On 27 June, the OECD published the Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In Vitro Testing Battery. This publication is the result of a major endeavour from the European Food Safety Agency (EFSA), the United States Environmental Protection Agency (US EPA) and researchers from the IUF – Leibniz Research Institute for Environmental Medicine and University of Konstanz over the last few years to standardise a battery of 17 assays and develop recommendations on the use of data in the context of hazard assessment or weight of evidence determination. The OECD supported the project leads in advancing the field of in vitro testing and assessment of chemicals to identify those with potential DNT properties. DNT is one of those hazards which happen early in the development of human brain and manifest in early years or later in life through either loss of cognitive function or sensory and motor deficits. Progress in knowledge, science and techniques have enabled the measurement of a number of fundamental neurodevelopmental processes using in vitro methods. Resources have been invested to also develop Adverse Outcome Pathways (AOPs), and apply this knowledge together with data derived from the battery in Integrated Approaches to Testing and Assessment (IATA) case studies. The project continues with activities to improve the document, test more chemicals, develop more assays, validate assays already available and propose more case studies to build confidence in these new approaches.
Why is the OECD working on in vitro assays for developmental neurotoxicity?
Approaches used so far to investigate potential developmental neurotoxicants
Developmentally neurotoxic chemicals are a diverse set of substances that have the potential to interfere with the normal development of the nervous system, which, if perturbed without compensation, may lead to adverse effects on nervous system structures and/or functions. OECD plays a central role in harmonising methods used for chemical safety testing, in the scope of the system of Mutual Acceptance of Data across countries. Current regulatory approaches to determine the potential developmental neurotoxicity of chemicals in humans are based on in vivo test protocols that require prenatal and postnatal exposure of pregnant rats followed by assessment of offspring for physical and neurodevelopmental landmarks, clinical observations, and behavioural and neuropathological endpoints (e.g., OECD Test Guideline No. 426: Developmental Neurotoxicity Study, 2007).
Why is this type of approach alone showing limitations?
As is the case with many in vivo test methods used to assess complex biological processes, these guideline studies have limitations that constrain their interpretation including high variability, low precision, reliance on apical endpoints with little, and uncertain human relevance. In addition, use of the in vivo developmental neurotoxicity test guideline has been limited due to its resource intensive nature in terms of cost, time and number of animals used, as well as a lack of chemical alerts that trigger it.
With increased mechanistic understanding and technological progress, other approaches become possible
In recognition of the challenges for traditional methods of toxicity testing (NRC, 2007; Kavlock et al., 2018) there has been a paradigm shift, moving away from chemical safety decisions based solely on in vivo animal tests using apical endpoints and towards the application of in vitro testing and within integrated approaches to testing and assessment IATA (OECD, 2016). This shift in approach relies on identifying and assessing the disruption of molecular events and cellular pathways associated with adverse outcomes in humans using in vitro models and higher throughput technologies. Thus, new approach methodologies (NAMs) including in vitro (omics, cell-based, tissue-based, etc) assays, in silico models, and other computational approaches, are being developed to provide information on chemical hazard and risk for humans while limiting the use of animals.
To address this evolution, the OECD Expert Group (EG) on developmental neurotoxicity in-vitro battery (DNT-IVB) is supporting and coordinating international efforts to develop test methods and foster regulatory acceptance of NAMs for DNT.
Current and planned OECD activities
OECD is coordinating a number of ongoing and future projects that aim at fostering development of new DNT in vitro assays and advancing acceptance of the DNT-IVB for regulatory use. As of June 2023, here is a brief description of current projects.
- OECD Initial Recommendations: The document titled: “Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery" was published in June 2023. The document describes the assays that comprise the battery in terms of neurodevelopment, provides criteria that allow evaluation of the relevance of the data to developmental neurotoxicity, and assists in the determination of the degree of certainty in any positive or negative findings to better inform the use of DNT in vitro data in regulatory hazard determinations by applying the IATA framework.
- Transparency: Most of the data collected to date using the assays in the DNT-IVB has been analysed with a common data pipeline, a common normalisation and dose-response modelling approach, and are now publicly accessible from the US EPA’s ToxCast database.
- Case Studies: A number of case studies on the use of DNT-IVB data have been completed already (see draft Guidance Document Appendix D, November 2022 for a summary). Some of these case studies can be accessed here.
- Tiered Testing Strategy: The OECD EG on DNT-IVB is engaged in the development of a tiered testing strategy for DNT that will provide guidance on how and when the DNT-IVB may be used in regulatory assessments. This will also put the DNT-IVB into the context of all available methods that range from In Silico to In Vivo methods and includes consideration for using in vitro in vivo extrapolation (IVIVE) specific to DNT.
- Reference Chemical List: The OECD EG has begun discussions of the development of a consensus-based list of reference chemicals that should be used in the development and inclusion of all new additions to the DNT-IVB.
- Additional Chemical Testing: There are a number of ongoing and planned efforts to fund and test additional chemicals using assays in the current DNT-IVB.
- EFSA: EFSA has supported chemical testing in the development of the current DNT-IVB. EFSA launched a new call for proposals in April 2023 to test additional chemicals of high importance in the EU, enhance interlaboratory transferability of the assays, compare in vitro results to in vivo guideline findings, investigate the use of the DNT-IVB to increase knowledge on human brain health and disease, and enhance development of AOPs for developmental neurotoxicity.
- US NIEHS/DTT: The DNT Health Effects Innovation (HEI) program within the Division of Translational Toxicology (DTT) is currently supporting chemical testing in the majority of the assays in the DNT-IVB. Testing is being done in phases. Phase 1, including chemicals nominated by multiple stakeholders, were distributed to assay developers for testing in 2022 with results expected later in 2023. Phase 2 chemical testing has recently begun with results expected in 2024 (see here)
- US EPA: In the current EPA Chemical Safety for Sustainability research program, EPA is continuing to test high-priority compounds from broad classes of chemicals, including pesticides and those regulated under the Toxic Substances Control Act.
- New Assay Development: Efforts are underway to develop new assays to address biological gaps identified in the DNT-IVB. Such gaps concern: (i) key neurodevelopmental processes currently not or insufficiently covered by the IVB; (ii) assays needing refinement with human cells or throughput; and (iii) gaps covering neurodevelopmental timing. Additional work is underway to assess novel methods, including high-throughput in vitro phenotypic profiling and high-throughput in vitro transcriptomics for DNT. Inclusion of these new methods into the DNT-IVB will depend on the outcome of a review by the EG.
- Interlaboratory Transferability of Test Methods in the DNT-IVB: Current efforts are underway to enable transferability of cell-based assays between testing laboratories. This involves work to both transfer assays between laboratories developing new assays, as well as transfers to commercial laboratories.
- In Vivo to In Vitro Comparisons: To enable comparisons between in vitro and in vivo findings both EFSA and USEPA are working to populate databases for Guideline DNT studies (OECD 426, USEPA 870.6300). Additional work is underway by USEPA and the NIEHS’s National Toxicology Program Interagency for the Evaluation of Alternative Toxicological Methods (NICEATM), to enhance physiological-based pharmacokinetic (PBPK) and in vitro to in vivo extrapolation (IVIVE) models to include components important to DNT e.g., blood brain and placenta barriers.
- Alternative Species: Whole organism models such as the zebrafish are recognised as valuable models to study DNT with the distinct advantage of complete brain development as well as the ability to assess chemical effects in an intact organism. Standardised DNT testing protocols for zebrafish eleutheroembryos have not been established yet. An international collaboration led by the Netherlands aims to establish a harmonised protocol for the behavioural testing in eleutheroembryos zebrafish. The objective is to evaluate reproducibility between laboratories, determine the predictivity of the assay, and explore the added value of the zebrafish whole organism model as an addition to the DNT-IVB. To date, four laboratories have screened ~28 chemicals, including positive and negatives. Future studies with this group might focus on additional behavioural assays in zebrafish for DNT.
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