Technical Report. Collaborative Study 2017 on TSNA, ph, and Moisture (Oven Volatiles)

Transcription

1 Tobacco and Tobacco Products Analytes Sub-Group Technical Report Collaborative Study 2017 on TSNA, ph, and Moisture (Oven Volatiles) July 2017 Study Project Leaders: David Ericson, Ph.D., Swedish Match, Sweden Johan Lindholm, Ph.D., Swedish Match, Sweden Anthony Brown, Altria Client Services LLC, U.S.A. Author: Anthony Brown, Altria Client Services LLC, U.S.A. Co-Author and Statistical Analysis: Michael Morton, Ph.D., Altria Client Services LLC, U.S.A.

2 Table of Contents 1. SUMMARY INTRODUCTION Objective ORGANISATION Participants Protocol DATA RAW DATA STATISTICAL ANALYSIS Exclusion of Outliers Calculation of Repeatability (r) and Reproducibility (R) DATA INTERPRETATIONS RECOMMENDATIONS APPENDICES... 10

3 1. Summary At the October 2016 CORESTA Smokeless Tobacco Sub-Group (STS) meeting (now named Tobacco and Tobacco Products Analytes Sub-Group, TTPA) held in Berlin, the Sub-Group initiated an interlaboratory collaborative study for the determination of TSNAs, ph, and moisture (oven volatiles or OV) in ground tobacco, cigarette filler, and cigar filler. The intent of this study was to update the scopes of the applicable CORESTA Recommended Methods (CRMs) to include these additional matrices as the CRMs currently only include smokeless tobacco products. CRM No. 72 (Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products by Liquid Chromatography - Tandem Mass Spectrometry) was submitted to ISO/TC 126 for development into an ISO standard in June This work item was approved as a Committee Draft on September 9, 2016 (ISO/CD 21766). The results of this study will be used to update the scope of ISO/CD to include these additional matrices. Tabulated data are presented along with repeatability (r) and reproducibility (R). The results of the study demonstrate that the CRMs for TSNAs, ph, and moisture are suitable for the analysis of ground tobacco, cigarette filler and cigar filler. The TTPA recommends that the three CRMs and ISO draft be updated to include the additional tobacco matrices. 2. Introduction In 2009, STS coordinated a collaborative study involving 23 laboratories to assess the repeatability and reproducibility of a selection of methods used to determine TSNAs, ph, and moisture in nine types of smokeless tobacco products 1. The STS recommended that the methods specified in the collaborative study for ph and TSNAs be the basis for two new CRMs and CRM No. 69 (Determination of ph in Smokeless Tobacco Products) and CRM No. 72 (Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products by LC-MS/MS) were published. In the 2009 study report, the STS also recommended that a follow-up collaborative study be conducted for moisture where standard conditions are specified. In 2010, the STS conducted the follow-up study 2. This study included the four CORESTA Reference Products (CRPs) and five commercial smokeless tobacco products. Thirteen laboratories provided data using a modified version of AOAC Official Method , Loss on Drying (Moisture) in Tobacco, Gravimetric Method. The results of this collaborative study were the basis for CRM No. 76 (Determination of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products). The CRMs for TSNAs, ph, and OV have been used in robust annual collaborative studies and the results of which are published at CORESTA.org. In recent years, the prioritization of CRM development has been driven by tobacco product regulation. Though the collaborative studies supporting the CRMs were focused on smokeless tobacco products, it was anticipated that the methods would also be appropriate for other tobacco products. At the STS meeting held in Berlin, Germany (October 2016), it was decided to conduct a collaborative study to support the expansion of the scope of the CRMs for TSNAs, ph, and moisture beyond smokeless tobacco to include ground tobacco, cigarette filler and cigar filler. 1 STS Technical Report: 2009 Collaborative Study (Smokeless Tobacco r&r Study), July updated Jan STS Technical Report: Analysis of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products, March TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

4 2.1 Objective This study was conducted to support scope expansion of the TSNAs, ph, and moisture CRMs beyond just smokeless tobacco to include ground tobacco, cigarette filler and cigar filler and to provide an assessment of inter-laboratory variability. The participating laboratories were to provide analytical results for the four TSNAs (N-nitrosonornicotine (NNN), N- nitrosoanatabine (NAT), N-nitrosoanabasine (NAB) 4-(N-nitrosomethylamino)-1-(3-pyridyl)- 1-butanone (NNK), ph, and moisture (oven volatiles). This work was conducted using the applicable CRMs referenced in Section 3. Data were collected from the participating laboratories and statistically evaluated in basic conformance with the recommendations of ISO :1994 and ISO/TR 22971: Organisation 3.1 Participants A list of the participating laboratories is provided in Table 1. Not all laboratories provided data for all analyses nor did all laboratories analyze all samples. The laboratories are listed in alphabetical order. Letter codes were assigned to each laboratory and do not correspond to the order shown in the table below. Table 1: List of Participating Laboratories in the 2017 WG4 Study 2017 WG4 Participants Altria Client Services LLC, United States American Snuff Company, LLC, United States British American Tobacco, Germany British American Tobacco Souza Cruz, Brazil British American Tobacco, Sweden China National Quality Supervision and Test Center, China Enthalpy Analytical, Inc., United States Essentra Scientific Services, United Kingdom Global Laboratory Services, United States Imperial Tobacco, Reemtsma, Germany ITC Limited, India ITG Brands, United States Japan Tobacco Inc., Leaf Tobacco Research Center, Japan KT&G Research Institute, South Korea Labstat International ULC, Canada Lauterbach & Associates, LLC, United States C.I.T. Montepaz S.A., Uruguay Philip Morris International, Switzerland R.J. Reynolds Tobacco Company, United States Shanghai Tobacco Group Co. Ltd., China Swedish Match Northern Europe, Sweden Swedish Match, Owensboro, United States Swisher International, United States University of Kentucky, United States TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

5 3.2 Protocol Specific details from the protocol are described below: Sample Shipment Laboratories were responsible for procuring 1R6F, 1R5F, RTDAC, and RT2 from University of Kentucky, NIST SRM 3222 from the United States National Institute of Standards and Technology, and Cigar filler #1 and CigarM16 from Altria Client Services LLC. Laboratories were requested to store the samples at approximately 20 C upon receipt. Laboratories were requested to conduct the study in December through March and report data by March 10, The samples are identified in Table 2. Table 2: Sample Identification Product Type 1R6F Ground Filler - American blended cigarette filler, ground 1R5F Ground Filler - American blended cigarette filler (high Burley), ground RTDAC Ground Tobacco - Dark air-cured tobacco, ground RT2 Ground Tobacco Flue-cured tobacco, ground NIST SRM Cigarette tobacco filler, cut Cigar Filler #1 - Flavored cigar filler, ground CigarM16 - Traditional dark-air-cured cigar filler, ground Within Laboratory Sample Preparation The laboratories were directed to remove samples from the 20 C freezer and place the unopened samples in a refrigerator for a minimum of 24 hours to ensure water was fully equilibrated. Samples could then be removed from the refrigerator for a minimum of 1 hour prior to opening for analysis. Once samples were opened, the samples could be stored in a tightly sealed container and stored at approximately 4 ºC for up to one week. Special handling requirements, which differ from those specified in the CRMs, are described below: - The 1R5F, RTDAC, Cigar Filler #1, and CigarM16 had high concentrations of TSNAs. Participants were instructed to weigh out 0.4g for each TSNA replicate instead of 1.0g, as is specified in the CRM for analysis. - The NIST SRM 3222 sample was cut cigarette filler. Participants were instructed to grind the contents of one bottle that contained 10g to ensure homogeneous aliquots were removed for testing. - The 1R6F, 1R5F, RTDAC, RT2, Cigar Filler #1, CigarM16, did not require sample grinding and were to be analyzed as received Sample Analysis and Data Reporting The participating laboratories were instructed to conduct triplicate replicate analyses (individual tobacco weighing) for the following: TSNAs, ph, and moisture (oven volatiles) using the current versions of the CRMs. - TSNAs: CRM N 72, Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products by Liquid Chromatography - Tandem Mass Spectrometry, February TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

6 - ph: CRM N 69, Determination of ph in Smokeless Tobacco Products, June Moisture (oven volatiles): CRM N 76, Determination of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products, April Participating laboratories were requested to document any deviations from the protocol and the CRMs and submit the deviations with their results. As stated in the protocol, data submitted with significant deviations from the applicable CRM would be excluded from the study. Deviations reported by the laboratories are identified below. Since the reported deviations were minor, no data sets were excluded from the study. - Lab M: Minor deviations were reported for TSNAs where a different column than that specified in the CRM was used. Specifically, a Gemini C18 3µm, (150mm x 3mm) was used instead of a C18 2.5µm, (50mm x 2.1mm). - Lab N: Minor deviations were reported for TSNAs where two internal standards (d4- NNN and d4-nnk) were used instead of four internal standards as is specified in the CRM. Specifically, NNN-d4 was used as the internal standard for NNN and NNK-d4 was used as the internal standard for NNK, NAT, and NAB. - Lab O: Minor deviations were reported for moisture where a fully automated Brabender Moisture Tester was used instead conventional forced air oven. The laboratory adhered to the recommended temperature of 100 C and a drying time of 3 hours. - Lab S: A deviation was reported where the laboratory removed the filler from 1R6F and 1R5F cigarettes instead of acquiring ground cigarette fillers as was stated in the study protocol. - Lab Y: Minor deviations were reported for TSNAs where two internal standards (d4- NNN and d4-nnk) were used instead of four internal standards as is specified in the CRM. Specifically, NNN-d4 was used as the internal standard for NNN and NNK-d4 was used as the internal standard for NNK, NAT, and NAB. All test results were to be reported on an as-is basis without correction for moisture content. The results were not to be rounded and ideally reported to at least one more digit than typically required. The study results and the comments were to be sent by to the study coordinators. 4. Data Raw The full data set for the study is provided in Appendix A. The results are presented on an asis basis, without correction for moisture. Each analysis includes three replicates. Not all laboratories provided data for all analyses or all samples. Data sets were removed from the repeatability (r) and reproducibility (R) (r & R) portion of the study if the data were identified as outlying data. Those data are included in Appendix A, but were eliminated prior to the r & R analysis. Raw data plots that include all replicates, without removal of outliers, are given in Appendix B. 5. Data Statistical Analysis The statistical analysis was conducted in basic conformance with ISO :1994 and ISO/TR 22971:2005. A summary of the results from outlier detection and the calculated results for repeatability (r) and reproducibility (R) are given below in sections 5.1 and 5.2, respectively. Raw data plots that include all replicates, without removal of outliers, are shown in Appendix B. TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

7 5.1 Exclusion of Outliers Procedures outlined in ISO :1994 and ISO/TR 22971:2005 were generally used for the exclusion of outliers. An adaptation of Levene s Test was used for eliminating laboratories with overly large repeatability standard deviations and Grubbs Test was used to eliminate laboratories with outlying mean values. ISO 5725(2) also recommends the use of Mandel s h and k plots. Mandel s h statistic is the same as the statistic used in Grubbs Test. Similarly Mandel s k statistic, associated with within lab standard deviation, is statistically equivalent to the c-value calculated in Cochran s Test ( k n c ). However, the critical values associated with Mandel s h and k statistics do not labs make allowance for multiple testing and can therefore, give a false impression of statistical significance. Thus, Mandel s h and k statistics do not add fundamentally new information and may lead to incorrect conclusions. For those reasons, we do not include Mandel s h and k plots. The intent of ISO :1994 is to eliminate outliers that exceed a 1% critical value. This was accomplished by an adaptation of Levene s Test. Levene s Test is preferable to Cochran s Test, which is recommended in ISO :1994, because of Cochran s Test s extreme sensitivity to deviations from normality. Grubbs Test and an adaptation of Levene s Test were applied at the standard nominal 1% significance level to determine outliers and the results are shown in Table 3. Levene s Test is mentioned in ISO/TR 22971:2005 as an alternative to Cochran s Test. However, Levene s Test does not directly apply without adaptation. For more details, see the footnote below 3. Table 3: Outliers Product Analyte Levene s Outlier Lab Grubbs Outlier Lab 1R6F Moisture Q 1R6F NAB D 1R5F NAB D 1R5F Moisture L 1R5F ph S RTDAC NAB D RT2 NNN A NIST-SRM-3222 Moisture H Cigar filler #1 NAB B Cigar M16 NAB D The ( ) symbol indicates an outlier was not detected. 3 Levene s Test is commonly used to determine if each of several subpopulations have the same variance. Since it was designed to test for overall differences, not to determine if the largest variance is significantly greater than the others, some adaptation is necessary to use the approach to eliminate laboratories whose within lab variation is too large. Levene s Test was adapted to this purpose by Morton, who presented the approach utilized in this report at the 2014 CORESTA Congress (Quebec, Canada, presentation ST28, October 14, 2014). Specifically, the approach taken here is a two-step process with a lab being eliminated as an outlier if both steps are statistically significant. First, Levene s Test was run at a nominal -level of Second a comparison of the largest variance to the remaining variances is carried out at a one-sided nominal level of =0.01/number of labs. Dividing by the number of labs is to account for multiple testing, since it is not known a priori which lab will have the largest variance. Simulation studies were carried out by Morton and presented at the 2014 CORESTA Congress and these results demonstrated that this process has an overall -level near 0.01 and is robust to deviations from normality. TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

8 5.2 Calculation of Repeatability (r) and Reproducibility (R) After removal of outlying data based on numerical data consistency methods (Grubbs Test and Levene s Test), the final repeatability and reproducibility (r & R) results were calculated. The r & R results are shown in Table 4. The r & R results reflect both laboratory variability and product consistency. Table 4: Repeatability (r) and Reproducibility (R) Limits Parameter Product N of Labs* Mean Repeatability Reproducibility r % r R % R NNN (µg/g) 1R6F % % NNN (µg/g) 1R5F % % NNN (µg/g) RTDAC % % NNN (µg/g) RT % % NNN (µg/g) NIST-SRM % % NNN (µg/g) Cigar filler # % % NNN (µg/g) CigarM % % NNK (µg/g) 1R6F % % NNK (µg/g) 1R5F % % NNK (µg/g) RTDAC % % NNK (µg/g) RT % % NNK (µg/g) NIST-SRM % % NNK (µg/g) Cigar filler # % % NNK (µg/g) CigarM % % NAT (µg/g) 1R6F % % NAT (µg/g) 1R5F % % NAT (µg/g) RTDAC % % NAT (µg/g) RT % % NAT (µg/g) NIST-SRM % % NAT (µg/g) Cigar filler # % % NAT (µg/g) CigarM % % NAB (µg/g) 1R6F % % NAB (µg/g) 1R5F % % NAB (µg/g) RTDAC % % NAB (µg/g) RT % % NAB (µg/g) NIST-SRM % % NAB (µg/g) Cigar filler # % % NAB (µg/g) CigarM % % TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

9 Parameter Product N of Labs* Mean Repeatability Reproducibility r % r R % R Moisture % 1R6F % % Moisture % 1R5F % % Moisture % RTDAC % % Moisture % RT % % Moisture % NIST-SRM % % Moisture % Cigar filler # % % Moisture % CigarM % % ph 1R6F NA 0.24 NA ph 1R5F NA 0.18 NA ph RTDAC NA 0.23 NA ph RT NA 0.22 NA ph NIST-SRM NA 0.32 NA ph Cigar filler # NA 0.18 NA ph CigarM NA 0.27 NA * This is the number of laboratory data sets reported as values and after removal of outliers. NA = Since ph is not a proportional scale, it is not appropriate to calculate % r or % R. 6. Data Interpretations Overall, the percent repeatability (%r) and percent reproducibility (%R) results from this study compare well to the results presented in the CRMs. Notable differences include the following: - Overall, we noted greater variability for the TSNA results for RT2 and NIST SRM 3222 than seen for other sample types. The cause for the higher variability is likely due to extremely low analyte concentrations that are at or below the limit of quantitation for many laboratories. - We also noted %R for moisture for NIST SRM 3222 was slightly greater than other study samples. A plausible cause for this higher variability was that the protocol stated this sample must be ground prior to analysis while all other samples were preground. Laboratories used their own grinding procedures and grinding may affect moisture content due to the generation of heat and loss of moisture due to the physical process of particle size reduction. Table 5 and Table 6 provides a comparison of the average results for this study to the certificates of analyses for 1R6F and NIST SRM 3222 where certified values are available 4,5. The values in this study are in good agreement with the certificates. 4 Certificate of Analysis for 1R6F Certified Reference Cigarette, Certificate Number CTRP, University of Kentucky Center for Tobacco Reference Products. 5 Certificate of Analysis for NIST Standard Reference Material 3222 Cigarette Tobacco Filler, National Institute of Standards and Technology. TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

10 Table 5: Comparison of Study Results to Certified Values for 1R6F Parameter Current Study Mean Certified Mean Values 4 Certified Uncertainty 4 NNN (µg/g) NNK (µg/g) Moisture % ph Table 6: Comparison of Study Results to Certified Values for NIST SRM Parameter Current Study Mean Certified Mean Values 5 Certified Uncertainty 5 NNN (µg/g) NNK (µg/g) Moisture % Recommendations The CRMs listed below were originally developed for the analysis of smokeless tobacco products: - TSNAs: CRM N 72, Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products by Liquid Chromatography - Tandem Mass Spectrometry, February ph: CRM N 69, Determination of ph in Smokeless Tobacco Products, June Moisture (oven volatiles): CRM N 76, Determination of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products, April The results of this study demonstrate that the CRMs are also fit for the analysis of ground tobacco, cigarette filler, and cigar filler. The TTPA recommends the scope of the CRMs be updated to include these additional matrices and that the r & R data from this study be added to the CRMs. The TTPA also recommends that ISO/CD Tobacco and tobacco products- Determination of tobacco-specific nitrosamines in tobacco and tobacco products- Method using LC-MS/MS be updated to include the r & R data supporting the inclusion of ground tobacco, cigarette filler and cigar filler. 8. Appendices Appendix A: Full Data Set Appendix B: Raw Data Plots TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

11 APPENDIX A: Full Data Set Full Data Set (results are presented on an as-is basis) NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % A 1R6F A 1R6F A 1R6F B 1R6F B 1R6F B 1R6F C 1R6F C 1R6F C 1R6F D 1R6F D 1R6F D 1R6F E 1R6F E 1R6F E 1R6F F 1R6F F 1R6F F 1R6F G 1R6F 5.23 G 1R6F 5.24 G 1R6F 5.23 H 1R6F H 1R6F H 1R6F I 1R6F I 1R6F I 1R6F J 1R6F J 1R6F J 1R6F K 1R6F K 1R6F K 1R6F ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

12 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L 1R6F L 1R6F L 1R6F M 1R6F M 1R6F M 1R6F N 1R6F N 1R6F N 1R6F O 1R6F O 1R6F O 1R6F P 1R6F P 1R6F P 1R6F Q 1R6F Q 1R6F Q 1R6F R 1R6F R 1R6F R 1R6F S 1R6F 5.55 S 1R6F 5.57 S 1R6F 5.58 T 1R6F T 1R6F T 1R6F U 1R6F U 1R6F U 1R6F V 1R6F V 1R6F V 1R6F X 1R6F X 1R6F X 1R6F ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

13 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y 1R6F Y 1R6F Y 1R6F A 1R5F A 1R5F A 1R5F B 1R5F B 1R5F B 1R5F C 1R5F C 1R5F C 1R5F D 1R5F D 1R5F D 1R5F E 1R5F E 1R5F E 1R5F F 1R5F F 1R5F F 1R5F G 1R5F 4.75 G 1R5F 4.75 G 1R5F 4.74 H 1R5F H 1R5F H 1R5F I 1R5F I 1R5F I 1R5F J 1R5F J 1R5F J 1R5F K 1R5F K 1R5F K 1R5F ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

14 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L 1R5F L 1R5F L 1R5F M 1R5F M 1R5F M 1R5F N 1R5F N 1R5F N 1R5F O 1R5F O 1R5F O 1R5F P 1R5F P 1R5F P 1R5F Q 1R5F Q 1R5F Q 1R5F R 1R5F R 1R5F R 1R5F S 1R5F 5.31 S 1R5F 5.34 S 1R5F 5.33 T 1R5F T 1R5F T 1R5F U 1R5F U 1R5F U 1R5F V 1R5F V 1R5F V 1R5F X 1R5F X 1R5F X 1R5F ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

15 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y 1R5F Y 1R5F Y 1R5F A RTDAC A RTDAC A RTDAC B RTDAC B RTDAC B RTDAC C RTDAC C RTDAC C RTDAC D RTDAC D RTDAC D RTDAC E RTDAC E RTDAC E RTDAC F RTDAC F RTDAC F RTDAC G RTDAC 5.82 G RTDAC 5.83 G RTDAC 5.82 H RTDAC H RTDAC H RTDAC I RTDAC I RTDAC I RTDAC J RTDAC J RTDAC J RTDAC K RTDAC K RTDAC K RTDAC ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

16 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L RTDAC L RTDAC L RTDAC M RTDAC M RTDAC M RTDAC N RTDAC N RTDAC N RTDAC O RTDAC O RTDAC O RTDAC P RTDAC P RTDAC P RTDAC Q RTDAC Q RTDAC Q RTDAC R RTDAC R RTDAC R RTDAC S RTDAC S RTDAC S RTDAC T RTDAC T RTDAC T RTDAC U RTDAC U RTDAC U RTDAC V RTDAC V RTDAC V RTDAC X RTDAC X RTDAC X RTDAC ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

17 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y RTDAC Y RTDAC Y RTDAC A RT A RT A RT B RT B RT B RT C RT C RT C RT D RT D RT D RT E RT2 E RT2 E RT2 F RT F RT F RT G RT G RT G RT H RT H RT H RT I RT I RT I RT J RT J RT J RT K RT K RT K RT ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

18 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L RT L RT L RT M RT M RT M RT N RT N RT N RT O RT J O RT J O RT J P RT P RT P RT Q RT Q RT Q RT R RT R RT R RT S RT2 S RT2 S RT2 T RT J U T RT J U T RT J U U RT U RT U RT V RT V RT V RT X RT X RT X RT ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

19 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y RT Y RT Y RT A SRM A SRM A SRM B SRM B SRM B SRM C SRM C SRM C SRM D SRM D SRM D SRM E SRM 3222 E SRM 3222 E SRM 3222 F SRM F SRM F SRM G SRM 3222 G SRM 3222 G SRM 3222 H SRM H SRM H SRM I SRM 3222 I SRM 3222 I SRM 3222 J SRM J SRM J SRM K SRM K SRM K SRM ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

20 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L SRM L SRM L SRM M SRM 3222 M SRM 3222 M SRM 3222 N SRM N SRM N SRM O SRM J J O SRM U O SRM J J P SRM 3222 P SRM 3222 P SRM 3222 Q SRM Q SRM Q SRM R SRM R SRM R SRM S SRM 3222 S SRM 3222 S SRM 3222 T SRM J J U T SRM J J U T SRM J J U U SRM U SRM U SRM V SRM 3222 V SRM 3222 V SRM 3222 X SRM X SRM X SRM ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

21 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y SRM Y SRM Y SRM A Cigar filler # A Cigar filler # A Cigar filler # B Cigar filler # B Cigar filler # B Cigar filler # C Cigar filler # C Cigar filler # C Cigar filler # D Cigar filler # D Cigar filler # D Cigar filler # E Cigar filler # E Cigar filler # E Cigar filler # F Cigar filler # F Cigar filler # F Cigar filler # G Cigar filler # G Cigar filler # G Cigar filler # H Cigar filler # H Cigar filler # H Cigar filler # I Cigar filler # I Cigar filler # I Cigar filler # J Cigar filler # J Cigar filler # J Cigar filler # K Cigar filler # K Cigar filler # K Cigar filler # ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

22 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L Cigar filler # L Cigar filler # L Cigar filler # M Cigar filler # M Cigar filler # M Cigar filler # N Cigar filler # N Cigar filler # N Cigar filler # O Cigar filler # O Cigar filler # O Cigar filler # P Cigar filler #1 P Cigar filler #1 P Cigar filler #1 Q Cigar filler # Q Cigar filler # Q Cigar filler # R Cigar filler # R Cigar filler # R Cigar filler # S Cigar filler # S Cigar filler # S Cigar filler # T Cigar filler # T Cigar filler # T Cigar filler # U Cigar filler # U Cigar filler # U Cigar filler # V Cigar filler #1 V Cigar filler #1 V Cigar filler #1 X Cigar filler # X Cigar filler # X Cigar filler # ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

23 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y Cigar filler # Y Cigar filler # Y Cigar filler # A Cigar M A Cigar M A Cigar M B Cigar M B Cigar M B Cigar M C Cigar M C Cigar M C Cigar M D Cigar M D Cigar M D Cigar M E Cigar M E Cigar M E Cigar M F Cigar M F Cigar M F Cigar M G Cigar M G Cigar M G Cigar M H Cigar M H Cigar M H Cigar M I Cigar M I Cigar M I Cigar M J Cigar M J Cigar M J Cigar M K Cigar M K Cigar M K Cigar M ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

24 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % L Cigar M L Cigar M L Cigar M M Cigar M M Cigar M M Cigar M N Cigar M N Cigar M N Cigar M O Cigar M O Cigar M O Cigar M P Cigar M16 P Cigar M16 P Cigar M16 Q Cigar M Q Cigar M Q Cigar M R Cigar M R Cigar M R Cigar M S Cigar M S Cigar M S Cigar M T Cigar M T Cigar M T Cigar M U Cigar M U Cigar M U Cigar M V Cigar M16 V Cigar M16 V Cigar M16 X Cigar M16 X Cigar M16 X Cigar M16 ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

25 NNK NNN NAT NAB Oven volatiles Lab Code Product µg/g µg/g µg/g µg/g % Y Cigar M Y Cigar M Y Cigar M The ( ) symbol indicates the laboratory did not submit a value for that sample analysis. The (J) symbol indicates the result was below the limit of quantitation. The (U) symbol indicates the result was below the limit of detection. ph TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

26 APPENDIX B: Raw Data Plots TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

27 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

28 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

29 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

30 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

31 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

32 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

33 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

34 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

35 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36

36 TTPA CTR 2017 Collaborative Study on TSNA, ph, and OV July /36