Nicotine-free vape liquids caused skull changes in mice fetuses. Effects appeared with “safer” chemical ratios used in modern products.
Exposure during pregnancy to two common liquids found in e-cigarettes, even without nicotine, can affect fetal skull development, according to a new study in mice.
Researchers exposed pregnant mice to a mix of the liquids responsible for vaping’s characteristic throat sensation and vapor. Offspring from mothers exposed to a particular ratio of these compounds were lighter at birth and showed narrower faces and shorter skulls compared to other test groups.
The results were unexpected. Not only was nicotine absent, but the chemical mixture linked to these changes was a formulation promoted by the e-cigarette industry as a safer alternative to older product blends.
“This had no nicotine, and it’s still having effects on the development of the skull in our model, which was not anything we expected,” said lead study author James Cray, professor of anatomy in The Ohio State University College of Medicine.
The research was published recently in the journal PLOS One.
Earlier research on nicotine and skull defects
Cray’s developmental biology lab has long examined how prenatal exposure to nicotine affects the development of the head and face. In a 2020 study, the team reported that nicotine delivered through breastmilk caused skull abnormalities in mice. According to the Centers for Disease Control and Prevention, about 3% of infants are born each year with a birth defect, with craniofacial anomalies—such as cleft lip and/or cleft palate—being among the most prevalent.
In the current research, the team aimed to develop a baseline model for comparison to animals exposed to nicotine-containing vape aerosols in utero.
Pregnant mice were exposed either to filtered air or to two common e-cigarette humectants—liquids that carry other substances—at different ratios: 50% propylene glycol and 50% glycerol (also known as vegetable glycerin), or a 30/70 mixture of propylene glycol to glycerol.
Cray explained that propylene glycol has been associated with enhanced nicotine absorption in e-cigarettes, prompting many companies to increase glycerol content in newer formulations in an effort to market them as safer.
The pregnant mice were exposed to these conditions at a rate of one puff per minute, for four hours a day, five days a week throughout the approximately 20-day gestation period.
Measured effects on skull width and weight
The cranial width and height of mouse pup skulls were scanned two weeks after birth. The analysis showed statistically significant reduced measures in the offspring of pregnant mice exposed to the presumed safer 30/70 PG/VG mixture compared to mice exposed to free air and the 50/50 formula.
“What we see is a consistent narrowing of all of the facial features, and the same thing as we move back into the cranium as well. So globally they’re narrower and a little bit shorter-headed, which does mimic some observable changes that we see in children,” Cray said. “We also saw a bit of a reduction in weight. These animals were within the normal range of an animal that age, but they still weighed less.”
The findings were consistent across several litters of mice and in both biological sexes.
“The 50/50 mixture had no dramatic statistical changes – and that’s where we were looking for the difference. We thought heavier propylene glycol should be causing more effects, and it was the exact opposite,” Cray said.
Regulation, legality, and rising use
The Food and Drug Administration began regulating electronic nicotine delivery systems in 2016, but has faced legal pushback from e-cigarette companies – which prevailed in a June 20 U.S. Supreme Court ruling that makes it easier to sue the FDA over efforts to block products from the market. In April, the court sided with the FDA’s crackdown on sweet-flavored products after a major increase in teen vaping.
Though researchers continue to study the health effects of multiple ingredients detected in e-cigarettes, many unknowns remain as the products grow in popularity.
“The majority of users are young adults and teenagers, so we are talking about people who are in peak reproductive years. And with development of the head happening very early in fetal development, people could be using these products and not even realize that they are pregnant, which is of great concern,” Cray said.
“This is a small study that speaks to the possibility that nicotine-free vaping is not safe. And it’s a sign that we probably should study the nicotine-free products as much as we study the nicotine-laden products.”
Reference: “In utero exposure to electronic cigarette carriers alters craniofacial morphology” by Ethan Richlak, Logan Shope, Ethan Leonard, Leslie Sewell, Tyler Maykovich, Amr Mohi, Roy A. Miller, Matthew W. Gorr, Loren E. Wold and James J. Cray, 30 June 2025, PLOS ONE.
DOI: 10.1371/journal.pone.0327190
This work was funded by the Ohio State colleges of Medicine and Dentistry.
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