51 Safety evaluation of natural flavour complexes

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Workshop W5. Safety evaluation of flavourings

programme a project on Food Safety in Europe (FOSIE): Risk Assessment of Chemicals in Food and Diet. One expert group within this project carried out a thorough and comprehensive investigation on the assessment of intake from the diet. In the case of chemicals in food this is based on three major aspects: (i) how to determine quantitatively the presence of a chemical in individual foods and diets including its fate during the processes within the food production chain; (ii) how to determine the consumption patterns of the individual foods containing the relevant chemicals and (iii) how to investigate both the likelihood of consumers eating large amounts of the given foods and of the relevant chemical being present in these foods at high levels. A critical review of the techniques used for the evaluation of these three aspects has been undertaken to determine those areas where the current approaches provide a solid basis for assessments and those areas where improvements are needed. In all three areas, the limitations of the approaches currently used lead to uncertainties, which can either cause an over- or underestimation of real intakes and thus risks. The expert group recommends using worst-case scenarios by applying conservative screening methods as a first step. In cases where these relatively crude tools predict a toxicologically significant exposure more sophisticated methods are needed to provide more accurate intake estimates.

W5 Safety evaluation of flavourings 49

THE SAFETY EVALUTATION OF FLAVOURS: CHALLENGES AND APPROACHES

Robert L. Smith. Biological Chemistry & Molecular Toxicology, Imperial College London, United Kingdom, SW7 2AZ There is nowadays general agreement that the conventional toxicological approaches are inappropriate for the safety evaluation of food flavour materials. The reasons for this are: (a) the large number of flavour substances in commercial use; massive resources would be needed should the evaluations be conducted using conventional procedures (b) the use-levels of many flavour substances amount to a few kg/year and there is inadequate economic base to support conventional assessment (c) human levels of exposure through the food are generally very low and self-limiting (d) there is a history of exposure to many naturally occurring flavour materials through food consumption (e) the majority of flavours belong to simple chemical classes devoid of ’structural alerts for toxicity’ although there are a few exceptions to this. With these facts in mind, several governmental (e.g. U.S.FDA, Europeans S.C.F. and JECFA) and non-governmental (FEMA Expert Panel) agencies have explored and developed non-traditional paradigms for the safety evaluation of food flavours. There is now a high degree of commonality in the various approaches but there are also some differences. These approaches take advantage of the fact that the majority of flavours belong to well-defined chemical groups where there exists a reasonable homology in terms of toxicity and metabolic fate. The goal of this symposium is to explore some of these differences and, in particular, to review the different ways at evaluating exposure and also to consider the applicability of some more recently derived concepts for assessing flavour safety. The key questions to be addressed are as follows: Assessment of exposure to flavours: What are the most appropriate methodologies; are there sub-groups of exposed people that require special attention? Thresholds and their utility: Are there robust thresholds for toxicity/carcinogenicity/saturation of metabolic pathways and metabolic switching and can these be used for safety assessment purposes? How can the safety of natural flavour complexes be evaluated: There are about 400 of these materials in current use as flavours; is there a need for a new paradigm for these materials?

1. Assessment of exposure to flavouring agents and natural flavour mixtures 50

NOVEL ESTIMATES OF THE EXPOSURE TO FLAVOURING SUBSTANCES

P. Cadby. Department of Product Safety and Regulatory Affairs, Firmenich SA, Geneva, Switzerland There are thousands of flavouring substances and hundreds of ways of consuming them. It is consequently impossible to carry out detailed analysis of the consumption patterns of each one, so as a result, it is necessary to find more practical and conservative methods for assessing exposure. Two studies have compared the suitability of one method: the Maximised Survey-derived Daily Intake (MSDI). In one of these studies, the MSDI-estimated intakes of 9 flavouring substances and one spice oleoresin were compared with detailed dietary intakes calculated over a 14 day period using frequency of eating, portion sizes, levels of the flavouring substance and probability of presence in a particularly food item. In a second study, the MSDI-estimated intakes of 12 flavouring substances were compared with intakes calculated using a stochastic model. This model used real levels in over 40,000 flavour formulae used in 31 different categories of food for which the intakes of males in the 16–24 year age group had been surveyed. In both of these studies, the ability of the MSDI to accurately but conservatively estimate upper percentile intakes was demonstrated. This stochastic method offers an opportunity to test whether the MSDI can also be used to estimate the intakes of the volatile constituents that are common to a number of different botanically-derived flavouring materials and food ingredients. 51

SAFETY EVALUATION OF NATURAL FLAVOUR COMPLEXES

V.J. Feron 1 , T.B. Adams 2 , S. Cohen 3 , J. Doull 4 , J.I. Goodman 5 , R.L. Hall 6 , L.J. Marnett 7 , I.C. Munro 8 , P.S. Portoghese 9 , R.L. Smith 10 , W.J. Waddell 11 , B.M. Wagner 12 . 1 TNO Nutrition and Food Research, Zeist, The Netherlands, 2 Scientific Secretary of the FEMA Panel Flavor & Extract Manufacturers Association, Washington, DC, USA, 3 Nebraska Medical Center, University of Nebraska, Omaha, NB, USA, 4 Dept. of Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, KS, USA, 5 Dept. of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA, 6 Towson, MD, USA, 7 Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA,8 CanTox Health Sciences International, Mississauga, ON, Canada, 9 Dept. of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA, 10 Div. of Biomedical Sciences, Section of Molecular Toxicology, Imperial College School of Medicine, South Kensington, London, UK, 11 Dept. of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA, 12 New York University School of Medicine, New York, B.M. Wagner Associates, Milburn, NJ, USA Natural flavour complexes (NFCs) are chemical mixtures obtained by applying physical separation methods to botanical sources such as pulp, peel, vegetables and spices. Many of the approximately 300 NFCs have a food origin. To date no scheme that allows the safety evaluation of NFCs has been developed by any agency, governmental or non-governmental. In this presentation, such a scheme, “The Naturals Paradigm”, will be discussed. The scheme is intended only for the safety evaluation of NFCs derived from higher plants to be used as flavouring substances for food and beverages. The scheme begins with a review of data on the history of dietary use of the NFC, and describes its chemical composition to ensure that the commercial product conforms to the composition limits that define the product. Next the paradigm assigns each known constituent to one of three structural classes of toxic potential, each class with its own exposure threshold of toxicological concern. Moreover, each constituent is assigned to one of some 30 congeneric groups. In subsequent steps, for each congeneric group

Symposium S5. Molecular epidemiology in occupational toxicology the method determines the daily per capita intake, considers the detoxification pathways and explores the availability of toxicological data. Toxicity testing may be needed. The paradigm also addresses constituents of unknown structure. As a conservative default assumption, the unknowns are placed in the structural class of greatest toxic potential and, thus, their total intake is compared to the most conservative threshold of toxicological concern. Further analytical data or toxicity tests may be needed. Finally, the paradigm considers the possibility of additive or synergistic interactive effects among congeneric groups. With the developed strategy, the overall objective of the paradigm can be attained: that no reasonably significant risk associated with the intake of NFCs goes unevaluated.

2. Thresholds in safety evaluation of flavouring agents 52

Since 1996 the FAO/WHO Joint Expert Committee on Food Additives (JECFA) has evaluated the safety of over 1100 flavouring substances, based on a decision tree related to chemical structure and intake. Safety conclusions are based no whether the estimated intake is above or below a threshold of toxicological concern that is relevant to that compound. The decision tree includes toxicity thresholds for the three structural classes determined by the Cramer et al decision tree. A review by Munro et al of the available data on compounds in the three structural classes defined the 5th percentile of the NOAEL values from chronic and sub-chronic studies. These values were divided by the normal 100-fold uncertainty factor to derive a threshold of toxicological concern for each structural class. The daily intake thresholds were 1800 micrograms for Class I (structures of low predicted toxicity), 540 micrograms for Class II (structures that cannot be assumed to be of low toxicity) and 90 micrograms for Class III (structural features indicative of high toxicity). If the substance is predicted to be metabolised to innocuous products there is no safety concern if the intake is below the threshold, but suitable toxicity data on the compound or structural analogues are required if the intake exceeds the threshold. If the substance is not predicted to be metabolised to innocuous products, and the intake is below the appropriate threshold, safety evaluation is based on data on the compound or structural analogues, but adequate toxicity data are required on the compound if the intake exceeds the threshold. An additional threshold of 1.5 micrograms per day, which was derived from an analysis of the estimated 1 in 1,000,000 risk for chemicals in the cancer potency database, is applied for compounds for which appropriate toxicity data are not available. 53

(Generally Recognized As Safe) and structurally related compounds have been reported to be carcinogenic in rodent studies. Four of these flavors had an increasing response at two doses; three had increasing responses at 3 doses; one had increasing responses at four doses. The three compounds with three doses fit this plot with a correlation coefficient of at least 0.9; the four doses of methyleugenol fit with a correlation coefficient of 0.999983. The intercept at zero percentage tumors of these was at least several orders of magnitude greater than the estimated daily dose of these flavoring agents to individuals in the United States. This is interpreted to indicate that these flavoring agents have a clear threshold for carcinogenicity in animals that is well above the levels currently approved for use in foods; consequently, these animal studies should not be a cause for concern for carcinogenicity of these compounds in humans. Rather, the animal studies should be viewed as providing evidence for the safety of these compounds at current levels of human exposure. 54

TOXICOLOGY DATABASES AND THE TOXICITY THRESHOLD CONCEPT USED BY JECFA

A.G. Renwick. Clinical Pharmacology Group, School of Medicine, University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK

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THE IMPACT OF DOSE-DEPENDENT METABOLIC SWITCHING UPON TOXICITY: THE ALLYLBENZENE FLAVOURS METHYLEUGENOL AND ESTRAGOLE AS CASE EXAMPLES

J. Caldwell. Faculty of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom The allylbenzenes are a important group of natural food flavours, present in a large number of widely consumed foods and beverages. Estragole and methyleugenol, the two most important congeners, are hepatocarcinogenic to rodents and we have employed a mechanism-based approach in which production of the hepatotoxic and hepatocarcinogenic sulfate conjugate of the 1’-hydroxy metabolite is used to interpret the pathological changes observed in laboratory rodents. Both the qualitative and quantitative aspects of the molecular disposition of methyleugenol and estragole and their associated toxicological sequelae have been relatively well defined from mammalian studies. Their profiles of metabolism, metabolic activation, and covalent binding are clearly dose dependent. The relative importance of metabolic activation and associated covalent binding to hepatic protein and DNA is markedly less at low levels of exposure (i.e. these events are not linear with respect to dose). In particular, rodent studies suggest that these events are negligible in the dose range 1–10 mg/kg body weight, approximately 100–1000 times the anticipated human exposure to these substances. This dose range contrasts markedly with the 37.5 – 150 mg/kg/day doses of methyleugenol used in the recent NTP study. For these reasons, it is concluded that present levels of dietary exposure to methyleugenol and estragole resulting from consumption of food, mainly in spices or 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 at low levels of exposure to methyleugenol and estragole.

S5 Molecular epidemiology in occupational toxicology

ANALYSIS OF THRESHOLDS FOR CARCINOGENICITY

William J. Waddell. Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, USA Extrapolation from studies of chemical carcinogenicity in rodents at high doses to humans at the typically low doses to which we are exposed has been one of the most controversial issues in toxicology. Current approaches usually assume that there is zero tumor production only at zero dose and doses are evaluated on a linear scale. Re-evaluations of large prominent studies, e.g., the ED01 study, N-nitrosodiethylamine, unequivocally demonstrate thresholds for carcinogenicity when the dose-response curves for animal studies done at high doses are calculated according to fundamental principles of chemistry (Waddell and Bates, 1969; Rozman, et al. 1996). This requires dose to be on a logarithmic scale and percent tumors on a linear scale. Fifteen compounds approved by the FEMA (Flavor and Extract Manufacturers Association) expert panel as GRAS

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EVALUATION OF OCCUPATIONAL EXPOSURE TO CARCINOGENS BY MOLECULAR EPIDEMIOLOGY METHODS

R.J. Sram, B. Binkova, O. Beskid, P. Rossner, I. Chvatalova, E. Biros, A. Milcova, Z. Stavkova, Z. Smerhovsky. Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Molecular epidemiology is a new and evolving area of research combining laboratory measurements of internal dose, biologically effective dose, biologic effects, and influence of individual susceptibility with epidemiologic methodologies. City policemen represent a group occupationally exposed to increased concentrations of carcinogenic polycyclic aromatic hydrocarbons (carcPAHs). Personal exposure to carcPAHs was evaluated by personal samplers during working shift prior to collection of biological samples. During the sampling