The safety evaluation of food flavoring substances: the role of genotoxicity studies.

The Flavor and Extract Manufacturers Association (FEMA) Expert Panel relies on the weight of evidence from all available data in the safety evaluation of flavoring substances. This process includes data from genotoxicity studies designed to assess the potential of a chemical agent to react with DNA or otherwise cause changes to DNA, either in vitro or in vivo. The Panel has reviewed a large number of in vitro and in vivo genotoxicity studies during the course of its ongoing safety evaluations of flavorings. The adherence of genotoxicity studies to standardized protocols and guidelines, the biological relevance of the results from those studies, and the human relevance of these studies are all important considerations in assessing whether the results raise specific concerns for genotoxic potential. The Panel evaluates genotoxicity studies not only for evidence of genotoxicity hazard, but also for the probability of risk to the consumer in the context of exposure from their use as flavoring substances. The majority of flavoring substances have given no indication of genotoxic potential in studies evaluated by the FEMA Expert Panel. Examples illustrating the assessment of genotoxicity data for flavoring substances and the consideration of the factors noted above are provided. The weight of evidence approach adopted by the FEMA Expert Panel leads to a rational assessment of risk associated with consumer intake of flavoring substances under the conditions of use.

Assessment of FD&C Yellow No. 6 (Sunset Yellow FCF) effects on sperm count, motility and viability in the rat in a 28-day toxicity study.

Sunset Yellow FCF was tested for 28-days in male Hsd:SD® rats for its potential effect on sperm quality parameters at dietary concentrations of 6,000, 12,000 and 18,000 ppm, corresponding to target doses of 500, 1000, and 1500 mg/kg bw/day. The measured average daily intake was 490, 944, and 1,475 mg/kg bw/day, based on feed consumption and stability of Sunset Yellow FCF in the diet. The animals fed diets with Sunset Yellow FCF presented no clinical signs of toxicity and no differences in feed consumption, body weights, organ weights, ophthalmology, hematology, clinical chemistry, urinalysis, or coagulation parameters that were considered adverse. No mortality or abnormalities were observed at necropsy, and no microscopic changes were observed in histopathology. Increased testes weights relative to body weight in animals of the middle and high intake groups were not associated with any abnormal findings in histopathology. Sperm quality evaluation presented no adverse effects on sperm motility, epididymal sperm count, homogenization-resistant spermatid count, or sperm morphological development. Therefore, in the absence of any adverse effects under the conditions of this study, the NOAEL for Sunset Yellow FCF was 1,475 mg/kg bw/day in male rats, corresponding to 18,000 ppm in the diet.

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.

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 natural flavor complexes: Asafetida oil, garlic oil and onion oil.

The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) applies its procedure for the safety evaluation of natural flavor complexes (NFCs) to re-evaluate the safety of Asafetida Oil (Ferula assa-foetida L.) FEMA 2108, Garlic Oil (Allium sativum L.) FEMA 2503 and Onion Oil (Allium cepa L.) FEMA 2817 for use as flavoring in food. This safety evaluation is part of a series of evaluations of NFCs for use as flavoring ingredients conducted by the Expert Panel that applies a scientific procedure published in 2005 and updated in 2018. Using a group approach that relies on a complete chemical characterization of the NFC intended for commerce, the constituents of each NFC are organized into well-defined congeneric groups and the estimated intake of each constituent congeneric group is evaluated using the conservative threshold of toxicological concern (TTC) concept. Data on the metabolism, genotoxic potential and toxicology for each constituent congeneric group are reviewed as well as studies on each NFC. Based on the safety evaluation, Asafetida Oil (Ferula assa-foetida L.), Garlic Oil (Allium sativum L.) and Onion Oil (Allium cepa L.) were affirmed as generally recognized as safe (GRASa) under their conditions of intended use as flavor ingredients.

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.

FEMA GRAS assessment of natural flavor complexes: Eucalyptus oil and other cyclic ether-containing 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, the sixth in the series, will summarize the re-evaluation of eight NFCs whose constituent profiles are characterized by significant amounts of eucalyptol and/or other cyclic ethers. This re-evaluation was based on a procedure first published in 2005 and subsequently updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and the organization of its chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of the constituents of the congeneric groups and the NFC under evaluation. Eight NFCs derived from the Eucalyptus, Melaleuca, Origanum, Laurus, Rosmarinus and Salvia 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 natural flavor complexes: Origanum oil, thyme oil and related phenol derivative-containing flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients, mostly consisting of a variety of essential oils and botanical extracts. This publication, seventh in the series, re-evaluates NFCs with constituent profiles dominated by phenolic derivatives including carvacrol, thymol and related compounds using a constituent-based procedure first published in 2005 and updated in 2018. The procedure is based on the chemical characterization of each NFC as intended for commerce and the estimated intake of the constituent congeneric groups. The procedure applies the threshold of toxicological concern (TTC) concept and evaluates relevant data on absorption, metabolism, genotoxic potential and toxicology of the constituent congeneric groups and the NFC under evaluation. Herein, the FEMA Expert Panel affirmed the generally recognized as safe (GRAS) status of seven phenolic derivative-based NFCs, Origanum Oil (Extractive) (FEMA 2828), Savory Summer Oil (FEMA 3013), Savory Summer Oleoresin (FEMA 3014), Savory Winter Oil (FEMA 3016), Savory Winter Oleoresin (FEMA 3017), Thyme Oil (FEMA 3064) and Thyme White Oil (FEMA 3065) under their conditions of intended use as flavor ingredients.

Dietary administration of ß-ionone epoxide to Sprague-Dawley rats for 90 days.

In a 90-day GLP-compliant study groups of Sprague-Dawley rats (10/sex/group) were fed diets containing ß-ionone epoxide, a fragrance material and a flavoring substance, at dietary concentrations providing target intakes of 0, 20, 40 and 80 mg/kg bw/day. There were no deaths and no adverse changes in clinical observations, ophthalmological examinations, body weight, body weight gain, food consumption, food efficiency; hematology, serum chemistry, urinalysis parameters; or in macroscopic findings attributable to ß-ionone epoxide administration. Increased absolute and relative liver weights in high dose females without correlating hepatic histopathological findings were considered non-adverse. Cortical vacuolation of adrenal zona fasciculata was observed in high-dose males but was considered non-adverse due to the nondegenerative nature of this alteration. ß-Ionone epoxide did not influence estrus cyclicity in females and did not affect sperm morphology or epididymal sperm count, homogenization-resistant spermatid count and motility measurements in male rats. The no-observed-adverse-effect level (NOAEL) for administration of ß-ionone epoxide in the diet was determined to be the highest dose tested of 80 mg/kg bw/day.

A chemical structure-based approach for estimating the added levels of flavourings to foods for the purpose of assessing consumer intake.

Intake assessment and hazard profile of chemical substances are the two critical inputs in a safety assessment. Human intake assessment presents challenges that stem either from the absence of data or from numerous sources of variability and uncertainty, which have led regulators to adopt conservative approaches that inevitably overestimate intake. Refinements of intake assessments produce more realistic estimates and help prioritise areas of concern and better direct investment of resources. However, use levels (ULs), which represent the usual added amount of flavourings to food products, are the starting point for refined intake assessments, are data-intensive, and data availability is often a limitation. The work presented here was undertaken to investigate the use level patterns of substances used as flavourings in foods and to develop a systematic tool for data extrapolation based on chemical structure. The available dataset consists of use levels reported through eight industry surveys and hence are representative of industry uses rather than regulatory limits, which are higher by design and not realistic. A systematic statistical analysis was undertaken to determine whether the industry-reported UL data can be used to estimate use levels of flavouring substances belonging to the same chemical group for which such data are not available. Predictive modelling approaches were explored to evaluate relationships in the data and utilised additional variables relevant to technological considerations, such as volatility losses upon heat treatment, and Tanimoto index-based pair-wise structural similarity scores to determine whether more granular similarity information can reduce the within-group variability. The analyses indicated that the use levels of flavouring substances can reasonably be estimated based on the available data using chemical group classifications stratified by food category. Source of uncertainty and limitations are discussed.

FEMA GRAS assessment of natural flavor complexes: Cinnamomum and Myroxylon-derived 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, third in the series, considers NFCs composed primarily of constituents with the 3-phenyl-2-propenyl or a cinnamyl functional group, using the procedure outlined in 2005 and updated in 2018 to evaluate the safety of naturally-occurring mixtures for their intended use as flavor ingredients. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of each NFC's chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of members of the congeneric groups and the NFC under evaluation. Six NFCs from the Myroxylon and Cinnamomum genera, Balsam Oil, Peru (FEMA 2117), Tolu Balsam Extract (FEMA 3069), Cassia Bark Extract (FEMA 2257), Cassia Bark Oil (FEMA 2258), Cinnamon Bark Extract (FEMA 2290) and Cinnamon Bark Oil (FEMA 2291) were evaluated and affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavor ingredients.

FEMA GRAS assessment of natural flavor complexes: Clove, cinnamon leaf and West Indian bay leaf-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association initiated the safety re-evaluation of over 250 natural flavor complexes (NFCs) used as flavor ingredients. This publication, 4th in a series focusing on the safety evaluation of NFCs, presents an evaluation of NFCs rich in hydroxyallylbenzene and hydroxypropenylbenzene constituents using a procedure initially published in 2005 and updated in 2018 that evaluates the safety of naturally occurring mixtures for their intended use as flavoring ingredients. The procedure requires the characterization of the chemical composition for each NFC and subsequent organization of the constituents into defined congeneric groups. The safety of each NFC is evaluated using the conservative threshold of toxicological concern (TTC) approach together with studies on absorption, metabolism and toxicology of the NFC and its constituent congeneric groups. By the application of this procedure, seven NFCs, derived from clove, cinnamon leaf and West Indian bay leaf were affirmed as "generally recognized as safe (GRAS)" under their conditions of intended use as flavor ingredients. An eighth NFC, an oleoresin of West Indian bay leaf, was affirmed based on its estimated intake, which is below the TTC of 0.15 µg/person per day for compounds with structural alerts for genotoxicity.

FEMA GRAS assessment of natural flavor complexes: Lavender, Guaiac Coriander-derived 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, fifth in the series, evaluates the safety of NFCs containing linalool and/or other characteristic mono- and sesquiterpenoid tertiary alcohols and esters using the safety evaluation procedure published by the FEMA Expert Panel in 2005 and updated in 2018. The procedure relies on a complete chemical characterization of the NFC intended for commerce and organization of the chemical constituents of each NFC into well-defined congeneric groups. The safety of each NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of both the constituent congeneric groups and the NFCs. Sixteen NFCs, derived from the Lavandula, Aniba, Elettaria, Daucus, Salvia, Coriandrum, Ribes, Guaiacum/Bulnesia, Citrus, Pogostemon, Melaleuca and Michelia 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 natural flavor complexes: Mint, buchu, dill and caraway derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavor ingredients. NFC flavor materials include a variety of essential oils and botanical extracts. The re-evaluation of NFCs is conducted based on a constituent-based procedure outlined in 2005 and updated in 2018 that evaluates the safety of NFCs for their intended use as flavor ingredients. This procedure is applied in the re-evaluation of the generally recognized as safe (GRAS) status of NFCs with constituent profiles that are dominated by alicyclic ketones such as menthone and carvone, secondary alcohols such as menthol and carveol, and related compounds. The FEMA Expert Panel affirmed the GRAS status of Peppermint Oil (FEMA 2848), Spearmint Oil (FEMA 3032), Spearmint Extract (FEMA 3031), Cornmint Oil (FEMA 4219), Erospicata Oil (FEMA 4777), Curly Mint Oil (FEMA 4778), Pennyroyal Oil (FEMA 2839), Buchu Leaves Oil (FEMA 2169), Caraway Oil (FEMA 2238) and Dill Oil (FEMA 2383) and determined FEMA GRAS status for Buchu Leaves Extract (FEMA 4923), Peppermint Oil, Terpeneless (FEMA 4924) and Spearmint Oil, Terpeneless (FEMA 4925).

Dietary administration of ß-caryophyllene and its epoxide to Sprague-Dawley rats for 90 days.

Two independent 90-day GLP-compliant studies were conducted in Sprague-Dawley rats with ß-caryophyllene or ß-caryophyllene epoxide, two common flavoring and fragrance materials. Dietary concentrations of ß-caryophyllene were 3500; 7000; and 21,000 ppm for males and 3500; 14,000; and 56,000 ppm for females. Dietary concentrations of ß-caryophyllene epoxide were 1750; 10,500; and 21,000 ppm. There were no deaths or clinical toxicity attributable to either substance administration. Statistically significant, dose-dependent reductions in body weight, body weight gain, food consumption, and food efficiency at the highest dietary concentrations of ß-caryophyllene, but not of ß-caryophyllene epoxide, were attributed to palatability issues. Neither ß-caryophyllene nor ß-caryophyllene epoxide influenced estrus cyclicity or sperm parameters. Macroscopic and microscopic findings were primarily related to changes in the kidneys of male rats, consistent with α2u-globulin nephropathy, and in the liver of male and female rats, including hepatocyte hypertrophy at the middle and high intake levels. These changes correlated with increased absolute and relative organ weights. Since the kidney findings were a species- and sex-specific effect, the NOAEL in each study was based on hepatocyte hypertrophy at the two highest dietary concentrations and were determined to be 222 mg/kg bw/day for ß-caryophyllene and 109 mg/kg bw/day for ß-caryophyllene epoxide.

FEMA GRAS assessment of natural flavor complexes: Citrus-derived flavoring ingredients.

In 2015, the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) initiated a re-evaluation of the safety of over 250 natural flavor complexes (NFCs) used as flavoring ingredients. This publication is the first in a series and summarizes the evaluation of 54 Citrus-derived NFCs using the procedure outlined in Smith et al. (2005) and updated in Cohen et al. (2018) to evaluate the safety of naturally-occurring mixtures for their intended use as flavoring ingredients. The procedure relies on a complete chemical characterization of each NFC intended for commerce and organization of each NFC's chemical constituents into well-defined congeneric groups. The safety of the NFC is evaluated using the well-established and conservative threshold of toxicological concern (TTC) concept in addition to data on absorption, metabolism and toxicology of members of the congeneric groups and the NFC under evaluation. As a result of the application of the procedure, 54 natural flavor complexes derived from botanicals of the Citrus genus were affirmed as generally recognized as safe (GRAS) under their conditions of intended use as flavoring ingredients based on an evaluation of each NFC and the constituents and congeneric groups therein.

2,4-Decadienal does not induce genotoxic effects in in vivo micronucleus studies.

2,4-Decadienal (E,E-) occurs naturally in foods and is also used as a flavoring ingredient. In vivo micronucleus studies were used to evaluate the potential for 2,4-decadienal to cause genotoxic effects. Male Han Wistar rats were dosed either by intraperitoneal injection or by gavage in two independent studies. The animals (12/group) received 25, 50, or 100 mg/kg bw of 2,4-decadienal via intraperitoneal injection, or 350, 700, or 1400 mg/kg bw via gavage. Dose-dependent decreases in the percentages of peripheral blood reticulocytes were observed in both studies, indicating that the target tissue was exposed to toxic levels of 2,4-decadienal. No induction of micronuclei in the bone marrow polychromatic erythrocytes or the peripheral blood reticulocytes was observed in either study. These results, coupled with previous mutagenicity studies, support the overall conclusion that 2,4-decadienal does not present a concern for genotoxicity.

Letter to the Editor in response to "Our unrequited love for natural ingredients," by Burdock and Wang.

This letter is in response to the review article "Our unrequited love for natural ingredients" authored by Burdock and Wang in the September edition of FCT. Our purpose is to clarify the procedure used by the FEMA Expert Panel for the safety evaluation of natural flavor complexes, as well as to note and/or correct inaccuracies that we have identified within the article.

Updated procedure for the safety evaluation of natural flavor complexes used as ingredients in food.

An effective and thorough approach for the safety evaluation of natural flavor complexes (NFCs) was published in 2005 by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). An updated procedure is provided here, which maintains the essential concepts of the use of the congeneric group approach and the reliance on the threshold of toxicological concern (TTC) concept. The updated procedure emphasizes more rigorous considerations of unidentified constituents and the genotoxic potential of constituents. The update of the previously established procedure is the first step in a multi-year project to conduct safety re-evaluations for more than 250 NFCs that have uses that are currently considered Generally Recognized as Safe (GRAS) by the FEMA Expert Panel. In addition, this procedure can be more generally employed in the safety evaluation of NFCs.