s7 ch30 food_toxicology

UNIT 7

APPLICATIONS OFTOXICOLOGY

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Copyright © 2001 by The McGraw-Hill Companies Retrieved from: www.knovel.com

CHAPTER 30

FOOD TOXICOLOGY

Frank N. Kotsonis, George A. Burdock,and W. Gary Flamm

TOLERANCE SETTING FOR SUBSTANCES IN FOODS

Pesticide ResiduesDrugs Used in Food-Producing AnimalsUnavoidable Contaminants

Heavy MetalsChlorinated OrganicsNitrosamines, Nitrosamides, and N-Nitroso

SubstancesFood-Borne Molds and Mycotoxins

SUBSTANCES FOR WHICH TOLERANCES MAY NOTBE SET

Toxins in Fish, Shellfish, and TurtlesDinoflagellate Poisoning (Paralytic Shellfish

Poisoning or PSP; Saxitoxin)Amnesic Shellfish Poisoning (Domoic Acid)Ciguatera PoisoningPuffer Fish Poisoning (Tetrodotoxin)Moray Eel PoisoningFish Liver PoisoningFish Roe PoisoningAbalone Poisoning (Pyropheophorbide A)Sea Urchin PoisoningSea Turtle Poisoning (Chelonitoxin)Haff Disease

Microbiologic Agents—Preformed Bacterial ToxinsClostridium botulinum and Clostridium butyricumClostridium perfringensBacillus cereusStaphylococcus aureusEscherichia coli

Bovine Spongiform EncephalopathySubstances Produced by CookingMiscellaneous Contaminants in Food

CONCLUSIONS

INTRODUCTION TO FOOD TOXICOLOGY

Uniqueness of Food ToxicologyNature and Complexity of FoodImportance of the Gastrointestinal Tract

SAFETY STANDARDS FOR FOODS, FOODINGREDIENTS, AND CONTAMINANTS

The Food, Drug and Cosmetics Act Provides for a Practicable ApproachThe Application of Experience: Generally

Recognized as Safe (GRAS)Use of Tolerances

Food and Color AdditivesMethods Used to Evaluate the Safety of Foods,

Ingredients, and ContaminantsSafety Evaluation of Direct Food and Color AdditivesSafety Determination of Indirect Food AdditivesSafety Requirements for GRAS Substances

Importance of the GRAS ConceptTransgenic Plant (And New Plant Varieties) PolicyMethods for Establishing Safe Conditions of Use

for Novel FoodsDietary Supplements

Assessment of CarcinogensCarcinogenicity as a Special ProblemBiological versus Statistical SignificanceCarcinogenic Contaminants

SAFETY OF FOOD

Adverse Reactions to Food or Food IngredientsFood AllergyFood Toxicity (Poisoning)Food IdiosyncrasyAnaphylactoid ReactionsPharmacologic Food ReactionsMetabolic Food Reactions

Importance of Labeling

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1050 UNIT 7 APPLICATIONS OF TOXICOLOGY

INTRODUCTION TO FOODTOXICOLOGY

This chapter describes the general principles of food toxicologyand explains how those principles have been shaped by existingfood laws and applied to the safety assessment of foods, food in-gredients, and food contaminants. Food toxicology is different fromother subspecialties in toxicology largely because of the nature andchemical complexity of food. The necessity for practical and work-able approaches to the assessment of food safety is addressedthroughout the chapter.

The typical western diet contains hundreds of thousands ofsubstances naturally present in food and many more which formin situ when food is cooked or prepared. Many of these substancesaffect the nutritional and esthetic qualities of food, including ap-pearance and organoleptic properties (i.e., conferring flavor, tex-ture, or aroma) that determine whether or not we will even try thefood or take a second bite, respectively. While these or other sub-stances present in food may be nutritional and/or gratifying, theymay not necessarily be “safe” in any amount or for any intendeduse. The Federal Food, Drug and Cosmetic (FD&C) Act gives thefederal government the authority to ensure that all food involvedin interstate commerce is safe. Congress, in writing the FD&C Act(and its subsequent amendments), understood that safety cannot beproved absolutely and indicated instead that the safety standard forsubstances added to food can be no more than a reasonable cer-tainty that no harm will occur. As pointed out in other sections ofthis chapter, the language of the FD&C Act effectively providesfor practical and workable approaches to the assessment of safetyfor food, food ingredients, and food contaminants. Because foodis highly complex, the legal framework provided by Congress forthe regulation of food and substances in food was kept simple sothat it would work. The basic element of the framework is thatfood, which is defined as articles or components of articles usedfor food or drink for humans or animals, bears the presumption ofsafety [sections 201(f) and 402(a)(1) of the FD&C Act]. This meansthat a steak or a potato is presumed to be safe unless it contains apoisonous or deleterious substance in an amount shown to make itordinarily injurious to health. In essence, this presumption of safetywas born of necessity. If the hundreds of thousands of substancesnaturally present in food were subject to the same strictures andlimitations that apply to added substances, food shortages couldeasily result. To avoid such crises, Congress developed a safetystandard that would not force regulatory authorities to ban com-mon, traditional foods.

In cases where the substance is not naturally present in foodbut is a contaminant or added ingredient, the safety standard isquite different. This standard decrees a food to be adulterated if itcontains any poisonous or deleterious substance that may render itinjurious. Therefore, the presence of a substance that is not a nat-ural component of a food demands a far higher standard of safety.The mere possibility that such a substance may render the food in-jurious to health is sufficient to ban the food containing that sub-stance. Thus, for additives and contaminants, Congress recognizedthat these substances are not as complex as food and must, there-fore, meet a higher standard of safety.

An understanding of the term safe is necessary in decidinghow many and what types of studies must be conducted to deter-mine that an added substance is safe. Wisely, the act does not giveexplicit instructions about how safety should be determined anddoes not explicitly define safety. Because neither the law nor the

U.S. Food and Drug Administration (FDA) or the U.S. Departmentof Agriculture (USDA) regulations explicitly define the term safetyfor substances added to food, scientists and their legal and regula-tory counterparts have worked out operational definitions for thesafety of food ingredients. The one principle on which there hasbeen nearly unanimous agreement is that safety concerns in regardto an added substance should focus on both the nature of the sub-stance and its intended conditions of use. It is recognized that sub-stances are not inherently unsafe, it is only the quantity at whichthey are presented in the diet that makes them unsafe. The quan-tity (or “level”) present in the diet is determined by the intendedconditions of use and limitations of use of the substance.

As with food, practical and workable solutions must be foundfor the constituents of additives, because all substances contain amyriad of impurities at trace and even undetectable levels. Deci-sions concerning the safety of impurities and the development ofappropriate specifications for food and color additives to assurethat they are of suitable purity must similarly constitute a work-able approach. In this case, the workable approach involves settingspecification limits on contaminants—limits that are intended toexclude the possibility that the level of contaminants present in anadditive may render the food to which the substance is added un-safe. As a practical matter and because of time and cost consider-ations, established specifications must be relatively simple andstraightforward and must provide reasonable assurance that an in-gredient is of suitable purity for its intended conditions of use.However, it generally is not necessary or practical to require ex-tensive analysis and identification of all individual impurities to es-tablish the fact that a substance is of “food-grade purity.” It shouldbe emphasized that specifications can serve their purpose of as-suring suitable purity only if the manufacturing processes used areadequately controlled to assure consistency in the quality and pu-rity of the product. It should be understood that the philosophy bywhich specifications are established for substances added to foodembodies the belief that not all risks are worthy of regulatory con-cern and control. Implicit in this philosophy is the concept ofthreshold of regulation, which is an important unifying concept infood safety assessment (Flamm et al., 1994).

To understand the meaning in the FD&C Act of the phrase“safe for intended conditions of use” as applied to a substanceadded to food, it is important to recognize that such a determina-tion must rest on a general understanding of the risks posed byfood itself. The requirement that substances added to food be safe(to a reasonable degree of certainty) demands consideration of thefar higher theoretical risk posed by food itself. Food, as stated ear-lier, contains hundreds of thousands of substances, most of whichhave not been fully characterized or tested. The presumption thata food is safe is based on a history of common use and on the factthat the consumption of certain foods is deeply rooted in tradition(e.g., “ethnic” foods or those foods traditionally consumed for aholiday celebration). When the uncertainty about the risk of theadded substance is small compared with the uncertainties attend-ing food itself, the standard of “reasonable certainty of no harm”for the added substance has been satisfied. Thus, for food-like sub-stances, the presumption is that the substance resembles food, isdigested and metabolized as food, and consequently raises fewertoxicologic and safety-related questions than do substances that arenot food-like. And such substances are added either directly or in-directly (substances that may migrate into food from packaging orother food contact surfaces) to foods in only very small or traceamounts, the low levels of exposure aid in demonstrating that the

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CHAPTER 30 FOOD TOXICOLOGY 1051

intended conditions of use of these substances are safe. These broadgeneralizations, however, do not suffice to exempt these food in-gredients from the requirements of thorough safety testing. TheFDA requires that such testing be done but it is tempered by con-siderations of (1) the basic nature of the substance, (2) the level towhich consumers will be exposed as the result of the intended use,and (3) the inherent safety of food and constituents of food.

Over the past several years, there has been increasing intereston the part of consumers in the health-enhancing properties offoods and the components they contain. Substances such as phy-tosterols from vegetable oils and isoflavones from soy have beenisolated and added to other foods at elevated levels to impartcholesterol-lowering abilities. Such products have raised regula-tory questions about whether these substances are functioning asdrugs and should be regulated as such or whether they should beviewed as new nutrients and allowed in foods, as are vitamin Cand iron. At a recent conference, experts in nutritional science con-cluded that the concept of nutrients should be expanded to includea growing number of desirable food constituents that produce quan-tifiable health benefits related to disease prevention (Sansalone,1999). This isolation of new food components and their use in for-tifying food products will necessitate a thorough evaluation ofsafety at the intended level of intake and for the population at large.

Finally, it should be recognized that in most of the world, mi-crobiologic contamination of food represents by far the greatestfood-borne risk facing consumers. Thus, while vigilance in assur-ing the safety of substances added to food under their intended con-ditions of use is appropriate, we should not lose sight of the ma-jor concern of food safety.

Uniqueness of Food Toxicology

The nature of food is responsible for the uniqueness of food toxi-cology. Food is not only essential to all life but also a major con-tributor to the quality of life. Food and drink are enjoyed for theirappearance, aroma, flavor, and texture. They are significant factorsin defining cultures and societies. For example, ethnic foods andgourmet foods have a status that far exceeds their nutritionalbenefits, but any proposal to ban an ethnic food because new datahave raised questions about its safety would be met with strongresistance.

As food occupies a position of central importance in virtuallyall cultures and because most food cannot be commercially pro-duced in a definable environment under strict quality controls, foodgenerally cannot meet the rigorous standards of chemical identity,purity, and good manufacturing practice met by most consumerproducts. The fact that food is harvested from the soil, the sea, orinland waters or is derived from land animals, which are subjectto the unpredictable forces of nature, makes the constancy of rawfood unreliable. Food in general is more complex and variable incomposition than are all the other substances to which humans areexposed. However, there is nothing to which humans have greaterexposure despite the uncertainty about its chemical identity, con-sistency, and purity. Experience has supported the safety of com-monly consumed foods, and the good agricultural practices underwhich food is produced mandate the need for quality controls. Nev-ertheless, it is clear that food is held to a different standard as apractical matter dictated by necessity.

Food also acquires uniqueness from its essential nutrients,which, like vitamin A, may be toxic at levels only 10-fold abovethose required to prevent deficiencies. The evaluation of food in-

gredient substances often must rely on reasoning unique to foodscience in the sense that such substances may be normal con-stituents of food or modified constituents of food as opposed to thetypes of substances ordinarily addressed in the fields of occupa-tional, environmental, and medical toxicology. Assessment of thesafety of such substances, which are added to food for their tech-nical effects, often focuses on digestion and metabolism occurringin the gastrointestinal (GI) tract. The reason for this focus is thatin many cases an ingested substance is not absorbed through theGI tract; only products of its digestion are absorbed, and these prod-ucts may be identical to those derived from natural food.

Nature and Complexity of Food

Food is an exceedingly complex mixture of nutrient and nonnutri-ent substances, whether it is consumed in the “natural” (un-processed) form or as a highly processed ready-to-eat microwave-able meal (Table 30-1). Among the “nutrient” substances, thewestern diet consists of items of caloric and noncaloric value; thatis, carbohydrates supply 47 percent of caloric intake, fats supply37 percent, and protein supplies 16 percent (all three of whichwould be considered “macronutrients”) (Technical AssessmentSystems, Inc., 1992), whereas minerals and vitamins, the “mi-cronutrients,” obviously have no caloric value but are no less es-sential for life.

Nonnutrient substances are often characterized in the popularliterature as being contributed by food processing, but nature pro-vides the vast majority of nonnutrient constituents. For instance,in Table 30-2 one can see that even among “natural” (or minimallyprocessed) foods, there are far more nonnutrient than nutrient con-stituents. Many of these nonnutrient substances are vital for thegrowth and survival of the plant, including hormones and naturallyoccurring pesticides (estimated at approximately 10,000 by Goldet al., 1992). Some of these substances may be antinutrient [e.g.,goiterogens in Brassica, trypsin and/or chymotrypsin inhibitors insoybeans, phytates that may bind minerals (present in soybeans),and antihistamines in fish and plants] or even toxic (e.g., tomatine,cycasin) to humans. An idea of the large number of substancespresent in food is given in the series edited by Maarse and associ-ates (1993), in which approximately 5500 volatile substances arenoted as occurring in one or more of 246 different foods. How-ever, this is only the tip of the iceberg, as the number of unidenti-fied natural chemicals in food vastly exceed the number that havebeen identified (Miller, 1991).

Nonnutrient substances are also added as a result of process-ing, and in fact, 21 CFR 170.3(o) lists 32 categories of direct ad-ditives, of which there are about 3000 individual substances. Ap-proximately 1800 of the 3000 are flavor ingredients, most of which

Table 30-1Food as a Complex Mixture

NUTRIENTS NONNUTRIENTS

Carbohydrates Naturally occurring substancesProteins Food additivesLipids ContaminantsMinerals Products of food processingVitamins

SOURCE: Smith 1991, with permission.

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already occur naturally in food and are nonnutritive. Of the 1800flavoring ingredients that may be added to food, approximatelyone-third are used at concentrations below 10 ppm (Hall and Oser,1968), about the same concentration as is found naturally.

Importance of the Gastrointestinal Tract

It is essential to appreciate the fact that the gut is a large, complex,and dynamic organ with several layers of organization and a vastabsorptive surface that has been estimated to be from 200 to 4500m2 (Concon, 1988). The GI transit time provides for adequate ex-posure of ingesta to a variety of environmental conditions (i.e.,variable pH), digestive acids and enzymes (trypsin, chymotrypsin,etc., from the pancreas and carbohydrases, lipases, and proteasesfrom the enterocytes), saponification agents (in bile), and a luxu-riant bacterial flora providing a repertoire of metabolic capabilitynot shared by the host (e.g., fermentation of “nondigestible” sug-ars such as xylitol and sorbitol) (Drasar and Hill, 1974). The en-

terocytes (intestinal epithelium) possess an extensive capacity forthe metabolism of xenobiotics that may be second only to that ofthe liver, with a full complement of phase (type) I and phase (type)II reactions present. The enteric monooxygenase system is analo-gous to the liver, as both systems are located in the endoplasmicreticulum of cells, require reduced nicotinamide adenine dinu-cleotide phosphate (NADPH) and O2 for maximum activity, are in-hibited by SKF-525A and carbon monoxide, and are qualitativelysimilar in their response to enzyme induction (Hassing et al., 1989).Induction of xenobiotic metabolism by the enteric monooxygenasesystem has been demonstrated in a number of substances, includ-ing commonly eaten foods and their constituents (Table 30-3). Di-etary factors may also decrease metabolic activity. For example, inone study, iron restriction and selenium deficiency decreased cy-tochrome P450 values, but a vitamin A rich diet had the same ef-fect (Kaminsky and Fasco, 1991).

The constituents of food and other ingesta (e.g., drugs, con-taminants, inhaled pollutants dissolved in saliva and swallowed)are physicochemically heterogeneous, and because the intestine hasevolved into a relatively impermeable membrane, mechanisms ofabsorption have developed that allow substances to gain access tothe body from the intestinal lumen. The four primary mechanismsfor absorption are passive or simple diffusion, active transport, fa-cilitated diffusion, and pinocytosis. Each of these mechanismscharacteristically transfers a defined group of constituents from thelumen into the body (Table 30-4). As noted in the table, xeno-biotics and other substances may compete for passage into thebody.

Aiding this absorption is the rich vascularization of the in-testine, with a normal rate of blood flow in the portal vein of ap-proximately 1.2 L/h/kg. However, after a meal, there is a 30 per-cent increase in blood flow through the splanchnic area (Concon,1988). It follows, then, that substances which affect blood flowalso tend to affect the absorption of compounds; an example is al-cohol, which tends to increase blood flow to the stomach and thus

Table 30-2Nonnutrient Substances in Food

NUMBER OF IDENTIFIED

FOOD NONNUTRIENT CHEMICALS

Cheddar cheese 160Orange juice 250Banana 325Tomato 350Wine 475Coffee 625Beef (cooked) 625

SOURCE: Smith, 1991, with permission.

Table 30-3Induction of Xenobiotic Metabolism in the Rat Intestine

INDUCER SUBSTRATE OR ENZYME REFERENCE

Butylated hydroxyanisole UDP-glucuronic acid Goon and Klaassen, 1992benzo[a]pyrene

Benzo[a]pyrene, cigarette smoke, Phenacetin Pantuck et al., 1974, 1975charcoal-broiled ground beef(vs. ground beef cooked on foil),

Purina Rat Chow (vs. semisyntheticdiet), chlorpromazine, chlorcyclizine

Cabbage or brussels sprouts Phenacetin, 7-ethoxycoumarin, Pantuck et al., 1976hexobarbital

Ethanol Benzo[a]pyrene Van de Wiel et al., 1992Indole-3-carbinol (present in Pentoxy- and ethoxyresorufin, Wortelboer et al., 1992b

brussels sprouts) testosteroneFried meat, dietary fat 7-Ethoxyresorufin O-deethylase Kaminsky and Fasco, 1991Brussels sprouts Aryl hydrocarbon hydroxylase, Kaminsky and Fasco, 1991

7-ethoxyresorufin O-deethylaseBrussels sprouts Ethoxyresorufin deethylation, Wortelboer et al., 1992a

glutathione S-transferase,DT-diaphorase

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enhances its own absorption. Few stimuli tend to decrease flow tothis area, with the possible exception of energetic muscular activ-ity and hypovolemic shock.

Lymph circulation is important in the transfer of fats, largemolecules (such as botulinum toxin), benzo[a]pyrene, 3-methyl-cholanthrene, and cis-dimethylaminostilbene (Chhabra and Eastin,1984). Lymph has a flow rate of about 1 to 2 mL/h/kg in humans,and few factors—with the exception of tripalmitin, which has beenshown to double the flow and therefore double the absorption ofp-aminosalicylic acid and tetracycline—are known to influence itsflow (Chhabra and Eastin, 1984). Another factor that lends impor-tance to lymph is the fact that the lymph empties via the thoracicduct into the point of junction of the left internal jugular and sub-clavian veins, preventing “first-pass” metabolism by the liver, un-like substances transported by the blood.

Many food ingredients are modified proteins, carbohydrates,fats, or components of such substances. Thus, an understanding ofthe changes these substances undergo in the GI tract, their possi-ble effect on the GI tract, and whether they are absorbed or affectthe absorption of other substances is critical to an understandingof food toxicology and safety assessment. Some of the factors thatmay affect GI absorption and the rate of absorption are listed inTable 30-5.

SAFETY STANDARDS FOR FOODS,FOOD INGREDIENTS, AND

CONTAMINANTS

The Food, Drug and Cosmetics ActProvides for a Practicable Approach

The FD&C Act presumes that traditionally consumed foods aresafe if they are free of contaminants. For the FDA to ban suchfoods, it must have clear evidence that death or illness can be tracedto consumption of a particular food. The fact that foods containmany natural substances, some of which are toxic at a high con-centration, is in itself an insufficient basis under the act for de-claring a food as being unfit for human consumption. Examples ofthis concept include acceptance of generally recognized as safe sta-tus and the implementation of tolerances.

The Application of Experience: Generally Recognized as Safe(GRAS) The FD&C Act permits the addition of substances tofood to accomplish a specific technical effect if the substance isdetermined to be GRAS by experts qualified by scientific trainingand experience to evaluate food safety. The FD&C Act does notrequire this determination be made by the FDA, though it does not

Table 30-4Systems Transporting Enteric Constituents

SYSTEM ENTERIC CONSTITUENT

Passive diffusion Sugars (e.g., fructose, mannose, xylose, which may also be transported by facilitated diffusion), lipid-soluble compounds, water

Facilitated diffusion D-xylose, 6-deoxy-1,5-anhydro-D-glucitol, glutamic acid, aspartic acid, short-chain fatty acids,xenobiotics with carboxy groups, sulfates, glucuronide esters, lead, cadmium, zinc

Active transport Cations, anions, sugars, vitamins, nucleosides (pyrimidines, uracil, and thymine, which may be incompetition with 5-fluorouracil and 5-bromouracil), cobalt, manganese (which competes for the irontransportation system)

Pinocytosis Long-chain lipids, vitamin B12 complex, azo dyes, maternal antibodies, botulinum toxin,hemagglutinins, phalloidins, E. coli endotoxins, virus particles.

Table 30-5Factors Affecting Intestinal Absorption and Rate of Absorption

FACTOR EXAMPLE

Gastric emptying rate Increased fat contentGastric pH Antacids, stress, H2-receptor blockersIntestinal motility Diarrhea due to intercurrent disease, laxatives, dietary fiber, disaccharide intolerance,

amaranthFood content Lectins of Phaseolus vulgaris (inhibition of glucose absorption and transport)Surface area of small intestine Short-bowel syndromeIntestinal blood flow AlcoholIntestinal lymph flow TripalmitinEnterohepatic circulation Chlordecanone (prevented by cholestyramine)Permeability of mucosa Inflammatory bowel disease, celiac diseaseInhibition of digestive processes Catechins of tea which inhibit sucrase and therefore glucose absorptionConcomitant drug therapy Iron salts/tetracycline

SOURCE: Modified from Hoensch and Schwenk, 1984, with permission.

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exclude the agency from making such decisions. The act insteadrequires that scientific experts base a GRAS determination on theadequacy of safety, as shown through scientific procedures orthrough experience based on common use in food before January1, 1958, under the intended conditions of use of the substance[FD&C Act, section 201(s)].

In addition to allowing GRAS substances to be added to food,the act provides for a class of substances that are regulated foodadditives, which are defined as “any substances the intended useof which results in its becoming a component . . . of any food . . .if such substance is not generally recognized . . . to be safe.” Hence,a legal distinction is drawn between regulated food additives andGRAS substances. Regulated food additives must be approved andregulated for their intended conditions of use by the FDA under21 CFR 172–179 before they can be marketed. In section 409 ofthe act, the requirements for data to support the safe use of a foodadditive are described in general terms. The requirements or rec-ommended methods for establishing safe conditions of use for anadditive are available in the form of a guideline issued by FDA(Toxicological Principles for the Safety Assessment of Direct FoodAdditives and Color Additives Used in Food). These guidelines, re-ferred to as “the Redbook,” provide substance and definition to thesafety standard applicable to regulated food additives: “reasonablecertainty of no harm under conditions of intended use.”

Use of Tolerances If a food contains an unavoidable contami-nant even with the use of current good manufacturing practices(CGMP), it may be declared unfit as food if the contaminant mayrender the food injurious to health. Thus, for a food itself to be de-clared unfit, it must be ordinarily injurious, while an unavoidablecontaminant in food need only pose the risk of harm for the foodto be found unfit and subject to FDA action. The reason for the di-chotomy is practicability. Congress recognized that if authoritywere granted to ban traditional foods for reasons that go beyondclear evidence of harm to health, the agency would be subject topressure to ban certain foods.

Foods containing unavoidable contaminants are not automat-ically banned because such foods are subject to the provisions ofsection 406 of the FD&C Act, which indicates that the quantity ofunavoidable contaminants in food may be limited by regulation forthe protection of public health and that any quantity of a contam-inant exceeding the fixed limit shall be deemed unsafe. This au-thority has been used by the FDA to set limits on the quantity ofunavoidable contaminants in food by regulation (tolerances) or byinformal action levels that do not have the force of law. Such ac-tion levels have been set for aflatoxin in peanuts, grain, and milk

(Table 30-6). Action levels have the advantage of offering greaterflexibility than is provided by tolerances established by regulation.Whether tolerances or action levels are applied to unavoidable con-taminants of food, the FDA attempts to balance the health riskposed by unavoidable contaminants against the loss of a portion ofthe food supply. In contrast, contaminants in food that are avoid-able by CGMP are deemed to be unsafe under section 406 if theyare considered poisonous or deleterious. Under such circumstances,the food is typically declared adulterated and unfit for human con-sumption. The extent to which consumers who are already in pos-session of such food must be alerted depends on the health riskposed by the contaminated food. If there is a reasonable probabil-ity that the use of or exposure to such a food will cause seriousadverse health consequences or death, the FDA will seek a class Irecall which provides the maximum public warning, the greatestdepth of recall, and the most follow-up. Classes II and III repre-sent progressively less health risk and require less public warning,less depth of recall, and less follow-up (21 CFR 7.3).

Food and Color Additives

An intentionally added ingredient, not considered GRAS, is eithera direct food additive or color additive. As with all ingredients in-tentionally added to food, there must be a specific and justifiablefunctionality. While a color additive has only one function, a foodadditive may have any one of 32 functionalities (Table 30-7).

The term color additive refers to a dye, pigment, or other sub-stance made by a process of synthesis or extracted and isolatedfrom a vegetable, animal, or mineral source [FD&C Act 201(t)].Blacks, whites, and intermediate grays are also included in this def-inition. When such additives are added or applied to a food, drug,or cosmetic or to the human body, they are capable of impartingcolor. Color additives are not eligible for GRAS status.

Two distinct types of color additives have been approved forfood use: those requiring certification by FDA chemists and thoseexempt from certification. Certification, which is based on chem-ical analysis, is required for each batch of most organic synthe-sized colors because they may contain impurities that may varyfrom batch to batch. Most certified colors approved for food usebear the prefix FD&C. They include FD&C Blue No. 1, FD&CBlue No. 2, FD&C Green No. 3, FD&C Red No. 3, FD&C RedNo. 40, FD&C Yellow No. 5, and FD&C Yellow No. 6. Orange Band Citrus Red No. 2 are the only certified food colors that lackthe FD&C designation (21 CFR 74 Subpart A).

The basis for the certification of these color additives is thefinding that each batch is of suitable purity and can be safely used

Table 30-6FDA Action Levels for Aflatoxins (Compliance Policy Guides 7120.26, 7106.10, and 7126.33)

COMMODITY LEVEL, ng/g

All products, except milk, designated for humans 20Milk 0.5Corn for immature animals and dairy cattle 20Corn for breeding beef cattle, swine, and mature poultry 100Corn for finishing swine 200Corn for finishing beef cattle 300Cottonseed meal (as a feed ingredient) 300All feedstuffs other than corn 20

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Table 30-7Direct Food Additives by Functionality

NUMBER DESIGNATION DESCRIPTION EXAMPLES

170.3(o) Anticaking agents Substances added to finely powdered or Glucitol, sodium ferrocyanide, silicon(1) and free-flow agents crystalline food products to prevent caking, dioxide

lumping, or agglomeration

(2) Antimicrobial agents Substances used to preserve food by Nisin; metyhyl-, ethyl-, propyl-, or butyl-preventing growth of microorganisms and ester of p-hydroxybenozoic acid;subsequent spoilage, including fungistats, sodium benzoate; sorbic acid and itsmold, and rope inhibitors and the effects saltslisted by the NAS/NRC under“preservatives”

(3) Antioxidants Substances used to preserve food by retarding Butylated hydroxyanisole (BHA),deterioration, rancidity, or discoloration due butylated hydroxytoluene (BHT),to oxidation propyl gallate

(4) Colors and coloring Substances used to impact, preserve, or FD&C Yellow No. 5 (tartrazine),adjuncts enhance the color or shading of a food, FD&C Red No. 4, �-carotene,

including color stabilizers, color fixatives, annatto, turmericcolor-retention agents

(5) Curing and pickling Substances imparting a unique flavor and/or Calcium chloride, glucitolagents color to a food, usually producing an

increase in shelf life

(6) Dough strengtheners Substances used to modify starch and gluten, Calcium bromate, baker’s yeast extract,producing a more stable dough, including calcium carbonatethe applicable effects listed by theNAS/NRC under “dough conditioners”

(7) Drying agents Substances with moisture-absorbing ability Calcium stearate, cobalt caprylate, cobaltused to maintain an environment of low tallatemoisture

(8) Emulsifiers and Substances that modify surface tension Phosphate esters of mono- andemulsifier salts in the component phase of an emulsion diglycerides, acetylated

to establish a uniform dispersion or monoglycerides, calcium stearateemulsion

(9) Enzymes Enzymes used to improve food processing Papain, rennet, pepsinand the quality of the finished food

(10) Firming agents Substances added to precipitate residual Calcium acetate, calcium carbonatepectin, strengthening the supporting tissueand preventing its collapse duringprocessing

(11) Flavor enhancers Substances added to supplement, enhance, Monosodium glutamate, inositolor modify the original taste and/or aromaof a food without imparting a characteristictaste or aroma of their own

(12) Flavor agents and Substances added to impart or help impart Cinnamon, citral, p-cresol, thymol,adjuvants a taste or aroma in food zingerone

(13) Flour-treating agents Substances added to milled flour at the Calcium bromatemill to improve its color and/or bakingqualities, including bleaching and maturingagents

(14) Formulation aids Substances used to promote or produce a Palm kernel oil, tallowdesired physical state or texture in food,including carriers, binders, fillers, plasticizers,film formers, and tableting aids

(continued)

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(15) Fumigants Volatile substances used for controlling insects Aluminum phosphide, potassiumor pests bromide

(16) Humectants Hygroscopic substances incorporated in food Arabic gum, calcium chlorideto promote retention of moisture, includingmoisture-retention agents and antidustingagents

(17) Leavening agents Substances used to produce or stimulate Carbon dioxide, adipic acidproduction of carbon dioxide in bakedgoods to impart a light texture, includingyeast, yeast foods, and calcium salts listedby the NAS/NRC under “dough conditioners”

(18) Lubricants and release Substances added to food contact surfaces Mineral oil, acetylated monoglyceridesagents to prevent ingredients and finished

products from sticking to them

(19) Nonnutritive Substances having less than 2 percent of the Acesulfame, aspartame, saccharinsweeteners caloric value of sucrose per equivalent unit

of sweetening capacity

(20) Nutrient supplements Substances that are necessary for the body’s Calcium carbonatenutritional and metabolic processes

(21) Nutritive sweeteners Substances that have greater than 2 percent Lactitol, hydrogenated starch hydrolysateof the caloric value of sucrose per equivalentunit of sweetening capacity

(22) Oxidizing and Substances that chemically oxidize or reduce Calcium peroxide, chloride, hydrogenreducing agents another food ingredient, producing a more peroxide

stable product, including the applicableeffects listed by the NAS/NRC under“dough conditioners”

(23) pH control agents Substances added to change or maintain Acetic acid, propionic acid, calciumactive acidity or basicity, including buffers, acetate, calcium carbonate, carbonacids, alkalis, and neutralizing agents dioxide

(24) Processing aids Substances used as manufacturing aids to Carbon dioxide, ammonium carbonate,enhance the appeal or utility of a food or ammonium sulfate, potassium bromidefood component, including clarifying agents,clouding agents, catalysts, flocculents, filleraids, and crystallization inhibitors

(25) Propellants, aerating Gases used to supply force to expel a Carbon dioxide, nitrous oxideagents, and gases product or reduce the amount of oxygen

in contact with the food in packaging

(26) Sequestrants Substances that combine with polyvalent Acetate salts, citrate salts, gluconate salt,metal ions to form a soluble metal complex metaphosphate, edetic acid, calciumto improve the quality and stability of acetateproducts

(27) Solvents and vehicles Substances used to extract or dissolve Acetic acid, acetylated monoglyceridesanother substance

(28) Stabilizers and Substances used to produce viscous Calcium acetate, calcium carbonatethickeners solutions or dispersions, to impart body,

improve consistency, or stabilize emulsions,including suspending and bodying agents,setting agents, jellying agents, and bulkingagents

(continued)

Table 30-7 (continued)


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as prescribed by regulation (FD&C Act, section 721). Certificationinvolves in-depth chemical analysis of major and trace componentsof each individual batch of color additives by FDA chemists andis required before any batch can be released for commercial use.Such color additives consist of aromatic amines or aromatic azostructures (FD&C Blue No. 1, FD&C Blue No. 2, FD&C GreenNo. 3, FD&C Red No. 40, FD&C Yellow No. 5, and FD&C Yel-

low No. 6) that cannot be synthesized without a variety of impu-rities.

Although aromatic amines are generally considered relativelytoxic substances, the FD&C colors are notably nontoxic. Table 30-8, which is adopted from a publication of the NationalAcademy of Sciences (NAS) (Committee on Food Protection,1971), shows that certified food colors have a low order of acute

(29) Surface-active agents Substances used to modify surface properties Sorbitan monostearate, mono- andof liquid food components for a variety diglycerides, polysorbate 60,of effects other than emulsifiers but acetostearinincluding solubilizing agents, dispersants,detergents, wetting agents, rehydrationenhancers, whipping agents, foaming agents,and defoaming agents

(30) Surface-finishing Substances used to increase palatability, Ammonium hydroxide, arabic gumagents preserve gloss, and inhibit discoloration

of foods, including glazes, polishes, waxes,and protective coatings

(31) Synergists Substances used to act or react with another Acetic acid, propionic acidfood ingredient to produce a total effectdifferent from or greater than the sum ofthe effects produced by the individualingredients

(32) Texturizers Substances that affect the appearance or Calcium acetatefeel of food

Table 30-7 (continued)


Table 30-8Data on Certified Food Colors Permanently Listed in the United States

NO ADVERSE EFFECT SAFE LEVEL ESTIMATED MAXIMUM

DIETARY LEVELS IN FOR HUMANS INGESTION

COLOR ANIMAL STUDIES mg/day mg/day PER PERSON

FD&C Blue No.1 5.0% rats 363 1.232.0% dogs

FD&C Blue No.2 1.0% rats, dogs 181 0.29FD&C Green No.3 5.0% rats 181 0.07

1.0% dogs2.0% mice

Orange B 5.0% rats 181 0.311.0% dogs5.0% mice

Citrus Red No.2 0.1% rats 18 Not applicableFD&C Red No. 3 0.5% rats 91 1.88

2.0% dogs2.0% mice

FD&C Yellow No.5 2.0% rats 363 16.32.0% dogs

FD&C Yellow No.6 2.0% rats 363 15.52.0% dogs2.0% mice

SOURCE: Committee on Food Protection, 1971.

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toxicity. The principal reason involves sulfonation of the aromaticamine or azo compound that constitutes a color additive. Such sul-fonic acid groups are highly polar, which, combined with their highmolecular weight, prevents them from being absorbed by the GItract or entering cells. All the FD&C food colors have been ex-tensively tested in all Concern Level (CL) tests (Table 30-9) andhave been found to be remarkably nontoxic.

Food colors that are exempt from certification typically havenot been subjected to such extensive testing requirements. The ex-empt food colors are derived primarily from natural sources. Whilesynthetic food colors have received the majority of public, scien-tific, and regulatory attention, natural color agents are also an im-portant class. Currently, 25 color additives have been given ex-emption from certification in 21 CFR 73. These agents consist ofa variety of natural compounds generally obtained by various ex-traction and treatment technologies. Included in this group of col-ors are preparations such as dried algae meal, beet powder, grapeskin extract, fruit juice, paprika, caramel, carrot oil, cochineal extract, ferrous gluconate, and iron oxide. A problem encounteredin attempts to regulate these additives is the lack of a precise chem-ical definition of many of these preparations. With a few excep-tions, such as caramel, which is the most widely used color, thenatural colors have not been heavily used. In part, this may be due to economic reasons, but these colors generally do not have the uniformity and intensity characteristic of the synthetic colors, therefore necessitating higher concentrations to obtain a specific color intensity. They also lack the chemical and colorstability of the synthetic colors and have a tendency to fade withtime.

Intake of color additives varies among individuals. The max-imal intake of food colors is estimated to be approximately 53.5mg/day, whereas the average intake per day is approximately 15mg (Committee on Food Protection, 1971). Only about 10 percentof the food consumed in the United States contains food colors.The foods that utilize food colors in order of the quantity of colorutilized are (1) beverages, (2) candy and confections, (3) dessertpowders, (4) bakery goods, (5) sausages (casing only), (6) cereals,(7) ice cream, (8) snack foods, and (9) gravies, jams, jellies, andso forth (Committee on Food Protection, 1971).

Methods Used to Evaluate the Safety of Foods, Ingredients, and Contaminants

Safety Evaluation of Direct Food and Color Additives The phi-losophy and approach to evaluating the safety of substances addedto foods has evolved over the past six decades. The concept thatforms the foundation for this evolution is the recognition that thesafety of any added substance to food must be established on thebasis of specific intended conditions of use or uses in food. In-tended conditions of use encompass (1) the foods to which the sub-stance is added, (2) the level of use in such foods, (3) the purposefor which the substance is used, and (4) the population expectedto consume the substance.Exposure: The Estimated Daily Intake Before 1958, the FDAemployed the philosophy that additives (and contaminants) shouldbe harmless per se; that is, an additive should not be harmful atany level. The impractical nature of this philosophy is illustratedby two examples: A reasonable person would assume that pure,distilled water is harmless, but if enough is ingested to cause elec-trolyte imbalance, death may result; similarly, sulfuric acid in itsconcentrated form can dissolve steel, but when used to control pHduring the processing of alcoholic beverages or cheeses, it is con-sidered GRAS by the FDA (Principles, 1991). Clearly, exposureshould be a major criterion for safety evaluation. This philosophyis reflected in the FDA’s Toxicological Principles for the Safety As-sessment of Direct Food Additives and Color Additives Used inFood (U.S. FDA, 1982),1 in which exposure is a key factor in analgorithm used to determine which types of testing should be car-ried out on a substance.

Exposure is most often referred to as an estimated daily in-take (EDI) and is based on two factors: the daily intake (I ) of thefood in which the substance will be used and the concentration (C)of the substance in that food:

EDI � C � I

Table 30-9Tests for Each Concern Level

CONCERN

LEVEL TESTS REQUIRED

I Short-term feeding study (at least 28 days in duration)Short-term tests for carcinogenic potential that can be used for determining priority for conduction of lifetime

carcinogenicity bioassays and may assist in the evaluation of results from such bioassays, if conducted

II Subchronic feeding study (at least 90 days in duration) in a rodent speciesSubchronic feeding study (at least 90 days in duration) in a nonrodent speciesMultigeneration reproduction study (minimum of two generations with a teratology phase) in a rodent speciesShort-term tests for carcinogenic potential

III Carcinogenicity studies in two rodent speciesA chronic feeding study at least 1 year in duration in a rodent species (may be combined with a carcinogenicity

study)Long-term (at least 1 year in duration) feeding study in a nonrodent speciesMultigenerational reproduction study (minimum of two generations) with a teratology phase in a rodent speciesShort-term tests for carcinogenic potential

1At the time of this writing, Redbook II remains in draft form and may undergo sig-nificant revision before finalizing. For this reason, the original (1982) version is usedin this chapter except where noted.

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In most cases, an additive is used in several different food cat-egories, but for the sake of simplicity, we can assume that the ad-ditive is used only in baked goods. If the additive is used at a levelthat does not exceed 10 ppm and the mean daily intake of bakedgoods is 137 g per person per day, the EDI for this substance is1370 �g per person per day.

Because most additives are used in more than one food, thetotal exposure (dose) is the sum of the exposures from each of thefood categories. The formula for exposure to substance X is

EDIx � (Cxf � If )� (Cxg � Ig) � (Cxh � Ih) � (C . . . )

where Cxf and Cxg are the concentration of X in food category fand the concentration of X in food category g, respectively. If andIg are the daily intake of food category f, food category g, and soon. Therefore, the EDI is the sum of the individual contributionsof X in each of the food categories.

Many of the same principles can be applied to an estimationof the consumption of residue from enzyme preparations [total or-ganic solids (TOS)], residue from secondary direct additives (sub-stances not intended to remain in a food after the technical effecthas been accomplished; this includes but is not limited to mold re-lease agents, solvents, defoaming agents, or chemicals used inwashing fruits), and contaminants.

These principles give rise to two questions: (1) How does onedetermine how much is added to each of the food categories? and(2) What are the food categories, and how are they determined?First, the agency will use as the basis for its calculations the high-est end of the range of use level for the new substance, but whatensures that these food group maximums will not be exceeded bya food manufacturer? This is also covered by a regulation—CGMPs (21 CFR 110)—in which a manufacturer is bound not toadd more of an additive than is reasonably required to achieve itsspecific technical effect. That is, if the desired red color of straw-berry ice cream is achieved with the addition of 1 ppm of red dyeand additional dye does not increase the color or the intensity ofthe color, it is in violation of CGMPs to add an amount greaterthan 1 ppm. (A discussion of CGMPs may be found in the FoodChemicals Codex, 1996.)

In regard to the second question on food categories, one setof 43 food categories is listed in 21 CFR 170.3(n), which grew outof a survey of food additives conducted by the National Academy

of Sciences/National Research Council and published in 1972. Asample of those categories is shown in Table 30-10. This surveypioneered the use of food categorization, but changing lifestyles ofthe population and shifts in food preferences have necessitated thegeneration of additional, more timely data.

The newer food consumption surveys provide more contem-porary data on food intake (Table 30-11). All these databases (andothers) have characteristics that serve a particular purpose. For ex-ample, one database may be only a 3-day “snapshot” of con-sumption, another may cover average consumption over 14 days,while yet another provides a detailed breakout of particular sub-populations (e.g., teenagers, the elderly, Hispanics). The majorityof these databases are available to the public but may not be par-ticularly “user-friendly,” and private industry has made some ofthem available on a fee basis.

In estimates of consumption and/or exposure, one must alsoconsider other sources of consumption for the proposed intendeduse of the additive if it already is used in food for another purpose,occurs naturally in foods, or is used in nonfood sources (e.g., drugs,toothpaste, lipstick). Thus, to estimate human consumption of aparticular food substance, it is necessary to know (1) the levels ofthe substance in food, (2) the daily intake of each food containingthe substance, (3) the distribution of intakes within the population,and (4) the potential consumption of or exposure to the substancefrom nonfood sources. (A discussion of chemical intake is coveredin Rees and Tennant, 1994.)

Before a food additive is approved, regulatory agencies re-quire evidence that it is safe for its intended use(s) and that its EDIis less than its acceptable daily intake (ADI). If such an estimateexceeds the ADI, regulatory agencies may impose restrictions onapprovals for certain uses or restrict future approvals for new cat-egories of use. The ADI is generally based on the results from an-imal toxicology studies, usually lifetime studies in rodents. Thesestudies are used to determine the no-observed-effect level (NOEL)for the additive. The NOEL is usually divided by 100 to determinethe ADI for a food additive, thus providing a 100-fold safety fac-tor to account for species differences and the inter-individual vari-ation among humans. This factor provides a reasonable certaintyin estimating safe doses in humans from animal studies (Butchkoand Kotsonis, 1996).Assignment of Concern Level (CL) and Required Testing Struc-ture-activity (SA) relationships are now the basis for developing

Table 30-10Food Categories


170.3(n)(1) Baked goods and Includes all ready-to-eat and ready-to bake Doughnuts, bread, croissants, cake

baking mixes products, flours, and mixes requiring mix, cookie doughpreparation before serving

(2) Beverages, alcoholic Includes malt beverages, wines, distilled Beer, malt liquor, whiskey, liqueurs,liquors, and cocktail mix wine coolers

(3) Beverages and beverage Includes only special or spiced teas, soft Herbal tea (non-tea-containing “teas”),bases, nonalcoholic drinks, coffee substitutes, and fruit- and soda pop, chicory

vegetable-flavored gelatin drinks

(4) Breakfast cereals Includes ready-to-eat and instant and Oatmeal (both regular and instant)regular hot cereals farina, corn flakes, wheat flakes

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many therapeutic drugs, pesticides, and food additives. These re-lationships are put to good use in the Toxicological Principles forthe Safety Assessment of Direct Food Additives and Color AdditivesUsed in Foods (U.S. FDA, 1982), which describes a qualitative“decision tree” that assigns categories to substances on the basisof the structural and functional groups in the molecule. Additiveswith functional groups with a high order of toxicity are assignedto category C, those of unknown or intermediate toxicity are as-signed to category B, and those with a low potential for toxicityare assigned to category A. For example, a simple saturated hy-drocarbon alcohol such as pentanol would be assigned to categoryA. Similarly, a substance containing an �,�-unsaturated carbonylfunction, epoxide, thiazole, or imidazole group would be assignedto category C. Thus, based on structure assignment and calculatedexposure, the CLs are assigned (Table 30-12). For example, a sub-stance in structure category B added to food at a level of 0.03 ppmwould be assigned to concern level II.

Once the CL is established, a specific test battery is prescribed,as shown in Table 30-9. The tests for CL III are the most demandingand provide the greatest breadth for the determination of adversebiological effects, including effects on reproduction. The tests arecomprehensive enough to detect nearly all types of observable tox-icity, including malignant and benign tumors, preneoplastic lesions,and other forms of chronic toxicity. The tests for CL II are of in-termediate breadth. These tests are designed to detect the most toxicphenomena other than late-developing histopathological changes.The short-term (genotoxicity) tests are intended to identify sub-stances for which chronic testing becomes critical. The CL I testbattery is the least broad, as is appropriate for the level of hazardwhich substances in this category may pose. However, if untoward

effects are noted, additional assessment becomes necessary. Stud-ies of the absorption, distribution, metabolism, and eliminationcharacteristics of a test substance are recommended before the ini-tiation of toxicity studies longer than 90 days’ duration. Of partic-ular importance for many proposed food ingredients is data on theirprocessing and metabolism in the GI tract.

Unique to food additive carcinogenicity testing is the contro-versial use of protocols that include an in utero phase. Under suchprotocols, parents of test animals are exposed to the test substancefor 4 weeks before mating and throughout mating, gestation, andlactation. Most countries and international bodies do not subscribeto the combining of an in utero phase with a rat carcinogenicitystudy, as this presents a series of logistic and operational problemsand substantially increases the cost of conducting a rat carcino-genicity study. The FDA began requesting in utero studies of thefood industry in the early 1970s, when it was discovered from life-time feeding studies that the artificial sweetener saccharin producedbladder tumors in male rats when in utero exposure was introduced.Subsequently, the FDA required the food, drug, and cosmetic colorindustries to conduct lifetime carcinogenicity feeding studies of 18color additives in rats using an in utero exposure phase. This test-ing has provided the largest database available to date on the per-formance of in utero testing.

Special note should also be made of genetic toxicity testing.Genetic toxicity tests are performed for two reasons: (1) to testchemicals for potential carcinogenicity and (2) to assess whethera chemical may induce heritable genetic damage. Currently, ge-netic toxicity assays can be divided into three major groups: (1)forward and reverse mutation assays (e.g., point mutations, dele-tions), (2) clastogenicity assays detecting structural and numerical

Table 30-11Databases for Estimating Food Intake

The Nationwide Food Consumption Survey, USDA, 1987–1988*Foods Commonly Consumed by Individuals, USDA (Pay et al., 1984)Continuing Survey of Food Intakes by Individuals, USDA, 1985, 1986, 1989*, 1990, 1991The 1977 Survey of Industry on the Use of Food Additives by the NRC/NAS, Volume IIIEstimates of Daily Intake (NRC/NAS), 1979 (Abrams, 1992)USDA Economic Research Service ReportsThe FDA Total Diet Study (Pennington and Gunderson, 1987)

*Indicates current use by FDA.SOURCE: Information kindly provided by Technical Assessment Systems, Inc. Washington, D.C.

Table 30-12Assignment of Concern Level

STRUCTURE CATEGORY A STRUCTURE CATEGORY B STRUCTURE CATEGORY C CONCERN LEVEL

�0.05 ppm in the total diet �0.025 ppm in the total diet �0.0125 ppm in the total diet I(�0.0012 mg/kg/day) (�0.00063 mg/kg/day) (�0.00031 mg/kg/day)

or or or0.05 ppm in the total diet 0.025 ppm in the total diet 0.0125 ppm in the total diet II

(0.0012 mg/kg/day) (0.00063 mg/kg/day) (0.00031 mg/kg/day)or or or

1 ppm in the total diet 0.5 ppm in the total diet 0.25 ppm in the total diet III(0.025 mg/kg/day) (0.0125 mg/kg/day) (0.0063 mg/kg/day)

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changes in chromosomes (e.g., chromosome aberrations, micronu-clei), and (3) assays that identify DNA damage (e.g., DNA strandbreaks, unscheduled DNA synthesis).

Because the correlation between carcinogens and mutagenshas proved to be less than desirable, as has been demonstrated byfalse-positive and false-negative findings when carcinogens andnoncarcinogens have been examined in genetic toxicity tests, it isrecommended that several tests be selected from a battery of tests.It should be kept in mind that as the number of tests employed in-creases, the possibility of false-negative results increases as well.Consequently, the National Toxicology Program (NTP) has advisedthat only a single gene mutational assay be used (Salmonella ty-phimurium) to optimize the prediction of carcinogenicity (Tennantand Zeiger, 1993).

Safety Determination of Indirect Food Additives Indirect foodadditives are substances defined as food additives that are not addeddirectly to food but enter food by migrating from surfaces that con-tact food. These surfaces may be from packaging material (cans,paper, plastic) or the coating of packaging materials or surfacesused in processing, holding, or transporting food.

Essential to demonstrating the safety of an indirect additiveare extraction studies with food-simulating solvents. The FDA rec-ommends the use of three food-simulating solvents—8% ethanol,50% ethanol, and corn oil or a synthetic triglyceride—for aqueousand acidic, alcoholic, and fatty foods, respectively (FDA, 1988).The conditions of extraction depend in part on the intended con-ditions of use. If the package material is intended to be retorted,the petitioner must conduct extractions for at least 2 hr at 275°F.For all conditions of use, high-temperature extraction (conductedat 275, 250, or 212°F for 2 h) is followed by a minimum of 238 hat 120°F except for refrigerated foods (in which 70°F is used) andfrozen food (in which only 120 h of extraction is required).

Extraction studies are used to assess the level or quantity ofa substance which might migrate and become a component of food,leading to consumer exposure. The prescribed extraction tests arebelieved to overestimate the amount of an indirect additive that islikely to migrate to food and thus are unlikely to underestimateconsumer exposure. To convert extraction data from packaging ma-terial into anticipated consumer exposure, the FDA has determinedthe fraction of the U.S. diet which comes into contact with differ-ent classes of material: glass, metal-coated, metal-uncoated, paper-uncoated, paper-coated, and polymers. For each class, FDA has as-signed a “consumption factor” (CF), which is the fraction of the

total diet that comes into contact with an individual class of ma-terial (Table 30-13).

The fraction of individual food types (aqueous, acidic, alco-holic, fatty) for which such packaging material is used is referredto as the food-type-distribution factor ( fT). To calculate consumerexposure (EDI), the following equation is used:

EDI � CF � [( fT aqueous � ppm in 8% ethanol) � ( fT acidic

� ppm in 8% ethanol) � ( fT alcohol � ppm in 50%ethanol) � ( fT fatty � ppm in corn oil)] � 3 kg perperson per day � mg per person per day2

For additives with virtually no migration (�0.05 ppm), inwhich the EDIs correspond to 0.15 mg per person per day, acutetoxicology data are considered sufficient to provide an assuranceof safety for the intended conditions of use of the additive.Migration levels, as determined by extraction studies, that aregreater than 0.05 to 1 ppm of exposure generally require subchronic(90-day) feeding studies in rodent and nonrodent (usually the dog)species. Where there is significant migration—that is, more than1 ppm exposure—carcinogenicity/chronic toxicity testing inrodents, at least a 1 year test in a nonrodent, and multigenerationreproduction testing to a minimum of two generations with a ter-atology phase in a rodent, are recommended by the FDA. Otherstudies may be indicated by the data or information available onthe substance.

Safety Requirements for GRAS Substances In spite of the factthat the FD&C Act and the relevant regulations (21 CFR 170.3,etc.) scrupulously avoid defining food except in a functionalsense—“food means articles used for food or drink for man orother animals . . . [and includes] chewing gum, and . . . articlesused for components of any such article”—it regards foods asGRAS when they are added to other food, for example, greenbeans in vegetable soup (Kokoski et al., 1990). It also regards anumber of food ingredients as GRAS, and these ingredients arelisted under 21 CFR 182, 184, and 186. However, it is importantto note that not all substances regarded as GRAS are listed as such.The language used in 21 CFR 182.1(a) acknowledges that thereare substances the FDA considers to be GRAS which are not listed.

Table 30-13Exposure Estimate Calculations (Package Type)

Food-Type Distribution (fT)

PACKAGE CATEGORY CF* AQUEOUS ACIDIC ALCOHOLIC FATTY

Glass 0.08 0.08 0.36 0.47 0.09Metal, polymer-coated 0.17 0.16 0.35 0.40 0.09Metal, uncoated 0.03 0.54 0.25 0.01† 0.20Paper, polymer-coated 0.21 0.55 0.04 0.01† 0.40Paper, uncoated 0.10 0.57 0.01† 0.01† 0.41Polymer 0.41 0.49 0.16 0.01 0.34

*As discussed in the text, a minimum CF of 0.05 is used initially for all exposure estimates.†1% or less.

2The 3 kg is the FDA’s value for daily food consumption which, when multiplied bymg/kg (ppm) and the weighting factors, reduces to milligrams of the additive per day.

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This accomplishes two things: (1) It leaves the door open for ad-ditional nonlisted substances to be affirmed as GRAS by theagency and (2) reinforces the concept that substances can bedeemed GRAS whether or not they are listed by the FDA or on apublicly available list. A list of examples of substances regardedas GRAS is given in Table 30-14. It is important to re-emphasizethat GRAS substances, though used like food additives, are notfood additives. Although the distinction may seem to be one ofsemantics, it allows GRAS substances to be exempt from the pre-market clearance restrictions enforced by the FDA and exemptfrom the Delaney clause, because that clause pertains only to foodadditives.

While the courts have ruled that GRAS substances must besupported by the same quantity and quality of safety data that sup-port food additives, this ruling should not be interpreted to meanthat the supporting data must be identical in nature and characterto those supporting a food additive. For uses of substances to beeligible for classification as GRAS, there must be common knowl-edge throughout the scientific community about the safety of sub-stances directly or indirectly added to food (21 CFR 170.30).

The studies relied on for concluding that a given use of a sub-stance is GRAS ordinarily are based on generally available dataand information published in the scientific literature. Such data areunlikely to be conducted in accordance with FDA-recommendedprotocols, as these studies often are conducted for reasons unre-lated to FDA approval. GRAS status also can be based on experi-ence with common use in food before January 1, 1958, which fur-ther distinguishes GRAS data requirements from those demandedof food additives. Such experience need not be limited to the UnitedStates; but if it comes from outside the United States, it must bedocumented by published or other information that is corroboratedby information from an independent source.

Importance of the GRAS Concept

The importance of the GRAS provision is obvious from its manyapplications. Many substances, for example, that are used in foodprocessing have never received formal FDA approval. The use ofthese substances in the manufacture of food products is consideredappropriate under CGMPs, while the substance itself is considered

GRAS for such purposes. Similarly, certain substances are per-mitted as optional ingredients in standardized foods [foods withstandards of identity specified by regulation (21 CFR 130–169)]even though they are not approved food additives and are not onany of the GRAS lists.

The GRAS concept as traditionally applied in the UnitedStates also has applicability to certain novel foods which may dif-fer only slightly from traditional foods or which, after careful con-sideration, can be regarded as raising no issues or questions ofsafety beyond that raised by the traditional foods they are intendedto replace. The GRAS approach may therefore permit the intro-duction of novel foods that contain less saturated fat and/or cho-lesterol or more fiber or are in other ways modified.

Transgenic Plant (and New Plant Varieties) Policy Crops havebeen genetically modified through conventional crop breeding formore than a hundred years to produce new plant varieties. This wasusually done by conventional breeding methods. Scientists todaycan use biotechnology to insert specific genes into a plant to giveit new characteristics. For example, approximately 25 percent ofthe corn crop planted in 1999 in the United States contains a genefrom the bacterium Bacillus thuringiensis that produces a Bt in-secticidal protein (James, 1999). Bt is a protein toxic to certaincaterpillar insect pests that destroy corn plants (EPA, 1988; Mc-Clintock et al., 1995). By enabling the corn plant to protect itselffrom this insect pest, the use of this product can reduce the needfor and use of conventional insecticides (Gianessi and Carpenter,1999).

Irrespective of the breeding method used to produce a newplant variety, tests must be done to ensure that the levels of nutri-ents or toxins in the plants have not changed and that the food isstill safe to consume. Clearly, new proteins produced in plant va-rieties must be nontoxic and not have the characteristics of pro-teins known to cause allergies. Thus, the proteins produced in ge-netically modified crops are evaluated for allergenicity (Metcalf etal., 1996). The DNA that is introduced into genetically modifiedplants to direct the production of such new proteins has been de-termined to be generally recognized as safe (FDA, 1992). The useof antibiotic resistance marker genes in genetically modified cropshas been determined by FDA and other regulatory agencies to be

Table 30-14Examples of GRAS Substances and Their Functionality

CFR NUMBER SUBSTANCE FUNCTIONALITY

Substances Generally Recognized as Safe21 CFR 182

182.2122 Aluminum calcium silicate Anticaking agent182.5065 Linoleic acid Dietary supplement

Direct Food Substances Affirmed as Generally Recognized as Safe21 CFR 184

184.1005 Acetic acid Several184.1355 Helium Processing aid

Indirect Food Substances Affirmed as Generally Recognized as Safe21 CFR 186

186.1025 Caprylic acid Antimicrobial186.1374 Iron oxides Ingredient of paper

and paperboard

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safe (FDA, 1992). Nonetheless, regulatory agencies have advisedresearchers to avoid using marker genes in crops that encode re-sistance to clinically important antibiotics.

The safety of new plant varieties (transgenic plants, geneti-cally modified plants) is regulated primarily under the FDA’s post-market authority [section 402(a)(1) of the FD&C Act]. This sec-tion, previously applied to occurrences of unsafe levels of toxicantsin food, is now applied to new plant varieties whose compositionhas been altered by an added substance. The new policy has beenapplied to plants containing substances that are GRAS [FederalRegister 57(104):22984–23005]. The Federal Register notice (May29, 1992) indicates that “[i]n most cases, the substances expectedto become components of food as a result of genetic modificationof a plant will be the same as or substantially similar to substancescommonly found in food, such as proteins, fats and oils, and car-bohydrates.” The notice also indicates the responsibility of the FDAto exercise the premarket review process when the “objective char-acteristics of the substance raise questions of safety.” In regard tosubstances within the new variety that are not similar to substancescommonly found in food, a food additive petition may have to befiled.

The Federal Register notice offers points of consideration forthe safety assessment of new plant varieties (Table 30-15).Accompanying these points of consideration are a decision flow-chart and advice that the FDA be consulted on certain findings, forexample, transference of allergens from one plant to another, achange in the concentration or bioavailability of nutrients, and theintroduction of a new macroingredient.

In the United States, new plant varieties are regulated not onlyby FDA but also by the EPA and USDA. FDA is responsible forthe safety and labeling of foods and feeds derived from crops, ir-respective of the method used to produce the new plant variety.EPA is responsible for assuring the safety of pesticides; thus in theexample cited above whereby a pesticide is produced in a new plantvariety, this product would also fall under the EPA’s jurisdiction.The USDA’s Animal and Plant Health Inspection Service has re-sponsibility for the environmental safety of field-testing and com-mercial planting of new plant varieties.

Methods for Establishing Safe Conditions of Use for NovelFoods Novel foods, including those derived from new plant va-rieties and macroingredient substitutes, present new challenges andmay require new methods of determining safety. For example, witheach new additive, it has been traditional (and rooted in a regula-tion such as 21 CFR 170.22) to establish an ADI, which is usually

based on 1�100 of the NOEL established in animal testing. Thisworks well for additives projected to be consumed at a level of 1.5g/day or less (which is equal to or less than 25 mg/kg), for this ex-trapolates at a 100-fold safety factor to consumption by a rat at alevel of 2500 mg/kg/day (about 5 percent of the rat’s diet). Theproblem arises when a new food or macroingredient substitute be-comes a substantive part of the diet (estimated to constitute as muchas 15 to 20 percent). For example, a macroingredient substitute orfood projected to be consumed at a level of just 5 percent of thediet (150 g/day) would require the test animal (rat) to consume 250g/kg/day, or slightly more than the rat’s body weight. This is anuntenable test requirement, for at those levels, the investigatorwould establish an effect level only for malnutrition, not for thetoxicity of the macroingredient. The converse is true for some es-sential nutrients, such as vitamins A and D and iron, which at doses100 times the nutritional use level would be toxic (Kokoski et al.,1990). The answer therefore lies in careful interpretation of toxi-cological data and the conduct, where appropriate, of special stud-ies to assess drug interactions, nutrient interactions, changes in gutflora, changes in gut activity, and the like (Borzelleca, 1992a,b;Munro, 1990). Also, it may be appropriate to consider what effect,if any, macroingredients may have on individuals with compro-mised digestive tracts, those dependent on laxatives, and those onhigh-fiber diets.

The regulatory approval of a new food additive is generallybased on traditional toxicology studies. The rationale is that datafrom such studies will adequately predict adverse effects that couldoccur in humans. However, such studies, especially for novel foods,may not be adequate. Therefore, although human studies are notgenerally required for food additives, in the case of novel foods,human studies are likely essential in evaluating their safety (Stargelet al., 1996).

Another useful tool in ensuring the safety of a food additiveis monitoring it after its approval, or postmarketing surveillance.With widespread use of a food additive, monitoring for consump-tion can determine whether actual consumption exceeds the EDIand monitoring for anecdotal complaints may identify adversehealth effects that escaped detection in earlier studies. This couldbe especially important for novel foods when traditional toxicol-ogy studies are not done at large multiples of the EDI (Butchko etal., 1994, 1996). Thus, the combination of traditional toxicity stud-ies, special animal and human studies, and possibly postmarketingsurveillance will ensure the safety of consumers and provide evi-dence to justify a safety factor different from 100.

Dietary Supplements Dietary supplements have a special statuswithin the law and the regulations; supplements are regarded asfoods or food-type substances but not food additives and not drugs.Although regarded as foods, they cannot be sold as conventionalfoods. Unique to dietary supplements (or dietary supplement in-gredients) is a lesser standard for safety than is required for foodingredients. That is, while a food ingredient must have demon-strated safety, a supplement ingredient must have no history of un-safe use, a much easier standard to meet. While this distinction issubtle, it permits the use of a substance as a dietary supplementthat cannot be used as a food ingredient (e.g., stevioside). In theexample of stevioside, because there is no history of unsafe use,the FDA has not objected to its use as a dietary supplement butfeels that the safety data are inadequate to support the use of ste-vioside as an ingredient added to food. Concomitantly, an unap-

Table 30-15Points of Consideration in the Safety Assessment of NewPlant Varieties

Toxicants known to be characteristic of the host and donorspecies

The potential that food allergens will be transferred fromone food source to another

The concentration and bioavailability of importantnutrients for which a food crop is ordinarily consumed

The safety and nutritional value of newly introducedproteins

The identity, composition, and nutritional value of modifiedcarbohydrates or fats and oils

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proved health claim cannot be made for the dietary supplement, asit is then regarded as a “drug” and subject to the rigorous drug ap-plication process with demonstrations of safety and effectiveness(Burdock, 2000).

Assessment of Carcinogens

Carcinogenicity as a Special Problem As discussed above,Congress provided the FDA with wide latitude in assessing safetyand assuring a safe food supply with—one exception. That ex-ception is a provision of the FD&C Act known as the Delaneyclause, which prohibits the approval of regulated food additives“found to induce cancer when ingested by man or animals” [sec-tion 409(c)(3)(A)]. The Delaney clause is found in two other sec-tions of the act. The three clauses—sections 409(c)(3)(A),706(b)(5)(B), and 512(d)(1)(H)—constitute the Delaney clause.

It must be emphasized that the Delaney prohibition appliesonly to the approval of food additives, color additives, and animaldrugs; it does not apply to unavoidable contaminants or GRAS sub-stances or ingredients sanctioned by the FDA or USDA before1958. The clause also does not apply to carcinogenic constituentsthat are present in food or color additives or animal drugs as non-functional contaminants provided that the level of such contami-nants can be demonstrated to be safe and the whole additive, in-cluding its contaminants (permitted by specification andregulations), is not found to induce cancer in humans or animals.This interpretation of the Delaney clause was set forth by the FDAin its so-called constituent policy published on April 2, 1982, asan Advanced Notice of Proposed Rulemaking (ANPR). The policymandates the development and use of animal carcinogenicity data

and probabilistic risk assessment to establish a safe level for thecontaminant in the additive under its intended conditions of use.

The constituent policy and, as discussed further on, the im-plementation of the so-called DES (diethylstilbestrol) proviso foranimal drugs under the Delaney clause, have forced the FDA todevelop a means for establishing safe levels for carcinogenic sub-stances. The DES proviso allows the addition of carcinogenic an-imal drugs to animal feed if they leave no residue in edible tissueas determined by an approved analytic procedure. To do this, theFDA has turned to the use of probabilistic risk assessment in whichtumor data in animals are mathematically extrapolated to an upper-bound risk in humans exposed to particular use levels of the addi-tive. The FDA takes the position that, considering the many con-servative assumptions inherent in the procedure, an upper-boundlifetime risk of one cancer in a million individuals is the biologi-cal equivalent of zero.

Much controversy surrounds the use of risk-assessment pro-cedures, in part because estimates of risk are highly dependent onthe many assumptions that must be made. The tendency is to be“risk-averse” and favor assumptions that exaggerate risk. As theseexaggerations are multiplicative, the total overestimation of riskcan be several orders of magnitude. Table 30-16 provides somerough estimates of potential ranges of uncertainty that might leadto large overestimates (Flamm and Lorentzen, 1988).

The common practice of testing at a maximum tolerated dose(MTD) (Williams and Weisburger, 1991) raises the question of ap-propriateness to human exposure. Do high test doses cause phys-iologic changes unlike those from human exposure? The basic as-sumption in quantitative risk assessment (QRA) that thedose-response curve is linear beneath the lowest observable effect

Table 30-16Uncertainty Parameters and Their Associated Range of Risk Factors

ESTIMATED RANGE,UNCERTAINTY PARAMETERS FACTOR

Extrapolation model 1–10,000Total dose vs. dose rate 30–45Most sensitive sex/strain vs. average sensitivity 1–100Sensitivity of human vs. test animal 1–1000Potential synergism or antagonism with other carcinogens or promoters 1–1000?Total population vs. target population, potential vs. actual market penetration 1–1000Absorptive rate (gut, skin, lung) for animals at high dose vs. humans at low dose 1–10Dose scaling: mg/kg body weight, ppm (diet, water, feed) surface area 1–15Upper confidence on users or exposed 1–10Specifics or tolerances 1–10Limits of detection vs. actual levels 1–1000Additivity vs. nonadditivity of multiple sites 1–3Survival or interim sacrifice adjustments 1–2Knowledge of only high-end plateau dose response 1–10Error or variation in detection methods 1–10Adjustments for less than lifetime bioassays 1–100Adjustments for intermittent and less than lifetime human exposure 1–100Use vs. nonuse of historical data 1–2Upper confidence and lower confidence limits vs. expected values in extrapolation level of acceptable risk 1–1000Level of acceptable risk 1–1000Adding or not adding theoretical risks from many substances 1–100

SOURCE: Flamm and Lorentzen, 1988.

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may result in the calculation of relatively high risks even at dosesthat are much lower than the lowest dose that produces cancer inexperimental animals (Flamm and Lorentzen, 1988). QRA is morea process than a science; many steps in the process are based onassumptions, not proven scientific facts. If only the most conser-vative assumptions are made throughout the process, many willrepresent overestimates of human risk by 10- or 100-fold, leadingto a combined overestimate of perhaps a millionfold or more.

Risk assessment cannot be used for either food additives orcolor additives because of the Delaney clause. If these additivesare found to induce cancer, they cannot be approved for foods orcolors no matter how small the estimated risk. Because of the harshconsequences of finding a food additive to be a carcinogen, theFDA has interpreted this clause as requiring an affirmative findingof a clear and unequivocal demonstration of carcinogenicity uponingestion. Historically, the FDA has employed a high threshold forestablishing that a food or color additive has been found to inducecancer when ingested by humans or animals. Very few substanceshave been disapproved or banned because of the Delaney clause.Two indirect food additives (Flectol H and mercaptoimidazoline)that migrate from packaging material were banned. Among directadditives, safrole, cinnamyl anthranilate, thiourea, and diethylpy-rocarbonate were banned because of the Delaney clause; di-ethylpyrocarbonate was banned because it forms urethane.

A number of substances [e.g., butylated hydroxyanisole(BHA), xylitol, methylene chloride, sorbitol, trichloroethylene, ni-trilotriacetic acid (NTA), diethylhexyl phthalate, melamine,formaldehyde, bentonite] listed in the Code of Federal Regulationas regulated food additives are also listed as carcinogens by Na-tional Toxicology Program (NTP), the International Agency for Re-search on Cancer (IARC), or the state of California (under the SafeDrinking Water and Toxic Enforcement Act of 1986, also knownas Proposition 65). How is this possible, and on what basis do thesefood additive listings continue?

Despite the fact that tests and conditions exist under whicheach of these substances will produce cancer in animals, the FDAhas found it possible to continue listing these substances as foodadditives. The reasoning applied in almost every case is based onsecondary carcinogenesis. The one exception is formaldehyde,which is carcinogenic only on inhalation, and there are compellingreasons to believe that inhalation is not an appropriate test in thiscase (Flamm and Frankos, 1985). Therefore formaldehyde is nottreated as a carcinogen prohibited by the Delaney clause.

For BHA, which induces forestomach cancer, the concept hasbeen advanced that its carcinogenicity is attributable primarily toirritation, restorative hyperplasia, and so on (Clayson et al., 1986).For xylitol, a sugar alcohol, an increase in bladder tumors and ad-renal pheochromocytomas is considered secondary to calcium im-balance resulting from the indigestibility of sugar alcohols and theirfermentation in the lower GI tract. Sorbitol, another sugar alcohol,behaves in a similar manner. For NTA, the argument is secondarycarcinogenesis, and although specific explanations vary, the mech-anism involving zinc imbalance has considerable scientific support.The review of diethylhexyl phthalate is ongoing, but the possibil-ity that peroxisome proliferation is involved has been offered, ashas the possibility that hepatocellular proliferation is primary tothe subsequent development of tumors.

Thus, the FDA has generally interpreted the phrase “found toinduce cancer when ingested by man or animals” as excluding can-cers that arise through many secondary means. Therefore, to be acarcinogen under the Delaney clause, a food or color additive must

be demonstrated to induce cancer by primary means when ingestedby humans or animals or to induce cancer by other routes of ad-ministration that are found to be appropriate. This is interpreted tomean that the findings of cancer must be clearly reproducible andthat the cancers found are not secondary to nutritional, hormonal,or physiologic imbalances. This position allows the agency to ar-gue that changing the level of protein or fat in the diet does not in-duce cancer but simply modulates tumor incidence (Kritschevsky,1994). Given the many modulating factors and influences con-nected with food and diet, the FDA must be careful about what itdeclares to be a carcinogen under the Delaney clause. Thus, theFDA has always had to look at the mechanistic question and hasbeen doing that for more than 30 years.

Biological versus Statistical Significance Much can be learnedabout the proper means of assessing carcinogenicity data by study-ing large databases for substances that have been tested for car-cinogenicity many times. The artificial sweetener cyclamate is anexample. The existence of more than a dozen studies on cyclamateand the testing of multiple hypotheses at dozens of different organand tissue sites in all these studies led to the awareness that theoverall false-positive error rate (i.e., higher cancer incidence at aspecific organ site in treated subjects versus controls as a result ofchance) could be inflated if individual findings were viewed out ofcontext (FDA, 1984). Therefore very careful attention must be paidto the totality of the evidence.

The possibility of false-negative error is always of concernbecause of the need to protect public health. However, it should berecognized that any attempt to prove absolutely that a substance isnot carcinogenic is futile. Therefore, an unrelenting effort to min-imize false-negative errors can produce an unacceptably high prob-ability of a false positive. Further, demanding certainty (i.e., a zeroor implicitly an extremely low probability of false-negative error)has negative consequences for an accurate decision-makingprocess. This is the case because it severely limits the ability todiscriminate between carcinogens and noncarcinogens on the ba-sis of bioassays (FDA, 1984).

In addition to the false-positive/false-negative trap, which isa statistical matter, there are many potential biological traps. Theappearance of a higher incidence of tumors at a specific organ sitein treated animals may not demonstrate by itself a carcinogenic ac-tion of the substance employed in the treatment. This is the casebecause the incidence of tumors at specific organ sites can be in-fluenced and controlled by many biological processes that may af-fect tumor incidence.

The test substance, typically administered at high MTDs, mayaffect one or more of the many biological processes known to mod-ulate tumor incidence at a specific organ site without causing aninduction of tumors at that or any other site. Nutritional imbalancessuch as choline deficiency are known to lead to a high incidenceof liver cancer in rats and mice. Simple milk sugar (lactose) isknown to increase the incidence of Leydig’s cell tumors in rats.Caloric intake has been shown to be a significant modifying fac-tor in carcinogenesis. Impairment of immune surveillance by a spe-cific or nonspecific means (stress) affecting immune responsive-ness and hormonal imbalance can result in higher incidences oftumors at specific organ sites. Hormonal imbalance, which can becaused by hormonally active agents (e.g., estradiol) or by othersubstances that act indirectly, such as vitamin D, may result in anincreased tumor incidence. Chronic cell injury and restorative hy-perplasia resulting from treatment with lemon flavor (d-limonene)

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probably are responsible for renal tumor development in male ratsby mechanisms that are of questionable relevance to humans(Flamm and Lehman-McKeeman, 1991).

In these examples, the increases in tumor incidence at spe-cific organ sites probably are secondary to significant changes innormal physiologic balance and homeostasis. Moreover, the in-creases in tumor incidence, and hence in the risk of cancer, prob-ably would not occur except at toxic doses (Ames and Gold, 1997).

To preserve the ability of a bioassay to discriminate, the pos-sibility of false-positive or false-negative results and of secondaryeffects must be considered. To be meaningful, evaluations must bebased on the weight of evidence, which must be reviewed as care-fully as possible. Particular attention must be given to the manyfactors used in deciding whether tumor incidences are biologicallyas well as statistically significant. These factors include (1) the his-torical rate of the tumor in question (is it a rare tumor, or does itoccur frequently in the controls?); (2) the survival histories of dosedand test animals (did dosed animals survive long enough to be con-sidered “at risk”? What effect did chemical toxicity and reducedsurvival have in the interpretation of the data?); (3) the patterns oftumor incidence (was the response dose-related?); (4) the biolog-ical meaningfulness of the effect (was it experimentally consistentwith the evidence from related studies? Did it occur in a target or-gan?); (5) the reproducibility of the effect with other doses, sexes,or species; (6) evidence of hyperplasia, metaplasia, or other signsof an ongoing carcinogenic process (is the effect supported by apattern of related nonneoplastic lesions, particularly at lowerdoses?); (7) evidence of tumor multiplicity or progression; and (8)the strength of the evidence of an increased tumor incidence (whatis the magnitude of the p value? for pairwise comparison? fortrend?).

A good discussion of the use of these factors by scientists indeciding whether a substance induces cancer in animals is con-tained in the notice of a final rule permanently listing FD&C Yel-low No. 6 (51 FR 41765–41783, 1988). An elevation of tumor in-cidence in rats was identified at two organ and/or tissue sites: (1)medullary tumors of the adrenal glands in female rats only and (2)renal cortical tumors in female rats only. Scientists at the FDA con-cluded that the increase in medullary tumors of the adrenal glandsin female rats did not suffice to establish that FD&C Yellow No.6 is a carcinogen. The basis for the decision was (1) a lack of doseresponse, (2) the likelihood of false positives, (3) the lack of pre-cancerous lesions, (4) morphologic similarity of adrenal medullarylesions in treated and control rats, (5) an unaffected latency period,(6) a lack of effect in male rats, and (7) a comparison with otherstudies in which there was no association between exposure toFD&C Yellow No. 6 and the occurrence of adrenal medullarytumors.

A similar judgment was made with respect to the cortical re-nal lesions in female rats, which were not found to provide a ba-sis for concluding that FD&C Yellow No. 6 can induce cancer ofthe kidneys. The main reasons leading to this conclusion were (1)the relatively common occurrence of proliferative renal lesions inaged male control rats (28 months or older), (2) the lack of renaltumors in treated males despite their usually greater sensitivity torenal carcinogens, (3) the lack of malignant tumors indicating noprogression of adenomas to a malignant state, (4) the lack of a de-creased latency period compared with controls, (5) the coincidenceof renal proliferative lesions and chronic renal disease, (6) the lackof genotoxicity, and (7) a lack of corroborative evidence from otherstudies that suggests a treatment-related carcinogenic effect ofFD&C Yellow No. 6 on the kidney. Both of these examples em-

phasize the importance of considering all the evidence in attempt-ing to decide the significance of any subset of data.

As essential elements, vitamins, sugars, and calories per secan increase tumor incidence in test animals; the mechanism bywhich tumors arise as the result of exposure to food or food in-gredients is critically important to assessing the relevance of thefinding to the safety of the substance under its intended conditionsof use in food. McClain (1994) provides an excellent discussionof mechanistic considerations in the regulation and classificationof chemical carcinogens.

Carcinogenic Contaminants The Delaney clause, which pro-hibits the addition of carcinogens to food, could ban many foodadditives and color additives if it were strictly interpreted to in-clude carcinogenic contaminants of additives within its definition.Clearly, this was not Congress’s intent, and just as clearly, the FDAneeded to develop a commonsense policy for addressing the prob-lem that all substances, including food and color additives, maycontain carcinogenic contaminants at some trace level.

Toward this end, the agency argued (FDA, 1982) that banningfood and color additives simply because they have been found orare known to contain a trace level of a known carcinogen does notmake sense because all substances may contain carcinogenic con-taminants. The agency asserted in its constituent policy that themere fact an additive contains a contaminant known to be car-cinogenic should not automatically lead the agency to ban that foodadditive under the Delaney clause but should instead cause theagency to consider the health risks it poses based on its level ofcontamination and the conditions of its use. The agency stated thatby using highly conservative scientific assumptions and a highlyconservative methodology for extrapolating cancer risk from highdose to low and from animals to humans, one could estimate suchrisks in a manner that would assure that the actual risks posed tohumans would not be underestimated. This reaffirmed the agency’sposition taken in the proposal on the addition of carcinogens to thefeed of food-producing animals (FDA, 1977).

SAFETY OF FOOD

Adverse Reactions to Food or Food Ingredients

In a survey of Americans, 30 percent indicated that they or some-one in their immediate families had a food sensitivity. Althoughthis number is likely too high, as much as 7.5 percent of the pop-ulation may be allergic to some food or component thereof (Tayloret al., 1989). Lactose intolerance (a deficiency of the disaccharideenzyme lactase) is very high among some groups; for example,there is an incidence of 27 percent in black children age 12 to 24months, which may increase to 33 percent by age 6 years(Juambeltz et al., 1993). The percentage of young (northernEuropean) children allegedly intolerant to food additives rangesfrom 0.03 to 0.23 percent (Wuthrich, 1993) to 1 to 2 percent(Fuglsang et al., 1993). Further, there are certain drug-food in-compatibilities about which physicians and pharmacists are obli-gated to warn patients, such as monoamine oxidase (MAO) in-hibitors and tyramine in cheese or benzodiazapenes and naringeninin grapefruit juice. People who are prescribed tetracycline also mustbe alerted not to take milk with this antibiotic. By any standard,there are large numbers of real and perceived adverse reactions toor incompatibilities with food (Thomas and Tschanz, 1994). Thefirst step in understanding these reactions is to define the nomen-

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clature, a task undertaken by the American Academy of Allergyand Immunology (Committee on Adverse Reactions to Foods) andthe National Institute of Allergy and Infectious Diseases (Andersonand Sogn, 1984). An adaptation of their attempt at definition andclassification is represented in Table 30-17. In the table, the defi-nitions proceed from general to most specific. Obviously, there islittle to distinguish the terms adverse reaction and sensitivity to a

food or a food intolerance except perhaps in the lexicon of the in-dividual, colored by his or her own experience. That is, an “ad-verse reaction” may indicate something as simple as an unpleas-ing esthetic or hedonic quality such as an unpleasant taste, whichmay in fact have a genetic basis, as in the ability to taste phenyl-thiocarbamide (Guyton, 1971), or may indicate a fatal outcome re-sulting from an immune or toxic reaction.

Table 30-17Adverse Reaction to Food: Definition of Terms

TERM DEFINITION CHARACTERISTICS/EXAMPLES

Adverse reaction General term that can be applied to a clinically Any untoward pathological reaction(sensitivity) to a food abnormal response attributed to an ingested resulting from ingestion of a food or

food or food additive. food additive. May be immune-mediated.

Food hypersensitivity An immunologic reaction resulting from Immune-mediated (cellular or humoral(allergy) the ingestion of a food or food additive. response), requires prior exposure to

This reaction occurs only in some patients, antigen or cross-reacting antigen. Firstmay occur after only a small amount of the exposure may have been asymptomatic.substance is ingested, and is unrelated toany physiological effect of the food or foodadditive.

Food anaphylaxis A classic allergic hypersensitivity reaction to A humoral immune response most oftenfood or food additives. involving IgE antibody and release of

chemical mediators. Mortality may result.

Food intolerance A general term describing an abnormal Any untoward pathologic reaction resultingphysiologic response to an ingested food from ingestion of a food or food additive.or food additive; this reaction may be an May be immune-mediated. Celiac diseaseimmunologic, idiosyncratic, metabolic, (intolerance to wheat, rye, barley, oats).pharmacologic, or toxic response.

Food toxicity (poisoning) A term use to imply an adverse effect caused Not immune-mediated. May be caused byby the direct action of a food or food additive bacterial endo- or exotoxin (e.g., hemorrhagicon the host recipient without the involvement E. coli) fungal toxin (e.g. aflatoxin), tetrodo-of immune mechanisms. This type of reaction toxin from pufferfish, domoic acid frommay involve nonimmune release of chemical mollusks, histamine poisoning from fishmediators. Toxins may be contained within (scombroid poisoning), nitrate poisoningfood or released by microorganisms or (i.e., methemoglobinuria).parasites contaminating food products.

Food idiosyncrasy A quantitatively abnormal response to a Not immune-mediated, Favism (hemolyticfood substance or additive; this reaction anemia related to deficiency of erythrocyticdiffers from its physiologic or pharma- glucose-6-phosphate dehydrogenase), fishcologic effect and resembles hypersensitivity odor syndrome, beetanuria, lactosebut does not involve immune mechanisms. intolerance, fructose intolerance, asparagusFood idiosyncratic reactions include those urine, red wine intolerance.which occur in specific groups of individualswho may be genetically predisposed.

Anaphylactoid An anaphylaxis-like reaction to a food or food Not immune-mediated. Scombroid poisoning,reaction to a food additive as a result of nonimmune release sulfite poisoning, red wine sensitivity.

of chemical mediators. This reaction mimicsthe symptoms of food hypersensitivity (allergy).

Pharmacological food An adverse reaction to a food or food additive Not immune-mediated. Tyramine in patientsreaction as a result of a naturally derived or added treated with MAO inhibitors, fermented

chemical that produces a drug-like or (alcohol-containing) foods in disulfiram-pharmacologic effect in the host. treated patients.

Metabolic food reaction Toxic effects of a food when eaten in excess Cycasin, vitamin A toxicity, goitrogens.or improperly prepared.

SOURCE: Adapted from Anderson and Sogn, 1984.

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Clinical descriptions of adverse reactions to food are not new.Hippocrates (460–370 B.C.) first recorded adverse reactions tocow’s milk that caused gastric upset and urticaria, and Galen (A.D.131–210) described allergic symptoms to goat milk. However, theimmunologic basis of many adverse reactions to food was not es-tablished until the passive transfer of sensitivity to fish was de-scribed in the early 1960s (Frankland, 1987; Taylor et al., 1989).This test, which evolved into the (skin) prick test and later the ra-dioallergosorbent (RAST) test, allowed a distinction to be madebetween immunologically based adverse reactions (true allergies)and adverse reactions with other causation.

Food Allergy Description Food hypersensitivity (allergy)refers to a reaction involving an immune-mediated response. Sucha response is generally IgE-mediated, although IgG4- and cell-mediated immunity also may play a role in some instances(Fukutomi et al., 1994). What generally distinguishes food allergyfrom other reactions is the involvement of immunoglobulins, ba-sophils, or mast cells (the latter being a source of mediating sub-stances including histamine and bradykinin for immediate reac-tions and prostaglandins and leukotrienes for slower-developingreactions) and a need for a prior exposure to the allergen or a cross-reactive allergen. An allergic reaction may be manifest by one ormore of the symptoms listed in Table 30-18. The list of foodsknown to provoke allergies is long and is probably limited only bywhat people are willing to eat. Although cutaneous reactions andanaphylaxis are the most common symptoms associated with foodallergy, the body is replete with a repertoire of responses that arerarely confined to only a few foods.

A curious type of food allergy, the so-called exercise-inducedfood allergy, is apparently provoked by exercise that has been im-mediately preceded or followed by the ingestion of certain foods(Kivity et al., 1994), including shellfish, peach, wheat, celery, and“solid” food (Taylor et al., 1989). The exact mechanism is un-known, but it may involve enhanced mast-cell responsiveness tophysical stimuli and/or diminished metabolism of histamine simi-lar to red wine allergy (Taylor et al., 1989). On the other hand,food intolerance in patients with chronic fatigue may have less todo with allergic response and has been shown to be a somatizationtrait of patients with depressive symptoms and anxiety disorders(Manu et al., 1993).Chemistry of Food Allergens Most allergens (antigens) in foodare protein in nature, and although almost all foods contain one ormore proteins, a few foods are associated more with allergic reac-tions than are others. For example, anaphylaxis to peanuts is more

common than is anaphylaxis to other legumes (e.g., peas,soybeans). Similarly, although allergies may occur from bonyfishes, there is no basis for cross-reactivity to other types of seafood(e.g., mollusks and crustaceans), although dual (and independent)sensitivities may exist (Anderson and Sogn, 1984). Interestingly,patients who are allergic to milk can usually tolerate beef and in-haled cattle dander, and patients allergic to eggs can usually toler-ate ingestion of chicken and feather-derived particles (Andersonand Sogn, 1984)—although in the “bird-egg” syndrome, patientscan be allergic to bird feathers, egg yolk, egg white, or any com-bination of the three (DeBlay et al., 1994; Szepfalusi et al., 1994).Some of the allergenic components of common food allergens arelisted in Table 30-19.

Table 30-19Known Allergenic Food Proteins

FOOD ALLERGIC PROTEINS

Cow’s milk Casein (Dorion et al., 1994; Stoger and Wuthrich, 1993)

�-Lactoglobulin (Piastra et al., 1994; Stoger and Wuthrich, 1993)

a-Lactalbumin (Bernaola et al., 1994; Stoger and Wuthrich, 1993)

Egg whites Ovomucoid (Bernhisel-Broadbent et al., 1994)

Ovalbumin (Fukotomi et al., 1994,Bernhiesel-Broadbent et al., 1994)

Egg yolks Livetin (de Blay et al., 1994; Szepfalusi et al., 1994)

Peanuts Ara h II (Dorion et al., 1994) Peanut I (Sachs et al., 1981)

Soybeans �-Conglycinin (7S fraction) (Rumsey et al., 1994)

Glycinin (11S fraction) (Rumsey et al., 1994)

Gly mIA (Gonzalez et al., 1992)Gly mIB (Gonzalez et al., 1992)Kunitz trypsin inhibitor (Brandon

et al., 1986)Codfish Gad cI (O’Neil et al., 1993)Shrimp Antigen II (Taylor et al., 1989)Green peas Albumin fraction (Taylor et al., 1989)Rice Glutelin fraction (Taylor et al., 1989)

Globulin fraction (Taylor et al., 1989)Cottonseed Glycoprotein fraction (Taylor et al.,

1989)Peach guava, 30-kDa protein (Wadee et al., 1990)

banana,mandarin,strawberry

Tomato Several glycoproteins (Taylor et al.,1989)

Wheat Gluten (Stewart-Tull and Jones, 1992)Gliadin (O’Hallaren, 1992)Globulin (O’Hallaren, 1992)Albumin (O’Hallaren, 1992)

Okra Fraction I (Manda et al., 1992)

SOURCE: Modified from Taylor et al., 1989, with permission.

Table 30-18Symptoms of IgE-Mediated Food Allergies

Cutaneous Urticaria (hives), eczema, dermatitis,pruritus, rash

Gastrointestinal Nausea, vomiting, diarrhea, abdominalcramps

Respiratory Asthma, wheezing, rhinitis, bronchospasmOther Anaphylactic shock, hypotension, palatal

itching, swelling including tongue andlarynx, methemoglobinemia*

*An unusual manifestation of allergy reported to occur in response to soy or cow milkprotein intolerance in infants (Murray and Christie, 1993).

SOURCE: Adapted from Taylor et al., 1989, with permission.

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Although food avoidance is usually the best means of pro-tection, it is not always possible because (1) the content of someprepared foods may be unknown (e.g., the presence of eggs or cot-tonseed oil), (2) there is the possibility of contamination of foodfrom unsuspected sources [e.g., Penicillium in cheeses or meat,Candida albicans (Dayan, 1993; Dorion et al., 1994), and cow’smilk antigens in the breast milk of mothers who have consumedcow’s milk (Halken, et al., 1993)], (3) an allergen may be presentin a previously unknown place [the insertion of Brazil nut DNAinto soybeans and subsequent appearance of the allergic 2S pro-tein in soybean products (Nordlee et al., 1996)], and (4) there is alack of knowledge about the phylogenetic relationships betweenfood sources (legumes include peas, soybeans, and peanuts; someAmericans are not aware that ham is a pork product). While avoid-ance is not always possible, promising research in the area of pro-biotics (i.e., promotion of the growth of beneficial intestinal bac-teria including lactobacilli or bifidobacteria) may help inmanagement of food allergy (Kirjavainen et al., 1999; Arunacha-lam, 1999).Demographics of Food Allergy Although children appear to bethe most susceptible to food allergy, with adverse reactionsoccurring in 4 to 6 percent of infants, the incidence appears to ta-per off with maturation of the digestive tract, with only 1 to 2 per-cent of young children (4 to 15 years) susceptible (Fuglsang et al.,1993). The increase in the number of adults exhibiting food allergy

may be due in part to an expanded food universe—that is, an in-creased willingness to try different foods. In one study, allergiesamong young children were most commonly to milk and eggs,while allergies that developed later in life tended to be to fruit andvegetables (Kivity et al., 1994).

Familial relationships also play a role. Schrander and col-leagues (1993) noted that among infants intolerant of cow’s milkprotein, 65 percent had a positive family history (first- or second-degree relatives) for atopy compared with 35 percent of healthycontrols.

Food Toxicity (Poisoning) See “Substances for which Toler-ances May Not Be Set” below.

Food Idiosyncrasy Food idiosyncrasies are generally defined asquantitatively abnormal responses to a food substance or additive;this reaction differs from the physiologic effect, and although itmay resemble hypersensitivity, it does not involve immune mech-anisms. Food idiosyncratic reactions include those that occur inspecific groups of individuals who may be genetically predisposed.Examples of such reactions and the foods that are probably re-sponsible are given in Table 30-20.

Probably the most common idiosyncratic reaction is lactoseintolerance, a deficiency of the lactase enzyme needed for themetabolism of the lactose in cow’s milk. A lack of this enzyme re-

Table 30-20Idiosyncratic Reactions to Foods

FOOD REACTION MECHANISM REFERENCE

Fava beans Hemolysis, sometimes Pyramidene aglycones in fava bean cause Chevion et al., 1985accompanied by jaundice and irreversible oxidation of GSH in G-6-PD-hemoglobinuria; also, pallor, deficient erythrocytes by blockingfatigue, nausea, dyspnea, fever and NADPH supply, resulting in oxidativechills, abdominal and dorsal stress of the erythrocyte and eventualpain hemolysis

Chocolate Migraine headache Phenylethylamine-related (?) Gibb et al., 1991;Settipane, 1987

Beets Beetanuria: passage of red urine Excretion of beetanin in urine after Smith, 1991(often mistaken for hematuria) consumption of beets

Asparagus Odorous, sulfurous-smelling urine Autosomal dominant inability to Smith, 1991metabolize methanthiol of asparagusand consequent passage of methanthiolin urine

Red wine Sneezing, flush, headache, diarrhea, Diminished histamine degradation: Wantke et al., 1994skin itch, shortness of breath deficiency of diamine oxidase (?)

Histamines present in wine

Choline- and Fish odor syndrome: foul odor Choline and carnitine metabolized to Ayesh et al., 1993carnitine- of body secretions trimethylamine in gut by bacteria,containing followed by absorption but inability tofoods metabolize to odorless trimethylamine

N-oxide

Lactose Abdominal pain, bloating, diarrhea Lactase deficiency Mallinson, 1987intolerance

Fructose- Abdominal pain, vomiting, diarrhea, Reduced activity of hepatic aldolase Frankland, 1987; Catto-containing hypoglycemia B toward fructose-1-phosphate Smith and Adams, 1993foods

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sults in fermentation of lactose to lactic acid and an osmotic effectin the bowel, with resultant symptoms of malabsorption anddiarrhea. Lactose intolerance is lowest in northern Europe at 3 to8 percent of the population; it reaches 70 percent in southern Italyand Turkey and nearly 100 percent in southeast Asia (Gudmand-Hoyer, 1994; Anderson and Sogn, 1984).

Anaphylactoid Reactions Anaphylactoid reactions are histori-cally thought of as reactions mimicking anaphylaxis (and other“allergic-type” responses, including though not limited to itching,chronic urticaria, angioedema, exacerbation of atopic eczema,rhinitis, bronchial obstruction, asthma, diarrhea and other intestinaldisturbances, and vasomotor headaches) through direct applicationof the primary mediator of anaphylactic reactions: histamine. In-gestion of scombroid fish (e.g., tuna, mackerel, bonito) as well assome nonscombroid fish (mahimahi and bluefish) that have beenacted upon by microorganisms (most commonly Proteus morganii,Proteus vulgaris, Clostridium spp., Escherichia coli, Salmonellaspp., and Shigella spp.) to produce histamine may result in an ana-phylactoid reaction also called scombrotoxicosis (Table 30-21)(Clark et al., 1999). The condition was reported to be mimickedby the direct ingestion of 90 mg of histamine in unspoiled fish (vanGeldern et al., 1992), but according to Taylor (1986), the effect ofsimply ingesting histamine does not produce the equivalent effect.Instead, Taylor stated that histamine ingested with spoiled fish ap-pears to be much more toxic than is histamine ingested in an aque-ous solution, as a result of the presence of histamine potentiatorsin fish flesh. These potentiators included putrefactive amines (pu-trescine and cadaverine) and pharmacologic potentiators such asaminoguanidine and isoniazid (histaminase inhibitors). The mech-anism of potentiation involves the inhibition of intestinal histamine-metabolizing enzymes (diamine oxidase), which causes increased

histamine uptake. Scombrotoxicosis in the absence of high hista-mine levels (less than the U.S. FDA action level for tuna of 50 mghistamine/100 g fish) was reported by Gessner et al., 1996. Melniket al., (1997) proposed that anaphylactoid responses may be thesum of several mechanisms: (1) an increased intake of biogenicamines (including histamine) with food, (2) an increased synthe-sis by the intestinal flora, (3) a diminished catabolism of biogenicamines by the intestinal mucosa, and (4) an increased release ofendogenous histamine from mast cells and basophils by histamine-releasing food. Further, improvement was observed in 50 percentof patients with histamine intolerance and atopic eczema whomaintained a histamine-depleted diet. Ijomah and coworkers (1991)claimed that dietary histamine is not a major determinant of scom-brotoxicosis, since potency is not positively correlated with thedose and volunteers tend to fall into susceptible and nonsuscepti-ble subgroups. Ijomah and coworkers (1991) suggested that en-dogenous histamine released by mast cells plays a significant rolein the etiology of scombrotoxicosis, whereas the role of dietaryhistamine is minor. An exception to this endogenous histamine the-ory was described by Morrow and colleagues (1991), who foundthe expected increase in urinary histamine in scombroid-poisonedindividuals but did not find an increase in urinary 9�,11�-dhydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid, the principalmetabolite of prostaglandin D2, a mast cell secretory product; thus,no mast-cell involvement was indicated. Rittweger et al. (1994)have reported an increase in urinary immunoreaction angiotensinI and angiotensin II following oral provocation tests to patientswith a history of anaphylactoid reactions to drugs, foods, and foodadditives, but unfortunately there are no reports describing urinaryangiotensin levels following oral histamine administration.

Smith (1991) described sulfite-induced bronchospasm (some-times leading to asthma), which was first noticed as an acute sen-

Table 30-21Anaphylactoid Reactions to Food


Western Australian Erythema and urticaria of the skin, Scombroid poisoning; high histamine Smart, 1992salmon (Arripis facial flushing and sweating, levels demonstrated in the fishtruttaceus) palpitations, hot flushes of the

body, headache, nausea, vomiting,and dizziness

Fish (spiked Facial flushing, headache Histamine poisoning; histamine Van Gelderen et al.,with histamine) concentration in plasma correlated 1992

closely with histamine dose ingested

Cape yellow tail Skin rash, diarrhea, palpitations, Scombroid poisoning, treated with Muller et al., 1992(fish) (Seriola headache, nausea and abdominal antihistamineslalandii) cramps, paresthesia, unusual taste

sensation, and breathing difficulties

Sulfite sensitivity Bronchospasm, asthma Sulfite oxidase deficiency to meta- Smith, 1991bisulfites in foods and wine

Tuna, albacore, Reaction resembling an acute allergic Scombroid poisoning treated with Lange, 1988mackerel, bonito, reaction antihistamines and cimetidinemahimahi, andbluefish

Cheese Symptoms resembling acute allergic Responds to antihistamines; histamine Taylor, 1986reaction poisoning?

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sitivity to metabisulfites sprayed on restaurant salads (and saladbars) and in wine. Sulfite is normally detoxicated rapidly to inor-ganic sulfate by the enzyme sulfite oxidase. In sensitive individu-als, there is apparently a deficiency in this enzyme, making themsupersensitive to sulfites. (The FDA has taken the position that theaddition of sulfite to food is safe only when properly disclosed onthe food label.)

Pharmacologic Food Reactions Also referred to as “false foodallergies” (Moneret-Vautrin, 1987), these adverse reactions arecharacterized by exaggerated responses to pharmacologic agentsin food (Table 30-22). These reactions are distinguished from otherclassifications because they are not associated with a specificanomaly of metabolism (e.g., lactose intolerance or favism) butmay be a receptor anomaly instead. These, then, are common phar-macologic agents acting in a very predictable manner, but at ex-ceptionally low levels.

Metabolic Food Reactions Metabolic food reactions are distinctfrom other categories of adverse reactions in that the foods aremore or less commonly eaten and demonstrate toxic effects onlywhen eaten to excess or improperly processed (Table 30-23). Thesusceptible population exists as a result of its own behavior—thatis, the “voluntary” consumption of food as a result of a limitedfood supply or an abnormal craving for a specific food. Such anabnormal craving was reported by Bannister and associates (1977),who noted hypokalemia leading to cardiac arrest in a 58-year-oldwoman who had been eating about 1.8 kg of licorice per week. In“glycyrrhizism,” or licorice intoxication, glycyrrhizic acid is theactive component, with an effect resembling that of aldosterone,which suppresses the renin-angiotensin-aldosterone axis, resultingin the loss of potassium. Clinically, hypokalemia with alkalosis,cardiac arrhythmias, and muscular symptoms, together withsodium retention, edema, and severe hypertension are observed.The syndrome may develop at a level of 100 g licorice per day butgradually abates upon withdrawal of the licorice (Tonnesen, 1979).

This category also includes the ingestion of improperly pre-pared food such as cassava or cycad, which, if prepared properlywill result in a toxin-free food. For example, cycad (Cacaos circi-nalis) is a particularly hardy tree in tropical to subtropical habitatsaround the world. Cycads often survive when other crops have beendestroyed (e.g., a natural disaster such as a typhoon or drought)and therefore may serve as an alternative source of food. Amongpeople who have used cycads for food, the method of detoxifica-tion is remarkably similar despite the wide range of this plant: theseeds and stems are cut into small pieces and soaked in water forseveral days; they are then dried and ground into flour. The effec-

tiveness of leaching the toxin (cycasin) from the bits of flesh ismost directly dependent on the size of the pieces, the duration ofsoaking, and the number of water changes. Shortcuts in process-ing may have grave consequences (Matsumoto, 1985).

Importance of Labeling

Food labeling allows susceptible individuals to avoid foods con-taining ingredients that may be harmful to them, such as allergensor substances they may be intolerant of, such as lactose. Thus, ifa food contained an allergy-causing protein, this would have to beindicated on the label. The FDA has indicated that, at this time,they are not aware of any information that foods developed throughgenetic engineering differ as a class in any attribute from foods de-veloped through conventional means, and that such foods wouldtherefore not warrant a special label (Thompson, 2000). FDA al-lows companies to include on the label of a product any statementas long as the statement is truthful and not misleading.

TOLERANCE SETTING FORSUBSTANCES IN FOODS

Pesticide Residues

A pesticide is defined under the FD&C Act as any substance usedas a pesticide, within the meaning of the Federal Insecticide, Fungi-cide and Rodenticide Act (FIFRA), in the production, storage, ortransportation of raw agricultural commodities (food in its raw ornatural state) [section 201(q)]. The Pesticide amendments of 1956to the FD&C Act (section 408) were the first amendments to theFD&C Act requiring premarket clearance evaluations of the safetyof chemicals added to food. Currently, the U.S. Environmental Pro-tection Agency (EPA) is responsible for evaluating the safety ofpesticides before issuing tolerances.

The regulation of pesticides and their safety is accomplishedunder both FIFRA and the FD&C Act. FIFRA governs the regis-tration of pesticides. Registration addresses specific uses of a pes-ticide, and without such registration a pesticide cannot be lawfullysold for such use in the United States. A major part of the regis-tration process involves tolerance setting. Pesticides intended foruse on food crops must be granted tolerances or exempted fromtolerances under the FD&C Act.

Tolerances for raw agriculture commodities (RAC’s) were es-tablished under section 408 of the FD&C Act. If the pesticide chem-ical was found to concentrate in any of the processed fractions fromthe processing studies on the food crops (usually at least 1.5 or2.0� of the residue concentration in the RAC), the residues in the

Table 30-22Pharmacologic Reactions to Food


Cheese, red wine Severe headache, hypertension Tyramine from endogenous or ingested Settipane, 1987tyrosine

Nutmeg Hallucinations Myristicin Anderson and Sogn, 1984Coffee, tea Headache, hypertension Methylxanthine (caffeine) acting as a Anderson and Sogn, 1984

noradrenergic stimulantChocolate Headache, hypertension Methylxanthine (theophylline) acting Anderson and Sogn, 1984

as a noradrenergic stimulant

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processed fraction(s) were considered to be intentional food addi-tives and were required to be assigned “Food Additive Tolerances”under Section 409 of the act. If the pesticide chemical in questionhad been classified by the EPA as a human or animal carcinogen,the Delaney clause would then be invoked, and the section 409food additive tolerance(s) could be denied on that basis.

Under the new Food Quality Protection Act (FQPA) of 1996,section 201(s) of the FD&C Act excludes pesticides from the def-inition of food additive—even in the case of concentration ofresidues in processed fractions. Consequently, the Delaney clauseis no longer applicable for pesticides. The Delaney clause has notbeen repealed from section 409 and continues to apply to inten-tional food additives other than pesticides. In the case of concen-tration of pesticide residues in a processed fraction above the sec-tion 408 tolerances established for the RAC, an additional tolerancefor that processed fraction is still established, but now under sec-tion 408 rather than section 409.

The FQPA requires that an additional tenfold safety factor“shall be applied for infants and children to take into account po-tential pre- and post-natal toxicity and completeness of the datawith respect to exposure and toxicity to infants and children.”Therefore the “default” assumption is that the additional 10� safety

factor will be applied to the chemical safety assessment resultingin a total safety factor of 1000�. It further states, however, that“the Administrator may use a different margin of safety for the pes-ticide chemical residue only if, on the basis of reliable data, suchmargin will be safe for infants and children.” Therefore, the addi-tional 10� safety factor may be reduced depending on such thingsas adequacy of exposure assessment, adequacy of the toxicologicdata-base, and the nature and severity of any adverse effects ob-served in these studies, the most important being the adequacy ofthe toxicologic data base.

Drugs Used in Food-Producing Animals

An animal drug “means any drug intended for use for animals otherthan man” [section 201(w) of the FD&C Act]. Animal drugs, whichtypically are used for growth promotion and increased food pro-duction, present a complex problem in the safety assessment of an-imal drug residues in human food. Determination of the potentialhuman health hazards associated with animal drug residues is com-plicated by the metabolism of an animal drug, which results inresidues of many potential metabolites. The sensitivity of modernanalytic methodologies designed to quantitate small amounts of

Table 30-23Metabolic Food Reactions


Lima beans, Cyanosis Cyanogenic glycosides releasing Anderson and Sogn, 1984cassava roots, hydrogen cyanide on contact withmillet (sorghum) stomach acidsprouts, bitteralmonds, apricotand peach pits

Cabbage family, Goiter (enlarged thyroid) Isothiocyanates, goitrin, or Anderson and Sogn, 1984;turnips, soybeans, S-5-vinyl-thiooxazolidone vanEtten and Tookey,radishes, rapeseed interferes with utilization of iodine 1985and mustard

Unripe fruit of Severe vomiting, coma, Hypoglycin A, isolated from the Evans, 1985the tropical tree and acute hypoglycemia fruit, may interfere with oxidationBlighia sapida, sometimes resulting in death, of fatty acids, so that glycogencommon in especially among the stores have to be metabolized forCaribbean and malnourished energy, with depletion of carbo-Nigeria hydrates, resulting in hypoglycemia

Leguminosae, Lathyritic symptoms: L-2-4-Diaminobutyric acid inhibition Evans, 1985 Cruciferae neurological symptoms of of ornithine transcarbamylase of the

weakness, leg paralysis, and urea cycle, inducing ammonia toxicitysometimes death

Licorice Hypertension, cardiac Glycyrrhizic acid mimicking Farese et al., 1991(glycyrrhizic acid) enlargement, sodium mineralocorticoids

retention

Polar bear and Irritability, vomiting, increased Vitamin A toxicity Bryan, 1984Chicken liver intracranial pressure, death

Cycads Amyotrophic lateral sclerosis Cycasin (methylazoxymethanol); Matsumoto, 1985; Sieber(cycad flour) (humans), hepatocarcino- primary action is methylation, et al., 1980

genicity (rats and nonhuman resulting in a broad range ofprimates) effects from membrane destruction

to inactivation of enzyme systems

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drugs and their various metabolites has made evaluation morecomplex.

The primary factors that must be considered in the evaluationof animal drugs are (1) consumption and absorption by the targetanimal, (2) metabolism of the drug by the target food animal, (3)excretion and tissue distribution of the drug and its metabolites infood animal products and tissues, (4) consumption of food animalproducts and tissues by humans, (5) potential absorption of thedrug and its metabolites by humans, (6) potential metabolism ofthe drug and its metabolites by humans, and (7) potential excre-tion and tissue distribution in humans of the drug, its metabolites,and the secondary human metabolites derived from the drug andits metabolites. Thus, the pharmacokinetic and biotransformationcharacteristics of both the animal and the human must be consid-ered in an assessment of the potential human health hazard of ananimal drug.

When an animal drug is considered GRAS, the safety assess-ment of the drug is handled as described under the section onGRAS, above. With respect to new animal drugs, safety assess-ment is concerned primarily with residues that occur in animal foodproducts (milk, cheese, etc.) and edible tissues (muscle, liver, etc.).Toxicity studies in the target species (chicken, cow, pig, etc.) shouldprovide data on metabolism and the nature of metabolites alongwith information on the drug’s pharmacokinetics. If this informa-tion is not available, these studies must be performed using the an-imal species that is likely to be exposed to the drug. During thisphase, the parent drug and its metabolites are evaluated both qual-itatively and quantitatively in the animal products of concern (eggs,milk, meat, etc.). This may involve the development of sophisti-cated analytic methodologies. Once these data are obtained, it isnecessary to undertake an assessment to determine potential hu-man exposure to these compounds from the diet and other sources.If adequate toxicity data are available, it is possible to undertakea safety assessment pursuant to the establishment of a tolerance.

To comply with the congressional intent regarding the use ofanimal drugs in food-producing animals as required in the “noresidue” provision of the Delaney clause, the FDA began to builda system for conducting risk assessment of carcinogens in the early1970s (FDA, 1977). In the course of developing a policy and/orregulatory definition for “no residue,” the FDA was compelled toaddress the issue of residues of metabolites of animal drugs knownto induce cancer in humans or animals. As the number of metabo-lites may range into the hundreds, it became apparent that, as apractical matter, not every metabolite could be tested with the samethoroughness as the parent animal drug. This forced the FDA toconsider threshold assessment for the first time. Threshold assess-ment combines information on the structure and in vitro biologi-cal activity of a metabolite for the purpose of determining whethercarcinogenicity testing is necessary (Flamm et al., 1994). If test-ing is necessary and the substance is found to induce cancer, theFDA’s definition states that a lifetime risk of one in a million asdetermined by a specified methodology is equivalent to the mean-ing of “no residue” as intended by Congress.

Unavoidable Contaminants

Certain substances—such as polychlorinated biphenyls (PCB’s) orheavy metals—are unavoidable in food because their widespreadindustrial applications or their presence in the earth’s crust haveresulted in their becoming a persistent and ubiquitous contaminantin the environment. As a result, foods and animal feeds, principally

those of animal and marine origin, contain unavoidable contami-nants at some level. Tolerances for residues of unavoidable con-taminants are established for foods and food ingredients to ensurethat they are safe under expected or intended conditions of use.

Heavy Metals There are 92 natural elements; approximately 22are known to be essential nutrients of the mammalian body and arereferred to as micronutrients (Concon, 1988). Among the mi-cronutrients are iron, zinc, copper, manganese, molybdenum, sele-nium, iodine, cobalt, and even aluminum and arsenic. However,among the 92 elements, lead, mercury, and cadmium are familiaras contaminants or at least have more specifications setting theirlimits in food ingredients (e.g., Food Chemicals Codex, 1996). Theprevalence of these elements as contaminants is due to their ubiq-uity in nature but also to their use by humans.Lead Although the toxicity of lead is well known, lead may bean essential trace mineral. A lead deficiency induced by feedingrats �50 ppb (versus 1000 ppb in controls) over one or more gen-erations produced effects on the hematopoietic system, decreasediron stores in the liver and spleen, and caused decreased growth(Kirchgessner and Reichmayer-Lais, 1981), but apparently not asa result of an effect on iron absorption (Reichmayer-Lais andKirchgessner, 1985). Although the toxic effects of lead are dis-cussed elsewhere in this text, it is important to note that the effectsare profound (especially in children) and appear to be long-lasting,since mechanisms for excretion appear to be inadequate in com-parison to those for uptake (Linder, 1991).

Over the years, recognition of the serious nature of lead poi-soning in children has caused the World Health Organization(WHO) and FDA to adjust the recommended tolerable total leadintake from all sources of not more than 100 �g/day for infants upto 6 months of age and not more than 150 �g/day for children from6 months to 2 years of age to the considerably lower range of 6 to18 �g/day as a provisional tolerable range for lead intake in a 10-kg child.

Initiatives to reduce the level of lead in foods, such as themove to eliminate lead-soldered seams in soldered food cans thatwas begun in the 1970s and efforts to eliminate leachable lead fromceramic glazes, have resulted in a steady decline in dietary lead in-take. Although food and water still contribute lead to the diet, datafrom the FDA’s Total Diet Study indicated a reduction in meandietary lead intake for adult males from 95 �g/day in 1978 to 9 �g/day in the period 1986–1988 (Shank and Carson, 1992).

Some lead sources are difficult to curtail, as lead often sur-vives food processing; for example, lead in wheat remains in thefinished flour (Linder, 1991). Therefore, reducing the contributionfrom dietary sources remains a challenge, but elimination of lead-soldered cans, lead-soldered plumbing, and especially the removalof tetraethyl lead as a gasoline additive have produced substantialreductions in lead ingestion. What is needed now is continuedvigilance of largely imported lead-based ceramic ware, lead-containing calcium supplements, and lead leaching into ground-water (Shank and Carson, 1992).Arsenic Arsenic is a ubiquitous element in the environment; itranks 20th in relative abundance among the elements of the earth’scrust and 12th in the human body (Concon, 1988). (Since arsenicis discussed in detail elsewhere in this text, the discussion here islimited to its relationship to foods.) There is some competition forarsenic absorption with selenium, which is known to reduce arsenictoxicity; arsenic is also known to antagonize iodine metabolismand inhibit various metabolic processes, as a result affecting a num-

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ber of organ systems. There are a number of sources of arsenic,including drinking water, air, and pesticides (Newberne, 1987), butarsenic consumed via food is largely in proportion to the amountof seafood eaten (74 percent of the arsenic in a market basket sur-vey came from the meat-poultry-fish group, of which seafood con-sistently has the highest concentration) (Johnson et al., 1981). Al-though arsenic is used as an animal feed additive, this source doesnot contribute much to the body burden, as 0.1% arsanilic acid ordocecylamine-p-chlorophenylarsonate fed to turkeys resulted intissue residues of only 0.31 and 0.24 ppm in fresh muscle (Un-derwood, 1973).

Acute poisoning with arsenic often results from mistaking ar-senic for sugar, baking powder, and soda and adding it to food.The time between exposure and symptoms is 10 min to severaldays, and the symptoms include burning of the mouth or throat, ametallic taste, vomiting, diarrhea (watery and bloody), borborigmi(rumbling of the bowles caused by movement of gas in the GItract), painful tenesmus (spasm of the anal or vesical sphincter),hematuria, dehydration, jaundice, oliguria, collapse, and shock.Headache, vertigo, muscle spasm, stupor, and delirium may occur(Bryan, 1984).Cadmium Cadmium is a relatively rare commodity in nature andis usually associated with shale and sedimentary deposites. It is of-ten found in association with zinc ores and in lesser amounts infossil fuel. Although rare in nature, it is a nearly ubiquitous elementin American society because of its industrial uses in plating, paintpigments, plastics, and textiles. Exposure of humans often occursthrough secondary routes as a result of dumping at smelters andrefining plants, disintegration of automobile tires (which containrubber-laden cadmium), subsequent seepage into the soil andgroundwater, and inhalation of combustion of cadmium-containingmaterials. The estimated yearly release of cadmium from automo-bile tires ranges from 5.2 to 6.0 metric tons (Davis, 1970; Lager-werff and Specht, 1971).

Although, like mercury, cadmium can form alkyl compounds,unlike mercury, the alkyl derivatives are relatively unstable andconsumption almost always involves the inorganic salt. Of two his-torical incidents of cadmium poisoning, one involved the use of

cadmium-plated containers to hold acidic fruit slushes before freez-ing. Up to 13 to 15 ppm cadmium was found in the frozen con-fection, 300 ppm in lemonade, and 450 in raspberry gelatin. Sev-eral deaths resulted. A more recent incident of a chronic poisoninginvolved the dumping of mining wastes into rice paddies in Japan.Middle-aged women who were deficient in calcium and had hadmultiple pregnancies seemed to be the most susceptible. Symptomsincluded hypercalciuria; extreme bone pain from osteomalacia;lumbago; pain in the back, shoulders, and joints; a waddling gait;frequent fractures; proteinuria; and glycosuria. The disease wascalled itai itai (“ouch-ouch disease”) as a result of the pain withwalking. The victims had a reported intake of 1000 �g/day, ap-proximately 200 times the normal intake in unexposed populations(Yamagata and Shigematsu, 1970). Cadmium exposure also hasbeen associated with cancer of the breast, lung, large intestine, andurinary bladder (Newberne, 1987).

Chlorinated Organics Chlorinated organics have been with usfor some time, and given their stability in water and resistance tooxidation, ultraviolet light, microbial degradation, and othersources of natural destruction, chlorinated organics will continueto reside in the environment for some time to come, albeit in minuteamounts. However, with the introduction of chlorinated hydrocar-bons as pesticides in the 1930s, diseases associated with an insectvector such as malaria were nearly eliminated. In the industrial-ized world, chlorinated organics brought the promise of nearly uni-versal solvents, and their extraordinary resistance to degradationmade them suitable for use as heat transfer agents, carbonless copypaper, and fire retardants (Table 30-24).

As persistent as these substances are in the environment anddespite the degree of toxicity that might be implied, the possiblehazard from chlorinated substances is relatively low. Ames et al.(1987) described a method for interpreting the differing potenciesof carcinogens and human exposures: the percentage HERP (hu-man exposure dose/rodent potency dose). Using this method, theydemonstrated that the hazard from trichloroethylene-contaminatedwater in Silicon Valley or Woburn, Massachusetts, or the daily di-etary intake from DDT (or its product, DDE) at a HERP of 0.0003

Table 30-24Examples of Levels of Chlorinated Hydrocarbons in British Food

Chlorinated Hydrocarbons, ug/kg

FOOD CHCl3 CCl4 TCE TCEY TTCE PCE HCB HCBD Per CE

Milk 5.0 0.2 0.3 — — 1.0 0.08 0.3Cheese 33.0 5.0 3.0 0.0 0.0 0.0 0.0 2.0Butter 22.0 14.0 10.0 — — — 2.0 13.0Chicken eggs 1.4 0.5 0.6 0.0 0.0 0.0 0.0 0.0Beef steak 4.0 7.0 3.0 16.0 0.0 0.0 0.0 0.0 0.9Beef fat 3.0 8.0 6.0 12.0 1.0Pork liver 1.0 9.0 4.0 22.0 0.5 0.4 5.0Margarine 3.0 6.0 — 0.8 7.0Tomatoes 2.0 4.5 — 1.7 1.0 70.1 0.8 1.2Bread (fresh) 2.0 5.0 2.0 7.0 — — — — 1.0Fruit drink (canned) 2.0 0.5 — 5.0 0.8 2.0

KEY: CHCl3 � chloroform; CCl4 � carbon tetrachloride; TCE � trichloroethane; TCEY � trichloroethylene; TTCE � tetrachloroethane; PCE � pentachloroethane; HCB �

hexachlorobenzene; HCBD � hexachlorobutadiene; Per CE � perchloroethylene.SOURCE: Modified from McConnell et al., 1975, with kind permission from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington OX5 1GB, UK.

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to 0.004 percent is considerably less than the hazard presented bythe consumption of symphytine in a single cup of comfrey herbtea (0.03 percent) or the hazard presented by aflatoxin in a peanutbutter sandwich (0.03 percent). The FDA’s authority to set toler-ances has been used only once in establishing levels for polychlo-rinated biphenyls (21 CFR 109.15 and 109.30).

Although the possibility always exists, there have been onlya few incidents of mass poisonings via food, two of which involvedcontaminated cooking oil. The first became known as yusho, orrice oil disease, from rice oil contamination by polychlorinatedbiphenyls (PCBs). The most vulnerable individuals were newborninfants of poisoned mothers. The liver and skin were the most se-verely affected. Symptoms included dark brown pigmentation ofnails; acne-like eruptions; increased eye discharge; visual distur-bances; pigmentation of the skin, lips, and gingiva; swelling of theupper eyelids; hyperemia of the conjunctiva; enlargement and el-evation of hair follicles; itching; increased sweating of the palms;hyperkeratotic plaques on the soles and palms; and generalizedmalaise. Recovery requires several years (Anderson and Sogn,1984). The second incident has become known as Spanish toxic oilsyndrome, and although details are still not fully known, it occurredafter aniline-contaminated rapeseed oil was distributed as cookingoil in Spain in 1981. Approximately 20,000 people were affectedand there were several deaths. Because symptoms were unique—including respiratory effects, eosinophilia, and muscle wasting—but not typical of aniline poisoning, the exact etiologic agent is stillunknown. Because the source of the aniline may have been im-properly cleaned tank trucks that had imported industrial chemi-cals, three hypotheses have been offered: the etiologic agent mayhave been (1) a contaminant in the aniline, (2) a contaminant in-troduced during transportation, or (3) a reaction product of normaloil components and the potential contaminants (the fraction of theoil most commonly associated with toxicity contained C18:3

anilide, also called oleyl anilide and “fatty acid anilide”) (Borda etal., 1998; Posada de la Paz et al., 1996; Wood et al., 1994).

Nitrosamines, Nitrosamides, and N-Nitroso Substances Ni-trogenous compounds such as amines, amides, guanidines, andureas can react with oxides of nitrogen (NOx) to form N-nitrosocompounds (NOCs) (Hotchkiss et al., 1992). The NOCs may bedivided into two classes: the nitrosamines, which are N-nitroso de-rivatives of secondary amines, and nitrosamides, which are N-nitroso derivatives of substituted ureas, amides, carbamates,guanidines, and similar compounds (Mirvish, 1975).

Nitrosamines are stable compounds, while many nitrosamideshave half-lives on the order of minutes, particularly at pH �6.5.Both classes have members that are potent carcinogens, but by dif-ferent mechanisms. In general, the biological activity of an NOCis thought to be related to alkylation of genetic macromolecules.N-nitrosamines are metabolically activated by hydroxylation at an�-carbon. The resulting hydroxyalkyl moiety is eliminated as analdehyde, and an unstable primary nitrosamine is formed. The ni-trosamine tautomerizes to a diazonium hydroxide and ultimatelyto a carbonium ion. Nitrosamides spontaneously decompose to acarbonium ion at physiologic pH by a similar mechanism(Hotchkiss et al., 1992). This is consistent with in vitro laboratoryfindings because nitrosamines require S9 for activity and ni-trosamides are mutagenic de novo.

NOCs originate from two sources: environmental formationand endogenous formation (Table 30-25). Environmental sourceshave declined over the last several years but still include foods (e.g.,

nitrate-cured meats) and beverages (e.g., malt beverages), cosmet-ics, occupational exposure, and rubber products (Hotchkiss, 1989).NOCs formed in vivo may actually constitute the greatest expo-sure and are formed from nitrosation of amines and amides in sev-eral areas, including the stomach, where the most favorable con-ditions exist (pH 2 to 4), although consumption of H2-receptorblockers or antacids decreases the formation of NOCs.

Environmentally, nitrite is formed from nitrate or ammoniumions by certain microorganisms in soil, water, and sewage. In vivo,nitrite is formed from nitrate by microorganisms in the mouth andstomach, followed by nitrosation of secondary amines and amidesin the diet. Sources of nitrate and nitrite in the diet are given inTable 30-26. Many sources of nitrate are also sources of vitaminC. Another possibly significant source of nitrate is well water; al-though the levels are generally in the range of 21 �M, average lev-els of 1600 �M (100 mg/L) have been reported (Hotchkiss et al.,1992). However, on the average, western diets contain 1 to 2 mmolnitrate per person per day (Hotchkiss et al., 1992). Nitrosation re-actions can be inhibited by preferential, competitive neutralizationof nitrite with naturally occurring and synthetic materials such asvitamin C, vitamin E, sulfamate, and antioxidants such as BHT,BHA, gallic acid, and even amino acids or proteins (Hotchkiss,1989; Hotchkiss et al., 1992).

N-nitrosoproline is the most common nitrosoamine present inhumans and is excreted virtually unchanged in the urine. The basalrate of urinary excretion of nitrosoproline, which is claimed to benoncarcinogenic, is 2 to 7 �g/day in subjects on a low-nitrate diet(Oshima and Bartsch, 1981). Epidemiologic studies have not pro-vided evidence of a causal association between nitrate exposureand human cancer, nor has a causal link been shown between N-nitroso compounds preformed in the diet or endogenously syn-thesized and the incidence of human cancer (Gangolli 1999).

Food-Borne Molds and Mycotoxins Molds have served humansfor centuries in the production of foods (e.g., ripening of cheese)and have provided various fungal metabolites with important me-dicinal uses; they also may produce metabolites with the potentialto produce severe adverse health effects. Mycotoxins represent adiverse group of chemicals that can occur in a variety of plantfoods. They also can occur in animal products derived from ani-

Table 30-25Sources of Dietary NOCs

The use of nitrate and/or nitrite as intentional food additives, both of which are added to fix the color ofmeats, inhibit oxidation, and prevent toxigenesis

Drying processes in which the drying air is heated by anopen flame source. NOx is generated in small amountsthrough the oxidation of N2, which nitrosates amines in the foods. This is the mechanism for contamination ofmalted barley products

NOCs can migrate from food contact materials such asrubber bottle nipples

NOCs can inhabit spices which may be added to foodCooking over open flames (e.g., natural gas flame) can

result in NOC formation in foods by the same mechanismas drying

SOURCE: Hotchkiss et al., 1992, with permission.

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mals that consume contaminated feeds. The current interest in my-cotoxicoses was generated by a series of reports in 1960–1963 thatassociated the death of turkeys in England (so-called turkey X dis-ease) and ducklings in Uganda with the consumption of peanutmeal feeds containing mold products produced by Aspergillusflavus (Stoloff, 1977). The additional discovery of aflatoxinmetabolites (for example, aflatoxin M1) led to more intensive stud-ies of mycotoxins and to the identification of a variety of thesecompounds associated with adverse human health effects, both ret-rospectively and prospectively.

Moldy foods are consumed throughout the world during timesof famine, as a matter of taste, and through ignorance of their ad-verse health effects. Epidemiologic studies designed to ascertainthe acute or chronic effects of such consumption are few. Data fromanimal studies indicate that the consumption of food contaminatedwith mycotoxins has a high potential to produce a variety of hu-man diseases (Miller, 1991).

With some exceptions, molds can be divided into two maingroups: “field fungi” and “storage fungi.” The former group con-tains species that proliferate in and under field conditions and donot multiply once grain is in storage. Field fungi are in fact su-perseded and overrun by storage fungi if conditions of moistureand oxygen allow. Thus, for instance, Fusarium spp., a field fun-gus commonly found on crops, is seldom found after about 6 weeksof storage, its place being taken by Aspergillus and Penicillium,both of which represent several species of storage fungi (Harrison,1971). However, the presence of mold does not guarantee the pres-ence of mycotoxin, which is elaborated only under certain condi-tions. Further, more than one mold can produce the same myco-toxin (e.g., both Aspergillus and Penicillium may produce themycotoxin cyclopiazonic acid) (Truckness et al., 1987; El-Bannaet al., 1987). Also, more than one mycotoxin may be present in anintoxication; that is, as in the outbreak of turkey X disease, there

is evidence that aflatoxin and cyclopiazonic acid both exerted aneffect, but the profound effects of aflatoxin overshadowed those ofcyclopiazonic acid (Miller, 1989). Although there are many dif-ferent mycotoxins and subgroups (Table 30-27), this discussion isconfined largely to two of the more toxicologically and economi-cally important ones: the aflatoxins and trichothecenes.Aflatoxins Among the various mycotoxins, the aflatoxins havebeen the subject of the most intensive research because of the ex-tremely potent hepatocarcinogenicity and toxicity of aflatoxin B1

in rats. Epidemiologic studies conducted in Africa and Asia sug-gest that it is a human hepatocarcinogen, and various other reportshave implicated the aflatoxins in incidences of human toxicity(Krishnamachari et al., 1975; Peers et al., 1976).

Generally, aflatoxins occur in susceptible crops as mixturesof aflatoxins B1, B2, G1, and G2, with only aflatoxins B1 and G1

demonstrating carcinogenicity. A carcinogenic hydroxylatedmetabolite of aflatoxin B1 (termed aflatoxin M1) can occur in themilk from dairy cows that consume contaminated feed. Aflatoxinsmay occur in a number of susceptible commodities and productsderived from them, including edible nuts (peanuts, pistachios, al-monds, walnuts, pecans, Brazil nuts), oil seeds (cottonseed, copra),and grains (corn, grain sorghum, millet) (Stoloff, 1977). In tropi-cal regions, aflatoxin can be produced in unrefrigerated preparedfoods. The two major sources of aflatoxin contamination of com-modities are field contamination, especially during times of droughtand other stresses, which allow insect damage that opens the plantto mold attack, and inadequate storage conditions. Since thediscovery of their potential threat to human health, progress hasbeen made in decreasing the level of aflatoxin in specific com-modities. Control measures include ensuring adequate storage con-ditions and careful monitoring of susceptible commodities for afla-toxin level and the banning of lots that exceed the action level foraflatoxin B1.

Table 30-26Nitrate and Nitrite Content of Food

NITRATE, NITRITE, NITRATE, NITRITE,VEGETABLES PPM PPM MEAT PPM PPM

Artichoke 12 0.4 Unsmoked side bacon 134 12Asparagus 44 0.6 Unsmoked back bacon 160 8Green beans 340 0.6 Peameal bacon 16 21Lima beans 54 1.1 Smoked bacon 52 7Beets 2400 4 Corned beef 141 19Broccoli 740 1 Cured corned beef 852 9Brussels sprouts 120 1 Corned beef brisket 90 3Cabbage 520 0.5 Pickled beef 70 23Carrots 200 0.8 Canned corn beef 77 24Cauliflower 480 1.1 Ham 105 17Celery 2300 0.5 Smoked ham 138 50Corn 45 2 Cured ham 767 35Radish 1900 0.2 Belitalia (garlic) 247 5Rhubarb 2100 NR* Pepperoni (beef) 149 23Spinach 1800 2.5 Summer sausage 135 7Tomatoes 58 NR Ukranian sausage (Polish) 77 15Turnip 390 NR German sausage 71 17Turnip greens 6600 2.3

*NR � not reported.SOURCE: Hotchkiss et al., 1992, with permission

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Table 30-27Selected Mycotoxins Produced by Various Molds

COMMODITIES

MYCOTOXIN SOURCE EFFECT CONTAMINATED

Aflatoxins B1, B2, G1, G2 Aspergillus flavus, parasiticus Acute aflatoxicosis, carcinogenesis Corn, peanuts, andand others

Aflatoxin M1 Metabolite of AFB1 Hepatotoxicity MilkFumonisins B1, B2, B3, Fusarium moniliforme Carcinogenesis Corn

B4, A1, A2

Trichothecenes Fusarium, Myrothecium Hematopoietic toxicity, meningeal Cereal grains, cornhemorrhage of brain, “nervous’’disorder, necrosis of skin,hemorrhage in mucosal epitheliaof stomach and intestine

T-2 toxin Trichoderma Corn, barley, sorghumTrichodermin CephalosporiumZearalenones Fusarium Estrogenic effect Corn, grainCyclopiazonic acid Aspergillus, Penicillium Muscle, liver, and splenic toxicity Cheese, grains, peanutsKojic acid Aspergillus Hepatotoxic? Grain, animal feed3-Nitropropionic acid Arthrinium sacchari, Central nervous system impairment Sugarcane

saccharicola, phaeospermumCitreoviridin Penicillium citreoviride, Cardiac beriberi Rice

toxicariumCytochalasins E, B, F, H Aspergillus and Penicillium Cytotoxicity Corn, cereal grainSterigmatocystin Aspergillus versiolar Carcinogenesis CornPenicillinic acid Penicillium cyclopium Nephrotoxicity, abortifacient Corn, dried beans,

grainsRubratoxins A, B Penicillium rubrum Hepatotoxicity, teratogenic CornPatulin Penicillium patulatum Carcinogenesis, liver damage Apple and apple

productsOchratoxin A. ochraceus, P. viridicatum Balkan nephropathy, carcinogenesis Grains, peanuts, green

coffeeErgot alkaloids Cladosporium purpurea Ergotism Grains

Aflatoxin B1 is acutely toxic in all species studied, with anLD50 ranging from 0.5 mg/kg for the duckling to 60 mg/kg for themouse (Wogan, 1973). Death typically results from hepatotoxicity.This aflatoxin is also highly mutagenic, hepatocarcinogenic, andpossibly teratogenic. A problem in extrapolating animal data to hu-mans is the extremely wide range of species susceptibility to afla-toxin B1. For instance, whereas aflatoxin B1 appears to be the mosthepatocarcinogenic compound known for the rat, the adult mouseis essentially totally resistant to its hepatocarcinogenicity.

Aflatoxin B1 is an extremely reactive compound biologically,altering a number of biochemical systems. The hepatocarcino-genicity of aflatoxin B1 is associated with its biotransformation toa highly reactive electrophilic epoxide that forms covalent adductswith DNA, RNA, and protein. Damage to DNA is thought to bethe initial biochemical lesion resulting in the expression of thepathologic tumor growth (Miller, 1978). Species differences in theresponse to aflatoxin may be due in part to differences in bio-transformation and susceptibility to the initial biochemical lesion(Campbell and Hayes, 1976; Monroe and Eaton, 1987).

Although the aflatoxins have received the greatest attentionamong the various mycotoxins because of their hepatocarcino-genicity in certain species, there is no compelling evidence thatthey have the greatest potential to produce adverse human healtheffects.

Trichothecenes Trichothecenes represent a group of toxic sub-stances of which it is likely that several forms may be consumedconcomitantly. They represent many different chemical entities allcontaining the trichothecene nucleus, and are produced by a num-ber of commonly occurring molds, including Fusarium, Myrothe-cium, Trichoderma, and Cephalosporium. The trichothecenes werefirst discovered during attempts to isolate antibiotics, and althoughsome show antibiotic activity, their toxicity has precluded theirpharmacologic use. Trichothecenes most often occur in moldy ce-real grains. There have been many reported cases of trichothecenetoxicity in farm animals and a few in humans. One of the more fa-mous cases of presumed human toxicity associated with the con-sumption of trichothecenes occurred in Russia during 1944 aroundOrenburg, Siberia. The disruption of agriculture caused by WorldWar II led to the overwintering of millet, wheat, and barley in thefield. Consumption of these grains resulted in vomiting, skin in-flammation, diarrhea, and multiple hemorrhages, among othersymptoms. This exposure was fatal to over 10 percent of the af-fected individuals (Ueno, 1977). The extent of toxicity associatedwith the trichothecenes in humans and farm animals is currentlyunknown owing to the number of entities in this group and the dif-ficulty of assaying for these compounds. The acute LD50s of thetrichothecenes range from 0.5 to 70 mg/kg, and though there havebeen reports of possible chronic toxicity associated with certain

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members of this group, more research will be needed before themagnitude of their potential to produce adverse human health ef-fects is understood (Sato and Ueno, 1977).Fumonisins Fumonisins are recently discovered mycotoxins pro-duced by Fusarium moniliforme and several other Fusariumspecies. Corn products contaminated with Fusarium moniliformeare responsible for several agriculturally important diseases inhorses and swine (Norred, 1993) and are being actively evaluatedto determine how great a threat they pose to public health. Initialevidence of the involvement of F. moniliforme produced toxins inhuman disease was reported by Marasas et al. (1988). They foundthat an increased incidence of esophageal cancer was associatedwith the consumption of contaminated corn (maize) by humans ina region in South Africa. Corn borer insect pests cause damage tothe developing grain, which enables spores of the toxin-producingFusarium fungi to germinate. The fungus then proliferates, whichleads to ear and kernel rot and the production of potentially haz-ardous levels of fumonisins. Corn varieties that express the Bt in-secticidal protein have recently been shown to contain significantlyreduced levels of fumonisin because the Bt protein significantly re-duces corn borer–induced tissue damage in corn products(Munkvold et al., 1997; 1999; Masoero et al., 1999).Zearalenone Another mycotoxin produced by Fusarium is zear-alenone. It was first discovered during attempts to isolate an agentfrom feeds that produced a hyperestrogenic syndrome in swine,characterized by a swollen and edematous vulva and actual vagi-nal prolapse in severe cases (Stob et al., 1962). Zearalenone canoccur in corn, barley, wheat, hay, and oats as well as other agri-cultural commodities (Mirocha et al., 1977). Zearalenone con-sumption can decrease the reproductive potential of farm animals,especially swine.

SUBSTANCES FOR WHICHTOLERANCES MAY NOT BE SET

All of the contaminants of food described to this point are thoseassociated with synthesis, growth, production, or storage and areregarded by the FDA as unavoidable. Because they are unavoid-able, the FDA sets limits rather than banning them, as describedearlier. The substances in this section are regarded as (1) avoid-able or of such hazard that a safe level cannot be set, therefore theFDA has determined that food containing such substances isbanned; or (2) beyond the control of the FDA and cannot be reg-ulated (for example, substances produced in the home).

Toxins in Fish, Shellfish, and Turtles

There are a number of seafood toxins (to be distinguished frommarine venoms), many of which are not confined to a single species(over 400 species have been incriminated in ciguatera toxicity) andare therefore most likely to be influenced by the environment. How-ever, some seafood toxins are specific to a single species or genus.A complicating factor in the study of seafood toxins is the spo-radic frequency and nonpredictability of the presence of the toxin.

Seafood toxins generally can be classified according to the lo-cation of the poison. For example: (1) ichthyosarcotoxin is con-centrated in the muscles, skin, liver, or intestines or is otherwisenot associated with the reproductive system or circulatory system,(2) ichthyootoxin is associated with reproductive tissue, (3) ichthy-ohemotoxin is confined to the circulatory system, and (4) ichthy-

ohepatotoxin is confined to the liver. In general, seafood toxins un-der FDA policy have a zero tolerance, with any detectable levelconsidered cause for regulatory action.

Dinoflagellate Poisoning (Paralytic Shellfish Poisoning or PSP;Saxitoxin) The etiologic agent in this type of poisoning is saxi-toxin or related compounds and is found in mussels, cockles, clams,soft-shell clams, butter clams, scallops, and shellfish broth. Bivalvemussels are the most common vehicles. Saxitoxin, originally iso-lated from toxic Alaskan butter clams (Saxidomus giganticus) isactually a family of neurotoxins and includes neosaxitin andgonyautoxins 1 through 4. All block neural transmission at the neu-romuscular junction by binding to the surface of the sodium chan-nels and interrupting the flow of Na� ions; atrioventricular nodalconduction may be suppressed, and there may be direct suppres-sion of the respiratory center and progressive reduction of periph-eral nerve excitability. The toxin produces paresthesias and neuro-muscular weakness without hypotension and lacks the emetic andhypothermic action of tetrodotoxin. Moderate symptoms are pro-duced by 120 to 180 �g per person and are reversible within hoursor days, while 80 �g of purified toxin per 100 g of tissue (0.5 to2 mg per person) may be lethal, due to asphyxiation, usually within12 h of ingestion. The toxin is an alkaloid and relatively heat sta-ble. The toxin is produced by several genera of plankton[Gonyaulax (now known as Alexandrium) catenella, Gonyaulaxacatenella, Gonyaulax tamarensis, Pyrodinium spp., Ptychodiscusbrevis, Gymnodinium catenaturm, and others]; and during red tides,blooms of these plankton may reach 20 to 40 million per milliliter.Toxic materials are stored in various parts of the body of shellfish.Digestive organs, liver, gills, and siphons contain the greatest con-centrations of poison during the warmer months. Distribution isworldwide (Bryan, 1984; Clark et al., 1999; Liston, 2000).

Amnesic Shellfish Poisoning (Domoic Acid) Consumption ofmussels harvested from the area off Prince Edward Island in 1987resulted in gastroenteritis, and many of the older individuals af-fected or those with underlying chronic diseases experienced neu-rologic symptoms including memory loss. Despite treatment, threepatients (71 to 84 years old) died within 11 to 24 days. The poi-soning was attributed to domoic acid produced by the diatomNitzschia pungens f. multiseries, which had been ingested by themussels during the normal course of feeding. Occurrence of do-moic acid has also been reported in California shellfish, producedby Nitzschia pseudodelicatissima, and in anchovies (resulting inpelican deaths), produced by Nitzschia pseudoseriata (now calledPseudonitzchia australis). Domoic acid has been reported in shell-fish in other provinces of Canada, Alaska, Washington, and Oregon;it may be as frequent as PSP toxins. Domoic acid has also beenreported in seaweed. Domoic acid was reported in Japan in 1958and was isolated from the red algae Chondria armata.

In the Canadian outbreak, mice injected with extracts (as inthe PSP assay) died within 3.5 h. The mice exhibited a scratchingsyndrome uniquely characteristic of domoic acid, followed by in-creasingly uncoordinated movements and seizures until the micefell on their sides, rolled over, and died. Levels of domoic acid40 �g/g wet weight of mussel meat caused the mouse symptoms(Canadian authorities require cessation of harvesting when levelsapproach 20 �g/g). Mice and rats can generally tolerate 30 to 50mg/kg (mouse NOEL via intraperitoneal injection is 24 mg/kg).Domoic acid is dose-responsive in humans, with no effect at 0.2to 0.3 mg/kg, mild (gastrointestinal) symptoms at 0.9 to 1.9 mg/kg,

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and the most serious symptoms at 1.9 to 4.2 mg/kg. Although ro-dents may appear to be more tolerant, the fatalities in humans wereassociated with underlying illness. Domoic acid is an analog ofglutamine, a neurotransmitter, and of kainic acid; the toxicity ofall three is similar as they are excitatory and act on three types ofreceptors in the central nervous system, with the hippocampus be-ing the most sensitive. Domoic acid may be a more potent activa-tor of kannic acid receptors than kannic acid itself. The stimula-tory action may lead to extensive damage of the hippocampus butless severe injury to the thalamic and forebrain regions (Clark etal., 1999; Todd, 1993).

Ciguatera Poisoning The cigua in ciguatera is derived from theSpanish name for the sea snail Turbo pica, in which the symptomswere first reported. Ciguatera and related toxins (scaritoxin andmaitotoxin) are ichthyosarcotoxic neurotoxins (anticholinesterase)and are found in 11 orders, 57 families, and over 400 species offish as well as in oysters and clams. The penultimate toxin (gam-biertoxin) is produced by the dinoflagellate Gambierdiscus toxi-cus, commonly isolated from microalgae growing on or near coralreefs that have ingested the dinoflagellate. The pretoxin appears topass through the food chain and is biotransformed upon transfer toor by the ingesting fish to the active form, which is consumed bymammals. Other toxins, including palytoxin and okadaic acid, un-related to gambiertoxin, may be present in ciguarteric fish and maynot contribute to toxicity. The asymptomatic period is 3 to 5 h af-ter consumption but may last up to 24 h. The onset of illness issudden, and symptoms may include abdominal pain, nausea, vom-iting, and watery diarrhea; muscular aches; tingling and numbnessof the lips, tongue, and throat; a metallic taste; temporary blind-ness; and paralysis. Deaths have occurred. Recovery usually oc-curs within 24 h, but tingling may continue for a week or more.The intraperitoneal LD50 of maitotoxin in mice is 50 ng/kg (Bryan,1984; Liston, 2000).

Puffer Fish Poisoning (Tetrodotoxin) Tetrodon or puffer fishpoisoning may be caused by the improper preparation and con-sumption of any of about 90 species of puffer fish (fugu, blowfish,globefish, porcupine fish, molas, burrfish, balloonfish, toadfish,etc.) and has been found in newts, frogs, octopus, starfish, flat-worms, various crabs, and gastropods. The toxin (tetrodotoxin) islocated in nearly all the tissues, but the ovaries, roe, liver, intes-tines, and skin are the most toxic. Toxicity is highest during thespawning period, although a species may be toxic in one locationbut not another. Tetrodotoxin is associated with the presence ofseveral bacteria on and in fish and shellfish and gives the fish anevolutionary advantage in providing protection against predators(i.e., they are endosymbiotic bacteria). A total of 21 species canproduce tetrodotoxins including Vibrio, Pseudomonas, E. coli, andat least two strains of red algae.

Tetrodotoxin is a neurotoxin and causes paralysis of the cen-tral nervous system and peripheral nerves by blocking the move-ment of all monovalent cations. The toxin is water-soluble and isstable to boiling except in an alkaline solution. A fatal dose maybe as little as 1 to 4 mg per person. The victim is asymptomaticfor 10 to 45 min but may have a reprieve for as long as 3 h ormore. Toxicity is manifest as a tingling or prickly sensation of thefingers and toes; malaise; dizziness; pallor; numbness of the lips,tongue, and extremities; ataxia; nausea, vomiting, and diarrhea; epi-gastric pain; dryness of the skin; subcutaneous hemorrhage anddesquamation; respiratory distress; muscular twitching, tremor,

incoordination, and muscular paralysis; and intense cyanosis.Fatality rates are high (Bryan, 1984; Liston, 2000).

Moray Eel Poisoning Although the moray eel (Gymnothorax ja-vanicus) and other carnivorous fish may accumulate ciguatoxin as the result of eating other contaminated fish, the Indo-Pacificmoray eel (Lycodontis nudivomer) has been shown to posses a mucous skin secretion with hemolytic, toxic, and hemagglutinat-ing properties. The hemolytic properties can be separated from thehemagglutinating properties. The hemolytic property is lost upontreatment with trypsin and is unstable in the presence of heat oracidic or alkaline media (Randall et al., 1981). The skin mucus of other species of eels, the common European eel (Anguilla an-guilla) and pike eel (Muraenesox cinereus), was found to have pro-teinaceous toxins immunologically similar to that of the skin mu-cous toxin from the Japanese eel (Anguilla japonica) (Shiomi etal., 1994).

Fish Liver Poisoning This type of poisoning involves an ichthy-ohepatotoxin and may be related to or cause hypervitaminosis A.It occurs after the consumption of the liver of sawara (Japanesemackerel) and ishingai (sea bass, sandfish, and porgy). After anasymptomatic period of 30 min to 12 h, the victim experiencesnausea, vomiting, fever, headache, mild diarrhea, rash, loss of hair,dermatitis, desquamation, bleeding from the lips, and joint pain(Bryan, 1984).

Fish Roe Poisoning This type of poisoning involves a group ofichthyootoxins found in the roe and ovaries of carp, barbel, pike,sturgeons, gar, catfish, tench, bream, minnows, salmon, whitefish,trout, blenny, cabezon, and other freshwater and saltwater fish. Poisonings have been reported in Europe, Asia, and North Amer-ica. Within this group of ichthyootoxins are heat-stable toxins and lipoprotein toxins. The asymptomatic period is 1 to 6 h, fol-lowed by a bitter taste, dryness of the mouth, intense thirst,headache, fever, vertigo, nausea, vomiting, abdominal cramps, di-arrhea, dizziness, cold sweats, chills, and cyanosis. Paralysis, con-vulsions, and death may occur in severe cases (Bryan, 1984; Fur-man, 1974).

Abalone Poisoning (Pyropheophorbide) Abalone poisoning iscaused by abalone viscera poison (located in the liver and diges-tive gland) and is unusual in that it causes photosensitization. Thetoxin, pyropheophorbide a, is stable to boiling, freezing, and salt-ing. It is found in Japanese abalone, Haliotis discus and Haliotissieboldi. The development of symptoms is contingent on exposureto sunlight. The symptoms are of sudden onset and include a burn-ing and stinging sensation over the entire body, a prickling sensa-tion, itching, erythema, edema, and skin ulceration on parts of thebody exposed to sunlight (Bryan, 1984; Shiomi, 1999). Paralyticshellfish toxin (PST) have been detected in abalone, probablythrough consumption of the mossworm, a plankton feeder that alsoclings to seaweed, and some shellfish (Takatani et al., 1997).

Sea Urchin Poisoning The etiologic agent forms during the re-productive season and is confined to the gonads. The sea urchinsinvolved include Paracentrotus lividus, Tripneustes ventricosus,and Centrechinus antillarum. The symptoms include abdominalpain, nausea, vomiting, diarrhea, and migraine-like attacks (Bryan,1984). The toxin has been shown to interfere with calcium uptakein nerve preparations (Zhang et al., 1998).

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Sea Turtle Poisoning (Chelonitoxin) The etiologic agent hereis chelonitoxin, which is found in the liver (greatest concentration)but also in the flesh, fat, viscera, and blood. Toxicity is describedas sporadic or even seasonal, indicating that the poison may be de-rived from toxic marine algae. Most outbreaks occur in the Indo-Pacific region. The turtles involved include the green sea turtle aswell as the hawksbill and leatherback turtles. Local custom in SriLanka is to first offer the liver to crows, and if the birds eat it, theflesh is regarded as safe. However, because the symptoms appearover a few hours to several days, this bioassay requires patience.Symptoms of intoxication in humans include vomiting; diarrhea;sore lips, tongue, and throat; foul breath; difficulty in swallowing;a white coating on the tongue, which may become covered withpin-sized, pustular papules; tightness of the chest; coma; and death.The toxin has been reported transferred to nursing infants from in-toxicated mothers. Postmortem examinations reveal congestion ofinternal organs, interstitial pulmonary edema, and necrosis of my-ocardial fibers. Fatality rates of 7 and 25 percent have been re-ported (Ariyananda and Fernando, 1987; Bryan, 1984; ChampetierDe Ribes et al., 1997; Chandrasiri et al., 1988).

Haff Disease Haff disease is a syndrome of unknown etiologythat occurs following consumption of certain types of fish. Thesyndrome consists of rhabdomyolysis with a release of muscle cellcontents into the blood. Patients are often rigid, sensitive to touch,and unable to move; their urine may have a dark brown color.Symptoms appear 18 h (with a range of 6 to 21 h) after consump-tion; they resolve within 2 to 3 days, and the fatality rate is ap-proximately 1 percent. “Haff disease” was first reported in the1920s along the Koenigsberg Haff, a brackish inlet on the BalticSea, although outbreaks have been reported in Sweden, the formerSoviet Union, and in the United States beginning in 1984. U.S.poisonings have been associated with buffalo fish (Ictiobus cypr-nellus) caught in California, Missouri (St. Louis), and Louisiana.No etiologic agent has been identified (Anonymous, 1998).

Microbiologic Agents—PreformedBacterial Toxins

Although the United States likely has the safest and cleanest foodsupply in the world, most U.S. food-related illness results from mi-crobial contamination. If all the food-borne health concerns couldbe divided into two large categories—poisonings and infections—the former would include chemical poisonings (e.g., contaminantssuch as chlorinated hydrocarbons) and intoxications, which mayhave a plant, animal, or microbial origin. In the infections cate-gory, food acts as a vector for organisms that exhibit their patho-genicity once they have multiplied inside the body. Infections in-clude the two subcategories enterotoxigenic infections (with therelease of toxins following colonization of the GI tract) and inva-sive infections, in which the GI tract is penetrated and the body isinvaded by organisms.

Food-borne disease outbreaks are tracked by the Centers forDisease Control and Prevention (CDC), which reports that thereare approximately 400 outbreaks of food-borne disease per year,involving 10,000 to 20,000 people. However, the actual frequencymay be as much as 10 to 200 times as high because (1) an out-break is classified as such only when the source can be identifiedas affecting two or more people and (2) most home poisonings aremild or have a long incubation time; they are therefore not con-

nected to the ingested food and go unreported. Naturally, becauseof differences in virulence and opportunity, some species are morelikely than others to cause outbreaks.

There are a number of food toxins of microbial origin; how-ever, discussion in this chapter is limited to preformed bacterialtoxins—that is, those toxins elaborated by bacteria concomitant totheir residence and growth in or on the food prior to ingestion.There are a number of different types of toxins. An enterotoxin isa toxin having action on the enteric cells of the intestine and anendotoxin is generally a lipopolysaccharide membrane constituentreleased from a dead or dying gram-negative becteria. These tox-ins are nonspecific and stimulate inflammatory responses frommacrophages, including but not limited to prostaglandins, throm-boxans, interleukins, and other mediators of immunity. Exotoxinsare synthesized and released (usually by gram-positive bacteria)and are not an integral part of the organism; however, they mayenhance its virulence. Some bacteria, such as Shigella, Staphylo-coccus aureus, and E. coli (which releases the shiga-like Verotoxin), can elaborate both endotoxin and exotoxin.

Clostridium botulinum and Clostridium butyricum Food botu-lism, although now relatively rare, still occurs and is important be-cause of its potency. All organisms of the Clostridium genus aregram-positive, spore-forming anaerobes. Botulism is due to the tox-ins A, B, E, and F, which may be produced by one or more strainsof C. botulinum and C. butyricum (type E only); toxins C and Dcause botulism in animals. Type G has not caused any human cases.C. botulinum organisms are categorized as group I to IV on the ba-sis of toxin produced; additionally, group I is proteolytic in culture(liquefying egg white, gelatin, and other solid proteins). The toxinis elaborated in foods, wounds, and infant gut and is neurotoxic,interfering with acetylcholine at peripheral nerve endings. Al-though the spores are among the most heat-resistant, the toxins areheat-labile (the toxin may be rendered harmless at 80 to 100°C for5 to 10 min). Botulinum toxins are large zinc-metalloproteins of~150,000 Da, composed of two parts: a 50,000-Da piece, the cat-alytic subunit, and the 100,000-Da piece, containing an N-termi-nal translocation domain and a C-terminal binding domain. Thestructural features are similar to those of tetanus toxin. For typesB, D, F, and G (and tetanus toxin), the target protein isVAMP/synaptobrevin, a protein associated with the synaptic vesi-cle. Types A and E cleave a protein associated with the presynap-tic memberane, ANAP25. Botulinum toxin C cleaves SNAP25 andsyntaxin, another protein involved in exocytosis. Although intra-cellular mechanisms of botulinum and tetanus toxins are similar,symptoms are different because different populations of neuronsare targeted. The symptoms may include respiratory distress andrespiratory paralysis that may persist for 6 to 8 months. The casefatality rate is 35 to 65 percent, and the poison is fatal in 3 to 10days; a lethal dose is approximately 1 ng. Sources and reservoirsinclude soil, mud, water, and the intestinal tracts of animals. Foodsassociated with botulinum toxin include improperly canned low-acid foods (green beans, corn, beets, asparagus, chili peppers,mushrooms, spinach, figs, baked potato, cheese sauce, beef stew,olives, and tuna). The toxin also may occur in smoked fish, fer-mented food (seal flippers, salmon eggs) and improperly home-cured hams. An increasing source of poisonings is from the use offlavored oils or oil infusion, most typically in garlic-in-oil prepa-rations; in 1993, FDA required acidification of such preparationsto prevent the growth of Clostridium. While a proteolytic strain ofC. botulinum (group I) may cause the food to appear and smell

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“spoiled” (by-products include isobutyric acid, isovaleric acid, andphenylpropionic acid), this is not the case with nonproteolyticstrains, many of which can flourish and elaborate toxin at temper-atures as low as 3°C (Belitz and Grosch, 1999; Bryan, 1984; Crane,1999; Hobbs, 1976; Loving, 1998; Lund and Peck, 2000).

The successful use of nitrates in meat to prevent spoilage byC. botulinum resulted in the petitioning of FDA by the USDA tohave sodium and potassium nitrate approved for addition by “priorsanction” (21 CFR 181.33). The mechanism of nitrates is believedto be due to an inactivation by nitric oxide of iron-sulfur proteinssuch as ferrodoxin and pyruvate oxidoreductase within the germi-nated cells. The activity is dependent on the pH and is proportionalto the level of free HNO2; 100 mg nitrate/kg of meat is necessaryfor the antimicrobial effect, although this effect can be enhancedwith ascorbates and chelating agents. Other antibacterials that pre-vent C. botulinum include nisin (used in cheese spreads), parabens,phenolic antioxidants, polyphosphates and carbon dioxide (Belitzand Grosch, 1999; Lund and Peck, 2000).

Clostridium perfringens The primary reservoir for C. perfrin-gens, unlike C. botulinum, is the intestinal tract of warm-bloodedanimals (including humans). Most incidences of C. perfringensfood poisoning are associated with the consumption of roasted meatthat has been contaminated with intestinal contents at slaughter,followed by roasting and inadequate storage, allowing C. perfrin-gens growth and enterotoxin (CPE) to be elaborated (although someCPE may actually be released during a “second sporulation”process in the stomach of the victim). Virtually all food poisoningis produced by type A strain, although a particularly severe form(called “pig-bel”) is produced by type C strain and is only seenamong natives of the New Guinea highlands. CPE is enterotoxicand follows an ordered series of events, first causing cellular ionpermeability, followed by macromolecular (DNA, RNA) synthesisinhibition, morphologic alteration, cell lysis, villus tip desquama-tion, and severe fluid loss. This is manifest by abdominal cramp-ing diarrhea occurring within 8 to 16 h, although symptoms are ofshort duration—1 day or less. Foods associated with C. perfrin-gens poisoning include cooked meat or poultry, gravy, stew, andmeat pies. The curious form called pig-bel follows feasting on porkby New Guinea highlanders, in whom low levels of proteases areinadequate to hydrolyze the toxins, which are subsequently ab-sorbed. C. perfringens is also associated with the production ofother 11 other toxins, including those associated with gas gangrene(Bryan, 1984; Crane, 1999; Duncan, 1976; Hauschild, 1971;Hobbs, 1979; Hobbs et al., 1953; Labbe, 2000; Walker, 1975).

Bacillus cereus B. cereus is also a gram-positive, spore-formingrod, but it is an aerobe. B. cereus is a causative agent of emetic ordiarrheagenic exo- and enterotoxins elaborated in food. The emeticthermostable toxin (surviving 259°F for 90 min) is called cerulide(a small cyclic peptide, of 1.2 kDa that acts on 5-HT3 receptors,stimulating the vagus afferent nerve) and is produced by serotypes1, 3, and 8. The diarrheagenic thermolabile toxin (133°F for 20min) is produced by serotypes 1, 2, 6, 8, 10, and 19 and may alsobe produced in situ in the lower intestine of the host. The diarrhealform may actually consist of three toxins, one of which is he-molytic. Reservoirs are soil and dust. Foods associated with thisorganism and its toxic properties include boiled and fried rice (prin-cipally the emetic form), while the diarrheal form has a wider oc-currence and may be found in meats, stews, pudding, sauces, dairyproducts, vegetable dishes, soups, and meat loaf (Bryan, 1984;

Crane, 1999; Gilbert, 1979; Goepfert et al., 1972; Granum andLund, 1997).

Evidence is accumulating that other species of Bacillus mayelaborate food toxins, including Bacillus thuringiensis, Bacillussubtilis, Bacillus licheniformis, and Bacillus pumilis (Crane, 1999;Granum and Baird-Parker, 2000).

Staphylococcus aureus Staphylococcal intoxication includesstaphlyloenterotoxicosis and staphylococcal food poisoning. S.aureus produces a wide variety of exoproteins, including toxicshock syndrome toxin-1 (TSST-1), the exfoliative toxins ETA andETB, leukociden, and the staphylococcal enterotoxins (SEA, SEB,SECn, SED, SEE, SEG, SHE and SEI). TSST-1 and the staphylo-coccal enterotoxins (SE) are also known as pyrogenic toxin su-perantigens (PTSAgs) on the basis of their biological characteris-tics. Although enterotoxemia develops only from the ingestion oflarge amounts of SE, emesis is produced as the result of stimula-tion of the putative SE receptors in the abdominal viscera, follow-ing which there is a cascade of inflammatory mediator release. Allthe SE toxins share a number of properties: an ability to cause eme-sis and gastroenteritis in primates, superantigenicity, intermediateresistance to heat and pepsin digestion, and tertiary structural sim-ilarity, including an intramolecular disulfide bond. Sources ofStaphylococcus include nose and throat discharges, hands and skin,infected cuts, wounds, burns, boils, pimples, acne, and feces. Theanterior nares of humans are the primary reservoirs. Other reser-voirs include mastitic udders of cows and ewes (responsible forcontamination of unpasteurized milk) and arthritic and bruised tis-sues of poultry. Foods are usually contaminated after cooking bypersons cutting, slicing, chopping, or otherwise handling them andthen keeping the foods at room temperature for several hours orstoring them in large containers. Foods associated with staphylo-coccal poisoning include cooked ham; meat products, includingpoultry and dressing; sauces and gravy; cream-filled pastry; pota-toes; ham, poultry, and fish salads; milk; cheese; bread pudding;and generally high-protein leftover foods (Bryan, 1976, 1984;Bergdoll, 1979; Cohen, 1972; Crane, 1999; Dinges et al., 2000;Minor and Marth, 1976).

Escherichia coli Although E. coli does not produce a preformedtoxin, it deserves mention because of the overwhelming publicitythe emergent strain O157:H7 has received (H and O refer to fla-gellar antigens and virulence markers). There are four categoriesof E. coli associated with diarrheal disease: enteropathogenic(EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and Verocytotoxin–producing E. coli (VTEC). The classification VTEC alsoincludes “shiga-like toxin”–producing E. coli (or SLTEC) and“shiga toxin”–producing E. coli (STEC). Enterohemorrhagic E.coli (EHEC) refers to those strains producing bloody diarrhea,which are a subset of VTEC. The reference to shiga toxin is theresult of the clinical similarity of the bloody diarrhea caused byEHEC to that caused by Shigella. Each of the diseases presentedby the four categories is also associated with one or more toxins(Willshaw et al., 2000).

Because cattle are a significant reservoir of E. coli, it is log-ical that most outbreaks in the United States have been associatedwith hamburgers and other beef products, although raw vegetables(often fertilized with manure) and unpasteurized apple cider andjuice have been reported as souces of outbreaks. Outbreaks inEurope are more often associated with contamination of recre-ational waters (swimming pools, lakes, etc.). Other sources of con-

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tamination include person-to-person contact (especially amonginstitutionalized persons and in families) and contact with farmanimals, especially following educational farm visits (Karch et al.,1999).

The subject of organic food has increasingly captured the pub-lic interest. Within this issue is a debate concerning the use in or-ganic and conventional farming of organic fertilizers (e.g., cow ma-nure) that may contain E. coli O157:H7 (Stephenson, 1997). Datareported to the U.S. Centers for Disease Control and Prevention(CDC) in 1996 and tabulated in a CDC document entitled “Clus-ters/Outbreaks of E. coli O157:H7 reported to CDC in 1996” showthat approximately 10 percent of all E. coli O157:H7 infections re-ported that year were from organically grown lettuce, although or-ganic foods apparently account for less than 1 percent of the totalfood supply. This information, although much too preliminary forany meaningful conclusions, nonetheless suggests the need forcareful evaluation of the use of manure in conventional and organicfarming.

At the basis of the potential problem is the use of inadequatelytreated manure for fertilizer. Human cases of E. coli O157:H7 in-fection have been reported from consumption of contaminated let-tuce, potatoes, radish sprouts, alfalfa sprouts, cantaloupe, and un-pasteurized apple cider and juice (Karch et al., 1999). Adequatetreating of manure requires composting the manure for a minimumof 3 months, during which the heap must reach a temperature of60°C; although this may be adequate to kill vegetative pathogens,it will not destroy spore formers such as Clostridium perfringensor Clostridium botulinum. Survival of viruses and protozoa duringcomposting is not known (Anonymous, 1999).

Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE) was first indentified inGreat Britain in 1986. BSE is a neurologic disease classified as atransmissible spongiform encephalopathy (TSE) and is similar toTSEs in other species, including scrapie (sheep and goats), trans-missible mink encephalopathy (ranch-bred mink), chronic wastingdisease (mule deer and elk), exotic ungulate encephalopathy (cap-tive exotic bovoids such as bison, orynx, and kudu), and felinespongiform encephalopathy (domestic cats, zoo Felidae). TSEsamong humans include kuru, Creutzfeldt-Jakob disease (CJD) and“new variant” CJD (nvCJD).

Clinically, these diseases all present neurologic deteriorationand wasting, with the incubation period and interval from clinicalonset to inexorable death determined by the dose of infective agent,its virulence, and the genetic makeup of the victim. The incuba-tion of BSE in cattle is generally 4 to 5 years (range of 20 monthsto 18 years) and an interval of 1 to 12 months from presentationof clinical signs to death. Characteristic histologic lesions in thebrain and spinal cord are vacuolation and “spongiform” changes.BSE fibrils (long strands of host glycoprotein called prion proteinor PrP) in spinal cord preparations may be seen with electron mi-croscopy following detergent extraction and proteinase K diges-tion. Scrapie tissues with highest infectivity are brain and spinalcord, followed by spleen, tonsil lymph nodes, distal ileum, andproximal colon. The infective agent can be transferred using prepa-rations of neural tissue from infected animals across species bar-riers. The most effective method of transfer is direct injection intothe brain or spinal cord, but transfer has been reported with in-traperitoneal injection and oral dosing. Vertical transfer (mother tooffspring) has been reported among domestic cattle, and lateral

transfer through biting or injury (especially among mink) has alsobeen reported. It is generally agreed that the infective agent is likelya variant of scrapie (endemic to sheep) and was transferred to cat-tle from rendered sheep via inadequately processed meat and bonemeal protein supplement. Disputes have arisen about other detailsof BSE, its relationship to other TSEs, and its effects in humansbecause of an expectation of conformation by BSE to historicalprinciples of disease.

There is mainstream agreement that the infective agents is aprion, a proteinaceous infective particle that does not possessnucleic acid. It is resistant to heat, animicrobials, ultraviolet rays,and ionizing radiation and is not consistently inactivated with al-cohol, formaldehyde, glutaraldehyde, or sodium hydroxide. Phe-nol and sodium hypochlorite disinfection have had variable suc-cess.

PrP protein is not the infectious agent, but rather the productof a TSE infection which has switched on the PrP gene. While theinfectious agent has not been elucidated, investigators have con-cluded that the agent in nvCJD and BSE is the same strain and thatthe same agent is also linked to feline spongiform encephalopathyand exotic ungulate encephalopathy. While this information mightindicate a simple mode of transmission, workers with the highestpotential incidence of exposure to BSE or TSE (sheep farmers,butchers, veterinarians, cooks, and abattoir workers) do not havean unusually high incidence of nvCJD (Collee, 2000; Prusiner,1991). Likewise, hemophilic patients have not reflected an in-creased incidence of nvCJD, although CJD transmission has beendocumented as the result of injections of human growth hormoneor gonadotrophin (derived from human pituitary gland), implanta-tion of dura mater and corneas, and even infected EEG electrodesand neurosurgical instruments (Collee, 2000; Lee et al., 1998;Prusiner, 1994).

The final chapter on BSE and other TSEs will not be writtenfor at least 15 to 20 years, the probable conclusion of the incuba-tion period for those exposed to BSE in the late 1980s and early1990s.

Substances Produced by Cooking

Tolerances cannot be set for contaminants produced as a result ofan action taken by the consumer. An example of this type of con-taminant is heterocyclic amines, which are generated during cook-ing. Heterocyclic amines (HCAs) were discovered serendipitouslyby Japanese investigators who, while examining the mutagenicityof smoke generated by charred foods, found that the extracts of thecharred surfaces of the meat and fish were quantitatively more mu-tagenic than could be accounted for by the presence of polycyclicaromatic hydrocarbons (Sugimura et al., 1989). Collectively, thereare more than 20 HCAs. They are formed as a result of high-temperature cooking of proteins (especially those containing highlevels of creatinine) and carbohydrates. Normally, as a result ofsuch heating, desirable flavor components are formed, for exam-ple, pyrazines, pyridines, and thiazoles. Intermediates in theformation of these substances are dihydropyrizines and dihy-dropyridines, which in the presence of oxygen form the flavor com-ponents; however, in the presence of creatinine, HCAs are formed(Table 30-28) (Chen and Chiu, 1998; Schut and Snyderwine, 1999).

These substances are rapidly absorbed by the GI tract, are dis-tributed to all organs, and decline to undetectable levels within 72h. HCAs behave as electrophilic carcinogens (Table 30-29). Theyare activated through N-hydroxylation by cytochrome P450 or

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P448, depending on the specific HCA. The N-hydroxy forms re-quire further activation by O-acetylation or O-sulfonation to reactwith DNA. DNA adducts are formed with guanosine in various or-gans, including the liver, heart, kidney, colon, small intestine,forestomach, pancreas, and lung. Unreacted substances are subjectto phase II detoxication reactions and are excreted via the urineand feces. In vitro, HCAs require metabolic activation, with somerequiring O-acetyltransferase and others not requiring it. Althoughmuch of the mutagenicity testing has been carried out in TA98 andTA100, these substances are mutagenic in mammalian cells bothin vitro and in vivo, Drosophila, and other strains of Salmonella(Skog et al., 1998; Sugimura and Wakahayashi, 1999)

Miscellaneous Contaminants in Food

Sometimes the items under the “miscellaneous” heading are themost interesting. For example, Rodricks and Pohland (1981)

pointed out an interesting historical case of the possible transfer ofa toxic botanic chemical from an animal to humans which was firstidentified by Hall (1979). It is found in the Bible, Book of Num-bers, 11:31–33, which describes hungry Israelites inundated withquail blown in from the sea; those who ate the quail quickly died.Hall speculated that the quail had consumed various poisonousberries, including hemlock, while they overwintered in Africa. Thehemlock berry contains coniine, a neurotoxic alkaloid to whichquail are resistant and that can accumulate in their tissue. Humansare not resistant to coniine, and consumption of large quantities ofquail tissue containing the neurotoxin could result in death as de-scribed in the biblical text.

Mountain laurel, rhododendron, and azaleas all possess an-dromedotoxin (now called acetylandromedol) and grayanotoxins(I, II, and II) in their shoots, leaves, twigs, and flowers. Honeymade from flowers of these plants is toxic to humans, and after anasymptomatic period of 4 to 6 h, salivation, malaise, vomiting, di-

Table 30-28Amounts of Heterocyclic Amines in Cooked Foods

Amount In Cooked Food, ng/g

SAMPLE IQ MeIQx 4,8-DIMeIQx Trp-P-1 Trp-P-2

Broiled beef 0.19 2.11 0.21 0.25Fried ground beef 0.70 0.64 0.12 0.19 0.21Broiled chicken 2.33 0.81 0.12 0.18Broiled mutton 1.01 0.67 0.15Food-grade beef extract 3.10

SOURCE: Sugimura et al., 1989; Adamson, 1990, with permission.

Table 30-29Mutagenicity and Carcinogenicity of Heterocyclic Amines

NUMBER OF CarcinogenicityREVERTANTS, u/g

HCA (STRAIN TA98) SPECIES STATISTICALLY SIGNIFICANT TUMORS

MeIQ 47,000,000 Mouse Liver, forestomachRat Zymbal gland, oral cavity, colon, skin, mammary gland

IQ 898,000 Mouse Liver, forestomach, lungRat Liver, mammary gland, Zymbal glandMonkey Liver, metastasis to lungs

MeIQx 417,000 Mouse Liver, lung, lymphoma, leukemiaRat Liver, Zymbal gland, clitoral gland, skin

Glu-P-1 183,000 Mouse Liver, blood vesselsRat Liver, small and large intestine, brain, clitoral gland, Zymbal gland

DiMeIQx 126,000 No dataTrp-P-2 92,700 Mouse Liver, lung

Rat Liver, clitoral glandTrp-P-1 8,990 Mouse Liver

Rat Liver, metastasis to lungsPhIP 1,800 Mouse Liver, lung, lymphoma

Rat Colon, mammary glandGlu-P-2 930 Mouse Liver, blood vessels

Rat Liver, small and large intestine, Zymbal gland, brain, clitoral gland

SOURCE: Adapted from Sugimura et al., 1989, with permission.

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arrhea, tingling of the skin, muscular weakness, headache, visualdifficulties, coma, and convulsions occur. Life-threatening brady-cardia and arterial hypotension may ensue. Needless to say, bee-keepers maintain apiaries well away from these species of plants.A similar poisoning occurs with oleander (Nerium oleander andNerium indicum), where honey made from the flowers, meatroasted on oleander sticks, or milk from a cow that eats the foliagecan produce prostrating symptoms. The oleander toxin consists ofa series of cardiac glycosides: thevetin, convallarin, steroidal, helle-borein, ouabain, and digitoxin. Sympathetic nerves are paralyzed;the cardiotoxin stimulates the heart muscles much as digitalis does,and gastric distress ensues (Anderson and Sogn, 1984; VonMalot-tki and Weichmann, 1996).

Other contaminations include contamination of milk withpyrrolizidine and other alkaloids after a cow has fed on tansy rag-wort (Senecio jacobaea) and tremetol contamination of milk fromwhite snakeroot (Eupatorium rugusum).

CONCLUSIONS

Food toxicology differs in many respects from other subspecialtiesof toxicology largely because of the nature and chemical com-plexity of food. Food consists of hundreds of thousands of chem-ical substances in addition to the macro- and micronutrients thatare essential to life. The federal law defining food safety in theUnited States, the FD&C Act, provides a workable scheme for es-tablishing the safety of foods, food ingredients, and contaminants.While the act does not specify how the safety of food and its com-

ponents and ingredients is to be demonstrated, it emphasizes theneed for reasonable approaches in both the application of tests andtheir interpretation. The specific examples of reasonable ap-proaches and interpretations of safety data discussed in this chap-ter illustrate both the means and the necessity for reasonableness.New policies, consistent with the safety provisions of the act, arebeing developed to provide guidance for determination of thesafety-in-use of novel foods and those foods derived from new plantvarieties.

Contaminants found in food may be divided into two largeclasses: those that are unavoidable by current good manufacturingpractice and those that are not. The former class is represented bysubstances such as certain chlorinated organic compounds, heavymetals, and mycotoxins that have been determined to be unavoid-able by current food manufacturing practice and for which toler-ances or action levels may be established. Additionally, pesticideresidues and residues of drugs used in food-producing animals mayhave tolerances established when necessary to protect public health.For an avoidable class of contaminants, tolerances are not set ei-ther because public health concerns dictates that the mere presenceof the substance or agent demands immediate regulatory action orbecause contamination results from food preparation in the home,which is beyond FDA control.

It is important to emphasize that the vast majority of food-borne illnesses in developed countries are attributable to microbi-ologic contamination of food arising from the pathogenicity and/ortoxigenicity of the contaminating organism. Thus, the overwhelm-ing concern for food safety in the United States remains directedtoward preserving the microbiologic integrity of food.

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