Evaluation of mixtures of flavor chemicals in a 90-day nose-only exposures in sprague-dawley rats.

BACKGROUND: One of the challenges to using some flavor chemicals in aerosol products is the lack of route of administration specific toxicology data. METHODS: Flavor chemicals (88) were divided into four different flavor mixtures based upon chemical compatibility and evaluated in 2-week dose-range-finding and subsequent 90-day nose-only rodent inhalation studies (OECD 413 and GLP compliant). Sprague-Dawley rats were exposed to vehicle control or one of three increasing concentrations of each flavor mixture. RESULTS: In the dose-range-range-finding studies, exposure to flavor mixture four resulted in adverse nasal histopathology in female rats at the high dose, resulting in this flavor mixture not being evaluated in a 90-day study. In the 90-day studies daily exposures to the three flavor mixtures did not induce biologically meaningful adverse effects (food consumption, body weights, respiratory physiology, serum chemistry, hematology, coagulation, urinalysis, bronchoalveolar lavage fluid analysis and terminal organ weights). All histopathology findings were observed in both vehicle control and flavor mixture exposed animals, with similar incidences and/or severities, and therefore were not considered flavor mixture related. CONCLUSION: Based on the absence of adverse effects, the no-observed-adverse-effect concentration for each 90-day inhalation study was the highest dose tested, 2.5 mg/L of the aerosolized high dose of the three flavor mixtures.

Subchronic inhalation of a novel electronic nicotine delivery system formulation and its corresponding base formulation.

BACKGROUND: Fresh Menthol 3% Nicotine (FM3) is a novel JUUL e-liquid formulation. Its potential toxicity and that of the corresponding base formulation relative to a filtered air (FA) control was studied in a subchronic inhalation study conducted in general accordance with OECD 413. METHODS: Aerosols generated with an intense puffing regime were administered to rats in a nose-only fashion at 1400 µg aerosol collected mass/L on a 6 hour/day basis for 90 days with a 42-day recovery. Exposure atmospheres met target criteria. Systemic exposure was confirmed by plasma measurement of nicotine. RESULTS: No test article-related mortality, clinical signs (other than reversible lower body weight gains in males), clinical pathology or gross findings were noted during this study. No microscopic lesions related to base formulation exposure were identified. Minimal microscopic lesions were observed in the FM3 6-hour exposure group. Microscopic lesions observed in the FM3 6-hour exposure group comprised only minimal laryngeal squamous metaplasia in one male and one female animal. No microscopic lesions related to FM3 exposure remained after the recovery period. CONCLUSION: Exposure atmosphere characterization indicated that conditions were achieved to permit thorough assessment of test articles and results indicate a low order of toxicity for the FM3 Electronic nicotine delivery systems (ENDS) formulation and its base formulation.

A Practical Framework for Novel Electronic Nicotine Delivery System Evaluation: Chemical and Toxicological Characterization of JUUL2 Aerosol and Comparison with Reference Cigarettes.

Electronic nicotine delivery systems (ENDSs) are designed as a non-combustible alternative to cigarettes, aiming to deliver nicotine without the harmful byproducts of tobacco combustion. As the category evolves and new ENDS products emerge, it is important to continually assess the levels of toxicologically relevant chemicals in the aerosols and characterize any related toxicology. Herein, we present a proposed framework for characterizing novel ENDS products (i.e., devices and formulations) and determining the reduced risk potential utilizing analytical chemistry and in vitro toxicological studies with a qualitative risk assessment. To demonstrate this proposed framework, long-term stability studies (12 months) analyzing relevant toxicant emissions from six formulations of a next-generation product, JUUL2, were conducted and compared to reference combustible cigarette (CC) smoke under both non-intense and intense puffing regimes. In addition, in vitro cytotoxicity, mutagenicity, and genotoxicity assays were conducted on aerosol and smoke condensates. In all samples, relevant toxicants under both non-intense and intense puffing regimes were substantially lower than those observed in reference CC smoke. Furthermore, neither cytotoxicity, mutagenicity, nor genotoxicity was observed in aerosol condensates generated under both intense and non-intense puffing regimes, in contrast to results observed for reference cigarettes. Following the proposed framework, the results demonstrate that the ENDS products studied in this work generate significantly lower levels of toxicants relative to reference cigarettes and were not cytotoxic, mutagenic, or genotoxic under these in vitro assay conditions.

Comparison of the toxicological potential of two JUUL ENDS products to reference cigarette 3R4F and filtered air in a 90-day nose-only inhalation toxicity study.

Electronic nicotine delivery systems (ENDS) are generally recognized as less harmful alternatives for those who would otherwise continue to smoke cigarettes. The potential toxicity of aerosols generated from JUUL Device and Virginia Tobacco (VT3) or Menthol (ME3) JUULpods at 3.0% nicotine concentration was assessed in rats exposed at target aerosol concentrations of 1400 µg/L for up to 6 h/day on a 5 day/week basis for at least 90 days (general accordance with OECD 413). 3R4F reference cigarette smoke (250 µg/L) and Filtered Air were used as comparators. JUUL ENDS product aerosol exposures at >5x the 3R4F cigarette smoke level resulted in greater plasma nicotine and cotinine levels (up to 2x). Notable cigarette smoke related effects included pronounced body weight reductions in male rats, pulmonary inflammation evidenced by elevated lactate dehydrogenase, pro-inflammatory cytokines and neutrophils in bronchoalveolar lavage fluid, increased heart and lung weights, and minimal to marked respiratory tract histopathology. In contrast, ENDS aerosol exposed animals had minimal body weight changes, no measurable inflammatory changes and minimal to mild laryngeal squamous metaplasia. Despite the higher exposure levels, VT3 and ME3 did not result in significant toxicity or appreciable respiratory histopathology relative to 3R4F cigarette smoke following 90 days administration.

Characterization of a rapid condensate collection apparatus for in vitro assays of electronic nicotine delivery systems.

In vitro testing of Electronic Nicotine Delivery System (ENDS) aerosol condensates is important in evaluating their potential toxicity. Collecting sufficient condensate for these tests is a time consuming and costly procedure. The "triple puff (TP)" is a novel system which collects the aerosol from three ENDS devices sequentially into a single filter pad and impinger. The TP substantially reduces condensate collection time relative to the conventional single ENDS, single puff (SP), device system. Both the TP and SP (using two puffing profiles) were used to generate condensates from JUUL ENDS e-liquid Mint 5.0% (nicotine by weight). Aerosols were collected using the filter pad and ethanol-containing impinger method. Condensates produced with the SP and TP were compared for concentrations of primary constituents and carbonyl compounds as well as for their cytotoxicity (OECD 129), mutagenicity (OECD 471) and genotoxicity (OECD 487). Condensates generated with the SP and TP, regardless of puffing regimen, were very similar chemically and equivalent in the biological assays tested (not cytotoxic, mutagenic, or genotoxic). The TP device significantly reduces production time of ENDS condensates relative to the standard SP method and thus may facilitate further research by reducing the time and effort required to collect ENDS condensates.