The natural basil flavonoid nevadensin protects against a methyleugenol-induced marker of hepatocarcinogenicity in male F344 rat.

The alkenylbenzene methyleugenol occurs naturally in a variety of spices and herbs, including basil, and their essential oils. At high dose levels methyleugenol induces hepatocarcinogenicity in rodents following bioactivation to 1'-sulfooxymethyleugenol which forms DNA adducts. This study investigated whether the inhibitory effect of the basil flavonoid nevadensin on sulfotransferase (SULT)-mediated bioactivation of methyleugenol observed in vitro would also be reflected in a reduction of DNA adduct formation and a reduction in an early marker for liver carcinogenesis in an 8-week rat study. Co-exposure to methyleugenol and nevadensin orally resulted in a significant inhibition of liver methyleugenol DNA adduct formation and in inhibition of hepatocellular altered foci induction, representing indicators for initiation of neoplasia. These results suggest that tumor formation could be lower in rodent bioassays when methyleugenol would be dosed in a matrix containing SULT inhibitors such as nevadensin compared to experiments using the pure methyleugenol.

GRASr2 evaluation of aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances used as flavoring ingredients.

This publication is the 1st in a series of publications by the Expert Panel of the Flavor and Extract Manufacturers Assoc. summarizing the Panel's 3rd re-evaluation of Generally Recognized as Safe (GRAS) status referred to as the GRASr2 program. In 2011, the Panel initiated a comprehensive program to re-evaluate the safety of more than 2700 flavor ingredients that have previously met the criteria for GRAS status under conditions of intended use as flavor ingredients. 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 acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances as flavoring ingredients are evaluated. The group of aliphatic acyclic and alicyclic terpenoid tertiary alcohols and structurally related substances was reaffirmed as GRAS (GRASr2) 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 studies and the lack of significant genotoxic and mutagenic potential.

In vivo validation and physiologically based biokinetic modeling of the inhibition of SULT-mediated estragole DNA adduct formation in the liver of male Sprague-Dawley rats by the basil flavonoid nevadensin.

SCOPE: The present work investigates whether the previous observation that the basil flavonoid nevadensin is able to inhibit sulfotransferase (SULT)-mediated estragole DNA adduct formation in primary rat hepatocytes could be validated in vivo. METHODS AND RESULTS: Estragole and nevadensin were co-administered orally to Sprague-Dawley rats, at a ratio reflecting their presence in basil. Moreover, previously developed physiologically based biokinetic (PBBK) models to study this inhibition in rat and in human liver were refined by including a submodel describing nevadensin kinetics. Nevadensin resulted in a significant 36% reduction in the levels of estragole DNA adducts formed in the liver of rats. The refined PBBK model predicts the formation of estragole DNA adducts in the liver of rat with less than twofold difference compared to in vivo data and suggests more potent inhibition in the liver of human compared to rat due to less efficient metabolism of nevadensin in human liver and intestine. CONCLUSION: Given the role of the SULT-mediated DNA adduct formation in the hepatocarcinogenicity of estragole, the results of the present study suggest that the likelihood of bioactivation and subsequent adverse effects in rodent bioassays may be lower when estragole is dosed with nevadensin compared to dosing of pure estragole.

Oral and dermal developmental toxicity studies of phenylethyl alcohol in rats.

Phenylethyl alcohol (PEA) was tested for developmental toxicity. Pregnant rats were fed 0, 83, 266, or 799 mg/kg/d PEA on gestation days (GDs) 6 to 15; only minimal, nonsignificant effects were observed. In dermal studies, PEA (neat) was applied to the skin on GDs 6 to 15 at dosages of 0, 140, 430, or 1400 mg/kg/d and at 0, 70, 140, 280, 430, or 700 mg/kg/d in a corroborative study. Observations included maternal and embryo-fetal toxicity/abnormalities at 1400 mg/kg/d, increased incidences of rudimentary cervical ribs at ≥430 mg/kg/d, and reduced fetal body weights at ≥140 mg/kg/d. Dermal maternal and developmental no-observed-adverse-effect levels are 70 mg/kg/d, based on dermal irritation and reductions (nonsignificant) in fetal body weights. Human exposure from fragrances is 0.02 mg/kg/d, resulting in a margin of safety >2600, when marked differences in dermal absorption between rats and humans are considered. Under normal fragrance use conditions, PEA is not a developmental toxicity hazard for humans.

Matrix modulation of the bioactivation of estragole by constituents of different alkenylbenzene-containing herbs and spices and physiologically based biokinetic modeling of possible in vivo effects.

The alkenylbenzene estragole is a constituent of several herbs and spices. It induces hepatomas in rodents at high doses following bioactivation by cytochrome P450s and sulfotransferases (SULTs) giving rise to the ultimate carcinogenic metabolite 1'-sulfooxyestragole which forms DNA adducts. Methanolic extracts from different alkenylbenzene-containing herbs and spices were able to inhibit SULT activity. Flavonoids including quercetin, kaempferol, myricetin, apigenin, and nevadensin were the major constituents responsible for this inhibition with Ki values in the nano to micromolar range. In human HepG2 cells exposed to the proximate carcinogen 1'-hydroxyestragole, the various flavonoids were able to inhibit estragole DNA adduct formation and shift metabolism in favor of glucuronidation which is a detoxification pathway for 1'-hydroxyestragole. In a next step, the kinetics for SULT inhibition were incorporated in physiologically based biokinetic (PBBK) models for estragole in rat and human to predict the effect of co-exposure to estragole and (mixtures of) the different flavonoids on the bioactivation in vivo. The PBBK-model-based predictions indicate that the reduction of estragole bioactivation in rat and human by co-administration of the flavonoids is dependent on whether the intracellular liver concentrations of the flavonoids can reach their Ki values. It is expected that this is most easily achieved for nevadensin which has a Ki value in the nanomolar range and is, due to its methyl ation, more metabolically stable than the other flavonoids.

Mode of action of pulegone on the urinary bladder of F344 rats.

Essential oils from mint plants, including peppermint and pennyroyal oils, are used at low levels as flavoring agents in various foods and beverages. Pulegone is a component of these oils. In a 2-year bioassay, oral administration of pulegone slightly increased the urothelial tumor incidence in female rats. We hypothesized that its mode of action (MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately leading to tumors. Pulegone was administered by gavage at 0, 75, or 150 mg/kg body weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy and scanning electron microscopy (SEM) and bromodeoxyuridine (BrdU) labeling index. Pulegone and its metabolites, piperitenone, piperitone, menthofuran, and menthone, were tested for cytotoxicity in rat (MYP3) and human (1T1) urothelial cells by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. No abnormal urinary crystals were observed by light microscopy. Urine samples (18-h) showed the presence of pulegone, piperitone, piperitenone, and menthofuran in both treated groups. By SEM, bladders from treated rats showed superficial necrosis and exfoliation. There was a significant increase in the BrdU labeling index in the high-dose group. In vitro studies indicated that pulegone and its metabolites, especially piperitenone, are excreted and concentrated in the urine at cytotoxic levels when pulegone is administered at high doses to female rats. The present study supports the hypothesis that cytotoxicity followed by regenerative cell proliferation is the MOA for pulegone-induced urothelial tumors in female rats.

Criteria for the safety evaluation of flavoring substances. The Expert Panel of the Flavor and Extract Manufacturers Association.

The current status of the GRAS evaluation program of flavoring substances operated by the Expert Panel of FEMA is discussed. The Panel maintains a rigorous rotating 10-year program of continuous review of scientific data related to the safety evaluation of flavoring substances. The Panel concluded a comprehensive review of the GRAS (GRASa) status of flavors in 1985 and began a second comprehensive review of the same substances and any recently GRAS materials in 1994. This second re-evaluation program of chemical groups of flavor ingredients, recognized as the GRAS reaffirmation (GRASr) program, is scheduled to be completed in 2005. The evaluation criteria used by the Panel during the GRASr program reflects the significant impact of advances in biochemistry, molecular biology and toxicology that have allowed for a more complete understanding of the molecular events associated with toxicity. The interpretation of novel data on the relationship of dose to metabolic fate, formation of protein and DNA adducts, enzyme induction, and the cascade of cellular events leading to toxicity provides a more comprehensive basis upon which to evaluate the safety of the intake of flavor ingredients under conditions of intended use. The interpretation of genotoxicity data is evaluated in the context of other data such as in vivo animal metabolism and lifetime animal feeding studies that are more closely related to actual human experience. Data are not viewed in isolation, but comprise one component that is factored into the Panel's overall safety assessment. The convergence of different methodologies that assess intake of flavoring substances provides a greater degree of confidence in the estimated intake of flavor ingredients. When these intakes are compared to dose levels that in some cases result in related chemical and biological effects and the subsequent toxicity, it is clear that exposure to these substances through flavor use presents no significant human health risk.

Issues and challenges in the safety evaluation of food flavors.

Within the context of safety assessment of "social chemicals" such as drugs, agrochemicals, major food additives and environmental chemicals, food flavors have conventionally been perceived to be of relatively low priority. The growing aspiration to achieve a "globally recognized list of flavors" and the necessity to achieve some degree of consensus as to the procedures to be used for their safety evaluation have changed this perception. The purpose of this workshop is to focus on the newer concepts and strategies applicable to the safety evaluation process and to evaluate these as part of a process to seek harmonization of approach and to ensure that up-to-date science-based procedures are incorporated in evaluation programs in the interest of both protecting the health of the public and helping the industry to continue to provide flavors that are safe.

The FEMA GRAS assessment of cinnamyl derivatives used as flavor ingredients.

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.

Studies on the metabolism of the thiofurans furfuryl mercaptan and 2-methyl-3-furanthiol in rat liver.

The metabolism of two thiofurans, namely furfuryl mercaptan (FM) and 2-methyl-3-furanthiol (MTF), to their corresponding methyl sulphide and methyl sulphoxide derivatives has been studied in male Sprague-Dawley rat hepatocytes and liver microsomes. Rat hepatocytes converted FM to furfuryl methyl sulphoxide (FMSO) and MTF to 2-methyl-3-(methylthio)furan sulphoxide (MMFSO). Liver microsomes catalysed the NADPH-dependent metabolism of furfuryl methyl sulphide (FMS) to FMSO and 2-methyl-3-(methylthio)furan sulphide (MMFS) to MMFSO. FMS and MMFS metabolism to their thiofuran methyl sulphoxide derivatives was induced by the treatment of rats with Aroclor 1254 and inhibited in liver microsomes treated with 1-aminobenzotriazole. The NADPH-dependent metabolism of FM to FMSO and MTF to MMFSO in liver microsomes was observed in the presence of S-adenosylmethionine. In summary, both thiofurans can be metabolised in rat liver to their thiofuran methyl sulphide derivatives which can be subsequently S-oxidised to form thiofuran methyl sulphoxides. FM and MTF appear to be substrates for rat hepatic microsomal thiol methyltransferase and the S-oxidation of FMS and MMFS appears to be primarily catalysed by cytochrome P450 forms.