The Next-Generation Developmental and Reproductive Toxicology (DART) Assay using High-Content Analysis of Genetically Diverse C. elegans Populations

1 Modern toxicology assessment of chemicals is under pressure from both scientific and social sources. Traditional 2 study models using small numbers of highly inbred mammals fail to reflect the wide genetic, geographic, and 3 demographic variation underlying differing population-specific responses to environmental toxicants and drugs. 4 Secondly, long-standing public pressure to reduce the use of animals in safety testing has resulted in regulatory 5 directives to ban the use of animal studies in approvals of new chemical entities and existing chemical product 6 re-registrations by 2035. This pressure along with continual advances in life science technology has led to the 7 development of new approach methods (NAMs) including in vitro, ethical in vivo, and in silico methods. 8 Environmental justice, especially for vulnerable fence line communities at much greater risk of environmental 9 exposure to chemicals, highlights the need to include vulnerable populations in toxicology studies. Modeling 10 population variation in toxic responses at the necessary throughputs is not feasible using outbred in vivo 11 mammalian models, and in vitro methods are unsuitable for assays requiring complete organisms such as 12 developmental and reproductive toxicity (DART). Using its novel vivoChip device, vivoVerse proposes a NAM 13 for DART testing based on the microscopic, soil-dwelling nematode, Caenorhabditis elegans. C. elegans has a 14 simple culturing protocol, ability to produce 300 progenies per adult, conserved toxicology pathways with 15 humans, intact germline with tractable in utero embryogenesis, is a non-sentient invertebrate with a 3-day life 16 cycle that is not subject to animal welfare legislation, and has well-characterized panels of several hundred 17 naturally occurring strains with diverse genetic backgrounds, making it a highly suitable small animal model for 18 DART assays. The vivoChip is a microfluidic-based imaging platform uniquely facilitating high-throughput 19 toxicology assays with C. elegans, using high-resolution imaging to quantify relevant phenotypic endpoints in 20 ~1,000 animals per chip. In Aim 1, we will develop a new vivoChip specifically for DART testing that allows rapid 21 immobilization of ~1,500 C. elegans of widely varying sizes. We will establish an AI/ML-assisted pipeline for 22 automated analysis of high-resolution, on-chip images of in utero, body, and organ phenotypes relevant to DART. 23 In Aim 2, we will develop a GLP-qualified DART assay that surveys 12 genetically diverse strains and 24 demonstrate strain and age-specific sensitivity for two reference chemicals. In Aim 3, we will compare DART 25 assessments with our panel of 12 strains and two sensitized mutants, with published data for chemicals of 26 significance to stakeholders, to demonstrate the value of our assay. With a more sensitive DART assay using 27 high-content readouts of in utero effects from twelve genetically diverse backgrounds, we expect to improve the 28 known safety prediction accuracies of C. elegans when compared to higher mammalian models. Armed with 29 such data, we will present our case study to the regulatory agencies for formal risk analysis, and in due course, 30 full acceptance of C. elegans as an alternative animal model for DART, making an impact on many industries.