ANNEX 5 A PROCEDURE FOR THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES I.C. Munro, Ph.D., FRCPath CanTox, Inc. Mississauga, Ontario Canada This paper was considered at the forty-fourth meeting of the Joint FAO/WHO Expert Committee on Food Additives. The conclusions of the Committee relating to its consideration of the paper are provided in the report, which has been published in the WHO Technical Report Series. This paper was not endorsed by the Committee, and the opinions expressed therein are those of the author. Comments on the approach outlined in the paper are invited, which should be addressed to the WHO Joint Secretary of JECFA, International Programme on Chemical Safety, 20, Avenue Appia, World Health Organization CH-1211 Geneva 27, Switzerland. ANNEX 5 A PROCEDURE FOR THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES Table of Contents 1. INTRODUCTION 2. CONCEPTS EMPLOYED IN THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES 2.1 Exposure to flavouring substances through food 2.2 Structure-activity relationships 2.3 Use of toxicity data 3. INTEGRATED METHOD FOR THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES 3.1 Structure-activity relationships and metabolic fate 3.2 Integrating information on exposure and toxicity 3.3 Evaluation criteria 3.4 Integrating data on the consumption ratio 4. CONCLUSIONS 5. REFERENCES APPENDICES Appendix A Estimating distribution of intakes of food ingredients across the population Appendix B Substances in reference database by order of structural class 1. INTRODUCTION The objective of this paper is to provide a procedure that can be used by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) for the safety evaluation of flavouring substances. The procedure extends principles and procedures recommended by IECFA in the past and is consistent with international concepts in safety evaluation. These principles have been discussed by JECFA (JECFA, 1972; 1974b; 1976a; 1978; 1980a,b; 1982a; 1983a; 1984a; 1986; 1987a,b; 1989; 1990a; 1991a; 1992) and have been recapitulated in a report, which outlines criteria for the safety evaluation of flavouring substances (WHO, 1987). This paper takes this previous background into account and provides a scientific basis for assessing the safety of flavouring substances used in food by presenting a procedure for evaluating this large group of compounds in a consistent, timely, and appropriate manner. 2. CONCEPTS EMPLOYED IN THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES Criteria for the safety evaluation of flavouring substances have been put forward by several national and international authorities. Included among these are JECFA, a special Task Group convened by the World Health Organization (WHO, 1987), the Committee of Experts on Flavoring Substances of the Council of Europe (CE), the Commission of the European Communities' Scientific Committee for Food (SCF, 1991), the British Industrial Biological Research Association (BIBRA), and the Flavor and Extract Manufacturers' Association of the United States Expert Panel (FEXPAN). In 1987, WHO published a health criteria document entitled Principles of the Safety Evaluation of Food Additives and Contaminants in Food, which contains a discussion of principles related to the safety evaluation of flavouring substances. This report recapitulated principles previously stated by JECFA on numerous occasions (JECFA, 1968; 1972; 1974b; 1976a; 1978; 1980a,b; 1982a; 1983a; 1984a; 1986; 1987a,b; 1989; 1990a; 1991a; 1992). Early on, it was recognized by JECFA that the safety evaluation of flavouring substances warranted special consideration in light of use patterns and typically low levels of human exposure (JECFA, 1972). This view also has been recognized by the (SCF) and has been recorded in their document entitled Guidelines for the Evaluation of Flavorings for Use in Foodstuffs. 1. Chemically, Defined Flavoring Substances (SCF, 1991). In addition, several organizations including JECFA (WHO, 1987), FEXPAN (Woods & Doull, 1991), SCF (1991), and the Council of Europe (CE, 1974, 1981, 1992) have noted that knowledge of structure-activity relationships and metabolism plays a key role along with exposure in the safety evaluation of flavouring substances. In this regard, JECFA (WHO, 1987) has used structure-activity relationships in evaluating groups of structurally related flavouring substances in a homologous series where toxicology studies exist on only one or a few members of the series. Structure-activity relationships can provide a useful means of assessing substances that lack toxicity data based on structurally related substances that have adequate toxicity data. 2.1 Exposure to flavouring substances through food Flavouring substances are used in processed foods and beverages to impart desirable organoleptic qualities and to provide the specific flavour profile traditionally associated with certain food products. Unlike many substances which are added to food to achieve a technological purpose, the use of flavouring substances is generally self-limiting and governed by the flavour intensity required to provide the necessary organoleptic appeal. Thus, flavouring substances are used generally in low concentrations resulting in human exposures that are very low. Estimates of the intake of flavouring substances by the population typically have involved the acquisition of data on poundage used in foods. Between 1970 and 1987, the U.S. National Academy of Sciences/National Research Council (NAS/NRC) conducted, under contract with the Food and Drug Administration (FDA), a series of poundage surveys of substances intentionally added to food (NAS, 1978, 1979, 1984, 1989). These surveys obtained information, both from ingredient manufacturers and from food processors, on the poundage of each substance committed to the food supply and on the usual and maximum levels at which each substance was added to foods in each of a number of broad food categories. Estimates of flavouring substance intake in the U.S. have been performed in two ways. One is to assume that the total poundage reported in food annually is completely consumed by the total population. Numerous checks using data from independent sources, such as imports, show that, in general, the reported poundage in the survey accounted for only 60% of the total used. Much more detailed and elaborate analyses (see Appendix A) led to the conclusion that it was conservative but reasonable to assume that each flavouring substance is consumed by only 10% of the population. A second method of calculating intake is to combine data on the level of use in specific food groups with data on food consumption to calculate the intake of each flavouring substance. Both methods tend to overestimate human intakes of flavouring substances because they deal with disappearance, that is the amount presumed to be used in food, and take no account of losses and waste during food manufacture, storage, preparation and consumption. Estimating intake of flavouring substances based on level of use data, coupled with data on the consumption of foods, typically leads to substantial overestimates of intake. This is because intake data for food commodities are stratified into broad categories of food products (e.g., baked products). Thus, cardamom, ordinarily used only in certain types of coffee cakes, would be assumed to be present in all breads, rolls, cakes and pastries In extreme cases, this may lead to overestimates of intake which are exaggerated several hundred-fold. Through a series of detailed studies conducted between 1970 and 1987 (see Appendix A) it has become clear that, while there is at present no perfect way to estimate intake of flavouring substances, poundage used provides a reasonable basis for calculating intakes. The intake data reported in this paper rely on annual poundage used in food and the estimates of intake reflect the assumptions that: i) the available survey data accounted for only 60% of production; and ii) the total amount produced is consumed by 10% of the population. Table 1 presents the intake data for 1323 chemically defined flavouring substances permitted for use in the U.S. calculated in this fashion. As can be seen from Table 1, most flavouring substances are consumed in amounts of less than 1 mg/person/day. The data are taken from the most recent U.S. NAS/NRC survey (NAS, 1989) of poundage used in food. For reasons previously stated, it can be assumed that these intake estimates are overestimated. Another important factor to consider in the evaluation of human exposure to flavouring substances is the extent to which flavouring substances intentionally added to foods also occur naturally in the food supply. The natural presence of flavouring substances in food is of course not necessarily indicative of safety. For many flavouring substances that occur naturally in foods, such natural occurrence, rather than intentionally added use, is the principal component of human exposure. The comparison of natural occurrence to intentional addition has been expressed as the consumption ratio (CR) (Stofberg & Kirschman, 1985). A CR of greater than one indicates food predominance (i.e. the flavouring substance is consumed at a higher level from foods than as an added substance). A CR greater than 10 indicates an almost insignificant contribution (-10%) of the flavouring substance as a food additive to the total intake (Stofberg & Grundschober, 1987). Stofberg and Grundschober (1987) calculated that out of 499 flavouring substances, 415 (83%) were food predominant (i.e. CR > 1) and 309 (62%) made an insignificant contribution to the food supply (i.e. CR > 10). As can be seen from this analysis, the use of natural occurrence as part of the safety evaluation provides an important perspective on the impact of intentional addition of flavouring substances to foods. If a flavouring substance is one of the few that for any reason, including high intake, is of relatively high safety concern, then a high consumption ratio likely enhances that concern because it indicates a much larger and uncontrolled exposure from natural occurrence than from intentional addition. If, on the other hand, a flavouring substance is one of the vast majority that have few, if any, safety issues and consequently low inherent concern, then a high consumption ratio reduces any concern still further because it indicates that intentionally added use is trivial. Stated in another way, if the added use of a flavouring substance amounts to less than 10% of its natural occurrence, this would indicate a minimal safety concern about added use. If added use is < 1% of natural use (i.e. CR > 100) then the added use can at most be of trivial safety concern. 2.2 Structure-activity relationships Toxicity is dependent on the chemical structure of a substance, its pharmacokinetics, and its metabolic reaction pathways. Available metabolic pathways are usually dose-dependent and, to a large extent, govern the magnitude of the toxic effect. Therefore, chemical structure, pharmacokinetics, metabolic fate and dose are key determinants of toxicity and play a critical role in safety evaluation of flavouring ingredients. Table 1. Number of flavouring substancesa within various intake categories Intake categoryb Cumulative (µg/day) No. of flavours frequency (% of total) < 0.01 349 26 0.01-0.1 93 33 0.1-1 274 54 1-10 224 71 10-100 204 86 100-1000 111 95 1000-10 000 45 98 10 000-100 000 16 99 100 000+ 7 100 TOTAL 1323 a Chemically defined flavouring substances permitted for use in the U.S. excluding botanicals. b Intake data calculated assuming: survey poundage reflects 60% of actual usage, 10% of population exposed, U.S. population in 1987 was 240 million. Formula: Intake (µg/person/day) = [(annual flavour usage in µg)‰0.6]‰(24×106 persons × 365 days). Poundage data from 1987 NAS/NRC survey data. Refinements to the initial concepts of structure-activity came as a result of increasing knowledge and confidence in predicting structure-activity relationships for flavouring substances. This formed the basis of a paper by Cramer et al. (1978) which, through the use of a "decision tree" approach, permitted the classification of flavouring substances into "classes of concern" based on structure and other considerations, similar in many respects to, but predating the "Concern Level" concept outlined by the U.S. Food and Drug Administration in its "Redbook" (FDA, 1982, 1993). The concept of establishing concern levels also has been investigated further by BIBRA to evaluate its applicability to food chemicals more generally (Phillips et al., 1987). This group evaluated and established concern levels for several food additives, plastic monomers, as well as flavouring substances. Although they reported that, in their opinion, the Cramer et al. (1978) decision tree misclassified a few substances, the decision tree was likely to be a more realistic approach for predicting toxicity than any other reported quantitative structure-activity relationship (QSAR) technique. Thus, there is general consensus, based on the work of Cramer et al. (1978), the subsequent work by BIBRA (Phillips et al., 1987), and the fact that the FDA (1982; 1993) uses structure-activity relationships in defining Concern Levels for food substances, that structure-activity has a solid basis in science when applied to substances of simple and closely related structures encountered at low exposures, a number of which are of known low toxicity and safe metabolic disposition. This is particularly the case for all but a very few flavouring substances. As will be discussed later in this paper, structure-activity relationships play an important role in the evaluation of flavouring substances. 2.3 Use of toxicity data Traditional safety evaluation procedures are only partially applicable to flavouring substances. Traditional approaches to the safety assessment of food additives typically involve the evaluation of considerable toxicological data, usually in an amount sufficient to establish a no-observed-effect-level (NOEL), permitting the establishment of an acceptable daily intake (ADI). Approximately half of the flavouring substances currently in use are naturally occurring simple acids, aldehydes, alcohols and esters. With few exceptions, these are rapidly metabolized to innocuous end products, the safety of which is well established or can be assumed from metabolic and toxicity data on the substance in question or on structurally related substances. Moreover, the acquisition of extensive toxicity data is unnecessary for the majority of flavouring substances because structure-activity relationships can be used as a means of assessing substances in a homologous series, in which only a few substances have toxicology data, to determine safety in use. This concept has been used by JECFA in the evaluation of structurally related flavouring substances, including the allyl esters, amyl acetate and isoamyl butyrate, benzyl compounds, citral compounds, alpha- and ß-ionones, and nonanal and octanal (JECFA, 1967: 1968; 1980a; 1984a,b; 1990a,b; 1991a,b; 1993a,b). In addition, as previously indicated in Table 1, 95% of flavouring substances are consumed at exposure levels less than 1 mg/person/day and in keeping with the safety evaluation procedure outlined in this paper, only limited toxicological data are required in such circumstances. When exposure is extremely low and there are organoleptic limitations on use levels, a primary consideration is whether there is a need to establish a numerical ADI for flavouring substances. There are several reasons why it is not appropriate or necessary to establish ADIs in the case of the majority of flavouring substances. ADIs are based on toxicological data and the establishment of a NOEL, an approach that differs from the concept that a safety evaluation can be performed in many instances on the basis of exposure and structure-activity relationships. Moreover, the organoleptic and gustatory properties of flavouring substances typically limit their use and, consequently, exposure to them. In addition, because a majority of flavouring substances occur in nature, there is a long history of human experience in flavouring substance consumption from traditional foods. Nearly fifty percent of flavouring substances given full ADIs by IECFA have consumption ratios greater than 1, indicating their predominant natural occurrence in food (Stofberg & Grundschober, 1987). The above factors have been noted by WHO (1987) as important in the evaluation of flavouring substances. It is also evident that very large safety margins exist for flavouring substances, as evidenced by the fact that the margin between the NOEL and the per capita intake of flavouring substances ranges from more than 50 to more than 1 000 000 times for the flavouring substances given full ADIs by JECFA (Table 2). Table 2. Safety margins between NOELs and per capita daily exposure for various flavouring substances given full ADIs by JECFAa Safety Margin Number of Flavours <100 1 100 - 1000 4 1000 - 10 000 13 10 000 - 100 000 9 100 000 + 7 TOTAL 34 a JECFA, 1967; 1968; 1970; 1971; 1972; 1974a,b; 1976a.b; 1978; 1980a,b,c; 1981a,b; 1982a,b; 1983a,b; 1984a,b; 1986; 1987a,b; 1989; 1990a,b; 1991a,b; 1992; 1993a.b,c. The factors discussed above lend support to the argument that it is unnecessary to establish ADIs for the majority of flavouring substances. Not establishing a numerical ADI for the great majority of flavouring substances is also consistent with the concept that presumed or known metabolism can be used as a basis for safety evaluation. 3. INTEGRATED METHOD FOR THE SAFETY EVALUATION OF FLAVOURING SUBSTANCES In a continuing effort to improve the basis for the safety evaluation of flavouring substances, this paper presents a procedure which integrates information on exposure, structure-activity relationships, metabolic fate and toxicity. It presents a safety evaluation procedure which allows a determination of the safety of flavouring substances under conditions of intended use. The key elements of the safety evaluation procedure are discussed below. 3.1 Structure-activity relationships and metabolic fate Without exception, flavouring substances are volatile organic chemicals. The vast majority of flavourings ingredients have simple, well characterized structures with a single functional group and low molecular weight (< 300). More than 700 of the 1323 chemically defined flavouring substances used in food in the U.S. are simple aliphatic acyclic and alicyclic alcohols, aldehydes, ketones, carboxylic acids, and related esters, lactones, ketals, and acetals. Other structural categories include aromatic (e.g., cinnamaldehydes and amhranilates), heteroaromatic (e.g., pyrazines and pyrroles) and heterocyclic (e.g., furanones and thiofurans) substances with characteristic organoleptic properties (e.g., furanones providing a strawberry note). For most flavouring substances, the structural differences are small. Incremental changes in carbon chain length and the position of a functional group or hydrocarbon chain typically describe the structural variation in groups of related flavouring substances. These systematic changes in structure provide the basis for understanding the effect of structure on the chemical and biological properties of a substance. Toxicity is dependent on the chemical structure and metabolism of a substance. The "decision tree" procedure (Cramer et al., 1978) relies primarily on chemical structure and estimates of total human intake to assess toxic hazard and to establish priorities for appropriate testing. The procedure utilizes recognized pathways of metabolic deactivation and activation, data on toxicity, and the presence of the substance as a component of traditional foods and as an endogenous metabolite. Substances are classified according to three categories: Class I - Substances of simple chemical structure and efficient modes of metabolism which would suggest a low order of oral toxicity (e.g., butyl alcohol or isoamyl butyrate). Class II - Contains structures that are intermediate. They possess structures that are less innocuous than substances in Class I, but do not contain structural features suggestive of toxicity like those substances in Class III. Members of Class II may contain reactive functional groups (e.g., furfuryl alcohol, methyl 2-octynoate, and allyl propionate). Class III - Substances of a chemical structure that permit no strong initial presumption of safety, or may even suggest significant toxicity (e.g., 2-phenyl- 3-carbethoxy furan and benzoin). The decision tree is a tool for classifying flavour ingredients according to levels of concern. The majority of flavouring substances fall into Class I because they are simple alcohols, aldehydes, ketones, acids or their corresponding esters, acetals and ketals that occur naturally in food and, in many cases, are endogenous substances. They are rapidly metabolized to innocuous products (e.g., carbon dioxide, hippuric acid, and acetic acid) by well recognized reactions catalyzed by cellular enzymes that exhibit high specificity and high catalytic efficiency (e.g., alcohol dehydrogenase and isovaleryl coenzyme A dehydrogenase). Substances that do not undergo detoxication via these highly efficient pathways (e.g., fatty acid pathway and citric acid cycle) are metabolized by reactions catalyzed by enzymes of low specificity and relatively low efficiency (e.g., cytochrome P-450 and glutathione transferase). For some groups of substances (e.g., branched-chain carboxylic acids, allyl esters, and linear aliphatic acyclic ketones), metabolic thresholds for intoxication have been identified (Krasavage et al., 1980; Deisinger et al., 1994; Jaeschke et al., 1987). The dose range, over which a well-defined change in metabolic pathway occurs, generally correlates with the dose range over which a transition occurs from a no-observed-adverse-effect level to an adverse-effect level. For such groups of substances the dose range at which this transition occurs is orders of magnitude greater than the level of exposure from use as flavour ingredients. Most substances in Class II belong to either of two categories; one includes substances with functional groups which are similar to, but somewhat more reactive than functional groups in Class I (e.g., allyl and alkyne); the other includes substances with more complex structures than substances in Class I, but that are common components of food. This category includes heterocyclic substances (e.g., 4-methylthiazole) and terpene ketones (e.g., carvone). The majority of the flavouring substances within Class III include heterocyclic and heteroaromatic substances and cyclic ethers. Many of the heterocyclic and heteroaromatic substances have sidechains with reactive functional groups. In a few cases, metabolism may destroy the heteroaromaticity of the ring system (e.g., furan). Although metabolism studies have been performed for Class III flavouring substances with elevated levels of exposure, the metabolic fate of many substances in this structural class cannot be confidently predicted. Review of the group of substances in each of the structural classes indicates that as structural complexity increases (Class I - III), the number of flavouring substances and the levels of exposure decrease significantly (Table 3). In all structural classes, one-quarter or more of the flavouring substances are consumed at levels below 0.01 µg/day or 0.2 µg/kg bw/day. Table 3. Number of flavouring substancesa divided by structural class within various intake categories Intake Categoryb No. of Flavours (% of Total) (µg/day) Class I Class II Class III <0.01 212 (24) 68 (28) 69 (34) 0.01-0.1 55 (6) 20 (8) 18 (9) 0.1-1 169 (19) 48 (20) 57 (28) 1-10 145 (17) 45 (19) 34 (17) 10-100 147 (17) 39 (16) 18 (9) 100-1000 95 (11) 12 (5) 4 (2) 1000-10 000 34 (4) 9 (4) 2 (1) 10 000-100 000 16 (2) 0 0 100 000+ 5 (0.6) 2 (0.8) 0 TOTAL 878 243 202 a Chemically defined flavouring substances permitted for use in the U.S. excluding botanicals. b Intake data calculated assuming: survey poundage reflects 60% of actual usage, 10% of population exposed, U.S. population in 1987 was 240 million. Formula: Intake (µg/person/day) = [(annual flavour usage in µg)‰0.6]‰(24×106persons × 365 days). Poundage data from 1987 NAS/NRC survey data. 3.2 Integrating information on exposure and toxicity One of the key elements of the safety evaluation procedure is based on the premise that intake levels can be specified for flavouring substances that would not present a safety concern. This paper presents a procedure which provides the scientific underpinnings for defining toxicologically inconsequential exposures for flavouring substances and expressing these as human exposure thresholds. The concept of specifying human exposure thresholds relies on principles that permit specifying the daily intake of a substance which can be considered, for practical purposes, as presenting no toxicological risks (and thus of no health or safety risk to consumers) even in the absence of specific toxicological data on the substance (Federal Register, 1993; Munro, 1990; Rulis, 1986; Frawley, 1967). The concept relies on knowledge of the range of toxicological risks for structurally related substances and on knowledge regarding the toxicological potency of relevant classes of chemicals for which good toxicity data exist. The principles underpinning the establishment of human exposure thresholds have been embodied in a recent United States government Federal Register (1993)1 notice emanating from the FDA, which provides the scientific basis for the conclusion that an exposure level to indirect food additives can be specified, below which no risk to public health would likely accrue. This exposure level has, in turn, been used by FDA to establish a proposed "threshold of regulation" for indirect food additives which precludes the need for toxicological evaluation of substances migrating into food from food-contact articles provided the amount that migrates does not lead to a dietary level in excess of 500 ppt (equivalent to 1.5 µg/person/day assuming a daily food intake of 3000 g). The FDA has noted that such a level would result in negligible risk to consumers even if the substance was shown later to be a carcinogen. This concept is in keeping with the well-established principle that resources should be directed to the safety evaluation of substances having high exposure and therefore greater potential for adverse effects and not toward substances with trivial exposure. The concept is particularly applicable to substances of low toxicity and with known or predictable metabolic fate. The scientific basis for the establishment of human exposure thresholds and the proposed FDA regulation are discussed below. Frawley (1967) initially suggested the concept of establishing human exposure thresholds. He showed, on the basis of studies conducted on several well-tested substances, including food additives, industrial and consumer chemicals, and pesticides that a generic "no-effect" level could be established that could preclude the need for toxicity studies and safety evaluation for a majority of substances intended for use as food packaging materials. Frawley constructed a reference database of non-tumorigenic endpoints using 220 two-year rodent studies. He presented the NOELs for all 220 compounds. Frawley (1967) reported that if he excluded heavy metals and pesticides from the analysis, there was no compound in the remaining database (except for acrylamide) which showed evidence of chronic toxicity at dietary concentrations of less than 100 ppm. Application of a typical 100-fold safety factor to the 100 ppm generalized NOEL would mean that humans could safely consume any of the materials provided the dietary concentration did not exceed 1 ppm. Frawley (1967), noting that his database was incomplete, proposed adding an additional safety factor of 10 which would translate to a toxicologically insignificant human exposure level of 0.1 ppm in the diet. Assuming an individual consumes 1500 grams of food per day, an exposure of 150 µg/person/day (approximately 2.5 µg/kg/day) or less to a chemical of unknown toxicity would be considered toxicologically insignificant. According to Frawley, such exposures could be considered of no safety concern. More recently, Rulis (1986) conducted a similar analysis of the FDA's Priority-Based Assessment of Food Additives (PAFA) database containing 159 compounds with subchronic or chronic toxicity data. He came to the same conclusion as Frawley (1967). Essentially there is no risk of toxicity in rodents exposed to certain food additives at dietary levels of less than 1 mg/kg bw/day or in human terms, approximately 1 to 10 µg/kg bw/day depending on the safety factor applied. Even twenty years apart, using different databases, the toxicologically inconsequential levels proposed by Frawley (1967) and Rulis (1986) were nearly identical. Munro (1990) used a database of approximately 350 substances compiled by Gold et al. (1984, 1989) to develop a human exposure threshold value to be applied to substances for which no presumption of safety can be made because of a complete lack of data on metabolism and potential toxicity. Munro (1990) proposed a threshold of regulation of up to 1000 ppt for indirect additives which would translate to a daily intake of 1.5 to 3.0 µg/person/day depending upon assumptions regarding food intake. The acceptable exposure of 1.5 µg/day is equivalent to that considered by FDA (Federal Register, 1993)1 to present no regulatory concern for a food packaging material even if later it was determined to be a carcinogen. 1 FDA has proposed a dietary concentration of 500 ppt as the threshold of regulation for substances used in food-contact articles. Assuming that an individual consumes 1500 g of solid food and 1500 g of liquid food per day, this threshold would equate to a toxicologically inconsequential level of 1.5 µg/day (Federal Register, 1993). The work conducted by the FDA (Federal Register, 1993), Frawley (1967), Rulis (1986) and Munro (1990) is expanded upon in this paper through the compilation of a large database of reference substances (Appendix B) from which a distribution of NOELs could be derived for chemicals of various structural types. The reference database describes the relationships between exposure, structure and toxicity for a wide variety of chemicals of divergent structure and it can be used as a reference point from which to judge the safety of flavouring substances. In compiling the database, strict criteria were applied in the selection of data sets. The objective of the exercise was to identify as many high quality toxicological studies as possible representing a variety of toxic endpoints and chemical structures. To accomplish this, the study types included those typically conducted in toxicology, such as subchronic, chronic, reproductive and teratology studies. Short term and acute studies were not included since these were considered not to be relevant for establishing chronic NOELs. The database consisted mainly of studies in rodents and rabbits. Very few studies in dogs and other species were found that met the established criteria. An evaluation of randomly selected dog and primate studies indicated that many had too few animals per group to derive a statistically valid NOEL. Moreover, for many dog studies, a common endpoint was reduced body weight and/or food consumption which was due, in many cases, either to palatability problems with the diet or vomiting. In addition, most studies in dogs and other non-rodent species were simply too short in duration to be classified as chronic studies. Only oral studies were included in the database. A further criterion for inclusion in the database was that a study had to have a demonstrated lowest-observed-effect level (LOEL) as well as a NOEL, thus ensuring that the study was rigorous enough to detect toxic effects. In some instances NOELs were included for studies not demonstrating a LOEL, and these were substances such as major food ingredients that were without toxicity at the highest dose tested in well-conducted studies. It should be noted that the inclusion of such substances in the database would not bias the database in favour of higher NOELs since the true NOEL for such substances probably would exceed the NOEL established from the available studies. In order to group NOELs for substances with only subchronic studies with those with chronic studies to derive the cumulative distribution of NOELs, subchronic NOELs were divided by a factor of three to approximate the most likely NOEL that would be derived from a chronic study. This conversion factor is based on research defining the relationship between subchronic and chronic NOELs. Weil and McCollister (1963) compared 3 month NOELs with 2 year NOELS for 33 different substances (including pharmaceuticals, pesticides, and food additives) fed to rats. They found that for most of the compounds (30), the ratio of the NOELs between subchronic and chronic studies was 5 or less and more than half of the compounds had a ratio equal to 2 or less. More recently, it has been discovered through further analysis of more chemical substances, that a more accurate adjustment factor for extrapolating NOELs derived from subchronic studies to lifetime was between 2 and 3 (Dourson, personal communication; Lewis et al., 1990; Beck et al., 1993). In compiling the database, emphasis was placed on retrieving data from certain databases known to contain well-validated toxicological endpoints for a series of well-defined chemical structures. An exhaustive search was made of compounds evaluated by JECFA. Other sources included the U.S. Environmental Protection Agency's (EPA) Integrated Risk Information System (IRIS) on-line database, the National Toxicology Program (NTP) studies, the Developmental and Reproductive Toxicity (DART) on-line database from EPA and the U.S. National Institute of Environmental Health Sciences and the published literature in general. The data entered into the database included the name of the chemical, Chemical Abstracts Service Registry Numbers (CAS No.), structural classification as assessed using the Cramer et al. (1978) decision tree and the FDA "Redbook", species, sex, route of administration, dose levels tested, study type, duration, endpoints reported, LOEL, NOEL and references. In an effort to be conservative in the construction of the reference database, NOELs selected by the author(s) of each study were used even though in some cases authors tended to over-interpret their data. In some instances, it was found that the stated NOEL may have been based on a misjudgment of an adverse effect by the author (e.g., physiological versus toxicological effects) or on artifactual effects (e.g., fetal toxicity as a result of maternal toxicity). An example of this is isopropyl alcohol, which has been reported to produce teratogenic effects at very low doses (0.018 mg/kg) in one study; however, its structure, known metabolism and other toxicological data provide no evidence for concluding teratogenicity. Even though scientifically, some of these author-derived NOELs were not thoroughly substantiated, they were included in the reference database, thereby increasing the degree of its conservative nature. NOELs selected by EPA for the IRIS database were entered without further review. In all, the database consists of 612 substances representing a range of industrial chemicals, pharmaceuticals, food substances and environmental and consumer chemicals likely to be encountered in commerce. Since the database was developed as a reference database for the evaluation of flavouring substances, all of which are organic chemicals, no organometallic or inorganic compounds were included in the database. For many of the substances, more than one NOEL was identified from the literature resulting from the fact that some substances were tested in more than one species and sex and/or demonstrated a range of endpoints suitable for establishing a NOEL. This led, in some cases, to multiple NOELs for individual substances. In all, the database contains 2944 entries. In order to correlate chemical structure with toxicity, the substances in the database were classified into three groups corresponding to the three structural classes outlined in the Cramer et al. (1978) decision tree. For each substance, the most conservative NOEL was selected from the reference database based on the most sensitive species, sex and endpoint. The cumulative distribution of the NOELs within each class is shown in Figure 1, along with the lognormal distributions fitted to these data. These results clearly delineate the effects of structural class on toxicity, with the median (50th percentile) NOEL decreasing from Class I through III. Similar differences among structural classes exist in the range between the 5th and 95th percentile. The human exposure threshold for each of the structural classes was calculated from the 5th percentile NOEL. The 5th percentile NOEL was chosen because this value would provide 95% confidence that any other substance of unknown toxicity but of the same structural class as those comprising the reference database would not have a NOEL less than the 5th percentile for that particular structural class within the reference database. The 5th percentile NOELs for each structural class are shown in Table 4. In converting the 5th percentile NOELs to human exposure thresholds (Table 4) for the various structural classes, a 100-fold safety factor was used since such a factor would inherently be applied in establishing safe intake levels for the substances comprising the database. The use of such a factor provides a substantive margin of safety since the human exposure thresholds are based on a large database of approximately 612 compounds with good supporting toxicological data. Furthermore, 5th percentile NOELs were used to calculate the thresholds, providing a more conservative figure than the arithmetic mean. Moreover, the estimated daily intakes of flavouring substances to which the human exposure threshold are compared are greatly overestimated as they represent the "eaters only" (10%) population. Thus, it is believed that a 100-fold safety factor provides a wide margin of safety in relating the results of the analysis of the reference database to flavouring substance exposure. It is evident from Table 4 that there are substantial differences in the 5th percentile NOELs for the various structural classes, indicating an obvious effect of structure on toxic potency. It is enlightening to compare the human exposure thresholds with present intakes of chemically defined flavouring substances in the U.S. As shown in Table 5, it is clear that for nearly all (93 to 97%) flavouring substances used in the U.S., intakes are below the human exposure threshold for their respective structural class. Because most flavouring substances possess simple structures and their metabolism is known or reasonably predictable, it can be concluded that it is highly improbable that they would present a toxicologicalrisk at exposure levels below the human exposure threshold for their respective structural class. However, even if information on structural class, metabolic fate and existing toxicity studies were not available, 743/1323 (56%) of flavouring substances used in the U.S. are consumed in amounts less than the 1.5 µg/person/day standard proposed by the FDA (Federal Register, 1993) and Munro (1990). This indicates that for approximately half of the existing list of 1323 chemically defined flavouring substances permitted for use in the U.S., exposure can be considered to be trivial. Table 4. Fifth percentile NOELs and human exposure thresholds for Cramer et al. (1978) structural classes in the reference database 5th Percentile NOELs, Human Exposure (µg/kg bw/day) Threshold,(µg/day)a,b I 137 2993 1800 II 28 906 540 III 447 147 88 a The human exposure threshold was calculated by multiplying the 5th percentile NOEL by 60 (assuming an individual weighs 60 kg) and dividing by a safety factor of 100, as discussed in the text. b Numbers rounded to two (2) significant figures. Table 5. Flavouring substances within each Cramer et al. (1978) structural class consumed in amounts below human exposure thresholds No. of No. of Flavours Human Exposure Flavours Under Human Structural Class Threshold Within Exposure (µg/day) Structural Threshold (%) Classa I 1,800 878 835 (95) II 540 243 227 (93) III 88 202 195 (97) a Adapted from the FEMA flavouring substance database of flavouring substances permitted for use in the U.S. 3.3 Evaluation criteria The evaluation criteria proposed for application to flavouring substances in this paper involve the integration of information on exposure, structure-activity relationships, metabolism and, as required, toxicity data. It should be noted that the safety evaluation criteria outlined below are not intended to be applied to any flavouring substances with unresolved toxicity problems or to substances that are presumed or known carcinogens. Such substances warrant special consideration and must be evaluated using more traditional methods of safety evaluation. While toxicity data exist on numerous flavouring substances and can be used as the basis for evaluation, there are many flavouring substances that lack toxicity data. The evaluation criteria are intended to provide a means of evaluating such substances. The criteria incorporate, where the data permit, the concept that metabolic fate can be predicted on the basis of presumed structure-activity relationships. The criteria also rely, in part, on the NOEL reference database which provides a human exposure threshold for each of the three structural classes of flavouring substances, as shown in Table 4. The evaluation criteria also incorporate, where available, toxicity data on flavouring substances and closely related structural analogues as a basis for safety evaluation. One of the criteria (number 5 below) incorporates the concept of a minimum human exposure threshold based on the 1.5 µg/person/ day standard proposed by the FDA (Federal Register, 1993) and Munro (1990). This standard can be applied to those flavouring substances for which metabolic fate is unknown and cannot be confidently predicted and for which there are no toxicity data on the flavouring substance or on a structurally related material from which to conclude any inference of safety in use. Flavouring substances that meet one of the five numbered criteria outlined below can be declared safe for their intended use without further evaluation: 1. a) The flavouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous end-products; and b) the conditions of intended use do not result in an exposure greater than the human exposure threshold for the relevant structural class, indicating a low probability of potential for adverse effects. 2. a) The conditions of intended use result in an exposure that exceeds the human exposure threshold for the relevant structural class; however b) the flavouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous end-products and it or its metabolites are endogenous human metabolites with no known biochemical regulating function. 3. a) The flavouring substance has a simple structure and will be metabolized and excreted through known detoxication pathways to innocuous end-products; and b) the conditions of intended use result in an exposure that exceeds the human exposure threshold for the relevant structural class; however c) toxicity data exist on the flavouring substance which provide assurance of safety under conditions of intended use, or there are toxicity data on one or more close structural relatives which provide a NOEL high enough to accommodate any perceived difference2 in toxicity between the flavouring substance and the structurally related substance having toxicity data. 4. a) The metabolic fate of the flavouring substance cannot be confidently predicted on the basis of structure; however b) the conditions of intended use result in an exposure below the human exposure threshold for the relevant structural class indicating a low probability of potential for adverse effects; and c) there are toxicity data on the substance, or on one or more structurally related substances with a NOEL at least 10 times greater than the 5th percentile NOEL for the relevant structural class. 5. a) The metabolic fate of the flavouring substance cannot be confidently predicted on the basis of structure; however b) the conditions of intended use result in an exposure below the human exposure threshold of 1.5 µg/day2, providing assurance that the substance will be safe under conditions of intended use. [NOTE: This criterion accommodates flavouring substances for which there are limited data on structure-activity and toxicity, but they would not be considered to present a safety concern below the human exposure threshold of 1.5 µg/day (Federal Register, 1993)]. The evaluation criteria listed above identify substances which are considered safe under conditions of intended use or those that may require additional data and further evaluation. Figure 2 presents the same criteria in the form of an evaluation sequence. The sequence contains a number of questions on structure, metabolism, exposure data and toxicity and provides an integrated mechanism to evaluate the safety of a flavour ingredient. Although the procedure incorporates relevant toxicity data on a substance or related substances where available, it does not require them. The procedure identifies those substances for which additional safety data may be required in order to perform an adequate safety evaluation. The effective application of this safety evaluation procedure depends on a substantial knowledge of toxicology, chemistry, metabolism, and exposure to flavouring substances. It can be applied most effectively when groups of structurally related flavouring substances are evaluated together. In a group evaluation, conclusions reached on the safety of individual substances are supported by similar conclusions for structurally related substances. For example, the results of the evaluation for butyl butyrate should be consistent with results for other esters formed from aliphatic acyclic linear saturated alcohols and acids having similar levels of exposure. These similar substances will pass through the same branch of the safety evaluation procedure because they fall into the same structural class, possess similar metabolic fate, and exhibit similar patterns of exposure from use as flavour ingredients and as components of food. 2 In most instances, groups of structurally related materials have toxicology data on at least one member of the group, usually the flavoring substance with the highest poundage. In most cases a large margin of safety ( i.e., 100- to 1000-fold) exists between the NOEL and the calculated exposure to the substance having toxicological data. Such margins of safety would be expected to accommodate any perceived difference between the toxicity of a flavouring substance having no toxicological data and its close structural relative for which a NOEL has been established. In the first step of the safety evaluation procedure (Figure 2) the user must assign a decision tree structure class (Cramer et al., 1978) to the substance. Following assignment of structure class, a question on metabolic fate appears. This question identifies those substances which are anticipated to be efficiently metabolized to innocuous products (e.g., 1-butanol) versus those which are transformed to more toxic metabolites (e.g., estragole) or have limited information on which to predict confidently the metabolic fate (e.g., 2-phenyl-3-carbethoxy furan). Once a substance has been sorted according to structure class and knowledge of metabolic fate, the next question compares the substance's daily per capita intake ("eaters only") from use as a flavour ingredient to the human exposure threshold (Table 4) for the same structure class. If the substance is metabolized to innocuous products (Step No. 2) and has an intake less than the human exposure threshold for the structure class (Step No. 3), the substance is considered safe (e.g., 1-octanol). If the intake is greater than the human exposure threshold (Step No. 3) and the substance or its metabolites are endogenous (Step No. 4), the substance is also considered safe, even though the intake is greater than the human exposure threshold (e.g., butyric acid). If the substance is not endogenous, then the substance or related substances must have a NOEL (Step No. 5) significantly greater than the intake of the substance in order to be considered safe (e.g., citral). If no such data exist or the NOEL is not significantly greater than the intake for the substance, then additional data are required in order to complete the safety evaluation. If metabolic fate cannot be confidently predicted and the intake (Step No. 2) is greater than the human exposure threshold (Step No. 3), additional data on metabolic fate or toxicity on the substance or structurally related substances are required to complete the safety evaluation (e.g., dihydro-coumarin). If the intake is less than the human exposure threshold, the substance or structurally related substances must have a NOEL significantly greater than the NOEL for the structure class (Step No. 6) in order for the substance to be considered safe (e.g., 2-ethyl-4-hydroxy-3(2H)-furanone). If an adequate toxicity study is not available and the substance has an intake less than 1.5 µg/day (Step No. 7), the substance is considered safe (e.g., 3-acetyl-2,5-dimethylthiophene). Otherwise additional data are required in order to complete the safety evaluation (e.g., 2-ethylfuran).
The principal objective of the safety evaluation procedure is to identify two groups of flavourings substances: i) those substances whose structure, metabolism, and relevant toxicity data clearly indicate that the substance would be expected not to be a safety concern under current conditions of intended use; and ii) those substances which may require additional data in order to perform an adequate safety evaluation. 3.4 Integrating data on the consumption ratio As pointed out by WHO (1987) and SCF (1991), natural occurrence is no guarantee of safety, but it is important to recognize that the safety evaluation of added use of flavouring substances needs to be conducted with an appreciation of the consumption ratio. Clearly, if added use of flavouring substances results in an exposure that exceeds that from natural sources, this will increase awareness of the need to consider carefully overall exposure in the light of existing data on toxicity and structure-activity relationships. On the other hand, if the added use is trivial with a consumption ratio of 10 to 100, that is, it increases total exposure by only 1 to 10%, then this fact needs to be taken into consideration when applying the criteria outlined above. The substances of primary concern are those which, in Figure 2, receive a "No" answer to Step No. 5, or a "Yes" answer to Step No. 7, indicating a possible need for additional data and evaluation beyond that included in the evaluation procedure outlined in this paper. In such an evaluation, as stated immediately above, the extent of natural occurrence should be given appropriate weight. Of much less concern with respect to consumption ratio are those substances that drop out of further consideration as a result of "No" answers to Step Nos. 3 or 7, or "Yes" answers to Step Nos. 4 or 6. The derivation of the thresholds, the estimation of intakes (see Appendix A), and the special factors applicable to the use of flavours (volatility, self-limiting use, etc.) build in multiple conservative assumptions more than adequate to cover additional exposure from natural occurrence to substances that in any case are of low inherent concern. The advances in analytical chemistry in the past 50 years provide virtual assurance that no flavouring substances of extensive natural occurrence remain unknown. Those of potential value yet to be discovered (e.g., as yet unknown components of roast beef, coffee, or chocolate flavour) are being sought at the ppb and ppt level. This does not suggest exposures above any of the thresholds discussed in this paper. 4. CONCLUSIONS It is neither possible nor necessary to conduct toxicological studies on all individual flavouring substances used in food. The vast majority of flavouring substances are members of groups of substances with common metabolic pathways, and typically, individual members of such a group display a similar toxicity profile. This is not surprising in light of the close structural similarity of the various flavouring substances comprising a chemical group. As demonstrated in this paper, exposure to flavouring substances is usually low and, in the majority of cases, below the human exposure threshold values presented in this paper. This knowledge, coupled with knowledge regarding structure-activity relationships, metabolic disposition, and toxicology data, permits the establishment of the proposed paradigm for safety assessment which provides a solid foundation to assure the safety of flavouring substances under conditions of intended use. 5. REFERENCES BECK, B.D., CONOLLY, R.B., DOURSON, M.L., GUTH, D., HATTIS, D., KIMMEL, C. & LEWIS, C. (1993). Symposium overview. 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JECFA (1993a). Evaluation of Certain Food Additives and Contaminants. Forty-first Report of the Joint FAO/WHO Expert Committee on Food Additives, Technical Report Series No. 837. JECFA (1993b). Toxicological Evaluation of Certain Food Additives and Contaminants. Forty-first Meeting of the Joint FAO/WHO Expert Committee on Food Additives, WHO Food Additive Series No. 32. JECFA (1993c). Toxicological Evaluation of Certain Food Additives and Naturally Occurring Toxicants. Thirty-Ninth Meeting of the Joint FAO/WHO Expert Committee on Food Additives, WHO Food Additive Series No. 30. KRASAVAGE, W.J., O'DONOGHUE, J.L., DIVINCENZO, G.D. & TERHAAR, C.J. (1980). The relative neurotoxicity of methyl-n-butyl ketone, n-hexane and their metabolites. Toxicol. Appl. Pharmacol., 52: 433-441. LEWIS, S.C., LYNCH, J.R. & NIKIFOROV, A.I. (1990). A new approach to deriving community exposure guidelines from "no-observed-adverse- effect levels". Regul. Toxicol. Pharmacol., 11: 314-330. MUNRO, I.C. (1990). Safety assessment procedures for indirect food additives: An overview. Regul. Toxicol. Pharmacol., 12: 001-0011. NAS (1978). Resurvey of the Annual Poundage of Food Chemicals Generally Recognized as Safe, National Technical Information Service (NTIS) PB288-081. NAS (1979). Comprehensive Survey of Industry on the Use of Food Additives, National Technical Information Service (NTIS) PB80-113418. NAS (1984). Poundage Update of Food Chemicals, National Technical Information Service (NTIS) PB84-162148. NAS (1989). 1987 Poundage and Technical Effects Update of Substances Added to Food. National Research Council, Washington, DC. Prepared for: Food and Drug Administration, Washington, D.C. NTIS Report No. PB91-127266. PHILLIPS, J.C., PURCHASE, R., WATTS, P. & GANGOLLI, S.D. (1987). An evaluation of the decision tree approach for assessing priorities for safety testing of food additives. Food Addit. Contam., 4(2): 109-123. RULIS, A.M. (1986). De Minimis and the Threshold of Regulation. In: Felix, C.W. (Ed.) Food Protection Technology. Lewis Publishers Inc., Chelsea, MI. pp. 29-37. SCF (1991). Guidelines for the Evaluation of Flavourings for Use in Foodstuffs: I. Chemically Defined Flavouring Substances. Commission of the European Communities, Scientific Committee for Food, Brussels, Belgium. STOFBERG, J. & KIRSCHMAN, J. (1985). The consumption ratio of flavouring materials: A mechanism for setting priorities for safety evaluation. Food Chem. Toxicol., 23: 857-860. STOFBERG, J. & GRUNDSCHOBER, F. (1987). Consumption ratio and food predominance of flavouring materials. Perfumer Flavourist, 12: 27-68. WEIL, C.S. & McCOLLISTER, D.D. 1963. Safety evaluation of chemicals. Relationship between short- and long-term feeding studies in designing an effective toxicity test. Agr. Food Chem., 11(6): 486-491. WHO (1987). Principles For The Safety Assessment Of Food Additives And Contaminants In Food. WHO Environmental Health Criteria. World Health Organization (WHO), International Program on Chemical Safety in Cooperation with the Joint FAO (Food & Agriculture Organization of the United Nations), WHO Expert Committee on Food Additives (JECFA), Geneva, Switzerland, Environmental Health Criteria 70. WOODS, L.A. & DOULL, J. (1991). GRAS evaluation of flavouring substances by the Expert Panel of FEMA. Regul. Toxicol. Pharmacol., 14: 48-58. ANNEX 5, Appendix A ESTIMATING DISTRIBUTION OF INTAKES OF FOOD INGREDIENTS ACROSS THE POPULATION In response to the Food Additives Amendment of 1958 to the U.S. Food, Drug and Cosmetic Act, the Flavor and Extract Manufacturers' Association (FEMA) in 1960 conducted a comprehensive survey of all flavoring materials then thought or claimed to be in use. Information included: identity; poundage used annually in food; year of first use; levels (in ppm) used in major food categories; toxicological data; and other information related to safety. After evaluation by a panel of external experts, this and subsequent information was published in a series of "FEMA GRAS Lists" (Hall and Oser, 1965, 1970; Oser and Hall, 1972; Oser and Ford, 1973ab, 1974, 1975, 1977, 1978, 1979; Oser et al., 1984, 1985; Burdock et al., 1990; Smith and Ford, 1993). FEMA determined in 1968 that another survey was needed to monitor trends and to improve on the previous effort. In addition, the U.S. Food and Drug Administration (FDA) began to plan to review its Generally Recognized as Safe (GRAS) lists, and in so doing, made use of the prior experience of FEMA. FEMA and FDA efforts merged into the first comprehensive survey of ingredients used in food, conducted by the National Academy of Sciences/National Research Council (NAS) in 1970 (NAS, 1973) with several subsequent surveys (NAS, 1978, 1979, 1984, 1989). Emerging from the 1960 and 1970 surveys was a clear indication that per capita intake of an ingredient (from annual poundage), although useful for comparison with other ingredients, has a significant limitation. As an average of intakes by those who consume foods containing the ingredient ("eaters") and by those who do not (non-eaters"), it does not represent the intake by the individuals of concern ( i.e., the "eaters"). This is not the case for ingredients commonly consumed by almost everyone ( e.g., sugar), but the difference between the average and "eaters" is significant for other ingredients consumed only by few people ( e.g. raspberry flavor). Because of these issues, an alternative method of calculating intakes was used in the first (1973) NAS report. This method involved the average level of use of an ingredient in a broad food category (e.g., baked goods) multiplied by the average daily consumption of that food category per person. The daily consumption for each category was obtained by multiplying the average portion size for the category, obtained from the U.S. Department of Agriculture (USDA) Food Consumption Survey by the average daily frequency of consumption of foods in that category over a 14-day survey period. The frequency of consumption data came from a 12,000+ member panel operated by the Market Research Corporation of America (MRCA; see note b to Table 1). Thus the average intake of an ingredient for all relevant food categories provided the "possible average daily intake" (PADI). These intakes were often highly exaggerated, as much as a hundred-fold, because an ingredient used in only a few foods in a food category was assumed to be at the average level in all foods in the category. Thus, for example, cardamom, used only in a few coffee cakes, was assumed to be used in each food in the entire category of baked goods ( i.e., all breads, rolls, crackers, and pastries). To address this issue, another method was developed to give the probable distribution of intakes for the entire population without exaggerating intake. This required an informed estimate of the probability that each food eaten would contain the ingredient of interest. The first series of such intake estimates used calculation "A", as shown in Table 1, and the results are summarized in Section "A" of Table 2. In Table 2, Column 1 ("Substance") lists the substances selected to test and improve the more refined method. In Section A, nutmeg and mace were chosen, among other reasons because they are not grown domestically in the U.S. and import data, therefore, provide a firm external check on pounds used. Furthermore, more than half of all nutmeg is consumed in four foods: bologna, pork sausage, frankfurters, and doughnuts. It thus presented an opportunity to see how heavy consumers of those foods would skew total intake distribution. The flavor ingredient 4-(p-hydroxyphenyl)butan-2-one was chosen because it is used in, and occurs naturally, almost entirely in only one flavor, raspberry flavor, and is far less widely consumed than chocolate, vanilla, or citrus flavors. Thus, it is a good example of an uncommon ingredient consumed by those who particularly like the few foods in which it is used (Hall, 1975). Column 2 [" per capita (External Check)"] lists per capita intake derived from import data, product manufacturer surveys, or tariff reports, all independent of the NAS survey to serve as an external check. Column 3 (" per capita [NAS(lbs)/0.6]") lists the per capita intake from the poundage reported in the NAS survey assumed to be 60% complete. Further research (see references to NRC surveys) showed this to be generally a valid assumption, but with certain qualifications. Ingredients such as sugar, corn syrup, and salt, could not be fully or reliably reported by bakers and other small food processors, whereas the flavor industry reported more fully, and the 60% assumption, while used, usually resulted in a poundage per capita overestimate. For Section A, Column 4 ["50th Percentile (Total Panel)"] presents the total panel 50th percentile for each substance, and Column 4a ("50th Percentile 3 S.D. Total Panel"), the 50th percentile plus 3 standard deviations. Column 4b ["very high (Total Panel)"] shows the so-called "very high intake". This is not a realistic figure, because no person could consume all foods using an ingredient at the highest level for every food in every major category. Note particularly, also, the earlier discussion of the exaggeration that arises from assuming that an ingredient used only in a few foods is used in all foods in a major food category. The "very high intake" serves simply as a wholly unreachable and therefore unrealistic value. Given the remaining and inevitable uncertainties due to possible errors in estimations, and possible non-food uses, the agreement between the total panel 50th percentile (Section A, Column 4), the NRC per capita, (Column 3), and the per capita from external sources (Column 2), is entirely satisfactory. However, calculation "A" did not provide data on only those who actually consumed the substance ("eaters only"), and the statistical treatment was not as solidly based as was desirable. This led to an improved calculation, "B", as shown in Table 3. Table 2, Section B, presents the results of these calculations. The substances shown in Column 1 of Section B were selected, as discussed in the referenced reports themselves, by the FDA or the NAS committee (NAS, 1976). An improved method of calculation was used, as described below, which provides the total sample mean (Column 4 ["Mean (Total Panel)"]) as a check against the per capita data in Columns 2 and 3. The external per capita figure for calcium propionate is twice the total panel mean, probably due primarily to usage by many small bakeries not included in the survey, and also to the high wastage of bread. The slightly smaller discrepancy for MSG may well reflect several added sources of error. Even so, comparison of the total panel mean with the per capita data from the NAS survey and the external checks shows satisfactory agreement. Food consumption surveys have significant and well-recognized sources of error in addition to those affecting the NRC surveys, and it seems unlikely that it is practical to expect intake estimates derived by different means to correspond more closely than within a factor of two. For Section B, Column 4 ["Mean (Total Panel)"] provides the total panel mean, Column 4a ["Mean (Eaters Only)"] the "eaters only" mean age, 2 and over and Column 4b ["S.D. (Eaters Only)"], the standard deviation for "eaters only". The procedure in calculation B does not eliminate all sources of error. Intake is still calculated from "disappearance figures" ( i.e, the levels introduced into food during processing, not the lower levels actually consumed). Thus, ignoring the often large losses due, for example, to volatilization or leaching in processing, preparation and storage result in exaggerated estimates of intake. In the other direction, heavy (frequent) eaters will often eat larger portion sizes, resulting in a tendency to underestimate higher intakes. The result overall, however, remains highly satisfactory. Table 1 Calculation "A" Step Operation Result For each specific food (SF)a e.g. bologna, within a broad food category, eg., meats, obtain from MRCAb the 1 total number of eatings of each SF by the total panel, in two weeks 14 days × 12,337 (persons in panel) = mean frequency of eating, p/d × 2 mean portion size in gc = mean consumption of SF, g/p/d × 3 probability of SF containing ingredient "I"d = probable mean consumption of SF containing I g/p/d × 4 usual use level of "I" in ppm of major food = 50th percentile of probable intake of I category containing SF ‰ 1,000e from SF in mg/p/d × 5 90th percentile MRCA frequency of eating = 90th percentile of probable intake of I 50th percentile MRCA frequency of eating from SF in mg/p/d 6 & 7 sum 4 and 5 separately across all SFs in that = 50th and 90th percentiles of probable major food category intake of I from that major food category Table 1 (cont'd) Calculation "A" Step Operation Result 8 fit a curve, assuming normal distribution, to = 50th percentile and standard deviation the points determined in 6 and 7 and calculate (SD) for each major food category 9 repeat steps 1 through 8 and sum across all = 50th percentile and SD in mg/p/d food categories probable total intake 10 repeat steps 4 and following, using "average = "very high intake" maximum use level" from the survey for each major food category a Specific food (SF), in this context, denotes a narrow category of very similar foods serving the same dietary and market niches (e.g. all bologna, all cola drinks). The eatings of each SF include in one total for each SF all eatings of similar brands or home-prepared versions of the same SF. There were approximately 10,000 such SFs. b The Market Research Corporation of America (MRCA) is a private market research company that surveys usage of food products and components It uses a nationally representative panel and records for each panel member the number of servings of each specific loud for each day of a two-week period. The panelists' reporting periods are distributed evenly throughout the year. The 1973 NAS report used data from the third Menu Census (1967-1968) involving 12,871 individuals. The dataused in calculations "A" and "B" came from the Fourth Menu Census (1972-1973) involving 12,337 individuals. c The USDA Food Consumption Survey (USDA, 1965) d Obtained from a consensus panel of experts knowledgeable about the ingredient and the food. e From NAS, 1973 Table 2 Distribution of MRCA 14-Day Average Daily Intakes (in mg/person/day)a of Selected Food Ingredients (1) (2) (3) (4) (4a) (4b) (5) "High Eaters Section Substance per capita per capita Mean Mean plus "Very High" Only" (External [NAS (lbs.)/0.6] (Total Panel) 3 S.D.b (Total Panel) [NAS Check) Total Panel (lbs.)/0.6]×10 A Nutmeg 23.6 10.3 15.7 51.3 71.1 103 Nutmeg Oil 1.2 1.0 2.0 5.7 7.1 10 A Mace 3.3 2.8 3.3 8.6 14.4 28 Mace, Oil NAc 0.26 0.19 0.67 0.46 2.6 Mace, Oleoresin NA 0.070 0.13 0.39 0.41 0.70 A 4-p-Hydroxyphenyl 0.048 0.086 0.041 0.16 0.37 0.86 Butan-2-one Table 2 (cont'd). (1) (2) (3) (4) (4a) (4b) (5) "High Eaters Section Substance per capita per capita Mean Mean plus "Very High" Only" (External [NAS (lbs.)/0.6] (Total Panel) 3 S.D.b (Total Panel) [NAS Check) Total Panel (lbs.)/0.6]×10 Mean Mean S.D. (Total Panel) (Eaters Only) (Eaters Only) B Calcium Propionate 92 33 44.8 45.1 20.7 330 Sodium Propionate NA 16 33,2 33.3 14.7 160 B Carob Bean Gum NA 26 35.6 36.1 35.0 260 Chondrus Extract 19.3 13 10.6 11.1 18.5 130 B Mono- and diglycerides NA 727 884 885 467 7,270 Monosodium Glutamate 186 194 98.8 99.0 88.5 1,940 (MSG) a See text for explanation of columns. b S.D. = Standard Deviation. Table 3 Calculation "B" 1 From the MRCA survey, obtain the total number of eatings of each SF, by each panel member, each day over the 14 - day period × 2 USDA mean portion size, for that person's = quantity of SF in g for that age group and the relevant major food person, that day category × 3 weighted mean of usual use level of I in = quantity of I in mg/d, that ppm ‰ 1,000 person, that day, if all of the SF contained I × 4 probability that particular SF contains I = expected intake of I for that person for that day, mg/d 5 Repeat steps 1 through 4 for that person for = expected intake of I for each of the 14 days that person for 14 days, mg 6 ‰ 14 = average daily intake over 14 days for that person, g/p/d 7 tabulate all intakes and compute = total panel mean intake of I mg/p/d 8 discard all persons with zero intake for the = "eaters only" mean and SD 14 day period, tabulate all "eaters only" of for I, mg/p/d I, age 2 and over, and compute Unfortunately, this more refined approach, while successful, is extremely elaborate, tedious, and expensive. The MRCA panel is expensive to maintain and operate, and the data are correspondingly costly. The approach requires extensive input from a broadly-based, necessarily large and well-informed panel of expert technologists. Such an effort can only be justified in those few cases where it is necessary to obtain the distribution of intakes with maximum accuracy. Given the wide safety margins food ingredients typically enjoy, and the uncertainties and assumptions inherent in interpreting toxicological data, such effort is rarely necessary. A much more appropriate, but quick and easy, method of estimating intake was needed, so long as it was conservative, but not excessively so. This led Rulis and others in FDA (Rulis et al., 1984; Rulis, 1987) to test and adopt the assumption that, except for an unusually narrow margin of safety or specific concern, the per capita "eaters only" intake could safely and conservatively be assumed - even for heavy eaters - simply by multiplying the best available estimate of per capita intake by ten (Column 5 ["High Eaters Only"]). Comparison of Column 5 with Column 4b, for Section B, makes clear that in every case the: "High Eaters Only" ( per capita × 10) estimate far exceeds the "Eaters Only mean plus 3 standard deviations", yet in no case are the estimates so high as to be unusable. With the special factors surrounding the use of flavoring ingredients (i.e., completeness of the surveys, typical volatility with consequent loss in processing, and self-limiting nature) the procedure in Column 5 is practical but highly conservative. It has been widely used in other publications (FDA, 1982, 1993; Easterday et al., 1993). It can be used safely in all instances except when a very narrow margin of safety requires maximally precise data for both toxicological effects and exposure. Recently, still more sophisticated methods for analyzing USDA Food Consumption Survey data have become available under license for those applications in which the need for relatively high precision of estimates justifies the cost (Helmbach, 1994). In nearly all instances where broadly- based ingredient disappearance data are available, per capita × 10, in actuality, a very high "eaters only" estimate, remains the approach that is by far the simplest and least expensive, while being conservative but realistic. REFERENCES Burdock, G.A., Wagner, B.M., Smith, R.L., Munro, I.C. and Newberne, P.M. 1990. Recent progress in the consideration of flavoring ingredients under the food additives amendment. 15. GRAS substances. A list of flavoring ingredient substances considered generally recognized as safe by the Flavor & Extract Manufacturers' Association Expert Panel. Food Technol 44(2):78, 80, 82, 84, & 86. Easterday, O.D., Ford, R.A., Hall, R.L., Stofberg, J., Cadby, P. and Grundschober, F. 1992. A Flavor Priority Ranking System, Acceptance and Internationalization. In: Finley, J.W., Robinson, S.F. and Armstrong, D.J. (Eds.) Food Safety Assessment. American Chemical Society, ACS Symposium Series No. 484. FDA. 1982. Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. Redbook. U.S. Food and Drug Administration, Bureau of Foods, Washington, DC. FDA. 1993. Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. Redbook II (Draft). U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Washington, DC. Hall, R.L. and Oser, B.L. 1965. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. III. GRAS substances. Food Technol 19(2):151-197. Hall, R.L. and Oser, B.L. 1970. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment. IV. GRAS substances. Food Technol 24(5):25-28, 30-32, 34. Hall, R.L. 1975. Unpublished data from personal communication to the NAS/NRC Committee on GRAS List Survey - Phase III, and to the FEMA. Heimbach, J.T. 1994. Personal communication re: TAS-DIET from J.T. Helmbach, Technical Assessment Systems, Inc., Washington, D.C. NAS. 1973. A comprehensive Survey on the Use of Food Chemicals Generally Recognized as Safe, National Technical Information Service (NTIS) PB-221949. NAS. 1976. Estimating Distribution of Daily Intakes of Certain GRAS Substances. National Research Council, Washington, DC. Prepared for: Food and Drug Administration, Washington, DC. December 1976. National Technical Information Service (NTIS) PB-299-381. NAS. 1978. Resurvey of the Annual Poundage of Food Chemicals Generally Recognized as Safe, National Technical Information Service (NTIS) PB288-081. NAS. 1979. Comprehensive Survey of Industry on the Use of Food Additives, National Technical Information Service (NTIS) PB80-113418. NAS. 1984. Poundage Update of Food Chemicals, National Technical Information Service (NTIS) PB84-162148. NAS. 1989. 1987 Poundage and Technical Effects Update of Substances Added to Food. National Research Council, Washington, DC. Prepared for: Food and Drug Administration, Washington, D.C. NTIS Report No. PB91-127266. Oser, B.L. and Ford, R.A. 1973a. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 6. GRAS substances. Food Technol 27(1):64-67. Oser, B.L. and Ford, R.A. 1973b. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 7. GRAS substances. Food Technol 27(11):56-57. Oser, B.L. and Ford, R.A. 1974. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 8. GRAS substances. Food Technol 28(9):76-80. Oser, B.L. and Ford, R.A. 1975. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 9. GRAS substances. Food Technol 29(8):70-72. Oser, B.L. and Ford, R.A. 1977. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 10. GRAS substances. Food Technol 31(1):65-74. Oser, B.L. and Ford, R.A. 1978. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 11. GRAS substances. Food Technol 32(2):60-70. Oser, B.L. and Ford, R.A. 1979. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 12. GRAS substances. Food Technol 33(7):65-73. Oser, B.L. and Hall, R.L. 1972. Recent progress in the consideration of flavoring Ingredients under the Food Additives Amendment 5. GRAS substances. Food Technol 26(5):35-42. Oser, B.L. and Hall, R.L. 1977. Criteria employed by the Expert Panel of FEMA for the GRAS evaluation of flavoring substances. Food Cosmet Toxicol 15:457-466. Oser, B.L., Ford, R.A. and Bernard, B.K. 1984. Recent Progress in the consideration of flavoring ingredients under the Food Additives Amendment 13. GRAS substances. Food Technol 38(10):66-89. Oser, B.L., Well, C.S., Woods, L.A. and Bernard, B.K. 1985. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 14. GRAS substances. Food Technol 39(11):108-117. Rulis, A.M., Hattan, D.G., and Morgenroth, V.H.,III. 1984. FDA's priority-based assessment of food additives. I. Preliminary results. Reg Toxicol Pharm 4:37-56. Rulis, A.M. 1987. Safety assurance margins for food additives currently in use. Reg Toxicol Pharm 7:160-168. Smith, R.L. and Ford, R.A. 1993. Recent progress in the consideration of flavoring ingredients under the Food Additives Amendment 16. GRAS substances. Food Technol-June 1993, pp. 104-117. ANNEX 5 Appendix B SUBSTANCES IN REFERENCE DATABASE BY ORDER OF STRUCTURAL CLASS Cramer et al. (1978) Structural Class I 1 acetic acid 2 acetoin 3 acetone 4 adipic acid 5 allura red AC 6 aminoundecanoic acid, 11- 7 ascorbic acid 8 ascorbic acid, 1- 9 benzaldehyde 10 benzoic acid 11 benzyl acetate 12 benzyl alcohol 13 bis(2-ethylhexyl)phthalate 14 brilliant black BN 15 butanediol, 1,3- 16 butanol, 2- 17 butanol, n- 18 butyl benzyl phthalate 19 butylated hydroxyanisole 20 butyrolactone, gamma- 21 calcium cyclamate 22 calcium formate 23 calcium stearoyl lactylate 24 carotenoic acid, beta-apo-8'-, methyl ester 25 cinnamaldehyde 26 citral 27 citranaxanthin 28 cumene 29 di(2-ethylhexyl)adipate 30 dibutyl phthalate 31 diethyl phthalate 32 diethylene glycol 33 diethylene glycol monoethyl ether 34 dimethoxane 35 dimethyl terephthalate 36 dimethylcarbonate 37 dimethyldicarbonate 38 dimethylphenol, 2,4- 39 dimethylphenol, 2,6- 40 dimethylphenol, 3,4- 41 dioctyl sodium sulfosuccinate 42 disodium 5'-guanylate 43 disodium 5'-inosinate 44 dodecyl gallate 45 ethanol 46 ethyl acetate 47 ethyl acrylate 48 ethyl butyrate 49 ethyl ether 50 ethyl formate 51 ethyl glycol monomethyl ether 52 ethyl heptanoate 53 ethyl nonanoate 54 ethyl-1-hexanol, 2- 55 ethylbenzene 56 ethylbutyric acid, 2- 57 ethylene glycol 58 ethylhexanoic acid, 2- 59 ethylphthalyl ethylglycolate 60 eugenol 61 FD & C blue No. 1 62 FD & C blue no. 2 63 formaldehyde 64 fumaric acid 65 geranyl acetate 66 glutamate, monosodium 67 glutamic acid hydrochloride 68 glutamic acid, 1- 69 glycerol 70 glyceryl tribenzoate 71 hexenal, 2- (trans) 72 hexylresorcinol, 4- 73 hydroquinone 74 hydroxybenzoic acid butyl ester, p- 75 hydroxybenzoic acid ethyl ester, p- 76 hydroxybenzoic acid methyl ester, p- 77 hydroxybenzoic acid propyl ester, p- 78 hydroxybenzyl acetone, p- 79 inosine monophosphate 80 ionone 81 isoamyl butyrate 82 isoamyl salicylate 83 isobutyl alcohol 84 isomaltitol 85 isopropyl alcohol 86 isovaleric acid 87 lactitol 88 limonene, d- 89 lithocholic acid 90 malonaldehyde, sodium salt 91 mannitol 92 mannitol, d- 93 menthol 94 methanol 95 methyl ethyl carbonate 96 methyl methacrylate 97 methyl salicylate 98 methyl-1-phenylpentan-2-ol, 4- 99 methylenebis, 2,2'- 100 methylphenol, 3- 101 methylphenylcarbinyl acetate 102 myrcene, beta- 103 nonalactone, gamma- 104 octyl acetate 105 octyl gallate 106 oleylamine 107 oxalic acid 108 phenol 109 phenoxyethanol 110 phenyl-1-propanol, 2- 111 phenylalanine 112 potassium sorbate 113 propyl gallate 114 propylene glycol 115 resorcinol 116 retinol 117 riboflavin 118 sodium benzoate 119 sodium erythorbate 120 sodium lauryl glyceryl ether sulfonate 121 sodium lauryl trioxyethylene sulfate 122 sodium stearoyl lactylate 123 sorbic acid 124 stearyl tartrate 125 styrene 126 sucrose monopalmitate 127 sucrose monostearate 128 tartaric acid 129 tertiary butyl hydroquinone 130 tocopherol, alpha- 131 tolualdehyde 132 toluene 133 triethylene glycol 134 triethylene glycol monomethyl ether 135 trimethylamine 136 undecalactone, gamma- 137 vanillin 138 xylitol Cramer et al. (1978) Structural Class II 1 acrylic acid 2 allyl alcohol 3 allyl heptanoate 4 allyl hexanoate 5 butylated hydroxytoluene 6 caffeine 7 carotene, beta- 8 carvone 9 carvone, d- 10 diacetyl 11 diallyl phthalate 12 diketopiperazine 13 ethyl maltol 14 ethyl vanillin 15 ethylhexyl phthalate, mono-2 16 etretinate 17 fenthion 18 furfural 19 isobornyl acetate 20 isophorone 21 maltol 22 methyl amyl ketone 23 methyl anthranilate 24 piperidine 25 piperonal 26 propargyl alcohol 27 pyridine 28 thujone Cramer et al, (1978) Structural Class III 1 (1-naphthyl)ethylene--diamine dihydro chloride, N- 2 (2-chloroethyl)trimethyl-ammonium chloride 3 (8-beta)N-cyclohexyl-6-methyl- 1 -(1 -methyl ethyl)ergoline-8 -carboxamide 4 (chloroacetyl)-acetanilide, 4'- 5 1,1'-(2,2,2-trichloroethylidene)bis(4-chloro)-benzene 6 11-oxo-11H-pyrido(2,1-b)quinazoline-2-carboxylic acid 7 2(2,4,5-trichlorophenoxy) propionic acid 8 2-(2-methyl--4-chlorophenoxy)propionic acid 9 4-(2-methyl-4-chlorophenoxy) butyric acid 10 acenaphthene 11 acephate 12 acesulfame potassium 13 acetaminophen 14 acetoacetamide 15 acetoacetamide-N-sulfonic acid 16 acetochlor 17 acetonitrile 18 acifluorin sodium 19 acrylamide 20 acrylonitrile 21 alachlor 22 albendazole 23 albendazole sulfoxide 24 aldicarb 25 aldicarb sulfone 26 alinidine hydrobromide 27 allyl isovalerate 28 amaranth 29 ameltolide 30 ametryn 31 amino-4-ethoxy-acetanilide, 3- 32 amino-4-nitrophenol, 2- 33 amino-5-nitrophenol, 2- 34 aminophenol, p- 35 amitraz 36 ammonium carmine 37 amphetamine sulfate, dl- 38 ampicillin trihydrate 39 anethole, trans- 40 anilazine 41 anisidine hydrochloride, p- 42 anthranilic acid 43 arochlor 1254 44 aspartame 45 asulam 46 atrazine 47 avermectin B1 48 azaperone 49 azinphos methyl 50 azorubine 51 azuletil (KT1-32) 52 baythroid 53 benomyl 54 bentazon 55 benzofuran 56 benzoic acid, 2-[[[[N-(4-methoxy-6-methyl -1,2,3-triazin-2-yl)-N-methylamino]carbonyl] amino]sulfonyl] methyl ester 57 benzoin 58 benzyl violet 4B 59 benzyl-p-chlorophenol, o- 60 betaxolol 61 bidrin 62 biphenthrin 63 biphenyl, 1,1- 64 biphenylamine hydrochloride, 2- 65 bis(2-chloro-1-methyl-ethyl)ether Technical Grade 66 bis(2-chloroisopropyl)ether 67 bisphenol A 68 bromoacetic acid, 2- 69 bromodichloromethane 70 bromomethane 71 bromoxynil 72 bromoxynil octanoate 73 brown FK 74 butyl chloride, n- 75 butylate 76 calcium cyanamide 77 canthaxanthin 78 caprolactam 79 captafol 80 captan 81 carazolol 82 carbaryl 83 carbendazim 84 carbofuran 85 carbon tetrachloride 86 carbosulfan 87 carboxin 88 carmoisine 89 chloramben 90 chlordane 91 chlorendic acid 92 chlorimuron-ethyl 93 chloro-p-toluidine, 3- 94 chloroacetic acid 95 chloroaniline hydrochloride, p- 96 chlorobenzene 97 chlorobenzilate 98 chlorodibromomethane 99 chloroform 100 chlorofructose, 6- 101 chloronaphthalene, beta- 102 chlorophenol, 2- 103 chloropropylate 104 chlorothalonil 105 chlorotoluene, o- 106 chlorpheniramine maleate 107 chlorpromazine 108 chlorsulfuron 109 chlorthal dimethyl 110 chocolate brown HT 111 C.I. Acid Orange 12 112 C.I. Acid Orange 3 113 C.I. Acid Red 18 114 C.I. Disperse Blue 1 115 C.I. acid red 14 116 C.I. disperse yellow 3 117 C.I. pigment red 23 118 C.I. solvent yellow 14 119 cimetidine 120 clofentezine 121 clonitralid 122 closantel 123 codeine 124 coumaphos 125 coumarin 126 crufomate 127 curcumin 128 cyclodextrin, beta- 129 cyclohexylamine 130 cyclophosphamide 131 cyhalothrin 132 cypermethrin 133 cyromazine 134 daminozide 135 decabromodiphenyl oxide 136 deoxyquinine 137 diamino-2,2'-stilbenedisulfonic acid, 4,4'-, disodium sa 138 diaminophenol dihydrochloride, 2,4- 139 diazinon 140 dibenzo-p-dioxin 141 dibromobenzene, 1,4- 142 dicamba 143 dichloro-2-propanol, 1,3- 144 dichloro-p-phenylenediamine, 2,6- 145 dichlorobenzene, 1,2- 146 dichlorobenzene, 1,4- 147 dichlorobenzilic acid 148 dichlorodifluoromethane 149 dichloroethane, 1,1- 150 dichloroethane, 1,2- 151 dichloroethylene, 1,1- 152 dichloroethylene, trans- 1,2- 153 dichloromethane 154 dichlorophenol, 2,4- 155 dichlorophenoxyacetic acid, 2,4- 156 dichloropropanol, 2,3- 157 dichloropropene, 1,3- 158 dichlorvos 159 dicofol 160 dieldrin 161 diethyldithiocarbamate 162 diethylthiourea, N,N'- 163 difenzoquat 164 diflubenzuron 165 dihydroavermectin-B1 a, 22,23- 166 dihydroavermectin-B1 b, 22,23- 167 dihydrocoumarin, 3,4- 168 diiodomethyl p-tolyl sulfone 169 dimethipin 170 dimethoate 171 dimethoxyaniline hydrochloride, 2,4- 172 dimethoxybenzidine-4,4'-diisocyanate, 3,3'- 173 dimethyl hydrogen phosphite 174 dimethyl methylphosphonate 175 dimethyl morpholinophosphoramidate 176 dimethylaniline, N,N'- 177 dinitrobenzene, m- 178 dinitrotoluene, 2,4- 179 dinocap 180 dinoseb 181 diphenamid 182 diphenhydramine hydrochloride 183 diphenylamine 184 diphenylhydantoin, 5,5'- 185 diphenylhydantoin, 5,5- 186 diquat 187 disulfoton 188 dithiobiurea, 2,5- 189 diuron 190 dodecylguanidine acetate, n- 191 EDTA, disodium 192 endothall 193 ephedrine sulfate 194 epichlorohydrin 195 erythromycin stearate 196 erythrosine 197 ethalfuralin 198 ethephon 199 ethion 200 ethyl dipropylthiocarbamate, s- 201 ethyl p-nitrophenyl phenylphosphorothioate 202 ethylene chlorohydrin 203 ethylene thiourea 204 ethylmethylphenylglycidate 205 fast green FCF 206 febantel 207 fenamiphos 208 fenbendazole 209 fenchlorphos 210 fluometuron 211 fluoranthene 212 fluorene 213 fluridone 214 flurprimidol 215 flutolanil 216 fluvalinate 217 folpet 218 fonofos 219 fosetyl-al 220 glufosinate-ammonium 221 glyphosate 222 haloxyfop-methyl 223 HC blue no. 1 224 HC blue no. 2 225 HC yellow 4 226 heptachlor 227 heplachlor epoxide 228 hexabromobenzene 229 hexachlorobenzene 230 hexachlorobutadiene 231 hexachlorocyclohexane, gamma- 232 hexachlorocyclopentadiene 233 hexachloroethane 234 hexachlorophene 235 hexahydro-1,3,5-trinitro-1,3,5-triazine 236 hexamethylenetetramine 237 hexazinone 238 hexythiazox 239 hydralazine 240 hydrazobenzene 241 hydrochlorothiazide 242 hydroxypropyl methanethiolsulfonate, 2- 243 hydroxyquinoline, 8- 244 imazalil 245 imazaquin 246 imazethapyr 247 iodinated glycerol 248 ipazilide 249 iprodione 250 ipronidazole 251 isopropalin 252 isoxaben 253 jervine 254 lactofen 255 lansoprazole 256 levamisole 257 linamarin 258 linuron 259 londax 260 malaoxon 261 malathion 262 maleic anhydride 263 maleic hydrazine 264 manidipine hydrochloride 265 melamine 266 mepiquat chloride 267 mercaptobenzothiazole, 2- 268 merphos 269 merphos oxide 270 meso-2,3-dimercaptosuccinic acid 271 metalaxyl 272 methamidophos 273 methidathion 274 methomyl 275 methoxychlor 276 methoxypsoralen, 8- 277 methyl carbamate 278 methyl ethyl ketoxime 279 methyl parathion 280 methyl-4-chlorophenoxyacetic acid, 2- 281 methyl-N-methylanthranilate 282 methyldopa sesquthydrate, alpha- 283 methylolacrylamide, N- 284 metolachlor 285 metribuzin 286 metsulfuron methyl 287 mexacarbate 288 miporamicin 289 mirex 290 molinate 291 monodiethanolamine salt of riboflavin-5'-phosphate 292 monuron 293 myclobutanil 294 naled 295 nalidixic acid 296 naphthoxy acetic acid, 2- 297 napropamide 298 natamycin 299 nitro-o-toluidine, 5- 300 nitro-p-phenylenediamine, 2- 301 nitroaniline, p- 302 nitroanthranilic acid, 4- 303 nitrofurantoin 304 nitrofurazone 305 nitroguanidine 306 nitronaphthalene, 1- 307 nitrosodiphenylamine, N- 308 norflurazon 309 ochratoxin A 310 octabromodiphenyl ether 311 octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine 312 olaquindox 313 oryzalin 314 oxadiazon 315 oxamyl 316 oxfendazole 317 oxyfluorfen 318 oxytetracycline 319 oxytetracycline hydrochloride 320 oxythioquinox 321 paclobutrazol 322 paraquat 323 parathion 324 patulin 325 pendimethalin 326 penicillin VK 327 pentabromodiphenyl ether 328 pentachloroethane 329 pentachloronitrobenzene 330 pentachlorophenol 331 pentaerythritol tetranitrate 332 permethrin 333 phenformin 334 phenyl-2-naphthylamine, n- 335 phenyl-3-methyl-5-pyrazolone, 1- 336 phenylbutazone 337 phenylenediamine, m- 338 phenylephrine hydrochloride 339 phosmet 340 phosphamidon 341 photodieldrin 342 phthalamide 343 phthalic anhydride 344 picloram 345 pilsicainide hydrochloride 346 pirimiphos-methyl 347 pravadoline 348 probenecid 349 prochloraz 350 proflavine monohydrochloride hemihydrate 351 promethazine hydrochloride 352 prometon 353 prometryn 354 pronamide 355 propachlor 356 propanil 357 propargite 358 propazine 359 propham 360 propiconazole 361 propiverine hydrochloride (P-4) 362 propoxur 363 propylene dichloride 364 pydrin 365 pyrazinamide 366 pyrene 367 pyrimidinone 368 quercetin 369 quinalphos 370 quinine hydrochloride 371 quinofop ethyl 372 red 2G 373 reserpine 374 resmethrin 375 thudamine 6G 376 ronidazole 377 rotenone 378 saccharin 379 sethoxydim 380 simazine 381 sodium 2,2-dichloropropionate 382 sodium cyclamate 383 sodium fluoroacetate 384 sodium naphthionate 385 sorbitan monolaurate 386 sorbitan monostearate 387 succinic anhydride 388 sucrose acetate isobutyrate 389 sulfadimidine 390 sulfisuxazole 391 sulfolene, 3- 392 sunset yellow FCF 393 suplatast tosilate 394 tebuthiuron 395 terbacil 396 terbutryn 397 tert-butyl-2-chlorophenol, 4- 398 tetrachlorobenzene, 1,2,4,5- 399 tetrachloroethane, 1,1,1,2- 400 tetrachloroethane, 1,1,2,2- 401 tetrachloroethylene 402 tetrachlorophenol, 2,3,4,6- 403 tetrachloropyridine, 2,3,5,6- 404 tetrachlorvinphos 405 tetracycline hydrochloride 406 tetraethyldithiopyrophosphate 407 tetraethylthiourea disulfide 408 tetrahydrocannabinol, delta-9- 409 tetrakis(hydroxymethyl)phosphonium chloride (THPC) 410 tetrakis(hydroxymethyl)phosphonium sulphate (THPS) 411 thiabendazole 412 thiameturon methyl 413 thiobencarb 414 thiophanate-methyl 415 thiram 416 tocopheryl acetate, dl-alpha- 417 toluenediamine hydrochloride, 2,6- 418 toluenediamine sulfate, 2,5- 419 tralomethrin 420 trenbolone acetate 421 trenbolone hydroxide, 17-alpha- 422 triadimefon 423 triallate 424 triamterene 425 tribromomethane 426 trichloroacetonitrile 427 trichlorobenzene, 1,2,4- 428 trichloroethane, 1,1,2- 429 trichloroethane, 1,1,1- 430 trichloroethylene 431 trichloroethylene, 1,1,2- 432 trichlorogalactosucrose 433 trichlorophenol, 2,4,5- 434 trichlorophenoxyacetic acid, 2,4,5- 435 tridiphane 436 triethylene tetramine dihydrochloride 437 trifluralin 438 trimethylaniline, 2,4,5- 439 trinitrotoluene, 2,4,6- 440 tris-(2-chloroethyl) phosphate 441 trisulfuron 442 tylosin 443 vinclozolin 444 vinyl chloride 445 zatosetron maleate 446 zearalenone 447 zeranol
See Also: Toxicological Abbreviations