In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells.

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 µM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Formation of potentially toxic metabolites of drugs in reactions catalyzed by human drug-metabolizing enzymes.

Data are presented on the formation of potentially toxic metabolites of drugs that are substrates of human drug metabolizing enzymes. The tabular data lists the formation of potentially toxic/reactive products. The data were obtained from in vitro experiments and showed that the oxidative reactions predominate (with 96% of the total potential toxication reactions). Reductive reactions (e.g., reduction of nitro to amino group and reductive dehalogenation) participate to the extent of 4%. Of the enzymes, cytochrome P450 (P450, CYP) enzymes catalyzed 72% of the reactions, myeloperoxidase (MPO) 7%, flavin-containing monooxygenase (FMO) 3%, aldehyde oxidase (AOX) 4%, sulfotransferase (SULT) 5%, and a group of minor participating enzymes to the extent of 9%. Within the P450 Superfamily, P450 Subfamily 3A (P450 3A4 and 3A5) participates to the extent of 27% and the Subfamily 2C (P450 2C9 and P450 2C19) to the extent of 16%, together catalyzing 43% of the reactions, followed by P450 Subfamily 1A (P450 1A1 and P450 1A2) with 15%. The P450 2D6 enzyme participated in an extent of 8%, P450 2E1 in 10%, and P450 2B6 in 6% of the reactions. All other enzymes participate to the extent of 14%. The data show that, of the human enzymes analyzed, P450 enzymes were dominant in catalyzing potential toxication reactions of drugs and their metabolites, with the major role assigned to the P450 Subfamily 3A and significant participation of the P450 Subfamilies 2C and 1A, plus the 2D6, 2E1 and 2B6 enzymes contributing. Selected examples of drugs that are activated or proposed to form toxic species are discussed.

Oxygen-18 Labeling Defines a Ferric Peroxide (Compound 0) Mechanism in the Oxidative Deformylation of Aldehydes by Cytochrome P450 2B4.

Most cytochrome P450 (P450) oxidations are considered to occur with the active oxidant being a perferryl oxygen (FeO3+, Compound I). However, a ferric peroxide (FeO2®, Compound 0) mechanism has been proposed, as well, particularly for aldehyde substrates. We investigated three of these systems, the oxidative deformylation of the model substrates citronellal, 2-phenylpropionaldehyde, and 2-methyl-2-phenylpropionaldehyde by rabbit P450 2B4, using 18O labeling. The formic acid product contained one 18O derived from 18O2, which is indicative of a dominant Compound 0 mechanism. The formic acid also contained only one 18O derived from H218O, which ruled out a Compound I mechanism. The possibility of a Baeyer-Villiger reaction was examined by using synthesized possible intermediates, but our data do not support its presence. Overall, these findings unambiguously demonstrate the role of the Compound 0 pathway in these aldehyde oxidative deformylation reactions.

Proteomics, modeling, and fluorescence assays delineate cytochrome b5 residues involved in binding and stimulation of cytochrome P450 17A1 17,20-lyase.

Cytochrome b5 (b5) is known to stimulate some catalytic activities of cytochrome P450 (P450, CYP) enzymes, although mechanisms still need to be defined. The reactions most strongly enhanced by b5 are the 17,20-lyase reactions of P450 17A1 involved in steroid biosynthesis. We had previously used a fluorescently labeled human b5 variant (Alexa 488-T70C-b5) to characterize human P450 17A1-b5 interactions, but subsequent proteomic analyses indicated that lysines in b5 were also modified with Alexa 488 maleimide in addition to Cys-70, due to disulfide dimerization of the T70C mutant. A series of b5 variants were constructed with Cys replacements for the identified lysine residues and labeled with the dye. Fluorescence attenuation and the function of b5 in the steroid lyase reaction depended on the modified position. Apo-b5 (devoid of heme group) studies revealed the lack of involvement of the b5 heme in the fluorescence attenuation. A structural model of b5 with P450 17A1 was predicted using AlphaFold-Multimer algorithms/Rosetta docking, based upon the individual structures, which predicted several new contacts not previously reported, that is, interactions of b5 Glu-48:17A1 Arg-347, b5 Glu-49:17A1 Arg-449, b5 Asp-65:17A1 Arg-126, b5 Asp-65:17A1 Arg-125, and b5 Glu-61:17A1 Lys-91. Fluorescence polarization assays with two modified b5 variants yielded Kd values (for b5-P450 17A1) of 120 to 380 nM, the best estimate of binding affinity. We conclude that both monomeric and dimeric b5 can bind to P450 17A1 and stimulate activity. Results with the mutants indicate that several Lys residues in b5 are sensitive to the interaction with P450 17A1, including Lys-88 and Lys-91.

Ninety-eight semesters of cytochrome P450 enzymes and related topics-What have I taught and learned?

This Reflection article begins with my family background and traces my career through elementary and high school, followed by time at the University of Illinois, Vanderbilt University, the University of Michigan, and then for 98 semesters as a Vanderbilt University faculty member. My research career has dealt with aspects of cytochrome P450 enzymes, and the basic biochemistry has had applications in fields as diverse as drug metabolism, toxicology, medicinal chemistry, pharmacogenetics, biological engineering, and bioremediation. I am grateful for the opportunity to work with the Journal of Biological Chemistry not only as an author but also for 34 years as an Editorial Board Member, Associate Editor, Deputy Editor, and interim Editor-in-Chief. Thanks are extended to my family and my mentors, particularly Profs. Harry Broquist and Minor J. Coon, and the more than 170 people who have trained with me. I have never lost the enthusiasm for research that I learned in the summer of 1968 with Harry Broquist, and I have tried to instill this in the many trainees I have worked with. A sentence I use on closing slides is "It's not just a laboratory-it's a fraternity."

Oxygen-18 Labeling Reveals a Mixed Fe-O Mechanism in the Last Step of Cytochrome P450 51 Sterol 14α-Demethylation.

The 14α-demethylation step is critical in eukaryotic sterol biosynthesis, catalyzed by cytochrome P450 (P450) Family 51 enzymes, for example, with lanosterol in mammals. This conserved three-step reaction terminates in a C-C cleavage step that generates formic acid, the nature of which has been controversial. Proposed mechanisms involve roles of P450 Compound 0 (ferric peroxide anion, FeO2 - ) or Compound I (perferryl oxygen, FeO3+ ) reacting with either the aldehyde or its hydrate, respectively. Analysis of 18 O incorporation into formic acid from 18 O2 provides a means of distinguishing the two mechanisms. Human P450 51A1 incorporated 88 % 18 O (one atom) into formic acid, consistent with a major but not exclusive FeO2 - mechanism. Two P450 51 orthologs from amoeba and yeast showed similar results, while two orthologs from pathogenic trypanosomes showed roughly equal contributions of both mechanisms. An X-ray crystal structure of the human enzyme showed the aldehyde oxygen atom 3.5 Šaway from the heme iron atom. Experiments with human P450 51A1 and H2 18 O yielded primarily one 18 O atom but 14 % of the formic acid product with two 18 O atoms, indicative of a minor contribution of a Compound I mechanism. LC-MS evidence for a Compound 0-derived Baeyer-Villiger reaction product (a 14α-formyl ester) was also found.

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Cytochrome P450 enzymes as drug targets in human disease.

Although the mention of cytochrome P450 (P450, CYP) inhibition usually brings to mind unwanted variability in pharmacokinetics, in several cases P450s are good targets for inhibition. These P450s are essential but in certain disease states it is desirable to reduce the concentrations of their products. Most of the attention to date has been with human P450s 5A1, 11A1, 11B1, 11B2, 17A1, 19A1, and 51A1. In some of those cases, there are multiple drugs in us, e.g., exemestane, letrozole, and anastrozole with P450 19A1, the steroid aromatase target in breast cancer. There are also several targets that are less developed, e. g. P450s 2A6, 8B1, 4A11, 24A1, 26A1, and 26B1. Significance Statement The selective inhibition of certain P450s that have major physiological functions has been shown to be very efficacious in certain human diseases. In several cases the search for better drugs continues.

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1).

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.

The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro.

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C.2. Compared with that seen at 37 °C, the intrinsic clearance rates (Vmax/Km) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A, .1B, .10, and .23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and .16 were decreased (79 ∼ 84%), those of CYP3A4.2 and .7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2, .7, .16, and .18 were decreased (58 ∼ 82%).3. These findings may be clinically useful for dose optimisation in patients with hypothermia or hyperthermia.

FEMA GRAS assessment of natural flavor complexes: Sage oil, Orris Root Extract and Tagetes Oil and related flavoring ingredients.

In recent years, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) has conducted a program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavor ingredients. This publication, twelfth in the series, details the re-evaluation of NFCs whose constituent profiles are characterized by alicyclic or linear ketones. In its re-evaluation, the Expert Panel applies a scientific constituent-based procedure for the safety evaluation of NFCs in commerce using a congeneric group approach. Estimated intakes of each congeneric group of the NFC are evaluated using the well-established and conservative Threshold of Toxicological Concern (TTC) approach. In addition, studies on the toxicity and genotoxicity of members of the congeneric groups and the NFCs under evaluation are reviewed. The scope of the safety evaluation of the NFCs contained herein does not include added use in dietary supplements or any products other than food. Thirteen (13) NFCs derived from the Boronia, Cinnamomum, Thuja, Ruta, Salvia, Tagetes, Hyssopus, Iris, Perilla and Artemisia genera are affirmed as generally recognized as safe (GRAS) under conditions of their intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

3,4-Desaturation of retinoic acid by cytochrome P450 27C1 prevents P450-mediated catabolism.

Cytochrome P450 (P450, CYP) 27C1 is expressed in human skin and catalyzes the 3,4-desaturation of retinoids. The enzyme has a relatively high specificity constant (kcat/Km), and ∼» of the retinoids in human skin are in the desaturated form but their function is unknown. 3,4-Dehydroretinoic acid (also didehydroretinoic acid, ddRA) has similar affinity as all-trans retinoic acid (atRA) for retinoid X and retinoic acid receptors (RXRs/RAR). The metabolism of ddRA is unknown, and we considered the hypothesis that desaturation might be a protective mechanism in maintaining active retinoid levels in the body. There are limited theoretical products that can result from ddRA oxidation. We optimized conditions for oxidation of atRA by human liver microsomes-a slow loss of atRA was seen due to 4-oxidation but no loss of ddRA was observed under the same conditions. We evaluated the HPLC peaks that were observed in microsomal incubations with ddRA using UV spectroscopy, NaBH4 and NaBD4 reduction, and mass spectrometry. None were potential ddRA oxidation products, and none were increased in the presence of the P450 cofactor NADPH. Known P450 inhibitors had no effects on the levels of these compounds. We conclude that ddRA is not readily oxidized by P450s and that one role of desaturation may be the maintenance of levels of functional retinoids.

Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1.

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4ß,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3ß-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Basis for the discrimination of supercoil handedness during DNA cleavage by human and bacterial type II topoisomerases.

To perform double-stranded DNA passage, type II topoisomerases generate a covalent enzyme-cleaved DNA complex (i.e. cleavage complex). Although this complex is a requisite enzyme intermediate, it is also intrinsically dangerous to genomic stability. Consequently, cleavage complexes are the targets for several clinically relevant anticancer and antibacterial drugs. Human topoisomerase IIα and IIß and bacterial gyrase maintain higher levels of cleavage complexes with negatively supercoiled over positively supercoiled DNA substrates. Conversely, bacterial topoisomerase IV is less able to distinguish DNA supercoil handedness. Despite the importance of supercoil geometry to the activities of type II topoisomerases, the basis for supercoil handedness recognition during DNA cleavage has not been characterized. Based on the results of benchtop and rapid-quench flow kinetics experiments, the forward rate of cleavage is the determining factor of how topoisomerase IIα/IIß, gyrase and topoisomerase IV distinguish supercoil handedness in the absence or presence of anticancer/antibacterial drugs. In the presence of drugs, this ability can be enhanced by the formation of more stable cleavage complexes with negatively supercoiled DNA. Finally, rates of enzyme-mediated DNA ligation do not contribute to the recognition of DNA supercoil geometry during cleavage. Our results provide greater insight into how type II topoisomerases recognize their DNA substrates.

FEMA GRAS assessment of natural flavor complexes: Lemongrass oil, chamomile oils, citronella oil and related flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, eleventh in the series, evaluates the safety of NFCs characterized by primary alcohol, aldehyde, carboxylic acid, ester and lactone constituents derived from terpenoid biosynthetic pathways and/or lipid metabolism. The scientific-based evaluation procedure published in 2005 and updated in 2018 that relies on a complete constituent characterization of the NFC and organization of the constituents into congeneric groups. The safety of the NFCs is evaluated using the threshold of toxicological concern (TTC) concept in addition to data on estimated intake, metabolism and toxicology of members of the congeneric groups and for the NFC under evaluation. The scope of the safety evaluation does not include added use in dietary supplements or any products other than food. Twenty-three NFCs, derived from the Hibiscus, Melissa, Ricinus, Anthemis, Matricaria, Cymbopogon, Saussurea, Spartium, Pelargonium, Levisticum, Rosa, Santalum, Viola, Cryptocarya and Litsea genera were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of derivatives of basil, nutmeg, parsley, tarragon and related allylalkoxybenzene-containing natural flavor complexes.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a program for the re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients in food. In this publication, tenth in the series, NFCs containing a high percentage of at least one naturally occurring allylalkoxybenzene constituent with a suspected concern for genotoxicity and/or carcinogenicity are evaluated. In a related paper, ninth in the series, NFCs containing anethole and/or eugenol and relatively low percentages of these allylalkoxybenzenes are evaluated. The Panel applies the threshold of toxicological concern (TTC) concept and evaluates relevant toxicology data on the NFCs and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s), the estimated intake of the constituent is compared to the TTC for compounds with structural alerts for genotoxicity and when exceeded, a margin of exposure (MOE) is calculated. BMDL10 values are derived from benchmark dose analyses using Bayesian model averaging for safrole, estragole and methyl eugenol using EPA's BMDS software version 3.2. BMDL10 values for myristicin, elemicin and parsley apiole were estimated by read-across using relative potency factors. Margins of safety for each constituent congeneric group and MOEs for each allylalkoxybenzene constituent for each NFC were determined that indicate no safety concern. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food. Ten NFCs, derived from basil, estragon (tarragon), mace, nutmeg, parsley and Canadian snakeroot were determined or affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

FEMA GRAS assessment of natural flavor complexes: Allspice, anise, fennel-derived and related flavoring ingredients.

The FEMA Expert Panel program to re-evaluate the safety of natural flavor complexes (NFCs) used as flavoring ingredients in food has resulted in the publication of an updated constituent-based procedure as well as publications on the safety evaluation of many botanical-derived NFCs. This publication, ninth in the series and related to the ninth publication, describes the affirmation of the generally recognized as safe (GRAS) status for NFCs with propenylhydroxybenzene and allylalkoxybenzene constituents under their conditions of intended use as flavoring ingredients added to food. The Panel's procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology for the NFCs themselves and their respective constituent congeneric groups. For NFCs containing allylalkoxybenzene constituent(s) with suspected genotoxic potential, the estimated intake of the individual constituent is compared to the TTC for compounds with structural alerts for genotoxicity and if exceeded, a margin of exposure is calculated using BMDL10 values derived from benchmark dose analyses using Bayesian model averaging, as presented in the tenth article of the series. Safety evaluations for NFCs derived from allspice, anise seed, star anise, sweet fennel seed and pimento leaves were conducted and their GRAS status was affirmed for use as flavoring ingredients. The scope of the safety evaluation contained herein does not include added use in dietary supplements or any products other than food.