SBIR-STTR Award

Developmental and reproductive toxicology (DART) studies are commonly conducted using a large number ofgenetically inbred rodent and rabbit strains to predict adverse effects of chemicals on human health. While invitro or ex vivo methods are used in early drug discovery fields, they face several challenges for DART studiesdue to lack of system-level biology, multi-organ physiology, and male/female reproductive organs. C. elegans isa genetically and molecularly attractive model organism that provides many advantages for high-throughputDART studies, including a high degree of conserved genes and biochemical pathways, a well-defined andcharacterized reproductive system, a rapid life and reproductive cycle, and low maintenance costs. While C.elegans based assays show a significant agreement with higher mammals (rat and rabbit), there is an urgentneed for more sensitive assays to improve both sensitivity and specificity for wide acceptance of C. elegans asan alternative animal model for DART testing. The goal of this proposal is to demonstrate the first C. elegansbased high-content DART assay to analyze multiple phenotypes that will reduce false negatives and captureinterindividual toxicology responses from a panel of genetically diverse strains. Newormics' vivoChip technologyis the only screening platform that can provide high-resolution imaging of C. elegans strains at high throughputs.It will allow us to assess accurately multiple endpoints, such as the feeding behavior, body size, vulvadevelopment, animal stress, and embryo health in non-anesthetized adults. With multi-parametric DART analysisusing genetic backgrounds, we can reduce the occurrence of seemingly disparate experimental results fromsingle-strain studies, enhance reproducibility, capture potential interindividual variabilities, and reduce false-negatives. In Aim 1, we will characterize the assay-quality of the proposed high-content DART assay (with 7phenotypes, including embryonic traits) by testing 6 known toxicants with 8 doses and in 10 replicates using theN2 wild-type strain. We will then screen 33 chemicals including 10 false negatives to demonstrate thepredictability of our multiple phenotype space and improved sensitivity of DART predictions using C. elegans. InAim 2, we will test a subset of chemicals against a panel of 12 divergent strains to capture the interindividualvariations in toxicological responses and improve interspecies predictability. We will calculate broad-senseheritability by measuring the variance in toxicology phenotypes and identify genetic contributions for suchvariances. In the Phase II, we plan to expand the application of our assay to test a larger library of chemicalsand incorporate a larger panel of genetically divergent strains to perform QTL analysis to pinpoint genes thatappear to be linked to DART effects. With the success of this proposal, Newormics will be in a unique positionto offer a comprehensive set of in vivo toxicology services to help reduce overall cost and number of animalsrequired for identifying toxicity of new chemicals.

Public Health Relevance Statement:


Project narrative:
Presently, toxicology studies rely on the use of genetically inbred laboratory animals to characterize hazard information for chemicals and cannot fully predict response to genetic variabilities in humans. To enhance the reliability of toxicology results to accurately reflect interindividual variations in humans while reducing the number of animals used in such studies, this project proposes an inexpensive and rapid screening assay utilizing an invertebrate small organism model, C. elegans, with a survey of hundreds of diverse genetic strains. This approach will facilitate an advanced toxicology testing of developmental and reproductive toxicology (DART) risks and thus reduce false-negatives and save millions of animals.

Project Terms:
Adult ; 21+ years old ; Adult Human ; adulthood ; Animal Testing Alternatives ; Animal Alternatives ; alternatives to animals in research ; research alternatives to animal use ; Animals ; Laboratory Animals ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Biology ; Body Size ; Confidence Intervals ; Pharmaceutical Preparations ; Drugs ; Medication ; Pharmaceutic Preparations ; drug/agent ; Embryo ; Embryonic ; Face ; faces ; facial ; Feeding behaviors ; Ingestive Behavior ; feeding-related behaviors ; nutrient intake activity ; Female ; Genes ; Goals ; Health ; Human ; Modern Man ; In Vitro ; Inbreeding ; Industry ; Invertebrates ; Invertebrata ; Life Cycle Stages ; Life Cycle ; life course ; Maintenance ; male ; Mammals ; Mammalia ; Methods ; Nematoda ; Nematodes ; roundworm ; Optics ; optical ; Pesticides ; Phenotype ; Physiology ; Oryctolagus cuniculus ; Domestic Rabbit ; Rabbits ; Rabbits Mammals ; Rattus ; Common Rat Strains ; Rat ; Rats Mammals ; Risk ; Rodent ; Rodentia ; Rodents Mammals ; Safety ; Sensitivity and Specificity ; Signal Transduction ; Cell Communication and Signaling ; Cell Signaling ; Intracellular Communication and Signaling ; Signal Transduction Systems ; Signaling ; biological signal transduction ; Stress ; Surveys ; Survey Instrument ; Technology ; Testing ; Time ; Toxicology ; Toxin ; Vulva ; vulvar ; Measures ; Caenorhabditis elegans ; C elegans ; C. elegans ; C.elegans ; base ; density ; improved ; Phase ; Variant ; Variation ; Biological ; Link ; Chemicals ; Evaluation ; insight ; Biochemical Pathway ; Metabolic Networks ; Genetic ; Heavy Metals ; Nature ; Life ; Principal Component Analyses ; Principal Component Analysis ; Dimensions ; System ; interest ; Services ; genetic strain ; Performance ; QTL ; Quantitative Trait Loci ; success ; chemical library ; small molecule libraries ; hazard ; Animal Models and Related Studies ; model of animal ; model organism ; Animal Model ; toxicant ; trait ; Toxicities ; Toxic effect ; novel ; Agreement ; Position ; Positioning Attribute ; reproductive ; Modeling ; response ; High Throughput Assay ; high throughput screening ; Adverse effects ; drug discovery ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; Genital system ; Reproductive system ; Diameter ; Caliber ; Length ; Dose ; Animal Testing ; Reproducibility ; Resolution ; in vivo ; Genomic Segment ; genomic region ; Whole Organism ; Molecular ; Development ; developmental ; Image ; imaging ; Pathway interactions ; pathway ; genome wide association study ; GWA study ; GWAS ; genome wide association ; genome wide association scan ; genome wide association studies ; genomewide association scan ; genomewide association studies ; genomewide association study ; whole genome association analysis ; whole genome association studies ; whole genome association study ; cost ; feeding ; next generation ; cost effective ; Population ; Heritability ; neurotoxicity ; neuron toxicity ; neuronal toxicity ; graphical user interface ; Graphical interface ; graphic user interface ; software user interface ; genome-wide analysis ; genome wide analysis ; genome wide studies ; genome-wide identification ; screening ; developmental toxicity ; predicting response ; prediction of response ; predictive response ; predictor of response ; response prediction ; inter-individual variation ; inter-individual variability ; interindividual variability ; interindividual variation ; reproductive organ ; high resolution imaging ; individual response ; individualized response ; Rapid screening ;

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. Modeling10 population variation in toxic responses at the necessary throughputs is not feasible using outbred in vivo11 mammalian models, and in vitro methods are unsuitable for assays requiring complete organisms such as12 developmental and reproductive toxicity (DART). Using its novel vivoChip device, vivoVerse proposes a NAM13 for DART testing based on the microscopic, soil-dwelling nematode, Caenorhabditis elegans. C. elegans has a14 simple culturing protocol, ability to produce 300 progenies per adult, conserved toxicology pathways with15 humans, intact germline with tractable in utero embryogenesis, is a non-sentient invertebrate with a 3-day life16 cycle that is not subject to animal welfare legislation, and has well-characterized panels of several hundred17 naturally occurring strains with diverse genetic backgrounds, making it a highly suitable small animal model for18 DART assays. The vivoChip is a microfluidic-based imaging platform uniquely facilitating high-throughput19 toxicology assays with C. elegans, using high-resolution imaging to quantify relevant phenotypic endpoints in20 ~1,000 animals per chip. In Aim 1, we will develop a new vivoChip specifically for DART testing that allows rapid21 immobilization of ~1,500 C. elegans of widely varying sizes. We will establish an AI/ML-assisted pipeline for22 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 and24 demonstrate strain and age-specific sensitivity for two reference chemicals. In Aim 3, we will compare DART25 assessments with our panel of 12 strains and two sensitized mutants, with published data for chemicals of26 significance to stakeholders, to demonstrate the value of our assay. With a more sensitive DART assay using27 high-content readouts of in utero effects from twelve genetically diverse backgrounds, we expect to improve the28 known safety prediction accuracies of C. elegans when compared to higher mammalian models. Armed with29 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.

Public Health Relevance Statement:
Product safety profiling, of which developmental and reproductive toxicology (DART) testing is an important component, is necessary to determine and continually ensure safe dosage or exposure levels of every chemical that comes into contact with humans. Toxicity testing is mostly done in genetically inbred mammals such as rats and rabbits, but public and regulatory pressure has led to directives eliminating the use of animals in safety testing where possible. In this proposal, we will develop a DART assay using genetically different wild strains of a microscopic soil worm, C. elegans, to provide a faster, cheaper, and more ethical alternative to testing on mammals, and investigate population-wide differences in responses to hazardous chemicals.

Project Terms:
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