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

1.现代化学品毒理学评估面临来自科学和社会的压力。传统 2使用少量高度近亲繁殖的哺乳动物的研究模型未能反映广泛的遗传、地理和生物多样性。 3.人口统计学差异是不同人群对环境毒物和药物的反应的基础。 第二，长期以来，公众要求减少在安全测试中使用动物的压力导致了监管机构的监管。 5项指令禁止在批准新化学实体和现有化学产品时使用动物研究 到2035年将有6个重新注册。这种压力沿着生命科学技术的不断进步， 7开发新方法（NAM），包括体外、伦理体内和计算机模拟方法。 8环境正义，特别是对环境风险大得多的脆弱围栏线社区 9暴露于化学品，强调需要在毒理学研究中纳入弱势群体。建模 10在必要的生产量下，使用远交系在体内进行毒性反应的群体差异是不可行的 11哺乳动物模型，体外方法不适用于需要完整生物体的测定， 发育和生殖毒性（DART）。vivoVerse使用其新颖的vivoChip器件，提出了一种NAM 13用于DART测试，基于显微镜下的土壤线虫，秀丽隐杆线虫。C. elegans有一个 14个简单的培养方案，每个成虫产生300个后代的能力，保守的毒理学途径， 15人类，完整的生殖系，子宫内胚胎发育容易，是一种无知觉的无脊椎动物，只有3天的生命 16个周期，不受动物福利立法的约束，并拥有数百个具有良好特征的小组 17种具有不同遗传背景的天然菌株，使其成为非常适合的小动物模型， 18个DART测定。vivoChip是一种基于微流体的成像平台， 19项毒理学试验表明，使用高分辨率成像来量化相关的表型终点， 每个芯片20 ~ 1，000只动物。在目标1中，我们将开发一种专门用于DART测试的新vivoChip， 21 ~ 1500 ℃的固定化。大小各异的秀丽线虫。我们将建立一个AI/ML辅助管道， 22对与DART相关的子宫内、身体和器官表型的高分辨率芯片图像进行自动分析。 在目标2中，我们将开发一种GLP合格的DART测定法，该测定法调查12种遗传多样性菌株， 24证明了两种参考化学品的菌株和年龄特异性敏感性。在目标3中，我们将比较DART 25评估与我们的小组的12个菌株和两个致敏突变体，与公布的数据的化学品， 26对利益相关者的意义，以证明我们的分析的价值。使用更灵敏的DART检测， 27个高内容的读数在子宫内的影响，从12个遗传不同的背景，我们希望提高 28个已知的C.与高等哺乳动物模型相比。手持 29这样的数据，我们将把我们的案例研究提交给监管机构进行正式的风险分析，并在适当的时候， 30完全接受C。作为DART的替代动物模型，对许多行业产生了影响。

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.