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7
Categories of Scientific Evidence—In Vitro Data

Guiding Principle: In vitro studies can serve as signals of potential harmful effects in humans. Validated1 in vitro studies can stand alone as independent indicators of risk to human health if a comparable exposure is attained in humans and the in vitro effects correlate with a specific adverse health effect in humans or animals. Alone, in vitro data from nonvalidated assays serve as hypothesis generators and as indicators of possible mechanisms of harm.

Numerous publications have described in vitro experiments on dietary supplement ingredients. Some experiments are specifically designed to examine safety endpoints, while others provide less specific information about an ingredient’s biological activity. Because no battery of tests is required on dietary supplement ingredients, results from safety tests common to other chemicals are not widespread, although they are available for some ingredients.

The first section of this chapter describes the unique power of in vitro studies and considerations for interpreting them. The next section describes various types of in vitro data that may be available, with descriptions of

1  

In this report, in vitro assays are considered validated when their results have been proven to predict a specific effect in animals and/or humans with reasonable certainty (not necessarily universally accepted or without detractors).



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Dietary Supplements: A Framework for Evaluating Safety 7 Categories of Scientific Evidence—In Vitro Data Guiding Principle: In vitro studies can serve as signals of potential harmful effects in humans. Validated1 in vitro studies can stand alone as independent indicators of risk to human health if a comparable exposure is attained in humans and the in vitro effects correlate with a specific adverse health effect in humans or animals. Alone, in vitro data from nonvalidated assays serve as hypothesis generators and as indicators of possible mechanisms of harm. Numerous publications have described in vitro experiments on dietary supplement ingredients. Some experiments are specifically designed to examine safety endpoints, while others provide less specific information about an ingredient’s biological activity. Because no battery of tests is required on dietary supplement ingredients, results from safety tests common to other chemicals are not widespread, although they are available for some ingredients. The first section of this chapter describes the unique power of in vitro studies and considerations for interpreting them. The next section describes various types of in vitro data that may be available, with descriptions of 1   In this report, in vitro assays are considered validated when their results have been proven to predict a specific effect in animals and/or humans with reasonable certainty (not necessarily universally accepted or without detractors).

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Dietary Supplements: A Framework for Evaluating Safety both the types of endpoints examined and the model systems used. Next is a description of validated in vitro assays—assays that are accepted for use in predicting effects on whole organisms. The seriousness of harm predicted by a given assay is then pointed out as an important consideration. Several annexes present information on genetic toxicology experiments, examples of validated in vitro assays, and a description of new in vitro methods that are evolving in toxicology. Finally, a spectrum of concern figure is presented to integrate the considerations discussed. CONSIDERATIONS AND RELEVANCE TO HUMANS A range of in vitro experimental systems is used to gain insight into the risk of adverse effects of compounds. These systems include isolated organs, isolated cells, microorganisms, subcellular organelles, and molecular entities such as enzymes, receptors, transport proteins, isolated membranes, and genes or gene fragments. A primary advantage of conducting in vitro studies is that their reductionist approach allows insight into a compound’s mechanisms of action that might be more difficult to obtain in a “whole-animal study.” The control possible with in vitro experiments enables examination of effect on the target process or structure in isolation from confounding factors. For example, control over the concentration of the chemical of interest or of one or more of its metabolites enables the interactions among chemicals or metabolites to be studied. In vitro experiments are also generally more rapid and less expensive to conduct than in vivo studies, thus in vitro studies are more likely than in vivo studies to be available for assessment of dietary supplement safety. While the reductionist approach of in vitro studies makes them powerful and inexpensive methods useful for learning about effects and mechanisms of actions of compounds, the reductionist approach also requires careful consideration of limitations. It is important to consider the degree to which the particular in vitro system replicates the biology of the human target cells and their responses to toxic substances, as discussed below. Another consideration is that in vitro procedures may be less informative if performed with substrate concentrations that are not comparable with those reached in vivo or if the substrate is not metabolized similarly in vitro and in vivo. It is important, for example, to consider the relationship between the compound applied directly to the in vitro system and the identity and concentration of the compound that reaches the target (e.g., tissue, receptor, subcellular component) following human ingestion of the dietary supplement ingredient. After a substance is ingested, the metabolic fate of the compound and the amount of the biologically active compound that actually reaches the target site is dependent on a multitude of processes, including absorption, distribution, metabolism, and excretion in

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Dietary Supplements: A Framework for Evaluating Safety what are often complex pathways. The various processes that influence what compound reaches the active site, and at what concentration, are collectively referred to as pharmacokinetics information. Knowledge of a dietary supplement ingredient’s pharmacokinetics and in vivo metabolism, if available from animal or human studies, will allow the most appropriate interpretation of the relevance of compound concentrations used in in vitro experiments to amounts ingested by humans. GENERAL TYPES OF IN VITRO ASSAYS As described above, in vitro assays are valuable because they allow scientists to answer questions that otherwise may not be answerable due to various constraints with whole-animal and human studies. It is not possible to describe the complete realm of in vitro assays in this report, but consideration of the value provided by those described here gives perspective to the value of in vitro assays as a scientific tool for predicting risk. Assay Types by Effect Observed Effects on Cellular Receptors A number of different assays focus on examining effects of supplement ingredients on cellular receptors. Knowledge of which receptors are activated or inhibited, combined with general knowledge about what physiological processes are stimulated by different types of receptors, will increase understanding of how dietary supplement ingredients will affect humans. General types of assays for examining receptor activity are: Receptor binding assays—used to measure how strongly a substance (such as a dietary supplement ingredient or its constituent) binds to various receptor types to determine if the substance is likely to activate or inhibit cellular receptor activity. Cell function assays—used to measure agonism or antagonism of receptor activity in the intact cell by examining effects that are downstream of the receptor itself. Receptor activation is often a critical event in biological pathways that leads to a functional effect, hence many manufactured pharmacological agents are selected for intentional receptor targeting. Strong agonist or antagonist action does not generally warrant particular attention in and of itself, but it may warrant attention when combined with general knowledge about the receptor. Many receptors are characterized well enough to know whether sufficient activation or inhibition in target tissues is associated

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Dietary Supplements: A Framework for Evaluating Safety with a pharmacological or toxicological process that presents a health risk. Thus whether cellular receptor effects should be considered as indicative of a risk depends on what is known about downstream effects of receptor activation and whether the agent in question reaches the receptor (the site of action) in a sufficient concentration (see Chapter 3 for a discussion about concentration at site of action). When activity at receptors is not associated with detrimental effects per se, understanding how the ingredient may affect the receptors may still prove valuable in providing a biological mechanism that can be used to understand adverse effects observed in intact (whole) and component biological systems (e.g., human, animal). Effects on Ion Channel Activity and Electrophysiology A number of assays can predict possible effects on the electrophysiology of the cells, either by directly measuring the membrane potential of the cells or by using other methods to examine effects on ion channels, the cellular proteins that control the cell’s ion distribution. For example, certain substances inhibit or block ion transport through particular types of ion channels, which, depending on the channel and the cells involved, can have a detrimental effect. Effects on some ion channels are associated with specific adverse physiological outcomes. For example, some drugs can induce what is called “long QT syndrome,” a rare, but potentially fatal heart disorder that may trigger arrhythmias including “torsades de pointes.” This syndrome results when drugs interact with potassium channels (usually with human ether-a-go-go-related gene (HERG) channels specifically), changing the action potential duration in cardiac cells. In the pharmaceutical world, concern about drugs that may induce this syndrome has led to the development of well-accepted in vitro assays to identify drugs that may exert this adverse effect on potassium channels (Roche et al., 2002). Such assays are a good example of validated assays (see discussion of validated assays below) and will also be helpful for identifying dietary supplement ingredients of concern. Effects on Enzyme Activity At the molecular level, enzyme activity assays measure substances’ abilities to inhibit or induce enzyme activity, either by directly affecting the enzyme’s activity or by affecting the amount of enzyme in the cell (e.g., affecting translation or enzyme stability). Whether the results of these assays in and of themselves warrant concern about risk depends on what is known about the enzyme and the resultant physiological impact if its func-

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Dietary Supplements: A Framework for Evaluating Safety tion is altered, as well the extent of the effect. For example, effects on cytochrome P450 enzymes important in metabolism of xenobiotics may suggest risk for pharmacokinetic interaction with other ingested substances (see Chapter 8). Effects on Genes and Nucleic Acids A number of techniques are capable of identifying genetic alterations that may be responsible for disease processes, alterations such as nucleic acid mutations, or effects on chromosomes. These assays are of growing importance for risk assessment because they measure events that could lead to delayed or latent adverse health effects. Annex 7-1 provides an expanded description of genetic toxicology information, and Annex 7-2 describes several specific types of validated genetic toxicity tests. The current challenge is to demonstrate a connection between changes in specific genes or combinations of genes and the underlying mechanism responsible for a given disease. New technology that may eventually aid in assessing risk of toxicity of compounds, including dietary supplement ingredients, is described in Annex 7-3. Assay Types by Experimental System The previous paragraphs organized in vitro assays by the effects assessed: effects on enzymes, ion channels, and nucleic acids. These and other in vitro tests may be conducted in a variety of experimental systems, such as isolated tissues or in isolated cells, each of which has its own considerations. Isolated Tissues In some experiments, animal tissues are isolated and then treated with test substances (e.g., dietary supplement ingredient). Tissues in many ways resemble an intact in vivo system because they contain a variety of cell types organized in their native structure and, because they are usually recently isolated from an animal, the protein expression is relatively similar to that in vivo. Detrimental effects observed in tissue or isolated organ assays may be predictive of effects in vivo. Nevertheless, the toxicological value of these effects can be limited by the inability of an isolated tissue to react with other reciprocating organ-body systems. For example, there are limitations inherent in excluding the gastrointestinal system in an experiment using isolated tissue. Botanical extracts provide an example of how important it is to consider how absorption of the ingested substance may affect which compounds reach the target tissue.

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Dietary Supplements: A Framework for Evaluating Safety Botanical extracts often contain polyphenolic compounds (e.g., tannin) that can reversibly or irreversibly bind to subcellular components, such as enzymes, signal transduction factors, and receptors. When administered orally to humans, however, these compounds may bind to food components or gastrointestinal cells, or they can be metabolized by gastrointestinal mucosal or bacteria enzymes and therefore not exert the same effects in vivo as seen in isolated tissue (Bravo, 1998; Yang et al., 2001). In summary, if the impact of excluding effects of other organ systems is considered or compensated for, isolated tissue can be a valuable tool for studying physiological effects of dietary supplement ingredients. Isolated Cells Suspensions of isolated cells or cultures of cells derived from animal tissue or from continuously replicating cell lines offer numerous advantages for studying chemical toxicity. The test substance can be examined in direct contact with the cell type of interest, the concentration of the test substance can be rigorously controlled, and the secondary effects of such extracellular influences as metabolic factors, matrix, and cell-to-cell contact can be excluded or controlled for. However, substances that are insoluble in culture media may not adequately reach the target cell in vitro, leading to misleading negative results. In addition, adaptation of cells to culture generally results in spontaneous alterations of gene expression so that caution must be exercised in extrapolating to whole organisms the results of chemical effects in culture. It is also important that the appropriate cell model is used. All cell types do not respond similarly to a single substance, even when the cells originate from the same organ. One cell type may exclude or excrete a compound, whereas another cell will not, and another may behave differently due to its unique biochemical pathways. Cell lines may have different activities than the parent tissue. For example, a problematic interpretation can be made using certain hepatocyte cultures that, unlike the liver, do not always support expression of metabolizing enzymes, causing some data generated in these assays to be misleading. A better choice for some tests might be cell cultures established specifically to evaluate metabolism of substances. Another approach is to add substrates to cell cultures that replace and/or activate metabolic enzymes, thus producing reactive metabolites that simulate in vivo metabolism of a given chemical/dietary supplement. VALIDATED IN VITRO ASSAYS In vitro studies vary in their value as predictors of harm. An in vitro assay will have the most direct predictive value when the measured effects

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Dietary Supplements: A Framework for Evaluating Safety are known to correspond to a specific functional change characteristic of an adverse health effect. It may often be appropriate to use in vitro data as hypothesis generators, as potential indicators of harmful health effects, or as information about biological plausibility or mechanism, rather than as standalone demonstrated indicators that in themselves indicate risk. However, some types of in vitro assays should be considered validated as predictors of possible harm, and thus when carefully conducted and interpreted, will provide valuable information beyond simply reinforcing observations from other categories of data or generating hypotheses. In vitro assays warrant attention and are appropriate to use as standalone indicators of risk to human health when the relationship between the results of an in vitro assay and actual clinical or animal outcomes has been demonstrated, thus validating the predictive value of the assay. A number of validated in vitro tests are in standard use for regulated materials and are often required for premarket approval by a regulatory authority. These form an important established battery of tests that are useful in predicting possible adverse effects. Although the regulatory situation is such that completion of these or other in vitro tests is not required for dietary supplement ingredients, the scientific value of these assays in predicting adverse effects in humans is as valuable in assessing the safety of dietary supplements as it is in assessing the safety of other substances. For example, specific types of in vitro tests are used by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) to identify potential pesticides and food additives that may lead to adverse effects (see Annex 7-2). In summary, it is recommended that in vitro assays—such as those contained in the Redbook (OFAS, 2001, 2003); Protection of Environment (40 C.F.R. § 150–189 [1998]); and the Consumer Product Safety Commission and its subacts, the Federal Hazardous Substances Act (15 U.S.C. § 1261–1278 [1960]) and the Labeling of Hazardous Art Materials Act (15 U.S.C. § 1277 [1988])—all be considered, among others, as important validated assays in the prediction of possible adverse effects in humans. Assays used by regulatory bodies are by no means the only assays or in vitro observations that should be considered as validated and thus independent predictors of risk. Other specific assays, such as certain receptors or ion channel assays, should also be considered validated. The important concept is that for an assay to stand alone as a predictor of risk, a connection between the observed biological effect and an adverse effect needs to have been made. (In Chapter 10, this concept is illustrated by the need for linkages between observed biological effects and adverse health effects.) In addition to the types of in vitro effects considered validated, other in vitro information is also valuable for assessing biological plausibility of concerns raised by other observations, such as observations of adverse

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Dietary Supplements: A Framework for Evaluating Safety effects in animals or humans. The assessment of biological plausibility becomes an issue when interpreting data and trying to predict the likelihood of causal relationships (see Chapter 10). While it is not necessary to establish a rational mechanism of harm to conclude that an ingredient poses an unreasonable risk, it is nonetheless valuable to identify possible mechanisms that explain the totality of the data. Many in vitro studies can be useful for this purpose. SERIOUSNESS OF HARM PREDICTED BY THE ASSAY In addition to considering the correlation of the particular in vitro assay with a particular adverse health outcome, it is of utmost importance to consider the seriousness of the outcome when evaluating the level of concern warranted by results of in vitro studies. Assays that warrant the most attention are those that predict biological effects/outcomes associated with toxicological manifestations, pathologies, or effects that could reasonably be hypothesized to lead to serious risks. This concept is analogous to Table 5-1, which categorizes the relative seriousness of various types of effects observed in animals. SUMMARY There is no quantitative method of assigning a weight to the outcome of each in vitro experiment and imputing an appropriate level of concern for public health risk from such data. Instead, it is important for experts to reach a judgment about the in vitro results based on the seriousness of the effect predicted, the validity of the assay in identifying substances that cause a particular adverse effect, the quality of the individual studies, and the consistency among the collection of studies. Considerations on study quality discussed in the human and the animal chapters (Chapters 4 and 5) apply to in vitro systems as well. Considerations include the strength of the association, its reproducibility in the same and corresponding systems, the specificity of the findings for both cause and effect, and the coherence of the evidence—all of which give more confidence that the findings are meaningful. When considering the information, it will be helpful to answer the following questions to decide how much concern for public health is warranted by the in vitro evidence: Is the in vitro test validated or commonly used to predict a serious adverse effect? For example, assays used in regulatory situations to predict possible carcinogenic effects are considered as such. Is the assay considered predictive of a particular adverse effect?

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Dietary Supplements: A Framework for Evaluating Safety How serious is the adverse effect that might be predicted? Mutagenicity in many assays, DNA damage in human cells, cell transformation, and production of DNA adducts in vitro are evidence of higher levels of concern, especially for potential carcinogenesis. Evidence of enzyme induction without DNA damage or mutagenesis is of lesser concern. Does the evidence provide mechanistic or mode of action information which lends biological plausibility to effects observed in humans or animals? Is the mechanism or mode of action consistent with the type of effect, caused by similar substances, such as plants in the same family? Is there information suggesting that the concentrations used in vitro are relevant or irrelevant? Concern should increase if the active ingredient and/or metabolite of concern reach relevant concentrations in blood or tissue (see Chapter 3). Is there consistency in more than one assay? The answers to these questions determine the appropriate level of concern, as described in Table 7-1. Information such as that in the right column warrants higher levels of concern about public safety and risk of consumption. In vitro information such as that described in the left column warrants lower levels of concern, while information described in the middle column warrants concern, but additional information may be required to warrant conclusion that a risk exists. ANNEX 7-1 USE OF GENETIC TOXICOLOGY INFORMATION Genetic toxicology is the study of the ability of substances to cause selective damage to the DNA of living organisms (or RNA in the case of RNA viruses). In addition to providing the raw materials for evolution, genetic alterations are associated with a large proportion of human diseases, including cancer. Chromosomal aberrations, such as deletions, inversions, and translocations, have been associated with leukemia, lymphoma, and some solid tumors. The term genotoxic is applied to substances (or physical agents like ultraviolet light or X-rays) that have an intrinsic ability to damage DNA (not simply due to gross toxicity that may secondarily result in damage to DNA). Different genotoxicants interact with DNA in different ways, cause different types of DNA alterations, and can be detected using different assay systems (Preston and Hoffmann, 2001). For example, substances that cause heritable changes in DNA sequence are called mutagens. A mutation may result from an alteration in a single DNA base or addition or deletion of one or more DNA bases (point muta-

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Dietary Supplements: A Framework for Evaluating Safety TABLE 7-1 Relative Spectrum of Concern for In Vitro Data Increasing Concern Standardizeda subcellular and cellular assays validated for the purpose of establishing in vivo toxic effect Standardized subcellular and cellular assays validated for the purpose of establishing in vivo toxic effect Standardized subcellular and cellular assays validated for the purpose of establishing in vivo toxic effect AND AND AND Multiple different assays suggesting the same pathological condition or endpoint Multiple different assays suggesting the same pathological condition or endpoint Multiple different assays suggesting the same pathological condition or endpoint AND AND AND Poor consistency/reproducibility in response AND No knowledge about concentration of toxicant in blood or tissue Consistency in response AND No knowledge about concentration of toxicant in blood or tissue Knowledge of presence of toxicant in blood or tissue enhanced by knowledge of concentrations comparable with those causing toxicity in vitro OR OR OR Standardized assays validated for the purpose of establishing organ toxicity Standardized assays validated for the purpose of establishing organ toxicity Standardized assays validated for the purpose of establishing organ toxicity AND AND AND Multiple different assays suggesting the same pathological condition or endpoint Multiple different assays suggesting the same pathological condition or endpoint Multiple different assays suggesting the same pathological condition or endpoint tions) that alters the amino acid in a protein coded for by a particular DNA triplet. This is the type of mutation that occurs in the heritable human disease sickle-cell anemia. Agents that cause these kinds of genetic effects are detected using assays that detect changes in specific genes, such as the Ames assay in Salmonella bacteria or the mouse lymphoma TK+/- assay in mammalian cells in vitro. Some genotoxicants cause visibly identifiable types of changes—alterations in chromosome structure or chromosome number (chromosome mutations, or clastogenicity). These types of changes can be detected by observation of cells (generally fixed and stained) with a microscope. The ability of some substances to cause DNA damage can also be assessed by determining whether the substance increases the normal level of activity of certain DNA repair processes or whether it is more lethal to cells

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Dietary Supplements: A Framework for Evaluating Safety Increasing Concern AND AND AND Poor consistency/reproducibility in response AND No knowledge about concentration of toxicant in blood or tissue OR Results obtained with nonstandardized, nonvalidated assays OR Results from microarray experiments show a gene expression pattern predictive of dangerous compoundsb Consistency in response AND No knowledge about concentration of toxicant in blood or tissue Knowledge of presence of toxicant in blood or tissue at concentrations comparable with those causing toxicity in vitro a Standardized in this context means that the assay is performed consistently across laboratories and often is officially promulgated by a standardization body, such as AOAC International (formerly the Association of Official Analytical Chemists), or the protocol is specified by a regulatory agency. b Toxicogenomics is a relatively new field, the impact of which is not possible to predict at this point. However, these types of data may become more important as the field progresses. If the value of genomics, proteomics, and other new technologies in identifying dangerous compounds is demonstrated in the future, then such results may warrant more concern than is indicated in this figure. deficient in a specific DNA repair system than to otherwise identical cells that have the DNA repair system. Because of the importance of genetic alterations in cancer development, and the observation that many carcinogens were also mutagenic, the original focus of genetic toxicology on evaluating potential hazards to the human gene pool shifted in the early 1970s to the use of genotoxicity assays as rapid screens for potential carcinogens (Ames et al., 1973, 1975; Bridges, 1976). The subsequent finding that many animal carcinogens are not genotoxic and that some chemicals show genotoxic activity in at least some assay systems, but do not show detectable carcinogenic activity in standard animal carcinogenicity studies, has somewhat lessened the attractiveness of genotoxicity assays (Tennant et al., 1987; Zeiger et al., 1990). Regulatory agencies that require safety testing of chemicals, for ex-

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Dietary Supplements: A Framework for Evaluating Safety ample, EPA for pesticides (40 C.F.R. § 158.340 [2001]) and FDA for food additives (21 C.F.R. § 170 [2000]) and drugs (FDA, 1997), currently require testing for genotoxicity (see Annex 7-2 for specific assays). The required battery of tests varies slightly from agency to agency, but includes at least one assay for gene mutations (generally the Ames assay), and an assay for chromosome damage (in vitro and/or in vivo). Some programs require a mammalian gene mutation assay in addition to the typical Ames bacterial gene mutation assay, and the test battery may include an assay for induction of DNA repair. Also, testing in additional assays may be triggered by positive results in the initial battery. While genotoxicity assays are no longer viewed as a quick and inexpensive way to detect chemical carcinogens, they do still have substantial utility. Under EPA’s recently revised cancer risk assessment guidelines (EPA, 2003a), for example, much greater emphasis is placed on using information on the mode of action of a carcinogen to guide how the risk assessment is performed. Evidence of lack of genotoxicity, together with positive evidence of another mode of carcinogenic action, may lead to use of a risk assessment model that is not linear at low doses and predicts lesser risk at low doses than the default low-dose linear model. In this regard, EPA’s approach has become more like that employed in many European countries, where nongenotoxic carcinogens have long been treated as presenting less risk at low doses than genotoxic carcinogens and are typically regulated in the same manner as noncarcinogens (identification of a no-effect level and application of an uncertainty factor to derive an allowable exposure level). Similarly, the International Agency for Research on Cancer (IARC) sometimes uses evidence of genotoxicity to modify its ranking of a chemical. For example, ethylene oxide was recently elevated to a class 1 human carcinogen despite there being only “limited evidence” in human studies, because the available positive animal carcinogenicity and clear evidence of genotoxicity in a wide variety of assay systems in vitro and in vivo was considered by the IARC reviewers as being convincing evidence that there was a clear human cancer risk from ethylene oxide exposure (IARC, 1994). In summary, genotoxicity data are not currently used alone in regulation of chemicals, and this is not likely to change. More commonly they are used to trigger a requirement for carcinogenicity testing. Internally, companies may use genotoxicity assays at an early point in product development as part of a screening system to identify promising leads—they may not wish to risk investing large amounts of time and money on a chemical that may turn out to be a carcinogen. This approach is consistent with Zeiger’s (1998) conclusion that “if a chemical is mutagenic in Salmonella, it should be considered a potential rodent carcinogen, unless ancillary information suggests otherwise.”

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Dietary Supplements: A Framework for Evaluating Safety While it is estimated that only about 60 to 70 percent of mutagens are carcinogens depending on the system (Tennant et al., 1987), current approaches are to require follow-up testing when genotoxic data are observed for other substances. It is consistent with these current approaches to consider genotoxic data about a dietary supplement ingredient as indicative of a possible carcinogen and a substantial risk of harm. If information suggests an ingredient is genotoxic, then responsible manufacturers will conduct additional research to determine if it is carcinogenic, which in many cases will provide mitigating information. ANNEX 7-2 IN VITRO TESTS VALIDATED FOR USE IN REGULATORY TOXICOLOGY CONTEXTS It is necessary to understand if chemicals have the potential to affect the genetic components of mammalian cells because of the importance of mutations and chromosomal aberrations to cancer development and genetic disorders, whether in somatic cells or germ cells, as discussed in Annex 7-1. Despite the controversy surrounding use of genotoxic tests to predict which chemicals are carcinogenic, EPA (2003b) has selected a battery of tests “to detect, with sensitive assay methods, the capacity of a chemical to alter genetic material in cells” and “to determine the relevance of these mutagenic changes to mammals.” “When mutagenic potential is demonstrated, these findings are incorporated in the assessment of heritable effect, oncogenicity, and possibly, other health effects” (EPA, 2003b). All of these tests are not universally accepted as fail-proof predictors of adverse human health effects, and the importance placed on each is not without debate (and, as indicated in the definition of “validated,” assays should be considered validated even if their value as predictors is not universally agreed upon), but tests held in such high regard by a governmental agency do, on their own, indicate a potential for significant or unreasonable risk if results are not mitigated by other information. In addition to mutagenicity tests, there is the opportunity to examine agents of interest in a battery of other in vitro tests that measure cytotoxicity, structural chromosomal aberrations, DNA damage, developmental toxicity, estrogenicity, and cell transformation (there are many other specific tests that could be used and these examples should not be viewed as inclusive): Cytotoxicity assays evaluate the ability of test materials to inhibit cellular proliferation in cells in culture. Often CHO-K1-B4 (Chinese hamster ovary) cells are used in this assay (Morita et al., 1992). For example, cytotoxicity of chromium picolinate was examined in Chinese hamster ovary cells (Stearns et al., 1995, 2002).

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Dietary Supplements: A Framework for Evaluating Safety The chromosomal aberration assay may also be conducted in CHO cells and detects the capacity of test agents to alter chromosomal structure (Buckton and Langlands, 1973; Gollapudi et al., 1986; Sinha et al., 1984). For example, genotoxicity assays assessing chromosomal aberrations were published for chromium picolinate (Stearns et al., 1995). Several methods are available for detection of developmental toxicity in vitro. One of these is the limb-bud micromass assay where fetal mouse limb-buds are allowed to differentiate via incubation in culture for 5 days with a test material. The test material’s effect on cytotoxicity and limb-bud differentiation are compared to determine if differentiation is inhibited at concentrations that are not cytotoxic, a sign of a potentially teratogenic compound (Flint and Orton, 1984). A popular and standardized method for assessing DNA damage (cells may be exposed either in vivo or in vitro) is the alkaline Comet Assay. This assay has been shown to be a sensitive and simple assay in monitoring DNA strand breaks in human leukocytes (Collins et al., 1997; Rojas et al., 1999; Tice and Strauss, 1995). Estrogenic activity of test materials in vitro may be assessed relative to the activity of 17-β-estradiol. One such assay is an estrogen receptor transactivation assay usually preformed in MCF-7 cells (Charles et al., 2000a, 2000b). For example, norhydroguaiaretic acid, a constituent of prototype monograph substance chaparral, was tested for estrogen-like activity in MCF-7 cells (Sathyamoorthy et al., 1994). The Syrian Hamster Embryo cellular transformation assay shares similarities with multistage carcinogenesis in vivo (Isfort et al., 1996). The assay may be used to study underlying mechanisms of genotoxic and nongenotoxic chemical carcinogenesis (Zhang et al., 2000). FDA also uses in vitro tests to identify potentially problematic chemicals of concern. In the Redbook 2000, it is recommended that a variety of short-term genetic toxicity tests be conducted for all chemicals that are direct food additives or color additives used in foods (OFAS, 2003). The data from genetic toxicity assays assist FDA in evaluating animal carcinogenicity data and in determining heritable effects of chemicals. Specifically, the guidelines for specific toxicity studies suggest the following short-term tests for genetic toxicity: Bacterial Reverse Mutation Test, In Vitro Mammalian Chromosome Aberration Test, In Vitro Mouse Lymphoma TK+/- Gene Mutation Assay, and In Vivo Mammalian Erythrocyte Micronucleus Test. FDA considers that positive results from these tests are sufficient to

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Dietary Supplements: A Framework for Evaluating Safety suggest risk unless there is further testing, in animals for example, that mitigates the concern. The value of such in vitro tests is no different if the test compound is a food additive or a dietary supplement. In fact, dietary supplement ingredients may sometimes be ingested in higher amounts than food additives. If these tests, or similarly accepted tests, are positive for dietary supplement ingredients or products, then it is scientifically justifiable to consider that a risk exists unless there is mitigating information. ANNEX 7-3 USE OF NEW TECHNOLOGIES IN PREDICTING RISKS Gene arrays, metabolomics, and proteomics are potential tools in the safety assessment of dietary supplements and many other materials. A number of recent publications discuss the use of these new technologies in their application to toxicology (Bartosiewicz et al., 2001; Farr and Dunn, 1999; Kuiper et al., 2001; Pennie et al., 2000; Robinson, 2001). One basic theme is that these technologies may provide a “fingerprint” of gene regulation associated with a toxic or adverse effect. Gene arrays (also known as genomics, transcriptomics, DNA chips, or microarrays) allow quantitative comparisons of the expression levels of potentially thousands of individual genes among different biological samples, allowing comparisons of normal with treated cells or tissues. Metabolomics provides a means to systematically analyze nonpeptide small molecules, such as vitamins, sugars, hormones, fatty acids, and other biochemicals, and is distinct from traditional analyses that target individual metabolites or pathways. An organism’s biochemical profile may be a better indicator of its cell physiology than a profile of its proteome or genome. Metabolomics may be able to detect differences in small molecules in cells, tissue, or fluid that are caused by chemical exposure. Such differences may eventually be used to understand mechanistic effects of substances and may provide insight into predicting potentially harmful substances. Proteomics is the analysis of many proteins and/or peptides simultaneously. Proteomics is used to identify proteins and their post-translational modifications, to quantify variation in protein content, and to study protein-protein interactions. Several efforts are specifically focused on developing these new technologies into approaches for predicting potential toxicities based on limited in vitro and in vivo data (Castle et al., 2002; Hamadeh et al., 2002; Pennie and Kimber, 2002; Ulrich and Friend, 2002; Waring and Halbert, 2002). In the arena of prescription pharmaceutical development, such an approach might enable resources to be focused on compounds predicted to have less safety concerns. If they prove successful in predicting toxicity, there is no biological reason that the same technologies will not provide similar infor-

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