Preclinical Testing of Flavors in E- vapor Products, Part 3: In Vitro Cytotoxicity and Genotoxicity of Representative Flavor Mixtures Utkarsh Doshi Tobacco Science Research Conference September 17, 2019 Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 1
Overview of Session ▪ Part 1: Selection of Representative Flavor Mixtures Using a Structural Grouping Approach (Kim Ehman) ▪ Part 2: Preparation and Stability Characterization of Representative Flavor Mixtures (Cameron Smith) ▪ Part 3: In Vitro Cytotoxicity and Genotoxicity of Representative Flavor Mixtures (Utkarsh Doshi) ▪ Part 4: Flavor Transfer from the Liquid to the Aerosol for Inhalation Exposure (Jingjie Zhang) Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 2
Preclinical Testing of Flavors in E-vapor Products: Overview Selection Process Preclinical Preparation, Application Characterization & Ketones Stability E-Vapor Industry Terpenes Acids 5000+ Flavors PG Part 1 In-vitro 38 Flavors Part 2 VG Acetals Phenols 200-300 Nicotine 38 Flavors Esters Pyridines Aldehydes In-vivo Test Formulation Exposure Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 3
Background ▪ Flavor compounds for oral consumption fall within “generally recognized as safe (GRAS)” category ▪ Limited safety data exists for inhalation route of exposure ▪ Many flavor compounds in e-vapor products are commonly used as mixtures which makes their hazard characterization resource and time-demanding ▪ Alternative approach (part 1): - Evaluate structural similarities to develop representative flavor mixtures for preclinical toxicity testing ▪ Representative flavor mixtures were tested for in vitro cytotoxicity and genotoxicity Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 4
Background (cont) 38 Representative Test Formulations Flavors (Flavor Mixtures) ± Nicotine & Carrier OECD Assays • Ames Mutagenicity • Micronucleus • Neutral Red Uptake Cytotoxicity ▪ Test Articles: - Carrier (PG:VG (80:20) + 2% Nicotine) - Test Formulation (18.6% flavor) - Test Formulation (18.6% flavor) + 2% Nicotine OECD: Organization for Economic Cooperation & Development Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 5
Mutagenicity Assessment ▪ Ames Assay: OECD 471 Test Guidance (1997). ▪ Detects compounds ability to cause mutations (point or frame-shift). ▪ Carrier & test formulations ± nicotine were tested in 5 strains of Salmonella typhimurium TA98, TA100, TA102, TA1535 & TA1537 in absence and presence of metabolic activation (Aroclor induced rat liver S9). Test Articles Mutagenicity Carrier (PG/VG/Nicotine) Negative Test Formulation Negative Test Formulation + Nicotine Negative Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 6
Genotoxicity Assessment ▪ Mammalian in vitro micronucleus assay: OECD 487 Test Guidance (2016). ▪ TK6, human lymphoblast cell line. ▪ Three treatment conditions: Short term ( ± S9), long term (-S9). Cytotoxicity in TK6 cells Cytotoxicity in TK6 cells Cytotoxicity in TK6 cells Test Formulation + Nic Test Formulation PG/VG/Nic 120 120 120 (Relative Population Doubling) (Relative Population Doubling) (Relative Population Doubling) 4h - S9 4h - S9 100 100 100 4h + S9 4h + S9 80 % Viability 80 80 27h - S9 27h - S9 % Viability % Viability 60 60 60 4h - S9 40 40 40 4h + S9 27h - S9 20 20 20 0 0 0 0.00 0.50 1.00 1.50 2.00 0.00 0.10 0.20 0.30 0.40 0.00 0.10 0.20 0.30 0.40 Concentration of E-liquid (%v/v) Concentration of E-liquid (%v/v) Concentration of E-liquid (%v/v) 7 Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final
Genotoxicity Assessment (cont) Test Articles Genotoxicity Carrier (PG/VG/Nicotine) Negative Test Formulation Equivocal Test Formulation + Nicotine Negative In Vitro Micronucleus Assay % Micronuclei (Test Formulation-Nicotine/4h+S9) Cytotoxicity 1.2 100 * 1.0 Upper limit of vehicle historical 80 Cytotoxicity (%) % Micronuclei control 0.8 60 0.6 40 0.4 20 0.2 0.0 0 DMSO 0.04 0.08 0.14 Concentration (%v/v)) Criteria For Positive Genotoxicity Call * p≤0.05, Fisher exact test All 3 criteria have to be met: • Statistical Significance (p≤0.05, Fisher exact) • Outside of vehicle historical control • Significant for trend Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 8
Cytotoxicity Assessment ▪ Neutral Red Uptake Assay: OECD 129 Test Guidance (2010) ▪ Murine fibroblast cell line (BALB/c 3T3 cells, clone 31) ▪ 48 hr treatment Neutral Red Uptake Cytotoxicity Assay 150 (Relative to Solvent Control) 125 Percent Viability 100 75 50 Carrier (PG/VG/Nicotine) 25 Test Formulation Test Formulation + Nicotine 0 0.0001 0.001 0.01 0.1 1 Concentration of E-liquid (% (v/v)) 50% Viability Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 9
Identifying Drivers of Cytotoxicity ▪ Cytotoxicity was common trend observed in all 3 assays. ▪ To understand the drivers of cytotoxicity, 38 flavor ingredients were divided into sub-group mixtures (called pre-blends) based on their solubility and chemical reactivity (part 2) and tested using NRU assay. Based on Solubility, Chemical Mixed Reactivity Pre-blends 38 Test (IA, IB, IC, II,III,IV) Representative Formulations Ingredients Neutral Red Uptake Cytotoxicity Assay Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 10
Cytotoxicity Assessment of Pre-blends Neutral Red Uptake Cytotoxicity Assay Cytotoxicity Potency of Pre-blends 60 140 (Relative to Vehicle Control) 120 50 Percent Viability 100 1/EC50 (%v/v) 40 80 60 30 40 20 20 0 10 0.0001 0.001 0.01 0.1 1 Concentration of E-liquid (% (v/v)) 0 Pre-blend Pre-blend Pre-blend Pre-blend Pre-blend Pre-blend Pre-blend IA Pre-blend IB Pre-blend IC IA IB IC II III IV Pre-blend II Pre-blend III Pre-blend IV ▪ Pre-blends IA, IB and II were the major contributors to toxicity. ▪ Examples of flavors reported to be in vitro cytotoxic/irritant: - IA (isopulegol) - II (furaneol, ethyl maltol) Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 11
Conclusions ▪ Representative flavor mixtures did not show mutagenicity and genotoxicity in the in vitro assays ▪ Representative flavor mixtures showed cytotoxicity in the in vitro assay, however the cytotoxicity was driven by few selected flavors or flavor groups ▪ Use of read across approach in combination with systematic toxicity evaluation (deconstructing mixtures into subsets of flavors) can reduce the list of compounds for thorough toxicological evaluation Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 12
Acknowledgements Altria Client Services, Richmond, Virginia, USA Jingjie Zhang Ashutosh Kumar K. Monica Lee Bioreliance (Millipore Sigma), Rockville, Maryland USA Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 13
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