Assessment of inhalation toxicity of cigarette smoke and aerosols from flavor mixtures: 5-week study in A/J mice

Abstract

Most flavors used in e-liquids are generally recognized as safe for oral consumption, but their potential effects when inhaled are not well characterized. In vivo inhalation studies of flavor ingredients in e-liquids are scarce. A structure-based grouping approach was used to select 38 flavor group representatives (FGR) on the basis of known and in silico-predicted toxicological data. These FGRs were combined to create prototype e-liquid formulations and tested against cigarette smoke (CS) in a 5-week inhalation study. Female A/J mice were whole-body exposed for 6 h/day, 5 days/week, for 5 weeks to air, mainstream CS, or aerosols from (1) test formulations containing propylene glycol (PG), vegetable glycerol (VG), nicotine (N; 2% w/w), and flavor (F) mixtures at low (4.6% w/w), medium (9.3% w/w), or high (18.6% w/w) concentration or (2) base formulation (PG/VG/N). Male A/J mice were exposed to air, PG/VG/N, or PG/VG/N/F-high under the same exposure regimen. There were no significant mortality or in-life clinical findings in the treatment groups, with only transient weight loss during the early exposure adaptation period. While exposure to flavor aerosols did not cause notable lung inflammation, it caused only minimal adaptive changes in the larynx and nasal epithelia. In contrast, exposure to CS resulted in lung inflammation and moderate-to-severe changes in the epithelia of the nose, larynx, and trachea. In summary, the study evaluates an approach for assessing the inhalation toxicity potential of flavor mixtures, thereby informing the selection of flavor exposure concentrations (up to 18.6%) for a future chronic inhalation study.

Keywords: E-cigarette; emphysema; flavor toolbox; harm reduction; inflammation; inhalation.

FIGURE 1

Body weight progressionThe average body weight measurements across the study period are shown for the (A) male and (B) female groups. As annotated above each graph, day 1 was the start of the exposure period; day 9 was the end of the acclimatization period; and days 36 to 47 were staggered scheduled dissection days. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavors; L, low; M, medium; H, high; SEM, standard error of the mean

FIGURE 2

Quantification of biomarkers of exposure in urineUrinary biomarkers of exposure in 24‐h urine samples are presented as (A) total levels and concentrations of five nicotine metabolites, (B) total urine volume, (C) proportions of five nicotine metabolites relative to the sum of all nicotine metabolites, (D) total levels of eugenol, and (E) total levels of ethyl vanillic acid. Nicotine metabolites were quantified from eight mice per group. Eugenol and ethyl vanillic acid were quantified from three mice per group, and inferential statistics were not performed. ** and *** represent statistically significant differences between the treatment and sham groups at p ≤ 0.01 and p ≤ 0.001, respectively. + and ++ represent statistically significant differences between the PG/VG/N/F and 3R4F groups at p ≤ 0.05 and p ≤ 0.01, respectively. #, ##, and ### represent statistically significant differences between the PG/VG/N/F and PG/VG/N groups at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavors; L, low; M, medium; H, high; LOD, limit of detection; LOQ, limit of quantification; LOD, limit of detection; SEM, standard error of the mean

FIGURE 3

Assessment of lung inflammationData shown are (A) relative lung weights, (B) neutrophil counts in BALF, (C) total protein concentration in BALF, and (D) fold changes in BALF inflammatory mediator levels relative to the concentrations in the sham groups. Data were derived from at least 10 mice per group. **, and *** represent statistically significant differences between the treatment and sham groups at p ≤ 0.01, and p ≤ 0.001, respectively. +++ represents statistically significant differences between the PG/VG/N/F and 3R4F groups at p ≤ 0.001. # represents statistically significant differences between the PG/VG/N/F and PG/VG/N groups at p ≤ 0.05. $ represents statistically significant differences between the high flavor and low flavor groups at p ≤ 0.05. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavors; L, low; M, medium; H, high; SEM, standard error of the mean

FIGURE 4

Organ weights and differential leukocyte countsResults shown are (A) relative spleen weights, (B) relative thymus weights, (C) absolute lymphocyte counts, (D) relative lymphocyte counts, (E) absolute neutrophil counts, and (F) relative neutrophil counts. Relative organ weights were from at least 10 mice per group. The average absolute and relative cell counts were from 6 to 10 mice per group. Relative counts are presented relative to the total leukocyte counts. *, **, and *** represent statistically significant differences between the treatment and sham groups at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. +, ++, and +++ represent statistically significant differences between the PG/VG/N/F and 3R4F groups at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. # represents statistically significant differences between the PG/VG/N/F and PG/VG/N groups at p ≤ 0.05. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavors; L, low; M, medium; H, high; SEM, standard error of the mean

FIGURE 5

Serum clinical chemistry parameters Results of quantification of serum analytes representative of liver function are shown for (A) alkaline phosphatase activity, (B) aspartate aminotransferase activity, (C) alanine aminotransferase activity, (D) total protein concentration, (E) albumin concentration, and (F) globulin concentration. The average analyte concentrations were from 10 to 11 mice per group. * and ** represent statistically significant differences between the treatment and sham groups at p ≤ 0.05 and p ≤ 0.01, respectively. +, ++, and +++ represent statistically significant differences between the PG/VG/N/F and 3R4F groups at p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. # and ## represent statistically significant differences between the PG/VG/N/F and PG/VG/N groups at p ≤ 0.05 and p ≤ 0.01, respectively. ! represents statistically significant differences between the high flavor and medium flavor groups at p ≤ 0.05. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavors; H, high; M, medium; L, low; SEM, standard error of the mean