Innovative Research for a Sustainable Future www.epa.gov/research Denise MacMillan l macmillan.denise@epa.gov l 919-397-8038 Introduction 5-Day In Vivo Repeat Exposure Study Results Conclusions Non-targeted Analysis for Identification of PFAS Biotransformation Products in Rat Plasma D. K. MacMillan,1 A. Renyer,2 M. DeVito,1 M. F. Hughes,1 and L. C. Wehmas1 1: U.S. Environmental Protection Agency, Office of Research and Development, Center for Computational Toxicology and Exposure, Durham, NC 2: Oak Ridge Institute for Science and Education, Durham, NC Per- and polyfluorinated substances (PFAS) are dispersed throughout the environment due to widespread commercial use, which leads to exposure and the potential for adverse health effects. Toxicological data are available for only a few of the nearly 10,000 currently known PFAS. The USEPA is using short term PFAS exposure studies to: o generate interim risk assessment data for priority chemicals concern to states and EPA Regions, o explore possible biotransformations in mammals, o and further understanding of the biological responses to exposure. Biotransformations of PFAS telomers have been observed in microbes and fish, but few other PFAS or species have been studied. Here we present results of non-targeted analysis (NTA) to identify potential biotransformation products (PBTPs) in plasma from rats exposed over five days to perfluoro(2,5,8-trimethyl-3,6,9-trioxadodecanoic) acid (PF-TODoA), a perfluoroether carboxylic acid oligomer of GenX, and begin to investigate biological response. PF-TODoA dose formulation o Sprague-Dawley, male/female o 8-10 weeks o n=4/sex/dose-level Day 1 2 3 4 5 6 * * * * * X * = Oral gavage. X = Rats euthanized. o Control + 8 Dose-levels o Vehicle = Water o Range: 0.3 – 335.2 mg/kg/day ~1/2 log10 spacing o Plasma (25 µL) prepared by protein precipitation with acidified acetonitrile • Chromatography: Phenomenex Kinetex XB-C18 (100 x 2.1 mm) • A: 95:5 H2O:MeOH and B: 95:5 MeOH:H2O, both containing 4 mM ammonium formate o NTA acquired with Sciex X500R QTOF • Negative ion electrospray ionization (-ESI) • Information Dependent Acquisition (IDA) and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) scanning Data processing, library searching (NIST 17, Sciex Fluorochemical, and in-house libraries), formula finding, peak picking and alignment, normalization, and statistical analysis were performed using Sciex OS 2.1.0 and MarkerView 1.3.1. Features were filtered by log fold change, p < 0.05, negative mass defect, and abundance. A screening list of PBTPs was generated using Biotransformer 3.0.1 MetFrag2 was used to generate in-silico spectra of potential candidates. Statistical Analysis NTA data from plasma of male and female rats exposed to 17 mg/kg/day PF-TODoA were normalized and analyzed by supervised principal component analysis (PCA-DA). The scores plot showed separation of the data for exposed samples from study controls. Data from male and female rats clustered apart from controls and distinct from each other. Differences in normalized responses of selected significant ions between groups are Control Male Female 0 -500 D1 Score -1000 500 D2 Score -600 0 600 PF-TODoA CASRN: 65294-16-8 DTXSID70191136 Data were screened for predicted PBTPs. Exact masses observed in MS scans suggested several predicted PBTPs were present, but only the ion of m/z 516.9542 gave an MS/MS spectrum. The fragment ions observed at m/z 350.9 and m/z 184.9 are characteristic of PF-TODoA and consistent with sequential cleavages of C3F6O. An in- silico spectrum generated for PubChem CID 13244841 (ion structure shown) with MetFrag matched all experimental fragment ions. The data suggest O-dealkylation of PF-TODoA to yield an alcohol with the formula C9HF19O3. F F F F F F F F F F F F F F F F F F F F F F F O O O O H O MS/MS of m/z 516.9542 F F F F F F F O – Predicted PBTPs The MS/MS spectrum of the in- source fragment of PF-TODoA, also known as HPFO-TeA, shows character