Preclinical Testing of Flavors in E- vapor Products, Part 3: In - - PowerPoint PPT Presentation

Preclinical Testing of Flavors in E- vapor Products, Part 3:

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 1

In Vitro Cytotoxicity and Genotoxicity of Representative Flavor Mixtures

Utkarsh Doshi Tobacco Science Research Conference September 17, 2019

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)

2 Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final

Preclinical Testing of Flavors in E-vapor Products: Overview

3

E-Vapor Industry 5000+ Flavors 200-300 Part 1

Aldehydes Acetals Ketones Phenols Terpenes Acids Pyridines Esters

38 Flavors

Selection Process

In-vivo Exposure

Preclinical Application

In-vitro

PG VG Nicotine

38 Flavors Preparation, Characterization & Stability

Part 2

Test Formulation

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final

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)

▪ Test Articles:

• Carrier (PG:VG (80:20) + 2% Nicotine)
• Test Formulation (18.6% flavor)
• Test Formulation (18.6% flavor) + 2% Nicotine

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 5

Test Formulations (Flavor Mixtures) ± Nicotine 38 Representative Flavors

&

Carrier OECD Assays

• Ames Mutagenicity
• Micronucleus
• Neutral Red Uptake

Cytotoxicity

OECD: Organization for Economic Cooperation & Development

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).

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 6

Test Articles Mutagenicity Carrier (PG/VG/Nicotine) Negative Test Formulation Negative Test Formulation + Nicotine Negative

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).

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 7 20 40 60 80 100 120

0.00 0.10 0.20 0.30 0.40

% Viability (Relative Population Doubling)

Concentration of E-liquid (%v/v)

Cytotoxicity in TK6 cells Test Formulation + Nic

4h - S9 4h + S9 27h - S9 20 40 60 80 100 120

0.00 0.50 1.00 1.50 2.00

% Viability (Relative Population Doubling)

Concentration of E-liquid (%v/v)

Cytotoxicity in TK6 cells PG/VG/Nic

4h - S9 4h + S9 27h - S9 20 40 60 80 100 120

0.00 0.10 0.20 0.30 0.40

% Viability (Relative Population Doubling)

Concentration of E-liquid (%v/v)

Cytotoxicity in TK6 cells Test Formulation

4h - S9 4h + S9 27h - S9

Genotoxicity Assessment (cont)

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 8

20 40 60 80 100 0.0 0.2 0.4 0.6 0.8 1.0 1.2 DMSO 0.04 0.08 0.14

Cytotoxicity (%) % Micronuclei Concentration (%v/v))

In Vitro Micronucleus Assay (Test Formulation-Nicotine/4h+S9)

% Micronuclei Cytotoxicity

*

Upper limit of vehicle historical control

Criteria For Positive Genotoxicity Call

All 3 criteria have to be met:

• Statistical Significance (p≤0.05, Fisher exact)
• Outside of vehicle historical control
• Significant for trend

* p≤0.05, Fisher exact test

Test Articles Genotoxicity Carrier (PG/VG/Nicotine) Negative Test Formulation Equivocal Test Formulation + Nicotine Negative

Cytotoxicity Assessment

▪ Neutral Red Uptake Assay: OECD 129 Test Guidance (2010) ▪ Murine fibroblast cell line (BALB/c 3T3 cells, clone 31) ▪ 48 hr treatment

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 9 25 50 75 100 125 150 0.0001 0.001 0.01 0.1 1

Percent Viability (Relative to Solvent Control) Concentration of E-liquid (% (v/v))

Neutral Red Uptake Cytotoxicity Assay

Carrier (PG/VG/Nicotine) Test Formulation Test Formulation + Nicotine

50% Viability

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

• n their solubility and chemical reactivity (part 2) and tested

using NRU assay.

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 10

Based on Solubility, Chemical Reactivity Mixed

Pre-blends

(IA, IB, IC, II,III,IV)

Test Formulations 38 Representative Ingredients Neutral Red Uptake Cytotoxicity Assay

20 40 60 80 100 120 140 0.0001 0.001 0.01 0.1 1

Percent Viability (Relative to Vehicle Control) Concentration of E-liquid (% (v/v))

Neutral Red Uptake Cytotoxicity Assay

Pre-blend IA Pre-blend IB Pre-blend IC Pre-blend II Pre-blend III Pre-blend IV

Cytotoxicity Assessment of Pre-blends

▪ 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

10 20 30 40 50 60 Pre-blend IA Pre-blend IB Pre-blend IC Pre-blend II Pre-blend III Pre-blend IV

1/EC50 (%v/v)

Cytotoxicity Potency of Pre-blends

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

Utkarsh Doshi l Regulatory Affairs l Altria Client Services l TSRC Sept 17, 2019 l Final 13

Acknowledgements

Altria Client Services, Richmond, Virginia, USA Jingjie Zhang Ashutosh Kumar

• K. Monica Lee

Bioreliance (Millipore Sigma), Rockville, Maryland

USA