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

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

The FEMA GRAS assessment of phenethyl alcohol, aldehyde, acid, and related acetals and esters used as flavor ingredients.

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.

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

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.

The FEMA GRAS assessment of hydroxy- and alkoxy-substituted benzyl derivatives used as flavor ingredients.

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.

A procedure for the safety evaluation of natural flavor complexes used as ingredients in food: essential oils.

A scientifically based guide has been developed to evaluate the safety of naturally occurring mixtures, particularly essential oils, for their intended use as flavor ingredients. The approach relies on the complete chemical characterization of the essential oil and the variability of the composition of the oil in the product intended for commerce. Being products of common plant biochemical pathways, the chemically identified constituents are organized according to a limited number of well-established chemical groups called congeneric groups. The safety of the intake of the each congeneric group from consumption of the essential oil is evaluated in the context of data on absorption, metabolism, and toxicology of members of the congeneric group. The intake of the group of unidentified constituents is evaluated in the context of the consumption of the essential oil as a food, a highly conservative toxicologic threshold, and toxicity data on the essential oil or an essential oil of similar chemotaxonomy. The flexibility of the guide is reflected in the fact that high intake of major congeneric groups of low toxicologic concern will be evaluated along with low intake of minor congeneric groups of significant toxicological concern (i.e., higher structural class). The guide also provides a comprehensive evaluation of all congeneric groups and constituents that account for the majority of the composition of the essential oil. The overall objective of the guide is to organize and prioritize the chemical constituents of an essential oil in order that no reasonably possible significant risk associated with the intake of essential oil goes unevaluated. The guide is, however, not intended to be a rigid checklist. The Flavor and Extract Manufacturers Association (FEMA) Expert Panel will continue to evaluate each essential oil on a case by case basis applying their scientific judgment to insure that each natural flavor complex is exhaustively evaluated.

Safety evaluation of natural flavour complexes.

Natural flavour complexes (NFCs) are chemical mixtures obtained by applying physical separation methods to botanical sources. Many NFCs are derived from foods. In the present paper, a 12-step procedure for the safety evaluation of NFCs, 'the naturals paradigm', is discussed. This procedure, which is not intended to be viewed as a rigid check list, begins with a description of the chemical composition of the commercial product, followed by a review of the data on the history of dietary use. Next, each constituent of an NFC is assigned to one of 33 congeneric groups of structurally related substances and to one of three classes of toxic potential, each with its own exposure threshold of toxicological concern. The group of substances of unknown structure is placed in the class of greatest toxic potential. In subsequent steps, for each congeneric group the procedure determines the per capita intake, considers metabolic pathways and explores the need and availability of toxicological data. Additional toxicological and analytical data may be required for a comprehensive safety evaluation. The procedure concludes with an evaluation of the NFC in its entirety, also considering combined exposure to congeneric groups. The first experiences with the use of this procedure are very promising. Future safety evaluations of larger numbers of NFCs will indicate the usefulness of the system, either in its present form or in a form modified on the basis of experience.

Rodent toxicity and nongenotoxic carcinogenesis: knowledge-based human risk assessment based on molecular mechanisms.

It is necessary to determine whether chemicals or drugs have the potential to pose a threat to human health. Chemicals that can damage DNA are detected in short-term assays, but the detection of nongenotoxic carcinogens relies upon bioassays in laboratory animals. However, there are marked differences between rodents and humans in response to nongenotoxic carcinogens, which makes the relevance of rodent data to human risk assessment questionable. Here, we address the background issues concerning rodent nongenotoxic carcinogenesis and then focus upon peroxisome proliferators, chloroform, and dioxins as examples of toxicants that cause rodent-specific oxidative stress, cell proliferation, and the suppression of apoptosis. In the case of peroxisome proliferators and dioxins, this response is receptor-mediated. The evidence presented suggests that, at least for some toxicants, the molecular mechanisms of the rodent carcinogenic responses do not operate in humans; this is discussed in the context of human risk assessment. Finally, consideration is given to incorporating mechanism-based information into risk assessment for regulatory purposes.

Safety assessment of allylalkoxybenzene derivatives used as flavouring substances - methyl eugenol and estragole.

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.

The FEMA GRAS assessment of pyrazine derivatives used as flavor ingredients. Flavor and Extract Manufacturers Association.

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.

A perspective on current and future uses of alternative models for carcinogenicity testing.

This perspective is based upon the data presented at the International Life Sciences Institute (ILSI), Health and Environmental Sciences Institute Workshop on the Evaluation of Alternative Methods for Carcinogenicity Testing (ILSI Workshop). It is important to understand that all models discussed at the Workshop have limitations and that they are not designed to be employed as stand-alone assays. Although they may have other, appropriate applications. I do not recommend use of the SHE cell assay and the Tg.AC model for the regulatory purposes of a safety assessment. In my view, the neonatal mouse, p53+/-, XPA-/-, XPA-/- and p53+/-, and the rasH2 models can, as a component of an overall assessment, provide information on potential carcinogenicity of a chemical that is appropriate for consideration in a regulatory context. Generally, these models exhibit the ability to detect genotoxic compounds. In most cases these compounds would be detected in a standard battery of genotoxicity tests and, therefore, quite often the use of an alternative is not necessary. Actually, I believe that a bioassay in rats will suffice most of the time, that is, in my view, a routine bioassay in mice is not necessary. Specific circumstances where data obtained from one of the "recommended" alternative models might be helpful are discussed. With regard to lessons for the future, there is a particular need for models that are responsive to chemicals that exhibit a nongenotoxic mode of action. Additionally, new models will continue to be developed and their half-life will likely be substantially shorter than the time required for traditional validation. The development of enhanced paradigms for validation should be a priority so that improved safety assessment decisions can be made more quickly. However, while evaluating and validating such models, it is important to consider the fundamental issues, for example, rational dose selection, evaluation of mode of action in the context of dose-response relationships including the existence of thresholds and secondary mechanisms, and species-to-species extrapolation. The alternatives to carcinogenicity testing project was a very major undertaking. In addition to the valuable information provided, it serves to illustrate the value of cooperation between academia, government, and industry. Furthermore, the involvement of the International Life Sciences Institute as the overall organizing, facilitating umbrella was crucial for the success of the project.

Operational reversibility is a key aspect of carcinogenesis.

The article highlighted in this issue is "Reversibility and Persistence of Di-2-Ethylhexyl Phthalate (DEHP)- and Phenobarbital-Induced Hepatocellular Changes In Rodents," by Jason S. Isenberg, Lisa M. Kamendulis, David C. Ackley, Jacqueline H. Smith, George Pugh, Jr., Arthur W. Lington, Richard H. McKee, and James E. Klaunig (pp. 192-199).

Repeated inhalation exposure to octamethylcyclotetrasiloxane produces hepatomegaly, transient hepatic hyperplasia, and sustained hypertrophy in female Fischer 344 rats in a manner similar to phenobarbital.

Octamethylcyclotetrasiloxane (D4) has been described as a phenobarbital-like inducer of hepatic enzymes. Phenobarbital (PB) and phenobarbital-like chemicals induce transient hepatic and thyroid hyperplasia and sustained hypertrophy in rats and mice. The extent to which these processes are involved with D4-induced hepatomegaly is not known. The present study has evaluated the effects of repeated inhalation exposure to D4 vapors on hepatic and thyroid cell proliferation and hypertrophy with respect to time and exposure concentration. Female Fischer 344 rats were exposed via whole body inhalation to 0 ppm D4, 700 ppm D4 vapors (6 h/day; 5 days/week), or 0.05% PB in drinking water over a 4-week period. Incorporation of 5'-bromo-2-deoxyuridine (BrdU) and the abundance of proliferating cell nuclear antigen were used as indicators of cell proliferation. Designated animals from each treatment group were euthanized on study days 6, 13, and 27. The effect of D4 exposure concentration on hepatic cell proliferation was evaluated at 0, 7, 30, 70, 150, 300, or 700 ppm. Liver-to-body weight ratios in animals exposed to 700 ppm D4 were increased 18, 20, and 22% over controls while PB-treated animals showed increases of 33, 27, and 27% over controls on days 6, 13, and 27 respectively. Hepatic incorporation of BrdU following exposure to D4 was highest on day 6 (labeling index = 15-22%) and was at or below control values by day 27. This pattern of transient hyperplasia was observed in all hepatic lobes examined and was similar to the pattern observed following treatment with PB.

Reevaluation of the cancer potency factor of toxaphene: recommendations from a peer review panel.

This reevaluation of the current U.S. EPA cancer potency factor for toxaphene is based upon a review of toxaphene carcinogenesis bioassays in mice conducted by Litton Bionetics (unpublished report, 1978) and the National Cancer Institute (NCI) (Technical Report Series No. 37, conducted by Gulf South Research Institute, 1979). The mechanistic data available for toxaphene, including consideration of the potential of the compound to induce genotoxicity, was examined with an emphasis on whether this information supports a change in the cancer potency factor. If a quantitative dose-response assessment for toxaphene is to be performed, the data from both the NCI and Litton cancer bioassays should be used. Additionally, liver tumor results from female mice, rather than male mice, should be used for estimating potential human cancer risk because the background rate of liver tumors in females is lower and less variable than that exhibited by males. An ED(10) was estimated as the point of departure. The mechanistic data were not sufficient to fully support a margin of exposure approach. Therefore, we believe that applying a linear extrapolation from the ED(10) to the origin is an appropriate means to estimate risk at low doses. This is a highly conservative approach and, when it is applied, we conclude that the current EPA cancer potency factor should be reduced from 1.1 (mg/kg/day)(-1) to 0.1 (mg/kg/day)(-1).

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Response to "Epigenetic mechanisms of carcinogenesis" by Klaunig JE, Kamendulis LM and Xu Y.

It is now quite clear that carcinogenesis involves more than mutagenesis and an increased focus on epigenetic events underlying the transformation of a normal cell into a frank malignancy is appropriate.

The FEMA GRAS assessment of trans-anethole used as a flavouring substance. Flavour and Extract Manufacturer's Association.

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.

Evaluation of methylated DNA binding protein-1 in mouse liver.

DNA methylation (DNA-5-methylcytosine [MeC]) plays a key role in regulation of gene expression. Highly methylated genes tend to be silenced whereas hypomethylated genes have an increased potential for expression. One way in which methylation may lead to inhibition of transcription is by permitting the binding of methylated DNA-binding proteins, which then block access of transcription factors to DNA. A particular methylated DNA-binding protein, MDBP-1, has been identified in nuclear extracts prepared from various human and rat tissues, and binds in a sequence-specific manner to DNA containing methylated cytosines (P. C. Supakar et al., 1988, Nucleic Acids Res. 16, 8029-8044). In the current study, an electrophoretic mobility shift assay (EMSA) was employed to assess the presence of MDBP-1 in protein isolated from mouse liver and to examine characteristics of its binding to DNA. The ligand used in our EMSA was a 32P-end-labeled, double-stranded, symmetrically methylated oligonucleotide containing the protein's binding site, 5'-CTAGATMGT-CAMGGMGAT-3' (M denotes 5-methyl-cytosine). Utilizing protein extracted from B6C3F1 mouse liver, we observed a complex that is competed by non-labeled, double-stranded, fully methylated ligand but not by an excess of a 26-mer, double-stranded oligonucleotide containing the binding site for AP-1. No complex was formed when a nonmethylated, double-stranded ligand, or our single-stranded ligands (methylated or unmethylated) were used as EMSA ligands. Complex formation was observed utilizing double-stranded, hemimethylated ligands; however, the affinity of MDBP-1 for these was one-tenth the affinity of MDBP-1 for fully methylated, double-stranded ligand. Additionally, protein isolated from C57BL/6 and C3H/He mouse liver was found to bind specifically to fully methylated ligand and hemimethylated ligands for MDBP-1, with similar characteristics as the binding of B6C3F1 mouse liver protein. These results indicate that MDBP-1 is present in mouse liver, and that the degree of methylation determines the strength of binding of MDBP-1 to DNA.