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Developmental Neurotoxicity (DNT)

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 neuro-developmental landmarks, clinical observations, and behavioral and neuropathological endpoints (e.g., OECD Test Guideline (TG) 426 ‘Developmental Neurotoxicity Study’, OECD 2007). For agrochemicals, the developmental neurotoxicity assessment is triggered if indications from adult neurotoxicity or other studies indicate potential concern.

In recognition of the challenges (complex study design with many endpoints, challenging interpretation, human relevance) and ethical considerations (high animal number required), there is a clear push to move away from DNT in vivo testing, particularly if compound prioritization or screening of a larger number of chemicals is envisaged.

Therefore, the Organisation for Economic Co-Operation and Development (OECD) has established a working group that recently published a document titled: “Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery” (Nov, 2023). The document describes in vitro assays that comprise an in vitro testing battery for neurodevelopment and provides criteria that allow evaluation of the relevance and uncertainty of the data to better inform the future use of DNT in vitro data in regulatory safety determinations.

However, more knowledge about the in vivo relevance of the in vitro outcomes in respect of adversity and contextualization with human exposure patterns is needed. Moreover, it is currently unclear how the different types of DNT data (in vivo, in vitro or epidemiological evidence) will be weighed as part of a weight of evidence (WoE) or tiered testing and assessment approach. The CropLife Europe Developmental Neurotoxicity (CLE DNT) subgroup has launched several activities to develop an industry position on these new approaches and contribute to the scientific and regulatory discussions.

1. CLE questionnaire CRO Assay Availabilities

Following publication of the EFSA technical report (Masjosthusmann et al. 2020), the CLE DNT sub-team has launched a survey to understand the capacity of CROs to conduct the described testing battery which comprises 5 test systems measuring 10 DNT-specific endpoints and additional 9 viability/cytotoxicity-related parameters.

The results of this survey indicated that the majority of CROs are not prepared for, or have had experience with, implementation of the assays defined in the EFSA technical report. In cases where some experience with DNT in vitro assays was indicated (though not specifically the EFSA assays), the experience was limited and considered by the CLE DNT sub-group to be insufficient with regard to addressing the totality of requirements outlined in the EFSA technical report. Moreover, cross-validation of the assays via method transfer into another lab would be one major milestone within a formal validation process.

2. Epi DNT review

The objective of the review was to understand the scope of epidemiology literature on neuro-developmental health outcomes and pesticide exposures. Among the 114 publications reviewed there were almost 100 unique assessment tools or variations of such for health outcomes conducted at different ages across an 18-year time span, 159 different chemicals or metabolites assessed, and 4 methods of exposure assessment. The observed heterogeneity in methodologies raises questions about whether results can and should be assessed in concert. Broad conclusions include: i) there is a need to “translate” terminologies among disciplines, ii) some study designs do not enable assessment of temporality, iii) health outcomes are often multifactorial, highlighting the need for replication across populations with harmonized methodologies, and iv) there is a need to pressure test assumptions implicit in exposure assessment methods.

CropLife Europe Contributions

Peer-reviewed paper: J. Reed, C. Burns, F. Pisa. 2023.Literature landscape of neurodevelopment and pesticides: A scoping review of methodologies.

3. SciPinion Peer Review Panel – Predictability of In Vitro DNT Assays to In Vivo Outcomes

For this project, the CLE DNT sub-group commissioned a consulting firm that engages experts from around the world to provide answers to specific scientific questions in an unbiased and scientifically sound manner. The primary goal of this initiative was to enhance the interpretation of results from the in vitro Developmental Neurotoxicity (DNT) battery in the context of human risk assessment. Specifically, the expert panel was tasked with addressing the following question: “Can the endpoints evaluated in a battery of in vitro Developmental Neurotoxicity (DNT) assays be correlated with specific apical endpoints in rodents and/or humans?”.

The outcome of this project is an independent review article that discusses the use of a battery of in vitro assays for assessing the impact of chemicals on the developing nervous system.

CropLife Europe Contributions

Peer-review paper: D. Juberg, D. Fox, P. Forcelli, S. Kacew, J.C. Lipscomb, S. Saghir, C. Sherwin, C. Koenig, S. Hays, C. Kirman. 2023. A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review