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1 Supplementary Online Content Goniewicz ML, Smith DM, Edwards KC, et al. Comparison of nicotine and toxicant exposure in users of electronic cigarettes and combustible cigarettes. JAMA etw Open. 2018;1(8):e doi: /jamanetworkopen efigure. Study Inclusion/Exclusion etable 1. Analytical Limits of Detection (), Biological Half-lives, and Assay Methods for icotine and Toxicants Assessed at Wave 1, PATH Study, etable 2. Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Current ever Users, e-cigarette Only-Users, Dual Users, and Cigarette-Only Smokers (ormalized for Creatinine, Geometric Means, 95% Confidence Intervals), PATH Wave 1 ( ) (n=5,105) emethods. Details on Model Building Procedures for Dual User Analysis and Sample Weighting etable 3. Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Everyday and Someday Users of e-cigarettes and Cigarettes (ormalized for Creatinine, Geometric Means, 95% Confidence Intervals), PATH Wave 1 ( ) (n=2,658) etable 4. Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Dual Users (ormalized for Creatinine, Geometric Means, 95% Confidence Intervals), PATH Wave 1 ( ) (n=792) ereferences This supplementary material has been provided by the authors to give readers additional information about their work.

2 efigure. Study Inclusion/Exclusion Wave 1 (W1) Adults, Population Assessment of Tobacco and Health (PATH) Study: =32,320 EXCLUDE: =10,519 W1 adult participants who did not provide urine samples =21,801 W1 adult participants who provided urine samples EXCLUDE: =10,279 W1 adult participants who provided urine samples but were not selected for biomarker analysis =11,522 W1 adult participants who provided urine samples and were selected for biomarker analysis EXCLUDE: =6,325 who did not meet product use inclusion criteria =5,197 W1 participants from biomarker sample who met inclusion criteria for product use EXCLUDE: =92 due to creatinine levels 10 mg/dl or >370 mg/dl FIAL SAMPLE n=5,105 Including: ever users (n=1,655) E cigarette only users (n=247) Dual users (n=792) Cigarette only smokers (n=2,411)

3 etable 1. Analytical Limits of Detection (), Biological Half-lives, and Assay Methods for icotine and Toxicants Assessed at Wave 1, PATH Study, Biomarker Limit of Detection Biological Half lifves Analytical Assay Method Urinary icotine Metabolites^1 (ng/ml) trans 3 Hydroxycotinine (HCTT) hrs All nicotine metabolites were assessed Cotinine (COTT) hrs using two separate isotope dilution icotine (ICT) hrs high performance liquid chromatography/ tandem mass Cotinine oxide (COXT) 2.02 /A spectrometric (HPLC MS/MS) methods. icotine 1 oxide (OXT) 2.50 /A 2,3 orcotinine (CCT) 1.11 /A ornicotine (CT) 2.50 /A Minor Tobacco Alkaloids (ng/ml) Anabasine (ABT) hrs All minor tobacco alkaloids were Anatabine (ATT) hrs assessed using two separate isotope dilution high performance liquid chromatography/tandem mass spectrometric (HPLC MS/MS) methods. 2,3 Arsenic and Arsenic Compounds (ug/l)^ Arsenous Acid hrs All arsenic compounds were assessed Arsenic Acid hrs using high performance liquid chromatography/inductively coupled Dimethylarsinic acid hrs plasma dynamic reaction cell mass Monomethylarsonic acid hrs spectrometry (HPLC ICP DRC MS). 4,5 Tobacco Specific itrosamines (TSAs) (ng/l) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) days 6 All TSAs were assessed using isotope nitrosonornicotine () 2.8 /A dilution high performance liquid nitrosoanatabine (AT) 4.2 /A chromatography/atmospheric pressure nitrosoanabasine (AB) 1.6 /A chemical ionization tandem mass spectrometry (HPLC MS/MS). 7

4 Biomarker Limit of Detection Biological Half lifves Analytical Assay Method Metals (µg/l) Beryllium (UBE) Several years All metals were assessed using Cadmium (UCD) years inductively coupled plasma mass Cobalt (UCO) Several days spectrometry (ICP MS). 8,9 Manganese (UM) days Lead (UPB) months in blood & soft tissues, years to decades in bone Strontium (USR) hrs Thallium (UTL) days Uranium (UUR) hrs Polycyclic Aromatic Hydrocarbons 10 (ng/l) 1 aphthol or 1 hydroxynaphthalene (1 AP) hrs All PAHs were assessed using 2 aphthol or 2 hydroxynaphthalene (2 AP) hrs enzymatic hydrolysis, on line solid 3 Hydroxyfluorene (3 FLU) hrs phase extraction, and isotope dilution liquid chromatography tandem mass 2 Hydroxyfluorene (2 FLU) hrs spectrometry Hydroxyphenanthrene (1 PHE) hrs 1 Hydroxypyrene (1 PYR) hrs 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) hrs Volatile Organic Compounds (VOCs) (ng/ml) 2 Methylhippuric acid (2MHA) (Xylene) hrs All VOCs were assessed using isotope 3, 4 Methylhippuric acid (34MH) (Xylene) hrs dilution UPLC MS/MS. 12,13 Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) or 17.4 hrs Acetyl S ( methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyanates) hrs Acetyl S (benzyl) L cysteine (BMA) (Toluene) 0.50 <10 hrs Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) 6.96 /A Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile) 2.60 /A Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) hrs Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) 5.25 /A Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) or 25.1 hrs Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl 0.79 >5 hrs

5 Biomarker Limit of Detection Biological Half lifves chloride, ethylene oxide) Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) 5.30 /A Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) 13.0 /A Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) 3.00 /A Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) 0.74 /A Mandelic acid (MADA) ,3.6, or 3.9 hrs Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) 0.60 >9 or <6 hrs* Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) ,8.8 or 10.5 hrs Acetyl S (phenyl) L cysteine (PMA) (Benzene) hrs 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) hrs Analytical Assay Method Footnotes: * Estimated from animal study. Multiple values indicate areas in which the literature lists multiple half-life values. /A = ot Available ^ Assays were conducted for each of the seven major nicotine metabolites listed in etable 1. In addition to the nicotine metabolites listed above, Total icotine Equivalents (TE-2) was computed and included in statistical analyses as a reflection of systemic nicotine levels for participants. TE-2 was calculated as the molar sum of cotinine and trans-3 -Hydroxycotinine. For arsenic and arsenic compounds, individual assays were conducted for each of the four listed compounds in etable 1. The analysis used a summary variable, Total Inorganic Arsenic, representing the sum of the arsenous acid, arsenic acid, dimethylarsinic acid, and monomethylarsonic acid levels in each urine sample. As these are summary variables, TE-2 and Total Inorganic Arsenic do not have listings for limits of detection.

6 etable 2: Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Current ever Users, e-cigarette Only-Users, Dual Users, and Cigarette-Only Smokers (ormalized for Creatinine, Geometric Means, 95% Confidence Intervals), PATH Wave 1 ( ) (n=5,105) ever users (1,655) (a) Current e cigarette only users (n=247) (b) Biomarker Geo Mean % Above Geo Mean % Above Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (TE2) (nmol/mg) 1, b % acd % trans 3 Hydroxycotinine (HCTT) 1, E 01 b 6.00E E 01 98% acd % Cotinine (COTT) 1, E 01 b 3.60E E 01 99% acd % icotine (ICT)^ E+01 b 1.20E E+01 64% acd % Cotinine oxide (COXT)^ E+01 b 6.80E E+01 99% acd % icotine 1 oxide (OXT)^ E+01 b 4.20E E+01 76% acd % orcotinine (CCT)^ E+00 b 2.30E E+00 95% acd % ornicotine (CT)^ E+00 b 1.80E E+00 35% acd % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ % cd % Anatabine (ATT)^ % cd % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic 1, % d % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) 1, b % ad % nitrosoanatabine (AT)* 1, b % ad % nitrosoanabasine (AB)* 1, b % ad % ' itrosonornicotine ()* 1, b % ad % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* 1, b % a % Cadmium (UCD) 1, b % ad % Cobalt (UCO) 1, % % Manganese (UM)* 1, % % Lead (UPB) 1, b % a % Strontium (USR) 1, % %

7 Thallium (UTL) 1, % % Uranium (UUR) 1, b % a % etable 2 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among current never users, e-cigarette only-users, dual users, and cigarette-only smokers (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=5,105) Current dual users (n=792) (c) Current cigarette only smokers (n=2,411) (d) Biomarker Geo Mean % Above Geo Mean % Above Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (TE2) (nmol/mg) bd % bc % trans 3 Hydroxycotinine (HCTT) bd % bc % Cotinine (COTT) bd % bc % icotine (ICT)^ b % b % Cotinine oxide (COXT)^ bd % bc % icotine 1 oxide (OXT)^ bd % bc % orcotinine (CCT)^ b % b % ornicotine (CT)^ b % b % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ b % b % Anatabine (ATT)^ b % b % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic % b % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) d % bc % nitrosoanatabine (AT)* d % bc % nitrosoanabasine (AB)* d % bc % ' itrosonornicotine ()* % b % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* % % Cadmium (UCD) % b % Cobalt (UCO) % %

8 Manganese (UM)* % % Lead (UPB) % % Strontium (USR) d % c % Thallium (UTL) % % Uranium (UUR) d % c % etable 2 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among current never users, e-cigarette only-users, dual users, and cigarette-only smokers (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=5,105) Biomarker Geo Mean ever users (1,655) (a) % Above Current e cigarette only users (n=247) (b) Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) 1, % d % 2 aphthol or 2 hydroxynaphthalene (2 AP) 1, % d % 3 Hydroxyfluorene (3 FLU) 1, % d % 2 Hydroxyfluorene (2 FLU) 1, % d % 1 Hydroxyphenanthrene (1 PHE) 1, % d % 1 Hydroxypyrene (1 PYR) 1, b % ad % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) 1, % d % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) 1, % d % 3, 4 Methylhippuric acid (34MH) (Xylene) 1, % d % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) 1, % d % Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyantes) 1, b % ad % Acetyl S (benzyl) L cysteine (BMA) (Toluene) 1, b % a % Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) 1, % d % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile)* 1, b % ad % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) 1, b % ad % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) 1, % d % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) 1, % d % Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) 1, % d % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) 1, % d % Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) 1, % d % Geo Mean % Above

9 Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) 1, % d % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) 1, % d % Mandelic acid (MADA) 1, % d % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) 1, % d % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) 1, % d % Acetyl S (phenyl) L cysteine (PMA) (Benzene) 1, % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) 1, % % etable 2 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among current never users, e-cigarette only-users, dual users, and cigarette-only smokers (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=5,105) Biomarker Current dual users (n=792) (c) Geo Mean % Above Current cigarette only smokers (n=2,411) (d) Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) d % bc % 2 aphthol or 2 hydroxynaphthalene (2 AP) % b % 3 Hydroxyfluorene (3 FLU) d % bc % 2 Hydroxyfluorene (2 FLU) d % bc % 1 Hydroxyphenanthrene (1 PHE) d % bc % 1 Hydroxypyrene (1 PYR) d % bc % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) % b % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) % b % 3, 4 Methylhippuric acid (34MH) (Xylene) d % bc % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) % b % Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyantes) d % bc % Acetyl S (benzyl) L cysteine (BMA) (Toluene) d % c % Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) d % bc % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile)* % b % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) d % bc % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) d % bc % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) % b % Geo Mean % Above

10 Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) d % bc % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) d % bc % Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) d % bc % Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) d % bc % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) d % bc % Mandelic acid (MADA) % b % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) d % bc % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) d % bc % Acetyl S (phenyl) L cysteine (PMA) (Benzene) % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) % % Footnotes: reflects total number of cases included in estimate (excluding participants with results outside of quality control standards.) *Indicates analyte with >40% of all values falling below Limit of Detection () for select group. % reflects proportion among cases included in estimate. ^ Detection rates for these metabolites only apply to participants who had urinary cotinine levels greater than 20 ng/l, which is reflected in the sample size for each biomarker. Weighted estimates; geometric mean calculated by exp(mean of log transformed [biomarker value/creatinine value]). Some VOC laboratory results were outstanding at the time these analyses were conducted, so weighted estimates may not accurately reflect values in the target population. Total icotine Equivalents (TE2) calculated by taking molar sum of trans-3'-hydroxycotinine and cotinine divided by urinary creatinine. Geometric means expressed to four significant figures if under 1000; geometric means over 1000 expressed to five significant figures. Red text denotes values with RSE>30% or denominator <50. Superscript letters denote statistically significant differences between tobacco use groups based on results from linear regression models adjusted for urinary creatinine, age, sex, ethnicity, education, SHS exposure, past 30 day marijuana use.(p<0.05). Analyses comparing dual users and cigarette-only smokers also included CPD as a control variable. a=significantly different from never users, b=significantly different from e-cigarette-only users, c=significantly different from dual users, d=significantly different cigarette-only users. $ VOC information presented as Biomarker ame (Lab Code) (Parent Compound)

11 "ever users"= "o" to lifetime use of any tobacco product; " E-cigarette-only Users"=Reported "Yes" to current everyday/someday use of e-cigarettes, "o" to current use of all other tobacco products; Dual Users"=Reported "Yes" to current everyday/someday use of tobacco cigarettes and e-cigarettes, "o" to current use of all other tobacco products; " Cigarette-only Smokers"=Reported "Yes" to current everyday/someday use of cigarettes, "o" to current use of all other tobacco products. All user groups reported no past three day use of icotine Replacement Therapies (RT).

12 emethods. Details on Model Building Procedures for Dual User Analysis and Sample Weighting Dual User Models: All models examining dual users adjusted for CPD in addition to covariates listed in Methods. Models utilized multivariable linear regression modelling, using log transformed biomarker data as outcomes, and adjusted for urinary creatinine, demographic covariates, CPD, SHS exposure, past 30 day marijuana use, and a categorical variable indicating dual user status (1- everyday smoker/everyday e-cigarette user, 2- everyday e-cigarette user/somedays smoker, 3- everyday smoker/somedays e-cigarette user, 4 - somedays smoker/somedays e-cigarette user). Models were run using the highest exposure level (Group 1, everyday smokers/everyday e-cigarette users) and Group 2, everyday smokers, somedays e-cigarette users as the referent categories. Weights: Since the biospecimen data represent a subsample of adults, specific urine weights were needed to account for potential differences between the full set of Adult Interview respondents and the set of adults with analyzed biospecimens. Weighting procedures are outlined in

13 etable 3. Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Everyday and Someday Users of e-cigarettes and Cigarettes (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=2,658) Current e cigarette only users (n=247) Biomarker Everyday users (n=147) Somedays users (n=100) Geo Mean % Above Geo Mean Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (nmol/mg) * % # % trans 3 Hydroxycotinine (HCTT) * % # % Cotinine (COTT) * % # % icotine (ICT)^ * % # % Cotinine oxide (COXT)^ * % # % icotine 1 oxide (OXT)^ * % # % orcotinine (CCT)^ * % # % ornicotine (CT)^ * % # % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ * % % Anatabine (ATT)*^ * % % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic * % % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) * % % nitrosoanatabine (AT)* * % % nitrosoanabasine (AB)* * % # % ' itrosonornicotine ()* * % # % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* % % Cadmium (UCD) % % Cobalt (UCO) * % % Manganese (UM)* % % Lead (UPB) % # % Strontium (USR) * % # % Thallium (UTL) * % % % Above

14 Uranium (UUR) % % etable 3 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among everyday and someday users of e-cigarettes and cigarettes (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=2,658) Current Cigarette only Smokers (n=2,411) Biomarker Everyday users (n=1,975) Somedays users (n=436) Geo Mean % Above Geo Mean Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (nmol/mg) 1, # % *# % trans 3 Hydroxycotinine (HCTT) 1, # % *# % Cotinine (COTT) 1, # % *# % icotine (ICT)^ # % *# % Cotinine oxide (COXT)^ # % *# % icotine 1 oxide (OXT)^ # % *# % orcotinine (CCT)^ # % *# % ornicotine (CT)^ # % *# % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ # % *# % Anatabine (ATT)*^ # % *# % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic # % * % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) # % *# % nitrosoanatabine (AT)* # % *# % nitrosoanabasine (AB)* # % *# % ' itrosonornicotine ()* # % * % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* % % Cadmium (UCD) % *# % Cobalt (UCO) # % # % Manganese (UM)* % % Lead (UPB) % # % % Above

15 Strontium (USR) # % # % Thallium (UTL) # % % Uranium (UUR) % % etable 3 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among everyday and someday users of e-cigarettes and cigarettes (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=2,658) Current e cigarette only users (n=247) Biomarker Everyday users (n=147) Somedays users (n=100) Geo Mean Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) * % % 2 aphthol or 2 hydroxynaphthalene (2 AP) * % % 3 Hydroxyfluorene (3 FLU) * % % 2 Hydroxyfluorene (2 FLU) * % % 1 Hydroxyphenanthrene (1 PHE) * % % 1 Hydroxypyrene (1 PYR) * % % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) * % % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) * % % 3, 4 Methylhippuric acid (34MH) (Xylene) * % % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) * % % Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyantes) * % % Acetyl S (benzyl) L cysteine (BMA) (Toluene) % % Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) * % % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile)* * % % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) * % # % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) * % % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) * % % Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) * % % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) * % % % Above Geo Mean % Above

16 Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) * % % Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) * % % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) * % % Mandelic acid (MADA) * % % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) * % % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) * % % Acetyl S (phenyl) L cysteine (PMA) (Benzene) % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) % % etable 3 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among everyday and someday users of e-cigarettes and cigarettes (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=2,658) Current Cigarette only Smokers (n=2,411) Biomarker Everyday users (n=1,975) Somedays users (n=436) Geo Mean Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) # % *# % 2 aphthol or 2 hydroxynaphthalene (2 AP) # % *# % 3 Hydroxyfluorene (3 FLU) # % *# % 2 Hydroxyfluorene (2 FLU) # % *# % 1 Hydroxyphenanthrene (1 PHE) # % * % 1 Hydroxypyrene (1 PYR) # % * % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) # % *# % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) # % *# % 3, 4 Methylhippuric acid (34MH) (Xylene) # % *# % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) # % *# % Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyantes) # % *# % Acetyl S (benzyl) L cysteine (BMA) (Toluene) % % Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) # % *# % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile)* # % *# % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) # % *# % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) # % *# % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) # % * % % Above Geo Mean % Above

17 Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) # % *# % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) # % * % Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) # % *# % Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) # % *# % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) # % *# % Mandelic acid (MADA) # % *# % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) # % *# % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) # % * % Acetyl S (phenyl) L cysteine (PMA) (Benzene) % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) % % Footnotes: reflects total number of cases included in estimate (excluding participants with results outside of quality control standards.) *Indicates analyte with >40% of all values falling below Limit of Detection () for select group. % reflects proportion among cases included in estimate. ^ Detection rates for these metabolites only apply to participants who had urinary cotinine levels greater than 20 ng/l, which is reflected in the sample size for each biomarker Weighted estimates, geometric mean calculated by exp(mean of log transformed [biomarker value/creatinine value]). Some VOC laboratory results were outstanding at the time these analyses were conducted, so weighted estimates may not accurately reflect values in the target population. Total icotine Equivalents (TE2) calculated by taking molar sum of trans-3'-hydroxycotinine and cotinine divided by urinary creatinine. Geometric means expressed to four significant figures if under 1000; geometric means over 1000 expressed to five significant figures. Red text denotes values with RSE>30% or denominator <50 $ VOC information presented as Biomarker ame (Lab Code) (Parent Compound) * Denotes statistically significant difference when compared to daily cigarette-only smokers based on results from linear regression models adjusted for urinary creatinine, age, sex, ethnicity, education, SHS exposure, past 30 day marijuana use (p<0.05). # Denotes statistically significant difference when compared to daily e-cigarette users based on results from linear regression models adjusted for urinary creatinine, age, sex, ethnicity, education, SHS exposure, past 30 day marijuana use, (p<0.05).

18 etable 4. Biomarkers of Exposure to Tobacco-Related Compounds and Other Chemicals, Among Dual Users (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=792) Biomarker Geo Mean Everyday smokers/everyday e cigarette users (n=90) (a) % Above Everyday e cigarette users/somedays smoker (n=55) (b) Geo Mean Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (TE2) (nmol/mg) d % d % trans 3 Hydroxycotinine (HCTT) bd % a % Cotinine (COTT) d % d % icotine (ICT)^ d % d % Cotinine oxide (COXT)^ d % d % icotine 1 oxide (OXT)^ d % d % orcotinine (CCT)^ d % d % ornicotine (CT)^ d % d % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ d % c % Anatabine (ATT)^ bd % ac % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic % % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) bd % ac % % Above

19 nitrosoanatabine (AT) bd % ac % nitrosoanabasine (AB) bd % ac % ' itrosonornicotine () bd % ac % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* c % % Cadmium (UCD) d % % Cobalt (UCO) % % Manganese (UM)* % % Lead (UPB) % % Strontium (USR) % % Thallium (UTL) % % Uranium (UUR) % % etable 4 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among dual users (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=792) Biomarker Geo Mean Everyday smokers/somedays e cigarette users (n=560) (c) % Above Somedays smokers/somedays e cigarette users (n=87) (d) Urinary icotine Metabolites (ng/mg creatinine) icotine Equivalence (TE2) (nmol/mg) d % abc % trans 3 Hydroxycotinine (HCTT) d % ac % Cotinine (COTT) d % abc % icotine (ICT)^ d % abc % Cotinine oxide (COXT)^ d % abc % icotine 1 oxide (OXT)^ d % abc % orcotinine (CCT)^ d % abc % ornicotine (CT)^ d % abc % Minor Tobacco Alkaloids (ng/mg creatinine) Anabasine (ABT)^ bd % ac % Anatabine (ATT)^ bd % ac % Arsenic and Arsenic Compounds (ug/g creatinine) Total Inorganic Arsenic % % Tobacco Specific itrosamines (TSAs) (pg/mg creatinine) Geo Mean % Above

20 4 methylnitrosamino) 4 (3 pyridyl) 1 butanol (AL) bd % ac % nitrosoanatabine (AT) bd % ac % nitrosoanabasine (AB) bd % ac % ' itrosonornicotine () bd % ac % Heavy Metals (ng/mg creatinine) Beryllium (UBE)* a % % Cadmium (UCD) d % ac % Cobalt (UCO) % % Manganese (UM)* % % Lead (UPB) % % Strontium (USR) % % Thallium (UTL) % % Uranium (UUR) % % etable 4 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among dual users (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=792) Biomarker Geo Mean Everyday smokers/everyday e cigarette users (n=90) (a) % Above Everyday e cigarette users/somedays smoker (n=55) (b) Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) d % c % 2 aphthol or 2 hydroxynaphthalene (2 AP) d % c % 3 Hydroxyfluorene (3 FLU) bd % ac % 2 Hydroxyfluorene (2 FLU) bd % ac % 1 Hydroxyphenanthrene (1 PHE) bd % ac % 1 Hydroxypyrene (1 PYR) bcd % ac % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) bd % ac % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) bd % ac % 3, 4 Methylhippuric acid (34MH) (Xylene) bd % ac % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) bd % ac % Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyanates) bd % ac % Acetyl S (benzyl) L cysteine (BMA) (Toluene) % % Geo Mean % Above

21 Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) bd % ac % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile) bcd % ac % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) bd % ac % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) % c % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) bd % ac % Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) bd % ac % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) % c % Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) bd % ac % Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) bcd % ac % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) bd % ac % Mandelic acid (MADA) bd % ac % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) bcd % ac % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) bcd % ac % Acetyl S (phenyl) L cysteine (PMA) (Benzene) % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) % % etable 4 cont : Biomarkers of exposure to tobacco-related compounds and other chemicals, among dual users (normalized for creatinine, geometric means, 95% confidence intervals), PATH Wave 1 ( ) (n=792) Everyday smokers/somedays e cigarette users (n=560) (c) % Biomarker Geo Mean Above Somedays smokers/somedays e cigarette users (n=87) (d) % Geo Above Mean Polycyclic Aromatic Hydrocarbons (ng/mg creatinine) 1 aphthol or 1 hydroxynaphthalene (1 AP) bd % ac % 2 aphthol or 2 hydroxynaphthalene (2 AP) bd % ac % 3 Hydroxyfluorene (3 FLU) bd % ac % 2 Hydroxyfluorene (2 FLU) bd % ac % 1 Hydroxyphenanthrene (1 PHE) bd % ac % 1 Hydroxypyrene (1 PYR) abd % ac % 2 Hydroxyphenanthrene and 3 Hydroxyphenanthrene (2 3PHE) bd % ac % Volatile Organic Compounds (VOC) $ (ng/mg creatinine) 2 Methylhippuric acid (2MHA) (Xylene) bd % ac % 3, 4 Methylhippuric acid (34MH) (Xylene) bd % ac % Acetyl S (2 carbamoylethyl) L cysteine (AAMA) (Acrylamide) bd % ac %

22 Acetyl S (methylcarbamoyl) L cysteine (AMCA) (, Dimethylformamide/isocyanates) bd % ac % Acetyl S (benzyl) L cysteine (BMA) (Toluene) % % Acetyl S (2 carboxyethyl) L cysteine (CEMA) (Acrolein) bd % ac % Acetyl S (1 cyano 2 hydroxyethyl) L cysteine (CYHA) (Acrylonitrile) abd % ac % Acetyl S (2 cyanoethyl) L cysteine (CYMA) (Acrylonitrile) bd % ac % Acetyl S (3,4 dihydroxybutyl) L cysteine (DHBM) (1,3 Butadiene) bd % c % Acetyl S (2 carbamoyl 2 hydroxyethyl) L cysteine (GAMA) (Acrylamide) bd % ac % Acetyl S (2 hydroxyethyl) L cysteine (HEMA) (Acrylonitrile, vinyl chloride, ethylene oxide) bd % ac % Acetyl S (2 hydroxypropyl) L cysteine (HPM2) (Propylene Oxide) bd % c % Acetyl S (3 hydroxypropyl) L cysteine (HPMA) (Acrolein) bd % ac % Acetyl S (3 hydroxypropyl 1 methyl) L cysteine (HPMM) (Crotonaldehyde) abd % ac % Acetyl S (4 hydroxy 2 methyl 2 buten 1 yl) L cysteine (IPM3) (Isoprene) bd % ac % Mandelic acid (MADA) bd % ac % Acetyl S (4 hydroxy 2 buten 1 yl) L cysteine (MHB3) (1,3 Butadiene) abd % ac % Phenylglyoxylic acid (PGHA) (Ethylbenzene, styrene) abd % ac % Acetyl S (phenyl) L cysteine (PMA) (Benzene) % % 2 Thioxothiazolidine 4 carboxylic acid (TTCA) (Carbon Disulfide) % % Footnotes: reflects total number of cases included in estimate (excluding participants with results outside of quality control standards.) *Indicates analyte with >40% of all values falling below Limit of Detection () for select group. % reflects proportion among cases included in estimate. ^ Detection rates for these metabolites only apply to participants who had urinary cotinine levels greater than 20 ng/l, which is reflected in the sample size for each biomarker Weighted estimates, geometric mean calculated by exp(mean of log transformed [biomarker value/creatinine value]). Some VOC laboratory results were outstanding at the time these analyses were conducted, so weighted estimates may not accurately reflect values in the target population. Total icotine Equivalents (TE2) calculated by taking molar sum of trans-3'-hydroxycotinine and cotinine divided by urinary creatinine. Geometric means expressed to four significant figures if under 1000; geometric means over 1000 expressed to five significant figures. Red text denotes values with RSE>30% or denominator <50. Superscript letters denote statistically significant differences between dual user groups based on results from linear regression models adjusted for urinary creatinine, CPD, age, sex, ethnicity, education, SHS exposure, past 30 day marijuana use, and tobacco use status (p<0.05).

23 $ VOC information presented as Biomarker ame (Lab Code) (Parent Compound)

24 ereferences 1. Li Z, Romanoff L, Bartell S, et al. Excretion Profiles and Half-Lives of Ten Urinary Polycyclic Aromatic Hydrocarbon Metabolites after Dietary Exposure. Chemical research in toxicology. 2012;25(7): Bernert JT, Harmon TL, Sosnoff CS, McGuffey JE. Use of continine immunoassay test strips for preclassifying urine samples from smokers and nonsmokers prior to analysis by LC-MS-MS. Journal of analytical toxicology. 2005;29(8): Wei B, Feng J, Rehmani IJ, et al. A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites. Clinica chimica acta; international journal of clinical chemistry. 2014;436: Caldwell KL, Jones RL, Verdon CP, Jarrett JM, Caudill SP, Osterloh JD. Levels of urinary total and speciated arsenic in the US population: ational Health and utrition Examination Survey Journal of exposure science & environmental epidemiology. 2009;19(1): Verdon CP, Caldwell KL, Fresquez MR, Jones RL. Determination of seven arsenic compounds in urine by HPLC-ICP-DRC-MS: a CDC population biomonitoring method. Analytical and bioanalytical chemistry. 2009;393(3): Goniewicz ML, Havel CM, Peng MW, et al. Elimination kinetics of the tobacco-specific biomarker and lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2009;18(12): Xia B, Xia Y, Wong J, et al. Quantitative analysis of five tobacco-specific -nitrosamines in urine by liquid chromatography-atmospheric pressure ionization tandem mass spectrometry. Biomedical chromatography : BMC. 2014;28(3): Caldwell KL, Hartel J, Jarrett J, Jones RL. Inductively coupled plasma mass spectrometry to measure multiple toxic elements in urine in HAES Atomic Spectroscopy. 2005;26(1): Jarrett JM, Xiao G, Caldwell KL, Henahan D, Shakirova G, Jones RL. Eliminating molybdenum oxide interference in urine cadmium biomonitoring using ICP-DRC-MS. Journal of Analytical Atomic Spectrometry. 2008;23(7): St Helen G, Goniewicz ML, Dempsey D, Wilson M, Jacob P, 3rd, Benowitz L. Exposure and kinetics of polycyclic aromatic hydrocarbons (PAHs) in cigarette smokers. Chemical research in toxicology. 2012;25(4): Wang Y, Meng L, Pittman E, et al. Quantification of urinary mono-hydroxylated metabolites of polycyclic aromatic hydrocarbons by on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. Analytical and bioanalytical chemistry. 2017;409(4): Alwis KU, Bailey TL, Patel D, Wang L, Blount BC. Measuring urinary -acetyl-s-(4-hydroxy-2-methyl-2- buten-1-yl)-l-cysteine (IPMA3) as a potential biomarker of isoprene exposure. Analitica chimica acta. 2016;941: Alwis KU, Blount BC, Britt AS, Patel D, Ashley DL. Simultaneous analysis of 28 urinary VOC metabolites using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI/MSMS). Analytica chimica acta. 2012;750:152.