Occupational and environmental exposure assessments often involve poorly characterized chemicals, and traditional toxicity testing is too slow to keep pace. Tiered testing strategies, recommended to the DOD by the National Research Council as early as 2015, aim to speed up risk decisions by applying increasingly complex new approach methodologies (NAMs) only when earlier, simpler tiers can't provide sufficient confidence.

In this webinar, Andrew Keebaugh will present a case study applying CNS-3D Brain Organoids and PNS-3D organoids within this tiered framework to evaluate the neurotoxicity of a broad panel of chemicals, including organophosphates and carbamates. Using calcium flux, acetylcholinesterase inhibition, and viability assays across both acute and three-week exposures, the team generated benchmark doses and points of departure, then converted these to human equivalent doses (HEDs) using reverse dosimetry models.

Across most chemicals tested, HED values derived from the CNS-3D organoid data were as conservative as, or more conservative than, in vivo toxicity benchmarks, with results tracking known potency relationships between parent compounds and their metabolites. In a separate arm of the study, PNS-3D organoids successfully reproduced hallmark features of organophosphate-induced delayed neuropathy, including axonal degeneration and impaired nerve conduction, in response to two model chemicals.

Together, these findings support the use of CNS-3D and PNS-3D organoids in later tiers of NAM-based screening strategies, enabling rapid, human-relevant risk assessment for data-poor chemicals.

Key Takeaways
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• CNS-3D organoid testing generated human equivalent doses that matched or exceeded the conservatism of in vivo toxicity values for most chemicals tested, using acetylcholinesterase inhibition as the most sensitive endpoint
• Longer (three-week) exposures revealed cumulative effects and lower-concentration sensitivity compared to 24-hour exposures, better reflecting real-world exposure risk
• PNS-3D organoids reproduced characteristic features of organophosphate-induced delayed neuropathy, demonstrating utility for mechanism-specific neurotoxicity screening

Speaker
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Andrew Keebaugh, PhD, DABT, Research Scientist, Health and Performance Technologies Division, AV, Inc. contracted to the Air Force Research Laboratory

Andrew Keebaugh is a research scientist working for AV, Inc., on a contract with the 711^thHuman Performance Wing of the Air Force Research Laboratory (AFRL). He is currently a member of the Predictive Risk Team within the Force Health Protection section of AFRL, whose focus is assessing and adapting new approach methodologies for human toxicological evaluations of existing and anticipated chemical exposures in the military environment. He received a Bachelor of Science degree in Biomedical Engineering and a Doctoral degree in Environmental Toxicology, both from the University of California, Irvine, and is certified as a Diplomate of the American Board of Toxicology.