Establishment of an a priori protocol for the implementation and interpretation of an in‐vitro testing battery for the assessment of developmental neurotoxicity

Abstract

In this project we set up a human cell-based DNT in vitro testing strategy that is based on test methods with high readiness and data generated therefrom. The methods underwent a fit-for-purpose evaluation that considered four key elements: 1. The test system, 2. the exposure scheme, 3. the assay and analytical endpoint(s) and 4. the classification model. This testing battery was challenged with 119 chemicals for which rich toxicological information was available (for some of them also on their DNT hazard). Testing was performed in 5 test systems measuring 10 DNT-specific endpoints and additional 9 viability/ cytotoxicity-related parameters. For approximately half of the compounds, additional and complementary data from DNT in vitro tests was added by the US-EPA. This extended battery was also evaluated. Testing results revealed that the test methods of this current DNT in vitro battery are reliable and reproducible. The endpoints had to a large extent low redundancy. Battery performance, as assessed with compounds well-characterized for DNT hazard had a sensitivity of 82.7 % and a specificity of 88.2 %. Gap analyses suggested that radial, astro-and microglia as well as myelination endpoints may be added to the battery. Two case studies, one for screening and prioritization of 14 flame retardants, and one on hazard characterization of 2 pesticides, were presented. Hypothetical AOPs were developed based on the latter case study. In conclusion, the DNT testing strategy explored here is a very promising first approach for DNT hazard identification and characterization. The performance is encouraging and may be improved by inclusion of further tests. Some uncertainties in DNT in vitro battery testing outcomes could be reduced by incorporating test data and modelling approaches related to in vitro and in vivo toxicokinetics of test compounds.

Toward a Better Testing Paradigm for Developmental Neurotoxicity: OECD Efforts and Regulatory Considerations

“…Although multiple tests have been developed, the critical step of their comparison using larger chemical libraries is still lacking. Recently, this issue has been reduced by the publication of more experimental work testing a consolidated list of compounds in the DNT-IVB [ 28 ]. These considerations led the OECD DNT experts to propose a significant step in the regulatory application of the DNT-IVB, namely the decision to develop guidance on the use and interpretation of data from in vitro DNT assays (with the inclusion of the zebrafish model).…”

“…The DNT-IVB (described in Section 3 ) was used to generate extensive experimental data initially by an EFSA-funded research project involving the Universities of Konstanz and Dϋsseldorf, with additional data to be generated by the US Environmental Protection Agency (US EPA). A protocol for the implementation and interpretation of the DNT-IVB has been published [ 28 ]. These efforts aim to support the development of an OECD guidance document on the interpretation and use of DNT-IVB data in regulatory decisions that is expected to be finalized in 2021.…”

“…Although approximately 30 or more potential assays have been developed, the ones selected for this battery have been evaluated for their readiness [ 22 ] and/or followed the recommendations of Crofton et al, 2011 [ 21 ] in their development. In addition, the laboratories that developed the assays in this proposed battery expressed a willingness and had the resources to pursue additional chemical testing with these assays [ 28 ]. Thus, it is important to note that an in vitro assay, which is not listed as part of this battery, might still be able to provide useful data for a decision regarding DNT.…”

“…Individual plates were made for each assay or group of assays based on how many of the 120 compounds had already been tested in that assay, and the untested compounds were plated and provided to the laboratories. The EFSA-funded researchers at the Universities of Konstanz and Dϋsseldorf have completed their testing and submitted a report to EFSA [ 28 ], while data collection for the US EPA assays should be complete by early 2021.…”

“…The data from the EFSA-funded project can be found in the report [ 28 ] and on the link to that report ( ; see “supporting information file: efs31938e-sup-0001-Annex-A.xlsx”). When it is complete, the data from the US EPA laboratory will be made available through the US EPA’s CompTox Chemicals Dashboard ( ), but this is not expected to be available to the public before mid-2021.…”