Technical Report Electronic Cigarette Aerosol Parameters Study

Transcription

1 E-Cigarette Task Force Technical Report 2014 Electronic Cigarette Aerosol Parameters Study March 2015 Task Force Coordinator: Chuck Garner, Ph.D. R. J. Reynolds, U.S.A. Task Force Secretary: Robert D. Stevens, Ph.D. Lorillard Tobacco Company, U.S.A. Author: Rana Tayyarah Lorillard Tobacco Company, U.S.A

2 Table of Contents 1. Summary 3 2. Introduction 3 3. Organisation Participants Protocol 4 4. Data - Raw 6 5. Data - Statistical Analysis Exclusion of Outliers Calculation of Repeatability and Reproducibility Calculation of Z-Scores 7 6. Data Interpretation Machine or product performance issues Impact of regime on overall yields Impact on relative yields of analytes Impact of regime on cumulative yield or puff trending Trend with flow rate among the test parameters Recommendations Additional Considerations Acknowledgements 12 APPENDIX A: Study Protocol and Data Template 13 APPENDIX D: Total Yield Graphs 80 APPENDIX E: Puff Set Graphs 84 APPENDIX F: ACM Variability - Percent Relative Standard Deviation for Puff Sets 88 ECIG-CTR Aerosol Parameters March /89

3 1. Summary In 2014, the CORESTA E-Cigarette Task Force (TF) conducted a collaborative study that included testing of commercial electronic cigarette products. This study was referred to as E- Cigarette Aerosol Parameters Study. The laboratory phase of this study was conducted from late 2013 to early 2014 and the results were presented to the Task Force in May The study included determinations of puff count, trapped aerosol, propylene glycol, nicotine, water, glycerin, and product weight loss. Four laboratories participated in the study. Each laboratory tested two products of choice. The goal of this work was to compare results from cigarette-machine puffing of e-cigarettes under a range of puffing parameters to make a recommendation for standardized parameters. From the previous TF meeting, parameters of 55mL volume taken over 3s every 30s using a square profile shape were favored. Thus, the more specific objective was to determine if this regime should be recommended as the standard or if any of the other regimes included in this study should be recommended. The study results demonstrate that any of the regimes evaluated would be suitable for machinetesting of e-cigarettes of a conventional design 1 for the determination of major constituents in trapped aerosol. Parameters of 55mL/3s/30s/square will be used for an upcoming proficiency test. These parameters will serve as a recommended machine regime for e-cigarette aerosol collection. 2. Introduction Though electronic cigarettes do not specifically contain tobacco, this is a relatively new product category that is of interest to many CORESTA members. A task force related to electronic cigarettes was formed by the Scientific Commission after a November 2012 workshop. One of the short term objectives of the Task Force is to gather and share preliminary data on analysis relevant to e-cigarettes worldwide with a view to making recommendations for product testing. As yet, there is no standardized method for puffing electronic cigarettes. Thus, evaluation of published literature and conductance of collaborative studies is hindered. At the September 2013 Task Force meeting, there was extensive discussion regarding puffing parameters 2. From this discussion it was clear that parameters of 55mL volume taken over 3s every 30s using a square profile shape were favored by the majority of the TF members but that there may be some benefit to an alternate volume (70mL) or an alternate puff duration (4s). Though typical for cigarette smoking, 35 ml puff volume, bell-shaped profiles, and 2s puff duration were each recommended against by researchers as being problematic from some e-cigarette products. The primary concerns discussed related to insufficient product trigger (time/heat), insufficient overlap between the machine (bell) profile and/or machine duration and the start and end times for some products puff time. Puff durations of 5s or longer were recommended against as being too long from some products. In order to prepare for a future proficiency study, the Task Force commissioned a study to determine the effect of puff profile on the yield of the major constituents in e-cigarette aerosols in order to recommend puffing parameters for aerosol collection. The study involved comparison of puffing parameters combinations including 55mL and 70mL puff volumes of 3s or 4s duration. Each participating laboratory tested two products of choice for glycerin, propylene glycol, water, and nicotine using six regimes. Since laboratories did not necessarily test the same products, no comparisons were made between any of the products 1 Self-actuating electronic cigarettes, approximately 25mm circumference with a cylindrical mouth-end 2 See Task Force meeting minutes from the September 2013 meeting for additional detail. ECIG-CTR Aerosol Parameters March /89

4 results. Comparisons were limited to understanding the impact of the regimes on yield, consistency of results, and reliability of the testing equipment for each product sample. Essentially, the study data were treated as eight individual studies of the impact of puffing parameters. 3. Organisation 3.1 Participants A list of the participating laboratories is provided in Table 1. The laboratories are listed in alphabetical order. The numerical laboratory codes (1-4) used in this report do not necessarily correspond to the same order as the list below. Table 1: List of Participating Laboratories Laboratory Altria Client Services, Inc. Arista Laboratories Enthalpy Analytical, Inc. Lorillard Tobacco Company 3.2 Protocol Participating laboratories supplied their own samples for testing. White Cloud and Red Kiwi supplied two of the laboratories with products. It was requested that two models, one disposable and one rechargeable, be tested. Sample identification was withheld with data reporting; samples were only identified as rechargeable or disposable. Participants were directed to store products at room temperature and that batteries be fully charged prior to use for the rechargeable products. Each laboratory conducted five replicate determinations for each product (Table 2) under each of the six regimes (Table 3). Test 1 (ISO) and Test 2 (Health Canada) were included in the study for informational purposes only. Products were tested for one battery charge to a maximum 300 puffs. Puffs were collected in 25 puff increments in a manner typical for each laboratory for e-cigarette aerosol collection. e-cigarettes were weighed between each set of 25 puffs for calculation of weight loss. Each laboratory tested the samples for the specified analytes using methods of choice. See Appendix A for the study protocol. ECIG-CTR Aerosol Parameters March /89

5 Table 2: Sample Identification Sample ID Description Lab1-Rechargeable Rechargeable product tested by Laboratory 1 Lab1-3 piece Rechargeable 3 piece-style product tested by Laboratory 1 Lab2-Rechargeable Rechargeable product tested by Laboratory 2 Lab2-Disposable Disposable product tested by Laboratory 2 Lab3-Rechargeable Rechargeable product tested by Laboratory 3 Lab3-Disposable Disposable product tested by Laboratory 3 Lab4-Rechargeable Rechargeable product tested by Laboratory 4 Lab4-Disposable Disposable product tested by Laboratory 4 Table 3: Collection Puffing Parameters Regime Code Puff Vol (ml) Duration (s) Shape Frequency Mean Flow (ml/s) Test 1 * 35/2/60/B 35 2 Bell Every 60s 17.5 Test 2 * 55/2/30/B 55 2 Bell Every 30s 27.5 Test 3 55/3/30/Sq 55 3 Square Every 30s 18.3 Test 4 55/4/30/Sq 55 4 Square Every 30s 13.8 Test 5 70/3/30/Sq 70 3 Square Every 30s 23.3 Test 6 70/4/30/Sq 70 4 Square Every 30s 17.5 *Note: Regime 1 and Regime 2 were not under evaluation because of previously reported issues with some products, but were included for informational purposes because they are based on standardized methods (ISO 3308 and HC-T-115) for traditional cigarette smoking. Where a bell shape profile has a non-constant flow and a square profile has constant flow ECIG-CTR Aerosol Parameters March /89

6 The study included determination of trapped aerosol, propylene glycol, nicotine, water, glycerin, and product weight loss. The aerosol collected mass (ACM) which is the measure of total particulates collected and the weight loss of the e-cigarette were determined gravimetrically. Analytes were determined using the method of choice by each laboratory. A comparison of the methods is shown below in Table 4. See Appendix B for summaries of each lab s methods. Table 4: Analytical Methods Overview Collection Sample Prep Analysis In Common Cerulean SM450 Standard Puff Engines ( 70mL puff) 44mm CFP, Conditioned to ISO std Room conditioned to ISO smoking std Organic Solvent 10-20mL Shake to extract Possible additional dilution for Gly/PG GC-TCD for water Packed column (most) GC-FID for other analytes Capillary column DB-Wax columns for most 6 12 calibration standards Linear (IS) calibration (most) r 2 >0.99 Unique Procedures Lab 4 used smaller labyrinth seals Lab 1 IPA (10mL) 30min Lab 2 EtOAc (20mL) 30min Lab 3 IPA (10mL) 20min Lab 4 n-propanol (20mL) 30min Lab 1 Internal Standard: EtOH (water), heptadecane (other analytes) Stabilwax column (nicotine, glycerin, PG) Lab 2 Internal Standard: 1,3-butanediol DBWax column for water Lab 3 Internal Standard: EtOH (water), anethole (other analytes), 3 GC runs, Linear (Ext) calibration for glycerin, PG Lab 4 Internal Standard: quinoline (nic); 1,4- butanediol (other analytes), quadratic (IS) calibration for water, glycerin 4. Data - Raw The summary descriptive statistics data are listed in Appendix C where each mean represents five replicates. Raw data plots are given in Appendix D. The data are displayed as mean with error bars representing one standard deviation. 5. Data - Statistical Analysis The purpose of the study was to gather information in preparation for a proficiency study. This study was not a formal proficiency test; there were four participating laboratories testing samples which were not in common using methodology which were not in common. Thus, many of the statistical analyses typically conducted following the recommendations given in ISO (1994) were not applicable to this data set. ECIG-CTR Aerosol Parameters March /89

7 5.1 Exclusion of Outliers Traditional outlier tests were not applied as discussed above. Data for devices that clearly did not function were removed by visual observation. When collecting puff-by-puff (or puff sets, in this case), puffs near the end of the device lifetime, as indicated by significant decrease in ACM yields or from a physical signal (such as a blinking LED) from the product, are typically of a very high variability. End-of-life puff sets yields were included in total yield calculations but the variability information was not included in the overall assessment of the product s variability under a given regime. In general, for puff set collection if a product or device had a lower than typical yield for a puff set the next puff set would have a higher than typical yield. Therefore, even though there may be puff set to puff set variability for a device, the cumulative yields may be consistent with the other replicates. This must be taken into account when reviewing the results. Also, analytes tend to track with total ACM; if the ACM is low for a measurement all other analytes are low by a similar magnitude. Thus, data were normalized to ACM in order to note any values that did not track with ACM for outlier investigation. Sample Lab1-3 piece was more variable overall compared to the other samples. The laboratory reported an experimental issue that was likely to be the cause of the apparent lower reliability of this sample. The sample s raw data are included here but the sample s results were not as heavily considered with regard to the overall conclusions drawn from the study. 5.2 Calculation of Repeatability and Reproducibility Each laboratory tested unique samples using non-standardized methodology. Thus, repeatability (r) and reproducibility (R) results were not calculated for this data set. 5.3 Calculation of Z-Scores Z-score values cannot be calculated for the data set. ECIG-CTR Aerosol Parameters March /89

8 6. Data Interpretation The objective of the study was to determine if 55mL of a 3s taken every 30s in a square profile (55/3/30/Sq) should be recommended as the standard OR if any of the other regimes should be recommended. To that end, we posed several questions to answer as we evaluated the data. 6.1 ISO and Canadian Intense Testing using ISO (Test 1) and Canadian Intense (Test 2) puffing parameters were included in the study for informational purposes but were not included in the evaluation for recommending a regime. Exclusion of these regimes was based on discussion at the September 2013 task force meeting during which multiple task force members discussed experience with device failure 3 when using bell shape puff profiles. In this study, there were no issues with product performance with either of these regimes. Simply based on the results of this study, either of these regimes would be viable choices if appropriate for a particular study design and product type. The 25 puff block yields were lower for these regimes. Thus, life-time puffing experiments with these parameters would require more puffs (longer collection times) than would be required for testing with the other regimes evaluated in this study. 6.2 Machine or product performance issues At least until e-cigarette-specific aerosol collection machines become more in use, it is important to ensure that the recommended regime will be compatible with conventional smoking machines. Indeed in this study, each participant used conventional 20-port linear smoking machines equipped with standard puff engines capable of a maximum puff volume of 70mL. No technical issues were noted with 55mL puff volume or with either duration used. There were a few technical issues noted for tests with 70mL puff volumes possibly because this is the maximum volume for the puff engines used. One lab reported issues with machine failure with 70mL puff volumes. Two laboratories reported that smoking machine modifications, such as air pressure modifications, were necessary for testing using 70mL puff volumes. No product failures were attributed to any of the smoking regimes. Based on machine performance, 55mL puff volume appears to be more favorable than 70mL. 6.3 Impact of regime on overall yields The aerosol collected mass (ACM) is an indication of overall performance. Results were compared for each sample type between the test regimes. Results were not compared between samples, as there is no expectation that any of the samples were in common between the laboratories. Overall yields were similar for the four regimes under consideration (Tests 3-6 Table 3); no practical or statistically significant differences were noted for any of the products. Precision for overall yields, as displayed in Appendix D, was similar for each of the regimes, as well. Additionally, a comparison between e-cigarette mass loss and ACM indicated that results were similar between the regimes tested. Ideally, the mass loss of the e-cigarette and the weight gain of the particulate trap would be virtually the same. As displayed below, the ratio of mass loss to ACM values were near 1 in most cases. 3 No or slow activation ECIG-CTR Aerosol Parameters March /89

9 Table 5: Ratio of e-cigarette Mass Loss to ACM Lab1- Rechargeable Lab2- Disposable Lab2- Rechargeable Lab3- Disposable Lab3- Rechargeable Lab4- Disposable Lab4- Rechargeable 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq Sample Lab1-3 piece was excluded from this comparison due to experimental issues; raw data are shown in Appendix C Thus, there is no advantage for any of the alternative regimes versus Test 3, 55/3/30/Sq, for overall (ACM) yields. ECIG-CTR Aerosol Parameters March /89

10 6.4 Impact on relative yields of analytes Another consideration is whether different regimes would give a different proportion of the analytes tested. Thus the percent of analyte in the ACM was calculated as Analyte (mg) / ACM (mg) x 100. As shown in the table below as the range and the percent difference in composition for each regime, in most cases the percent composition varied by no more than 10% among the regimes for a given sample except for water. For example, for sample Lab1- Rechargeable, the percent composition of nicotine was 1.22%, 1.24%, 1.21%, and 1.24% for Tests 3, 4, 5, and 6, respectively. This is a range of 0.03pt (as an absolute value) or 2% (calculated as percent difference). Thus, there appears to be no impact on the composition of the ACM based on regime choice for the regimes tested. Table 6: Range in Analyte Percent Composition across the Regimes for each Sample As % Composition Total PG Total Nicotine Total Glycerin Total Water Lab1-Rechargeable Lab2-Disposable Lab2-Rechargeable Lab3-Disposable NA Lab3-Rechargeable NA Lab4-Disposable Lab4-Rechargeable As % Difference* in % Composition Total PG Total Nicotine Total Glycerin Total Water Lab1-Rechargeable 3% 2% 4% 21% Lab2-Disposable 4% 4% 5% 33% Lab2-Rechargeable 3% 3% 4% 32% Lab3-Disposable NA 1% 6% 3% Lab3-Rechargeable NA 2% 7% 9% Lab4-Disposable 10% 22% 16% 21% Lab4-Rechargeable 19% 8% 6% 17% *Calculated as (2(Max %Composition-Min %Composition))/(Max+Min)x100 Sample Lab1-3 piece was excluded from this comparison due to experimental issues; raw data are shown in Appendix C ECIG-CTR Aerosol Parameters March /89

11 6.5 Impact of regime on cumulative yield or puff trending The ACM was collected discretely rather than in total; 25 puffs were collected per pad up to 300 puffs. Thus delivery profile trends may be assessed. While the overall yields were similar among the regimes, the delivery profiles were impacted by puff duration (2s vs 3s), but not by puff volume. This trending is exemplified below for one of the samples. Trend lines for the regimes using 3s duration, 55/3/30/Sq and 70/3/30/Sq, are virtually identical. While trend lines for the 4s duration regimes, 55/4/30/Sq and 70/4/30/Sq, also group together. Note also that the 4s trends have higher yields and fewer puff sets than the 3s trends. All trend lines are displayed in Appendix E. While there appears to be a pattern to the puff set profiling based on duration, there is no advantage for any of the alternative regimes versus Test 3, 55/3/30/Sq, for cumulative yield. ACM (mg/pad) Test 3-55/3/30/Sq Test 4-55/4/30/Sq Test 5-70/3/30/Sq Test 6-70/4/30/Sq Puff Number Figure 1: Puff Set Trending for Sample Lab 2 Rechargeable ACM (mg) Yield per pad. 6.6 Difference in yield variability The relative standard deviation (%RSD) was calculated for each pad set to determine if any of the regimes appeared to have improved precision. End-of-unit life puffs would be expected to be highly variable no matter the regime. Thus partial last puff sets were not considered in the evaluation of variability. Collection variability results calculated as the percent relative standard deviation (%RSD) of the ACM delivery for each pad set are shown in Appendix F. Puff set precision was relatively high and generally similar among and between the sets. For all products tested, 80% of the RSD values were less than 15% regardless of regime. There is no advantage for any of the alternative regimes versus Test 3, 55/3/30/Sq, for variability. 6.7 Trend with flow rate among the test parameters One of the questions in the case of product failure for machine vaping relates to whether the flow rate is sufficient to activate the device. At the September 2013 meeting, TF participants discussed device failure issues for some products if the flow rate was not sufficient for device activation. A flowrate of 13.8 ml/s (55mL/4s) was specifically discussed as problematic. In this study, flow rates ranged from 13.8 ml/s to 23.3 ml/s and there were no apparent product failures due to regime (i.e. flow rate). There was no clear trending in yield when compared based on flow rate. Note that the number and range of products tested was limited. Thus, flow rate may still be a concern for less conventional products. Thus, the task force would be hesitant to recommend a regime using 55mL with 4s without additional testing. ECIG-CTR Aerosol Parameters March /89

12 7. Recommendations Based on discussions during TF meetings: A 70-mL puff volume may be problematic for smoking machine designs with 70mL as the maximum volume. Parameters which result in a low flow rate (i.e. 55mL with a 4s duration) may result in device failure depending on the product being tested. Puffing parameters using bell profile shapes may be problematic for some product types due to air flow issues. Based on results from this study: Overall, yields were similar for the four test regimes. Delivery profile trends were dependent on puff duration and not on puff volume. Precision was acceptable and similar for all regimes. There were no technical advantages among the regimes tested. Thus, based on TF discussion and on the results of this study, Test Regime 3 (a square 55mL puff, 3s in duration, taken twice a minute), was recommended for adoption as an interim standard vaping regime for e-cigarette testing. Choice of regime parameters may need to be refined as additional product types are tested. 8. Additional Considerations Limitations to the work that should be considered include: Only products of a conventional design were included in the study. Puff volume and combinations of volume and duration were limited by capabilities of conventional smoking machines in standard configuration. Reference products and standardized analytical methodology do not exist for e- cigarette aerosol collection at this time. Reporting units that may be useful for fostering a collective understanding of the research include mg/pad for incremental collection, mg/ total puffs for total collection on a single pad. In addition to analytes of interest collection and reporting of ACM, puff counts, and e- cigarette weight change are useful quality measures and would aid collaborative understanding of results. 9. Acknowledgements The study team, Drs. Rob Stevens, Narendra Meruva, and Gene Gillman; the participating laboratories; and White Cloud and Red Kiwi that supplied some of the e-cigarettes for testing are greatly appreciated. ECIG-CTR Aerosol Parameters March /89

13 APPENDIX A: Study Protocol and Data Template CORESTA E-CIGARETTE ANALYTICAL TESTING SUB TEAM (Team Leader Dr. Rob Stevens, Lorillard Tobacco) Project Title: 2013 Major constituents in e-cigarette aerosol Date: Nov 11, 2013 Written by: Gene Gillman, Ph.D. (Enthalpy Analytical) Phone: Confidentiality Notice: All data generated in the course of this analysis should be handled in the strictest of confidence by all participating laboratories and other taskforce members. ECIG-CTR Aerosol Parameters March /89

14 Study Protocol to Determine the Effect of Puff Profile on the Yield of the Major Constituents in E-Cigarette Aerosol 1. Objective The main objective of this study to determine the effect of puff profile on the yield of the major constituents in e-cigarette aerosol. This study will include six puff profiles or test conditions with various puff volumes, durations and puff shapes. 2. Study coordinator Dr. Robert Stevens Director, R&D Testing Services Lorillard Tobacco Company, R&D 420 N. English Street P.O. Box Greensboro, N.C Bus. (336) FAX (336) Analytes and Methods 3.1 Puff Count Puff count for each puffing increment (each pad) will be recorded. For most data points the puff count will be 25. For products which extinguish prior to 300 puffs there may be pads with fewer than 25 puffs. The actual puff count should be recorded. Record as count/pad in the data template. 3.2 Trapped Aerosol The total collected aerosol mass (ACM) for each sample will be determined by measuring the trapping system before and after each collection event. The total amount of aerosol collected will be determined by difference in mass (Pad weight after collection pad weight before collection). Record as mg/pad in the data template. This test measurement is akin to total particulate matter (TPM) for conventional cigarette testing. 3.3 Weight loss The amount of material lost from each e-cigarette will be determined by measuring the e- cigarette before and after each collection event. The total amount of weight loss will be determined by difference in mass (e-cigarette weight after collection e- cigarette weight before collection). Record as mg in the data template. 3.4 Propylene Glycol The amount of propylene glycol per sample will be determined by the method currently in use in each laboratory. Record as mg/pad in the data template. 3.6 Glycerin The amount of glycerin per sample will be determined by the method currently in use in each laboratory. Record as mg/pad in the data template. 3.7 Water The amount of water per sample will be determined by the method currently in use in each laboratory. Record as mg/pad in the data template. ECIG-CTR Aerosol Parameters March /89

15 4. Samples 4.1 Required materials Each participating laboratory shall supply product for their own testing. Two models shall be tested. These should include both a rechargeable model and disposable model. These two brands do not have to be products from the same manufacturer. A minimum of 30 e-cigarettes for each brand is required to fulfill the study design. 4.2 Sample Handling All samples should be stored at room temperature. Rechargeable devices should be charged before use. A single battery charge will be used for each new cartomizer. Note: Only one battery charge will be used for each cartomizer. 5. Experimental plan Five replicates for each e-cigarette under each test condition is required. If possible, it is requested that all samples for a test condition be analyzed on a single day. A series of discreet samples will be collected from each e-cigarette (disposable or cartomizer). For each e-cigarette, samples will be collected in 25 puffs increments (1-25, and etc.) until the battery is depleted. Five e-cigarettes should be tested under each test condition. For each aerosol sample, laboratories are asked to determine weight lost from the device, amount of ACM trapped, nicotine, propylene glycol, glycerin, and water. Data will be reported on a per mg basis. Aerosol should be generated from each type of e-cigarette using the test conditions given in Table 1. Note that Tests 1 and 2 use puffing parameters specified in for ISO smoking (ISO 3308) and Canadian Intense smoking (HC-T-114) of conventional cigarettes. This protocol does not attempt to make any other specifications with regard to these two standard smoking regimes (i.e. room conditions). Note: Five e-cigarettes should be tested under each test condition. Table 1: Test Conditions to be used for this study Test Puff Volume Shape Frequency Duration Not 1 35 Bell* Every 60 Sec 2 Sec ISO 3308 Puffing Parameters 2 55 Bell* Every 30 Sec 2 Sec HC-T-114 Puffing Parameters (without tape) 3 55 Square # Every 30 Sec 3 Sec 4 55 Square # Every 30 Sec 4 Sec 5 70 Square # Every 30 Sec 3 Sec 6 70 Square # Every 30 Sec 4 Sec * Bell Shape is an ISO 3308 type puff profile # Square Shape is a constant flow rate profile Note: Rechargeable devices must be tested using a new cartomizer and fully charged battery. Samples will be collected until the battery is discharged. Only one battery charge is to be used per cartomizer. ECIG-CTR Aerosol Parameters March /89

16 6. Data Submission The attached templates should be used for data submission. Please supply data in the requested format without creating new cells or rows in the spreadsheet. Results should be reported back to Rana Tayyarah on or before Feb 14 th, 2014 in order to allow sufficient time to evaluate the data. 7. Data Analysis The data will be analyzed statistically according to ISO Timescale First week of December 2013 All documents will be distributed to participating laboratories o Protocol of the study o Report form December 2013/January 2014 Laboratories will perform the study Mid February The results must be sent to the data coordinator on or before February 14 th 2014 in order to give to the statistician sufficient time to evaluate the data. Data received after February 14 th will not be included in the draft report discussed at the May meeting. March/April 2014 The statistician will perform the data analysis and the Task Force coordinators will format the data for presentation. May 2014 The taskforce member will assess the results of the collaborative study during the next taskforce meeting. ECIG-CTR Aerosol Parameters March /89

17 Data Sheet for 2013 Major Constituents in e-cigarette Aerosol Parameters Study Laboratory name Company Representative (Title, Name) Address Tel Fax address Note: Please don't modify formatted data sheet for the convenience of data analyses. Note: Laboratory names will not be used for reporting. All labs will be coded. Each lab will know their own code. Note: In this spreadsheet, cells that are shaded blue require input. Cells that are not shaded are calculations or information and should not be edited. Note: Please return this completed spreadsheet to by February 14, ECIG-CTR Aerosol Parameters March /89

18 0 Method Synopsis: please type an overall summary of your method in this box and provide specific details below Method Details (example) ACM Nicotine Water Glycerin PG Sample Handling Storage prior to testing Additional Details Type of Aerosol Collection Machine Style (Include a description of features if non-standard equipment was used) unopened at 65% RH 72F Linear Smoking Machine Make Vaping was Concurent for Which analytes? Room Conditions during aerosol collection Cerulean SM450 All were concurrent 65 %RH, 72F Additional Details for Aerosol Collection Trapping System Filter Pad / Diameter Were filter pads conditioned? Trap Type + Number of Traps CFP 44 mm Yes, 62+/-%RH, 75F Fritted Impinger Solvent Extraction Additional Filtration Additional sample processing prior to analysis Additional Details 20 ml MeOH (sub-ambient) wrist-action shaker, 30min PTFE: 0.45um extracts were diluted 1:2 for Gly/PG extracts analyzed same day for Nic/Water next day for Gly/PG Analytical Equipment Description Agilent GC/MS, EI in SIM mode using detuerated IS; D-5 pyridine (84), pyridine(79) & D-7 quinoline(136) & quinoline(129); Detection Systems + Conditions Column: DBWax 30mX0.25mmX0.25Um, 50oC for 6min, 4oC/min to 160oc, 30oC to 250oC for 5.5min: 10:1 split, flow 15mL/min, total flow 20ml/min, 1ul injection extracts were stored 4C prior to analysis Additional Details Reference -- Quantitation Information Limit of Quantitation (mg/pad) Limit of Detection (mg/pad) Number of standards Concentration of each Standard Internal Standard Calibration Type r 2 Additional Details Ethanol (water), Anethole (others) linear, with IS *Note: the example listed is not an e-cig method. It is just meant to show the requested level of detail for you to list for your aerosol method. ECIG-CTR Aerosol Parameters March /89

19 0 Please paste in a picture of an example chromatogram for a calibration standard and for one or more samples for chromatographic methods Please paste pictures relevant to any unusual observations noted during testing. Please include text explanation as necessary. ECIG-CTR Aerosol Parameters March /89

20 0 Condition Volume Shape Frequency Puff Duration NOTES Test 1 35 Bell Every 60 Sec 2 Sec ISO 3308 Puffing Parameters Test 2 55 Bell Every 30 Sec 2 Sec HC-T-114 Puffing Parameters (without tape) Test 3 55 Square Every 30 Sec 3 Sec Test 4 55 Square Every 30 Sec 4 Sec Test 5 70 Square Every 30 Sec 3 Sec Test 6 70 Square Every 30 Sec 4 Sec ECIG-CTR Aerosol Parameters March /89

21 0 Sample 1 - Test 1 Sample Description Note: e-cigarette brand names are not needed. At a minimum, indicate "rechargable" or "disposable" as a sample description. Test Conditions Puff Volume (ml) Shape Frequency Duration Test 1 35 Bell Every 60 Sec 2 Sec Testing Dates Test 1 Sample 1 Collection Extraction Analyses Observations Note: Please include under "observations" information about anything unusual in the behavior, etc of the samples. For example, unusual colors, smells, "spitting" (droplets inside pad holder and/or on pad). Note: If photograph(s) would be helpful to demonstrate an unusual observation, please paste in on the chromatogram and photos tab with text to explain. Testing Summary Total Puffs Total ACM Total PG Total Nicotine Total Glycerin Total Water mg mg mg mg mg ECIG-CTR Aerosol Parameters March /89

22 0 Sample 1 - Test 1 Test Conditions Puff Volume Shape Frequency Duration Test 1 35 Bell Every 60 Sec 2 Sec Note: Puffing should be ceased after one battery discharged OR after 300 puffs, whichever comes first. Puff Count Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate Note: Later puff sets may not be a full 25 puffs. Record the actual puff counts for these pads. Weight Loss Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/device) ECIG-CTR Aerosol Parameters March /89

23 0 Sample 1 - Test 1 Test Conditions Puff Volume (ml) Shape Frequency Duration Test 1 35 Bell Every 60 Sec 2 Sec ACM Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/pad) PG Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/pad) ECIG-CTR Aerosol Parameters March /89

24 0 Sample 1 - Test 1 Test Conditions Puff Volume (ml) Shape Frequency Duration Test 1 35 Bell Every 60 Sec 2 Sec Nicotine Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/pad) Glycerin Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/pad) Water Puffs Replicate 1 Replicate 2 Replicate 3 Replicate 4 Replicate 5 (mg/pad) ECIG-CTR Aerosol Parameters March /89

25 APPENDIX B: Laboratory Method Summaries and Example Chromatograms Laboratory 1 Method Summary Nicotine Water Glycerin PG Sample Handling Storage prior to testing unopened at 65% RH 72F unopened at 65% RH 72F unopened at 65% RH 72F unopened at 65% RH 72F Additional Details Type of Aerosol Collection Machine Style (Include a description of features if non-standard equipment was used) Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Make Cerulean SM450 Cerulean SM450 Cerulean SM450 Cerulean SM450 Vaping was Concurrent for Which analytes? All were concurrent All were concurrent All were concurrent All were concurrent Room Conditions during aerosol collection 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F Additional Details for Aerosol Collection ECIG-CTR Aerosol Parameters March /89

26 Laboratory 1 Method Summary (continued) Trapping System Nicotine Water Glycerin PG Filter Pad / Diameter CFP 44 mm CFP 44 mm CFP 44 mm CFP 44 mm Were filter pads conditioned? Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Trap Type + Number of Traps Solvent 10 ml 2-Propanol 10 ml 2- Propanol 10 ml 2-Propanol 10 ml 2-Propanol Extraction wrist-action shaker, 30min wrist-action shaker, 30min wrist-action shaker, 30min wrist-action shaker, 30min Additional Filtration Additional sample processing prior to analysis Additional Details extracts analyzed same day for Nic/Water /Gly/PG extracts analyzed same day for Nic/Water /Gly/PG extracts analyzed same day for Nic/Water /Gly/PG extracts analyzed same day for Nic/Water /Gly/PG ECIG-CTR Aerosol Parameters March /89

27 Laboratory 1 Method Summary (continued) Analytical Equipment Nicotine Water Glycerin PG Description Agilent 58/90 GC Series II FID/TCD Agilent 5890 GC Series II FID/TCD Agilent 5890 GC Series II FID/TCD Agilent 5890 GC Series II FID/TCD Detection Systems + Conditions Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Column: Supelco Porapak R 6ft. X 1/8 inch ID Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Column: Restek Stabilwax 30mX0.32mmX1.0Um, 1ul injection Additional Details Reference ECIG-CTR Aerosol Parameters March /89

28 Laboratory 1 Method Summary (continued) Quantitation Information Nicotine Water Glycerin PG Limit of Quantitation (mg/pad) Limit of Detection (mg/pad) Number of standards Concentration of each Standard , 0.103, 0.257, 0.514, 0.771, 1.03, 1.23 mg/ml 0.495, 1.24, 2.48, 3.71, 4.95, 5.94 mg/ml 0.331, 0.662, 1.66, 3.31, 4.97, 6.62, 7.95, 9.93, 13.2, 33.1 mg/ml 0.255, 0.510, 1.27, 2.55, 3.82, 5.10, 6.11, 7.64, 10.2, 25.5 mg/ml Internal Standard Ethanol (water), Heptadecane (others) Ethanol (water), Heptadecane (others) Ethanol (water), Heptadecane (others) Ethanol (water), Heptadecane (others) Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS r Additional Details ECIG-CTR Aerosol Parameters March /89

29 Figure 1: Laboratory 1 Example Chromatogram Aerosol Sample Figure 2: Laboratory 1 Example Chromatogram Aerosol Sample ECIG-CTR Aerosol Parameters March /89

30 Figure 3: Laboratory 1 Example Chromatogram Calibration Standard Figure 4: Laboratory 1 Example Chromatogram Calibration Standard ECIG-CTR Aerosol Parameters March /89

31 Laboratory 2 Method Summary Nicotine Water Glycerin PG Sample Handling Storage prior to testing Unopened; room temp. Unopened; room temp. Unopened; room temp. Unopened; room temp. Additional Details N/A N/A N/A N/A Type of Aerosol Collection Machine Style (Include a description of features if non-standard equipment was used) Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Make Cerulean SM450 Cerulean SM450 Cerulean SM450 Cerulean SM450 Vaping was Concurrent for Which analytes? All were concurrent All were concurrent All were concurrent All were concurrent Room Conditions during aerosol collection 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F Additional Details for Aerosol Collection N/A N/A N/A N/A ECIG-CTR Aerosol Parameters March /89

32 Laboratory 2 Method Summary (continued) Trapping System Nicotine Water Glycerin PG Filter Pad / Diameter CFP 44 mm CFP 44 mm CFP 44 mm CFP 44 mm Were filter pads conditioned? Yes, 60±2% RH; 22±2 C Yes, 60±2% RH; 22±2 C Yes, 60±2% RH; 22±2 C Yes, 60±2% RH; 22±2 C Trap Type + Number of Traps N/A N/A N/A N/A Solvent 20 ml EtOAc 20 ml EtOAc 20 ml EtOAc 20 ml EtOAc Extraction wrist-action shaker, 30min wrist-action shaker, 30min wrist-action shaker, 30min wrist-action shaker, 30min Additional Filtration N/A N/A N/A N/A Additional sample processing prior to analysis N/A N/A N/A N/A Additional Details N/A N/A N/A N/A ECIG-CTR Aerosol Parameters March /89

33 Laboratory 2 Method Summary (continued) Analytical Equipment Nicotine Water Glycerin PG Description Agilent 7890 GC-FID Agilent 6890 GC-TCD Agilent 7890 GC-FID Agilent 7890 GC-FID Detection Systems + Conditions Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 C to 250 C (11 C/min ramp); 1 minute hold; 1 µl injection volume Column: DBWax (30mx0.53mmx1µm); 50 C to 240 C (20 C/min ramp); 1 minute hold; 0.5 µl injection volume Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 C to 250 C (11 C/min ramp); 1 minute hold; 1 µl injection volume Column: RTXWax (30mx0.32mmx1µm); Split 20:1; 120 C to 250 C (11 C/min ramp); 1 minute hold; 1 µl injection volume Additional Details Reference ECIG-CTR Aerosol Parameters March /89

34 Laboratory 2 Method Summary (continued) Quantitation Information Nicotine Water Glycerin PG Limit of Quantitation (mg/pad) Limit of Detection (mg/pad) N/A N/A N/A N/A Number of standards Concentration of each Standard µg/ml µg/ml µg/ml µg/ml Internal Standard 1,3-butanediol 1,3-butanediol 1,3-butanediol 1,3-butanediol Calibration Type Linear; 1/x2 Linear; 1/x2 Linear; 1/x2 Linear; 1/x2 r Additional Details N/A N/A N/A N/A ECIG-CTR Aerosol Parameters March /89

35 Figure 1: Laboratory 2 Example Chromatogram Aerosol Sample Figure 2: Laboratory 2 Example Chromatogram Aerosol Sample ECIG-CTR Aerosol Parameters March /89

36 Figure 3: Laboratory 2 Example Chromatogram Calibration Standard Figure 4: Laboratory 2 Example Chromatogram Calibration Standard ECIG-CTR Aerosol Parameters March /89

37 Laboratory 3 Method Summary Nicotine Water Glycerin PG Sample Handling Storage prior to testing unopened at 65% RH 72F unopened at 65% RH 72F unopened at 65% RH 72F unopened at 65% RH 72F Additional Details Type of Aerosol Collection Machine Style (Include a description of features if non-standard equipment was used) Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Linear Smoking Machine Make Cerulean SM450 Cerulean SM450 Cerulean SM450 Cerulean SM450 Vaping was Concurrent for Which analytes? All were concurrent All were concurrent All were concurrent All were concurrent Room Conditions during aerosol collection 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F 65 %RH, 72F Additional Details for Aerosol Collection One vape run, nic water concurrent, gly/pg concurrent One vape run, nic water concurrent, gly/pg concurrent One vape run, nic water concurrent, gly/pg concurrent One vape run, nic water concurrent, gly/pg concurrent ECIG-CTR Aerosol Parameters March /89

38 Laboratory 3 Method Summary (continued) Trapping System Nicotine Water Glycerin PG Filter Pad / Diameter CFP 44 mm CFP 44 mm CFP 44 mm CFP 44 mm Were filter pads conditioned? Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Yes, 62+/-%RH, 75F Trap Type + Number of Traps NA NA NA NA Solvent 10 ml IPA 10 ml IPA Extraction 20min shake 20min shake 20min shake 20min shake Additional Filtration NA NA NA NA Additional sample processing prior to analysis Additional Details extracts analyzed same day for Nic/Water next day for Gly/PG extracts analyzed same day for Nic/Water next day for Gly/PG Additional dilution for Gly based on ACM Samples were diluted if nec for gly Samples were diluted if nec for gly Laboratory 3 Method Summary (continued) Analytical Equipment Nicotine Water Glycerin PG Description GC-FID GC-TCD (dual column with Nic analysis) GC-FID GC-FID Detection Systems + Conditions Agilent DB-Wax (10 m x 0.18 mm x 0.3 µm film) Splitless (purge time 0.5 min, 50mL/min) psi 40 ml/min 400 ml/min 160 C hold for 0.5 minutes 80 C/minute to 200 C 50 C/ minute to 225 C hold 2.0 min. Stainless steel Packed column Haye Sep-Q 60/80 mesh; 3' x 1/4", or equivalent); on-column injection; He pressure 9psi; initial temp 160C DB-Wax Analytical GC column (15m X 0.25mm ID)(0.25 µm film thickness) DB-Wax Analytical GC column (15m X 0.25mm ID)(0.25 µm film thickness) Additional Details Reference ECIG-CTR Aerosol Parameters March /89

39 Laboratory 3 Method Summary (continued) Quantitation Information Nicotine Water Glycerin PG Limit of Quantitation (mg/pad) Limit of Detection (mg/pad) Number of standards Concentration of each Standard (mg/ml) 0, 0.05, 1.15, 0.20, 0.25, 0.50, 1.0 (mg/ml) 0, 0.25, 0.75, 1.25, 2.5, 5.0, 1.0 (mg/ml) 0.025, 0.099, 0.25, 1.2, 2.5 (mg/ml) , , 0.04, 0.16, 0.40, 2.0, 4.0 Internal Standard Ethanol (water), Anethole (others) Ethanol (water), Anethole (others) Ethanol (water), Anethole (others) Ethanol (water), Anethole (others) Calibration Type linear, with IS linear, with IS linear, with IS linear, with IS r 2 > > > > Additional Details ECIG-CTR Aerosol Parameters March /89

40 Figure 1: Laboratory 3 Example Chromatogram Calibration Standard Figure 2: Laboratory 3 Example Chromatogram Calibration Standard ECIG-CTR Aerosol Parameters March /89

41 Figure 3: Laboratory 3 Example Chromatogram Calibration Standard ECIG-CTR Aerosol Parameters March /89

42 Laboratory 4 Method Summary Nicotine Water Glycerin PG Sample Handling Storage prior to testing Samples stored under ambient conditions Samples stored under ambient conditions Samples stored under ambient conditions Samples stored under ambient conditions Additional Details Type of Aerosol Collection Machine Style (Include a description of features if non-standard equipment was used) Linear smoking machine Linear smoking machine Linear smoking machine Linear smoking machine Make Cerulean Cerulean Cerulean Cerulean Vaping was Concurrent for Which analytes? All were concurrent All were concurrent All were concurrent All were concurrent Room Conditions during aerosol collection 60 ± 4 %RH, 72 ± 5 F 60 ± 4 %RH, 72 ± 5 F 60 ± 4 %RH, 72 ± 5 F 60 ± 4 %RH, 72 ± 5 F Additional Details for Aerosol Collection Based on the collected aerosol mass, the amount of extraction solution to be added is adjusted Based on the collected aerosol mass, the amount of extraction solution to be added is adjusted Based on the collected aerosol mass, the amount of extraction solution to be added is adjusted Based on the collected aerosol mass, the amount of extraction solution to be added is adjusted ECIG-CTR Aerosol Parameters March /89

43 Laboratory 4 Method Summary (continued) Trapping System Nicotine Water Glycerin PG Filter Pad / Diameter CFP 44mm CFP 44mm CFP 44mm CFP 44mm Were filter pads conditioned? 60 ± 3 %RH, 72 ± 2 F 60 ± 3 %RH, 72 ± 2 F 60 ± 3 %RH, 72 ± 2 F 60 ± 3 %RH, 72 ± 2 F Trap Type + Number of Traps N/A N/A N/A N/A Solvent 20 ml of dry n-propanol extraction solution 20 ml of dry n-propanol extraction solution 20 ml of dry n- propanol extraction solution 20 ml of dry n- propanol extraction solution Extraction 30 min mixing time on an orbital shaker 30 min mixing time on an orbital shaker 30 min mixing time on an orbital shaker 30 min mixing time on an orbital shaker Additional Filtration N/A N/A N/A N/A Additional sample processing prior to analysis Additional Details Samples were diluted 1:10 with extraction solution for PG and Glycerin Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water. Samples were diluted 1:10 with extraction solution for PG and Glycerin Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water. Samples were diluted 1:10 with extraction solution for PG and Glycerin Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water. Samples were diluted 1:10 with extraction solution for PG and Glycerin Care must be taken to ensure all glassware used for standard and sample preparation is free of residual water. ECIG-CTR Aerosol Parameters March /89

44 Laboratory 4 Method Summary (continued) Description Analytical Equipment Nicotine Water Glycerin PG Detection Systems + Conditions Additional Details Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD). Columns: Two DB- ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90 C for 1min, 15 C/min to 120 c, 40 C to 280 C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250 C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min. n-propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination. Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD). Columns: Two DB-ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90 C for 1min, 15 C/min to 120 c, 40 C to 280 C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 225 C, GC-TCD - 2µL injection at 10:1 split and carrier gas flow of 1.5mL/min. n-propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination. Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD). Columns: Two DB- ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90 C for 1min, 15 C/min to 120 c, 40 C to 280 C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250 C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min. n-propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination. Agilent GC equipped with dual hot split/splitless injection ports and dual detectors (FID and TCD). Columns: Two DB- ALC1 capillary columns (front and back), 30m x 0.32 mm ID, 1.8 µm film thickness. Oven Programming: 90 C for 1min, 15 C/min to 120 c, 40 C to 280 C for 2min, Bake time 2 min, total run time 9 min, Injection port temperatures of 250 C, GC-FID - 1µL injection at 25:1 split and carrier gas flow of 3mL/min. n-propanol extraction solvent is stored dried with molecular sieves (5 g per liter of extraction solution) to remove any trace levels of water contamination. Reference N/A N/A N/A N/A ECIG-CTR Aerosol Parameters March /89

45 Laboratory 4 Method Summary (continued) Quantitation Information Nicotine Water Glycerin PG Limit of Quantitation (mg/pad) 51.1 µg/ml 516 µg/ml 50.7 µg/ml 49.3 µg/ml Limit of Detection (mg/pad) 16.9 µg/ml 170 µg/ml 16.7 µg/ml 16.3 µg/ml Number of standards Concentration of each Standard 50, 100, 250, 500, 1000, , 1000, 2500, 5000, 10000, , 100, 250, 500, 1000, , 100, 250, 500, 1000, 2000 Internal Standard Quinoline (FID) 1,4-Butanediol (TCD) 1,4-Butanediol (FID) 1,4-Butanediol (FID) Calibration Type Linear, with IS Quadratic, with IS Quadratic, with IS Linear, with IS r 2 >0.990 >0.990 >0.990 >0.990 Additional Details N/A N/A N/A N/A ECIG-CTR Aerosol Parameters March /89

46 Figure 1: Laboratory 4 Example Chromatogram Aerosol Sample Figure 2: Laboratory 4 Example Chromatogram Aerosol Sample ECIG-CTR Aerosol Parameters March /89

47 Figure 3: Laboratory 4 Example Chromatogram Calibration Standard Figure 4: Laboratory 4 Example Chromatogram Calibration Standard ECIG-CTR Aerosol Parameters March /89

48 APPENDIX C: Raw Data Means Raw data are presented as means ± 1 standard deviation for puff sets and for total yields Results for puff sets with average ACM values <6mg are not reported. Lab1-3 piece Puff Set Data Puff Count Lab1-3 piece Puff Set Data Weight Loss (mg) ECIG-CTR Aerosol Parameters March /89

49 Lab1-3 piece Puff Set Data ACM (mg) Lab1-3 piece Puff Set Data Propylene Glycol (mg) ECIG-CTR Aerosol Parameters March /89

50 Lab1-3 piece Puff Set Data Glycerin (mg) Lab1-3 piece Puff Set Data Nicotine (mg) ECIG-CTR Aerosol Parameters March /89

51 Lab1-3 piece Puff Set Data Water (mg) Lab1-Rechargeable Puff Set Data Puff Count ECIG-CTR Aerosol Parameters March /89

52 Lab1-R Puff Set Data Weight Loss (mg) Lab1-Rechargeable Puff Set Data ACM (mg) ECIG-CTR Aerosol Parameters March /89

53 Lab1-Rechargeable Puff Set Data Propylene Glycol (mg) Lab1-Rechargeable Puff Set Data Glycerin (mg) ECIG-CTR Aerosol Parameters March /89

54 Lab1-Rechargeable Puff Set Data Nicotine (mg) Lab1-Rechargeable Puff Set Data Water (mg) ECIG-CTR Aerosol Parameters March /89

55 Lab2-Disposable Puff Set Data Puff Count Lab 2-D Puff Set Data Weight Loss (mg) ECIG-CTR Aerosol Parameters March /89

56 Lab2-Disposable Puff Set Data ACM (mg) Lab2-Disposable Puff Set Data Propylene Glycol (mg) ECIG-CTR Aerosol Parameters March /89

57 Lab2-Disposable Puff Set Data Glycerin (mg) Lab2-Disposable Puff Set Data Nicotine (mg) ECIG-CTR Aerosol Parameters March /89

58 Lab2-Disposable Puff Set Data Water (mg) Lab2-Rechargeable Puff Set Data Puff Count ECIG-CTR Aerosol Parameters March /89

59 Lab 2-R Puff Set Data Weight Loss (mg) Lab2-Rechargeable Puff Set Data ACM (mg) ECIG-CTR Aerosol Parameters March /89

60 Lab2-Rechargeable Puff Set Data Propylene Glycol (mg) Lab2-Rechargeable Puff Set Data Glycerin (mg) ECIG-CTR Aerosol Parameters March /89

61 Lab2-Rechargeable Puff Set Data Nicotine (mg) Lab2-Rechargeable Puff Set Data Water (mg) ECIG-CTR Aerosol Parameters March /89

62 Lab3-Disposable Puff Set Data Puff Count Lab 3-D Puff Set Data Weight Loss (mg) ECIG-CTR Aerosol Parameters March /89

63 Lab3-Disposable Puff Set Data ACM (mg) Lab3-Disposable Puff Set Data Propylene Glycol (mg) ECIG-CTR Aerosol Parameters March /89

64 Lab3-Disposable Puff Set Data Glycerin (mg) Lab3-Disposable Puff Set Data Nicotine (mg) ECIG-CTR Aerosol Parameters March /89

65 Lab3-Disposable Puff Set Data Water (mg) Lab3-Rechargeable Puff Set Data Puff Count ECIG-CTR Aerosol Parameters March /89

66 Lab 3-R Puff Set Data Weight Loss (mg) Lab3-Rechargeable Puff Set Data ACM (mg) ECIG-CTR Aerosol Parameters March /89

67 Lab3-Rechargeable Puff Set Data Propylene Glycol (mg) Lab3-Rechargeable Puff Set Data Glycerin (mg) ECIG-CTR Aerosol Parameters March /89

68 Lab3-Rechargeable Puff Set Data Nicotine (mg) Lab3-Rechargeable Puff Set Data Water (mg) ECIG-CTR Aerosol Parameters March /89

69 Lab4-Disposable Puff Set Data Puff Count Lab 4-D Puff Set Data Weight Loss (mg) ECIG-CTR Aerosol Parameters March /89

70 Lab4-Disposable Puff Set Data ACM (mg) Lab4-Disposable Puff Set Data Propylene Glycol (mg) ECIG-CTR Aerosol Parameters March /89

71 Lab4-Disposable Puff Set Data Glycerin (mg) Lab4-Disposable Puff Set Data Nicotine (mg) ECIG-CTR Aerosol Parameters March /89

72 Lab4-Disposable Puff Set Data Water (mg) Lab4-Rechargeable Puff Set Data Puff Count ECIG-CTR Aerosol Parameters March /89

73 Lab 4-R Puff Set Data Weight Loss (mg) Lab4-Rechargeable Puff Set Data ACM (mg) ECIG-CTR Aerosol Parameters March /89

74 Lab4-Rechargeable Puff Set Data Propylene Glycol (mg) Lab4-Rechargeable Puff Set Data Glycerin (mg) ECIG-CTR Aerosol Parameters March /89

75 Lab4-Rechargeable Puff Set Data Nicotine (mg) Lab4-Rechargeable Puff Set Data Water (mg) ECIG-CTR Aerosol Parameters March /89

76 Lab1-3 piece Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq 150 NA NA NA NA 3.61 NA 64.5 NA 70/3/30/Sq /4/30/Sq Lab1-Rechargeable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq ECIG-CTR Aerosol Parameters March /89

77 Lab2-Disposable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq Lab2-Rechargeable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq ECIG-CTR Aerosol Parameters March /89

78 Lab3-Disposable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq Lab3-Rechargeable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq ECIG-CTR Aerosol Parameters March /89

79 Lab4-Disposable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq Lab4-Rechargeable Total Yields Puff Count Weight Loss (mg) ACM (mg) PG (mg) Glycerin (mg) Nicotine (mg) Water (mg) Test Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev Average Stdev 35/2/60/B /2/30/B /3/30/Sq /4/30/Sq /3/30/Sq /4/30/Sq ECIG-CTR Aerosol Parameters March /89

80 APPENDIX D: Total Yield Graphs Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 1: Lab1-3 piece Total Yields Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 2: Lab1-Rechargeable Total Yields Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

81 Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 3: Lab2-Disposable Total Yields Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 4: Lab2-Rechargeable Total Yields Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

82 Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 5: Lab3-Disposable Total Yields Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 6: Lab3-Rechargeable Total Yields Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

83 Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 7: Lab4-Disposable Total Yields Total ACM (mg) Test 3 Test 4 Test 5 Test 6 Figure 8: Lab4-Disposable Total Yields Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

84 APPENDIX E: Puff Set Graphs ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Puff Number Figure 1: Lab1-3 piece Total Yields ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Figure 2: Lab1-Rechargeable Total Yields Puff Number Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

85 ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Puff Number Figure 3: Lab2-Disposable Total Yields ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Figure 4: Lab2-Rechargeable Total Yields Puff Number Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

86 ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Puff Number Figure 5: Lab3-Disposable Total Yields ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Figure 6: Lab3-Rechargeable Total Yields Puff Number Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

87 ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Puff Number Figure 7: Lab4-Disposable Total Yields ACM (mg/pad) Test 3 Test 4 Test 5 Test 6 Puff Number Figure 8: Lab5-R Total Yields Regime Test 3 Test 4 Test 5 Test 6 Code 55/3/30/Sq 55/4/30/Sq 70/3/30/Sq 70/4/30/Sq ECIG-CTR Aerosol Parameters March /89

88 APPENDIX F: ACM Variability - Percent Relative Standard Deviation for Puff Sets Rechargeable Products Results n=5 ECIG-CTR Aerosol Parameters March /89