Novel genotoxicity assay incorporating the toxicogenomics biomarker TGx-28.65 with a direct digital counting

Regulatory agencies including EPAFDAECHAand EMA require thorough assessment of the health effects of chemicals present in the environment and marketplaceInterpretation of positive genotoxicity findings using the current standard in vitro testing battery is a major challenge to both industry and regulatory agenciesThese tests have high sensitivitybut suffer from low specificityleading to high rates of irrelevant positive findingsi epositive results in vitro that are not relevant or reproduced in vivothis leads to unnecessary and costly followup as well as exclusion of potentially beneficial agents from further developmentWe have developed an in vitro transcriptomic biomarker based approach that provides pathway based mechanistic context to positive genotoxicity assay dataparticularly for in vitro chromosome damage assays that suffer from a high frequency of irrelevantfalse positiveresultsOur transcriptomic biomarkerTGxreadily distinguishes DNA damage inducing agents from other agents with much higher accuracyIn Phase I we have successfully integrated TGxwith nCountertechnologywhich is based on direct multiplexed measurement of gene expressionnCounter offers high levels of precisionlinearityreproducibilityand sensitivityandltcopy per cellThese unique characteristics of nCounter make it an ideal technology platform for our biomarker based genotoxicity screening assayWe now show that that our nCounter approach can be used directly with cell lysates and adapted to high throughput screeningHTSthis addresses another critical need since the standard in vitro genotoxicity panel is typically not amenable to HTSThe objective of this application is to develop and commercialize this high throughput genotoxicity screening system using our well validated TGxtoxicogenomic biomarkerWe will further assess our approach with priority environmental agents usingchemicals from the Toxcast collectionWhile many aneugens are not DNA damagingthey can also trigger chromosome aberrations and micronucleus formationso there is a need to also assess for this propertyWe have already observed transcriptomic responses for a limited number of aneugens and will now assess with a larger panel of aneugens with known modes of actionThe proposed approach will employ our standard human TKcell approach with or without metabolic activationand will be extended to HepaRGD spheroid culture to more closely model in vivo exposureprimary cellssuch as human hepatocyteswill also be employed using the conditionally reprogramed cell approach developed at GeorgetownOur proposed approach can be integrated into genetic safety hazard assessment as a follow up to positive chromosome damage findingsas well as a stand alone for in vitro genotoxicity assessmentConsidering the high cost of animal testing and now an E Uregulatory ban for some product applicationsdevelopment of accurate and cost effective in vitro approaches is criticalThis proposal should significantly benefit safety assessment by providing highly specific genotoxicity HTSand is poised to become a commercially successful screening service Project Narrative Describe the relevance of this research to public health inat mostthree sentencesGenotoxicity testing is an essential component of the safety assessment paradigm required by regulatory agencies world wide for environmental pollutantsindustrial chemicals and drug candidatesHoweverthe current genotoxicity testing battery features a high incidence of false positive findingsparticularly for in vitro chromosome damage assaysand ToxCast lacks sensitivity in identifying genotoxins with their current highthroughput screenThe Phaseof this project will enable a broad application of the first toxicogenomics assay for specific genotoxicityi eDNA damage assessmentand will address a critical need for high throughput mechanism based evaluation of the vast number of chemicals with potential for human exposures