If you were to look for a nice, easy place to develop an in vitro testing strategy, Developmental Neurotoxicity (DNT) probably wouldn’t be at the top of your list. The development of the brain and nervous system involves multiple specific processes, both pre- and postnatal, that are both time and location-dependent, and can be perturbed by chemical exposures. On top of that, there are both placental and blood-brain barriers to think about. DNT is the very definition of a complex endpoint.

Challenges in Current DNT Investigation Methods

But the current investigation of DNT poses serious problems. While neurodevelopmental disorders are common, and rising (currently affecting about 10-15% of all human births), and chemical exposures during pregnancy and early life are an important risk factor (EFSA, 2021), animal testing is generally only triggered by neurotoxicity seen in (possibly far less susceptible) adults (Shafer, 2021). As noted in a recent report from the Organisation for Economic Cooperation and Development, only a few chemicals (<200) have been evaluated in guideline DNT studies. To protect public health, further testing may be needed for hundreds of pesticides and thousands of industrial chemicals (OECD, 2023a).

A Shift Towards In Vitro Test Battery (IVB)

In vivo testing for DNT is covered by two OECD test guidelines [footnote 1]. Both propose the prenatal and postnatal exposure of animals, followed by the assessment of their offspring. They require large numbers of animals (about 1000/test), normally rats, and are labour-intensive, time-consuming (lasting 1-2 years) and very expensive (OECD, 2023a,b; Shafer, 2021). In the real world of stretched budgets, a more manageable and efficient approach is needed.

Development of the In Vitro Test Battery for DNT

In 2016, a workshop of the OECD and European Food Safety Authority met to discuss the possible use of a DNT In Vitro Test Battery [footnote 2] (IVB) (Fritsche et al., 2017; OECD, 2017). This formed the basis of several global projects to develop and validate DNT test methods, led by EFSA and OECD, and by the US Environmental Protection Agency, Danish EPA and US National Toxicology Program.

This year, an OECD Working Group, including members from EFSA and the US EPA, released its initial recommendations on the evaluation of data from the DNT IVB. Although the test battery is still in its early stages, the report provides a very useful background, and recommendations for next steps. As few Adverse Outcome Pathways (AOPs) are available for DNT, the IVB was developed based on the key biological events in neurodevelopment (OECD, 2023a; Shafer, 2021), namely:

  • The proliferation and differentiation of neural stem cells into Neural Progenitor Cells (NPCs);
  • The proliferation and differentiation of NPCs;
  • Cell maturation, with neurite outgrowth and synapse formation;
  • Apoptosis of surplus cells;
  • Cell-cell interactions resulting in a functional neuronal network

Limitations of the In Vitro Test Battery for DNT

Anyone hoping for a single screening assay – the DNT equivalent of the Ames test – is going to be disappointed. The current DNT IVB consists of 17 assays, and there is still some of what is assumed to be the vital biology unaccounted for. This latest OECD report notes that 81 chemicals had been tested in all 17 assays, 97 in 14 or more, 331 in at least four, and 476 in at least one. In most cases, chemicals were active in more than one test. While full validation of the DNT IVB has not yet been conducted, the sensitivity of different groups of DNT tests ranged from 61-87% and specificity from 71-93%, with the predictive power of the battery “similar to other in vitro” test methods. For future improvement, the Working Group emphasised the importance of establishing a consensus set of positive and negative controls for validation, and developing new test methods to fill remaining biological data gaps. Some of the test systems use rat (rather than human) cells, a possible major weakness if, as many of the 3R community believe, the rat is not a reliable human surrogate, and there is no accounting for more indirect toxicity driven by endocrine disruption or immune signalling. There are also more general problems associated with in vitro testing, such as a lack of ADME [footnote 3] insights, and no real confidence that any observed perturbation will directly translate to an adverse effect in humans (OECD, 2023a).

OECD Work on In Vitro Assays for DNT

The OECD Working Group concluded that, given the limitations of the DNT IVB, “at this time, negative results shouldn’t be interpreted as a lack of DNT potential”, but that it can be used to prioritise chemicals for further testing (as the US EPA is currently doing for organophosphates) or strengthen in vivo results as part of a weight-of-evidence approach. The development of a tiered decision framework for DNT has also recently begun; a draft framework is currently under OECD review (OECD, 2023a), and EFSA has developed AOP-based IATAs [footnote 4] for deltamethrin and flufenacet. The process allowed EFSA to conclude on a lack of DNT potential for flufenacet, while a DNT concern was supported for deltamethrin and other pyrethroids (EFSA, 2021).

Ongoing Developments in DNT Testing

There is clearly a lot of work going on in this area, and certainly some way to go before the DNT IVB can bring any quick, easy or cheap answers to the complex question of DNT. The test battery was a frequent topic of discussion at this year’s EUROTOX Congress in Slovenia, and we will be keeping an eye out for any future developments, particularly on regulatory acceptance and therefore human health risk assessment.

If you require professional advice or further guidance on DNT testing, reach out to bibra directly.

References and footnotes

EFSA (2021). European Food Safety Authority. Development of Integrated Approaches to Testing and Assessment (IATA) case studies on developmental neurotoxicity (DNT) risk assessment. EFSA Journal 19(6), e06599. https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2021.6599

Fritsche E, Crofton KM, Hernandez AF, Hougaard Bennekou S, Leist M, Bal-Price A, Reaves E, Wilks MF, Terron A, Solecki R, Sachana M and Gourmelon A (2017). OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. Altex 34, 311-315 (cited in OECD, 2017).

OECD (2005). Organisation for Economic Cooperation and Development. Guidance document on the validation and international acceptance of new or updated test methods for hazard assessment. OECD Series on Testing and Assessment Number 34. ENV/JM/MONO(2005)14. https://one.oecd.org/document/ENV/JM/MONO(2005)14/en/pdf

OECD (2007). Organisation for Economic Cooperation and Development. Developmental Neurotoxicity Study. OECD Guideline for the Testing of Chemicals 426. Adopted: 16 October 2007. https://www.oecd-ilibrary.org/docserver/9789264067394-en.pdf?expires=1697101980&id=id&accname=guest&checksum=D10A9D79C1618D5950AB06E607EA4C3F

OECD (2017). Organisation for Economic Cooperation and Development. Annex 1. Report of the OECD/EFSA workshop on developmental neurotoxicity (DNT): The use of non-animal test methods for regulatory purposes. Background document on integrated testing strategies for the identification and evaluation of chemical hazards associated with the developmental neurotoxicity (DNT), to facilitate discussions at the joint EFSA/OECD workshop on DNT. OECD Series on Testing and Assessment Number 261. ENV/JM/MONO(2017)4/ANN1 (cited in OECD, 2023a). https://one.oecd.org/document/env/jm/mono(2017)4/ann1/en/pdf

OECD (2018). Organisation for Economic Cooperation and Development. Extended one-generation reproductive toxicity study. OECD Guideline for the Testing of Chemicals 443. Adopted: 25 June 2018. https://www.oecd-ilibrary.org/docserver/9789264185371-en.pdf?expires=1697117569&id=id&accname=guest&checksum=068EEBC8932EBBFA9EF1F18C6E62C5A2

OECD (2023a). Organisation for Economic Cooperation and Development. Initial recommendations on evaluation of data from the developmental neurotoxicity (DNT) in-vitro testing battery. Series on Testing and Assessment No. 377. ENV/CBC/MONO(2023)13. https://one.oecd.org/document/ENV/CBC/MONO(2023)13/en/pdf

OECD (2023b). Organisation for Economic Cooperation and Development. OECD work on in vitro assays for developmental neurotoxicity. Last updated June 2023. https://www.oecd.org/env/ehs/testing/developmental-neurotoxicity.htm

Shafer T (2021). Developmental neurotoxicity (DNT) in vitro battery as an alternative to DNT in vivo guideline studies used by OPP. US Environmental Protection Agency ORD Board of Scientific Counselors Subcommittee Chemical Safety for Sustainability and Health and Environmental Risk Assessment National Research Programs, RTP, North Carolina, February 02 - 05, 2021. https://doi.org/10.23645/epacomptox.13922930


1] Test Guidelines 426 (Developmental Neurotoxicity Study) (OECD, 2007) and 443 (the Extended One-Generation Reproductive Toxicity (EOGRT) test) (OECD, 2018).

2] A test battery being “a number of test methods that are generally performed at the same time or in close sequence” where each test within the battery is designed to complement the others (OECD, 2005).

3] Absorption, Distribution, Metabolism and Excretion.

4] “Pragmatic, science-based approaches for chemical evaluations in the context of hazard or risk assessments that rely on an integrated analysis of existing information, with optional use of the adverse outcome pathway (AOP) framework, coupled with the generation of new information if necessary. IATAs follow an iterative approach to answer a defined question in a specific regulatory context, taking into account the acceptable level of uncertainty associated with the decision context” (OECD, 2023a).

Contact us