Altria Group, Inc. · NYSE:MO
Altria — Technical Presentation (September 2019)
13 slides from Altria's technical presentation, Sep 17, 2019
Slide 1: Preclinical Testing of Flavors in E-vapor Products, Part 3
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
Slide 2: Overview of Session
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
Slide 3: Preclinical Testing of Flavors in E-vapor Products: Overview
Preclinical Testing of Flavors in E-vapor Products: Overview Selection Process Preclinical Preparation, Application Characterization & Ketones Stability E-Vapor Industry Terpenes Acids 5000+ Flavors PG1 Part In-vitro 38 Flavors Part 2 VG Acetals Phenols 200-300 Nicotine 38 Flavors Esters Pyridines A
Slide 4: Background
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
Slide 5: Background (cont)
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% f
Slide 6: Mutagenicity Assessment
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 metabo
Slide 7: Genotoxicity Assessment
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 PG/VG/Nic Test Formulatio
Slide 8: Genotoxicity Assessment (cont)
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 Cyto
Slide 9: Cytotoxicity Assessment
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 Form
Slide 10: Identifying Drivers of Cytotoxicity
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 as
Slide 11: Cytotoxicity Assessment of Pre-blends
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 P
Slide 12: Conclusions
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 com
Slide 13: Acknowledgements
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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