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Tobacco and Tobacco Products Analytes Sub-Group

Technical Report

2017 Collaborative Study on and Benzo[a]pyrene in Tobacco Products

March 2018

Study Project Leaders: Regina Ballentine, Altria Client Services LLC, U.S.A. Anthony Brown, Altria Client Services LLC, U.S.A. Author: Regina Ballentine, Altria Client Services LLC, U.S.A. Co-Author and Statistical Analysis: Michael Morton, Ph.D., Altria Client Services LLC, U.S.A. Table of Contents

1. SUMMARY ...... 3

2. INTRODUCTION ...... 3

2.1 Objective ...... 3

3. ORGANISATION ...... 3

3.1 Participants ...... 3

3.2 Protocol ...... 4

4. DATA – RAW ...... 6

5. DATA – STATISTICAL ANALYSIS ...... 6

5.1 Exclusion of Outliers ...... 6

5.2 Calculation of Repeatability (r) and Reproducibility (R) ...... 7

6. DATA INTERPRETATIONS ...... 8

7. RECOMMENDATIONS ...... 9

8. APPENDICES ...... 9

1. Summary

At the May 2017 CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA) meeting held in Charlottesville, USA, the Sub-Group initiated a collaborative study for the determination of ammonia and benzo[a]pyrene in smokeless tobacco, cigarette filler, and ground cigars (wrapper, binder, and filler). The intent of this study was to support laboratory accreditation and to expand the scopes of CORESTA Recommended Methods (CRMs) N° 79 and N° 82 to include ground cigars.

2. Introduction

At the CORESTA Tobacco and Tobacco Products Analytes Subgroup (TTPA) meeting held in Charlottesville, USA (May 2017), it was decided to conduct a collaborative study for Ammonia and Benzo[a]pyrene (B[a]P) using the following CRMs:  CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015)  CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017)

The collaborative study involved 17 laboratories and will support laboratory accreditation and a scope expansion of the CRMs to include ground cigars (wrapper, binder, and filler). Tabulated data are presented along with repeatability (r) and reproducibility (R). The results of the study demonstrate that the CRMs for Ammonia and B[a]P are suitable for the analysis of ground cigars. The TTPA recommends that the two CRMs be updated to include ground cigars.

2.1 Objective This study was conducted to support laboratory accreditation and scope expansion of the Ammonia and B[a]P CRMs beyond just tobacco, cigarette filler, and smokeless tobacco to include ground cigars. This work was conducted using the samples listed in Table 2. Data were collected from the participating laboratories and statistically evaluated in basic conformance with the recommendations of ISO 5725-2:1994 and ISO/TR 22971:2005.

3. Organisation

3.1 Participants A list of the participating laboratories is provided in Table 1. Not all laboratories provided data for both methods nor did all laboratories analyze all samples. The laboratories are listed in alphabetical order. Identifying number codes were assigned to each laboratory to protect confidentiality and do not correspond to the order shown in the table below.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 3/29 Table 1: List of Participating Laboratories in the 2017 Collaborative Study

Participating Laboratories Ammonia B[a]P

Altria Client Services LLC, United States X X American Snuff Company, United States X C.I.T. Montepaz S.A., Uruguay X X China National Tobacco Quality Supervision and Test Center, X X China CNTC, Zhengzhou Tobacco Research Institute, X X Key Laboratory of Tobacco Flavor Basic Research, China CNTC, Yunnan Tobacco Quality Supervision and Test Station, X X China Enthalpy Analytical, Inc., (Richmond) United States X X Global Laboratory Services, United States X X ITG Brands, United States X X Japan Tobacco Inc., Japan X X JTI-Ökolab, Austria X KT&G Research Institute, South Korea X X Imperial Tobacco, Reemtsma, Germany X RJ Reynolds Tobacco Company, United States X X Swedish Match, North America, United States X Swedish Match, Northern Europe, Sweden X University of Kentucky, United States X X

3.2 Protocol Specific details from the protocol are described below:

3.2.1 Sample Shipment Laboratories were responsible for procuring 1R6F ground cigarette filler from the University of Kentucky, CRP1.1, CRP2.1, CRP3.1, and CRP4.1 from North Carolina State University, and Cigar filler #1-05/17 and Cigar M16-05/17 from Altria Client Services LLC. Laboratories were requested to store the samples at approximately 4 °C upon receipt. If samples were not analysed within one week, they were to be stored in a freezer at approximately −20 °C until the analyses were performed. Laboratories were requested to conduct the study July through August and report data by August 15, 2017. The samples are identified in Table 2. The protocol incorrectly identified Cigar Filler #1-05/17 as only ground filler; however, this sample was a ground finished cigar that included the wrapper, binder, and filler.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 4/29 Table 2: Sample Identification

Product Type CRP1.1 - Swedish-style snus pouch CRP2.1 - American-style loose moist snuff CRP3.1 - American-style loose dry snuff powder CRP4.1 - American-style chopped loose-leaf chewing tobacco 1R6F Ground Filler-RT1 - American blend cigarette filler Cigar Filler #1-05/17 - Flavoured ground cigar (wrapper, binder, and filler) Cigar M16-05/17 - Traditional dark-air cured ground cigar (wrapper, binder, and filler)

3.2.2 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 that the in the sample had been fully equilibrated. Samples could then be removed from the refrigerator for a minimum of 2 hours prior to opening for analysis. Once samples were opened, the samples could be stored in a tightly sealed container at approximately 4 ºC for up to one week. Additional handling requirements are described below:  CRP1.1: The Snus pouches should be cut into 2 halves and both parts added directly into the extraction vessel. Both Snus and pouch material (paper) are to be analyzed.  All other samples should be mixed with a spatula before removing aliquots for analysis.

3.2.3 Sample Analysis and Data Reporting The participating laboratories were instructed to conduct three replicate analyses (individual tobacco weighing) for Ammonia and B[a]P using the current versions of the CRMs.  Ammonia: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015)  B[a]P: CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) 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. The following deviations for Ammonia CRM N° 79 were reported:  Lab 1 – 1) No suppressor was used. 2) Elution was done with 3 mM Oxalic acid solution containing 1 mM 18-Crown-6 ether.  Lab 2 – A segmented flow analyzer was used for analysis.  Lab 3 – The amount of sample and volume of extraction solution was changed.  Lab 5 – A chilled auto-sampler tray was not used.  Lab 8 – No suppressor was used.  Lab 14 – 1) Modified the mobile phase gradient because a CS19 column was used. 2) Several sample dilutions were performed prior to analysis.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 5/29 The following deviations for B[a]P: CRM N° 82 were reported:  Lab 1 – 1) ultrasonic extraction was used instead of an orbital shaker or wrist action shaker. 2) was used for calibration standards instead of 50:50 :isooctane. 3) There was no filtration of the sample extract 4) Evaporated solvent before washing the SPE cartridge 5) Hexane was the only wash solvent used for solvent phase extraction even though : water and isopropyl alcohol were also specified.  Lab 2 – Used a DB-5MS (30 m x 0.25 mm x 1.0 µm) column.  Lab 10 – Used a DB-35MS column.  Lab 11 – Used a DB-35MS column.  Lab 12 – 1) Used a DB-5MS Ultra Inert column. 2) Concentration differed from protocol because neat B[a]P-d12 material was used to prepare the Working Internal Stock Solutions. The protocol deviations for ammonia analysis of Labs 1 and 2 were considered significant (i.e. likely to impact testing results) and their ammonia data were excluded from the r&R portion of the study. Similarly the protocol deviation for B[a]P analysis of Lab 1 was considered significant and their B[a]P data were also excluded from the r&R portion of the study. All other deviations were considered of minor significance and therefore, the data were included. All test results were to be reported to three decimal places. The study results and the comments were sent by e-mail to the study coordinators.

4. Data – Raw

The full data set for the study is provided in Appendix B. The results are presented on an as- is 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 or the lab was excluded for protocol deviations. All data are included in Appendix B, but the excluded data were eliminated prior to the r&R analysis. Raw data plots that include all replicates, without removal of outliers, are given in Appendix C.

5. Data – Statistical Analysis

The statistical analysis was conducted in basic conformance with ISO 5725-2:1994 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 C.

5.1 Exclusion of Outliers Procedures outlined in ISO 5725-2:1994 and ISO/TR 22971:2005 were generally used for the exclusion of outliers. An adaptation of Levene’s Test1 was used for eliminating laboratories with overly large repeatability standard deviations and Grubbs’ Test was used to eliminate laboratories with outlying mean values.

1 The approach is discussed in detail by Michael Morton in “Within-Laboratory Variance Outlier Detection: An Alternative to Cochran’s Test” in Beitrage zur Tabakforschung International, Vol 27 No. 7, pp 135-144.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 6/29 ISO 5725-2:1994 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  nlabsc) . However, the critical values associated with Mandel’s h and k statistics do not 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 results of the outlier tests are shown in Table 3. In addition to the outliers shown in Table 3, one of the Lab 16 Cigar Filler #1-05/17 B[a]P replicates was a single-point outlier and that B[a]P replicate was deleted and not included in the r&R calculations.

Table 3: Outliers

Product Analyte Levene’s Outlier Lab Grubbs’ Outlier Lab CRP3.1 B[a]P ̶ 17 Cigar M16-05/17 B[a]P 17 ̶ The (–) symbol indicates an outlier was not detected.

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 for ammonia are shown in Table 4 and B[a]P r&R results are shown in Table 5. The r&R results reflect both laboratory variability and product consistency.

Table 4: Ammonia Repeatability (r) and Reproducibility (R) Limits

Mean Repeatability Reproducibility N° of Product Ammonia Labs* (µg/g) r r (%) R R (%) CRP1.1 - Swedish-style snus pouch 12 982 63,6 6,5 687 70 CRP2.1 - American-style loose moist snuff 12 2400 161 6,7 1248 52 CRP3.1 - American-style loose dry snuff 12 7159 469 6,5 1647 23 powder CRP4.1 - American-style chopped loose- 12 2003 142 7,1 536 27 leaf chewing tobacco 1R6F Ground Filler-RT1 - American blend 12 1005 85,1 8,5 260 26 cigarette filler Cigar Filler #1-05/17 - Flavoured cigar 9 786 117 15 203 26 (ground wrapper, binder, and filler) Cigar M16-05/17 - Traditional dark-air cured cigar (ground wrapper, binder, and 9 2813 262 9,3 1026 36 filler) * This is the number of laboratory data sets reported as values and after removal of outliers.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 7/29 Table 5: Benzo[a]pyrene Repeatability (r) and Reproducibility (R) Limits

Mean Repeatability Reproducibility N° of Product B[a]P Labs* (ng/g) r r (%) R R (%) CRP1.1 - Swedish- style snus pouch 14 0,86 0,496 58 0,902 105 CRP2.1 - American-style loose moist snuff 14 154 13,1 8,5 42,4 28 CRP3.1 - American-style loose dry snuff 13 147 9,94 6,8 41,7 28 powder CRP4.1 - American-style chopped loose- 14 1,21 0,638 53 0,82 67 leaf chewing tobacco 1R6F Ground Filler-RT1 - American blend 14 4,88 1,161 24 3,44 71 cigarette filler Cigar Filler #1-05/17 - Flavoured cigar 11 10,4 2,20 21 5,89 57 (ground wrapper, binder, and filler) Cigar M16-05/17 - Traditional dark-air cured cigar (ground wrapper, binder, and 10 4,38 0,42 10 1,97 45 filler)

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:  The ammonia % reproducibility values were higher than anticipated for CRP1.1 and CRP2.1. However that seemed largely a result of Lab 12 being near outlier classification. Excluding Lab 12 would result in reproducibility values for CRP1.1 and CRP2.1 similar to the previous study.  Similarly the B[a]P % reproducibility value is somewhat larger for Cigar Filler #1-05/17 than would have been expected. That is largely driven by Lab 17 that is near outlier classification for this sample and was classified as an outlier or near outlier classification for several other products. If that laboratory were excluded from the study, the % reproducibility for Cigar Filler #1-05/17 would be approximately 38 %, in line with the expected levels. The ammonia value for 1R6F Ground Filler-RT1 is in good agreement with the certified value of 956 μg/g published by the University of Kentucky Center for Tobacco Reference Products2.

2 Certificate of Analysis for 1R6F Certified Reference Cigarette, Certificate Number 2017-002CTRP, University of Kentucky Center for Tobacco Reference Products.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 8/29 7. Recommendations

The CRMs listed below were originally developed for the analysis of tobacco, cigarette filler, and smokeless tobacco products:  Ammonia: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015)  B[a]P: CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) The results of this study demonstrate that the methods are also fit for purpose for the analysis of cigar filler. The TTPA recommends the scope of the CRMs to be updated to include the additional matrices not included in the previous studies and that the applicable r&R data for these new matrices be added to the CRMs.

8. Appendices

Appendix A: Study Protocol Appendix B: Full Data Set Appendix C: Raw Data Plots

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 9/29 APPENDIX A: Study Protocol

CORESTA TOBACCO AND TOBACCO PRODUCTS ANALYTES SUB-GROUP

Project Title: Collaborative study for Ammonia and Benzo[a]pyrene in Tobacco Products

Type of Document: Collaborative Study Protocol

Date: July 5, 2017

Study Coordinators: Regina Ballentine and Anthony Brown

Confidentiality Notice: All data submitted by participating laboratories will be coded and kept confidential.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 10/29 1. Introduction

At the CORESTA Tobacco and Tobacco Products Analytes Sub-Group (TTPA) meeting held on May 2017 in Charlottesville, the group decided to conduct a collaborative study for ammonia and benzo[a]pyrene (B[a]P) in tobacco products.

2. Objective

The objectives of the study are: (1) support laboratory accreditation and (2) expand the scope of the following CORESTA Recommended Methods (CRMs) to include cigars: • Ammonia: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015) • B[a]P: CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) The final output will be one technical report and the corresponding updates to CRM N°79 and CRM N° 82 to include cigars. Results will be discussed at the next TTPA meeting scheduled for October 7, 2017 in Kitzbühel, Austria. Note: Use of methods other than those specified will not support the study objectives.

3. Time schedule

Table 1: Study timeline

Date Activity

Laboratories state their intention to participate and provide the shipping May 31, 2017 information in order to receive two cigar fillers from Altria Distribute the study protocol and data reporting sheet (the methods are the May 31, 2017 CRMs) June 2017 Altria distributes ground cigar samples to participants Participants order 1R6F ground cigarette filler (RT1) from the University of July 2017 Kentucky. July-August Laboratories conduct the study August 15, 2017 Laboratories submit results by this date October 7, 2017 Discuss results at TTPA meeting in Kitzbühel, Austria

4. Participating Laboratories:

The laboratories listed in Table 2 have kindly agreed to take part in the study using the most recent versions of the specified CRMs. Other laboratories are encouraged to participate and should notify Regina Ballentine [email protected] and Anthony Brown [email protected] of their interest to participate.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 11/29 Table 2: Participating Laboratories

Participating Laboratories Ammonia B[a]P

Altria Client Services LLC, United States X X American Snuff Company, United States X British American Tobacco, Brazil X British American Tobacco, Germany X C.I.T. Montepaz S.A., Uruguay X X China National Tobacco Quality Supervision and Test Center, X X China CNTC, Zhengzhou Tobacco Research Institute, X X Key Laboratory of Tobacco Flavor Basic Research, China CNTC, Yunnan Tobacco Quality Supervision and test Station, X X China Enthalpy Analytical, United States X X Eurofins, Sweden X X Global Laboratory Services, United States X X ITG Brands, United States X X Japan Tobacco Inc., Japan X JTI-Ökolab AUSTRIA TABAK, Austria X X KT&G Research Institute, South Korea X X Labstat International ULC, Canada X X Reemtsma (Imperial Tobacco Limited) X X RJ Reynolds tobacco Company, United States X X Swedish Match, Northern Europe, Sweden X X University of Kentucky, United States X

5. Samples

The samples should be ordered from the suppliers listed in Table 3. Altria will provide the two ground cigar samples to all participants. For long-term storage, it is critical that the samples be stored at the recommended temperature of −20 °C. Upon receipt, the samples may be stored at approximately 4 °C if the analyses will be conducted within 1-week. If samples will not be analysed within 1-week, they shall be stored in a freezer at approximately −20 °C until the analyses are performed.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 12/29 Table 3: Samples

Quantity to Sample Name Description Supplier Request

1 Flavoured cigar filler, Cigar Filler #1-05/17 Altria Client Services 1 bottle ground Traditional dark-air cured Cigar M16-05/17 1 cigar, ground wrapper, and Altria Client Services 1 bottle filler

2 American blended cigarette 1R6F Ground Filler-RT1 University of Kentucky 1 bottle filler North Carolina State 2016 CRP1.1 Swedish-style snus pouch 4 cans University American-style loose moist North Carolina State 2016 CRP2.1 4 cans snuff University American-style loose dry North Carolina State 2016 CRP3.1 4 cans snuff powder University American-style chopped North Carolina State 2016 CRP4.1 4 cans loose-leaf chewing tobacco University 1: The cigar filler distributed for the previous study should not be used for this study. The sample name provided above should match that on the sample bottle. 2: The 1R6F ground filler must be ordered from the University of Kentucky. Do not use filler that has been removed from cigarettes.

6. Analysis

6.1 Methods: Report ammonia and B[a]P in each sample using the current versions of the specified CRMs. Data collected with other methods, or with methods that have significant deviations from the CRMs will not be included in the study. • Ammonia: CRM N° 79, Determination of Ammonia in Tobacco and Tobacco Products by Ion Chromatographic Analysis (April 2015) • B[a]P: CRM N° 82, Determination of Benzo[a]pyrene in Tobacco Products by GC-MS (July 2017) Note: The following three samples will exceed the calibration range for ammonia and will need to be diluted as described in CRM N° 79: 1R6F ground filler (RT1), Cigar Filler #1-05/17, and Cigar M16-05/17. Note: CRP2.1 and CRP3.1 will exceed the calibration range for B[a]P and will need to be prepared with a reduced sample mass as described in the current version of CRM N° 82. 6.2 Replicates: Conduct three (3) independent replicate analyses for each sample. The replicates should be determined from independent tobacco extractions.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 13/29 6.3 Sample equilibration: Samples held at −20 °C should be placed unopened in a refrigerator for a minimum of 24 hours to ensure water has fully equilibrated within the product. Samples shall be removed from the refrigerator a minimum of 2 hours prior to opening for analysis. The samples should not be opened during the time the samples are equilibrating to ambient temperature. Once samples are equilibrated to ambient temperature, the samples shall be stored at approximately 4 °C for up to one week if the analyses will not be conducted immediately. Sample retains shall be stored at −20 °C. Additional handling requirements are described below: • CRP1.1: The Snus pouches shall be cut into 2 halves and added directly into the extraction vessel. Both Snus and pouch material (paper) are to be analyzed. • All other samples should be mixed with a spatula before removing aliquots for analysis. 6.4 Data Reporting: The data report spreadsheet provided with this protocol should be used for data reporting. Send the completed data reporting spreadsheet to the study coordinators (Regina Ballentine [email protected], Anthony Brown [email protected]) and Karl Wagner [email protected]) Additionally: • Data shall be reported to three decimal places for Ammonia and Benzo[a]pyrene. • If data are below the LOQ, but above the LOD, report the estimated analytical result and note that the analytical result is an estimate beside the cell. • If the result is below the LOD, report the actual LOD value using the format (e.g.

7. Statistical Analysis

A statistical analysis in general conformance with ISO 5725-2:1994 and ISO/TR 22971:2005 will be conducted.

8. Presentation of the Results

The results will be presented for discussion at the TTPA meeting, which is scheduled to be held in Kitzbühel, Austria on October 7, 2017.

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 14/29 APPENDIX B: Full Data Set

Full Data Set (results are presented on an as-is basis)

Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 1 1R6F Ground Filler 1045,77 4,964 1 1R6F Ground Filler 1033 4,997 1 1R6F Ground Filler 1024,28 4,947 2 1R6F Ground Filler 1071,49 3,914 2 1R6F Ground Filler 1078,71 3,301 2 1R6F Ground Filler 1081,21 4,979 3 1R6F Ground Filler 969,66 4,999 3 1R6F Ground Filler 962,95 5,401 3 1R6F Ground Filler 968,65 4,683 4 1R6F Ground Filler 882,45 5,279 4 1R6F Ground Filler 882,63 5,147 4 1R6F Ground Filler 882,88 4,712 5 1R6F Ground Filler 953,71 – 5 1R6F Ground Filler 889,51 – 5 1R6F Ground Filler 922,17 – 6 1R6F Ground Filler – 4,609 6 1R6F Ground Filler – 4,199 6 1R6F Ground Filler – 3,976 7 1R6F Ground Filler 1063,88 4,79 7 1R6F Ground Filler 1193,19 4,997 7 1R6F Ground Filler 1085,16 4,69 8 1R6F Ground Filler 965,26 4,505 8 1R6F Ground Filler 941,39 4,544 8 1R6F Ground Filler 958,31 5,297 9 1R6F Ground Filler – 5,536 9 1R6F Ground Filler – 5,371 9 1R6F Ground Filler – 4,882 10 1R6F Ground Filler 1029,59 5,86 10 1R6F Ground Filler 1088,03 6,144 10 1R6F Ground Filler 1083,32 6,351 11 1R6F Ground Filler 1108,58 7,042 11 1R6F Ground Filler 1096,52 6,584 11 1R6F Ground Filler 1097,36 7,25 12 1R6F Ground Filler 1196,69 5,218

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 15/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 12 1R6F Ground Filler 1088,59 5,801 12 1R6F Ground Filler 1176,35 5,32 13 1R6F Ground Filler 912,53 – 13 1R6F Ground Filler 924,61 – 13 1R6F Ground Filler 945,98 – 14 1R6F Ground Filler 1054,22 4,013 14 1R6F Ground Filler 1051,15 4,057 14 1R6F Ground Filler 1065,4 4,801 15 1R6F Ground Filler – 5,078 15 1R6F Ground Filler – 6,161 15 1R6F Ground Filler – 5,395 16 1R6F Ground Filler 973,46 4,757 16 1R6F Ground Filler 979,91 5,472 16 1R6F Ground Filler 1002,88 4,563 17 1R6F Ground Filler 938,8 1,641 17 1R6F Ground Filler 925,58 1,893 17 1R6F Ground Filler 919,46 1,657 1 Cigar Filler #1-05/17 810,17 9,704 1 Cigar Filler #1-05/17 821,71 9,603 1 Cigar Filler #1-05/17 823,48 9,973 2 Cigar Filler #1-05/17 937,05 8,416 2 Cigar Filler #1-05/17 1039,44 8,116 2 Cigar Filler #1-05/17 981,92 9,264 3 Cigar Filler #1-05/17 716,54 11,212 3 Cigar Filler #1-05/17 710,65 10,528 3 Cigar Filler #1-05/17 707,63 10,972 4 Cigar Filler #1-05/17 859,17 11,186 4 Cigar Filler #1-05/17 854,51 11,393 4 Cigar Filler #1-05/17 862,93 11,174 5 Cigar Filler #1-05/17 786,16 – 5 Cigar Filler #1-05/17 720,11 – 5 Cigar Filler #1-05/17 737,72 – 6 Cigar Filler #1-05/17 – 11,307 6 Cigar Filler #1-05/17 – 10,772 6 Cigar Filler #1-05/17 – 10,891 7 Cigar Filler #1-05/17 813,01 8,986

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 16/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 7 Cigar Filler #1-05/17 875,25 9,029 7 Cigar Filler #1-05/17 988,79 9,101 8 Cigar Filler #1-05/17 740,81 11,033 8 Cigar Filler #1-05/17 735,09 11,163 8 Cigar Filler #1-05/17 766,94 10,871 9 Cigar Filler #1-05/17 – 10,906 9 Cigar Filler #1-05/17 – 10,018 9 Cigar Filler #1-05/17 – 9,988 10 Cigar Filler #1-05/17 – – 10 Cigar Filler #1-05/17 – – 10 Cigar Filler #1-05/17 – – 11 Cigar Filler #1-05/17 – – 11 Cigar Filler #1-05/17 – – 11 Cigar Filler #1-05/17 – – 12 Cigar Filler #1-05/17 – – 12 Cigar Filler #1-05/17 – – 12 Cigar Filler #1-05/17 – – 13 Cigar Filler #1-05/17 721,59 – 13 Cigar Filler #1-05/17 726,28 – 13 Cigar Filler #1-05/17 779,94 – 14 Cigar Filler #1-05/17 843,63 10,429 14 Cigar Filler #1-05/17 819,47 10,872 14 Cigar Filler #1-05/17 831,79 10,742 15 Cigar Filler #1-05/17 – 11,855 15 Cigar Filler #1-05/17 – 11,562 15 Cigar Filler #1-05/17 – 11,264 16 Cigar Filler #1-05/17 808,15 26,731 16 Cigar Filler #1-05/17 814,46 14,139 16 Cigar Filler #1-05/17 815,62 13,718 17 Cigar Filler #1-05/17 811,68 8,331 17 Cigar Filler #1-05/17 703,52 4,537 17 Cigar Filler #1-05/17 678,07 4,079 1 Cigar M16-05/17 2881,75 4,652 1 Cigar M16-05/17 2856,41 4,945 1 Cigar M16-05/17 2895,77 4,83 2 Cigar M16-05/17 2796,85 3,252

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 17/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 2 Cigar M16-05/17 2842,69 3,6 2 Cigar M16-05/17 2859,52 3,827 3 Cigar M16-05/17 2525,26 4,718 3 Cigar M16-05/17 2509,2 4,51 3 Cigar M16-05/17 2482,86 4,736 4 Cigar M16-05/17 2665,28 4,354 4 Cigar M16-05/17 2724,03 4,358 4 Cigar M16-05/17 2716,98 4,393 5 Cigar M16-05/17 2416,82 – 5 Cigar M16-05/17 2479,34 – 5 Cigar M16-05/17 2387,09 – 6 Cigar M16-05/17 – 3,643 6 Cigar M16-05/17 – 3,843 6 Cigar M16-05/17 – 3,696 7 Cigar M16-05/17 3233,03 3,499 7 Cigar M16-05/17 3078,61 3,442 7 Cigar M16-05/17 2854,04 3,516 8 Cigar M16-05/17 2706,31 4,601 8 Cigar M16-05/17 2703,03 4,475 8 Cigar M16-05/17 2768,89 4,725 9 Cigar M16-05/17 – 4,569 9 Cigar M16-05/17 – 4,75 9 Cigar M16-05/17 – 4,66 10 Cigar M16-05/17 – – 10 Cigar M16-05/17 – – 10 Cigar M16-05/17 – – 11 Cigar M16-05/17 – – 11 Cigar M16-05/17 – – 11 Cigar M16-05/17 – – 12 Cigar M16-05/17 – – 12 Cigar M16-05/17 – – 12 Cigar M16-05/17 – – 13 Cigar M16-05/17 2592,97 – 13 Cigar M16-05/17 2573,59 – 13 Cigar M16-05/17 2544,75 – 14 Cigar M16-05/17 3707,77 4,13

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 18/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 14 Cigar M16-05/17 3386,25 4,049 14 Cigar M16-05/17 3653,2 4,061 15 Cigar M16-05/17 – 4,725 15 Cigar M16-05/17 – 5,01 15 Cigar M16-05/17 – 4,913 16 Cigar M16-05/17 2726,03 6,044 16 Cigar M16-05/17 2684,11 5,686 16 Cigar M16-05/17 2742,67 5,529 17 Cigar M16-05/17 3117,13 1,777 17 Cigar M16-05/17 2999,46 1,395 17 Cigar M16-05/17 2965,94 2,496 1 CRP1.1 1117,11 0,774 1 CRP1.1 1100,79 0,724 1 CRP1.1 1105,95 0,82 2 CRP1.1 1210,06 1,256 2 CRP1.1 1201,53 1,393 2 CRP1.1 1226,61 1,203 3 CRP1.1 708,65 0,709 3 CRP1.1 739,93 0,738 3 CRP1.1 710,64 0,695 4 CRP1.1 1201,23 0,672 4 CRP1.1 1133,95 0,707 4 CRP1.1 1182,06 0,876 5 CRP1.1 945 – 5 CRP1.1 911,71 – 5 CRP1.1 888,49 – 6 CRP1.1 – 0,246 6 CRP1.1 – 0,467 6 CRP1.1 – 0,405 7 CRP1.1 854.14 0,551 7 CRP1.1 897,3 0,497 7 CRP1.1 898,73 0,513 8 CRP1.1 1058,02 0,726 8 CRP1.1 1020,61 1,31 8 CRP1.1 1016,34 1,138 9 CRP1.1 – 0,782

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 19/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 9 CRP1.1 – 0,958 9 CRP1.1 – 0,678 10 CRP1.1 1144,67 1,001 10 CRP1.1 1113,59 1,17 10 CRP1.1 1095,14 0,964 11 CRP1.1 1095,62 0,811 11 CRP1.1 1107,86 0,869 11 CRP1.1 1060,79 0,96 12 CRP1.1 512,3 1,11 12 CRP1.1 515,73 1,85 12 CRP1.1 520,57 1,34 12 CRP1.1 523,28 – 12 CRP1.1 530,87 – 12 CRP1.1 519,24 – 13 CRP1.1 1103,42 – 13 CRP1.1 1112,02 – 13 CRP1.1 1140,03 – 14 CRP1.1 1308,67 0.881 14 CRP1.1 1368.87 1,099 14 CRP1.1 1327,8 1,193 15 CRP1.1 – 0.696 15 CRP1.1 – 0,689 15 CRP1.1 – 0,678 16 CRP1.1 957,53 0,641 16 CRP1.1 963,7 0,986 16 CRP1.1 915,96 0,756 17 CRP1.1 949,59 0,736 17 CRP1.1 947,28 0,374 17 CRP1.1 926,71 0,917 1 CRP2.1 2520,15 145,236 1 CRP2.1 2511,12 148,117 1 CRP2.1 2520,71 150,547 2 CRP2.1 2672,21 133,016 2 CRP2.1 2727,74 133,632 2 CRP2.1 2634,37 131,035 3 CRP2.1 2009,55 163,373

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 20/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 3 CRP2.1 2014,95 170,672 3 CRP2.1 1980,89 152,943 4 CRP2.1 2523,4 159,776 4 CRP2.1 2541,8 159,071 4 CRP2.1 2527,55 158,191 5 CRP2.1 2083,32 – 5 CRP2.1 2094,26 – 5 CRP2.1 2146,41 – 6 CRP2.1 – 157,203 6 CRP2.1 – 152,759 6 CRP2.1 – 147,352 7 CRP2.1 2624,88 123,418 7 CRP2.1 2431,03 122,48 7 CRP2.1 2694,11 126,62 8 CRP2.1 2399,37 162,087 8 CRP2.1 2387,88 159,981 8 CRP2.1 2343,25 163,741 9 CRP2.1 – 159,369 9 CRP2.1 – 165,754 9 CRP2.1 – 155,566 10 CRP2.1 2666,11 164,219 10 CRP2.1 2644,22 164,466 10 CRP2.1 2606,03 168,755 11 CRP2.1 2577,02 171,31 11 CRP2.1 2529,91 175,78 11 CRP2.1 2635,55 163,369 12 CRP2.1 1456,94 134,767 12 CRP2.1 1487,69 126,206 12 CRP2.1 1487,32 128,646 12 CRP2.1 1559,84 – 12 CRP2.1 1577,55 – 12 CRP2.1 1569,83 – 13 CRP2.1 2482,97 – 13 CRP2.1 2431,96 – 13 CRP2.1 2474,68 – 14 CRP2.1 2991,42 153,808

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 21/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 14 CRP2.1 3017,1 152,262 14 CRP2.1 3175,13 146,656 15 CRP2.1 – 157,562 15 CRP2.1 – 167,926 15 CRP2.1 – 163,784 16 CRP2.1 2405,32 165,799 16 CRP2.1 2352,07 160,052 16 CRP2.1 2418,71 175,17 17 CRP2.1 2580,9 152,001 17 CRP2.1 2522,26 153,986 17 CRP2.1 2526,41 153,764 1 CRP3.1 7344,96 142,849 1 CRP3.1 7329,82 146,41 1 CRP3.1 7319,63 145,202 2 CRP3.1 7294,05 119,106 2 CRP3.1 7287,65 122,879 2 CRP3.1 7224,06 125,733 3 CRP3.1 6532,32 156,368 3 CRP3.1 6361,06 154,617 3 CRP3.1 6436,62 161,26 4 CRP3.1 7082,86 146,702 4 CRP3.1 7080,79 142,284 4 CRP3.1 7123,86 143,024 5 CRP3.1 6676,37 – 5 CRP3.1 6702,43 – 5 CRP3.1 6843,66 – 6 CRP3.1 – 151,207 6 CRP3.1 – 145,27 6 CRP3.1 – 147,931 7 CRP3.1 6440,07 120,235 7 CRP3.1 6713,6 122,277 7 CRP3.1 6801,99 120,154 8 CRP3.1 7091,37 145,096 8 CRP3.1 7176,77 147,63 8 CRP3.1 7346,6 144,275 9 CRP3.1 – 145,21

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 22/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 9 CRP3.1 – 150,198 9 CRP3.1 – 155,745 10 CRP3.1 7951,23 160,023 10 CRP3.1 7744,26 171,229 10 CRP3.1 7343,13 173,402 11 CRP3.1 7729,27 168,759 11 CRP3.1 7814,94 164,649 11 CRP3.1 7726,82 171,533 12 CRP3.1 6433,61 135,382 12 CRP3.1 6453,56 138,869 12 CRP3.1 6447,31 132,973 13 CRP3.1 6673,57 – 13 CRP3.1 6787,51 – 13 CRP3.1 6780,75 – 14 CRP3.1 7975,7 139,686 14 CRP3.1 8393,99 140,034 14 CRP3.1 8125,24 137,056 15 CRP3.1 – 153,822 15 CRP3.1 – 153,017 15 CRP3.1 – 153,694 16 CRP3.1 7234,61 155,941 16 CRP3.1 7232,5 147,217 16 CRP3.1 7297,75 151,32 17 CRP3.1 7900,98 70,969 17 CRP3.1 7328,67 72,195 17 CRP3.1 7950,23 72,595 1 CRP4.1 2115,08 1,194 1 CRP4.1 2128,45 1,302 1 CRP4.1 2137,17 1,175 2 CRP4.1 2075,87 0,808 2 CRP4.1 2044,87 1,038 2 CRP4.1 2042,05 0,945 3 CRP4.1 1778,65 0,927 3 CRP4.1 1797,27 1,026 3 CRP4.1 1719,34 1,057 4 CRP4.1 2141,95 1,262

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 23/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 4 CRP4.1 2144,86 1,284 4 CRP4.1 2178,76 1,139 5 CRP4.1 1712,11 – 5 CRP4.1 1692,45 – 5 CRP4.1 1762,69 – 6 CRP4.1 – 0,921 6 CRP4.1 – 0,951 6 CRP4.1 – 1,041 7 CRP4.1 2108,25 0,972 7 CRP4.1 2322,89 1,403 7 CRP4.1 2281,23 1,118 8 CRP4.1 2008,75 1,397 8 CRP4.1 2082,33 1,684 8 CRP4.1 2000,75 1,451 9 CRP4.1 – 1,097 9 CRP4.1 – 0,95 9 CRP4.1 – 1,056 10 CRP4.1 2080,32 1,295 10 CRP4.1 2072,56 1,751 10 CRP4.1 2133,38 1,498 11 CRP4.1 2124,74 1,564 11 CRP4.1 2168 1,366 11 CRP4.1 1998,32 1,559 12 CRP4.1 1724,8 1,215 12 CRP4.1 1727,8 0,973 12 CRP4.1 1731,19 1,106 13 CRP4.1 1892,9 – 13 CRP4.1 1882,51 – 13 CRP4.1 1900,89 – 14 CRP4.1 2245,51 0,982 14 CRP4.1 2278,07 0,98 14 CRP4.1 2194,66 0,792 15 CRP4.1 – 1,347 15 CRP4.1 – 1,688 15 CRP4.1 – 1,273 16 CRP4.1 1992,05 1,161

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 24/29 Ammonia B[a]P Lab Code Sample (µg/g) (ng/g) 16 CRP4.1 1977,55 1,65 16 CRP4.1 1969,84 1,292 17 CRP4.1 2067,97 2,027 17 CRP4.1 2075,96 0,785 17 CRP4.1 2141,72 1,102

The (–) symbol indicates the laboratory did not submit a result for that sample analysis

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 25/29 APPENDIX C: Raw Data Plots

TTPA-150-1-CTR 2017 Collaborative Study on Ammonia and Benzo[a]pyrene – March 2018 26/29

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 27/29

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 28/29

TTPA-150-1-CTR 2017 Coll. Study on Ammonia and Benzo[a]pyrene – March 2018 29/29