TSNA Task Force for Air-cured Tobacco
(Report to the Scientific Commission May 2005)

Coordinator:  Gary Palmer, University of Kentucky , USA

Liaison:  Mark Nielsen, GenApps , USA

Objectives

Objective 1: Develop a standardized nornicotine screening protocol so that baseline levels of nornicotine are comparable in tobacco seed varieties used by investigators.

 Objective 2: Develop guidelines or suggested critical survey questions for farmer practice surveys so that results can be compared within and between tobacco origins.

 Objective 3: Develop a collaborative study to investigate standard deviation for moisture content within farmer marketing packages among origins.

Objective 4: Develop a collaborative study, which uses hobo loggers or a suitable substitute to collect curing conditions and possible impact of TSNA levels for tobaccos of diverse origins and curing environments. Attempt to standardize placement of equipment and sample protocols.

The TSNA Task Force has determined that no action is required on Objective 1.  Three cured powder tobacco samples were supplied to six laboratories for nicotine and nornicotine analysis.  Those collaborating on the project included Lowell Bush, Coordinator ( University of Kentucky ), Christian de Roton (Altadis Institut du Tabac), Hitoshi Saito (Japan Tobacco International), Cliff Bennett (US Smokeless Tobacco), Cui Ming wu (Profigen), and Harold Burton ( University of Kentucky ).  Each laboratory used their normal routine protocol for analysis.  Dr. Bush tabulated the results and checked deviation and variability across all laboratories.  He reported that the standard deviation and coefficient of variation were low enough among laboratories that developing a standard screening protocol is not necessary.  Other collaborators concurred with these findings and the committee agreed not to recommend that the Scientific Committee adopt a standardized nornicotine screening protocol.

The TSNA Task Force asked that the Scientific Commission of CORESTA review a grower survey developed by the Task Force with the leadership of George Scott, Universal Leaf Tobacco Company, USA .  If accepted this will complete Objective 2.

Objective 3 was reassessed in Japan and the Task Force proposed that this objective be dropped.  Although moisture content is known to be a contributing factor in the development of TSNA, deviation within a marketing package is though to be of less importance than average moisture content. (Attachment 1)

 Christian de Roton, Altadis – Institut du Tabac , France coordinated a second year of research on Objective 4.  The protocol for the 2004 collaborative study was as follows:  Using a low nornicotine line of Burley, ms KY 14 x L8, or Dark Air Cured tobacco; conduct studies in 5-10 different curing barns;  place hobo-loggers at representative points in the barn, at the upper-middle leaf level;  place another hobo-logger outside the barn or, failing that, take the data from the nearest weather station;  record dates of harvest, end of yellowing, end of browning, and end of cure; observe the appearance of molds (white, grey, etc.), darkening, fermentation, and other curing incidents at taking down; take a representative sample of 2-3 upper-middle leaves from 15-20 plants closely surrounding the hobo-logger; freeze dry or use force air at 35oC, grind, separate midrib and lamina and analyze for TSNA and, if possible, NO₂ and nornicotine; obtain another and larger sample of upper-middle leaves surrounding the hobo-logger to evaluate the grade and quality.

            In 2004 there were four participating collaborators: Francisco Palacio (Colombiana de Tabaco S.A., Columbia) with three sites; Marlene Adams (R.J. Reynolds Tobacco, USA) with one site; Hitoshi Saïto (JTI, Japan) with two sites and Christian de Roton and Bruno Fontaine (Altadis/ITB and ANITTA, France) with ten sites.
            Christian recommended a third year of study, but emphasized the need for more study sites due to a high location effect seen in previous studies.  Christian announced that he will retire on Dec. 15 of 2004; however, he agreed to develop changes in protocol for studies to be conducted in 2005 prior to his departure with assistance from Bruno Fontaine, Anitta , France .  Due to the close association between Christian and Bruno during the course of pursing Objective 4, Bruno was asked to become coordinator and chair of this objective upon the retirement of Christian.  A completion date of October 2006 is projected for Objective 4 with an appropriate report presented at the 2006 CORESTA Congress, in Paris , France .

Proposed Objectives

 Two new protocols were proposed at the CORESTA Congress in Kyoto .

 Objective 5:  Resolve sample handling of post cure tobacco for TSNA determination.

Marlene Adams, R.J. Reynolds Tobacco, was appointed chair and coordinator.  Her charge was to make contact with Elui Krugel, Coordinator of the Sampling Task Force, to prevent duplicate effort, to make data available to the group regarding sample handling, and to determine with the aid of other interested members of the TSNA task force if sampling guidelines should be developed.  Elui reported that the Sampling Task Force did not consider a TSNA protocol.  Marlene submitted a sample protocol and suggestions for modification was solicited from TSNA Task Force members twice before a final proposal was developed for consideration by the Scientific Commission. (Attachment 2)   

 Objective 6:  Review the issues of post cure tobacco storage and ventilation parameters.

 It was recommended that Stewart Livesay be asked to chair and coordinator this objective.  No action has been taken on this objective.

 Members of the TSNA Task Force respectfully requests that the Scientific Commission consider changing the TSNA Task Force to a Study Group, since new areas of pursuit may continue to evolve.   We further request approval of the Grower Survey, TSNA Sampling Protocol which completes Objective 5, and addition of Objective 6. 

                          CORESTA TSNA Grower Survey                                            (Attachment 1 )                                                                 Compiled by TSNA Task Force

                          George Scott, Chair, Universal Leaf , USA

 Date: _________________

 I.  Grower Information

II. General Crop Information

III. Curing and Barn Information  

                                      TSNA Sampling Protocol            (Attachment 2 )

 ·         For all samples regardless of type (stem, lamina, green, or cured) the most critical features to insure accurate data are:

o       Moisture content

o       Temperature (length of exposure time can interact to increase TSNA levels)

o       Sample size

·         Stems should always be separated from lamina prior to analysis.

·         For a representative sample, take a leaf from multiple plants at the 4 to 6 leaf position from the top.

·         For sampling cured leaf, ensure the sample is as dry as possible, < 18%; however, excess heat should be avoided.  (In processing this may be done prior to re-drying.)

o       Freeze-drying is the preferred method, but may not be available to all

o       If freeze-drying is not available, dry with low humidity not heat

o       Air dry in low humidity environments but ambient temperatures should not exceed 35 C

o       35 C should be the absolute maximum temperature, but should include forced air in humid areas or, if oven dried, as temperature approaches 35 C

o       Even samples with low moisture may have significant increases in TSNA if temperatures exceed 35 C

o       Do not over fill dryer, allow space for air flow

 ·         A ground sample is best, since it is more homogeneous and easily mixed, and it also cuts down on turn-around time for sample results.

 ·         At least 100 grams is required for analysis. Ten to fifteen leaves should provide approximately 100 grams depending on leaf size of the tobacco being sampled. Replicate analysis is standard practice; this mass provides enough material for repeat analysis if necessary.

 ·         Never store samples for long periods of time, especially at high temperatures.

 ·         For sampling a re-dried lamina: a composite of a grade taken during moisture sampling should be sufficient for TSNA analysis. TSNA can be variable from container to container.

 ·         Ship samples in breathable containers such as brown paper bags - never use plastic containers to store or ship samples destined for TSNA analysis.