Determining Toxic Levels in Pre‐implantation Mouse Embryo Development from Exposure to Electronic Nicotine Delivery System (e‐Cig) Carrier Fluid

Electronic Nicotine Delivery Systems (ENDS or e‐cig), are increasingly popular, despite not well FDA regulated. With increased trends in ENDS usage, especially as a “safer” alternative to cigarettes, the potential damage due to other chemicals in the ENDS carrier fluid (CF) must be determined in the absence of nicotine for both non‐vaporized (NCF) and vaporized (VCF) material. The main components of this CF are propylene glycol, vegetable glycerin, and flavor additives. Gas Chromatography/Mass Spectroscopy was used to determine the components of ENDS/NCF and ENDS/VCF. Our data establish a relationship between pre‐implantation embryo exposure to ENDS NCF and VCF relative to embryo development in a murine model. ENDS/NCF was placed in the e‐cig and then removed; ENDS/VCF was placed in the e‐cig, vaporized, and then condensed back to fluid using a glass‐bead‐filled, 7mm diameter × 7m long, copper coil at 2°C. A single 2‐cell embryo was placed in each well of a 96 well culture plate, in triplicate experiments, with modified Krebs bicarbonate‐buffered embryo growth medium (mKBBM) as mKBBM/NCF or mKBBM/VCF mixtures. The mKBBM/NCF was initially tested at 0.0% (control), 0.1%, 0.3%, and 0.5% concentrations by volume. The mKBBM/VCF was tested at 0.0, 0.001, 0.01, 0.6, 1.6, 2.6, and 3.6 percent concentrations by volume. Embryos were incubated for 96 hours (hrs) during continuous exposure and morphological observations were made every 24 hrs. This mouse embryo assay (MEA) has been used for IVF toxicity testing with passing minimum values of 20% morula‐65% blastocyst (85% combined) growth rate at 96 hrs. In the NCF group, control embryos passed the MEA (89%) while 0.1%, 0.3%, and 0.5% failed (84%, 0%, and 0% respectively). In the VCF group, 0.0, 0.001, 0.01 and 0.6% embryos passed the MEA (100% for all but 0.6% which was 87%) while the 1.6%, 2.6%, and 3.6% failed the MEA (80%, 80%, and 16% respectively). Experiments with identical percentages for both NCF and VCF are currently being performed. Additionally, a one‐way ANOVA with TUKEY posthoc, α = 0.05, was performed to evaluate null hypothesis of no difference based on exposure and viability. This revealed significant differences between groups at 24, 72, and 96, but not at 48 exposure hrs. These results indicate an initial developmental delay in the first 24 hrs with an adaptive catch‐up growth phase from 24–48 hrs followed by a drop‐off in growth post 48 hrs of CF exposure. Farsalinos, et al ., 2015, estimated 3mL of e‐liquid/day; 0.375% per 80kg man, well below our concentrations but we did not include nicotine in our experiments. Study animals were on IACUC‐approved protocol #11.161107. Support or Funding Information Center for Research, Liberty University College of Osteopathic Medicine