feedback, and reduces duplication of effort\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E will be a valuable resource for researchers who want to learn about current research methodologies and how to write a winning grant proposal\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E creates an early scientific record for researchers who have developed novel methodologies, software, innovations or elaborate protocols\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E facilitates subsequent publication of results demonstrating that the methodology has already been reviewed, and reduces the effort of writing up the results, as the protocol can be easily referenced\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003E\u003Cem\u003EJRP\u003C\u002Fem\u003E demonstrates to reviewers of subsequent results papers that authors followed and adhered to carefully developed and described a priori methods\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003EIn an effort to make research more reproducible and to avoid problems such as switched outcomes, many journals now require publication of research protocols (even for non-RCTs)\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003EStudies whose protocols or grant proposals have been accepted in \u003Cem\u003EJRP\u003C\u002Fem\u003E are \"in principle accepted\" for subsequent publication of results in other JMIR journals as long as authors adhere to their original protocol, regardless of study results (even if they are negative), reducing publication bias in medicine\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003EPublished protocols will receive a Registered Report Identifier which will facilitate publication of the subsequent results paper; see \u003Ca href=\"https:\u002F\u002Fsupport.jmir.org\u002Fhc\u002Fen-us\u002Farticles\u002F360003450852\" class=\"search-result-link\" style=\"font-family: -apple-system, BlinkMacSystemFont, 'Segoe UI', Roboto, Oxygen, Ubuntu, Cantarell, 'Open Sans', 'Helvetica Neue', sans-serif;\"\u003EWhat is a Registered Report?\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin: 7px 0px; line-height: 2rem;\"\u003EAuthors publishing their protocols in \u003Cem\u003EJRP\u003C\u002Fem\u003E will receive a 20% discount on the article processing fee if they publish their results in another journal of the JMIR journal family (for example, JMIR for ehealth studies, i-JMR for others)\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003EFor more information on why to publish protocols or proposals see our Knowledge Base article \u003Ca href=\"https:\u002F\u002Fjmir.zendesk.com\u002Fhc\u002Fen-us\u002Fsearch\u002Fclick?data=BAh7CjoHaWRsKwiMY7TGGgA6CXR5cGVJIgxhcnRpY2xlBjoGRVQ6CHVybEkiWC9oYy9lbi11cy9hcnRpY2xlcy8xMTUwMDI4NjA0MjgtV2h5LXNob3VsZC1JLXB1Ymxpc2gtbXktcHJvdG9jb2wtb3ItZ3JhbnQtcHJvcG9zYWwtBjsHVDoOc2VhcmNoX2lkSSIpZDQ3NTBkOTMtNzZiMC00MjE3LWI2YTUtYTYyN2MyNjM3Nzc2BjsHRjoJcmFua2kG--17df9da20604c029389a64624f977a83e294542b\" class=\"search-result-link\"\u003EWhy should I publish my protocol or grant proposal?\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003EIn 2024, \u003Ci\u003EJMIR Research Protocols \u003C\u002Fi\u003Ereceived a \u003Ca href=\"https:\u002F\u002Fjmir.org\u002Fannouncements\u002F476?__hstc=178719527.ed0fa05e2bd0080000bda414da03537c.1715194607001.1719500535822.1719509057133.89&__hssc=178719527.25.1719509057133&__hsfp=904621610\"\u003E\u003Cspan class=\"s1\"\u003EJournal Impact Factor™ of 1.4\u003C\u002Fspan\u003E\u003C\u002Fa\u003E (5-Year Journal Impact Factor™: 1.5) according to the latest release of the Journal Citation Reports™ from Clarivate, 2024.\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003EWith a \u003Ca href=\"https:\u002F\u002Fwww.scopus.com\u002Fsourceid\u002F21100967335\"\u003ECiteScore of 2.4,\u003C\u002Fa\u003E\u003Cem\u003E JMIR Research Protocols\u003C\u002Fem\u003E ranks in the 66th percentile (#211 of 636) as a Q2 journal in the field of General Medicine, according to Scopus data.\u003C\u002Fp\u003E\r\n\u003Cp class=\"p1\"\u003E\u003Cem\u003EJMIR Research Protocols \u003C\u002Fem\u003Eis indexed in \u003Ca href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F?term=JMIR+Res+Protoc[jour]\"\u003EPubMed\u003C\u002Fa\u003E, \u003Ca href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fpmc\u002Fjournals\u002F2066\u002F\"\u003EPubMed Central (PMC),\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fnlmcatalog\u002F?term=jmir+research+protocols\"\u003EMEDLINE,\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fv2.sherpa.ac.uk\u002Fid\u002Fpublication\u002F32254\"\u003ESherpa Romeo,\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fdoaj.org\u002Ftoc\u002F1929-0748?source=%7B%22query%22%3A%7B%22bool%22%3A%7B%22must%22%3A%5B%7B%22terms%22%3A%7B%22index.issn.exact%22%3A%5B%221929-0748%22%5D%7D%7D%5D%7D%7D%2C%22size%22%3A100%2C%22sort%22%3A%5B%7B%22created_date%22%3A%7B%22order%22%3A%22desc%22%7D%7D%5D%2C%22_source%22%3A%7B%7D%2C%22track_total_hits%22%3Atrue%7D\"\u003EDOAJ, \u003C\u002Fa\u003E\u003Ca href=\"https:\u002F\u002Fwww.scopus.com\u002Fsourceid\u002F21100967335\"\u003EScopus,\u003C\u002Fa\u003E \u003Ca href=\"https:\u002F\u002Fmjl.clarivate.com\u002Fjournal-profile\"\u003EWeb of Science(WoS)\u002FESCI\u002FSCIE,\u003C\u002Fa\u003E and EBSCO.\u003C\u002Fp\u003E",googleAnalyticsId:"UA-186918-7",impactFactor:G,journalDescription:"\u003Cp\u003E\u003Cstrong\u003EProtocols, grant proposals, \u003Ca href=\"https:\u002F\u002Fsupport.jmir.org\u002Fhc\u002Fen-us\u002Farticles\u002F360003450852-What-is-a-Registered-Report\" target=\"_blank\" rel=\"noopener\"\u003Eregistered reports (RR1)\u003C\u002Fa\u003E\u003C\u002Fstrong\u003E\u003C\u002Fp\u003E",journalInitials:"JRP",footer:"\u003Cul style=\"display: flex; flex-wrap: wrap; justify-content: center; list-style: none;\"\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener\"\u003E\u003Cimg src=\"https:\u002F\u002Fasset.jmir.pub\u002Fresources\u002Fimages\u002Fpartners\u002Fcrossref.jpg\" alt=\"Crossref Member\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Cimg src=\"https:\u002F\u002Fasset.jmir.pub\u002Fresources\u002Fimages\u002Fpartners\u002Fopen-access.jpg\" alt=\"Open Access\" \u002F\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener\"\u003E\u003Cimg src=\"https:\u002F\u002Fasset.jmir.pub\u002Fresources\u002Fimages\u002Fpartners\u002Foaspa.jpg\" alt=\"Open Access Scholarly Publishers Association\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E \u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E \u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener\"\u003E\u003Cimg src=\"https:\u002F\u002Fasset.jmir.pub\u002Fresources\u002Fimages\u002Fpartners\u002Ftrend-MD.jpg\" alt=\"TrendMD Member\" \u002F\u003E\u003Cimg src=\"https:\u002F\u002Fasset.jmir.pub\u002Fresources\u002Fimages\u002Fpartners\u002FORCID.jpg\" alt=\"ORCID Member\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E \u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cdiv data-v-d2fa5e4c=\"\" data-v-44a23348=\"\"\u003E\r\n\u003Csection class=\"partners-wrapper\" data-test=\"partnerships-section\" data-v-d2fa5e4c=\"\"\u003E\r\n\u003Ch2 class=\"text-center\" style=\"text-align: center;\" aria-label=\"Indexed in\" data-v-d2fa5e4c=\"\" tabindex=\"0\"\u003E\u003C\u002Fh2\u003E\r\n\u003Ch2 class=\"text-center\" style=\"text-align: center;\" aria-label=\"Indexed in\" data-v-d2fa5e4c=\"\" tabindex=\"0\"\u003EThis journal is indexed in\u003C\u002Fh2\u003E\r\n\u003Cdiv class=\"green-underline\" style=\"background: #367c3a; height: 3px; margin: 0 auto 40px; width: 100px;\" data-v-30c6e183=\"\"\u003E\u003C\u002Fdiv\u003E\r\n\u003Cul style=\"display: flex; flex-wrap: wrap; justify-content: center; list-style: none;\"\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener\" href=\"https:\u002F\u002Fpubmed.ncbi.nlm.nih.gov\u002F?sort=date&size=100&term=%22JMIR+Res+Protoc%22%5Bjour%5D&sort_order=desc\"\u003E\u003Cimg src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FPubMed.jpg\" alt=\"PubMed\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener\" href=\"https:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fnlmcatalog\u002F101599504\"\u003E\u003Cimg class=\"image_resized\" style=\"aspect-ratio: 461\u002F81; width: 64.99%;\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FPMC.jpg\" alt=\"PubMed Central\" width=\"461\" height=\"81\" \u002F\u003E\u003Cimg style=\"aspect-ratio: 240\u002F81;\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FMEDLINE.jpg\" alt=\"MEDLINE\" width=\"240\" height=\"81\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E \u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E \u003C\u002Fli\u003E\r\n\u003Cli style=\"width: 100%;\"\u003E\r\n\u003Cp style=\"text-align: center;\"\u003E\u003Ca target=\"_blank\" href=\"https:\u002F\u002Fwww.scopus.com\u002Fsourceid\u002F21100967335\" rel=\"noopener\"\u003E\u003Cimg class=\"image_resized\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FScopus-1.jpg\" width=\"239\" height=\"87\" style=\"aspect-ratio: 419\u002F153;\" \u002F\u003E\u003C\u002Fa\u003E\u003Cimg class=\"image_resized\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FDOAJ-1.jpg\" alt=\"DOAJ\" width=\"253\" height=\"84\" style=\"aspect-ratio: 461\u002F153;\" \u002F\u003E\u003Cimg class=\"image_resized\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FDOAJ%20seal.png\" alt=\"DOAJ Seal\" width=\"173\" height=\"75\" style=\"aspect-ratio: 110\u002F110;\" \u002F\u003E\u003Cbr \u002F\u003E\u003Ca target=\"_blank\" href=\"https:\u002F\u002Fv2.sherpa.ac.uk\u002Fid\u002Fpublication\u002F32254\" rel=\"noopener\"\u003E\u003Cimg style=\"aspect-ratio: 287\u002F81;\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FSherpa%20Romeo.jpg\" alt=\"Sherpa Romeo\" width=\"287\" height=\"81\" \u002F\u003E\u003C\u002Fa\u003E\u003Cimg style=\"aspect-ratio: 287\u002F81;\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FEBSCO%20Essentials.jpg\" alt=\"EBSCO\u002FEBSCO Essentials\" width=\"395\" height=\"81\" \u002F\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fli\u003E\r\n\u003Cli\u003E \u003C\u002Fli\u003E\r\n\u003Cli\u003E \u003C\u002Fli\u003E\r\n\u003Cli style=\"width: 74.64%;\"\u003E\r\n\u003Cp style=\"text-align: center;\"\u003E\u003Ca target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\u002F\u002Fdoaj.org\u002Ftoc\u002Fc9b232aa100e40f6bb8aa3f0189f08a5\"\u003E\u003Cimg class=\"image_resized\" style=\"aspect-ratio: 498\u002F100;\" src=\"https:\u002F\u002F19668141.fs1.hubspotusercontent-na1.net\u002Fhubfs\u002F19668141\u002F00%20Marketing\u002FLogos\u002FExternal%20logos%20for%20journal%20pages\u002FESCI.png\" alt=\"Web of Science - ESCI\" width=\"136\" height=\"135\" \u002F\u003E\u003C\u002Fa\u003E\u003C\u002Fp\u003E\r\n\u003C\u002Fli\u003E\r\n\u003Cli style=\"margin-bottom: 10px; margin-right: 10px; margin-top: 10px;\"\u003E\r\n\u003Cp style=\"text-align: center;\"\u003E \u003C\u002Fp\u003E\r\n\u003C\u002Fli\u003E\r\n\u003Cli\u003E \u003C\u002Fli\u003E\r\n\u003Cli\u003E\u003Ca target=\"_blank\" rel=\"noopener\"\u003E \u003C\u002Fa\u003E\u003C\u002Fli\u003E\r\n\u003C\u002Ful\u003E\r\n\u003Cp\u003E \u003C\u002Fp\u003E\r\n\u003C\u002Fsection\u003E\r\n\u003C\u002Fdiv\u003E",onlineIssn:"1929-0748",searchDescription:F,searchKeywords:"publish, journal, publication, open access, grant proposals, protocols, formative research, early results, randomized trial, registry",submissionChecklist:[{order:aj,content:"\u003Cp\u003EThe submission has not been previously published nor is it before another journal for consideration; or an explanation has been provided in Comments to the Editor. Related\u002Foverlapping published or submitted work will be uploaded as supplementary files so reviewers and editors can determine the degree of overlap with previous\u002Fother papers under consideration. Salami slicing of research is discouraged.\u003C\u002Fp\u003E"},{order:"2",content:"\u003Cp\u003EThe submission file is in Microsoft Word (.doc\u002F.docx) file format.\u003C\u002Fp\u003E"},{order:"3",content:"\u003Cp\u003EThe text meets this journal's formatting requirements, in particular those summarized in the \u003Ca href=\"http:\u002F\u002Fwww.jmir.org?Instructions_for_Authors:Instructions_for_Authors_of_JMIR#checklist\" target=\"_blank\"\u003EAuthor Checklist\u003C\u002Fa\u003E found in Instructions for Authors. The text employs \u003Cem\u003Eitalics\u003C\u002Fem\u003E, rather than \u003Cspan style=\"text-decoration: underline;\"\u003Eunderlining\u003C\u002Fspan\u003E or bold as emphasis; with figures and tables (portrait only, no landscape format) placed within the text, rather than at the end. Additional information has been put in separate files to be uploaded as Multimedia Appendix.\u003C\u002Fp\u003E"},{order:az,content:"\u003Cp\u003EI have read and understood the \u003Ca href=\"..\u002F..\u002F\u002F?Instructions_for_Authors:Instructions_for_Authors_of_JMIR#Open_Access\" target=\"instr\"\u003Efee schedule\u003C\u002Fa\u003E. In particular, I understand and agree that unless my department\u002Forganization is a \u003Ca href=\"..\u002F..\u002Fsupport.htm\" target=\"member\"\u003Einstitutional member\u003C\u002Fa\u003E BEFORE submission (see dropdown-list in step 1 of the submission process), I\u002Fmy department will be billed for the article processing fee (see Instructions for authors) in case of acceptance. PLEASE MENTION IN THE COVER LETTER ON SUBMISSION THAT YOU 1) AGREE TO PAY THE APF, OR 2) IF YOU THINK THAT THE APF SHOULD BE WAIVED DUE TO MEMBERSHIP OR FOR ANY OTHER REASONS. Journal sections marked with * may be eligible for a fee waiver or reduction under certain circumstances (must be justified in the comments field for the editor on submission). APFs may not apply for article categories marked with * (check instructions for authors). ** Special fees (in particular a submission fee) apply for \u003Ca href=\"..\u002F..\u002F\u002F?Instructions_for_Authors:Protocol_review\"\u003Eresearch protocols and grant proposals\u003C\u002Fa\u003E. Note that the APF will also be billed if the author retracts the manuscript after acceptance, or if a case of scientific misconduct prevents us from publishing a manuscript after acceptance. \u003C\u002Fp\u003E\r\n\u003Cp\u003EPlease note the price increase for JMIR in July 2015.\u003C\u002Fp\u003E"},{order:"6",content:"\u003Cp\u003EAll cited webreferences (webpages, online available PDF reports) which are NOT journal articles or which do not have a DOI have been cached using WebCite (\u003Ca href=\"http:\u002F\u002Fwww.webcitation.org\"\u003Ewww.webcitation.org\u003C\u002Fa\u003E) . Instead of citing the \"live\" webpage\u002Fwebsite, the author should cite the WebCite archived webpage. No URLs in the body of the manuscript are allowed - all URLs are cited as references.\u003C\u002Fp\u003E"},{order:"8",content:"\u003Cp\u003E(please check this checkbox even if you do not wish to fast-track as an indication that you read this). I understand that if I wish to fast-track the paper, I will pay the Fast-Track-Fee immediately after submission (a payment link will be provided after submission) or at a later stage. The FTF guarantees an editorial decision within 15 working days (see website for further instructions)\u003C\u002Fp\u003E"},{order:"9",content:"\u003Cp\u003EI understand that all author names and their affiliations for the final publication will be taken from the database (metadata form), not the submitted manuscript, thus all author names must be entered in the metadata form during submission. Authors may remove author names from the manuscript if they prefer blind review. All coauthors have been\u002Fwill be entered in the metadata form, and all coauthors fulfill ICMJE criteria in that they made 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published. Authors should meet conditions 1, 2, and 3.\u003C\u002Fp\u003E"},{order:"10",content:"\u003Cp\u003EP-values are reported in accordance with our \u003Ca href=\"..\u002F..\u002F\u002F?Instructions_for_Authors:Instructions_for_Authors_of_JMIR#p\" target=\"instr\"\u003Einstructions for authors\u003C\u002Fa\u003E.\u003C\u002Fp\u003E"},{order:"11",content:"\u003Cp\u003ESince 26 Oct 2006, we now require payment of a \u003Cstrong\u003EUS$ 90 submission fee\u003C\u002Fstrong\u003E for ALL articles submitted to the \u003Cem\u003EJ Med Internet Res\u003C\u002Fem\u003E (=this journal) EXCEPT letters or invited articles (there is no submission fee for sister journals - please change the journal in the drop down list above before proceeding). You can use Paypal or a credit card immediately after submission. Authors will not be able to complete the submission process without payment. This fee cannot be waived (only exception: invited articles), needs to be paid also by institutional members, and is non-refundable. This fee is in addition to other potential fees such as the optional fast-track fee (FTF) and the article processing fee (APF) for non-members. Authors should understand that the submission fee is non-refundable, even if the manuscript is promptly rejected without peer-review (we do send out the majority of papers for peer-review, but we reserve the right to reject papers without peer-review for any reason, including the topic not being deemed interesting enough, which is a subjective decision by the editor).\u003C\u002Fp\u003E"},{order:"12",content:"\u003Cp\u003EAuthors agree that the manuscript and peer-review reports may be transferred to a JMIR sister\u002Fpartner journal (e.g. \u003Ca href=\"http:\u002F\u002Fwww.i-jmr.org\" target=\"_new\"\u003Ei-JMR\u003C\u002Fa\u003E, \u003Ca href=\"http:\u002F\u002Fwww.researchprotocols.org\" target=\"_new\"\u003EJMIR Res Protoc\u003C\u002Fa\u003E, JMIR mHealth, JMIR Human Factors and others), if the paper is not found suitable for publication in JMIR, but is publishable in another journal. The submission fee for that partner journal (if any) will be waived, and transfer of the peer-review reports may mean that the paper does not have to be re-reviewed. Authors will receive a notification when the manuscript is transferred, and at that time can decide if they want to pursue publication in a sister\u002Fpartner journal. If authors do NOT wish an automatic transfer to an alternative journal after rejection for JMIR, this should be noted in the cover letter.\u003C\u002Fp\u003E"}],articlesWidget:{enabled:i,count:v,label:"Recent Articles"},openReviewWidget:{enabled:i,count:v,label:"\u003Ca href=\"https:\u002F\u002Fpreprints.jmir.org\"\u003EPreprints\u003C\u002Fa\u003E Open for Peer-Review"},searchWidget:{enabled:i},partnershipsWidget:{enabled:i},submitButton:{enabled:i,label:"Submit Article"},editorInChief:"\u003Cp\u003E\u003Cspan\u003EAmy Schwartz, MSc, Ph.D., Scientific Editor at JMIR Publications, Ontario, Canada\u003C\u002Fspan\u003E\u003C\u002Fp\u003E"}}},journals:{data:[{journal_id:b,title:"Journal of Medical Internet Research",tag:"The leading peer-reviewed journal for digital medicine and health and health care in the internet age. June 2024 - Journal Impact Factor: 5.8. Q1 journal in \"Medical Informatics\" and \"Health Care Sciences & Services\" categories.(Source: Journal Citation Reports™ 2024 from Clarivate™)",description:a,path:aA,slug:aA,seq:b,enabled:b,environment:e,url:"https:\u002F\u002Fwww.jmir.org",batch:b,year:1999,colour:"#247CB3",impact:"5.8",order:b,published:8973,transfers:a,cite_score:"14.4"},{journal_id:f,title:F,tag:at,description:au,path:B,slug:av,seq:c,enabled:b,environment:e,url:aw,batch:b,year:t,colour:E,impact:G,order:u,published:ax,transfers:a,cite_score:ay},{journal_id:aB,title:"JMIR Formative Research",tag:"Process evaluations, early results and feasibility\u002Fpilot studies of digital and non-digital interventions. June 2024 - Journal Impact Factor: 2.0 (Source: Journal Citation Reports™ 2024 from Clarivate™)",description:d,path:aC,slug:aC,seq:w,enabled:b,environment:e,url:"https:\u002F\u002Fformative.jmir.org",batch:c,year:H,colour:"#605959",impact:"2.0",order:n,published:3044,transfers:a,cite_score:"2.7"},{journal_id:n,title:"JMIR mHealth and uHealth",tag:"Focused on health and biomedical applications in mobile and tablet computing, pervasive and ubiquitous computing, wearable computing and domotics. June 2024 - Journal Impact Factor: 5.4. Q1 journal in \"Health Care Sciences & Services\" and \"Medical Informatics\" categories. 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