Exploring in vitro to in vivo extrapolation for exposure and health impacts of e-cigarette flavor mixtures.

In vitro to in vivo extrapolation (IVIVE) leverages in vitro biological activities to predict corresponding in vivo exposures, therefore potentially reducing the need for animal safety testing that are traditionally performed to support the hazard and risk assessment. Interpretation of IVIVE predictions are affected by various factors including the model type, exposure route and kinetic assumptions for the test article, and choice of in vitro assay(s) that are relevant to clinical outcomes. Exposure scenarios are further complicated for mixtures where the in vitro activity may stem from one or more components in the mixture. In this study, we used electronic cigarette (EC) aerosols, a complex mixture, to explore impacts of these factors on the use of IVIVE in hazard identification, using open-source pharmacokinetic models of varying complexity and publicly available data. Results suggest in vitro assay selection has a greater impact on exposure estimates than modeling approaches. Using cytotoxicity assays, high exposure estimates (>1000 EC cartridges (pods) or > 700 mL EC liquid per day) would be needed to obtain the in vivo plasma levels that are corresponding to in vitro assay data, suggesting acute toxicity would be unlikely in typical usage scenarios. When mechanistic (Tox21) assays were used, the exposure estimates were much lower for the low end, but the range of exposure estimate became wider across modeling approaches. These proof-of-concept results highlight challenges and complexities in IVIVE for mixtures.

Systems pharmacology-based approach for dissecting the mechanisms of pyrazine components in Maotai liquor.

Maotai liquor is a typical representative of sauce aroma-style flavor liquors and has been considered to be a precious cultural heritage of the oriental spirit culture. Aroma components are largely responsible for the characteristic aroma of liquor. Pyrazine compound is one of the most important categories of aroma components that affect the flavor of Maotai liquor. However, limited information is available regarding the systemic analysis of pyrazine compounds, especially the pharmacological effects of bioactive pyrazine components. Therefore, in the current study, a systemic analysis approach was provided by integrating absorption, distribution, metabolism, and excretion (ADME) screening, target identification, pharmacological evaluation and pathway analysis to explore the pharmacological mechanism of pyrazine compounds in Maotai liquor. As a result, 17 pyrazine components with adequate pharmacokinetic properties were filtered out using ADME models. Thirty eight potential targets of these active compounds were identified through target prediction. The pharmacological evaluation was proposed to uncover the pharmacological effect of pyrazine compounds in Maotai liquor from the holistic perspective. Finally, the pharmacological effects of the pathways perturbed by potential targets were interpreted based on the pathway analysis. Our study lays the foundation for formulating a comprehensive understanding of the pyrazine compounds in Maotai liquor, which would contribute to the development of Chinese liquor.

Controlled release of flavor oil nanoemulsions encapsulated in filled soluble hydrogels.

The goal of this project was to create hydrogels, a type of soluble biopolymer delivery system to encapsulate flavored nanoemulsions that are released under artificial saliva conditions. Low methoxyl (LM) pectin and whey protein isolate (WPI) at pH 4.0 were used to form the hydrogels at a ratio of 4:1 (w/w), respectively. Orange oil, medium-chain triglyceride (MCT) oil, and WPI were used to make stable nanoemulsions loaded with flavor oil. The nanoemulsions were encapsulated into hydrogels with a mean diameter of 768 ±â€¯36 nm. The ability of the hydrogels to encapsulate the orange oil and release the flavor in the presence of artificial saliva was determined using size distribution data, confocal microscopy, and the release of limonene as assessed by solid-phase microextraction using gas chromatography mass spectrometry. Results showed that the encapsulation of flavor nanoemulsions in filled hydrogels reduces the release of limonene.

Mechanisms of toxicity and biomarkers of flavoring and flavor enhancing chemicals in emerging tobacco and non-tobacco products.

Tobacco products containing flavorings, such as electronic nicotine delivery devices (ENDS) or e-cigarettes, cigars/cigarillos, waterpipes, and heat-not-burn devices (iQOS) are continuously evolving. In addition to increasing the exposure of teenagers and adults to nicotine containing flavoring products and flavoring enhancers, chances of nicotine addiction through chronic use and abuse also increase. These flavorings are believed to be safe for ingestion, but little information is available about their effects on the lungs. In this review, we have discussed the in vitro and in vivo data on toxicity of flavoring chemicals in lung cells. We have further discussed the common flavoring agents, such as diacetyl and menthol, currently available detection methods, and the toxicological mechanisms associated with oxidative stress, inflammation, mucociliary clearance, and DNA damage in cells, mice, and humans. Finally, we present potential biomarkers that could be utilized for future risk assessment. This review provides crucial parameters important for evaluation of risk associated with flavoring agents and flavoring enhancers used in tobacco products and ENDS. Future studies can be designed to address the potential toxicity of inhaled flavorings and their biomarkers in users as well as in chronic exposure studies.

Toxicity in food flavorings at the cellular level associated with each other at different doses / Toxicidade em nível celular de aromatizantes alimentares associados entre si em diferentes doses

This study aimed to examine the cytotoxicity and genotoxicity of synthetic flavorings, nature identical, Chocolate, Strawberry and Condensed Milk. This evaluation was performed in root meristem cells of Allium cepa L., in exposure times of 24 and 48 hours and using doses of 0.2; 0.4 and 0.6 mL, in combination, in which one of the three doses of a flavoring was combined with a different dose of one of the two other flavor additives studied. Roots were fixed in Carnoys solution, hydrolyzed in hydrochloric acid, stained with acetic orcein and then analyzed, under light microscopy, 5,000 cells for each treatment. For data analysis, it was used Chi-square test at 5%. All the treatments with combinations between the flavorings Chocolate/Strawberry and Strawberry/Condensed Milk reduced, in both exposure times considered, cell division of A. cepa roots, proving to be cytotoxic. In turn, the treatments with the association of Chocolate/Condensed Milk did not change significantly the mitotic index of the cells analyzed. The Strawberry flavoring was the most cytotoxic among the additives tested. None of the evaluated associations was genotoxic under the study conditions.

Objetivou-se nesta pesquisa avaliar a citoxicidade e genotoxicidade de aromatizantes alimentares sintéticos de chocolate, morango e leite condensado. Esta avaliação ocorreu por meio das células meristemáticas de raízes de A. cepa L., nos tempos de exposição de 24 e 48h e nas doses de 0,2; 0,4 e 0,6 mL, em associação, em que para uma das três doses de um dos aromatizantes associou-se uma dose diferente de um dos outros dois aditivos de aroma em estudo. Em seguida, as raízes foram fixadas em solução de Carnoy, hidrolisadas em ácido clorídrico e coradas com orceína acética. Analisaram-se, em microscópio óptico, 5.000 células para cada grupo tratamento, e utilizou-se o teste estatístico Qui-quadrado a 5% para análise dos dados. A partir dos resultados, verificou-se que todos os tratamentos decorrentes das associações entre chocolate/morango e morango/leite condensado reduziram, nos dois tempos de exposição considerados, a divisão celular das raízes A. cepa, mostrando-se citotóxicos. Já os tratamentos provenientes da associação chocolate/leite condensado não alteraram de forma significativa os índices mitóticos das células do tecido em análise. Foi possível inferir que o aditivo de morango foi o mais citotóxico dos aditivos em estudo. Nenhuma das associações avaliadas foi genotóxica nestas condições de estudo.

Determination of Menthol in Plasma and Urine by Gas Chromatography/Mass Spectrometry (GC/MS).

Menthol, a monoterpene, is a principal component of peppermint oil and is used extensively in consumer products as a flavoring aid. It is also commonly used medicinally as a topical skin coolant; to treat inflammation of the mucous membranes, digestive problems, and irritable bowel syndrome (IBS); and in preventing spasms during endoscopy and for its spasmolytic effect on the smooth muscle of the gastrointestinal tract. Menthol has a half life of 3-6 h and is rapidly metabolized to menthol glucuronide which is detectable in urine and serum following menthol use. We describe a method for the determination of total menthol in human plasma and urine using liquid/liquid extraction, gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode and menthol-d4 as the internal standard. Controls are prepared with menthol glucuronide and all samples undergo enzymatic hydrolysis for the quantification of total menthol. The method has a linear range of 5-1000 ng/mL, and coefficient of variation <10%.

A physiologically based in silico model for trans-2-hexenal detoxification and DNA adduct formation in rat.

trans-2-Hexenal (2-hexenal) is an α,ß-unsaturated aldehyde that occurs naturally in a wide range of fruits, vegetables, and spices. 2-Hexenal as well as other α,ß-unsaturated aldehydes that are natural food constituents or flavoring agents may raise a concern for genotoxicity due to the ability of the α,ß-unsaturated aldehyde moiety to react with DNA. Controversy remains, however, on whether α,ß-unsaturated aldehydes result in significant DNA adduct formation in vivo at realistic dietary exposure. In this study, a rat physiologically based in silico model was developed for 2-hexenal as a model compound to examine the time- and dose-dependent detoxification and DNA adduct formation of this selected α,ß-unsaturated aldehyde. The model was developed based on in vitro and literature-derived parameters, and its adequacy was evaluated by comparing predicted DNA adduct formation in the liver of rats exposed to 2-hexenal with reported in vivo data. The model revealed that at an exposure level of 0.04 mg/kg body weight, a value reflecting estimated daily human dietary intake, 2-hexenal is rapidly detoxified predominantly by conjugation with glutathione (GSH) by glutathione S-transferases. At higher dose levels, depletion of GSH results in a shift to 2-hexenal oxidation and reduction as the major pathways for detoxification. The level of DNA adduct formation at current levels of human dietary intake was predicted to be more than 3 orders of magnitude lower than endogenous DNA adduct levels. These results support that rapid detoxification of 2-hexenal reduces the risk arising from 2-hexenal exposure and that at current dietary exposure levels, DNA adduct formation is negligible.

Evaluation of human interindividual variation in bioactivation of estragole using physiologically based biokinetic modeling.

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1'-hydroxyestragole, due to variation in two key metabolic reactions involved in the formation and detoxification of this metabolite, namely 1'-hydroxylation of estragole and oxidation of 1'-hydroxyestragole. Formation of 1'-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1, and results of the present study support that oxidation of 1'-hydroxyestragole is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2). In a first approach, the study defines physiologically based biokinetic (PBBK) models for 14 individual human subjects, revealing a 1.8-fold interindividual variation in the area under the liver concentration-time curve (AUC) for 1'-hydroxyestragole within this group of human subjects. Variation in oxidation of 1'-hydroxyestragole by 17beta-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1'-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99th percentile divided by the 50th percentile of the predicted distribution of the AUC of 1'-hydroxyestragole in the liver. The CSAF was estimated to range between 1.6 and 4.0, depending on the level of variation that was taken into account for oxidation of 1'-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the population.

The FEMA GRAS assessment of alpha,beta-unsaturated aldehydes and related substances used as flavor ingredients.

This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear alpha,beta-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume alpha,beta-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.

The FEMA GRAS assessment of aromatic substituted secondary alcohols, ketones, and related esters used as flavor ingredients.

This publication is the 11th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. The list of GRAS substances has now grown to more than 2100 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. In this monograph, a detailed interpretation is presented on the renal carcinogenic potential of the aromatic secondary alcohol alpha-methylbenzyl alcohol, aromatic ketone benzophenone, and corresponding alcohol benzhydrol. The relevance of these effects to the flavor use of these substances is also discussed. The group of aromatic substituted secondary alcohols, ketones, and related esters was reaffirmed as GRAS (GRASr) based, in part, on their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential.