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Smokeless Tobacco Sub-Group Technical Report CORESTA Reference Products 2016 Analysis January 2017 Study Coordinator: John Bunch, Ph.D. American Snuff Company, USA Author and Sub-Group Coordinator: Karl Wagner, Ph.D. Altria Client Services LLC, USA Co-Author and Statistical Analysis: Michael Morton, Ph.D. Altria Client Services LLC, USA

Smokeless Tobacco Sub-Group · Volatiles) of Smokeless Tobacco Products, April 2014. TSNAs: CRM N° 72, Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products

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Text of Smokeless Tobacco Sub-Group · Volatiles) of Smokeless Tobacco Products, April 2014. TSNAs: CRM N°...

  • Smokeless Tobacco Sub-Group

    Technical Report

    CORESTA Reference Products

    2016 Analysis

    January 2017

    Study Coordinator:

    John Bunch, Ph.D.

    American Snuff Company, USA

    Author and Sub-Group Coordinator:

    Karl Wagner, Ph.D.

    Altria Client Services LLC, USA

    Co-Author and Statistical Analysis:

    Michael Morton, Ph.D.

    Altria Client Services LLC, USA

  • Table of Contents

    1. Summary ........................................................................................................................... 3

    2. Introduction ....................................................................................................................... 3

    2.1 Objective ................................................................................................................. 3

    3. Organisation ...................................................................................................................... 4

    3.1 Participants .............................................................................................................. 4

    3.2 Protocol ................................................................................................................... 4

    3.2.1 Sample Shipment ......................................................................................... 4

    3.2.2 Within Laboratory Sample Preparation ....................................................... 5

    3.2.3 Sample Analysis and Data Reporting .......................................................... 5

    4. Data – Raw ........................................................................................................................ 7

    5. Data – Statistical Analysis .............................................................................................. 12

    5.1 Exclusion of Outliers ............................................................................................. 12

    5.2 Calculation of Repeatability (r) and Reproducibility (R) ...................................... 13

    5.3 Calculation of Z-Scores ......................................................................................... 15

    6. Data Interpretations ......................................................................................................... 19

    7. Recommendations ........................................................................................................... 20

    APPENDIX A: Composition of the CRPs ............................................................................... 21

    APPENDIX B: Raw Data Plots for 2016 WG4 Study ............................................................. 25

    APPENDIX C: Comparison of Repeatability and Reproducibility ......................................... 34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 3/34

    1. Summary

    In May 2016, the CORESTA Smokeless Tobacco Sub-Group (STS) initiated an inter-

    laboratory study designed to characterize the four new CORESTA Reference Products (CRPs)

    manufactured in 2016. Nineteen laboratories participated in the study. The participating

    laboratories reported the levels of nicotine, pH, moisture (oven volatiles), tobacco specific

    nitrosamines (TSNAs), ammonia, and benzo[a]pyrene (B[a]P) in the CRPs using CORESTA

    Recommended Methods (CRMs). Tabulated data are presented along with repeatability (r),

    reproducibility (R) and z-scores. The results of the 2016 study establish a starting point for

    future stability studies of the 2016 CRPs.

    2. Introduction

    In November 2008, the STS was established by recommendation of the CORESTA Scientific

    Commission. In 2009, STS Working Group Three (WG3) cooperated to design and

    manufacture four CORESTA Reference Products (CRPs) referred to as CRP1, CRP2, CRP3,

    and CRP4. These products were intended as replacements for the Smokeless Tobacco

    Research Products: 2S3 (Moist Snuff), 1S2 (Dry Snuff) and 2S1 (Loose-leaf Chewing

    Tobacco), which were more than ten years old. The STS recommended monitoring the

    stability of the 2009 CRPs on an annual basis, by determining the levels of nicotine, pH,

    moisture (oven volatiles) and TSNAs using CRMs or draft CRMs1. These studies were

    conducted through 2015 and demonstrated that the CRPs were stable when held at the

    recommended storage conditions of -20 °C.

    In 2015 the supply of the 2009 CRPs decreased to the point that the STS decided to

    remanufacture the CRPs. Similar to the 2009 production, four CRPs were manufactured in

    2016 and included Swedish snus pouch (CRP1.1), American-style loose moist snuff

    (CRP2.1), American-style loose dry snuff powder (CRP3.1), and American-style chopped

    loose-leaf chewing tobacco (CRP4.1). CRP4.1 was produced in a chopped format in 2016 to

    improve within package sample homogeneity as compared to the 2009 loose leaf format.

    Even though the 2016 CRPs included the four product styles that were produced in 2009, it

    was anticipated the chemistry would differ between the two productions considering different

    tobaccos were used and different manufacturers produced three of the reference products.

    The composition of the CRPs supplied by the manufacturers is provided in Appendix A. This

    report serves as the initial characterization of the 2016 CRPs by reporting the levels of

    nicotine, pH, moisture, TSNAs, ammonia and B[a]P using the applicable CRMs.

    The 2009 and 2016 CRPs are stored at -20 °C and distributed by the North Carolina State

    University (NCSU) Tobacco Analytical Services Laboratory.

    2.1 Objective

    The 2016 participating laboratories were to provide analytical results for nicotine, pH,

    moisture (oven volatiles), the four TSNAs (N-nitrosonornicotine (NNN), N-nitrosoanatabine

    (NAT), N-nitrosoanabasine (NAB) 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone

    (NNK)), ammonia, and benzo[a]pyrene (B[a]P). This work was conducted using the

    applicable CRMs referenced in Section 3.

    This study was conducted to support the characterization of the 2016 CRPs and to provide an

    assessment of inter-laboratory variability. Data were collected from the participating

    1 STS Technical Report: CORESTA Reference Products 2010 Analysis, February 2014.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 4/34

    laboratories and statistically evaluated in basic conformance with the recommendations of

    ISO 5725-2:1994 and ISO/TR 22971:2005. Additionally, z-scores were calculated as a

    measure of each laboratory’s performance as compared to the results of other laboratories.

    3. Organisation

    3.1 Participants

    A list of the participating laboratories is provided in Table 1. Not all laboratories provided

    data for all analyses. The laboratories are listed in alphabetical order. Letter codes were

    assigned to each laboratory and do not correspond to the order in the table below.

    Table 1: List of Participating Laboratories

    Participating Laboratories

    Altria Client Services LLC, United States

    American Snuff Company, United States

    Enthalpy Analytical Richmond, United States

    Essentra Scientific Services, United Kingdom

    Fiedler & Lundgren, Sweden

    General Chemistry State Laboratory, Greece

    Global Laboratory Services, United States

    Imperial Tobacco Group, Seita, France

    ITC Limited, India

    ITG Brands, United States

    Japan Tobacco, Inc., Japan

    JTI-Ökolab, Austria

    Labstat International ULC, Canada

    Montepaz S.A., Uruguay

    R.J. Reynolds Tobacco Company, United States

    Swedish Match Northern Europe, Sweden

    Swedish Match, Owensboro, United States

    Swisher International, United States

    University of Kentucky, United States

    3.2 Protocol

    Specific details from the protocol are described below.

    3.2.1 Sample Shipment

    Laboratories were responsible for procuring each of the CRPs from NCSU immediately

    before starting the study. Laboratories were requested to store the samples at approximately

    −4 °C upon receipt if the analyses would be conducted within one week or to store the

    samples at approximately −20 °C if the analyses would be delayed. Laboratories were

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 5/34

    requested to conduct the study in May through July and report data by July 22, 2016. The

    samples are identified in Table 2.

    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

    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 water was 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 and stored at approximately 4 °C for up to one week. Handling

    requirements for the CRPs are described below:

    The snus pouch (CRP1.1) was to be cut into two halves and placed directly into the extraction vessel. Both the tobacco and pouch material were included in the analysis.

    The moist snuff (CRP2.1), dry snuff product (CRP3.1), and chopped loose-leaf (CRP4.1) did not require sample grinding and were to be analyzed as received.

    3.2.3 Sample Analysis and Data Reporting

    The participating laboratories were instructed to conduct triplicate analyses for the following:

    nicotine, pH, moisture (oven volatiles), TSNAs, ammonia, and B[a]P. The laboratories were

    requested to use the current versions of the following CRMs:

    Nicotine: CRM N° 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005. Either the methyl tert-butyl ether

    (MTBE) or n-hexane extraction may be used. The extraction method shall be specified

    in the data reporting sheet.

    pH: CRM N° 69, Determination of pH in Smokeless Tobacco Products, June 2010

    Moisture (oven volatiles): CRM N° 76, Determination of Moisture Content (Oven Volatiles) of Smokeless Tobacco Products, April 2014.

    TSNAs: CRM N° 72, Determination of Tobacco Specific Nitrosamines in Smokeless Tobacco Products by Liquid Chromatography - Tandem Mass Spectrometry, February

    2016.

    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, March 2016.

    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 were excluded from the r&R

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 6/34

    portion of the study, except for ammonia. All ammonia results were included in the statistical

    analysis due to the relatively small number of labs that submitted data without significant

    deviations from CRM N° 79. Deviations reported by the laboratories are identified below as

    well as if the data were excluded from the study.

    Lab A: (1) The CRM was not followed for ammonia. Specifically, a non-suppressed, isocratic method was used with a different mobile phase. (2) The CRM was not

    followed for B[a]P; therefore, the data were excluded from the r&R portion of the

    study.

    Lab C: Minor deviations were reported for nicotine where 2N NaOH was used instead of 5N NaOH. Minor deviations were reported for B[a]P where the concentration of

    the internal standard in the standards and samples differed from that specified.

    Lab E: Minor deviations were reported for nicotine where 5 ml of extraction solution was used instead of 7 ml. Additionally, minor deviations in the GC temperature

    program were also noted.

    Lab G: Minor deviations were reported for TSNAs where two internal standards (d4-NNN and d4-NNK) were used instead of four. Deviations for ammonia were reported

    where unsuppressed conductivity detection was used, the extraction solution was

    10mM MSA, not 0.025N H2SO4, and the analytical column was an Ion Pac CS14.

    Lab H: Minor deviations were reported for nicotine where a different column than that specified in the CRM was used. Specifically, a CP-Wax 52 CB (Polyethylene

    glycol) (30 m × 0.25 mm) was used instead of a 50 % phenyl methylpolysiloxane

    column (15 m × 0.53 mm). Deviations for ammonia were reported where non-

    suppressed conductivity was used instead of suppressed conductivity.

    Lab I: Minor deviations were reported for ammonia where 0.25 g of tobacco was extracted instead of 0.50 g. Minor deviations were reported for pH where the samples

    were left to stand for 1 hour in the dark post shaking prior to measuring pH.

    Lab K: Significant deviations were reported for ammonia where a method other than CRM N° 79 was used.

    Lab N: Minor deviations were reported for ammonia where the calibration type used for ammonia was cubic with offset instead of quadratic.

    Lab O: The laboratory reported that the CRPs were stored at 4 ºC prior to analysis instead of the recommended temperature of -20 ºC.

    Lab Q: Significant deviations were reported for ammonia where a colorimetric method was used instead of CRM N° 79.

    All test results were to be reported on an as-is basis with no 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 e-mail to the study coordinators (John

    Bunch and Karl Wagner).

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 7/34

    4. Data – Raw

    The full data set for the study is listed in Table 3. 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. Raw data plots that include all replicates, without removal of

    outliers, are given in Appendix B.

    As mentioned above, data sets were removed from the r&R portion of the study due to

    significant deviations from the study protocol (except for ammonia) or if the data were

    identified as outlying data. Those data are included in Table 3, but were eliminated prior to

    the r&R analysis.

    Table 3: Full Data Set (results are presented on an as-is basis)

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    A CRP1.1 0.750 8.33 53.70 0.192 0.046 0.141 0.010 1086 0.51

    A CRP1.1 0.740 8.34 53.73 0.191 0.047 0.141 0.009 1087 0.47

    A CRP1.1 0.742 8.33 53.76 0.188 0.047 0.143 0.009 1088 0.52

    B CRP1.1 0.777 8.45 53.47 0.185 0.066 0.159 0.012 772 1.05

    B CRP1.1 0.767 8.49 53.52 0.195 0.065 0.154 0.014 752 0.92

    B CRP1.1 0.766 8.49 53.35 0.199 0.078 0.153 0.012 781 0.79

    C CRP1.1 0.697 8.42 54.29 0.206 0.050 0.145 0.008 – 0.76

    C CRP1.1 0.698 8.45 53.70 0.212 0.050 0.150 0.008 – 0.68

    C CRP1.1 0.702 8.44 53.94 0.197 0.050 0.149 0.008 – 0.65

    D CRP1.1 – 8.33 53.56 – – – – – 0.63

    D CRP1.1 – 8.36 53.73 – – – – – 0.66

    D CRP1.1 – 8.34 53.39 – – – – – 0.66

    E CRP1.1 0.756 8.34 53.69 – – – – 1244 –

    E CRP1.1 0.716 8.36 53.26 – – – – 1094 –

    E CRP1.1 0.733 8.38 53.46 – – – – 1237 –

    F CRP1.1 0.740 8.11 53.78 0.420 0.050 0.130 0.010 – –

    F CRP1.1 0.730 8.12 54.44 0.370 0.050 0.130 0.010 – –

    F CRP1.1 0.740 8.11 53.28 0.430 0.040 0.120 0.010 – –

    G CRP1.1 0.753 8.30 53.12 0.178 0.044 0.130 0.008 893 2.95

    G CRP1.1 0.754 8.27 53.57 0.133

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 8/34

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    K CRP1.1 0.780 8.50 52.24 0.189 0.054 0.144 0.009 1160 0.38

    L CRP1.1 0.670 7.80 53.99 – – – – 974 –

    L CRP1.1 0.690 7.80 54.06 – – – – 912 –

    L CRP1.1 0.800 7.86 53.51 – – – – 965 –

    M CRP1.1 – 8.09 54.00 – – – – – –

    M CRP1.1 – 8.09 54.50 – – – – – –

    M CRP1.1 – 8.19 54.00 – – – – – –

    N CRP1.1 – – – 0.242 0.071 0.202

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 9/34

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    G CRP2.1 1.037 7.74 51.34 3.798 2.293 4.817 0.324 2205 145

    G CRP2.1 1.022 7.72 51.36 3.558 2.162 4.514 0.297 2338 155

    H CRP2.1 1.217 7.51 51.05 3.340 1.869 4.195 0.235 2423 128

    H CRP2.1 1.271 7.53 51.10 3.375 1.924 4.305 0.217 2427 120

    H CRP2.1 1.249 7.55 51.20 3.277 1.819 4.326 0.222 2568 133

    I CRP2.1 – 7.64 51.00 3.450 1.755 4.995 0.258 2228 –

    I CRP2.1 – 7.68 51.14 3.435 1.815 4.785 0.256 2264 –

    I CRP2.1 – 7.65 51.14 3.410 1.775 4.800 0.254 2283 –

    J CRP2.1 0.890 7.75 51.39 3.133 2.234 3.952 0.242 – 122

    J CRP2.1 0.880 7.73 51.75 3.251 1.921 4.042 0.271 – 123

    J CRP2.1 0.820 7.76 51.25 3.156 2.044 3.945 0.282 – 117

    K CRP2.1 1.120 7.76 51.09 3.168 1.953 3.786 0.255 2830 111

    K CRP2.1 1.120 7.77 51.57 3.153 1.959 3.807 0.267 2870 112

    K CRP2.1 1.120 7.74 51.42 3.165 1.959 3.795 0.267 2870 110

    L CRP2.1 1.220 7.65 51.72 – – – – 1928 –

    L CRP2.1 1.260 7.68 51.82 – – – – 1933 –

    L CRP2.1 1.230 7.67 51.68 – – – – 1895 –

    M CRP2.1 – 7.66 51.50 – – – – – –

    M CRP2.1 – 7.61 51.80 – – – – – –

    M CRP2.1 – 7.64 51.50 – – – – – –

    N CRP2.1 – – – 4.081 2.303 4.614 0.282 2703 –

    N CRP2.1 – – – 4.132 2.363 4.474 0.280 2445 –

    N CRP2.1 – – – 4.118 2.297 4.487 0.278 2512 –

    O CRP2.1 1.081 7.94 50.97 – – – – – –

    O CRP2.1 1.083 7.96 50.95 – – – – – –

    O CRP2.1 1.081 8.00 51.10 – – – – – –

    P CRP2.1 – 7.69 51.43 3.750 2.091 4.057 0.238 – 185

    P CRP2.1 – 7.69 51.71 3.686 2.123 4.086 0.236 – 179

    P CRP2.1 – 7.68 51.54 3.645 2.057 3.986 0.242 – 175

    Q CRP2.1 1.040 7.70 51.29 – – – – 2486 –

    Q CRP2.1 1.040 7.76 51.40 – – – – 2695 –

    Q CRP2.1 1.040 7.71 51.38 – – – – 2571 –

    R CRP2.1 1.059 7.86 51.64 3.318 2.133 4.442 0.257 2430 146

    R CRP2.1 1.053 7.83 51.59 3.323 2.211 4.367 0.260 2380 142

    R CRP2.1 1.035 7.85 51.76 3.319 2.108 4.382 0.261 2350 138

    S CRP2.1 1.080 7.75 51.61 – – – – 1950 120

    S CRP2.1 1.070 7.76 51.54 – – – – 1943 128

    S CRP2.1 1.140 7.76 51.55 – – – – 2004 133

    A CRP3.1 1.665 6.89 7.77 5.527 2.257 4.092 0.329 7330 130

    A CRP3.1 1.667 6.88 7.74 5.607 2.289 4.131 0.313 7274 126

    A CRP3.1 1.667 6.89 7.72 5.485 2.234 4.055 0.310 7274 127

    B CRP3.1 1.690 6.96 7.34 5.470 2.871 4.432 0.307 6803 117

    B CRP3.1 1.695 6.94 7.42 5.317 2.799 4.358 0.294 6767 122

    B CRP3.1 1.703 6.92 7.40 5.465 2.852 4.268 0.267 6780 124

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 10/34

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    C CRP3.1 1.633 6.96 7.67 4.798 2.340 3.902 0.319 – 153

    C CRP3.1 1.626 6.97 7.64 4.832 2.514 4.205 0.316 – 160

    C CRP3.1 1.626 6.88 7.63 4.749 2.535 4.107 0.313 – 152

    D CRP3.1 – 6.89 7.30 – – – – – 190

    D CRP3.1 – 6.89 7.35 – – – – – 174

    D CRP3.1 – 6.90 7.21 – – – – – 174

    E CRP3.1 1.659 6.78 7.35 – – – – 7764 –

    E CRP3.1 1.667 6.82 7.28 – – – – 7092 –

    E CRP3.1 1.672 6.78 7.29 – – – – 7903 –

    F CRP3.1 1.700 7.35 7.72 5.870 2.390 4.190 0.340 – –

    F CRP3.1 1.710 7.36 7.31 5.940 2.380 4.190 0.340 – –

    F CRP3.1 1.700 7.36 7.94 5.970 2.390 4.220 0.340 – –

    G CRP3.1 1.652 6.88 7.24 5.852 2.697 4.643 0.354 7139 137

    G CRP3.1 1.653 6.86 7.19 5.686 2.645 4.609 0.348 7106 139

    G CRP3.1 1.657 6.87 7.30 5.840 2.674 4.680 0.342 7128 138

    H CRP3.1 1.851 6.80 7.25 5.470 2.246 4.169 0.244 7527 118

    H CRP3.1 1.869 6.88 7.40 5.409 2.278 4.149 0.241 7604 121

    H CRP3.1 1.859 6.85 7.40 5.372 2.229 4.105 0.239 7500 127

    I CRP3.1 – 6.91 6.67 5.550 2.330 4.930 0.295 7051 –

    I CRP3.1 – 6.94 6.86 5.550 2.405 4.835 0.285 6935 –

    I CRP3.1 – 6.92 6.85 5.600 2.385 4.885 0.302 6763 –

    J CRP3.1 1.340 7.07 7.43 5.062 2.580 4.098 0.331 – 106

    J CRP3.1 1.360 7.06 7.46 5.030 2.447 4.188 0.285 – 108

    J CRP3.1 1.340 7.09 7.48 4.806 2.404 4.175 0.274 – 106

    K CRP3.1 1.790 6.89 7.37 5.598 2.475 3.858 0.303 8180 112

    K CRP3.1 1.770 6.87 7.32 5.616 2.460 3.864 0.300 8120 111

    K CRP3.1 1.780 6.86 7.28 5.673 2.475 3.834 0.303 8160 113

    L CRP3.1 1.780 7.02 8.47 – – – – 6150 –

    L CRP3.1 1.850 6.94 8.65 – – – – 6296 –

    L CRP3.1 1.840 6.89 8.71 – – – – 6477 –

    M CRP3.1 – 6.95 7.60 – – – – – –

    M CRP3.1 – 6.85 7.40 – – – – – –

    M CRP3.1 – 6.87 7.30 – – – – – –

    N CRP3.1 – – – 7.010 2.877 4.594 0.317 6320 –

    N CRP3.1 – – – 6.794 2.861 4.654 0.324 6695 –

    N CRP3.1 – – – 7.005 2.859 4.611 0.327 6346 –

    O CRP3.1 1.749 7.11 6.63 – – – – – –

    O CRP3.1 1.745 7.15 6.74 – – – – – –

    O CRP3.1 1.749 7.08 6.79 – – – – – –

    P CRP3.1 – 6.89 7.43 5.696 2.554 4.057 0.280 – 163

    P CRP3.1 – 6.90 7.42 6.245 2.562 4.085 0.274 – 159

    P CRP3.1 – 6.88 7.40 6.088 2.456 4.140 0.270 – 161

    Q CRP3.1 1.660 6.93 7.35 – – – – 7354 –

    Q CRP3.1 1.650 6.95 7.40 – – – – 7191 –

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 11/34

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    Q CRP3.1 1.650 6.92 7.38 – – – – 7210 –

    R CRP3.1 1.700 6.95 7.42 5.572 2.581 4.578 0.287 6520 133

    R CRP3.1 1.701 6.88 7.35 5.453 2.564 4.558 0.300 6870 130

    R CRP3.1 1.699 6.89 7.49 5.710 2.554 4.546 0.300 6470 133

    S CRP3.1 1.810 7.20 7.45 – – – – 6309 122

    S CRP3.1 1.810 7.22 7.49 – – – – 5961 121

    S CRP3.1 1.810 7.19 7.36 – – – – 6365 133

    A CRP4.1 0.879 6.09 23.82 3.275 0.683 1.377 0.108 2079 1.00

    A CRP4.1 0.867 6.08 23.79 3.211 0.680 1.412 0.107 2074 1.05

    A CRP4.1 0.877 6.07 23.80 3.249 0.675 1.406 0.105 2118 1.09

    B CRP4.1 0.872 6.18 24.73 3.357 0.998 1.713 0.122 1738 1.43

    B CRP4.1 0.869 6.19 24.43 3.177 0.923 1.743 0.117 1787 1.20

    B CRP4.1 0.877 6.13 24.19 3.116 0.875 1.582 0.109 1862 1.03

    C CRP4.1 0.815 6.03 24.82 3.166 0.783 1.662 0.122 – 1.36

    C CRP4.1 0.811 6.02 25.22 3.161 0.750 1.642 0.119 – 0.91

    C CRP4.1 0.814 6.03 25.05 3.253 0.791 1.651 0.112 – 1.07

    D CRP4.1 – 6.14 23.56 – – – – – 1.81

    D CRP4.1 – 6.13 23.30 – – – – – 1.54

    D CRP4.1 – 6.13 23.73 – – – – – 1.61

    E CRP4.1 0.904 6.01 23.42 – – – – 2137 –

    E CRP4.1 0.901 6.02 23.56 – – – – 2172 –

    E CRP4.1 0.907 6.01 24.03 – – – – 2016 –

    F CRP4.1 0.930 6.35 23.34 4.810 0.730 1.600 0.150 – –

    F CRP4.1 0.890 6.35 24.36 4.760 0.720 1.620 0.150 – –

    F CRP4.1 0.890 6.34 24.14 4.730 0.730 1.580 0.150 – –

    G CRP4.1 0.862 6.02 23.52 3.540 0.868 1.615 0.126 1965 2.13

    G CRP4.1 0.854 6.02 23.54 3.576 0.873 1.650 0.132 1975 2.41

    G CRP4.1 0.857 6.02 23.76 3.496 0.802 1.679 0.130 1919 2.68

    H CRP4.1 0.940 5.91 23.80 3.251 0.727 1.563 0.095 1975 1.38

    H CRP4.1 0.949 5.92 23.80 3.292 0.784 1.586 0.098 1957 1.40

    H CRP4.1 0.956 5.94 23.55 3.315 0.706 1.554 0.094 2074 1.29

    I CRP4.1 – 6.05 22.67 3.290 0.710 1.640 0.104 1803 –

    I CRP4.1 – 6.04 22.98 3.365 0.750 1.705 0.110 1829 –

    I CRP4.1 – 6.05 22.96 3.230 0.715 1.640 0.103 1809 –

    J CRP4.1 0.930 6.11 23.97 3.352 0.795 1.451 0.116 – 1.12

    J CRP4.1 0.840 6.10 24.16 3.205 0.772 1.409 0.105 – 1.14

    J CRP4.1 0.790 6.13 24.39 3.098 0.765 1.475 0.091 – 1.01

    K CRP4.1 0.940 6.04 23.16 3.273 0.702 1.437 0.102 2520 0.88

    K CRP4.1 0.910 6.04 23.26 3.264 0.699 1.437 0.108 2510 0.92

    K CRP4.1 0.900 6.06 23.51 3.297 0.714 1.437 0.111 2480 0.90

    L CRP4.1 1.030 6.04 24.86 – – – – 1850 –

    L CRP4.1 1.100 6.03 25.23 – – – – 1809 –

    L CRP4.1 1.100 5.90 25.08 – – – – 1803 –

    M CRP4.1 – 6.01 24.40 – – – – – –

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 12/34

    Lab code

    Product Nicotine pH Moisture NNN NNK NAT NAB Ammonia B[a]P

    % % µg/g µg/g µg/g µg/g µg/g ng/g

    M CRP4.1 – 5.98 24.30 – – – – – –

    M CRP4.1 – 5.98 24.30 – – – – – –

    N CRP4.1 – – – 3.953 0.942 1.895 0.119 1833 –

    N CRP4.1 – – – 4.077 0.978 1.820 0.122 1869 –

    N CRP4.1 – – – 4.022 0.999 1.858 0.126 1781 –

    O CRP4.1 0.894 6.24 24.55 – – – – – –

    O CRP4.1 0.898 6.25 24.51 – – – – – –

    O CRP4.1 0.899 6.19 24.48 – – – – – –

    P CRP4.1 – 6.07 24.66 3.696 0.784 1.439 0.098 – 1.34

    P CRP4.1 – 6.05 24.61 3.670 0.795 1.522 0.098 – 1.23

    P CRP4.1 – 6.06 24.73 3.751 0.795 1.461 0.105 – 1.28

    Q CRP4.1 0.860 6.09 25.79 – – – – 2114 –

    Q CRP4.1 0.860 6.01 25.83 – – – – 2016 –

    Q CRP4.1 0.870 6.01 25.87 – – – – 2054 –

    R CRP4.1 0.873 6.13 23.73 3.379 0.867 1.690 0.116 2050 0.86

    R CRP4.1 0.890 6.07 23.82 3.263 0.806 1.586 0.110 1990 0.85

    R CRP4.1 0.873 6.08 23.80 3.389 0.754 1.641 0.111 2040 0.76

    S CRP4.1 0.880 6.18 24.79 – – – – 1717 1.34

    S CRP4.1 0.950 6.15 25.24 – – – – 1698 1.45

    S CRP4.1 0.960 6.15 25.27 – – – – 1712 1.57

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

    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. Even though ISO 5725-2:1994 does not suggest calculation of z-scores, z-scores

    are presented in section 5.3 so that the participating laboratories would have an additional

    measure of their performance compared to their peers. Raw data plots that include all

    replicates, without removal of outliers, are shown in Appendix B.

    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 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 ( cnk labs ). 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.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 13/34

    The intent of ISO 5725-2:1994 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 5725-2:1994, 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 4. 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.2

    Table 4: Outliers among 2016 Data

    Analyte Product Levene’s Outlier Lab Grubbs’ Outlier Lab

    Nicotine CRP1.1 − J

    Nicotine CRP3.1 − J

    Nicotine CRP4.1 − L

    Moisture CRP3.1 F −

    Moisture CRP3.1 − L

    NNN CRP1.1 − F

    NNN CRP2.1 − F

    NNN CRP4.1 − F

    NAB CRP2.1 − F

    NAB CRP4.1 J −

    NAT CRP3.1 C −

    B[a]P CRP1.1 − G

    B[a]P CRP4.1 G −

    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 limits (r & R) were calculated.

    The r & R results are shown in Table 5. The r & R results reflect both laboratory variability

    and product consistency.

    2 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 0.02. 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.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 14/34

    Table 5: Repeatability (r) and Reproducibility (R) Limits

    Parameter Product N° of

    Labs* Mean

    Repeatability Reproducibility

    r % r R % R

    Nicotine (%) CRP1.1 13 0.762 0.084 11.0% 0.128 16.8%

    Nicotine (%) CRP2.1 14 1.069 0.053 5.0% 0.265 24.8%

    Nicotine (%) CRP3.1 13 1.722 0.033 1.9% 0.211 12.2%

    Nicotine (%) CRP4.1 13 0.886 0.070 7.9% 0.116 13.1%

    pH CRP1.1 18 8.296 0.080 NA 0.497 NA

    pH CRP2.1 18 7.737 0.047 NA 0.282 NA

    pH CRP3.1 18 6.960 0.084 NA 0.399 NA

    pH CRP4.1 18 6.082 0.074 NA 0.283 NA

    Moisture (%) CRP1.1 18 53.95 1.11 2.1% 1.84 3.4%

    Moisture (%) CRP2.1 18 51.41 0.33 0.6% 0.74 1.4%

    Moisture (%) CRP3.1 16 7.33 0.19 2.6% 0.74 10.1%

    Moisture (%) CRP4.1 18 24.19 0.60 2.5% 2.15 8.9%

    NNN (µg/g) CRP1.1 11 0.190 0.042 22.1% 0.068 35.8%

    NNN (µg/g) CRP2.1 11 3.391 0.212 6.3% 0.874 25.8%

    NNN (µg/g) CRP3.1 12 5.631 0.324 5.7% 1.536 27.3%

    NNN (µg/g) CRP4.1 11 3.394 0.196 5.8% 0.741 21.8%

    NNK (µg/g) CRP1.1 11 0.052 0.013 25.2% 0.030 56.6%

    NNK (µg/g) CRP2.1 12 2.059 0.210 10.2% 0.525 25.5%

    NNK (µg/g) CRP3.1 12 2.512 0.137 5.5% 0.567 22.6%

    NNK (µg/g) CRP4.1 12 0.790 0.090 11.4% 0.261 33.1%

    NAT (µg/g) CRP1.1 12 0.140 0.032 22.9% 0.071 50.7%

    NAT (µg/g) CRP2.1 12 4.237 0.371 8.8% 0.888 21.0%

    NAT (µg/g) CRP3.1 11 4.327 0.116 2.7% 0.876 20.2%

    NAT (µg/g) CRP4.1 12 1.588 0.108 6.8% 0.378 23.8%

    NAB (µg/g) CRP1.1 10 0.009 0.003 35.9% 0.006 63.2%

    NAB (µg/g) CRP2.1 11 0.265 0.027 10.2% 0.067 25.4%

    NAB (µg/g) CRP3.1 12 0.303 0.033 10.8% 0.085 28.0%

    NAB (µg/g) CRP4.1 11 0.115 0.011 9.4% 0.043 37.9%

    Ammonia (µg/g) CRP1.1 12 1034 116 11.2% 416 40.2%

    Ammonia (µg/g) CRP2.1 12 2356 190 8.1% 860 36.5%

    Ammonia (µg/g) CRP3.1 12 7020 504 7.2% 1681 23.9%

    Ammonia (µg/g) CRP4.1 12 1976 122 6.2% 603 30.5%

    B[a]P (ng/g) CRP1.1 9 0.716 0.410 57.2% 0.581 81.1%

    B[a]P (ng/g) CRP2.1 10 143.9 17.5 12.2% 73.1 50.8%

    B[a]P (ng/g) CRP3.1 10 135.2 12.1 9.0% 65.9 48.7%

    B[a]P (ng/g) CRP4.1 9 1.210 0.350 28.9% 0.789 65.2%

    *This is the number of laboratory data sets reported above the LOQ and after removal of outliers.

    NA = Since pH is not a proportional scale, it is not appropriate to calculate % r or % R.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 15/34

    5.3 Calculation of Z-Scores

    Although calculation of z-scores is not suggested in ISO 5725-2:1994, z-scores were

    calculated so that the participating laboratories could compare their results to those of their

    peers. As mentioned above, data sets were removed from the r&R portion of the study due to

    significant deviations from the study protocol (except for ammonia) or if the data were

    identified as outlying data. Those data are included in the calculation of z-scores.

    The z-scores were calculated using methods suggested in ISO 13528: 2005. The calculations

    were based on Algorithm A given in Annex C of ISO 13528: 2005. It is expected that most of

    the z-scores should fall within the range of ±2, and that laboratories having values with |z|>3

    should be treated as an “action signal” to investigate laboratory performance. Final summary

    tables of z-scores are presented in Tables 6a through 6e for the study.

    Table 6a: Z-Scores

    Nicotine pH

    Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1 CRP1.1 CRP2.1 CRP3.1 CRP4.1

    A -0.25 -0.47 -0.49 -0.41 0.15 -0.35 -0.50 0.08

    B 0.35 -0.34 -0.16 -0.45 1.02 2.06 0.01 1.06

    C -1.32 -1.04 -0.91 -1.88 0.78 -0.19 0.01 -0.55

    D − − − − 0.21 0.99 -0.43 0.71

    E -0.47 0.03 -0.49 0.30 0.31 -0.27 -1.43 -0.71

    F -0.43 -0.25 -0.08 0.28 -1.21 -0.31 4.22 3.23

    G -0.07 -0.59 -0.63 -0.81 -0.14 0.01 -0.66 -0.63

    H 2.12 3.22 1.65 1.36 -1.21 -2.46 -0.93 -1.77

    I − − − − -1.15 -0.92 -0.13 -0.32

    J -5.81 -3.56 -4.02 -0.92 0.46 0.18 1.38 0.47

    K 0.58 0.99 0.77 0.60 0.97 0.30 -0.63 -0.32

    L -0.82 3.06 1.25 4.44 -3.01 -0.80 0.14 -0.99

    M − − − − -1.15 -1.16 -0.46 -0.99

    N − − − − − − − −

    O 0.60 0.31 0.41 0.12 0.48 2.85 1.78 1.81

    P − − − − -0.34 -0.55 -0.46 -0.16

    Q 1.36 -0.43 -0.63 -0.68 0.60 -0.11 -0.03 -0.44

    R 0.27 -0.27 -0.12 -0.31 1.25 1.40 -0.31 0.27

    S 0.19 0.58 1.10 0.92 0.46 0.30 2.68 1.02

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 16/34

    Table 6b: Z-Scores

    Moisture NNN

    Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1 CRP1.1 CRP2.1 CRP3.1 CRP4.1

    A -0.26 -0.60 1.59 -0.48 -0.02 -0.68 -0.10 -0.62

    B -1.08 0.75 -0.12 0.37 0.10 -0.56 -0.38 -0.72

    C 0.46 0.41 1.14 1.13 0.66 -1.08 -1.81 -0.81

    D -0.75 -0.43 -0.59 -0.84 − − − −

    E -1.02 -1.44 -0.50 -0.65 − − − −

    F 0.04 1.42 1.19 -0.29 10.23 11.16 0.78 4.98

    G -0.78 -0.23 -0.80 -0.74 -1.36 0.53 0.48 0.46

    H 3.05 -1.04 -0.29 -0.59 -0.11 -0.26 -0.38 -0.47

    I -0.51 -1.12 -2.97 -1.70 0.09 0.04 -0.04 -0.43

    J 0.47 0.20 0.23 0.01 -0.41 -0.71 -1.42 -0.71

    K -0.22 -0.17 -0.42 -1.13 -0.32 -0.77 0.10 -0.50

    L 0.10 1.18 5.79 1.17 − − − −

    M 1.01 0.68 0.11 0.22 − − − −

    N − − − − 2.39 2.07 3.10 2.22

    O 3.65 -1.43 -3.33 0.45 − − − −

    P -0.97 0.54 0.03 0.65 -1.19 0.82 0.97 1.08

    Q 4.53 -0.18 -0.16 2.18 − − − −

    R -0.54 0.91 0.05 -0.50 -0.41 -0.29 -0.01 -0.25

    S -0.40 0.56 0.11 1.22 − − − −

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 17/34

    Table 6c: Z-Scores

    NNK NAT

    Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1 CRP1.1 CRP2.1 CRP3.1 CRP4.1

    A -0.53 -0.92 -1.14 -1.21 0.13 -0.72 -0.62 -1.37

    B 2.43 1.41 1.52 1.74 0.74 -0.19 0.17 0.73

    C -0.10 0.29 -0.21 -0.10 0.41 -0.35 -0.69 0.52

    D − − − − − − − −

    E − − − − − − − −

    F -0.52 -0.53 -0.56 -0.66 -0.53 -0.29 -0.30 0.14

    G -0.86#

    0.62 0.75 0.76 -0.90 1.09 1.06 0.49

    H -0.73 -0.94 -1.18 -0.51 -0.14 0.18 -0.48 -0.11

    I 0.59 -1.38 -0.62 -0.68 1.02 2.12 1.79 0.60

    J 0.21 0.04 -0.15 -0.07 -1.11 -0.80 -0.44 -1.02

    K 0.04 -0.51 -0.18 -0.91 0.05 -1.41 -1.36 -1.08

    L − − − − − − − −

    M − − − − − − − −

    N 2.61 1.30 1.63 2.22 2.38 1.01 0.98 2.06

    O − − − − − − − −

    P -1.07 0.16 0.07 0.10 -1.46 -0.59 -0.62 -0.80

    Q − − − − − − − −

    R * 0.46 0.26 0.31 0.29 0.59 0.80 0.43

    S − − − − − − − −

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

    The (#) symbol indicates one of the three replicate values was reported as less than the LOQ and was omitted.

    The (*) symbol indicates all three replicates were less than the LOQ.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 18/34

    Table 6d: Z-Scores

    NAB Ammonia

    Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1 CRP1.1 CRP2.1 CRP3.1 CRP4.1

    A 0.66 0.30 0.44 -0.46 0.32 0.41 0.47 0.77

    B 2.29 -0.54 -0.54 0.28 -1.70 -1.30 -0.35 -0.88

    C -0.39 0.77 0.39 0.40 − − − −

    D − − − − − − − −

    E − − − − 0.98 0.75 0.94 0.87

    F 0.83 9.93 1.25 2.96 − − − −

    G 0.12#

    1.23 1.53 1.36 -1.20 -0.31 0.20 0.00

    H -0.59 -1.53 -2.25 -1.31 0.30 0.34 0.87 0.28

    I 0.10 -0.45 -0.39 -0.55 -0.19 -0.28 -0.14 -0.78

    J -1.39 -0.13 -0.28 -0.65 − − − −

    K 0.22 -0.20 -0.10 -0.42 0.73 1.46 1.86 3.09

    L − − − − -0.55 -1.28 -1.12 -0.74

    M − − − − − − − −

    N * 0.40 0.64 0.77 0.92 0.58 -0.89 -0.70

    O − − − − − − − −

    P -1.06 -1.04 -1.08 -0.93 − − − −

    Q − − − − 0.59 0.67 0.40 0.61

    R * -0.32 -0.32 0.02 0.69 0.09 -0.62 0.42

    S − − − − -1.09 -1.14 -1.28 -1.37

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

    The (#) symbol indicates one of the three replicate values was reported as less than the LOQ and was omitted.

    The (*) symbol indicates all three replicates were less than the LOQ.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 19/34

    Table 6e: Z-Scores

    B[a]P

    Lab CRP1.1 CRP2.1 CRP3.1 CRP4.1

    A -1.01 -0.40 -0.26 -0.60

    B 0.84 -0.29 -0.55 -0.07

    C -0.14 0.58 0.97 -0.39

    D -0.35 1.84 2.04 1.25

    E − − − −

    F − − − −

    G 10.57 0.33 0.21 3.55

    H 0.40 -0.59 -0.50 0.35

    I − − − −

    J 0.21 -0.81 -1.19 -0.46

    K -1.47 -1.20 -0.95 -1.05

    L − − − −

    M − − − −

    N − − − −

    O − − − −

    P 0.28 1.45 1.23 0.13

    Q − − − −

    R -0.91 0.00 -0.07 -1.28

    S 0.64 -0.56 -0.36 0.64

    The (–) symbol indicates the laboratory did not submit data for that sample analysis.

    6. Data Interpretations

    The averages, percent repeatability, and percent reproducibility from this study are compared

    in Appendix C to the most recent WG4 study (2015) for nicotine, moisture, pH and TSNA

    and to the 2014 ammonia Technical Report and the 2016 B[a]P Technical Report. The

    comparisons of this year’s study are made to the corresponding 2009 CRPs. It can be seen

    that the chemistry differs in many instances between the 2009 and 2016 CRP productions.

    This was anticipated considering different crop-year tobaccos were used and different

    manufacturers produced three of the reference products. Although many small differences

    exist between the two productions, notable differences include the following:

    • B[a]P is significantly higher in CRP2.1 and CRP3.1 as compared to the corresponding 2009 CRPs

    • Ammonia is significantly higher in CRP3.1 as compared to CRP3

    We also noted several instances where variability from this year’s study was greater from the

    previous studies. With B[a]P the difference seems to correlate with the significantly higher

    concentration of B[a]P in both CRP2.1 and CRP3.1. The levels of B[a]P in these CRPs far

    exceeds that from the 2015 B[a]P collaborative study, which is the basis for the CRM. At this

    time it is not clear why the variation is greater than previously seen. We also noted increased

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 20/34

    variability for the TSNAs in CRP1.1 as compared the previous study. The concentrations of

    the TSNAs in CRP1.1 are extremely low and a plausible cause for this increased variability is

    that the concentrations are near the lower analytical working range for many laboratories.

    7. Recommendations

    The results of this study suggest the 2016 CRPs are characteristic of the product styles and are

    suitable for use as reference products. This study will also serve as a baseline for future

    analyses. At the STS meeting held in Berlin, Germany October 9, 2016, the STS decided to

    conduct a follow-up collaborative study to investigate the increased reproducibility values

    seen in this study for B[a]P as compared to the 2015 B[a]P collaborative study.

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 21/34

    APPENDIX A: Composition of the CRPs

    CRP1.1- Swedish Snus

    Manufactured in January 2016

    CRP1.1 is a Swedish-style snus smokeless tobacco product that was produced without added

    flavorings, except for those required to produce a product that is characteristic of the style.

    The reference product is packaged in plastic cans that contain twenty four 1g pouches.

    The target product composition supplied by the manufacturer is given below:

    Component % (wwb)

    Air-cured tobacco 8.4

    Air-cured stem 0.2

    Sun-cured tobacco 5.4

    Sun-cured stem 9.7

    Flue-cured stem 12.8

    Propylene glycol 2.2

    Sodium carbonate 1.8

    Sodium chloride 4.0

    Water 51.4

    Pouch paper 4.1

    Total 100

    wwb: wet weight basis

    The following parameters were determined at the time of manufacture:

    Parameter Value

    Moisture content 53.7 %

    pH 8.4

    Nicotine content 0.8 % (wwb)

    wwb: wet weight basis

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 22/34

    CRP2.1- Moist Snuff

    Manufactured in April 2016

    CRP2.1 is an American-style loose moist snuff smokeless tobacco product that was produced

    without added flavorings, except for those required to produce a product that is characteristic

    of the style. The reference product is packaged in plastic cans that contain 34 grams of

    product.

    The target product composition supplied by the manufacturer is given below:

    Component % (wwb)

    Dark-fired tobacco 26.6

    Air-cured tobacco 8.1

    Burley Stem 3.8

    Sodium carbonate 0.5

    Sodium chloride 8.0

    Water 53.0

    Total 100

    wwb: wet weight basis

    The following parameters were determined at the time of manufacture:

    Parameter Value

    Moisture content 53 %

    pH 7.7

    Nicotine content 1.2 % (wwb)

    wwb: wet weight basis

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 23/34

    CRP3.1- Dry Snuff

    Manufactured in April 2016

    CRP3.1 is an American-style loose dry snuff smokeless tobacco product that was produced

    without added flavorings, except for those required to produce a product that is characteristic

    of the style. The reference product is packaged in plastic cans that contain 34 g of product.

    The target product composition supplied by the manufacturer is given below:

    Component % (wwb)

    Dark-fired tobacco 54.8

    Burley Stem 33.6

    Sodium carbonate 1.1

    Sodium chloride 1.1

    Ammonium carbonate 1.4

    Water 8.0

    Total 100

    wwb: wet weight basis

    The following parameters were determined at the time of manufacture:

    Parameter Value

    Moisture content 8.1%

    pH 7.1

    Nicotine content 1.7% (wwb)

    wwb: wet weight basis

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 24/34

    CRP4.1- Chopped Loose-leaf

    Manufactured in January 2016

    CRP4.1 is a chopped American-style loose-leaf smokeless tobacco product that was produced

    without added flavorings, except for those required to produce a product that is characteristic

    of the style. The reference product is packaged in plastic cans that contain 28g of product.

    The target product composition supplied by the manufacturer is given below:

    Component % (wwb)

    Air-cured tobacco 32.4

    Air-cured stem 5.8

    Sodium chloride 1.8

    Sodium propionate 0.3

    Glycerin 6.8

    Sucrose 10.9

    Dextrose 3.5

    Maltose 1.6

    Corn syrup solids 13.9

    Water 23.0

    Total 100

    wwb: wet weight basis

    The following parameters were determined at the time of manufacture:

    Parameter Value

    Moisture content 23 %

    pH 6.2

    Nicotine content 0.9 % (wwb)

    wwb: wet weight basis

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 25/34

    APPENDIX B: Raw Data Plots for 2016 WG4 Study

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 26/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 27/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 28/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 29/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 30/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 31/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 32/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 33/34

  • STS-105-CTR 2016 Analysis of CRPs – January 2017 34/34

    APPENDIX C: Comparison of Repeatability and Reproducibility

    Parameter Product N° of

    Labs*

    Current Study

    1

    Previous Studies

    2

    Current Study

    1

    Previous Studies

    2

    Current Study

    1

    Previous Studies

    2

    Mean Mean % r % r % R % R

    Nicotine (%) CRP1.1 13 0.762 0.98 11.00% 12.30% 16.80% 21.50%

    Nicotine (%) CRP2.1 14 1.069 1.29 5.00% 4.50% 24.80% 12.60%

    Nicotine (%) CRP3.1 13 1.722 2.17 1.90% 2.30% 12.20% 10.30%

    Nicotine (%) CRP4.1 13 0.886 1.09 7.90% 8.10% 13.10% 22.90%

    pH CRP1.1 18 8.296 7.85 NA NA NA NA

    pH CRP2.1 18 7.737 7.65 NA NA NA NA

    pH CRP3.1 18 6.96 6.75 NA NA NA NA

    pH CRP4.1 18 6.082 5.91 NA NA NA NA

    Moisture (%) CRP1.1 18 53.95 51.06 2.10% 8.20% 3.40% 10.60%

    Moisture (%) CRP2.1 18 51.41 54.34 0.60% 1.30% 1.40% 1.40%

    Moisture (%) CRP3.1 16 7.33 10.05 2.60% 2.90% 10.10% 12.80%

    Moisture (%) CRP4.1 18 24.19 22.39 2.50% 2.60% 8.90% 7.90%

    NNN (µg/g) CRP1.1 11 0.19 0.67 22.10% 11.20% 35.80% 16.80%

    NNN (µg/g) CRP2.1 11 3.391 1.81 6.30% 6.80% 25.80% 16.20%

    NNN (µg/g) CRP3.1 12 5.631 8.2 5.70% 7.60% 27.30% 15.80%

    NNN (µg/g) CRP4.1 11 3.394 1.92 5.80% 7.60% 21.80% 19.80%

    NNK (µg/g) CRP1.1 11 0.052 0.21 25.20% 14.20% 56.60% 27.10%

    NNK (µg/g) CRP2.1 12 2.059 0.44 10.20% 7.20% 25.50% 22.50%

    NNK (µg/g) CRP3.1 12 2.512 4.15 5.50% 11.80% 22.60% 25.50%

    NNK (µg/g) CRP4.1 12 0.79 0.44 11.40% 14.20% 33.10% 24.40%

    NAT (µg/g) CRP1.1 12 0.14 0.51 22.90% 12.60% 50.70% 29.70%

    NAT (µg/g) CRP2.1 12 4.237 1.72 8.80% 5.70% 21.00% 22.90%

    NAT (µg/g) CRP3.1 11 4.327 5.54 2.70% 7.70% 20.20% 27.40%

    NAT (µg/g) CRP4.1 12 1.588 1.2 6.80% 7.80% 23.80% 36.30%

    NAB (µg/g) CRP1.1 10 0.009 0.035 35.90% 33.60% 63.20% 42.50%

    NAB (µg/g) CRP2.1 11 0.265 0.152 10.20% 10.60% 25.40% 20.90%

    NAB (µg/g) CRP3.1 12 0.303 0.398 10.80% 10.40% 28.00% 26.20%

    NAB (µg/g) CRP4.1 11 0.115 0.059 9.40% 11.10% 37.90% 31.50%

    Ammonia (µg/g)

    CRP1.1 12 1034 951 11.20% 11.90% 40.20% 55.60%

    Ammonia (µg/g)

    CRP2.1 12 2356 2581 8.10% 4.00% 36.50% 40.80%

    Ammonia (µg/g)

    CRP3.1 12 7020 4786 7.20% 3.10% 23.90% 18.20%

    Ammonia (µg/g)

    CRP4.1 12 1976 2769 6.20% 16.70% 30.50% 24.80%

    B[a]P (ng/g) CRP1.1 9 0.716 0.7 57.20% 20% 81.10% 79%

    B[a]P (ng/g) CRP2.1 10 143.9 56.2 12.20% 6% 50.80% 22%

    B[a]P (ng/g) CRP3.1 10 135.2 40.3 9.00% 12% 48.70% 22%

    B[a]P (ng/g) CRP4.1 9 1.21 1.11 28.90% 32% 65.20% 64%

    1. The columns labelled “Current Study“ include data from the current 2016 study for the 2016 CRPs. 2. The columns labelled “Previous Studies“ includes data for the 2009 CRPs3,4,5.

    3 STS Technical Report: CORESTA Reference Products (Smokeless Tobacco) - 2015 Analysis, December 2015.

    4 STS Technical report: Determination of Ammonia in Tobacco and Smokeless Tobacco Products, August 2014.

    5 STS Technical report: 2015 Collaborative Study on Benzo[a]pyrene in Tobacco Products, March 2016.

    1. Summary2. Introduction2.1 Objective

    3. Organisation3.1 Participants3.2 Protocol3.2.1 Sample Shipment3.2.2 Within Laboratory Sample Preparation3.2.3 Sample Analysis and Data Reporting

    4. Data – Raw5. Data – Statistical Analysis5.1 Exclusion of Outliers5.2 Calculation of Repeatability (r) and Reproducibility (R)5.3 Calculation of Z-Scores

    6. Data Interpretations7. RecommendationsAPPENDIX A: Composition of the CRPsAPPENDIX B: Raw Data Plots for 2016 WG4 StudyAPPENDIX C: Comparison of Repeatability and Reproducibility