Covid-19 has been shown to increase risk associated with vaping. To increase participant safety and use real world vaping habits, the investigators plan to continue studying vaping effects on the airways in an observational format.

While e-cigs are commonly represented as safer alternatives to tobacco cigarettes, little is known regarding the health effects of their short- or long-term use. The responses and the e-cig components exerting these effects on the airways are largely unknown. This study will identify if specific e-cig flavors modify respiratory immune responses. This study will determine the effects of cinnamaldehyde (CA)-containing e-cigarettes on airway epithelial cell ciliary function (i.e., MCC) in humans. Additionally the study will determine the effects of CA-containing e-cigarettes on airway immune cells obtained through induced sputum (SI) after inhalation of CA-containing e-cig aerosols to determine CA-induced effects on a) immune cell function (e.g., phagocytosis, respiratory burst), b) immune cell surface phenotype, and c) mediator production in humans in vivo.

Investigators will evaluate the acute effect of CA-flavored e-cigs on MCC and IS immune cells in up to 32 healthy, young adults who are current e-cig users with a total of less than 10 pack-years cigarette smoking history. MCC will be measured by gamma scintigraphy at baseline and following controlled vaping of e-liquids with and without cinnamon flavoring. Two different e-liquids (one completely devoid and one containing at least 30 mM CA similar to "Hot Cinnamon Candies" which is commercially available) will be used for two separate randomized vaping sessions.

The randomization scheme for the two different e-liquids (e-liquids with and without CA) will be generated by using the Web site Randomization.com (http://www.randomization.com), assigned treatment Regimen A and B by an assigned study team member, and provided to the study team. This individual will also be responsible for loading the e-cigarette with the appropriate solution for that session prior to the vaping sessions.

Participants will undergo baseline testing during the screening visit, which will occur 2-3 weeks prior to the first controlled vaping session. Investigators will also recruit non-vaping control subjects (n=32), who will only undergo the baseline testing and thus serve as a non-exposed/non-vaping control group. will aim to recruit similar numbers of males and females in both cohorts. While investigators cannot guarantee age-matching and sex-matching in these cohorts, based on our previous studies, investigators do not expect to find significant age and sex differences in the two cohort. In addition, potential confounders, such as age, sex, and BMI will be included as covariates in our multivariate analysis.

Observations obtained from the non-vaping control group will provide necessary information on potential baseline differences in the two cohorts (i.e. current vapers versus non-vaping controls). These data from the non-vaping control group are important to provide a reference for any potential CA-induced changes in the vaping group. Hence, there are two stages of the study:

Stage 1. A cross sectional observational cohort comparison of baseline MCC and IS immune cells in a reference cohort of n=32 non-vaping control subjects and E-cig cohort of n=32 currently vaping subjects (confounding based on other variables such as BMI, sex, age is possible for this stage).

Stage 2. A randomized comparison of changes in MCC and IS immune cells after Regimen A (e-cig us without CA) and Regimen B (e-cig use with CA). The cohort of e-cig users will undergo a randomized 2-treatment, 2-period, 2-sequence crossover study of CA exposure.

For stage 1, baseline measurements of Tc99m-SC clearance will be used to measure each subject's normal baseline MCC and IS immune cell characteristics. For both stages, subjects will be asked to complete a vaping diary to record information on the device and e-liquids (name/vendor/e-liquids/puffs/device settings) used during their normal vaping sessions for the entire duration of the study. In addition, for stage 2, participants will be asked to maintain their current habits for the duration of the study, not to significantly increase or decrease their vaping patterns, including the nicotine concentrations of their e-liquids.

For stage 2, for each e-cig vaping session (Training and MCC Test Days), subjects will be asked to follow a laboratory-based protocol involving 6, 5-minute paced vaping segments (1 puff/minute) over a 1 hour time period, vaping the e-liquid with and without CA provided by us. On each Test Day, participants will undergo the vaping protocol immediately prior to inhalation of the Tc99m-SC (10 min between end of vaping and inhalation of Tc99m-SC). An initial deposition scan of Tc99m-SC will then be obtained followed by dynamic imaging of the lung with subjects seated in front of the gamma camera to determine potential changes in MCC induced by acute exposure to CA-flavored e- cigarettes. Induced sputum samples will be collected at baseline, and after each MCC scan.

24 hours after completion of the MCC scans. The two randomized vaping sessions will be separated by 2-3 weeks. While there are no data providing specific information on the duration needed to washout the effects of CA on MCC, previous studies examining changes in MCC following inhalation of other aerosols have shown that this washout period is sufficient to prevent potential carryover between the two treatments.

Inclusion Criteria:

• An equal number of participants who currently use a vaping device and those who do not use a vaping device
• Age 18-40
• Must have a Forced Vital Capacity (FVC) and Forced Expiratory Volume in 1 second (FEV₁) of at least 80% of predicted. Participants who fall out of the normal range will be offered a copy of the test to share with their personal physician.

Exclusion Criteria:

• Any pre-existing lung disease (asthma, cystic fibrosis, etc.)
• Any significant chronic illness, such as, but not limited to, heart disease, uncontrolled hypertension, diabetes, auto-immune disease
• Any use of tobacco products (other than e-cig) in the past 3 months, or a greater than 10 pack year history of smoking cigarettes
• Pregnant or nursing women
• Participants with a history of radiation exposure in the past year which exceeds annual safe limits.

Individual participant data that underlie the results reported in this article, after deidentification (text, tables, figures, and appendices).

Beginning 9 months and ending 36 months following article publication

Investigators whose proposed use of the data has been approved by an independent review committee ("learned intermediary") identified for this purpose.

Proposals may be submitted up to 36 months following article publication. After 36 months the data will be available in our University's data warehouse but without investigator support other than deposited metadata

Requests for access to individual participant data should be sent to bring44@email.unc.edu or carole.robinette@med.unc.edu. Access will be granted after a data access agreement has been signed with UNC.

From the time a participant signed consent through completion of the second intervention, a total of up to 3 months.

Adverse event data included all participants who received at least one dose of Cinnamaldehyde (CA) or Propylene Glycol/Vegetable Glycerin (PG/VG).

The e-liquids will be maintained by an individual not active in the study procedures or analysis.