Monday, 19 August 2019 07:12

Replacement of the Year 2019: Testing Strategy for Developmental Neurotoxicity Tests Featured

This year's "Replacement of the Year" issue examines the current development and acceptance of a testing strategy to replace rodents in tests for brain development disorders (developmental neurotoxicity tests). Scientists have been working for some time with regulatory authorities such as the European Food and Safety Agency (EFSA) on a cost-effective testing strategy based on a reliable in vitro test battery to identify DNT hazards to reduce exposure to these chemicals. For this purpose, the complex processes of brain development are to be broken down into individual spatially and temporally separate development steps. For each step, individual tests are to be developed.

During pregnancy, various environmental influences can disrupt the development of nerve cells in the human fetus. Environmental toxins can inhibit the division, differentiation or migration of nerve cells (1). In later life, these disruptions can lead to learning disabilities, developmental delays, abnormalities from the autism spectrum or attention deficit and hyperactivity disorders. Indeed, such disorders can be attributed to a combination of several causes, including genetic ones. However, environmental chemicals and drugs are also discussed as determinants (2) as shown in laboratory and human studies (3).

Photo: Volker_Pietzonka_Pixabay


To test the safety of chemicals, such as pesticides, animal experiments are carried out with rats following the OECD guideline. These tests must be conducted when there is evidence of neurotoxicity, cholinesterase disorders (liver damage), changes in thyroid hormones or estrogen balance, gained from previous studies. In pesticide production, the United States generally prescribes development neurotoxicity studies to assess safety (4). The three most important OECD guidelines investigating life-phase-dependent neurotoxicity are OECD guidelines 424 (neurotoxicity), 426 (developmental neurotoxicity study) and 443 (extended one-generation reproductive toxicity study) with rodents (mostly rat).

The test is administered to the mother animals daily from the time of implantation of the fertilized egg until the 21st day of prenatal development (during the suckling phase), if possible, the route should be used, via which pregnant women may also be exposed. Offspring are thus exposed to the test substance during pre- and postnatal neurological development (5). According to OECD guideline 426, about 80 dams and up to 960 offspring are tested. In addition to body weight, the brain weight, neuropathological changes, sexual maturation, behavior, motor activity, motor, and sensory functions, learning and memory of the young animals are examined.

Pesticides are discussed as possible triggers of neurotoxic influences.
Photo: Erich Westendarp, Pixabay.

However, the animal studies are of limited significance and controversial concerning the number of animals used, time and costs. For instance, scientists such as Prof. Marcel Leist, who is researching DNT himself, criticize the fact that most developmental neurotoxic phenomena, such as speech disorders, impairment of attention duration or IQ cannot be measured at all on animals (6). Besides, studies have already revealed significant differences in developmental neurotoxicity between rodents and humans using in vitro test systems (7).

On the horizon: New in vitro and in silico methods in a graduated testing strategy

A team of researchers led by Prof. Dr. Ellen Fritsche from the Institute for Environmental Medicine Research (IUF) in Düsseldorf used so-called neurospheres from neural stem cells and induced pluripotent stem cells of humans (hiPSCs), mice and rats to investigate the influence of environmental chemicals on the early development of the brain. When cultured in vitro, human neural progenitor cells form a spherical shape (neurospheres) and simulate the process of brain development (8). Cell proliferation, cell differentiation, cell migration as well as apoptosis - all processes that take place in the developing brain - are tested in the working group (9).

Normal human neural progenitor cells differentiate into neurocytes, astrocytes, and oligodendrocytes. Neurocytes (stained green with the marker ß(III)tubulin) and astrocytes (stained red with the marker GFAP) can be seen.
Photo: Leibniz Institute of Environmental Research (IUF).

Since 2005, international researchers have been developing concepts for the application and interpretation of alternative methods with the ultimate goal of regulatory acceptance (10). Regulatory authorities such as the European Food and Safety Agency (EFSA) have been working with scientists for some time to develop a cost-effective testing strategy based on a reliable in vitro test battery to identify DNT hazards and initiate measures to reduce exposure to these chemicals (9). The complex processes of brain development are to be broken down into individual spatially and temporally separate development steps to develop individual tests for each step.

Key events in brain development include stem cell differentiation, cell division,  apoptosis of neuronal stem cells, migration of neuronal stem cells to the site of their identification, differentiation, maturation of nerve and glial cells, synapse formation as well as network formation. With a battery of several in vitro tests, substances can be investigated for their influence on all these developmental steps as well as on region-specific properties of the brain or on molecular aspects of hormonally influenced, respectively gender-specific neuronal cell functions.

The new method for a replacement of animal experiments consists roughly of the following steps:

Step 0: Toxicokinetic modeling
Step 1: In vitro tests with human cells
Step 2: Tests on alternative model organisms
Step 3: In vitro tests on rodent cells
Stage 4 (optional): in vivo tests with rodents (this animal experiment is intended only as of the very last instrument, (11)).

Scientists are considering the scientific development status of the test battery itself still as being immature. Important key events such as nerve cell division, apoptosis as well as the migration of the nerve precursor cells can already be well described. The differentiation and investigation of the function of glial cells, an important immune cell type of the brain, the formation and electrical activity of neuronal networks, as well as the relationship between hormones, brain development and interfering chemicals, still require further research.

Endocrine-disrupting chemicals are suspected of interfering with neurodevelopment. Hormonal influence is multifaceted and disturbances of estrogen, androgen, progesterone, endocannabinoid signaling pathways may affect the developing brain at certain stages of development (12).

Support from regulatory authorities

There is an agreement among different regulatory authorities to support a standardized DNT testing strategy with a series of in vitro tests before in vivo testing. This means that animal testing as such is not completely excluded but largely reduced.

The current non-animal testing methods are not yet suitable for deriving health-related limit values and ultimately ending animal testing. Confidence in the non-animal testing methods is not yet sufficient; there are still uncertainties.

Currently, performance standards and a testing strategy are being established for the tiered test system, which will consist of a plurality of in vitro methods and an in vivo experiment with the zebrafish (9) (see scheme). But also many in silico methods are available or under development to investigate human-specific, molecular and cellular effects of chemicals. They should be integrated into predictive models for developmental neurotoxicity (13).

Simplified scheme for building a test battery based on the gradual development of the brain. The diagram shows which steps are already covered by tests (+) and for which procedures still development is needed (-). The development steps of the brain and nervous system are considered separately. The red lines indicate in which temporal phase of the development the process mainly takes place (but not exclusively). At this time the process is particularly vulnerable, which can lead to an interference of the brain development.
Scheme according to Hessel et al. (2018). Toxicology and Applied Pharmacology: 136-152.

Together with the European Food Safety Administration (EFSA), the Organisation for Economic Cooperation and Development (OECD), the American Environmental Protection Agency (EPA) and international scientists, the European validation authority EURL ECVAM is currently developing the strategy with a focus on a number of in vitro methods, preferably based on human-induced pluripotent stem cells. With these methods, the evaluation of chemicals concerning their effect on critical neurodevelopmental processes during individual stages of brain development should be possible. The available in vitro assays will be incorporated into Integrated Approaches to Testing and Assessment (IATA) together with in silico methods, non-mammalian models and existing animal and human data. This approach has also been adopted in an OECD project that is developing a guideline for in vitro methods for DNT testing.

The project is co-managed by EFSA, EURL ECVAM and US-EPA (3). EFSA plays an important role as the competent pesticides monitoring authority.

17 in vitro methods are currently in the validation process at EURL ECVAM (3). It is strongly expected that the use of currently available human-relevant in vitro models from induced pluripotent stem cells, in combination with the other proposed animal-free models, will provide approaches for the development of predictive models for DNT effects (3).

Read in the pdf below the interview with Prof. Dr. Ellen Fritsche (in German).

(1) Aschner, M., Ceccatelli, S., Daneshian, M., Fritsche, E., Hasiwa, N., Hartung, T., Hogberg, H. T., Leist, M., Li, A., Mundi, W. R., Padilla, S., Piersma, A. H., Bal-Price, A., Seiler, A., Westerink, R. H., Zimmer, B. & Lein, P. J. (2017). Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use. ALTEX 34 (1): 49-74.
(2) Lena Smirnova, Helena T. Hogberg, Marcel Leist & Thomas Hartung (2014). Food for Thought ...: Developmental Neurotoxicity – Challenges in the 21st Century and In Vitro Opportunities. ALTEX 31 (2): 129–156.
(3) EURL ECVAM (2018). Status report on the development, validation and regulatory acceptance of alternative methods and approaches 2018. Publications Office oft he European Union, DOI: 10.2760/818599 (online).
(4) Bal-Price, A., Crofton, K. M., Leist, M. Allen,S., Arand, M., Buetler, T., Delrue, N., Fitzgerald, R. E., Hartung, T., Heinonen, T., Hogberg, H., Hougaard Bennekou, S., Lichtensteiger, W., Oggier, D., Paparella, M., Axelstad, M., Piersma, A., Rached, E., Schilter, B., Schmuck, G., Stoppini, L., Tongiorgi, E., Tiramani, M., Monnet-Tschudi, F., Wilks, M. F., Ylikomi, T. & Fritsche, E. (2015). International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes. Arch Toxicol 89: 269–287. DOI 10.1007/s00204-015-1464-2
(6) Leist, M. (2016). How to link test system to the prediction of developmental neurotoxicity (DNT). EFSA-Workshopbeitrag OECD/EFSA Workshop on Developmental Neurotoxicity (DNT): the use of non-animal test methods for regulatory purposes. online 18 October 2016.
(7) Dach, K., Bendt, F., Huebenthal, U., Giersiefer, S., Lein, P. J., Heuer, H. & Fritsche, E. (2017) BDE-99 impairs differentiation of human and mouse NPCs into the oligodendroglial lineage by species specific modes of action. Scientific Reports 7: 44861. DOI: 10.1038/srep44861
(8) Moors, M. et al. (2009): Human Neurospheres as Three-Dimensional Cellular Systems for Developmental Neurotoxicity Testing. Environmental Health Perspectives 117 (7): 1131-1138.
(9) Fritsche, E., Crofton, K. M., Hernandez, A. F., Hougaard Bennekou, S., Leist, M., Bal-Price, A., Reaves, E., Wilks, M. F., Terron, A., Solecki, R., Sachana, M., Gourmelon, A. (2017). OECD/EFSA Workshop on Developmental Neurotoxicity (DNT): The Use of Non-Animal Test Methods for Regulatory Purposes. Meeting Report. ALTEX 34(2): 311-315. doi:10.14573/altex.1701171
(10) Barenys, M. & Fritsche, E. (2018). A Historical Perspective on the Use of Stem/Progenitor Cell-Based In Vitro Methods for Neurodevelopmental Toxicity Testing. Toxicological Sciences 165(1): 10–13.
(12) Fritsche, E., Barenys, M., Klose, J., Masjosthusmann, S., Nimtz, L., Schmuck, M., Wuttke, S. & Tigges, J. (2018). Current Availability of Stem Cell-Based In Vitro Methods for Developmental Neurotoxicity (DNT) Testing. Toxicological Sciences 165/1: 21-30. doi: 10.1093/toxsci/kfy178.
(13) Fritsche, E., Grandjean, P., Crofton, K. M., Aschner, M., Goldberge, A., Heinonen, T., Hessel, E. V. S., Hogberg, H. T., Hougaard Bennekou, S., Lein, P. J., Leist, M., Mundy, W. R., Paparella, M., A. H. Piersma, Sachana, M., Schmuck, G., Solecki, R., Terron, A., Monnet-Tschudi, F., Wilks, M. F., Witters, H., Zurich M.-G. & Bal-Price, A. (2018). Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes. Toxicology and Applied Pharmacology 354: 3–6.