N-nitrosonornicotine reduction in dark tobacco varieties and ......Ramsey S. Lewis, Steven W. Bowen, Matthew R. Keogh, Ralph E. Dewey Phytochemistry71 (2010) 1988 -1998 e4e4, e5e5,

TSRC2017

N-nitrosonornicotine reduction in dark tobacco varieties and smokeless product prototypes.M. Lusso; A. Adams; B. Lewis; T. Poyner; K. Lion; L. DeLoach; T. Danielson; J. Franke; U. Warek and J. Strickland

Altria Client Services l Biotechnology l 09/18/2017 l Draft l Confidential 1

Altria Client Services (ALCS) authors should use Altria Client Services LLC., Research, Development & Reg Affairs, 601 East Jackson Street, Richmond VA 23219.

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Overview

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Background- Gene discovery and proof of concept

Breeding of Stable Reduced Converter varieties

NNN levels in LC and SRC dark tobacco varieties

Expected impact of SRC varieties on NNN levels of DFC leaf crop

NNN Levels in Smokeless Tobacco Prototypes with and without ZYVERTTM technology

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Background

3Altria Client Services l Biotechnology l 09/18/2017 l Draft l Confidential

Three nicotine demethylase genes mediate nornicotinebiosynthesis in Nicotiana tabacum L.: Functional characterization of the CYP82E10 gene

Ramsey S. Lewis, Steven W. Bowen, Matthew R. Keogh, Ralph E. Dewey

Phytochemistry 71 (2010) 1988 - 1998

e4e4, e5e5, e10e10 = ZYVERTTM Technology

SRC Varieties = with ZYVERTTM Technology

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ALCS Breeding of SRC Varieties


ALCS Proprietary Dark Tobacco VarietiesPD 7305 LC – PD 7305 SRCPD 7309 LC – PD 7309 SRCPD 7312 LC – PD 7312 SRCPD 7318 LC – PD 7318 SRCPD 7319 LC – PD 7319 SRC

All five dark tobacco SRC varieties are eligible for seed certification

Experimental Burley and Flue-cured SRC varieties were also developed

SRC Varieties withZYVERTTM Technology

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NNN Levels in LC and SRC Tobacco Varieties Grown in Research Plots


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NN

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LC SRC

Dark Tobacco Varieties

3 Seasons & Multiple Locations

74% NNNReduction

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NNN Distribution in DFC Crop 05-13


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NNN Range - BLOQ to 78 ppm

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Expected Impact of SRC Varieties on NNN Levels of DFC Leaf Crop


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NNN Distribution in DFC Crop 05-13

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3.8ppm74%

Reduction

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SRC Dark Tobacco Varieties Summary

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SRC dark tobacco varieties had NNN reduction of ̴74% compared to LC varieties when grown in research plots.

Between 2005 and 2013, depending on the year, 9% to 32% of the DFC tobacco crop had NNN levels below 1 ppm.

Between 2005 and 2013, depending on the year, 68% to 91% of the DFC tobacco crop had NNN levels below 3.8 ppm.

With the implementation of SRC varieties, depending on the year, 68% to 91% of the DFC tobacco crop could be below 1 ppm, with 9% to 32% in any given year being unusable.


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Smokeless Product PrototypesControl

2014 regular production of LC varieties

- Dark Fire-Cured- Dark Air-Cured- Burley Stems

ZYVERTTM2014 on-farm production SRC varieties with ZYVERTTM Technology- Dark Fire-Cured- Dark Air-Cured- Burley Stems

Altria Client Services l Biotechnology l 09/18/2017 l Draft l Confidential 9

Aged for 2 yearsBlended (dry flour)

Fermented (3 cures each)Finished

Similar to CRP2 – Moist Snuff Reference ProductSimilar to CRP2 – Moist Snuff Reference Product

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NNN Levels in Blend Components

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DFC HHD DAC HHD Burley Stem

NNN

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ControlZyvert

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NNN Levels in DF, OOC & Finished Product Prototypes

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ControlZyvert

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Smokeless Tobacco Prototype Summary

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A NNN reduction of 68% to 78% was observed in the individual blend components, Dry Flour and Finished products when SRC tobacco containing the ZYVERTTM technology was used to make smokeless tobacco prototypes.

NNN levels in the finish product prototypes were comparable to the raw material.


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Questions

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Documents

N-nitrosonornicotine reduction in dark tobacco varieties and ......Ramsey S. Lewis, Steven W. Bowen, Matthew R. Keogh, Ralph E. Dewey Phytochemistry71 (2010) 1988 -1998 e4e4, e5e5,