Is the Harvard E-Cigarette Buttery Flavor Study Credible?

A month ago, I warned vapers to avoid e-liquids containing buttery flavorings diacetyl (DA) and acetyl propionyl (AP, also known as 2,3-pentanedione) – chemicals associated with bronchiolitis obliterans, a serious, sometimes fatal lung disease seen in workers producing buttered popcorn. 

This week, faculty at the Harvard School of Public Health reported that DA, AP and/or acetoin (a similar flavoring) are present in popular e-cigarettes’ aerosols.  The study, whose lead author is Joseph G. Allen, appears in Environmental Health Perspectives (abstract here).

If true, the report would implicate major e-cigarette manufacturers, but there are irregularities in the researchers’ methods and findings. 

Allen used a custom lab device “that drew air through the e-cigarette for eight seconds at a time with a resting period of 15 or 30 second [sic] between each draw. Eight seconds was chosen to make certain that each draw had adequate time for the entire contents to be forced out of the smoking device and through the sampling media…The samples were collected until the e-cigarette cartridges or cartomizers were exhausted, determined by the lack of visible emissions in the chamber.”  (emphasis mine)  In other words, the researchers used an aggressive procedure that vaped the samples dry.  Each “puff” was eight seconds long – an unrealistic length of time that could have produced higher-than-normal temperatures, combustion and smoke.

This is significant because at least two previous studies by Fujioka and Shibamoto (here) and Pierce et al. (here)  found that cigarette smoke contains both DA and AP.  Pierce reported that the smoke of a single cigarette has up to 1,037 micrograms of DA and 165 micrograms of AP.  She noted that DA is approved as a cigarette tobacco additive in Germany and the UK, and AP is also permitted in the UK.  However, according to Pierce, this “strongly suggests that pyrolysis products of tobacco and other cigarette components comprise a substantial fraction of the [DA] and [AP] concentrations in mainstream smoke.”  If the Allen e-cigarette methodology generated smoke, their results could have been compromised.

Another quirk of the Allen study was the resampling of “several of the same flavors from the same package (ie, testing two e-cigarette cartridges from the same pack)” for six of the flavors in the table at left.  It is troubling that the researchers failed to adequately explain why they ran these extra samples on only some products. 

More importantly, they didn’t offer a valid explanation for a wide divergence in values.  The researchers found no acetoin in one Pina Colada sample, but it was present in other samples at 17, 46 and 130 micrograms.  One Classic sample had no detectable acetoin, while another had 38 micrograms.  Allen suggested that these differences might be due to “variable chemical doses.” 

That explanation would be questionable if the sampled products were produced by major manufacturers with tight quality control systems, which seems to be the case here.

While, inexplicably, the Harvard researchers did not identify the e-cigarette aerosol brands under study, the brands can be inferred from the data. They are likely:

A – Mark Ten

B – Vuse

C – blu

D – NJOY

E – White Cloud

F – South Beach Smoke

G – Volcano

H – Tasty Vapor

I – Unknown

 

Since Pina Colada is a blu e-cigarette from Lorillard, and Classic is a Mark Ten product from Altria, ingredients can be expected to be highly consistent among samples taken “from the same pack”, as was done in this study.  

Allen and colleagues also suggested that variability could be related to the fact the researchers “relied on a visual determination of emissions of the e-cigarette in the chamber” to determine that the e-cigarette was “fully spent.”  A maxim in toxicology is to use methods that have been validated for the products and agents under investigation.  Eyeballing a stop point is not a validated method.    

As I advised previously, vapers should only use liquids that are certified to be free of buttery flavors that are suspected respiratory toxicants.  However, laboratory investigations of e-cigarettes should use validated methods to assure credibility.  The results of the Harvard Buttery Flavor Study do not meet this standard.

Red Flag for Vapers: Avoid Some Flavoring Agents

As a health professional, I started telling smokers to switch to smokeless tobacco in 1994, based on decades of definitive epidemiologic evidence for the relative safety of smokeless tobacco.  But I was slow to endorse e-cigarettes as a reduced-risk option for smokers. 

There is no parallel body of evidence for e-cigarettes, but then, there is no scientific evidence that would link vapor inhalation to cancer, heart attacks or strokes.  That is significant, but as a pathologist, I must consider whether long-term vapor consumption can cause respiratory problems.  There is little human experience with intense, long-term inhalation of propylene glycol, vegetable glycerin and other agents, including flavorings. 

Konstantinos Farsalinos and colleagues in 2014 reported a laboratory analysis of 159 e-cigarette liquids from 36 manufacturers/retailers in six European countries and the U.S.  They found that almost 70% of the samples contained varying amounts of diacetyl (DA) and/or a similar flavor compound, acetyl propionyl (AP).  Although these substances naturally occur in fermented products like cheese and beer, they are also added to foods to provide a butter-like flavor.  They are generally recognized as safe by the FDA when added in small quantities to foods.

DA and AP are known, however, to cause bronchiolitis obliterans, a serious, sometimes fatal lung disease in exposed workers (here, here, here), most notably in plants producing buttered popcorn (here).

Farsalinos estimated the amount of DA and AP that vapers would inhale using the tested liquids.  He concluded, “The median daily exposure levels were slightly lower than the strict [National Institute on Occupational Safety and Hazards]-defined safety limits for occupational exposure and 100 and 10 times lower compared with smoking respectively; however, 47.3% of DA and 41.5% of AP-containing samples exposed consumers to levels higher than the safety limits.”

Farsalinos’ study should have prompted e-liquid suppliers to abandon those agents. They have not.

Raquel Rutledge, a reporter for the Milwaukee Journal Sentinel, recently worked with a Marquette University chemist to test liquids sold in local vape shops (here).  She “bought five e-liquids dubbed top-sellers by sales clerks… and had them tested for [DA and AP]… All five contained both chemicals.”  According to her story, some had high levels.

Staff at the vape shops selling the liquids were unaware of the presence of these toxins.

It is unacceptable for any vape shop to sell liquids with flavoring ingredients that are proven respiratory toxicants.  Vapers should only use liquids that are certified to be free of these agents.

Formaldehype vs. Fact: Levels Are Far Lower in E-Cigarettes Than In Cigarettes

Researcher Naoki Kunugita at Japan’s National Institute of Public Health recently fueled anti-e-cig hysteria with this unverifiable claim: “In one brand of e-cigarette the team found more than 10 times the level of carcinogens contained in one regular cigarette.”

The charge came not in a peer-reviewed study, but in comments to the press publicizing favorable e-cig research.  His group looked at 13 Japanese e-cigs and reported in the International Journal of Environmental Research and Public Health that they had lower levels of formaldehyde than cigarettes (here).

Formaldehyde is everywhere, even in the air we breathe.  Individuals inhale about 1,000 micrograms per day, according to the World Health Organization (here), and a microgram is a very small amount – one-millionth of a gram.

The Japanese researchers collected from 0 to 34 micrograms from 10 puffs of e-cigarettes – at most, about one thirtieth of normal daily exposure from air.  In contrast, 10 puffs of a cigarette deliver 150 to 200 micrograms.

Apparently, after the controlled research was completed, Kunugita recorded 1,600 micrograms (10 times the cigarette level) using another e-cig brand.  That single observation generated the worldwide headlines.  Not only is the finding completely out of range with respect to all other studies, the claim is unverifiable. 

Publishing a scientific report showing low formaldehyde levels and then publicizing an unsubstantiated claim of 10-fold carcinogenicity is irresponsible.  The announcement was rightfully condemned by e-cigarette expert Konstantinos Farsilanos in his research blog (here).

The unvalidated claim undercuts legitimate science and sends smokers a horrific message: keep lighting up, because e-cigarettes are more dangerous. 

Mouse, Rat & Cell Studies Don’t Make E-Cigarettes Carcinogenic

In determining what causes cancer in humans, epidemiologic and public health research is far superior to lab studies based on cells, mice or rats.  While the latter can provide important information about the biology of cancer, the vast majority of carcinogens have been discovered in studies of human exposures. 

Although numerous epidemiologic studies prove that smokers are more likely to contract a variety of cancers, decades of research on cells and animals have failed to establish which of the thousands of toxins in cigarette smoke cause human lung, bladder or esophagus cancer. 

Of the two major components of e-cigarette juice, we know this: Nicotine, the subject of thousands of studies, has never been shown to be a cancer-causing agent, and propylene glycol is generally recognized as safe for human use by the FDA. 

Regrettably, these facts haven’t stopped some researchers from scaremongering about e-cigarettes.  A study published in January (here) has led to a media frenzy suggesting that e-cigarette liquid may be as dangerous as smoke (here).  This is nonsense. 

Normal cells do not live forever.  But cancer cells are “immortalized” and are able to proliferate indefinitely.  The experiments reported in this study were conducted in immortalized cell cultures, which also included mutations of two important genes: p53, an anticancer gene that is active in normal cells, was “silenced”; and k-ras, a well-characterized oncogene, was “activated.” 

The researchers were essentially using a cancer cell line.  They measured the effect of two (unquantified) concentrations of nicotine e-cig solution and some sort of smoke extract on assays of growth and invasiveness after 10 days of exposure. 

Exposure of the cells to the low-nicotine e-cig solution and to the smoke extract had no effect on the invasiveness of the cells (a cancer trait).  They reported, “We will next examine the effects of high nicotine conditioned media on cell invasion,” indicating a future experiment.

The researchers noted that after 96 hours of exposure to e-cig solution, the cells showed changes in gene expression.  This is not particularly newsworthy.  Genes are the bits of DNA that tell cells what to do.  At any given time cells have many thousands of active genes.  Any environmental change can produce changes in the expression of large numbers of genes. 

In their effort to implicate nicotine, the researchers omitted information as to whether they had established appropriate experimental controls, such as exposure of the cells to other common agents such as caffeine or coffee extracts.

Cellular and molecular research explores the incredibly complicated biology of cancer, but it is of limited value in identifying carcinogens.  There are well established tests to determine if an agent is a possible mutagen, which is an indication that it might be cancer-causing.  A 2007 study of American smokeless products was essentially negative (here), which is completely consistent with epidemiologic studies.  It is likely that tests of e-cigarette liquids would produce similar results.   

Undistinguished research on smokeless tobacco products routinely generates headlines and soundbites best suited for the tabloids.  From a public health standpoint, it is shameful that researchers and media conflate vague, exaggerated and highly theoretical claims about e-cigarette juice to the very real risks of cigarettes.