A significant shift in the safety assessment of new drug candidates and environmental chemicals that canrevolutionize the practice of regulatory toxicology is ongoing. This shift includes a reduction, or in some caseselimination, of traditional toxicity testing in animals with the implementation of higher-throughput testing schemesusing human cell systems. Knowledge regarding the potential of new chemicals, food additives, andpharmaceuticals to damage the human genome and cause mutations remains critical to public health. Mutationsare heritable changes in the cellular genome and are key events in the induction of cancer, birth defects, andneurological diseases. Screening chemicals for their potential to cause mutation in a human relevant assay offersan effective strategy for improving public health. The lack of a mutagenicity bioassay in human cells is a majordata gap in the genetic toxicology test battery used by regulatory agencies for hazard identification andquantitative risk assessments. Our novel methods, initially developed to identify genetic subclones withincancers, permits analytical assessment of mutagenicity and precise quantification of these very rare events (1in 100,000 - 1,000,000). These methods can be integrated with human cells as a genetic toxicology assay thatcan replace 50 yr. old clonal selection techniques to assess mutation. Although duplex sequencing is highlyinnovative and a "game changer", without integration into a well-defined human cell-based system and carefulvalidation studies, its application in regulatory Genetic Toxicology will be limited. The focus of this SBIR PhaseI application is to develop a New Approach Methodologies (NAM) combining human cells and duplex sequencingas an in vitro alternative to in vivo mutation assays. We will accomplish this by conducting "proof of principle"experiments using a well-established human cell line, as outlined in two specific aims. In Specific Aim 1, we willdetermine the time course for the induction of mutations at multiple loci using duplex sequencing for a prototypicmutagenic compound. In Specific Aim 2, we will determine the dose response for the induction of mutations atmultiple loci using duplex sequencing for two prototypic mutagenic compounds. Completion of this Phase I SBIRwill lead to development of a human cell-based mutation assay that can be used as a follow up to bacterialmutation assays and as a NAM to reduce reliance on current in vivo gene mutation assays in rodents. This datawill also support a Phase II application to validate this assay and adapt these methods to quantify mutation inhuman CD34+ cells and HepaRG⢠cells.
Public Health Relevance Statement: Project Narrative
The practice of regulatory toxicology is shifting towards a reduction, or in some cases elimination, of traditional
toxicity testing in animals with the implementation of higher-throughput testing schemes using human cell
systems. DNA damage and mutation have a key role in a broad spectrum of human disease. The purpose of
this SBIR application is to develop a mutational profiling assay in human cells to fill a data gap in the Genetic
Toxicology testing programs and as an alternative to animal studies.
Project Terms: Congenital Abnormality ; Birth Defects ; Congenital Anatomic Abnormality ; Congenital Anatomical Abnormality ; Congenital Defects ; Congenital Deformity ; Congenital Malformation ; Animals ; Ursidae Family ; Bears ; Ursidae ; bear ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Biological Sciences ; Biologic Sciences ; Bioscience ; Life Sciences ; Malignant Neoplasms ; Cancers ; Malignant Tumor ; malignancy ; neoplasm/cancer ; Cell Culture Techniques ; cell culture ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; Cells ; Cell Body ; Chromosome abnormality ; Aberrant Chromosome ; Chromosomal Aberrations ; Chromosomal Abnormalities ; Chromosomal Alterations ; Chromosome Aberrations ; Chromosome Alterations ; Chromosome Anomalies ; Cytogenetic Aberrations ; Cytogenetic Abnormalities ; chromosomal defect ; chromosome defect ; Client ; Cosmetics ; cosmetic product ; DNA Damage ; DNA Injury ; European Union ; European Community ; Food Additives ; Genes ; Genome ; Human Genome ; human whole genome ; Human ; Modern Man ; In Vitro ; Methods ; Methodology ; Mutagenicity Tests ; Genetic Toxicity Tests ; Genotoxicity Tests ; Mutagen Screening ; mutagen testing ; Mutation ; Genetic Alteration ; Genetic Change ; Genetic defect ; genome mutation ; nervous system disorder ; Nervous System Diseases ; Neurologic Disorders ; Neurological Disorders ; neurological disease ; Poison ; Toxic Chemical ; Toxic Substance ; toxic compound ; Public Health ; Reaction Time ; Response RT ; Response Time ; psychomotor reaction time ; Rodent ; Rodentia ; Rodents Mammals ; Safety ; Sales ; stem cells ; Progenitor Cells ; Family suidae ; Pigs ; Suidae ; Swine ; porcine ; suid ; Technology ; Testing ; Time ; Toxicology ; Measures ; Risk Assessment ; base ; improved ; Phase ; Chemicals ; Toxicity Tests ; Toxicity Testing ; Genetic ; Nature ; Knowledge ; programs ; Event ; Techniques ; System ; Cancer Induction ; carcinogenesis ; Gene Alteration ; Gene Mutation ; genotoxicity ; transgenic ; Transgenic Organisms ; novel ; Amendment ; validation studies ; Human Cell Line ; Single-Cell Gel Electrophoresis ; Comet Assay ; response ; Genetic Toxicology ; Toxicology Genetics ; Toxicogenetics ; Pharmaceutical Agent ; Pharmaceuticals ; Pharmacological Substance ; Pharmacologic Substance ; CD34 ; HPCA1 ; CD34 gene ; Dose ; Animal Testing ; DNA Alteration ; DNA mutation ; Genetic mutation ; Sequence Alteration ; genomic alteration ; DNA Sequence Alteration ; Data ; Mammalian Cell ; in vivo ; Ames Assay ; AMES mutagen test ; Ames Salmonella/microsome mutagenicity assay ; Ames Test ; Scheme ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Validation ; transmission process ; Transmission ; follow-up ; Active Follow-up ; active followup ; follow up ; followed up ; followup ; Development ; developmental ; Hazard Identification ; cost ; novel strategies ; new approaches ; novel approaches ; novel strategy ; innovation ; innovate ; innovative ; Heritability ; daughter cell ; human disease ; novel therapeutics ; new drug treatments ; new drugs ; new therapeutics ; new therapy ; next generation therapeutics ; novel drug treatments ; novel drugs ; novel therapy ; environmental chemical ; in vitro testing ; drug candidate ; next generation sequencing ; NGS Method ; NGS system ; next gen sequencing ; nextgen sequencing ; screening ; experimental study ; experiment ; experimental research ; safety assessment ; mutation assay ;
