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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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2019 The Author(s).)

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 ± 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Physiologically based kinetic (PBK) models and the virtual cell based assay can be linked to form so called physiologically based dynamic (PBD) models. This study illustrates the development and application of a PBK model for prediction of estragole-induced DNA adduct formation and hepatotoxicity in humans. To address the hepatotoxicity, HepaRG cells were used as a surrogate for liver cells, with cell viability being used as the in vitro toxicological endpoint. Information on DNA adduct formation was taken from the literature. Since estragole induced cell damage is not directly caused by the parent compound, but by a reactive metabolite, information on the metabolic pathway was incorporated into the model. In addition, a user-friendly tool was developed by implementing the PBK/D model into a KNIME workflow. This workflow can be used to perform in vitro to in vivo extrapolation and forward as backward dosimetry in support of chemical risk assessment., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vapor dosimetry models provide a means of assessing the role of delivered dose in determining the regional airway response to inspired vapors. A validated hybrid computational fluid dynamics physiologically based pharmacokinetic model for inhaled diacetyl has been developed to describe inhaled diacetyl dosimetry in both the rat and human respiratory tracts. Comparison of the distribution of respiratory tract injury with dosimetry estimates provides strong evidence that regional delivered dose rather than regional airway tissue sensitivity to diacetyl-induced injury is the critical determinant of the regional respiratory tract response to this water soluble reactive vapor. In the rat, inhalation exposure to diacetyl causes much lesser injury in the distal bronchiolar airways compared to nose and large tracheobronchial airways. The degree of injury correlates very strongly to model based estimates of local airway diacetyl concentrations. According to the model, regional dosimetry patterns of diacetyl in the human differ greatly from those in the rat with much greater penetration of diacetyl to the bronchiolar airways in the lightly exercising mouth breathing human compared to the rat, providing evidence that rat inhalation toxicity studies underpredict the risk of bronchiolar injury in the human. For example, repeated exposure of the rat to 200ppm diacetyl results in bronchiolar injury; the estimated bronchiolar tissue concentration in rats exposed to 200ppm diacetyl would occur in lightly exercising mouth breathing humans exposed to 12ppm. Consideration of airway dosimetry patterns of inspired diacetyl is critical to the proper evaluation of rodent toxicity data and its relevance for predicting human risk., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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 toxicological evaluation of two structurally related flavors with modifying properties, 3-((4-amino-2,2-dioxido-1H- benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2,2-dimethyl-N-propylpropanamide (S6973; CAS 1093200-92-0) and (S)-1-(3-(((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)methyl)piperidin-1-yl)-3-methylbutan-1-one (S617; CAS 1469426-64-9), was completed for the purpose of assessing their safety for use in food and beverage applications. Both compounds exhibited minimal oxidative metabolism in vitro, and in rat pharmacokinetic studies, were poorly absorbed and rapidly eliminated. Neither compound exhibited genotoxic concerns. S6973 and S617 were not found to be mutagenic or clastogenic, and did not induce micronuclei in vitro or in vivo. In subchronic oral toxicity studies in rats, the no-observed-adverse-effect-levels (NOAELs) were 20 mg/kg/day and 100 mg/kg/day (highest doses tested) for S6973 and S617, respectively, when administered as a food ad-mix for 90 consecutive days. Furthermore, S617 demonstrated a lack of maternal toxicity, as well as adverse effects on fetal morphology at the highest dose tested, providing a NOAEL of 1000 mg/kg/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sodium salt of glutamate (monosodium glutamate; MSG) imparts a savory/meaty taste to foods, and has been used as a flavoring agent for millennia. Past research on MSG/glutamate has evaluated its physiologic, metabolic and behavioral actions, and its safety. Ingested MSG has been found to be safe, and to produce no remarkable effects, except on taste. However, some recent epidemiologic and animal studies have associated MSG use with obesity and aberrations in fat metabolism. Reported effects are usually attributed to direct actions of ingested MSG in brain. As these observations conflict with past MSG research findings, a symposium was convened at the 13th International Congress on Amino Acids, Peptides and Proteins to discuss them. The principal conclusions were: (1) the proposed link between MSG intake and weight gain is likely explained by co-varying environmental factors (e.g., diet, physical activity) linked to the "nutrition transition" in developing Asian countries. (2) Controlled intervention studies adding MSG to the diet of animals and humans show no effect on body weight. (3) Hypotheses positing dietary MSG effects on body weight involve results from rodent MSG injection studies that link MSG to actions in brain not applicable to MSG ingestion studies. The fundamental reason is that glutamate is metabolically compartmentalized in the body, and generally does not passively cross biologic membranes. Hence, almost no ingested glutamate/MSG passes from gut into blood, and essentially none transits placenta from maternal to fetal circulation, or crosses the blood-brain barrier. Dietary MSG, therefore, does not gain access to brain. Overall, it appears that normal dietary MSG use is unlikely to influence energy intake, body weight or fat metabolism.

A number of α,β-unsaturated aldehydes are present in food both as natural constituents and as flavouring agents. Their reaction with DNA due to their electrophilic α,β-unsaturated aldehyde moiety may result in genotoxicity as observed in some in vitro models, thereby raising a safety concern. A question that remains is whether in vivo detoxification would be efficient enough to prevent DNA adduct formation and genotoxicity. In this study, a human physiologically based kinetic/dynamic (PBK/D) model of trans-2-hexenal (2-hexenal), a selected model α,β-unsaturated aldehyde, was developed to examine dose-dependent detoxification and DNA adduct formation in humans upon dietary exposure. The kinetic model parameters for detoxification were quantified using relevant pooled human tissue fractions as well as tissue fractions from 11 different individual subjects. In addition, a Monte Carlo simulation was performed so that the impact of interindividual variation in 2-hexenal detoxification on the DNA adduct formation in the population as a whole could be examined. The PBK/D model revealed that DNA adduct formation due to 2-hexenal exposure was 0.039 adducts/10⁸ nucleotides (nt) at the estimated average 2-hexenal dietary intake (0.04 mg 2-hexenal/kg bw) and 0.18 adducts/10⁸ nt at the 95th percentile of the dietary intake (0.178 mg 2-hexenal/kg bw) in the most sensitive people. These levels are three orders of magnitude lower than natural background DNA adduct levels that have been reported in disease-free humans (6.8-110 adducts/10⁸ nt), suggesting that the genotoxicity risk for the human population at realistic dietary daily intakes of 2-hexenal may be negligible.

trans-2-Hexenal (2-hexenal) is an α,β-unsaturated aldehyde that occurs naturally in a wide range of fruits, vegetables, and spices. 2-Hexenal as well as other α,β-unsaturated aldehydes that are natural food constituents or flavoring agents may raise a concern for genotoxicity due to the ability of the α,β-unsaturated aldehyde moiety to react with DNA. Controversy remains, however, on whether α,β-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,β-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.

Food materials designated as "Generally Recognized as Safe" (GRAS) are attracting the attention of researchers in their attempts to systematically identify compounds with putative health-related benefits. In particular, there is currently a great deal of interest in exploring possible secondary benefits of flavor ingredients, such as those relating to health and wellness. One step in this direction is the comprehensive characterization of the chemical structures contained in databases of flavoring substances. Herein, we report a comprehensive analysis of the recently updated FEMA GRAS list of flavoring substances (discrete chemical entities only). Databases of natural products, approved drugs and a large set of commercial molecules were used as references. Remarkably, natural products continue to be an important source of bioactive compounds for drug discovery and nutraceutical purposes. The comparison of five collections of compounds of interest was performed using molecular properties, rings, atom counts and structural fingerprints. It was found that the molecular size of the GRAS flavoring substances is, in general, smaller cf. members of the other databases analyzed. The lipophilicity profile of the GRAS database, a key property to predict human bioavailability, is similar to approved drugs. Several GRAS chemicals overlap to a broad region of the property space occupied by drugs. The GRAS list analyzed in this work has high structural diversity, comparable to approved drugs, natural products and libraries of screening compounds. This study represents one step towards the use of the distinctive features of the flavoring chemicals contained in the GRAS list and natural products to systematically search for compounds with potential health-related benefits.

We describe a method for expanding the linear dynamic range for multiple reaction monitoring (MRM) in quantitative liquid chromatography/tandem mass spectrometry (LC-MS/MS) using additional transitions for isotopologues. In addition to the regular transition for the highest possible sensitivity, a transition corresponding to the less abundant isotopologue ions was utilized. This decreases saturation at the ion detector; the sensitivity reduction increases the upper dynamic limit. We demonstrated this for a rat plasma assay for a candidate flavor compound; the linear dynamic range increased by an order of magnitude from 3 to 6,000 ng/mL with the regular MRM alone to 3-60,000 ng/mL using additionally the isotopologue transition., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This publication is the thirteenth 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 2600 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. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; 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 potential., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The metabolism of 1,8-cineole after ingestion of sage tea was studied. After application of the tea, the metabolites 2-hydroxy-1,8-cineole, 3-hydroxy-1,8-cineole, 9-hydroxy-1,8-cineole and, for the first time in humans, 7-hydroxy-1,8-cineole were identified in plasma and urine of one volunteer. For quantitation of these metabolites and the parent compound, stable isotope dilution assays were developed after synthesis of [(2)H(3)]-1,8-cineole, [9/10-(2)H(3)]-2-hydroxy-1,8-cineole and [(13)C,(2)H(2)]-9-hydroxy-1,8-cineole as internal standards. Using these standards, we quantified 1,8-cineole by solid phase microextraction GC-MS and the hydroxyl-1,8-cineoles by LC-MS/MS after deconjugation in blood and urine of the volunteer. After consumption of 1.02 mg 1,8-cineole (19 μg/kg bw), the hydroxycineoles along with their parent compound were detectable in the blood plasma of the volunteer under study after liberation from their glucuronides with 2-hydroxycineole being the predominant metabolite at a maximum plasma concentration of 86 nmol/L followed by the 9-hydroxy isomer at a maximum plasma concentration of 33 nmol/L. The parent compound 1,8-cineole showed a low maximum plasma concentration of 19 nmol/L. In urine, 2-hydroxycineole also showed highest contents followed by its 9-isomer. Summing up the urinary excretion over 10 h, 2-hydroxycineole, the 9-isomer, the 3-isomer and the 7-isomer accounted for 20.9, 17.2, 10.6 and 3.8% of the cineole dose, respectively.

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.

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 present study describes the isolation of microorganisms capable of producing alpha-pinene from beta-pinene. 24 (15 fungi, 9 bacteria) microorganisms were isolated from galbanum, gum and soil, were screened for their ability to transform beta-pinene to alpha-pinene. Biotransformation products were extracted with n-hexan and analyzed by gas chromatography. One microorganism (bacterial strain) were found. The biotransformation medium involved, phosphate buffer pH 6, 100 microL beta-pinene, 1 g biomass of microorganism, 37 degrees C, 150 rpm and 22 h. The experiments were performed in conical flasks. The optimum cell growth were obtained when 30 g L(-1) glycerin applied. The optimum conversion beta-pinene to alpha-pinene was obtained when 20 g L(-1) glycerin applied as carbon source for bacterial strain.

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.

This publication is the ninth 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. 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. Scientific data relevant to the safety evaluation of the use of phenethyl alcohol, aldehyde, acid, and related acetals and esters as flavoring ingredients is evaluated. The group of phenethylalcohol, aldehyde, acid, and related acetals and esters was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food, 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. This evidence of safety is supported by the fact that the intake of phenethyl alcohol, aldehyde, acid, and related acetals and esters as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

This publication is the eighth 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. 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. Scientific data relevant to the safety evaluation of the use of benzyl derivatives as flavoring ingredients is evaluated. The group of benzyl derivatives was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; 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. This evidence of safety is supported by the fact that the intake of benzyl derivatives as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

This publication is the ninth 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. 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. Scientific data relevant to the safety evaluation of the use of hydroxy- and alkoxy-substituted benzyl derivatives as flavoring ingredients is evaluated. The group of hydroxy- and alkoxy-benzyl derivatives was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; 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. This evidence of safety is supported by the fact that the intake of hydroxy- and alkoxy-substituted benzyl derivatives as natural components of traditional foods is greater than their intake as intentionally added flavoring substances.

This publication is the seventh 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. 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. Scientific data relevant to the safety evaluation of the use of cinnamyl derivatives as flavoring ingredients is evaluated.

The use of condoms to prevent sexually transmitted diseases, especially HIV, is widely encouraged. Condoms contain latex, nonspermicidal lubricants (such as dimethylsiliconium) and other nonspecified compounds, such as colorants and flavorings. Latex causes allergy reaction in susceptible individuals but little is known regarding the cytotoxic effects of other additives. The objective of this study was to develop a sensitive in vitro system to determine the toxic effects of condom material. The modified L929 FDA method and a more specific cell type, such as the cervical epithelial tumor cell line HeLa, was used. Lubricated (LC), lubricated and flavored (LFC), and lubricated, flavored and colored condoms (LFCC) were evaluated. Washings containing condom surface material were prepared by washing condom fragments in medium for different time intervals. Changes in cell number, viability and lysosome integrity in the L929 and HeLa cell lines was determined using the Crystal Violet, MTT and Neutral Red assays, respectively. The condom type affected cell viability and lysosome integrity, with LC inducing an increase in cell viability and LFC a decrease in lysosome integrity. The HeLa cell line in combination with the MTT and NR assay was themost sensitive in vitro system to determine the toxic effects of condom material.

This publication is the seventh 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 flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of the allylalkoxybenzene derivatives methyl eugenol and estragole is critically evaluated by the FEMA Expert Panel. The hazard determination uses a mechanism-based approach in which production of the hepatotoxic sulfate conjugate of the 1'-hydroxy metabolite is used to interpret the pathological changes observed in different species of laboratory rodents in chronic and subchronic studies. In the risk evaluation, the effect of dose and metabolic activation on the production of the 1'-hydroxy metabolite in humans and laboratory animals is compared to assess the risk to humans from use of methyl eugenol and estragole as naturally occurring components of a traditional diet and as added flavouring substances. Both the qualitative and quantitative aspects of the molecular disposition of methyl eugenol and estragole and their associated toxicological sequelae have been relatively well defined from mammalian studies. Several studies have clearly established that the profiles of metabolism, metabolic activation, and covalent binding are dose dependent and that the relative importance diminishes markedly at low levels of exposure (i.e. these events are not linear with respect to dose). In particular, rodent studies show that these events are minimal probably in the dose range of 1-10 mg/kg body weight, which is approximately 100-1000 times the anticipated human exposure to these substances. For these reasons it is concluded that present exposure to methyl eugenol and estragole resulting from consumption of food, mainly spices and added as such, does not pose a significant cancer risk. Nevertheless, further studies are needed to define both the nature and implications of the dose-response curve in rats at low levels of exposure to methyl eugenol and estragole.

This is the fifth 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. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of pyrazine derivatives as flavoring ingredients is evaluated.

Unlabelled: Acetylated distarch phosphate (ADiSP) is a modified starch used in some baby foods. The bioavailability of ADiSP and a native (unmodified) starch was evaluated in 20 normal infants and 21 toddlers aged 8-24 mo with chronic non-specific diarrhea. Formulae contained 8% native or 8% modified waxy maize starch. No infant or toddler consuming Formula N (native starch) had elevated peak breath hydrogen levels (20 ppm or greater), stools clinically positive for reducing substances (0.75% or greater) or loose stools. Fourteen infants received formula M (modified starch): 2 had elevated breath hydrogen, 1 had positive stools and another had loose stools. Of the 21 toddlers fed formula M, 2 had elevated breath hydrogen, but none had positive stools or loose stools. Formula NS (native starch with 2% sorbitol) had little effect on breath hydrogen in the infants but significantly increased it in the toddlers. Formula NS produced loose stools in 2 toddlers but no clinically positive stools in any infant or toddler. Formula MS (modified starch with 2% sorbitol) elevated breath hydrogen in 3 infants and 8 toddlers, and produced positive stools in 2 infants and 2 toddlers, and loose stools in 4 infants and 7 toddlers. Formula MSF (modified starch with 2% sorbitol and 5% fructose) elevated breath hydrogen in 7 infants and 10 toddlers, positive stools in 7 infants and 6 toddlers, and loose stools or diarrhea in 7 infants and 11 toddlers., Conclusion: ADiSP modified starch can increase breath hydrogen and produce loose stools. Sorbitol and fructose aggravate the malabsorption, in some cases leading to frank diarrhea.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA) has assessed the safety of furfural for its continued use as a flavour ingredient. The safety assessment takes into account the current scientific information on exposure, metabolism, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. Furfural was reaffirmed as GRAS (GRASr) as a flavour ingredient under conditions of intended use based on: (1) its mode of metabolic detoxication in humans; (2) its low level of flavour use compared with higher intake levels as a naturally occurring component of food; (3) the safety factor calculated from results of subchronic and chronic studies, (4) the lack of reactivity with DNA; and (5) the conclusion that the only statistically significant finding in the 2-year NTP bioassays, an increased incidence of hepatocellular adenomas and carcinomas in the high-dose group of male mice, was secondary to pronounced hepatotoxicity. Taken together, these data do not indicate any risk to human health under conditions of use as a flavour ingredient. This evidence of safety is supported by the occurrence of furfural as a natural component of traditional foods, at concentrations in the diet resulting in a 'natural intake' that is at least 100 times higher than the intake of furfural from use as a flavour ingredient.

This study describes the metabolic fate of trans-4'-methoxyprop-[1-14C]enylbenzene, the natural flavor compound trans-anethole, in rats and mice given single doses of 250 mg/kg body weight. In both rats and mice, an essentially quantitative (> 95% of dose) recovery of 14C was obtained with the majority in the 0-24 hr urine. Separation and identification of 18 urinary anethole metabolites were achieved by radio-HPLC, chemical derivatization, and GC/ MS. Anethole undergoes three primary oxidation pathways-O-demethylation, omega-side chain oxidation, and side chain epoxidation-followed by a variety of secondary pathways of oxidation and hydration, the products of which are extensively conjugated with sulfate, glucuronic acid, glycine, and glutathione. A novel major metabolite has been characterized in the rat, apparently originating from conjugation of the epoxide with glutathione, namely S-[1-(4'-methoxyphenyl)-2-hydroxypropane]-N-acetylcysteine. These metabolites are discussed in terms of the pathways responsible for and the toxicological consequences of their formation.

A method has been developed and validated for the analysis of methyleugenol, a volatile flavoring agent, in rodent plasma at concentrations from 0.050 to 10.0 micrograms/ml. The method involves the addition of acetonitrile to the plasma, removal of the protein precipitate, and analysis of the supernatant by reversed-phase (C18) HPLC using an acetonitrile-water mixture with UV detection. The precision, accuracy, sensitivity, and specificity of the method were assessed. The stability of methyleugenol in plasma, during freeze-thaw cycles, refrigerated at the extract stage, and during the analysis was evaluated. The recovery of methyleugenol from plasma was also determined. This method was found to be acceptable for plasma concentrations in toxicokinetic studies of methyleugenol in rodents. Over 400 samples from toxicokinetic studies have been successfully analyzed to date. Kinetic data from a preliminary single administration intravenous and oral study in rats is also presented.

Racemic trans-anethole epoxide [1-(4'-methoxyphenyl)-propane-1,2-oxide] was incubated with water, buffers, and rat liver microsomes and cytosol and the stereochemistry of the diols produced was determined by HPLC as their dicamphanyl esters. The diol metabolites were isolated by HPLC from the urine of rats administered [1'-14C] trans-anethole and their stereochemistry determined after derivatization to their camphanyl esters. The stereochemical course of the metabolism of trans-anethole by rat liver microsomes and cytosol is discussed.

Previous work suggests that fuel oxidation in the liver provides an unconditioned stimulus for "calorie-based" flavor preference learning. To investigate this possibility in more detail, we manipulated liver metabolism by taking advantage of the greater specificity to the liver of fructose than glucose. Intact rats preferred flavored food ingested with a drink of 35% fructose solution to flavored food eaten with either no sweet drink (Experiment 1) or an equicaloric drink of glucose (Experiment 2). The effect on food preference did not depend on the taste of the sugar solutions: when given a choice between glucose and fructose solutions, the rats drank the same volume of each. Moreover, a preference for fructose-paired flavored food was obtained when fructose and glucose solutions were given by gavage (Experiment 3). Unlike intact rats, rats with hepatic vagotomy preferred equally flavored food paired with fructose solution and flavored food paired with no sugar solution (Experiment 1). They also avoided flavored food paired with gavage of fructose slightly, relative to flavored food paired with gavage of glucose (Experiment 3). These results suggest that the unconditioned stimulus for calorie-based conditioning is transduced in the liver, and that an intact hepatic vagus nerve is required for conditioning to occur.

1. The metabolic fates of the naturally occurring food flavours trans-anethole and estragole, and their synthetic congener p-propylanisole, have been investigated in human volunteers using the [methoxy-14C]-labelled compounds. The doses used were close to those encountered in the diet, 1 mg, 100 micrograms and 100 micrograms respectively. 2. In each case, the major routes of elimination of 14C were in the urine and in the expired air as 14CO2. 3. Urinary metabolites were separated by solvent extraction, t.l.c. and h.p.l.c., and characterized by comparison of chromatographic mobilities with standards and by radioisotope dilution. Nine 14C urinary metabolites were found after trans-anethole administration, four after p-propylanisole and five after estragole. All were products of side chain oxidations. 4. The principal metabolites of p-propylanisole were 4-methoxyhippuric acid (12%) and 1-(4'-methoxyphenyl)propan-1-ol (2%) and -2-ol (8%). 5. The major metabolite of trans-anethole was 4-methoxyhippuric acid (56% of dose), accompanied by much smaller amounts of the two isomers of 1-(4'-methoxyphenyl)propane-1,2-diol (together 3%). 6. After estragole administration, the two volunteers eliminated 0.2 and 0.4% of the dose respectively as 1'-hydroxyestragole. 7. The human metabolic data is discussed with reference to the comparative metabolic disposition of these compounds in the mouse and rat, species commonly used in their safety assessment.