INTRODUCTION TO FOOD TOXICOLOGY

Uniqueness of Food Toxicology

Nature and Complexity of Food

Importance of the Gastrointestinal Tract

SAFETY STANDARDS FOR FOODS, FOOD INGREDIENTS, AND CONTAMINANTS

The Food, Drug and Cosmetics Act Provides for a Practicable Approach

The Application of Experience: Generally Recognized as Safe (GRAS)

Use of Tolerances

Food and Color Additives

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

Safety Evaluation of Direct Food and Color Additives Safety Determination of Indirect Food Additives Safety Requirements for GRAS Substances

Importance of the GRAS Concept

Transgenic Plant (And New Plant Varieties) Policy Methods for Establishing Safe Conditions of Use

for Novel Foods Dietary Supplements

Assessment of Carcinogens

Carcinogenicity as a Special Problem Biological versus Statistical Significance Carcinogenic Contaminants

SAFETY OF FOOD

Adverse Reactions to Food or Food Ingredients

Food Allergy

Food Toxicity (Poisoning) Food Idiosyncrasy Anaphylactoid Reactions Pharmacologic Food Reactions Metabolic Food Reactions

Importance of Labeling

TOLERANCE SETTING FOR SUBSTANCES IN FOODS

Pesticide Residues

Drugs Used in Food-Producing Animals

Unavoidable Contaminants

Heavy Metals

Chlorinated Organics

Nitrosamines, Nitrosamides, and N-Nitroso

Substances

Food-Borne Molds and Mycotoxins

SUBSTANCES FOR WHICH TOLERANCES MAY NOT BE SET

Toxins in Fish, Shellfish, and Turtles

Dinoflagellate Poisoning (Paralytic Shellfish Poisoning or PSP; Saxitoxin)

Amnesic Shellfish Poisoning (Domoic Acid) Ciguatera Poisoning

Puffer Fish Poisoning (Tetrodotoxin) Moray Eel Poisoning

Fish Liver Poisoning

Fish Roe Poisoning

Abalone Poisoning (Pyropheophorbide A) Sea Urchin Poisoning

Sea Turtle Poisoning (Chelonitoxin) Haff Disease

Microbiologic Agents—Preformed Bacterial Toxins

Clostridium botulinum and Clostridium butyricum Clostridium perfringens

Bacillus cereus Staphylococcus aureus Escherichia coli

Bovine Spongiform Encephalopathy Substances Produced by Cooking Miscellaneous Contaminants in Food

CONCLUSIONS

INTRODUCTION TO FOOD

TOXICOLOGY

This chapter describes the general principles of food toxicology and explains how those principles have been shaped by existing food laws and applied to the safety assessment of foods, food ingredients, and food contaminants. Food toxicology is different from other subspecialties in toxicology largely because of the nature and chemical complexity of food. The necessity for practical and workable approaches to the assessment of food safety is addressed throughout the chapter.

The typical western diet contains hundreds of thousands of substances naturally present in food and many more which form in situ when food is cooked or prepared. Many of these substances affect the nutritional and esthetic qualities of food, including appearance and organoleptic properties (i.e., conferring flavor, texture, or aroma) that determine whether or not we will even try the food or take a second bite, respectively. While these or other substances present in food may be nutritional and/or gratifying, they may not necessarily be “safe” in any amount or for any intended use. The Federal Food, Drug and Cosmetic (FD&C) Act gives the federal government the authority to ensure that all food involved in interstate commerce is safe. Congress, in writing the FD&C Act (and its subsequent amendments), understood that safety cannot be proved absolutely and indicated instead that the safety standard for substances added to food can be no more than a reasonable certainty that no harm will occur. As pointed out in other sections of this chapter, the language of the FD&C Act effectively provides for practical and workable approaches to the assessment of safety for food, food ingredients, and food contaminants. Because food is highly complex, the legal framework provided by Congress for the regulation of food and substances in food was kept simple so that it would work. The basic element of the framework is that food, which is defined as articles or components of articles used for food or drink for humans or animals, bears the presumption of safety [sections 201(f) and 402(a)(1) of the FD&C Act]. This means that a steak or a potato is presumed to be safe unless it contains a poisonous or deleterious substance in an amount shown to make it ordinarily injurious to health. In essence, this presumption of safety was born of necessity. If the hundreds of thousands of substances naturally present in food were subject to the same strictures and limitations that apply to added substances, food shortages could easily result. To avoid such crises, Congress developed a safety standard that would not force regulatory authorities to ban common, traditional foods.

In cases where the substance is not naturally present in food but is a contaminant or added ingredient, the safety standard is quite different. This standard decrees a food to be adulterated if it contains any poisonous or deleterious substance that may render it injurious. Therefore, the presence of a substance that is not a natural component of a food demands a far higher standard of safety. The mere possibility that such a substance may render the food injurious to health is sufficient to ban the food containing that substance. Thus, for additives and contaminants, Congress recognized that these substances are not as complex as food and must, therefore, meet a higher standard of safety.

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

U.S. Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA) regulations explicitly define the term safety for substances added to food, scientists and their legal and regulatory counterparts have worked out operational definitions for the safety of food ingredients. The one principle on which there has been nearly unanimous agreement is that safety concerns in regard to an added substance should focus on both the nature of the substance and its intended conditions of use. It is recognized that substances are not inherently unsafe, it is only the quantity at which they are presented in the diet that makes them unsafe. The quantity (or “level”) present in the diet is determined by the intended conditions of use and limitations of use of the substance.

As with food, practical and workable solutions must be found for the constituents of additives, because all substances contain a myriad of impurities at trace and even undetectable levels. Decisions concerning the safety of impurities and the development of appropriate specifications for food and color additives to assure that they are of suitable purity must similarly constitute a workable approach. In this case, the workable approach involves setting specification limits on contaminants—limits that are intended to exclude the possibility that the level of contaminants present in an additive may render the food to which the substance is added unsafe. As a practical matter and because of time and cost considerations, established specifications must be relatively simple and straightforward and must provide reasonable assurance that an ingredient is of suitable purity for its intended conditions of use. However, it generally is not necessary or practical to require extensive analysis and identification of all individual impurities to establish the fact that a substance is of “food-grade purity.” It should be emphasized that specifications can serve their purpose of assuring suitable purity only if the manufacturing processes used are adequately controlled to assure consistency in the quality and purity of the product. It should be understood that the philosophy by which specifications are established for substances added to food embodies the belief that not all risks are worthy of regulatory concern and control. Implicit in this philosophy is the concept of threshold of regulation, which is an important unifying concept in food 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 substance added to food, it is important to recognize that such a determination must rest on a general understanding of the risks posed by food itself. The requirement that substances added to food be safe (to a reasonable degree of certainty) demands consideration of the far higher theoretical risk posed by food itself. Food, as stated earlier, contains hundreds of thousands of substances, most of which have not been fully characterized or tested. The presumption that a food is safe is based on a history of common use and on the fact that the consumption of certain foods is deeply rooted in tradition (e.g., “ethnic” foods or those foods traditionally consumed for a holiday celebration). When the uncertainty about the risk of the added substance is small compared with the uncertainties attending food itself, the standard of “reasonable certainty of no harm” for the added substance has been satisfied. Thus, for food-like substances, the presumption is that the substance resembles food, is digested and metabolized as food, and consequently raises fewer toxicologic and safety-related questions than do substances that are not food-like. And such substances are added either directly or indirectly (substances that may migrate into food from packaging or other food contact surfaces) to foods in only very small or trace amounts, the low levels of exposure aid in demonstrating that the

intended conditions of use of these substances are safe. These broad generalizations, however, do not suffice to exempt these food ingredients from the requirements of thorough safety testing. The FDA requires that such testing be done but it is tempered by considerations of (1) the basic nature of the substance, (2) the level to which 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 interest on the part of consumers in the health-enhancing properties of foods and the components they contain. Substances such as phytosterols from vegetable oils and isoflavones from soy have been isolated and added to other foods at elevated levels to impart cholesterol-lowering abilities. Such products have raised regulatory questions about whether these substances are functioning as drugs and should be regulated as such or whether they should be viewed as new nutrients and allowed in foods, as are vitamin C and iron. At a recent conference, experts in nutritional science concluded that the concept of nutrients should be expanded to include a growing number of desirable food constituents that produce quantifiable health benefits related to disease prevention (Sansalone, 1999). This isolation of new food components and their use in fortifying food products will necessitate a thorough evaluation of safety at the intended level of intake and for the population at large.

Finally, it should be recognized that in most of the world, microbiologic contamination of food represents by far the greatest food-borne risk facing consumers. Thus, while vigilance in assuring the safety of substances added to food under their intended conditions of use is appropriate, we should not lose sight of the major concern of food safety.

Uniqueness of Food Toxicology

The nature of food is responsible for the uniqueness of food toxicology. Food is not only essential to all life but also a major contributor to the quality of life. Food and drink are enjoyed for their appearance, aroma, flavor, and texture. They are significant factors in defining cultures and societies. For example, ethnic foods and gourmet foods have a status that far exceeds their nutritional benefits, but any proposal to ban an ethnic food because new data have raised questions about its safety would be met with strong resistance.

As food occupies a position of central importance in virtually all cultures and because most food cannot be commercially produced in a definable environment under strict quality controls, food generally cannot meet the rigorous standards of chemical identity, purity, and good manufacturing practice met by most consumer products. The fact that food is harvested from the soil, the sea, or inland waters or is derived from land animals, which are subject to the unpredictable forces of nature, makes the constancy of raw food unreliable. Food in general is more complex and variable in composition than are all the other substances to which humans are exposed. However, there is nothing to which humans have greater exposure despite the uncertainty about its chemical identity, consistency, and purity. Experience has supported the safety of commonly consumed foods, and the good agricultural practices under which food is produced mandate the need for quality controls. Nevertheless, it is clear that food is held to a different standard as a practical matter dictated by necessity.

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

gredient substances often must rely on reasoning unique to food science in the sense that such substances may be normal constituents of food or modified constituents of food as opposed to the types of substances ordinarily addressed in the fields of occupational, environmental, and medical toxicology. Assessment of the safety of such substances, which are added to food for their technical effects, often focuses on digestion and metabolism occurring in the gastrointestinal (GI) tract. The reason for this focus is that in many cases an ingested substance is not absorbed through the GI tract; only products of its digestion are absorbed, and these products may be identical to those derived from natural food.

Nature and Complexity of Food

Food is an exceedingly complex mixture of nutrient and nonnutrient substances, whether it is consumed in the “natural” (unprocessed) form or as a highly processed ready-to-eat microwaveable meal (Table 30-1). Among the “nutrient” substances, the western diet consists of items of caloric and noncaloric value; that is, carbohydrates supply 47 percent of caloric intake, fats supply 37 percent, and protein supplies 16 percent (all three of which would be considered “macronutrients”) (Technical Assessment Systems, Inc., 1992), whereas minerals and vitamins, the “micronutrients,” obviously have no caloric value but are no less essential for life.

Nonnutrient substances are often characterized in the popular literature as being contributed by food processing, but nature provides the vast majority of nonnutrient constituents. For instance, in Table 30-2 one can see that even among “natural” (or minimally processed) foods, there are far more nonnutrient than nutrient constituents. Many of these nonnutrient substances are vital for the growth and survival of the plant, including hormones and naturally occurring pesticides (estimated at approximately 10,000 by Gold et al., 1992). Some of these substances may be antinutrient [e.g., goiterogens in Brassica, trypsin and/or chymotrypsin inhibitors in soybeans, 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 substances present in food is given in the series edited by Maarse and associates (1993), in which approximately 5500 volatile substances are noted as occurring in one or more of 246 different foods. However, this is only the tip of the iceberg, as the number of unidentified natural chemicals in food vastly exceed the number that have been identified (Miller, 1991).

Nonnutrient substances are also added as a result of processing, and in fact, 21 CFR 170.3(o) lists 32 categories of direct additives, of which there are about 3000 individual substances. Approximately 1800 of the 3000 are flavor ingredients, most of which

Table 30-1

Food as a Complex Mixture

SOURCE: Smith 1991, with permission.

Table 30-2

Nonnutrient Substances in Food

SOURCE: Smith, 1991, with permission.

already occur naturally in food and are nonnutritive. Of the 1800 flavoring ingredients that may be added to food, approximately one-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 vast absorptive surface that has been estimated to be from 200 to 4500 m2 (Concon, 1988). The GI transit time provides for adequate exposure 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 proteases from the enterocytes), saponification agents (in bile), and a luxuriant bacterial flora providing a repertoire of metabolic capability not shared by the host (e.g., fermentation of “nondigestible” sugars such as xylitol and sorbitol) (Drasar and Hill, 1974). The en-

Table 30-3

Induction of Xenobiotic Metabolism in the Rat Intestine

terocytes (intestinal epithelium) possess an extensive capacity for the metabolism of xenobiotics that may be second only to that of the liver, with a full complement of phase (type) I and phase (type) II reactions present. The enteric monooxygenase system is analogous to the liver, as both systems are located in the endoplasmic reticulum of cells, require reduced nicotinamide adenine dinucleotide phosphate (NADPH) and O2 for maximum activity, are inhibited by SKF-525A and carbon monoxide, and are qualitatively similar in their response to enzyme induction (Hassing et al., 1989). Induction of xenobiotic metabolism by the enteric monooxygenase system has been demonstrated in a number of substances, including commonly eaten foods and their constituents (Table 30-3). Dietary factors may also decrease metabolic activity. For example, in one study, iron restriction and selenium deficiency decreased cytochrome P450 values, but a vitamin A rich diet had the same effect (Kaminsky and Fasco, 1991).

The constituents of food and other ingesta (e.g., drugs, contaminants, inhaled pollutants dissolved in saliva and swallowed) are physicochemically heterogeneous, and because the intestine has evolved into a relatively impermeable membrane, mechanisms of absorption have developed that allow substances to gain access to the body from the intestinal lumen. The four primary mechanisms for absorption are passive or simple diffusion, active transport, facilitated diffusion, and pinocytosis. Each of these mechanisms characteristically transfers a defined group of constituents from the lumen into the body (Table 30-4). As noted in the table, xenobiotics and other substances may compete for passage into the body.

Aiding this absorption is the rich vascularization of the intestine, with a normal rate of blood flow in the portal vein of approximately 1.2 L/h/kg. However, after a meal, there is a 30 percent increase in blood flow through the splanchnic area (Concon, 1988). It follows, then, that substances which affect blood flow also tend to affect the absorption of compounds; an example is alcohol, which tends to increase blood flow to the stomach and thus

enhances its own absorption. Few stimuli tend to decrease flow to this area, with the possible exception of energetic muscular activity and hypovolemic shock.

Lymph circulation is important in the transfer of fats, large molecules (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 been shown to double the flow and therefore double the absorption of p-aminosalicylic acid and tetracycline—are known to influence its flow (Chhabra and Eastin, 1984). Another factor that lends importance to lymph is the fact that the lymph empties via the thoracic duct into the point of junction of the left internal jugular and subclavian veins, preventing “first-pass” metabolism by the liver, unlike substances transported by the blood.

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

Table 30-5

Factors Affecting Intestinal Absorption and Rate of Absorption

SAFETY STANDARDS FOR FOODS, FOOD INGREDIENTS, AND CONTAMINANTS

The Food, Drug and Cosmetics Act

Provides for a Practicable Approach

The FD&C Act presumes that traditionally consumed foods are safe if they are free of contaminants. For the FDA to ban such foods, it must have clear evidence that death or illness can be traced to consumption of a particular food. The fact that foods contain many natural substances, some of which are toxic at a high concentration, is in itself an insufficient basis under the act for declaring a food as being unfit for human consumption. Examples of this concept include acceptance of generally recognized as safe status and the implementation of tolerances.

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

exclude the agency from making such decisions. The act instead requires that scientific experts base a GRAS determination on the adequacy of safety, as shown through scientific procedures or through experience based on common use in food before January 1, 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 food additives, which are defined as “any substances the intended use of 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 and GRAS substances. Regulated food additives must be approved and regulated for their intended conditions of use by the FDA under 21 CFR 172–179 before they can be marketed. In section 409 of the act, the requirements for data to support the safe use of a food additive are described in general terms. The requirements or recommended methods for establishing safe conditions of use for an additive are available in the form of a guideline issued by FDA (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food). These guidelines, referred to as “the Redbook,” provide substance and definition to the safety standard applicable to regulated food additives: “reasonable certainty of no harm under conditions of intended use.”

Use of Tolerances If a food contains an unavoidable contaminant even with the use of current good manufacturing practices (CGMP), it may be declared unfit as food if the contaminant may render the food injurious to health. Thus, for a food itself to be declared unfit, it must be ordinarily injurious, while an unavoidable contaminant in food need only pose the risk of harm for the food to be found unfit and subject to FDA action. The reason for the dichotomy is practicability. Congress recognized that if authority were granted to ban traditional foods for reasons that go beyond clear evidence of harm to health, the agency would be subject to pressure to ban certain foods.

Foods containing unavoidable contaminants are not automatically banned because such foods are subject to the provisions of section 406 of the FD&C Act, which indicates that the quantity of unavoidable contaminants in food may be limited by regulation for the protection of public health and that any quantity of a contaminant exceeding the fixed limit shall be deemed unsafe. This authority has been used by the FDA to set limits on the quantity of unavoidable contaminants in food by regulation (tolerances) or by informal action levels that do not have the force of law. Such action levels have been set for aflatoxin in peanuts, grain, and milk

Table 30-6

(Table 30-6). Action levels have the advantage of offering greater flexibility than is provided by tolerances established by regulation. Whether tolerances or action levels are applied to unavoidable contaminants of food, the FDA attempts to balance the health risk posed by unavoidable contaminants against the loss of a portion of the food supply. In contrast, contaminants in food that are avoidable by CGMP are deemed to be unsafe under section 406 if they are considered poisonous or deleterious. Under such circumstances, the food is typically declared adulterated and unfit for human consumption. The extent to which consumers who are already in possession of such food must be alerted depends on the health risk posed by the contaminated food. If there is a reasonable probability that the use of or exposure to such a food will cause serious adverse health consequences or death, the FDA will seek a class I recall which provides the maximum public warning, the greatest depth of recall, and the most follow-up. Classes II and III represent 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 either a direct food additive or color additive. As with all ingredients intentionally added to food, there must be a specific and justifiable functionality. While a color additive has only one function, a food additive may have any one of 32 functionalities (Table 30-7).

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

Two distinct types of color additives have been approved for food use: those requiring certification by FDA chemists and those exempt from certification. Certification, which is based on chemical analysis, is required for each batch of most organic synthesized colors because they may contain impurities that may vary from batch to batch. Most certified colors approved for food use bear the prefix FD&C. They include FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 3, FD&C Red No. 3, FD&C Red No. 40, FD&C Yellow No. 5, and FD&C Yellow No. 6. Orange B and Citrus Red No. 2 are the only certified food colors that lack the FD&C designation (21 CFR 74 Subpart A).

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

Glucitol, sodium ferrocyanide, silicon dioxide

Nisin; metyhyl-, ethyl-, propyl-, or butylester of p-hydroxybenozoic acid; sodium benzoate; sorbic acid and its salts

Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate

FD&C Yellow No. 5 (tartrazine),

FD&C Red No. 4, -carotene,

annatto, turmeric

Calcium chloride, glucitol

Calcium bromate, baker’s yeast extract, calcium carbonate

Calcium stearate, cobalt caprylate, cobalt tallate

Phosphate esters of monoand diglycerides, acetylated monoglycerides, calcium stearate

Papain, rennet, pepsin

Calcium acetate, calcium carbonate

Monosodium glutamate, inositol

Cinnamon, citral, p-cresol, thymol, zingerone

Calcium bromate

Palm kernel oil, tallow

Aluminum phosphide, potassium bromide

Arabic gum, calcium chloride

Carbon dioxide, adipic acid

Mineral oil, acetylated monoglycerides

Acesulfame, aspartame, saccharin

Calcium carbonate

Lactitol, hydrogenated starch hydrolysate

Calcium peroxide, chloride, hydrogen peroxide

Acetic acid, propionic acid, calcium acetate, calcium carbonate, carbon dioxide

Carbon dioxide, ammonium carbonate, ammonium sulfate, potassium bromide

Carbon dioxide, nitrous oxide

Acetate salts, citrate salts, gluconate salt, metaphosphate, edetic acid, calcium acetate

Acetic acid, acetylated monoglycerides

Calcium acetate, calcium carbonate

(29) Surface-active agents Substances used to modify surface properties of liquid food components for a variety

of effects other than emulsifiers but including solubilizing agents, dispersants, detergents, wetting agents, rehydration enhancers, whipping agents, foaming agents, and defoaming agents

Sorbitan monostearate, monoand diglycerides, polysorbate 60, acetostearin

as prescribed by regulation (FD&C Act, section 721). Certification involves in-depth chemical analysis of major and trace components of each individual batch of color additives by FDA chemists and is required before any batch can be released for commercial use. Such color additives consist of aromatic amines or aromatic azo structures (FD&C Blue No. 1, FD&C Blue No. 2, FD&C Green No. 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 impurities.

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