Literature Review on Nicotine’s Role in Human Health

94 Males:

• 33 Smokers (CC), 41.3 y, CC use since 5.4 y, 13.3 cig/d

• 31 Vapers (EC), 37.6 y, EC use since 2.2 y, 6.8 sessions/d

• 30 NU, 40.7 y

Periodontal parameters, sign. diff.: < / >:

PI : CC > EC ≈ NU

BOP: CC ≈ EC < NU

CAL: CC ≈ EC ≈ NU

MBL: CC ≈ EC ≈ NU

Perceived oral symptoms (OS):

• Gingival pain: CC > EC ≈ NU

• Gingival bleeding: CC ≈ EC > NU

• Gingival swelling: CC > EC ≈ NU

AO: periodontal inflammation and self-perceived OS were poorer among CC than among EC.

ARO: Dual users were excluded, but no verification of EC only users.

The authors cite evidence that N leads to vasoconstriction and therefore low BOP.

ARO: Study data do not allow to deduce N's role.

L: Small groups.

G: No verification of EC only use; no N-free EC group.

• 45 Former smokers (FCC), mean 47 y

• 45 EC users (EC), mean age 47 y, ECs for at least 6 months, N content: 0–34 mg/mL

Oral mucosa lesions, sign. higher in EC:

• Nicotine stomatitis

• Hairy tongue

• Hyperplastic candidiasis

Not sign. diff.:

• Melanosis

• Rhomboid glossitis

• Lichen planus

• Erythematous candidiasis

• Leukoplakia

• Squamous cell carcinoma

• Total

AO: EC use is linked to three types of inflammatory lesions, but not in precancerous lesions.

ARO: Only self-reports, no objective check for dual use.

ARO: N's role cannot be deduced from the study.

L: Small group sizes, only medium- to short-term use of EC.

G: No check for dual use; no N-free EC group.

P: Larger long-term study with EC only use verification and an N-free EC group would be of interest.

154 Males:

• 39 Smokers (CC), 42.4 y, duration of habit: 17.2 y, 16.2 cig/d, 4.8 min/cig

• 40 Waterpipe (WP), 44.7 y, duration of habit: 14.6 y, 4.3 WP/d, 17.1 min/WP

• 37 Vapers (EC), 28.3 y, duration of habit: 3.1 y, 9.2 cig/d, 8.1 min/cig

• 38 NU, 40.6 y

Oral health parameters, > / <: sign. diff.:

• PI: CC ≈ WP > EC* ≈ NU

* not sign. diff. from all other groups

• BoP: CC ≈ WP > EC ≈ NU

• PD: CC ≈ WP > EC ≈ NU

• CAL: CC ≈ WP > EC ≈ NU

• MBL: CC ≈ WP > EC ≈ NU

BMs in saliva:

• Cot: CC ≈ WP ≈ EC > NU

• IL-1ß: CC ≈ WP > EC ≈ NU

• IL-6: CC ≈ WP > EC ≈ NU

AO: Parameters of oral health were poorer in CC and WP than EC and NU.

ARO: Dual users were excluded, but no verification of EC only use.

EC group is significantly (?) younger!

Authors cite constricting effect of N on gingival blood vessels.

ARO: N's role cannot been deduced from the study.

L: Small group sizes; EC only use not objectively verified.

G: No inclusion of an N-free EC group.

Male subjects with teeth implant :

• 40 Smokers (CC), 45.8 y, CC since 21.3 y

• 40 Water pipe users (WP), 43.5 y, WP use for 19.5 y

• 40 Vapers (EC), 35.6 y, EC use for 8.7 y, 6.5 sessions/d, 37.7 min/session

• 40 Non-smokers (NS), 42.6 y

Peri-implant parameters:

• PI: Sign. higher in CC, WP, EC vs NS

• BOP: Sign. lower in CC, WP, EC vs NS

• PD: Sign. higher in CC, WP, EC vs NS; also sign, higher in CC, WP vs EC

Bone loss:

• Sign, higher in CC, WP, EC vs NS; also sign. higher in CC, WP vs EC

TNF-α, IL-6, IL-1ß in PISF:

• Sign. higher in CC, WP, EC than in NS

ARO: The authors do not interpret their results with EC users.

No information on possible dual use of EC and other products.

The authors state that N is reported to exert vasoconstriction on gingival blood vessels.

ARO: A role of N cannot be deduced from the study.

G: No N-free EC group; no information on possible dual use EC/other products (CC, WP).

P: A study with these gaps filled would be of interest.

Subjects with teeth implants:

• 47 Male vapers (EC), 35.8 y, mean EC use: 4.4 y, 6.5 sessions/d, 37.7 min/session

• 45 Male never smokers (NS), 42.6 y

Peri-implant parameters:

• PI: not sign. diff.

• BOP: sign. lower in EC than NS

• PD: sign. higher in EC than NS

• PIBL: sign. higher in EC than NS

TNF-α and IL-1ß in PISF: sign. higher in EC than NS

ARO: No information on possible additional CC use.

The authors state that N is reported to reduce the healing process and exerts vasoconstriction on gingival blood vessels.

ARO: A role of N cannot be deduced from the study.

G: No N-free EC group; no CC group (positive control); no information on possible dual use EC/CC.

P: A study with these gaps filled would be of interest.

• 9,632 Never EC users (nEC)

• 329 Longitudinal EC users (LEC) for at least 1 year

• 8,298 Occasional EC users (OEC)

PATH study, waves 1, 2, 3

LEC sign. increased compared to nEC at wave 3 for:

• New gum disease (compared to wave 1)

• Bone loss around teeth

• any periodontal disease

nEC and OEC were not sign. different

AO: EC use may be harmful to oral health.

ARO: All data on behavior and endpoints were self-reports.

ARO: N's role or involvement in the effects cannot be deduced from the study.

L: Only self-reports; EC use might be too short.

G: No objective verification of EC only use; no N-free EC group.

• 46 CC users (14.2 pack × years)

• 44 EC users (duration of use: 9.4 y)

• 45 NU (never used CC or EC)

Periodontal health status:

CC < EC < NU

Indicators: PI, BOP, PD, CAL, MBL

Cytokine profile in gingival crevicular fluid (IL1ß, IL6, INFγ, TNF-α, MMP-8); CC sign. diff. from EC and NU; EC closer to NU

AO: Periodontal status is poorer and GCF levels of proinflam. cytokines are higher in CC compared with EC and NU. There is evidence for increased periodontal inflammation in EC users.

ARO: Compliance of EC use not approved (could be questionable).

ARO: N's role cannot be deduced.

L: Small group sizes; EC compliance questionable (given the long duration of use).

102 Males with tooth implants:

• 35 Smokers, 36.3 y, daily CC use for > 12 months

• 33 Waterpipe (WP) users, 34.1 y, daily WP use for > 12 months

• 34 Vapers, 33.5 y, daily EC use for > 12 months, 8.4 mg N/mL

• 35 NU, 32.2 y

Peri-implant (inflamm.) variables:

• PI, PD, cotinine sign. increased, BOP decreased in CC, WP, EC compared to NU

AO: Cotinine peri-implant in fluid was increased in users of N products (ARO: trivial!).

ARO: Dual users were excluded, but not verification.

Authors suggest that N is the main effector.

ARO: N's role, however, cannot be deduced from the study data.

L: Small group sizes, unclear compliance (particularly of EC group).

G: No N-free EC group, no objective verification of compliance.

95 Males with tooth implants:

• 32 Smokers (CC), 40.4 y, CC since 13.7 y, 11.3 cig/d

• 41 Vapers (EC), 35.8 y, EC since 4.4 y, 6.5 sessions/d, 37.7 min/session

Peri-implant sign diff:

• PI: CC > EC > NU

• BOP: CC ≈ EC < NU

• PD: CC ≈ EC < NU

• MBL: CC > EC > NU

• MMP-9: CC > EC > NU

• IL-1ß: CC > EC > NU

AO: Peri-implant is compromised by CC and (to smaller degree) also by EC.

ARO: Dual users were excluded, but not verification of status.

The authors point out that N can reduce healing and BOP and also AGEs.

ARO: N's role cannot be deduced from the study data.

L: Small group sizes; unclear compliance.

G: No N-free EC group; no objective verification of EC only use.

89 Males, at least 30% BOP:

• 28 Vapers (EC use daily since at least 12 months, no history of tobacco use), 32.5 y

• 30 Smokers (CC, > 5 cig/d on the previous 12 months), 36.4 y

• 31 NU, 32.6 y

Investigations at BL, 3 and 6 months; FMUS after BL

Gingival inflammation (as indicated by PI, PD, BOP) were similar in EC and NU group and worse in CC group (at all time points).

AO: State to be cautious with the interpretation of the results due to weaknesses in the study design.

ARO: Dual users excluded, but no verification of EC only use.

AO: It is stated that N has a vasoconstrictive effect reducing the microcirculation.

ARO: N's role cannot be deduced from the study.

L: Low number of subjects, short duration of product use.

G: No N-free EC group, no verification of EC only use.

• 42 EC users, 28 y, CotP: 115 ng/mL

• 24 CC users, 42 y, CotP: 122 ng/mL

• 27 NU, 24 y, CotP: 2.5 ng/mL

Gene dysregulation in oral epithelial cells compared to NU: ~50% higher in CC (1,725 vs 1,152; 299 in common) “cancer pathway” was highest in EC and CC

AO: The findings have significant impact on public health.

ARO: Possible dual use not considered.

N's role not discussed by the authors.

ARO: cannot be deduced from the study results.

L: Small group sizes; dual use is possible.

G: No N-free group.

P: Study with the weaknesses eliminated would be of interest.

71 Subjects with chronic periodontitis:

• 36 Vapers (EC), 47.7 y, mean duration of EC: 3.3 y, 17.6 sessions/d, 8.4 puffs/session, all were former smokers

• 35 NU, 46.5 y

Investigations at BL and 3 months, in between: SRP

BL: no sign. diff. between EC and NU in PI, PD, CAL, MBL, GCF IL-4, IL-9, IL-10, and IL-13; 3-months: no sign. diff. in PI, PD, CAL, MBL in EC compared to BL, but sign. reduction in PI, GI and PD in NU; GCF IL-4, IL-9, IL-10, and IL-13 elevated in both groups compared to BL (higher in NU than EC)

AO: The anti-inflammatory effect of SRP was higher in NU than in EC.

ARO: No verification of EC only use.

ARO: N's role cannot be deduced, although N may be involved mechanistically in the inflammation processes.

L: Small group sizes.

G: No verification of EC only use; no N-free EC group.

Periodontitis patients:

• 19 Smokers (CC), 35.3 y, smoking for 14.0 y

• 19 Vapers (EC), 34.7 y, CC use for 12.1 y than switched to EC since at least 12 months

• 19 Former smokers (FS), 35.6 y, CC use for 12.1 y then stopped since at least 12 months

• PD and PI not sign. diff. between groups

• GI: sign. diff., CC < EC < FS

• BMs in GCF:

• IL-8: CC < EC < FS, sign.

• TNF-α: CC > EC > FS, sign.

• GSH-Px: CC ≈ EC < FS, sign.

• 8-OHdG: not sign. diff.

AO: CCs and ECs had the same unfavourable effects on the markers of oxidative stress and inflammatory cytokines.

ARO: Dual smokers (CC and EC) were excluded. No information on compliance.

ARO: Role of nicotine cannot be deduced from the study.

L: Small group sizes, no long-term use of ECs, no information on unique EC use in vapers.

G: No N-free EC group.

• 12 NU, 35.7 y, CotS: 0.56 ng/mL

• 12 CC, 40.3 y, CotS: 142.5 ng/mL

• 12 EC, 34.9 y, CotS: 180.2 ng/mL, N in e-liquid: 0–24 mg/mL

• 12 Dual (EC, CC) users (DU), 39.4 y, CotS: 299.0 ng/mL

Sign. diff. between groups:

CotS: DU > NU

Inflamm BMs in saliva:

• IL-1ß: none

• PGE2: CC > NU, CC > EC, CC > DU

GCF BMs for inflamm, ox stress and growth factors:

• RAGE: DU > EC

• MMP-9: NU > EC

• MPO: NU < CC, NU < EC, EC < DU

Various growth factors: none

AO: EC/CC induced differential changes on oral health.

ARO: Strange sign. diff. for CotS and some other BMs.

No detailed assessment of product use.

ARO: N's role cannot be deduced.

L: Small group sizes; EC compliance questionable.

G: No N-free EC group.

105 Males:

• 28 Smokers (CC), 33.3 y, 6.1 pack-years

• 26 EC users, 31.6 y, 0.9 y EC use, 30.2 sessions/d

• 25 JUUL (JU) users, 32.1 y, 0.8 y JU use, 25.3 sessions/d

• NU, 33.5 y

Self-rated oral symptoms, sign. diff.:

• Pain in teeth: CC > JU, CC > NU, EC > NU

• Bleeding gums: EC > NU

• Bad breath: CC > EC, CC > JU, CC > NU

• Pain in gums: CC > EC, CC > JU, CC > NU

Periodontal parameters, sign. diff.:

• PI: CC > EC ≈ JU ≈ NU

• BOP: none

• PD: CC > EC ≈ JU ≈ NU

CAL: none

• MBL: none

AO: Pain in teeth and gums are more often perceived by CC than EC and JUUL users and NU.

ARO: Dual users were excluded, but no verification of EC or JUUL only use.

Authors cite evidence that N is involved in gingival AGE formation and gum blood flow.

ARO: Study results do not provide evidence for N's role in oral health.

L: Small group sizes; only short use of EC and JU.

G: No N-free EC group.

Male subject N habit since > 12 months, mean age ~45 y in each subgroup:

• 30 Smokers (CC), CC habit: 18.3 y, 12.6 cig/d, 8.3 min/cig

• 30 WP users, WP habit: 15.6 y, 5.5 WP/d, 22.6 min/WP

• 30 EC users, EC habit: 6.4 y, 15.4 sessions/d, 20.5 min/session

• 30 NU

Oral health, sign. diff. (</>):

• PI/PD/CAL/MBL: CC ≈ WP ≈ EC > NU

• BOP: CC ≈ WP ≈ EC < NU

Markers in GCF:

• RANKL:CC ≈ WP ≈ EC > NU

• OPG: CC ≈ WP ≈ EC > NU

AO: All product users (including EC) show impairment of oral health.

ARO: Dual users were excluded. No verification of possible dual use in subgroups.

The authors cite some evidence that N can increase RANKL and OPG levels.

ARO: From this study, no involvement of N can be deduced.

L: Small group sizes; unclear, whether only EC were used (no objective verification).

G: No N-free EC group.

P: A larger study without these weaknesses would be worthwhile.

• 39 NU, (m/f): 29/38 y, COex: 1.8 ppm, CotS: 11.9 ng/mL

• 40 EC users, (m/f): 36/36 y, COex: 5.1 ppm, CotS: 104 ng/mL

• 40 CC users, (m/f): 46/45 y, COex: 18.8 ppm, CotS: 536 ng/mL

Oral microbiome:

Sign. diff. in EC from NU and also from CC

AO: EC users are more prone to infections.

ARO: Dual use not unlikely (see COex in EC).

The authors mention evidence that N could be involved.

ARO: N's role cannot be deduced from the data.

L: Small group sizes; long-term product use not well characterized, misclassification possible.

G: No N-free EC group.

64 Subjects, 28–83 y:

• 15 Controls (NU)

• 18 CC users

• 16 Mixed (CC, EC)

• 15 EC users

Pro-inflamm BMs in saliva:

• IL-6, IL-8, IL1ß*, TNF-α*

Anti-inflamm BMs in saliva:

• IL-10, IL-1RA, CRP

*: sign. diff. in ANOVA

EC closer to CC and mixed than to NU.

AO: The combined findings of this study and previous studies put into question the safety of ECs as a smoking cessation mechanism.

ARO: No statement on duration of EC use, no check of self-reported product use.

ARO: N's role cannot be deduced.

L: Small group sizes.

G: No information on duration of use of products.

• 30 Male vapers (EC), ≥ 2 y EC use

• 30 Male smokers (CC), ≥ 2 y CC use

• Investigations at BL, 3 and 6 months

Sign. differences

• BOP: higher in EC

• PD: higher in CC at 6 month

Sign. dose-response in CC group (pack-years) for:

• PD, CAL, MMP-8,CTX (at BL, 3 and 6 months)

Sign. dose-response in EC group (session-years) for:

• PD (all time points), MMP-8 (3 and 6 months)

Effects higher in CC than EC.

AO: CC showed higher periodontal worsening than EC. MMP-8, CTX are prognostic factors for clinical attachment loss in CC and EC users.

ARO: No verification of pure EC or CC status.

ARO: N can lead to reduced gingival blood flow (vasoconstriction, lower BOP in CC).

N's role and participation cannot be deduced distinctly from this study.

L: Small groups, 6 months might be too short.

G: No N-free group.

P: Larger study with verified long-term users would be of interest.

• 27 CC users, (m/f): 48/51 y

• 28 EC users, (m/f): 36/40 y

• 29 NU, (m/f): 29/39 y

Investigations at BL and 6 months later

Subgingival microbiome:

• consistent for 3 groups over time

• Unique in EC group, higher agreement with CC

• No clear group diff. for cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, others)

• PD sign. higher in CC

AO: Suggest that there is a unique periodontal risk associated with e-cig use (similar to CC).

ARO: EC only use not verified.

The authors assume that N could have specific selection pressure on the microbiome.

ARO: N's role is not deducible from this study.

L: Small group sizes; duration of EC use too short?

G: No N-free EC group.

• 20 NU

• 20 EC users

• 8 CC users

Acrolein-DNA adducts in buccal cells (measured monthly, 3 time points), medians in fmol/μmol Gua:

CC / EC / NU:

446 /179 / 21.0

AO: This is the first identification of a carcinogen-DNA adduct in any tissue of EC users.

ARO: No involvement of N to be expected.

L: Small group sizes; dual use cannot be completely excluded.

P: Larger study with a long-term verification marker would be of interest.

76 Subjects, ~25 y :

• 12 Snus (including N pouch users)

• 19 Smokers (CC)

• 8 Vapers (EC)

• 37 NU

Oral mucosa changes:

• Only seen in snus group

Inflammation BMs in saliva:

• IL-6: Snus > CC ≈ EC > NU

• IL-1: Snus > CC ≈ EC > NU

• IL-8: Snus > CC ≈ EC > NU

• TNF-α: Snus > CC ≈ EC > NU

AO: Saliva is a suitable matrix for detecting oral mucosa changes. Product use relies on self-reports.

ARO: N's role cannot be deduced from the data.

L: Small groups; not well characterized product use history; snus and N pouch use not separated.

G: No N-free EC group.

72 Healthy, young subjects:

• 24 EC only users, 24.3 y, CotP: 84.9 ng/mL

• 24 CC only users, 26.0 y, CotP: 76.7 ng/mL

• 24 NU (no CC, no EC), 25.3 y, CotP: 2.6 ng/mL

DNA lesions in oral epithelial cell (LA-PCR methods), (≈: not sign.)

• POLP gene: CC ≈ EC > NU

• HPRT gene: CC ≈ EC > NU

EC: dose-dependent and device-dependent

AO: DNA damage was shown for the first time.

ARO: EC/CC use only short-term verified.

The authors state that N-content in e-liquid was not a predictor for DNA damage.

ARO: No role of N deducable.

L: Product use not verified (long-term); small group sizes; DNA lesions are unspecific (strand breaks, bulky adducts, oxidation, etc.).

P: A larger study, avoiding the weaknesses with a specific DNA analysis would be of interest.

35–64 y old men:

• 585 Cases with MI

• 589 Controls

Duration of SLT use: 10–30 y

ORs (CI) vs NU for MI:

• CC: 1.87 (1.40–2.48)

• SLT: 0.89 (0.62–1.29)

AO: Snuff dipping for middle aged men is associated with a lower risk for MI than smoking (CC).

ARO: No verification of SLT use.

The authors suggest that CO and PAH, rather than N, might be more important for MI formation.

N's role is probably small and cannot be deduced.

L: Relative low number of cases/controls using SLT (10–15%).

G: Dual use (SLT+CC) not assessed.

P: Larger study which avoids these weaknesses would be of interest.

WHO MONICA study, 25–64 y old men:

• 687 MI cases

• 687 Controls

Duration of SLT use: 5–30 y

ORs (CI) for first MI (fatal and non-fatal) compared to NU:

• SLT only (N=59): 0.96 (0.65–1.41)

• CC only (N=248): 3.65 (2.67–4.99)

SLT risk slightly (not sign.) higher for fatal MI (OR=1.50)

AO: MI is not increased in snuff dippers.

ARO: Dual use was evaluated separately, but no verification for SLT only users.

L: Small group sizes; only men, relatively young population.

G: No other NGPs; no N-free ECs.

MI, RR for ever snuff users vs NU:

• Non-fatal: 0.91 (0.81–1.02)

• Fatal: 1.28 (1.06–1.55)

• Fatal, highest consumption: 1.96 (1.08–3.58)

AO: Snuff use increases the risk of fatal MI.

ARO: Tobacco use information was obtained from questionnaires at entry.

The authors cite evidence that N is a risk factor for MI.

Participation of N in MI generation cannot be excluded.

L: RR are very low and only sign. for fatal MIs; no clear dose-response; tobacco habits rely on self-reports; changes in habits are not assessed.

9 Epi studies on MI

6 Epi studies on stroke

in Sweden (Swe) and USA

Duration of use: 20 y (estimate)

RR (CI) of current SLT users (number of studies):

Any MI:

• Swe (6): 0.87 (0.75–1.02)

• USA (3): 1.11 (1.04–1.19)

Fatal MI:

• Swe (5): 1.27 (1.07–1.52)

• USA (3): 1.11 (1.04–1.19)

Any stroke:

• Swe (3): 1.02 (0.93–1.13)

• USA (3): 1.39 (1.22–1.60)

Fatal stroke:

• Swe (3): 1.25 (0.91–1.70)

• USA (3): 1.39 (1.22–1.60)

AO: Use of SLT increases the risk of fatal MI and stroke, which does not seem to be explained by chance.

ARO: Dual use (SLT + CC) is a general issue.

The authors cite evidence that N might be involved in the development of MI and stroke.

ARO: From the data, the role of N cannot be deduced.

L: Heterogeneity; SLT products in USA and Sweden are different, limited or no dose-response shown.

G: No other NGPs.

• ULSAM: 1,056 elderly men, never smoking, median FU 8.9 y

• CWC: 118,425 construction workers, never smoking, median FU 18 y

Duration of snus use: 50 y (estimate)

Heart failure, harm ratio (CI) snus users vs NU:

• ULSAM: 2.08 (1.03–4.22)

• CWC: 1.28 (1.00–1.64)

AO: Use of snus increases the risk of heart failure.

ARO: Snus only use was not verified.

The authors assume that N per se could be detrimental for the CV tissue.

ARO: N's role cannot be deduced from the studies.

L: Low case numbers (particularly in ULSAM study); no reliable information on tobacco use habits (misclassification possible); no dose-response observed; men only.

Survey of 2014 and 2016

• 60,100 NU, 51.9 y, 2,309 MIs

• 7,093 Former EC users, 39.9 y, 225 MIs

• 1,483 EC some days, 41.4 y, 61 MIs

• 776 EC daily (41 never smoked), 44.2 y, 47 MIs

Duration of EC use: < 10 y (?)

Adjusted ORs (CI) for MI (vs NU):

• Former EC: 1.06 (0.86–1.30)

• EC some days: 1.16 (0.83–1.62)

• EC daily: 1.79 (1.20–2.16)

AO: Daily use of EC increases the MI risk.

ARO: Dual use was adjusted in the ORs, no verification.

ARO: N's role cannot be deduced from this study.

L: All data rely on self-reports.

G: No N-free EC group.

Causality between EC use and MI was questioned in a letter to the editor (47).

National Health Interview Survey, 2014, 2016, 2017, 2018; 16,855 participants:

• 2,848 NU, 30.3 y

• 401 Vapers (EC), 26.7 y

• 7,291 Tobacco users (mostly CC), 44.0 y

• 2,240 Dual, 42.2 y

Duration of EC use: < 10 y (estimate)

Adjusted ORs (CI) vs NU:

MI:

• EC: 4.09 (1.29–12.98)

• CC: 4.52 (2.49–8.21)

• Dual: 5.44 (2.90–10.22)

Stroke:

• EC: 1.22 (0.36–4.18)

• CC: 2.15 (1.38–3.35)

• Dual: 2.32 (1.44–3.74)

CHD:

• EC: 0.67 (0.18–2.44)

• CC: 1.90 (1.20–3.11)

• Dual: 2.27 (1.37–3.77)

AO: EC users have an increased risk for MI. The highest risk for MI, stroke and CHD were observed for dual users.

ARO: Grouping relies on self-reports (danger of misclassification).

L: All data rely on self-reports (misclassification of product use is possible).

G: No N-free EC group.

118,395 healthy, never-smoking men (construction workers), enrolled 1978–1993, follow-up 2003

• 3,248 cases of stroke

Duration of snuff use: 10–30 y (estimate)

RR for ever snuff users:

• All strokes: 1.02 (0.92–1.13)

• Fatal strokes: 1.27 (0.92–1.76)

• Fatal ischemic strokes: 1.63 (1.02–2.62)

AO: Snuff use increases the risk of fatal ischemic stroke.

ARO: Tobacco use information was obtained from questionnaires at entry.

The authors cite evidence that N is a risk factor for stroke.

ARO: Participation of N in ischemic stroke generation cannot be excluded.

L: RR are very low and only sign. for fatal ischemic strokes; no clear dose-response; tobacco habits rely on self-reports; changes in habits are not assessed.

Telephone survey, 161,529 young adults (18–44 y):

• 13,3077 NU

• 13,318 CC only

• 3,437 EC only

• 4,204 EC/exCC

• 7,493 Dual

Duration of EC use: < 10 y (estimate)

OR (CI) for stroke (vs NU, adjusted):

• CC only: 1.59 (1.14–2.22)

• EC only: 0.69 (0.34–1.42)

• EC/exCC): 2.54 (1.16–5.56)

• Dual: 2.91 (1.62–5.25)

AO: Sole EC use is not a risk factor for stroke.

ARO: All data rely on self-reports (no verification of product use).

The authors cite evidence that N may be involved in stroke and CVD.

ARO: Absence of risk in EC only would suggest no role of N.

L: Misclassification due to self-report is possible; switching to EC due to early symptoms possible.

G: No N-free EC group.

Behavior and risk factor survey (BRFSS), 2016, 486,303 participants:

• G1: EC every day, m/f: 2,778/2,229

• G2: EC some day, m/f: 5,018/5,151

• G3: Former EC, m/f: 29,014/29,815

• G4: NU, m/f:164,605/226,937, older than G1–G3

Duration of EC use: < 10 y (estimate)

Adjusted OR (CI) for stroke vs NU:

• G1: 1.62 (1.18–2.31)

• G2: 1.28 (1.02–1.61)

• G3: 1.09 (0.98–1.23)

AO: EC use is of potential concern for CVD.

Self-reports and cross-sectional design can lead to confounding and bias.

ARO: N's role cannot be deduced from this study.

L: Only short use of EC (2007–2016); most participants were former CC users; dual use possible; temporality (due to cross-sectional design) not determined.

1,134,896 Subjects, groups used for meta-analysis:

• EC1: EC users (all, including only, dual, former CC)

• EC2: EC only users

• EC3: Current dual users

• EC4: EC only users, former CC users

• CC: CC only users

• NU: no EC, no CC

Duration of EC use: ?

OR for prevalence of stroke:

• EC1 vs NU: 1.25 (1.01–1.55) (no publ. bias, high heterogen.)

• EC2 vs NU: 1.13 (0.99–1.29)

• EC3 vs CC: 1.39 (1.19–1.64)

• EC4 vs NU: 1.59 (1.22–2.07)

Subgroup analysis had lower heterogeneity

AO: The role of EC use in stroke development is inconclusive due to the strong effect of former CC use.

ARO: General problems with misreports of product use.

L: Partly low quality studies; stroke types not differentiated; misclassification of product use possible; temporality unclear in cross-sectional studies.

G: No prospective studies available; no N-free EC groups available.

NHANES 2015–2018, 79,825 users:

• 7,756 EC users, 48y

• - EC1: EC use in last 30d

• - EC2: No EC use in last 30d

• 23,444 Dual users, 50y

• 48,625 CC users, 59y

EC users are sign. younger

Adjusted ORs (CI) for stroke:

• EC vs CC: 1.15 (1.15–1.16)

• Dual vs CC: 1.14 (1.14–1.15)

• EC1 vs EC2: 1.60 (1.60–1.61)*

* discrepant to the text!?

AO: Stroke in EC users was earlier in onset than in smokers.

ARO: Usual problems with cross-sectional approach (temporality, recall bias).

L: Temporality in cross-sectional studies; misreports in product use; stroke not further classified.

G: No N-free EC group.

577 young men (18–19 y):

• 377 NU

• 43 CC only

• 127 Snuff only

• 30 Dual (Snuff + CC)

Sign. diff. in CVD-related BMs in urine:

• Tx-M: CC > NU, Dual > NU

PGI-M not sign. diff. between groups

AO: CC but not snuff use facilitates TBX-A2 formation, reflecting platelet activation and potential CVD development.

ARO: No verification of snuff only use.

The authors cite evidence that N might be involved in CVD formation, the snuff only results appear to disfavor this.

ARO: N's role cannot be deduced from the data.

L: Small user group sizes; young men only.

G: No other NGPs (EC, HTP), N-free EC.

P: Larger study with older subjects, including additional NGPs would be of interest.

Male construction workers, up to 65 y (1970/71), follow-up after 12 years for mortalities:

• 32,546 NU

• 6,297 SLT users

• 14,983 Smokers (CC1), < 15 cig/d

• 13,518 Smokers (CC2), ≥ 15 cig/d

Duration of SLT use: 10–40 y (estimate)

RR (CI) compared to NU:

• CVD:

– SLT: 1.4 (1.2–1.6)

– CC1: 1.8 (1.6–2.0)

– CC2: 1.9 (1.7–2.2)

AO: Both CC and SLT users have an increased risk for CVD, risk for SLT is lower.

ARO: SLT (only) use was not verified, dual use is not unlikely.

The authors cite evidence that N could be involved in atherosclerotic process and that the result show that N is partially involved in CVD generation.

ARO: N's role not deducible from the data.

L: Only male workers (healthy-worker effect?); dual use (SLT+CC) is possible.

G: Other NGPs; ECs without N missing.

143 Men, 35–60 y:

• 40 NU, 43.1 y, CotP: 3.8 ng/mL

• 28 SLT users, 44.4y, median 25y of SLT use, CotP: 338 ng/mL

• 29 Smokers (CC), 48.0y, median 30y of CC use, CotP: 248 mg/mL

Markers for atherosclerosis sign. diff. (↑/↓) in CC, SLT were not sign. diff. from NU:

• Carotid intima thickness: ↑

• Plaques in intima: ↑

• Plasma cholesterol: ↑

• HDL: ↓

• TG: ↑

• Apo A-1: ↑

• Apo B: ↑

• Fibrinogen: ↑

• Alcohol consumption: ↑

AO: The increased occurrence of atherosclerosis in smokers is caused by other components of tobacco smoke than nicotine.

ARO: SLT only use not verified.

L: Small group sizes; SLT group might contain dual users.

G: No other NGPs included.

P: A study avoiding these weaknesses would be worthwhile.

391 Healthy men, 58 y

• 139 NU (never SLT, never CC)

• 48 SLT users (29% also smoked)

• 96 CC users

Risk factors for atherosclerosis sign. diff. from NU:

• SLT: TG

• CC: TG, CRP, IMT (carotis and femoral), plaques (femoral), HI

AO: Smoking, but not SLT is an import risk for atherosclerosis.

ARO: No exclusive SLT group evaluated.

L: Only men, only one age.

G: No exclusive SLT group, no other NGPs.

• NU (no SLT, no CC): Total: 9,906; 1,510 CVD cases

• SLT (no CC): Total: 354; 102 CVD cases; SLT = Snuff + chewing tobacco

Harm ratio (CI) for CVD vs NU:

• SLT: 1.31 (1.06–1.61)

• CC (only): lower CVD risk than SLT!?

AO: SLT use increases the risk CVD and is no alternative to CC use.

ARO: SLT use only was not verified.

No statement on role of N from the authors.

ARO: A role of N is possible, but cannot be deduced from the study data.

L: Misclassification of SLT (and CC) is possible (also conceded by the authors).

40 Subjects (20 S and 20 NS) were investigated under 2 conditions, separated by 1 week:

• C1: Vaping, 9 puffs, EC with 16 mg N/mL

• C2: Smoking, 1 CC (0.6 mg N/cig);

Blood samples for BM analysis were taken right before and 5 min after product use

Sign. diff. between S and NS at baseline:

• sCD40L: higher in S

• sP-Selectin: higher in S

Changes pre/post:

Elevation in NS and S under both conditions (C1, C2) for the 3 BMs

• sCD40L

• sP-Selectin

• Platelet aggregation

AO: Both CC and EC use acutely increase platelet activation.

ARO: Involvement of N is possible but cannot be deduced from the study (no EC without N included).

L: Low number of subjects.

G: No ECs without N included.

P: Results suggest a chronic effect of smoking on sCD40L, sP-Selectin. Therefore a larger study with long-term (> 12 months) use of CC and EC would be worthwhile.

17 occasional smokers were assigned to 2 conditions, separated by 1 week:

• C1: EC with N (19 mg/mL), 30 puffs in 30 min

• C2: EC without N, 30 puffs in 30 min

BM measurements at 0, 2, 4, 6 h post vaping

C1: sign. increase in:

• endothelial cell-derived EVs (BM1)

• platelet-derived EVs (BM2)

• platelet-derived EVs expressing P-selectin (BM3)

• platelet-derived EVs expressing CD40 (BM4)

C2: sign. increase only in

• BM4 (smaller than in C1)

AO: As few as 30 puffs of nicotine-containing EC vapor caused an increase in levels of circulating EVs of endothelial and platelet origin, which may signify underlying vascular changes.

ARO: Nicotine likely to be involved in the acute increase of all 4 BMs.

L: Low number of subjects.

G: BM levels after chronic use of EC and other NGP use.

Young adults:

• 20 Smokers (CC), 27.0 y

• 20 EC users (EC use only for the past 3 months, 80 % were previous smokers), 25.7 y,

• 20 NS, 24.6 y

Plaque burden (wall area and thickness) sign. higher in CC and EC than in NS (EC about in the mid between CC and NS); vascular inflammation not sign. diff. between the 3 groups

AO: CC and EC users had significantly more carotid plaque burden compared to NU. Results further indicate that vaping does not cause an increase in vascular inflammation.

ARO: Product use was self-reported (no biochemical validation).

ARO: Role of N cannot be deduced.

L: Small sample sizes; EC use probably too short and compliance not approved.

G: No N-free EC group.

17 Men, 18–75 y

• 7 NU, 25.6 y, ≥2 containers SLT/week, CotP: < 10 ng/mL

• 5 SLT users, 28.2 y, CotP: 226 ng/mL

• 5 CC users, 21.2 y, ≥10 cig/d, CotP: 170 ng/mL

Sign. differences (>/<):

• FMD (endothel-dependent): NU > SLT ≈ CC

• FMD (nitroglycerine-dependent): NU ≈ SLT ≈ CC

AO: Endothelial function is sign. impaired by CC and SLT.

ARO: SLT only use not verified.

The authors cite evidence (including their results) that N may impair endothel-dependent FMD.

L: Small sample size; only men; low average age (short product use period); dual use possible.

G: No other NGPs, no N-free EC group.

20 Snus users (m/f=18/2), mean age: 34 y:

• All 20 performed a session with 1 g snus

• 10 performed a similar session with placebo

Measurements at 0 (BL), 20 and 35 min

Sign. changes:

• FMD (brachial artery): Snus after 35 min

• HR: Snus after 20 and 35 min

• SBP: Snus after 20 min

• DBP: Snus after 20 min

AO: Oral moist snuff significantly impaired FMD of the brachial artery, predicting an increased CVD risk.

The authors cite evidence that N can impair endothelial function via ROS formation.

L: Small group size; mostly men, relatively young, only acute effects detected.

G: No other NGPs, no N-free EC group.

P: A study on chronic FMD impairment would be of interest.

1,592 Healthy men (from HUNT3 Study):

• 886 NU, 47.4 y

• 238 Snuff only users, 42.8 y

• 447 Smokers (CC), 47.4 y

• 21 Dual users, 44.0 y

FMD tended to be lower in snuff users compared to NU.

The diff. in FMD was larger in SLT users with low fitness.

Sign. changes:

• FMD (brachial artery): Snus after 35 min

• HR: Snus after 20 and 35 min

• SBP: Snus after 20 min

• DBP: Snus after 20 min

The authors conclude that snus acutely impairs FMD and is a risk factor for CVD.

AO: The possible role of N is not discussed.

ARO: Role of N cannot be deduced from the provided information.

L: Partly small group sizes; only men; duration of SLT use not provided.

15 Smokers, 22.9 y, were randomly allocated to 3 conditions:

• EC(+) with N: 10 4s-puffs, every 30s, 24 mg N/mL

• EC(−) without N: same vaping pattern, 0 mg N/mL

• CC: 1 CC

48 h washout period between conditions; measurements before and every 15 min after vaping/smoking up to 2 h

Sign. changes in CC and EC(+) conditions between 15 and 60 min:

HR, SBP, DBP, AI, PWV; effects larger in CC

No change in EC(−)

AO: Speculate that long-term EC(+) use may increase the risk for CVD.

ARO: The results suggest the CV effects were caused by N.

L: Very small number of subjects; changes after only one use of product was investigated.

G: Data allow no deduction of a dose-response relationship for N.

P: A study with long-term EC users avoiding the weaknesses would be of interest.

70 Smokers (CC) in cessation clinic:

Acute study:

• G1: 35 vaped ECs with N

• G2: 35 vaped ECs without N

Chronic study (1 month):

• G3: 24 were dual users (CC/EC)

• G4: 42 only used EC

• G5: 20 only used CC (control)

Acute study:

• PWV (marker for arterial stiffness): increase CC > EC with N > EC no N > sham

• AI: CC > EC with N ≈ EC no N > sham

• MDA: CC > EC with N ≈ EC no N > sham

Chronic study:

• PWV, AIX75, MDA: decrease in G3 and G4 (larger in G4), unchanged in G5

AO: The findings suggest that ECs may be used in a medically supervised smoking-cessation program.

AO: EC use can reduce effect of smoking on arterial stiffness and oxidative stress (acute and chronic).

ARO: Effect of N (if any) on arterial stiffness and oxidative stress is only marginal.

L: Small group sizes, short duration.

P: Perform larger and longer studies.

25 Occasional smokers, 24 y, were allocated to 3 conditions:

• Vaping (EC) without N

• Vaping (EC) with N (3 mg N/mL)

• Sham vaping (EC switched off)

Vaping session: 25 puffs (4 s, 30 s intervals). Measurements before (BL) and up to 120 min after vaping

Changes when vaping with N (before vs after):

• Impaired vasodilution

• Increased arterial stiffness (AI, PWV)

• Increase in HR, SBP and DBP

• Increase in ox. stress (MPO)

These parameters did not change upon vaping without N or sham vaping

AO: The observed effects were solely attributable to N and that PG/VG aerosol (without N) does not alter micro- and macrovascular function and oxidative stress.

ARO: Measured (acute) effects are caused by N.

L: Only acute effects were measured, small group size.

P: Similar but larger study with long-term users would be of interest.

• G1: 40 CC (> 2 years)

• G2: 37 CC switched to EC with N for 4 weeks

• G3: 37 CC switched to EC without N for 4 weeks

• FMD: sign. improved in G2 (1.44%) and G3 (1.52%) vs G1; no sign. diff. between G2 and G3.

• PWV: Sign. improved in G2/3 combined vs G1

• AI: No sign. diff. between groups

• oxLDL: no sign. diff. between G1, G2, G3

• CRP: No sign. diff. between G1, G2, G3

• BP: No sign. diff. between groups

AO: Improvements in women higher than in men.

ARO: More compliant groups G2 and G3 (COex < 6 ppm) show higher effect of switching to EC.

ARO: No evidence that nicotine is measurably involved in the observed effects.

L: Too short use of ECs; low numbers of subjects in all groups.

P: Long-term study (> 12 months) with larger group sizes (> 100/group).

Sign. changes by condition:

• LTA EPI: CC: sign. increase, EC: no change

• PFA: CC: sign. increase, EC: no change

• PWV: CC: sign. increase, EC: sign. decrease

• MDA: CC: sign. increase, EC: sign. decrease

AO: Switching to ECs for 4 months has a neutral effect on platelet function while it reduces arterial stiffness and oxidative stress compared to CC smoking.

ARO: Role of N cannot be deduced because no N-free EC was applied as additional condition.

L: Small sample size, 4 month probably too short to assess long-term effects.

G: N-free EC would be of interest.

P: Perform study which overcomes the deficiencies (L, G).

16 NU (no nicotine products in the last 6 months) were assigned to 3 vaping conditions:

• V1: 18 puffs, 5.4 % nicotine

• V2: 18 puffs, no nicotine

• V3: 18 sham puffs

Measurements 0, 1 and 2 h after vaping

Biomarkers: FMD, SBP, DBP were not significantly different under all conditions at all time points.

AO: Vaping ECs regardless of nicotine content are not significantly different from each other and do not produce lasting effects over the course of a 2-hour trial.

ARO: No involvement of nicotine can be deduced, since no effects were observed.

L: Study duration too short (single use of product), too few subjects/conditions.

P: Long-term study (> 12 months) with larger group sizes (> 100/group).

• G1:49 Vapers (EC for > 1 year)

• G2: 40 Smokers (CC for > 1 year)

• G3: 47 NU (non-smokers or ex-smokers for > 1 year)

FMD

• Baseline FMD not sign. diff. between G1, G2 and G3

• Acute CC use of G2: FMD sign. lower (impaired)

• Acute EC use of G1 (with/without nicotine, N-inhaler, sham): no sign. diff.

• Acute EC use of G3 (NU) (with/without nicotine, N-inhaler, sham): no sign. diff.

HR, SBP, DBP:

• Acute use of CC (in G2): sign. increase of all three

• Acute use of EC with nicotine, N-inhaler (in G1 and G3: sign. increases

• Acute use of EC without nicotine, sham (in G1 and G3: partly decreases, mostly not sign.

AO: Although it is reassuring that acute EC vaping did not acutely impair FMD, it would be premature and dangerous to conclude that ECs do not lead to atherosclerosis or increase cardiovascular risk.

L: Small group sizes.

P: Perform study with larger and well defined (in terms of product use) groups.

Changes in vapers (pre/post):

• FMD: sign. decrease

• PTT: sign. increase

• PWV: sign. increase

• Ox stress via NOX-2: sign. increase

AO: EC vapour exposure increases vascular, cerebral, and pulmonary oxidative stress via a NOX-2-dependent mechanism. Our study identifies the toxic aldehyde acrolein as a key mediator of the observed adverse vascular consequences.

Experiments with mice show that:

• EC without N cause larger detrimental effects on endothelial function, ox stress, inflammation and lipid peroxidation;

• acrolein is mostly responsible for the effects of ECs.

L: (Human study): low subject number, too short.

G: (Human study): No condition without N.

• 94 NS, 29 y

• 285 CC, 32 y

• 36 EC, 29 y

• 52 Dual, 33 y

Vascular measures sign. diff. between groups:

• Carotid-femoral PWV

• Carotid-radial

• AI

• Central SBP

• Central DBP

No sign. diff.: FMD

• NO production reduced in CC and EC

• eNOS activity reduced in EC compared to CC and NS

AO: EC use is not associated with a more favorable vascular profile.

ARO: No information on EC use duration.

L: Most groups too small.

G: No information on EC use duration; EC without N should be included.

• 51 Smokers (CC), 21.3 y, CC duration 3 y, 3 cig/d

• 22 Vapers (EC), 21.4 y, EC duration 4 y (2–6 y), 1 (0.8–1.6)mg N/mL

• 197 NU, 21.1 y

Sign. diff. in BMs:

• Albuminuria: EC > CC > NU

• Arterial stiffness:

– AI: NU < EC ≈ CE

AO: No association of BMs with N consumed was found.

ARO: Albuminuria measured with dipstick only.

Compliance of EC group not approved.

ARO: N's role cannot be deduced from the data, although no correlation was reported.

L: Only very young subjects; EC use somewhat questionable; albumin method doubtful.

G: No N-free EC group.

P: Larger study without the weaknesses would be of interest.

31 NS vaped 1 EC without N (16 puffs of 3 s)

Blood samples were drawn for BM analysis pre and post vaping

Sign. changes (post vs pre):

Increases:

• CRP

• s-ICAM-1

• HMGB1

• ASC

• ROS

Decreases:

• NO production

• FMD

• PWV

AO: The findings indicate that a single episode of vaping has adverse impacts on vascular inflammation and function.

ARO: No statement on the role of N possible by the authors.

L: Sample size is low, only acute effects investigated.

G: No condition with N for comparison.

P: Larger study including ECs with N. Also investigate these BMs in long-term users of CC, EC and possibly other products.

• 24 Snus users (males, healthy), 44.8 y; ≥15 y snus, < 1 y CC use

• 26 NU (males, healthy), 43.4 y

Arterial stiffness (sign. diff.):

• PWV: higher in snus users

• AI: higher in snus users

Snus users reported sign. higher alcohol consumption at BL.

AO: Long-term snus use may alter endothelial function and increase CVD risk.

L: Snus only use not verified; small group sizes; only males (women could have different CVD risks).

P: Larger study with both sexes and product use verification would be of interest.

372 Subjects, conditions compared:

• Vaping without N (control, EC−)

• Vaping with N (EC+)

• Smoking (CC)

Endothelial function, acute changes:

EC+ vs EC−:

• FMD: no sign. change

• PWV: sign. increase

• AI: sign. increase

EC+ vs CC:

• FMD: no sign. change

• PWV: sign. decrease

• AI: sign. decrease

AO: EC use negatively changes the endothelial function.

ARO: Observed effects with EC+ are caused by N.

L: Relatively small number of studies and subjects; only acute effects evaluated (long-term use would be of interest).

Subjects with chronic product use:

• 42 Vapers (EC), 29 y, mean duration of EC use: 1.7 y, CotU: 923 ng/mL

• 28 Smokers (CC), 34 y, mean duration of CC use: 10.2 y, CotU: 1,735 ng/mL

• 50 NU, 28 y, CotU: 2 ng/mL

Endothelial function (≈: not sign.)

• FMD: EC ≈ CC < NU

• NO release (unstimulated and stimulated) : EC ≈ CC < NU

• Endothelial cell permeability in user sera: EC < CC ≈ NU

AO: EC use can increase CVD risk and dual use can further increase the CVD risk.

ARO: Misreports of product use history is possible.

ARO: The authors’ in vitro experiments show no difference with and without N in the EC aerosol, except for cell viability.

N's role cannot be deduced from the data.

L: Small group sizes; chronic EC use only 1.7 y (on average).

G: No N-free EC group.

97,586 Male construction workers:

• 23,885 NU

• 5,014 SLT users

• 8,823 Smokers (CC), ≥15 cig/d

Duration of SLT use: 10–30 y (estimate)

Sign. OR (vs NU):

• Hypertension (cause for disability pension): SLT: OR=3.0, CC: OR=2.2

• SBP > 160 mm Hg: SLT: OR=1.8/1.3 (in age groups: 46–55/56–65); CC: OR=0.8/0.7

AO: Also SLT use is associated with elevated risk for CVD.

ARO: No verification of SLT only use.

The authors cite evidence and conclude from their results that N is causally related to high BP and CVD risk in SLT users.

ARO: N's role cannot be deduced from the study data.

L: Only males included; healthy-worker-effect? No objective verification of SLT group.

G: No other NGPs, no N-free group.

Hypertension (HT): SBP > 160 or DBP > 100 mm Hg; RR for ever snuff users:

• BL: 1.23 (1.15–1.33)

• Follow-up: 1.39 (1.07–1.72)

No clear dose-response

AO: Snuff use increases the risk of hypertension.

L: No clear dose-response; tobacco habits rely on self-reports only; changes in habits are not assessed.

PATH study 2015–2016, 19,147 participants:

• 8,783 NS (never CC or EC)

• 183 CEV-NS (current EC, never CC)

• 334 CEV-FS (current EC, former CC)

• 3,938 FS (former CC)

• 5,056 CES (current CC only)

• 581 CDU (current dual)

Duration of EC use: ~ 5 y (estimate)

Prevalence of self-reported hypertension (≈: not sign.): NS ≈ CEV-NS < CEV-FS ≈ FS ≈ CES ≈ CDU

AO: Current EC use is similar to CC in terms of hypertension.

ARO: Misclassification in product use possible (not verified)

The authors cite evidence that use of N-free ECs were associated with higher BP reduction.

ARO: N's role in BP appears plausible.

L: Authors consider a number of limitations including dietary factors (not assessed) and (unreliable) self-reports.

Community Health Survey, 2019, groups:

• Dual users (EC and CC)

• EC only users

• CC only users

Duration of EC use: 5–10 y (estimate)

OR for hypertension (SBP > 140 or DBP > 90):

Males:

• Dual: 1.24*

• EC only: 1.22*

• CC only: 1.16*

Females:

• Dual: 1.44

• EC only: 1.41

• CC only: 1.35*

*: p < 0.05

AO: Also EC and dual user have an increased risk for hypertension.

ARO: Bias for misreport (particularly in female) is possible.

No verification of product use.

The authors cite evidence that N could be responsible for hypertension.

ARO: Involvement of N can be assumed.

L: Small group sizes for EC users (particularly female); misreport of product use cannot be excluded.

G: No N-free EC group.

10 Healthy volunteers (smokers, 24–61y):

• Smoking CC, 12 puffs, intervals of 45 s (1–1.3 cig), 9 min

• Oral snuff, 2.5 g, 30 min

• Chewing tobacco, avg 7.9 g, 30 min

• Nicotine gum (NG), 2 pieces, 30 min

Random order, 24 h between conditions

AO: N-related adverse effects also to be expected with the other N-products.

(See left cell)

ARO: All effects caused by N.

L: Low number of subjects.

G: No ‘sham’ experiments.

15 SLT users, 18–33 y performed exercises under 2 conditions:

• SLT, 2.5 g SLT: rest, 60%, 85% V_O2max

• Placebo: same exercises

Cardio-respiratory responses: (SLT vs placebo):

V_O2: No diff.

Qc: No diff.

HR: sign. increase

SV: sign. decrease

Lactate: sign. increase

AO: SLT compared to placebo under workload increases HR and anaerobic energy production.

L: Only few and young males were investigated; only acute effects were studied.

P: A study with long-term, older users including other NGPs would be of interest.

135 Healthy men, 35–60 y :

• 59 NU, 45.1 y

• 47 SLT users, 44.3 y

• 29 Smokers (CC); 47.2 y

24 h-BP monitoring:

• Daytime SBP, DBP and HR sign. higher in CC and SLT compared to NU

Sign. positive correlation between CotP and BP in SLT, negative (not sign.) in CC.

AO: Increases in HR and BP in CC and SLT were most likely due to the effects of Nicotine, in CC additional influences play a role.

ARO: A few dual users were in the CC group. No verification of SLT only use.

The author cite evidence that N can cause increase in HR and BP and maybe also chronic hypertension.

L: Small group sizes; only men.

G: No other NGPs; no N-free EC.

P: Studies avoiding these weaknesses would be of interest.

33 Users of CC or EC were assigned to 3 conditions with > 4 weeks separation:

• C1: EC with 1.2% N, vaped with 60 puffs at 30 s intervals

• C2: EC without N, similar vaping conditions

• C3: Sham (vaping without e-liquid)

HRV (indicator for sympathomimetic effect) was sign. increased in C1, but not C2 and C3.

Ox stress (measured by plasma paraoxonase activity) was unchanged under all 3 conditions.

Fibrinogen?

AO: Habitual EC use was associated with a shift in cardiac autonomic balance toward sympathetic predominance and increased oxidative stress, both associated with increased cardiovascular risk.

Sympathomimetic effect after acute EC use is causally related to N.

L: Small subject numbers; low N uptake in C1.

Selected from 31 healthy subjects:

• 9 NU (29 y)

• 9 EC users (28 y, 2.1 y EC use)

• 9 CC users (27.1 y, 7.3 pack x years)

• SBP, DBP, HR, glucose, fibrinogen, oxLDL, HOI, SBP not sign. diff. between groups

• FDG uptake in spleen and aorta sign. trend of increase NU < EC < CC (indicator for spleenocardiac axis)

ARO: Compliance status of EC users is unclear (although authors exclude dual use).

ARO: Sympathomimetic effect of N, role of other constituents in CC or EC unclear.

L: Very small group sizes; compliance unclear.

• 9 Vapers (EC for > 3 months, no CC in last months), 28.5 y ; 4 conditions (separated by 1 week)

– 3 Types of CL (ciglike EC), 18 mg N/mL, 10 3-s-puffs at 26 s intervals

– TEC (tank EC), 18 mg N/mL, same puffing pattern

• Nicotine in plasma: CC >> TEC > CL

• HR: CC >> TEC > CL

AO: TEC are potential cessation products but also have an addiction potential (like CC and unlike CL).

ARO: The results suggest that N dose-dependently and acutely cause an increase in HR.

L: Small group sizes, only short-term EC users.

G: No N-free condition, only HR no other physiological changes.

30 Dual users (< 5 cig/d, > 1 mL e-liquid/d), performed 4 conditions (EC with 18 mg N/mL, 2 sessions, each 10 puffs every 30 s, PG/VG varied:

• 1. 100% PG

• 2. 55% PG

• 3. 20% PG

• 4. 2% PG

• Nicotine in plasma: Higher in condition 1 and 2

• HR: Increased under all 4 conditions (no sign. diff.)

100% PG less pleasant and satisfying

AO: The PG/VG ratio must be also considered when evaluating the N delivery from ECs.

ARO: N's role on HR cannot be deduced, but results are compatible with an involvement of N.

L: Small group sizes, only short-term EC users.

G: No N-free condition, only HR no other physiological changes.

Changes to BL at week 6:

• −37% reduced CPD

• 7% stopped

• SBP: no sign. diff.

• DBP: no sign. diff.

AO: The provision of ECs is a potentially useful harm reduction intervention in smokers with a psychotic disorder.

ARO: N's role cannot deduced

L: Small group size, only short-term effects were assessed.

G: No N-free group.

P: Patients with mental disorders might be a suitable group to investigate the long-term effects of EC (relatively high smoking rate).

Acute changes after EC use (* = sign.):

• HR: 2.27 bpm* (11 studies)

• SBP: 2.02 mm* (7 studies)

• DBP: 2.01 mm* (7 studies)

Changes after switching to EC:

• HR: −0.03 bpm (3 studies)

• SBP: −7.00 mm (3 studies)

• DBP: −3.65 mm* (3 studies)

AO: EC should not be labelled as CV safe.

ARO: Effects could be caused partly of completely by N.

L: Low number of studies and subjects.

G: No separate evaluation for N-effects possible.

186 Smokers (CC), African Americans/Latinx: 92/94, 43.3 y, 12.1 cig/d; randomized to

• 125 EC use, 5% N

• 61 Controls (CC use as usual)

Sign. changes EC vs control (CC) on week 2 and 6:

• NNAL

• COex

No sign. changes EC vs CC:

• Cotinine in urine

• Respiratory symptoms (weeks 2 and 6)

• FEV_25–75% (w 2 and 6)

• SBP (w 2 and 6)

• DBP (w 2 and 6)

Significances similar in EC only users

AO: ECs may be an inclusive harm reduction strategy for this population.

ARO: 58–68% in EC group were dual users, 4% were CC only users in EC group. Compliance was not enforced.

ARO: All EC contained 5% N. The role of N cannot be deduced from this study.

L: Only short- to medium-term study (6 weeks).

G: No N-free EC group.

P: Long-term study (> 1 year) including an N-free EC group would be of interest.

Biomarkers in blood were measured before and after product use:

• s-NOX2-dp: increase

• NO: decrease

• H₂O₂ production: increase

• 8-Isoprostane: increase

• Vitamin E: decrease

• s-CD40 ligand: increase

• s-P-Selectine: increase

• FMD: decrease

• SBP: increase

• DBP: increase

All changes were sign., except NO and Vitamin E after HTP; CC use showed the largest changes

AO: Acute effects of HTPs, ECs, and CCs are different on several oxidative stress, antioxidant reserve, platelet function, cardiovascular, and satisfaction dimensions, with CCs showing the most detrimental changes in clinically relevant features.

ARO: Involvement of N in observed effects is possible but cannot be evaluated (EC without N is lacking).

L: Too short study duration.

G: Condition EC without nicotine is missing.

P: Long-term study (> 12 months) with larger group sizes (> 100/group) and an additional condition (EC without nicotine).

24 Smokers (CC for > 1 y), 30.9 y, were allocated to 4 conditions:

• 1. EC (36 mg N/mL), 2 sessions with 10 puff, separated by 20 min

• 2. EC (0 N), same regimen

• 3. CC (10 puffs, own brand)

• 4. Nicotine inhaler (10 mg N, 10 puffs)

• HR: Increase in conditions 1, 3, 4; ranking: 3 > 1 > 4; no increase in condition 2 (not mentioned)?

• BP: measured but not included in results

ARO: Incomplete reporting, abstinence (prior to experimental sessions) doubtful (according to the authors).

ARO: It can be deduced that N increases HR upon acute use.

L: Only smokers were investigated, small group, incomplete reporting (only HR, no results on BP).

P: A larger study with (long-term) EC users and more physiological measurements would be of interest.

36 Dual users of CC and EC were investigated in the clinic under three conditions:

• C1: ad lib use of CC

• C2: ad lib use of EC

• C3: abstinence from nicotine

• HR (bpm, 24 h): 72.5 (C1), 68.7 (C2), 66.8 (C3)

• SBP (mm Hg, 24 h): 119 (C1), 120.2 (C2), 116.6 (C3)

• DBP (mm Hg, 24 h): 76.8 (C1), 76.7 (C2), 73.2 (C3); C2 sign. higher than C3.

Urinary biomarkers:

• Epinephrine, norepinephrine, dopamine, 8-isoprostane, 11-dh-TXB2; ratios between groups not sign., except for C1/C3 for epinephrine and 8-isoprostane.

Blood biomarkers:

• IL6 and IL8: sign. for C1/C3 and C2/C3

AO: CC and EC had similar patterns of hemodynamic effects compared with NU, with a higher average HR with CC vs EC, and similar effects on biomarkers of inflammation. EC may pose some CV risk, particularly to smokers with underlying CVD, but may also provide a harm reduction opportunity for smokers willing to switch entirely to EC.

ARO: Involvement of nicotine in observed effects is possible but cannot be evaluated (EC without nicotine is lacking).

L: Too short study duration.

G: Condition EC without nicotine is missing.

P: Long-term study (> 12 months) with larger group sizes (> 100/group) and an additional condition (EC without nicotine).

19 Studies evaluated,:

• Smokers (CC)

• Vapers (1. generation ECs, with N, (EC+))

• Vapers (1. generation EC−, without N (EC−))

Acute CV effects (sign. diff.) CC vs EC+:

• ΔHR (10 studies): +1.86 bpm

• ΔSBP (11 stud.): +1.68 mm Hg

• ΔDBP (11 stud.): +2.09 mm Hg

EC+ vs EC−:

• ΔHR (4 studies): +6.44 bpm

• ΔSBP (5 stud.): +3.73 mm Hg

• ΔDBP (5 stud.): +3.25 mm Hg

HRV for CC vs NU:

Sign. increased

AO: EC are sympatho-excitatory, the effect is lower for the 1. generation ECs compared to CCs.

ARO: The effects are caused (completely?) by N.

L: Studies were done with 1. generation of ECs only.

P: Similar studies with newer generations of ECs and also other NGPs.

32 Vapers (EC use since > 3 months, partly CC), 25.6 y, were allocated to 4 EC conditions, separated by 48 h:

• 1: 0.5 Ω/3 mg N/mL

• 2: 0.5 Ω/8 mg N/mL

• 3: 1.5 Ω/3 mg N/mL

• 4: 1.5 Ω/8 mg N/mL

10 puffs at 30 s intervals, from min. 70 to 130: ad lib vaping

Changes by condition:

• Plasma N: 2 > 1 > 4 > 3

• HR: 1 > 2 ≈ 4 > 3

• Puff number (ad lib): 2 ≈ 4 < 1 ≈ 3

ARO: Changes in topography and physiology (HR) follow the nicotine delivery.

ARO: Increase in HR directly dependent on N uptake.

L: Only acute effects were studied; relatively young subjects; 14 vapers were naive to sub-ohm ECs.

P: Study with older, long-term users, extension to other CV paramters: BP, FMD, PWV, AI.

• 37 CC users, 26.7 y

• 43 EC users (self-reported), 28.0 y

• 65 NU, 21–45 y

Groups allocated to

• CC (1 CC in 7 min)/straw (CC group only)

• ECN (with 1.2% N)/EC0 (without N) (EC and NU); 60 4s-puffs at 30 s intervals

• Nicotine inhaler (NI)/straw (EC and NU)

• HR: no diff. at BL; increase by acute CC use, partly by ECN, not by NI, straw and EC0

• ECG-indices (indicating ventricular repolarization, risk for sudden death): no diff. at BL; increase by acute CC use, partly by ECN (in EC users), not by NI, straw and EC0

AO: If one does not currently smoke, one should not use ECs due to adverse CV effects.

NI only slightly increase plasma N (therefore no effect to be expected).

ARO: Unclear how long EC was used in EC group.

ARO: Use of EC with N but not without N increased HR.

L: Small groups, only young subjects, EC use duration not provided.

P: Larger study with long-term EC users would be of interest.

ECG-indices: only partial influence of N.

15 NU, healthy, 21 y, were assigned to 2 conditions:

• EC(+) with N, 20 puffs at 30 s intervals, 59 mg N/mL

• EC(−) without, same puffing profile, 0 mg N/mL

Conditions were separated by ~1 month; measurements were done before (BL), during and after vaping (recovery), each for ~10 min

Sign. changes occurred only in EC(+) condition:

• HR: increase

• SBP, DBP, MAP: increase

• MSNA: decrease

All changes lasted into the recovery phase

AO: Suggest that the decrease in MSNA is mediated by an intact baroreflex (resulting from the increase in MAP) in the healthy young subjects. In older population N may cause exaggerated sympatho-excitation being detrimental to CV health.

ARO: The results suggest the CV effects were caused by N.

L: Very small number of subjects; changes after only one use of product was investigated; very young population.

G: Data allow no deduction of a dose-response relationship for N.

P: A study with long-term EC users avoiding the weaknesses would be of interest.

37 vapers decreased CPD from 28 (BL) to 6.4 cig/d (12 weeks and 6 months);

HR, SBP and DBP sign. decreased after 12 weeks

ARO: EC group was actually a dual user group.

ARO: Role of N cannot be deduced from this study.

L: Small group size, only medium term effects can be observed.

G: No N-free group.

P: Schizophrenics might be suitable group to investigate the long-term effects of EC (very high smoking rate).

• 18 NU, male, 24.4 y

• 21 Snuff users, male, 24.1 y, duration of snuff use: 7.0 y

• 19 Smokers (CC), male, 25.3 y, duration of CC: 9.1 y

Sign. diff. in CVD-related BOBEs (≈ : not sign.):

• Hb: CC > Snuff ≈ NU

• WBC: CC > NU, Snuff > NU

• Fibrinogen: CC > NU

• Serum insulin: CC > NU, Snuff > NU

• Serum cholesterol: CC > Snuff ≈ NU

• TG: CC > NU, Snuff > NU

Not sign. diff. between groups: LDL, HDL, LDL/HDL, Lp(a)

AO: Snuff use has similar but lower effects on CVD-related BOBEs, except for lipids.

ARO: Use of snuff only not verified.

The authors cite evidence that NG use does not affect lipids and that CC-related hyperlipidemia is not due to N.

ARO: N's role cannot be deduced from this study.

L: Small group sizes; only very young men included.

G: Other NGPs (EC, HTP), N-free EC.

P: Larger study with older subjects, including additional NGPs would be of interest.

1,061 Baseball players (mostly 20–29 y):

• 477 SLT users

• 584 NU

CC excluded

No sign. difference SLT vs NU in:

• HR

• SBP

• DBP

• HDL

• Cholesterol

WBC sign. lower in SLT vs NU

AO: SLT use has at most a modest effect on CVD risk factors.

ARO: No verification of SLT use.

The authors cite evidence that N might contribute to CVD development.

ARO: Results suggest that N uptake does not increase CVD-related risk factors.

L: Only young and fit subjects included.

G: No other NGPs.

Swedish men, 25–64 y :

• 124 Smokers (CC)

• 130 Ex-CC

• 92 Snuff dippers

• 38 Snuff+CC

• 220 NU

BMs for fibrinolysis (t-PA, PAI-1, fibrinogen, pre-/post-load insulin):

• CC: Fibrinogen sign. increased

No sign. diff. for other groups and BMs

AO: Snuff use does not appear to affect potential CVD risk factors.

ARO: Possibility of mis-report and -classification of product use.

The authors cite evidence that N is not involved in fibrinogenesis.

ARO: N's role not deducible from the data.

L: Relatively small group sizes; only male snuff users.

HDL-C/HDL2-C/HDL3-C), sign. diff. or changes:

• Day 0: NU > CC

• Day 35: no change in HDL

• Day 77: CC: increase in HDL to NU levels

Weight gain:

• Day 35: no changes

• Day 77: increase in CC (f)

AO: N inhibits HDL normalization in m/f quitters and weight gain in f quitters.

AO: see left column. Evidence is cited that weight gain occurs in long-term NRT users.

ARO:

HDL increase: 1

ARO:

Weight gain: 1/0

L: Small sample sizes; only short-term NRT use.

G: No other NGPs tested, in particular EC with/without N.

P: Long-term study including NGPs would be of interest.

• 16 Habitual EC users, 28.6 y, EC use: 241 min/d, EC use duration: 1.6 y

• 18 NU, 26.6 y

BMs for CVD risk factors sign. increased in EC vs NU:

• CAB (shift to sympathetic predominance)

• oxLDL and HDL: not sign. diff. between groups

AO: EC use increases the risk for CVD.

ARO: Some CC use cannot be excluded (also conceded by the authors).

The authors cite evidence that N could cause the observed effects (CAB shift, ox stress).

ARO: An effect of N in CAB shift is likely.

L: Small sample sizes; only young subjects; short period of EC use.

G: no other NGPs studied; no group with N-free ECs.

P: A study avoiding these weaknesses would be of interest.

160 Smokers (CC menthol), randomized to:

• 78 HTP (menthol, 1.21 mg N/stick), 39.2 y

• 42 CC (menthol), 33.7 y

• 40 Smoking abstinent (SA), 38.8 y

5 d confined, 85 d ambulatory conditions

Sign. improvement after 90 d vs CC:

• 8-epi-PGF_2α, s-ICAM-1, WBC, HDL,

No sign. diff. HTP vs CC after 90 d:

• 11-dh-TXB2, fibrinogen, homocysteine, hs-CRP, BG, HbA1c, LDL, TG, cholesterol, apolipoprotein A1, SBP, DBP, FEV1

All BOBEs were not sign. diff. between HTP vs SA on day 90 (except BG and TG, higher in HTP than SA)

Compliance (checked with COex < 10 ppm for HTP and SA) was high: 89.7% (HTP), 97.6% (CC), 92.5% (SA).

Dual use (HTP + CC): 2.6%

AO: The reductions in HTP users were promising and reached almost the levels in the SA group.

ARO: N's role cannot be deduced from this study.

L: Low actual compliance in HTP and SA group after 90 d (could have spoiled the results); 3 months might be too short for biological effects.

P: Larger and longer study with better compliance would be of interest.

206 Users of CCs and ECs were switched to vaping (EC, 1.6 % N, ad lib use).

Follow-ups (FUs) at 1, 3, 6, 12, 18 and 24 months; 102 subjects completed the study.

No clinically relevant adverse effects (AE) were observed during the 24 months study.

No consistent changes over time were observed for EC-compliant subjects:

WBC, LDL, HDL, FVC, FEV1, PEF

Authors’ definition: “EVP-compliant subjects” were defined as subjects who were abstinent from CCs for “at least 80% of the completed study days.”

ARO: N's role cannot be deduced.

L: Compliance criteria not sufficient to see effects.

G: Control groups are missing: CC, NU.

160 Smokers (CC), randomized to:

• 80 HTP (menthol, 1.21 mg N/stick), 39.2 y

• 41 CC (menthol), 33.7 y

• 39 Smoking abstinent (SA), 38.8 y

5 d confined, 86 d ambulatory conditions

Sign. improvement after 90 d compared to CC:

• 8-epi-PGF_2α, s-ICAM-1

No sign. diff. HTP vs CC after 90 d:

• 11-dh-TXB2, fibrinogen, homocysteine, hs-CRP, BG, HbA1c, LDL, HDL, TG, cholesterol, apolipoprotein A1, WBC, SBP, DBP, FEV1

All BOBEs were not sign. diff. between HTP vs SA on day 90 (except WBC, higher in HTP than SA).

Compliance (checked with COex < 10 ppm): 51% in HTP group, 18% in SA group.

AO: The reductions were promising with respect to health risk reduction.

ARO: N's role cannot be deduced from this study.

L: Low actual compliance in HTP and SA group after 90 d (could have spoiled the results); 3 months might be too short for biological effects.

P: Larger and longer study with better compliance would be of interest.

• 94 NS, 29 y

• 285 CC, 32 y

• 36 EC, 29 y

• 52 Dual, 33 y

Vascular measures sign. diff. between groups:

• Carotid-femoral PWV

• Carotid-radial

• AI

• Central SBP

• Central DBP

No sign. diff.: FMD

• NO production reduced in CC and EC

• eNOS activity reduced in EC compared to CC and NS

AO: EC use is not associated with a more favorable vascular profile.

ARO: No information on EC use duration.

ARO: N can possibly play a role in the observed effect (but not to be deduced from the data).

L: Most groups too small.

G: No information on EC use duration; EC without N should be included.

All men:

• 337 Dual users (CC + EC), 36.7 y, CotU: 1,303 ng/mL, 15.1 cig/d

• 4079 CC only, 46.3 y, CotU: 1,236 ng/mL, 14.8 cig/d

• 3,027 Never smokers (NS), 39.8 y, CotU: 0.7 ng/mL

Sign. diff. of dual users to other groups:

• WBC: higher than CC and NS

• SBP: lower than CC

• FBG: higher than NS

• TG: higher than NS

• HDL: lower than NS

• Metabolic syndrome: higher than NS

AO: Given that most EC users are dual users and dual users are more vulnerable to CV risk factors than CC-only smokers and NU, more active treatment for smoking cessation should be considered with priority.

ARO: Proportion of EC use in dual users appears low.

ARO: Role of N cannot be decuced.

L: EC only group is missing.

• 104 Never users, 29 y, CotU: 3 ng/dL

• 290 CC users, 33 y, CotU: 927 ng/dL

• 42 Sole EC users, 28 y, CotU: 686 ng/dL

• 47 Dual (EC+CC) users, 33 y, CotU: 851 ng/dL

• 23 Sole pod users, 26 y, CotU: 970 ng/dL (pods are new generations of ECs)

• 19 Dual pod users, 24 y, CotU: 508 ng/dL

Sign. diff.:

• NU vs CC: FBG,TGs, HDL, VLDL

• NU vs sole EC: FBG, TGs, HDL

• NU vs dual EC: TGs, HDL, VLDL

• CC vs sole EC: none

• NS vs sole pod: none

• NS vs dual pod: none

AO: Overall, users of early generation electronic cigarettes display adverse metabolic profiles. In contrast, pod-based electronic cigarette users have similar lipid profiles to never users.

ARO: Duration of use of ECs and pods not provided (probably shortest in pod users).

ARO: Relative high cotinine levels in pod users and lack of sign. diff. to NS suggest only a minor (if any) role of N in lipid profile.

L: Duration of EC/pod use was least 3 months, but might be too short.

G: Role of N difficult to elucidate (no EC/pod group without N included).

Healthy current smokers (CC) were allocated to 3 groups:

• 59 CC (continue smoking), 17.9 cig/d (at 180 d)

• 127 HTP use, 21.9 sticks/d (at 180 d)

• 109 Cessation (NU) 40

Never-smokers also included

Sign. diff. HTP vs CC (improvement) at 180 d:

• FeNO, WBC, 11-dh-TXB2, 8-epi-PGF_2α, s-ICAM-1

No sign. diff. HTP vs CC at 180 d:

• HDL, FEV1

AO: Larger studies are needed for evaluating the risk reduction.

ARO: Compliance was approved by CEVal: 76% of HTP and 73 % of NU group (180 d).

ARO: N's role cannot be deduced from the study results.

L: Too short durations o HTP use.

G: No comparisons of NU vs HTP groups included.

P: A larger and longer study would be of interest.

324 Healthy participants, 21–45 y

• 65 NU, CotU: 3 mg/dL

• 19 EC users, CotU: 826 mg/dL

• 212 Smokers (CC), CotU: 854 mg/dL

• 28 Dual users, CotU: 910 mg/dL

Number of sign. changes in CAC subpopulation (15) vs NU:

• EC: 2

• CC: 4

• Dual: 6

No sign. diff. when EC and Dual groups were compared to CC

AO: EC use is associated with higher endothelial inflammation.

ARO: Product habits rely on self-reports (no verification).

ARO: N's role cannot be deduced from the study.

L: Small group sizes; self-reports only for product habits; temporal issues (cross-sectional study).

G: No N-free EC group.

Male construction workers, up to 65 y (1970/71), follow-up after 12 y for mortalities:

• 32,546 NU

• 6,297 SLT users

• 14,983 Smokers (CC1), < 15 cig/d

• 13,518 Smokers (CC2), ≥ 15 cig/d

Duration of SLT use: 10–30 y (estimate)

RR (CI) compared to NU:

• CVD:

– SLT: 1.4 (1.2–1.6)

– CC1: 1.8 (1.6–2.0)

– CC2: 1.9 (1.7–2.2)

AO: Both CC and SLT users have an increased risk for CVD, risk for SLT is lower.

ARO: SLT (only) use was not verified, dual use is not unlikely.

The authors cite evidence that N could be involved in atherosclerotic processes and that the results show that N is partially involved in CVD generation.

ARO: N obviously has no role in risk for all cancers.

L: Only male workers (healthy-worker effect?); dual use (SLT + CC) is possible.

G: Other NGPs; ECs without N.

NHANES I (BL), 20 y-follow-up:

• 5,192 NU (no tobacco), 47.8 y

• 505 SLT (exclusive), 54.0 y

• 5,523 CC (exclusive), 44.9 y

Hazard ratio for SLT vs NU (=1.1, not sign.) for

• All causes

• All cancers

• All CVD

Dual use did not increase all cause mortality beyond the sum of CC and SLT risks.

• Borderline increase in SLT (f) for all cancers (not sign.)

AO: See a limitation that only ever use of SLT could be included in the study.

ARO: Use pattern could have changed during the 20 y of follow-up.

ARO: Overall results suggest that N is not a contributor to the reported mortalities.

L: SLT use not completely assessed.

G: No other NGPs included.

P: Future evaluations of NHANES data could include NGPs.

• 123 Pancreatic cancer cases

• 682 Matched controls

All non-CC users (life-long)

Duration of SLT use: 10–30 y (estimate)

OR for pancreatic cancer vs NU:

• Cigar (ever/only): 1.7/1.9

• Pipe (ever/only): 0.6/0.3

• SLT (ever/only): 1.4/1.1

All ORs not sign.

SLT: sign. (p = 0.04) trend with amount/d

AO: Suggest that heavy use of SLT may increase risk for pancreas cancer.

ARO: Compliance with product use was not checked.

ARO: Most probably, TSNAs play a role. Involvement of N is possible.

L: Product use relies on self-reports, past tobacco history may be questionable

Lung Health Study (substudy), 3,320 subjects (smoking intervention with NRT, N gum), FU at 7.5 years:

• 1,986 NRT users, mean age 48 y

• 1,329 no NRT users, mean age 48 y

• Unclear what the CC group during 5-year surveillance is

Cancer risks:

Lung cancer:

• CC: sign. increase

• NRT: not sign.

• GI (incl. oral): CC and NRT not sign.

• All cancer: CC and NRT not sign.

AO: Despite short FU time period, smoking predicted cancer in this analysis and nicotine replacement therapy did not.

ARO: Danger of misreports of product use (CC, NRT) and abstinence.

The authors cite genetic evidence that N might contribute to cancer risk, which the data failed to prove (ARO).

L: Small number of cancer cases; short FU period; unclear CC group; low N doses from gum.

G: Nicotine inhaler not studied (of interest for lung cancer).

• 9 Smokers (CC), 42.2 y, CotU: 5.87 ng/mL

• 15 Vapers (EC, all containing N), 35.7 y, CotU: 5.25 ng/mL, quit CC since 8.7 months

• 21 Ex-smokers, 43.0 y, CotU: 1.23 ng/mL, quit CC since 67.0 months

Gene expression in bronchial epithelial cells (obtained by brushing):

468 (of 3,165) genes varied between EC and Ex-CC, 79 of these were in accordance with CC; downregulated genes in EC were mostly also downregulated in cells exposed to EC aerosol.

AO: Pattern of EC is closer to former CC than to current CC users.

ARO: No check of dual use (CC and EC) was performed.

ARO: CotU in CC and EC appears much too low (μg/mL?).

ARO: Role of N cannot be deduced.

L: Small group sizes.

G: No N-free EC group; also no initial EC user group (only former CC users who switched to EC).

At BL:

• 469 CC users, ≥1 cig/d, ~50 y

• 228 EC users, ≥50 puffs/week, ~50 y

• 215 Dual users, ~50 y

FU after 6 y

Incidence of ‘possibly smoking-related diseases’ (included cancer):

Not sign. diff. between 3 groups

AO: That there was no evidence for harm reduction in EC only or dual users after 6 y.

ARO: Possibility of mis-reports of product use (COex in only 50% of subjects).

ARO: N's role not deducable.

L: Small group sizes; misclassification not excluded.

G: No N-free EC group.

• 15 Vapers (EC), 29.3 y, EC but no CC use for at least 6 months

• 15 Smokers (CC), 29.5 y, CC use for at least 1 year

• 15 Controls (NU), 28.9 y

Epigenetic measurements in peripheral blood leukocytes

• 5-mC in LINE-1: NU* > EC ≈ CC

• 5-hmC (global): NU* > EC ≈ CC

*: sign. diff. from other 2 groups

Expression levels of various DNA methyl transferases: not sign. diff. between groups.

AO: In conclusion, we have demonstrated, for the first time, key epigenetic modifications, including hypomethylation of LINE-1 repeat elements and global loss of DNA hydroxymethylation, in a well-defined population of exclusive vapers (EC) and smokers (CC) relative to controls.

ARO: Dual users excluded (but only COex and COHb applied for approval).

ARO: N's role cannot be deduced from the present study.

L: Small group sizes; no objective check of dual use; changes in peripheral leukocytes may differ from those in target cells.

G: No inclusion of N-free EC group.

P: Larger study with similar endpoints but inclusion of an N-free EC group.

• 116 EC users, 20.9 y, e-liquid consumption: 7.8 mg/d, 5.6 mg N/mL

• 117 CC users, 22.8 y

• 117 NU, 20.6 y

Epigenome-wide DNA methylation profiles in saliva:

• EC were distinct from CC

• Biological aging profile: EC more similar to NU than CC

• EC profile did not discriminate between lung cancer from normal tissue (CC profile did)

• EC profiles did not replicate in independent samples

AO: DNA methylation profiles are clearly distinct from CC.

ARO: Relationship to chronic effects are not yet clear.

ARO: Role of N cannot be deduced from the present study.

L: Very young population; only short durations of products use possible.

G: No N-free EC group.

P: Endpoints may be of interest when more data are collected.

TP53 gene was upregulated, MPG gene was downregulated.

AO: A single EC use can modify gene expressions (towards a cancer risk).

ARO: Role of N cannot be deduced.

L: Extremely small group.

G: No N-free EC group.

P: Perform larger study including CC group, N-free EC group; investigate effects after long-term (chronic) use.

• 269 Controls, NU, 30.4 y

• 22 Control subgroup, NU, 33.5 y (= controls who had BOEs measured in urine)

• 112 Smokers (CC), 41.2 y

• 35 Vapers (EC), 23.5 y

• 19 SLT users, 36.6 y

DNA methylation was measured in blood samples.

CC had sign. lower methylation rate in cg05575921 (CpG residue in AHRR) than all other groups.

AO: Methylation extent of cg05575921 together with CEMA is suitable to distinguish CC from other NGP users.

ARO: The same can be achieved by CEMA alone!

ARO: N not involved in the demethylation of cg05575921.

L: Small group sizes; short product use durations.

Healthy adults, 18–50 y

• 13 NU, 30.4 y, CotS: 0.08 ng/mL

• 12 EC users, 28.7y, CotS: 200.7 ng/mL, predominantly used EC in last 6 months

• 14 Smokers (CC), 30.7 y, CotS: 159.0 ng/mL

Gene expression changes (decreases vs NU) in nasal biopsies, immune-related:

• 53 genes common in CC and EC

• 305 additional genes in EC

AO: EC use leads to immune suppression in the nasal mucosa.

ARO: Dual users were excluded, but not verified.

ARO: N's role cannot be deduced from the data.

L: Small group sizes; probably only short use duration of EC; relatively young subjects.

G: No N-free EC group.

134 Smokers (CC) were assigned to ECs with:

• A: 49 subjects, 2.4% N over 12 weeks

• B: 45 subjects, 2.4% N (6 weeks) and 1.8% N (following 6 weeks)

• C: 40 subjects 0% N (12 weeks)

For final evaluation, subjects were classified into:

• Failures (F): continued CC, not meeting criteria for reducers (R) or quitters (Q)

• R: reduced CC by ≥50%

• Q: no CC, COex<7ppm

FeNO and COex measurements at BL, 12, 34, 52 weeks:

• FeNO: stayed at BL level for F and R groups, sign. increased for Q

• COex after 52 weeks: F > R > Q

AO: Switching from CC to EC can revert harm in the lung.

ARO: Unfortunately, data were not evaluated to EC N-content.

N's role cannot be deduced from the data.

L: Small group sizes; no evaluation by N-content.

P: A larger study avoiding the weaknesses would be of interest.

2,086 Adolescents (16–18 y)

• 502 Ever EC users (past users)

• 201 Current EC users (EC use in last 30d)

OR (CI) for bronchitic symptoms:

• Past EC: 1.85 (1.37–2.49)

• Current EC: 2.02 (1.42–2.88), become insign. after adjustment for life-time CC use

OR not increased for wheeze after adjustment for CC.

AO: EC use increases risk for bronchitis in adolescents.

ARO: EC only use not verified (authors also concede confounding by CC).

ARO: N's role cannot be deduced from this study.

L: Possibly insufficient assessment of CC use in EC groups.

• 9 Vapers (EC), 26.6 y, 0.0–1.8% N

• 12 NU, 27.8 y

No sign. changes between BL, 12, 24 and 42 months for:

• BW

• HR

• SBP

• DBP

• FEV1

• FVC

• FEV1/FVC

• FEF_25–75%

• FeNO

AO: EC long-term use is not associated with health concern in young users.

ARO: Non-compliant subjects were excluded (but no objective prove for compliance).

ARO: N's role cannot be deduced from the data.

L: Very small groups; use of only 1. generation of EC with probably low N uptake.

G: No N-free EC group.

P: A larger study including N-free EC group would be of high interest.

• 22 Smokers with COPD, switched to EC, 65.2 y

• 22 Smokers with COPD, continued CC use, 66.5 y

BL, and follow-up (FU) at 12, 24 and 36 months

• CC group: CPD almost unchanged at 20 cig/d; no sign. change in lung function and walk distance

• EC group: decrease in CPD (20 to 1.5 cig/d); sign. improvements in lung function and walk distance

Also improvements in dual users

AO: EC use ameliorates COPD outcomes and might also reverse the harm of some smoking effects.

ARO: Although dual use was evaluated separately, compliance is an open question.

The authors speculate that N is not the compound responsible for COPD in smokers.

L: Small group sizes.

See also Polosa et al. 2020 (223).

ARO: From the study data, the role of N in COPD cannot be deduced.

• 27 healthy smokers (H-CC), 23.0 y

• 27 smokers with mild asthma (MA-CC), 23.0 y

Both groups performed a control (EC without liquid) and vaping session (e-liquid with 12 mg/mL N) of 10 puffs with 30 s intervals

• Control session: no changes

• EC session: acute increase in resp. resistance (larger effect in MA-CC); acute decrease in FeNO

AO: One EC session has acute mechanical and inflammation effect on the respiratory tract (larger in smokers with MA).

ARO: N's role cannot be deduced from this study.

L: Only acute effects of EC smokers were investigated.

G: No N-free group included.

10 NS were assigned to one of 2 conditions:

• C1 (N=7): EC with N, 10 puffs, after 30 min again 10 puffs

• C2 (N=3): EC without N, same regime as in C1

BM measurements pre and 30 min after vaping

Observed changes (post vs pre vaping):

• C1: EMPs elevated, transcriptoms of SAE and AE changed

• C2: No changes found

AO: This study provides in vivo human data demonstrating that acute inhalation of EC aerosols dysregulates normal human lung homeostasis in healthy naive individuals.

ARO: Effects were found only after exposure to EC with N.

L: Very low subject numbers, only acute effects were investigated, which were completely reversible.

G: No information available whether chronic use of N-products can lead to persistent changes.

• 30 Vapers (EC), males, 27.1 y, former and current CC or tobacco users were excluded; self-reported EC use for > 6 months

• 30 NU, males, 25.9 y, former CC and tobacco users were excluded; self-reported

Sign. lower in EC vs NU:

• FEV1

• FEV1/FVC

• FEF 25, 50, 75, 25–75, 75–85%

Not sign. diff.:

• FeNO

• FVC

• PEF

AO: EC use impairs lung function.

ARO: Product use status replies on self-reports, not objectively approved.

ARO: N's role cannot be deduced from this study.

L: Small groups, only short- to medium term use.

G: No N-free EC group; no CC group (as a positive control).

30 NS performed one

• 5 min EC session (1.8% N, 15 puffs)

• 5 min CC session (0.6 mg N/cig)

Lung function measured after 1 and 15 min sign. differences (decrease) from BL;

• FEV1: EC only after 1 min; CC after 1 and 15 min

• FEV1/VC: EC only after 1 min; CC after 1 and 15 min

• FEV_25–75: EC and CC both after 1 and 15 min

AO: EC use may be dangerous for CC smokers.

ARO: AO appears at least questionable.

ARO: Only short term use and effects were observed.

ARO: N's role cannot be deduced.

L: Only short-term use was investigated.

G: No N-free EC condition included.

P: A study with long-term EC users (included N-free) would be warranted.

• 16 NU, 29.6 y, CotP: 0.06 ng/mL

• 17 CC, 31.8 y, 10.5 cig/d, CotP: 184 ng/mL

• 16 EC, 28.3 y, 218 puffs/d, CotP: 218 ng/mL

BMs in induced sputum, sign. diff. in CC and EC vs NU:

• Elastase

• MMP-9

• Myeloperoxidase

• MUC5AC

• ADH3A1

• Thioredoxin

• GST

AO: EC use alters the profile of innate defense proteins in airway secretions.

ARO: Only self-reports for product use, not objectively verified.

ARO: N's role cannot be deduced from the study.

L: Small group sizes, no verification of product use, short EC use?

G: No other NGPs, no N-free ECs.

P: Improved study with these endpoints would be of interest.

• 18 NU, 27.3 y

• 13 CC, 34.0 y: 9.5 pack-years, 10.1 cig/d (last 2 weeks)

• 10 EC, 26.8 y: last 2 weeks: 44.1 puffs/d, 11.4 mL e-liquid/d

Bronchial brush biopsies and lavage (BAL) samples: 300 proteins alter in CC and EC, 78 in both groups, 113 uniquely in EC (e.g. CYP1B1, MUC5AC and MUC4); PG/VG aerosol altered human resp. cell cultures

AO: EC use has long-term effects on the lung, possibly mediated by PG/VG.

ARO: Unclear for how long ECs were used.

ARO: Effect of N cannot be excluded. Effects of PG/VG alone also possible.

L: Small group sizes, long-term use (in EC users) is not well defined.

P: Proteome study with long-term users would be of interest.

206 Users of CCs and ECs were switched to vaping (EC, 1.6% N, ad lib use)

Follow-ups (FUs) at 1, 3, 6, 12, 18 and 24 months; 102 subjects completed the study

No clinically relevant adverse effects (AE) were observed during the 24 months study

No consistent changes over time were observed for EC-compliant subjects: WBC, LDL, HDL, FVC, FEV1, PEF

Authors’ definition: “EVP-compliant subjects” were defined as subjects who were abstinent from CCs for “at least 80% of the completed study days.”

ARO: N's role cannot be deduced

L: Compliance not sufficient to see effects.

G: Control groups are missing: CC, NU.

• 14 NU, 25.8 y

• 14 CC, 29.5 y: 9.5 pack-years, 10.1 cig/d (last 2 weeks)

• 14 EC, 26.1 y: last 2 weeks: 44.1 puffs/d, 11.4 mL e-liquid/d

Bronchial brush biopsies and lavage (BAL) samples: Release of proteases (elastase, MMP-2, MMP-9) and gelantinolytic activity higher in CC and EC users than NU, N-dependent

AO: EC users are at increased risk for chronic lung disease.

ARO: Unclear for how long ECs were used.

ARO: N might play a role in protease release of lung cells and can disturb protease-antiprotease balance.

L: Small group sizes, long-term use (in EC users) is not well defined.

P: Study of anti-protease/protease balance in NGP long-term users (EC, HTP) would be of interest.

20 Smokers (CC), assigned to two conditions, separated by 24 h:

• C1: Vaping, 15 puff, EC with N (18 mg/mL)

• C2: Smoking 1 CC ad lib

Acute biomarker changes pre/post, sign. for C1 and C2:

• HR (C2 > C1)

• PWA (pulse wave amplitude) (C2 > C1)

• PMPs (platelet microparticles) (C1 >C2);

Sign. change only in C2:

• PECAM-1, MPs, EMPs:

Sign. change only in C1:

• RHI, AI, s-P-selectin, PEF;

No sign. change (C1 or C2):

• SBP, DBP, s-ICAM-1, s-VCAM-1, s-E-selectin, FEV1, FVC, FEV1/FVC

AO: These findings suggest that both electronic cigarettes and tobacco smoking negatively impact vascular function.

ARO: Involvement of N possible for HR, PWA and PMPs, RHI, AI, s-P-selectin.

L: Small group sizes, only acute changes.

G: No condition EC without N.

Sign. role of N unlikely for PECAM-1, MPs, EMPs.

Role of N unclear for SBP, DBP, s-ICAM-1, s-VCAM-1, s-E-selectin, FEV1, FVC and FEV1/FVC.

15 Occasional smokers (< 10 CC/month):

Cross over of two conditions separated by 1 week:

• EC with N (19 mg/mL), 30 puffs in 30 min

• EC without N (0 mg/mL), 30 puffs in 30 min

Vascular measurements over 4 h:

• DBP, HR and PWV increase over 2 h compared to BL, sign. higher in EC with N compared to EC without N (sign. for HR and PWV)

Respir. measurements over 6 h:

• No sign. diff. + vs −N for VC, FEV1 FeNO;

• FeNO sign. increased in EC (+/−N) vs BL

• VC sign. decreased in EC (+/−N) vs BL;

Airway resistance sign. increased 0.5 h post exposure in EC with N

AO: ECs with N have an acute impact on vascular and respiratory functions. Long-term effects require further studies.

ARO: N clearly involved in acute effects.

L: Small group sizes; only acute effects investigated.

P: Investigate long-term effects in users of EC with and without N.

25 Occasional smokers (CC) were assigned to 3 conditions (random order):

• Sham vaping (EC switched off)

• EC with N

• EC without N

20 Heavy smokers (CC):

• 10 assigned to sham EC

• 10 assigned to EC without N

Vaping: 25 puffs at 30 s intervals, 4 s inhalation

• PO₂: sign. decrease vs BL in EC without N (in both occasional and heavy smokers)

• Serum CC16: sign. increase vs BL in EC without and with N (in occasional smokers)

• Serum SP-D: no changes

• FEV1: sign. decrease in EC without N vs BL

AO: Effect on PO₂ is caused by PG/VG in ECs rather than N.

ARO: N appears to play no or only a minor role in the decrease of PO₂.

L: Results are valid for intense vaping under acute conditions only with a low number of subjects.

P: A larger study under long-term, realistic use conditions would be of interest.

• 12 Never-smokers (NS), 26 y

• 15 Vapers (EC), 27 y: 80% were former smokers, duration of EC use (average, range): 3 (0.5–4) y, 127 puff/d, 7 mL e-liquid/d, N-content: 12 (1.5–36) mg/mL

• 16 Smokers (CC), 26 y

Cell counts in BAL fluid:

Sign. diff.: a: NS vs EC, b: NS vs CC, c: CC vs EC

Total cells: b

Macrophages: b

Lymphocytes: –

Neutrophils: b

Inflammasome components (ASC, caspase-1): EC more like NS

AO: Suggest to also investigate NS who started EC use (in order to avoid the influence of former smoking) and to study the role of N.

ARO: EC were relatively well characterized. EC use was relatively short.

ARO: The observation that EC users were close to never-smokers suggests that N has no major impact on the BMs investigated.

L: Small group sizes; relatively short duration of EC use.

G: No cessation group, no group using N-free ECs.

263 Smokers (CC) at baseline assigned to replace CC progressively by

• EC (N = 191, 3 groups: 0, 6, 36 mg/mL N), 19.1 cig/d at BL: after 1 month: −8 cig/d, after 3 months: −9 cig/d

• EC-dummy (fresh air) (N = 72), 18.1 cig/d at BL: after 1 month: −5 cig/d, after 3 months: −6 cig/d

Lung function parameters

• FEV1

• FVC

• FEV1%

• FEF25–75

• FET

• and

• HR

• DBP

• SBP

were not sign. diff. between EC and fresh air at BL, 1 and 3 months

AO: EC use sign. contributes to health outcome.

ARO: Amount of CC used by the two groups might be too high to see an effect.

ARO: No effects were observed, therefore, role of N cannot be deduced.

L: Sensitivity of the measured study parameters might be too low; study duration might be too short.

402,822 Never CC users:

• 399,719 NU (no CCs or ECs), median age group: 45–49 y

• 3,103 EC only users, median age group: 18–25 y (!)

Duration of EC use: ~ 5 y (estimate)

OR for self-reported asthma:

• Occasional EC: 1.31 (1.05–1.62)

• Daily EC: 1.73 (1.21–2.48)

AO: EC use is a risk factor for asthma (to be approved in future longitudinal studies).

ARO: Dual use cannot be excluded.

ARO: N's role cannot be deduced from the study data.

L: EC use not further characterized; EC users much younger than NU; self-selection (to use ECs because of asthma) possible.

32,320 Adults:

Propensity matching:

• 2,727 EC users

• 2,727 NU

Duration of EC use: < 10 y (estimate)

OR (CI) for self-reported COPD diagnosis (EC vs NU): 1.43 (1.12–1.85)

AO: EC use is associated with COPD in adults, long-term studies are required.

ARO: Confounding is possible.

Possible N involvement not discussed by the authors.

ARO: N's role cannot be deduced from the study.

L: Data rely on self-reports; confounding possible (EC used started after diagnosis?); duration and dose of EC not considered.

G: No long-term use of ECs.

• 30 Smokers (CC), 23.2 y, CC since 50 months, 0.6 mg/cig, 6.2 cig/d

• 30 Vapers (EC), 22.2 y, EC since 29 months, 12 mg N/mL, 15.6 sessions/d

• 30 Dual (CC/EC), 22.3 y, since 67.3/27.7 months, 0.6/12 mg N, 8.0/14.7 /d

• 30 NU, 22.9 y

• FeNO (BL, 1, 30 min)

– BL: NU > CC ≈ Dual ≈ EC

– 1 m: NU > CC ≈ Dual ≈ EC

– 30 m: CC ≈ Dual < EC

AO: EC use is similar in terms of reduced airflow (PEF) and FeNO as smoking.

ARO: Long-term use was not well assessed. Changes in terms of lung function are variable.

ARO: N's role cannot be deduced

L: Long-term use of EC (> 2 y), but compliance not well assessed.

G: No long-term, N-free EC group.

P: Long-term study with these groups (+ N-free EC) would be of interest.

58,336 Adolescents, 12–18 y Self-reported tobacco/N use:

• 49,542 NU

• 4,496 CC only users

• 540 EC only users

• 51 HTP only users

• 2,344 Dual users (CC + EC)

Sign. increased OR (NU = 1):

• Asthma: CC, HTP

• Allergic rhinitis: none

• Atopic dermatitis: CC, (EC), dual

AO: Any product use might be risk for the indicated allergic diseases, however, longitudinal studies are required.

ARO: No verification of product (only) use.

ARO: N involvement cannot be deduced from the study data

L: Some groups are very small (EC, HTP); high probability of dual/multi use in the “only” groups; very young subjects and short product uses.

G: No N-free EC group.

Review of 6 epidemiological studies with former CC users who switched to ECs

Duration of EC use: < 10 y (estimate)

EC vs CC:

• 3 Studies on RD (COPD, chronic bronchitis, emphysema, asthma, and wheezing): all ORs < 1.0 (sign.)

• 3 Studies on CVD (MI, CHD, stroke): all ORs ≈ 1.0 (not sign.)

AO: Switching to ECs reduces risk for RD but does not change risk for CVD.

ARO: General issue with misclassification in epidemiological studies.

ARO: N's role cannot be deduced from the presented data.

L: Low number of studies, problem of misclassification.

G: No N-free EC group.

P: Authors emphasize need for prospective studies and RCTs.

• G1: 15 NU (< 100 CC in lifetime, no EC in past year)

• G2: 15 NU switched to EC without N or flavor for 4 weeks

Biomarkers in BAL:

• Total cell yield, cell concentration, macrophages, lymphocytes, neutrophils, eosinophils, IL1ß, IL2, IL4, IL6, IL8, IL10, IL12p70, IL13, IFNγ, TNF-α: no sign. diff. between baseline and 4 weeks follow-up in G1 and G2;

Biomarkers in bronchial brush:

• mRNA, miRNA: also no sign. diff.

AO: Although limited by study size and duration, this is the first experimental demonstration of an impact of e-cig use on inflammation in the human lung among never-smokers.

ARO: G2 had sign. elevated PG levels in urine after EC use.

ARO: Role of N cannot be evaluated (due to study design).

L: Too short use of ECs; low numbers of subjects in both groups.

P: Long-term study (> 12 months) with larger group sizes (> 100/group).

• 42 Never-smokers, 25 y

• 15 EC users, 27 y: 80% were former smokers, duration of EC use (average, range): 2.6 (0.5–4) y, 163 puff/d, 8.3 mL e-liquid/d, N-content: 10.7 (1.5–36) mg/mL

• 16 Smokers (CC), 26 y

Level in EC group mostly between NS and CC (closer to NS): Cells in BAL fluid:

Sign. diff: a: NS vs EC, b: NS vs CC, c: CC vs EC

• Total cells: b

• Macrophages: b, c

• Lymphocytes: b, c

• Neutrophils: –

• Eosinophils: –

• Differential gene expression and DNA methylation: EC more like NS

AO: Suggest to also investigate former smokers without switching to ECs.

ARO: EC were relatively well characterized. EC use relatively short.

ARO: The observation that EC users were close to never-smokers suggests that N has no major impact on the BMs investigated.

L: Small group sizes; relatively short duration of EC use.

G: No cessation group, no group using N-free ECs.

61 College students:

• 32 self-reported EC use in the last 30 d, 34.4% reported CC use in last month

• 29 NU in the last 30 d, 10.3% reported CC use in last month

EC vs NU:

Recent cough: 25.0 vs 3.4% * Sign. diff. cytokines in saliva: IL2, IL4, INFγ

Not sign. diff.:

IL6, IL8, IL10, IL12p70, IL13, TNF-α

AO: Findings reveal dysregulation of salivary immune profiles toward a TH1 phenotype in emerging adult EC users and short-term immune function may be dysregulated in young adult EC users.

ARO: No pure EC users of NU (almost multi-product users).

ARO: N's role cannot be deduced.

L: Small groups, no ‘pure’ product use; probably very short time of product use (< 30 d).

G: No data to evaluate role of N.

• 6 Vapers (EC), EC use since ≥ 6 months

• 6 Smokers (CC), CC since ≥ 6 months

• 6 WP smokers, WP since ≥ 6 months

• 6 Dual CC/WP smokers

• 6 NU

Age range (all groups) 18–65 y

lncRNAs in plasma exosome: Distinct profile in the user groups with some overlap

AO: lncRNAs allow risk estimates for COPD, asthma, IPF.

ARO: No verification of EC only use.

ARO: N's role cannot be deduced.

L: Small group sizes; no verification of product compliance (especially EC).

G: No N-free EC group.

• 22 Vapers (EC), 35.5 y

• 26 Smokers (CC), 32.8 y

• 12 WP smokers, 32.8 y

• 10 Dual CC/WP smokers, 35.5 y

• 26 NU, 33.9 y

microRNA from plasma exosome:

The 4 user groups show common differential expression of micro-RNAs which are different from NU.

AO: The exosomes/microRNAs are BMs to understand the lung injury caused by smoking and vaping.

AO: Suggest that the differences between the user groups and NU demonstrate “nicotine-specific” effects.

ARO: Given the study design, the role of N remains open.

L: Small group sizes; no verification of product compliance.

G: No N-free EC group.

30 EC users (former CC, EC since 38 months), 38 y, randomly allocated to 3 conditions, separated by 7 d):

• (1) EC with N, 5 d

• (2) EC without N, 5 d

• (3) Cessation (EC stop), 5 d

Sign. diff. between conditions:

• PG in serum: 1 ≈ 2 > 3

• PG in urine: 1 ≈ 2 > 3

• TCO₂: none

• HR: 1 > 2 ≈ 3

• SBP: 1 > 2 ≈ 3

• DBP: 1 > 2 ≈ 3

• Serum CC16: 1 ≈ 2 < 3

• Serum SP-D: none

• FEV1, PEF, FEF25/50/75: none

AO: Short-term EC cessation can lead to decrease in lung inflammation (indicated by change in CC16). Effects appear to be related to a disturbance of the lung gas exchange.

ARO: N is not involved in the observed pulmonary changes.

L: Only acute effects in a limited population were investigated.

P: Investigations of long-term EC use (with and without N) with larger groups would be worthwhile.

N is causally related to the observed CV effects (HR, SBP, DBP):

• 19 Smokers with COPD, switched to EC, 65 y, 22.1 cig/d at BL

• 20 Smokers with COPD (control), continued CC use, 65.9 y, 20.2 cig/d at BL

BL, and follow-up (FU) at 12, 24, 48 and 60 months

• CC group: CPD almost unchanged at 20 cig/d; no sign. change or slight improvement in lung function and walk distance, COPD exacerbations, CAT score

• EC group: decrease in CPD (20 to 1.5 cig/d); sign. improvements in lung function and walk distance, COPD exacerbations, CAT score

Also improvements in dual users (about 3 cig/d)

AO: EC use ameliorates COPD outcomes and might also reverse the harm of some smoking effects.

ARO: Although dual use was evaluated separately, compliance is an open question.

The authors speculate that N is not the compound responsible for COPD in smokers.

ARO: From the study data, the role of N in COPD cannot be deduced.

L: Small group sizes.

P: A larger study over longer time periods including a N-free EC group would be of value.

Of note: N strength in e-liquid was reduced over time.

• Cohort 1: 26 NU, 22 vapers (EC)

• Cohort 2: 25 NU, 26 CC, 12 Waterpipe (WP), 10 Dual (CC/WP)

Grouping according to self-reports

Self-reported resp. symptoms:

• EC and CC reported the most symptoms

IgE: EC sign. higher than NU; dual users higher than other groups of Cohort 2

IgG: EC and NU not diff.; dual users higher than other groups of Cohort 2

AO: Our pilot study showed that users have a preference toward fruit and more sweet flavors and that EC and dual use resulted in an augmented systemic immune response.

ARO: Only self-reported data (except IgE and IgG). No verification of product use (particularly EC only).

ARO: Role of N cannot be deduced from the data.

L: Small group sizes; probably too short product use durations.

G: No N-free EC group.

25 healthy and 25 asthmatic smokers (aged 40 y) vaped 1 EC (with nicotine)

Acute changes (after vs before, sign.):

Healthy subjects:

• FeNO: 3.6 ppb (↑)

• FEV1: unchanged

• Inflam. BM in EBC (IL-1ß, IL-4, IL-6, IL-8, IL-10, TNF-α, LTB4, 8-isopr: unchanged

All BM sign changed in asthmatics after 1 EC

AO: EC vaping resulted in acute alteration of both pulmonary function and airway inflammation in stable moderate asthmatic patients.

The authors cite evidence that EC use without N had no effects.

ARO: N's role cannot be deduced from the data.

L: Small group sizes; only acute effects.

G: No N-free EC group; no long-term observations.

P: A study without these weaknesses would be of interest.

Evaluation of RNA expression data sets from:

• Smokers (CC)

• Vapers (EC with N)

• Vapers (EC without N and flavors)

Gene expressions:

• ACE2: upreg. in CC

• Pro-inflammatory cytokines: upreg. in CC and EC with N

• Inflammasome genes: upreg. in CC and EC with N

AO: CC and EC (with N) increase susceptibility to COVID-19

ARO: Product use not well characterized

Authors do not clearly separate effects of N and flavors.

ARO: N possibly involved in proinflamm. gene expression

L: Product use not well characterized, misclassification possible

64 Healthy, young adults, mean age: 26 y

• 28 NU, PG in urine: 2.1 mg/L

• 13 Vapers (EC for > 1 y, no CCs for > 5 month), PG in urine: 25.5 mg/L

• 27 Smokers (CC), PG in urine: 6.6 mg/L

Lipid laden macrophages (LLM) in BAL:

• NU: 18%

• EC: 54%

• CC: 96%

AO: LLM are related to EVALI, relation to inflammation is open.

ARO: Dual use cannot be excluded

ARO: N's role cannot be deduced.

L: Small group sizes; dual use in EC group is possible; not clear what LLM indicates.

G: No N-free EC group.

186 Smokers (CC), African Americans/Latinx: 92/94, 43.3 y, 12.1 cig/d; randomized to

• 125 EC use, 5% N

• 61 Controls (CC use as usual)

Sign. changes EC vs control (CC) on week 2 and 6:

• NNAL

• COex

No sign. changes EC vs CC:

• Cotinine in urine

• Respiratory symptoms (weeks 2 and 6)

• FEV_25–75% (w 2 and 6)

• SBP (w 2 and 6)

• DBP (w 2 and 6)

• Significances similar in EC only users

AO: ECs may be an inclusive (suitable) harm reduction strategy for this population.

ARO: 58–68% in EC group were dual users, 4% were CC only users in EC group. Compliance was not enforced.

ARO: All EC contained 5% N. The role of N cannot be deduced from this study.

L: Only short- to medium-term study (6 weeks).

G: No N-free EC group.

P: Long-term study (> 1 year) including an N-free EC group would be of interest.

• 9 Vapers (EC since > 1 y), 23 y, range EC use: 1.5–4 y, 3.3 pack-year equivalents

• 7 NU, 21 y

Sign. diff. at BL:

• FEV1: EC < NU

• FEV1/FVC: EC < NU

• VA/Q mismatch: EC > NU

BL vs post acute EC use:

• V/Q mismatch: gets worse

No sign. diff. at BL:

• SPO₂ (oxygen saturation)

• HR (but sign. increase after acute vaping)

AO: EC use leads to early lung changes. Authors could not exclude some dual use. The authors suppose that nicotine is involved in the VA/Q changes.

ARO: No direct involvement of N can be deduced, but can theoretically be possible.

L: Very small groups, dual use cannot be excluded.

G: No N-free EC group.

P: Larger, long-term study with N-free EC group would be of interest.

• 76 Vapers (EC), 20 min EC use with 5% N

• 73 NU, passively exposed to EC for 20 min

Sign. changes after 20 min (active) vaping:

• HR,↑

• ventilation frequency, ↑

• oral temperature, ↑

• SPO₂, ↓

Passive exposure:

• oral temp, ↑

Blood sugar and FVC not changed (both groups)

AO: Vaping with mint-flavored ECs with 5% N for 20 min resulted in significant immediate physiological changes. Exposure to EC vapor significantly increased oral temperature.

ARO: N's role cannot be deduced from this study.

L: Only short-term effects.

• 60 Smokers (CC), 39.1 y, reduced CC and increased EC use (still 25% CC use after 3 months)

• 20 Smokers (CC), 44.2 y; stopped CC use

Changes after 3 months:

• Bronchial reactivity (BHR) (mannitol provocation test) decrease in both groups

• FEV1 small change in both groups

• FeNO small change in both groups

No sign. diff. in the changes between both groups

AO: Whether the decrease in BHR observed after 3 months is maintained when using ECs over longer time periods or has an individual prognostic value, must be clarified in long-term studies.

ARO: No clear improvements by EC use or stopping smoking were observed. Both groups contain CC users after 3 months.

ARO: Role of N cannot be deduced from the study.

L: Use of CCs in both groups.

G: No N-free EC group.

1,039,203 Subjects

• Current EC users

• Ever EC users

• NU

Duration of EC use: < 10 y (estimate)

Pooled OR (CI) for asthma vs NU:

• Current EC: 1.36 (1.21–1.52)

• Ever EC: 1.24 (1.13–1.36)

AO: EC use is correlated with asthma (however, limitations are considered).

ARO: N's role cannot be deduced from this study.

L: All weaknesses of cross-sectional studies: temporality, misclassifications by self-reports.

Groups:

• EC ever

• EC current

• EC former

• Dual

• CC

• NU

Duration of EC use: < 10 y (estimate)

OR (CI) for asthma vs NU:

• EC ever: 1.27 (1.17–1.37)

• EC current: 1.30 (1.17–1.45)

• EC former: 1.22 (1.08–1.39)

• Dual: 1.47 (1.13–1.91)

• CC current: 1.33 (1.19–1.49)

AO: Current and former EC use is associated with asthma.

ARO: Usual weaknesses of cross-sectional studies.

ARO: N's role not discussed by authors.

Cannot be deduced from this study.

L: Cross-sectional study: no causality, temporality open; mis-report of product use possible.