Choice of power setting

The “upper limit of realistic usage,” of the CE4 product was recently suggested as 4.0 V (7.3 W), based on the subjective perceptions of vapers4, in a human subjects study included in the “reinvestigation” of our 2015 report6. However, the study was not completely blinded. Subjects were asked if they had knowledge of and ability to detect “dry puffs,” the experimental endpoint. Up to seven distinct power levels were increased during the vaping sessions in order, from lowest to highest power, rather than in a randomized manner. The justification was that experienced vapers in the study could detect the higher power levels due to their perception of concomitantly larger inhaled aerosols, so it was suggested that there was no need to randomize. However, this assumption contradicts the researchers’ own data4, as well as earlier findings by others14 showing that aerosol volumes can contract at higher power levels when using the CE4 e-cigarette. Despite the concerns, herein we note the findings of the “replication study”, defining 4.0 V (7.3 W) as the recommended “safe” power setting.

Collecting and analyzing HCHO and 1a-d from the same sample

Recently, we described a tandem cold-trap/DNPH impinger/smoking machine set-up for separating and trapping both aerosol particulate matter as well as gas-phase aldehydes derived from the same puff12. The aerosol initially is pulled through a pair of cold-traps (−77 °C) that aid in inhibiting possible unwanted further reactions, allowing for effectively trapping relatively unstable materials such as intact 1a-d. In the current study, we added additional washes of the cold-traps with acetonitrile to improve the recovery accuracy of 1a-d. The NMR quantification standard versus the internal standard signal ratio was used to calculate the yield of the acetonitrile extraction. The amount of formaldehyde hemiacetal observed in the spectra was subsequently factored up to account for transfer losses. No gaseous HCHO is found in the cold-traps12, which is instead found as its DNPH adduct in the impingers, connected in series after the cold traps. The impingers were set up according to the CORESTA recommended method, as described previously12. The aerosol components found in the cold-traps were dissolved and analyzed by 1H NMR. The HCHO-DNPH adducts formed in the impinger were analyzed by HPLC.

Aerosol levels of 1a-d are formed in several-fold excess of those of gaseous HCHO

Table 1 shows that the levels of 1a-d are formed in excess compared to those of gaseous HCHO. This is in agreement with our investigations using other e-cigarettes12,15. Interestingly, the amount of 1a-d found in the current investigation is higher, in each of the four runs, compared to our initial 2015 study (380 μg/10 puffs), despite the higher power (5 V) used in the older study6. One reason the levels of 1a-d are higher herein is that we used the harsher 50 puff “replication study” puffing regimen, which the researchers performing the latter study had modified4 (without explanation) from our original milder 10 puff experiments. In addition, in our prior investigation6, the aerosol was pulled from the e-cigarette manually via syringe, collected as it passed over the surface of DMSO-d 6 in an NMR tube, at room temperature. One could visibly see a significant amount of aerosol lost to the ambient atmosphere using this older method, prompting us to state in the 2015 paper that the levels of 1a-d were underestimated6. Others have independently replicated the use of this latter syringe and non-destructive 1H NMR method in a relatively newer e-cigarette model, and found that 1a-d comprised a significant percentage of the total formaldehyde in the aerosol of a different e-cigarette16.

Table 1 Levels of 1a-d, HCHO and e-liquid consumed (commercial Café Mocha brand, the same used in our 2015 study, ref.6) from four vaping sessions with two CE4 atomizers at a power level of 4.0 V. Full size table

The levels of gaseous HCHO and 1a-d in the context of the recent literature

In addition to Farsalinos et al.4, Gillman and co-workers have also investigated the formation of formaldehyde in a CE4 device (Table 2)14. They found that the CE4 emitted formaldehyde levels that were above OSHA (TWA) workplace limits, as well as above those from 20 traditional cigarettes. Importantly, these concerning levels of formaldehyde were observed at every CE4 power level used, including those below the 7.3 W (4.0 V) “defined” safe threshold4, and even at the lowest power level studied (5.3 watts)14.

Table 2 Comparison of formaldehyde levels in the current and two recent studies using a CE4 e-cigarette. Full size table

Intriguingly, there is a >10-fold discrepancy between the levels of formaldehyde reported between the Farsalinos4 and Gillman groups14, within the 6.5–7.3 W range (Table 2). However, it should be noted that Gillman used a different e-liquid. Moreover, neither of these two studies explicitly accounted for levels of 1a-d. Our finding of 13.5 ± 3.2 μg of gaseous HCHO/puff at 7.3 W embodies an intermediate value of formaldehyde. It is above the OSHA (TWA) workplace limits (5.3 mg/day)14, calculated as described previously (13.5 μg HCHO/puff ÷ 6 mg aerosol/puff = 2.25 mg gaseous HCHO/g aerosol)14. Using a conservative value of 4 g e-liquid consumed/day17 affords 10.0 mg gaseous HCHO inhaled/day, nearly double the OSHA limit.

The levels of gaseous HCHO and 1a-d in the context of self-regulation of toxin intake

In the “replication study,” which involved human subjects (Table 2, first column), it was reported that, at the 4.2 V (8 W) power level, 88% of participants detected “dry puffs”4. This means that 12% of the subjects would have been exposed to ca. 10 μg of formaldehyde/puff at 4.2 V (8 W)4, without any sensory awareness that they should cease usage at an exposure level that corresponds to a daily intake of 8.3 mg of formaldehyde per day, which is above the OSHA limit. Notably, this was calculated using the data from the relatively lowest reported levels (those from ref.4, the “replication study”). The levels of gaseous HCHO obtained in the current study are approximately six-fold higher at lower power (7.3 W, 4.0 V), without factoring in any contribution to total formaldehyde from 1a-d. The levels of formaldehyde from Gillman’s study14 were 2- and 3-fold higher than those of ref.4 even when obtained at the relatively lower power levels of 6.5 W and 7.8 W (Table 2).

The inconsistencies in the inter-laboratory data displayed in Table 2 is in keeping with the literature, and not just in studies that have involved CE4 e-cigarettes. Several researchers, including us, have noted elsewhere the concerning interlaboratory differences in reported e-cigarette toxin levels as well as the factors exacerbating this issue18,19,20,21,22. When care is taken to avoid the drying of heating coils and/or burning e-liquids in laboratory studies, such as using single puff samples15, using power levels described by vapers as popular for specific devices19 and/or applying settings corresponding to manufacturers’ recommendations23, etc., wide variations in levels of aerosol toxins can still be observed. Interestingly, in a recent, related “re-investigation” of work by Sleiman et al., Farsalinos and Gillman reported that, even under “dry puff” conditions, they found formaldehyde levels that were not only below OSHA thresholds but also were >12-fold lower than those reported in the original manuscript by Sleiman (under the same conditions)5. Thus, measurements of toxicant levels can differ by orders of magnitude between labs for reasons other than “dry puff”; moreover, “dry puff” conditions should not always be cited as the sole factor promoting elevated levels of carbonyls.

Limitations

Limitations of the study herein include the fact that we did not use human subjects. However, using e-cigarette data acquired based on recommended settings, as opposed to concomitant human testing, is well-precedented, for example, by the same authors23 who published the “replication study”4 of our 2015 findings. Regardless, had we reported the results described herein at the intermediate 4.0 V power level three years ago, it would have been concluded by those performing the “replication study”4, that they were obtained under “normal,” “non-averse” vaping conditions. Importantly, even if one assumes the unlikely scenario wherein the CE4 device produced all “dry-puffs” at 7.3 W in every experiment (Table 1) in the current study, thereby inflating the levels of HCHO and 1a-d under conditions “averse” to users, the results of the “replication study”4 show that a concerning percentage (12%) of human subjects could not detect dry puffs under conditions affording levels of formaldehyde that are above the OSHA threshold. Moreover, the aforementioned results do not account for the facts that the harsh taste of formaldehyde and other aldehydes is dulled due to the nicotine drive24, as well as by the cross-desensitization of transient receptor potential ankyrin subtype 1 (TRPA1) channels in sensory neurons25,26.