Risk Assessment, Regulation, and the Role of Biomarkers for the Evaluation of Dietary Ingredients Present in Dietary Supplements

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39 Risk Assessment, Regulation, and the Role of Biomarkers for the Evaluation of Dietary Ingredients Present in Dietary Supplements Sandra A. James-Yi1, Corey J. Hilmas2, Daniel S. Fabricant3 1

Associate Principle Scientist, Product Safety/Nutritional Toxicology, Mary Kay Inc., Addison, Texas, United States; Senior Vice President of Scientific and Regulatory Affairs, Natural Products Association, Washington D.C., United States; 3President and CEO, Natural Products Association, Washington D.C., United States

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INTRODUCTION Dietary supplements in the United States are an FDA-regulated commodity used by an increasing number of individuals on a daily basis. This topic brings up a wide range of opinions from both supplement users and nonusers alike, in regard to regulation, safety, and efficacy. There are reports in the literature suggesting that the use of certain dietary supplement products and ingredients, specifically, those with a known or predictable pharmacological activity (nutraceuticals), have the potential for adverse events even if a preemptive risk assessment is performed prior to market distribution. Despite the long-held standing that a particular dietary ingredient is "safe" due to a substantial history of traditional use, information and research substantiating present-day usage and form may be lacking. There are considerable drivers in the marketplace that contribute to or potentially compound risk. These include a public perception that these products are inherently safe because they are advertized as “all-natural”; have a history of traditional use but in a different form or manufactured by a different process; and are sold to the consumer as a capsule or tablet, similar to prescription drugs they would get from a medical professional, and thus deemed to have undergone the same process, scrutiny, and guidance as pharmaceuticals prior to release in the marketplace or there may be the belief that they have passed regulatory standards for product safety or efficacy, by a regulatory body such as FDA. In addition,

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because of the passage of time between the original use of a supplement and present day suggested use, and the evolution of modern-day medicine, there is a significant loss of historical knowledge and familiarity with many products, their nutritive value, and recommended usages within the traditional medical community. Because of customer demand, many health-care practitioners are becoming more familiar with many of the dietary supplements on the market. The industry has grown at such a rapid pace that all of the products and combination products that are available to the average consumer may be difficult for medical practitioners and the general public to follow. Inherent in the science of nutrition is the difficulty in developing and performing robust studies. The scale with which products are coming onto the market makes this even more of a challenge for health-care professionals, industry, government regulators, and consumers alike. Communication is essential between health-care practitioners and their patients with respect to what is known about individual and combination dietary supplement products and the applicable cautions that need to be tailored to an individual’s specific needs. A contributing factor to the risk associated with the use of dietary supplements is consumer misinformation or lack of education for their intended use. Consumers of supplements often “self-prescribe” over-the-counter dietary supplements based on information from webbased public domains, bloggers, media outlets, wordof-mouth within their communities, manufacturers, and distributors of products.

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The goal of this chapter is to review the process of assessing risk associated with the use of dietary supplements; the role that biomarkers play in the risk assessment and identification of hazards associated with the use of dietary supplements by different populations of users; and how science informs FDA regulatory decisions with respect to FDA’s regulatory tools for application to dietary supplements. Although not exhaustive, listed below are factors to take into consideration when evaluating comprehensive safety profiles for dietary supplements as part of the process of risk assessment and hazard identification. Although the goal of this chapter is to address the biomarkers of physiological and biochemical changes corresponding with or directly caused by dietary supplements and ingredients with pharmacological activity (nutraceuticals) or toxicity, this subject is immense and incorporates all of the chapters in this text as they apply to biomarkers of multiple organ system functions. Dietary supplements are commonly mixtures and many times they are derived from the extrapolation of botanicals used in traditional or folk medicine for modern-day applications. As such they will have multiple target sites for activity that cannot be easily condensed into a simple algorithm. Factors that need to be considered by toxicologists in performing risk assessments for these products include but are not limited to the following: • Inappropriate application of the known pharmacology of an ingredient or supplement for uses that differ from their traditional or historical applications. For example, ephedra has an extensive history of use for treatment of clinical symptoms associated with respiratory disease. Its traditional use was never intended to be for weight loss. Thus, the known pharmacology of the ingredient, especially safety and efficacy, was not applicable to products marketed or intended to be used for weight loss. • Changes in the duration of use of a dietary supplement from the traditional posology of acute or intermittent usage in folk medicine to an intended current usage of prolonged, continuous, or chronic usage, typical of nutritional supplementation to an individual’s diet. • Changes in dose causing a larger, possibly excessive dose to be administered to achieve some particular result(s). Maximum levels of product, close to the tolerable upper limit for administration, may be delivered to consumers to make sure that a product on a store shelf has a labeled amount of constituent ingredients at the time of purchase and use. Some products that may have a short shelf-life of active constituent(s) have resulted in industry maximizing

the amount per serving for dosing, based on tolerable, upper limits for a reasonable expectation of safe use rather than the minimal amount used to effect as is practiced within the pharmaceutical industry. • Changes in processing that are significantly different from traditional or well-established methods. Herbs that historically have been consumed in one specific form, or in a particular cultural tradition for use, may now be processed or manufactured differently for the mass market. An example would be a botanical supplement that historically used a particular part of the plant for the basis of a tea preparation in folk medicine, may now be extracted with an organic solvent to extract and concentrate a specific, physiologically active component of the same plant. This significantly changes the chemical profile being delivered to consumers. The inherent synergist or antagonistic activity of constituents in the original tea may be gone and the resulting, concentrated extract may have an inherently different activity, posology, and risk assessment for a reasonable expectation for safe use within the general population for its intended use. • Concurrent use of dietary supplements by consumers taken in conjunction with pharmaceuticals, especially drugs with a narrow therapeutic index. The medicinal drugs and supplements may have similar mechanistic actions or pathways of metabolism. Health-care professionals and the general population may not be educated adequately to think of dietary supplements contributing to potential problems with this type of polypharmacy. The concurrent use of multiple dietary supplements or herbal medicines with additive, synergistic, potentiating, or antagonistic pharmacological and toxicological activity can have serious consequences in individuals being treated for other medical conditions. As science-based professionals, there needs to be a heightened level of communication between regulatory, academia, industry, and health-care professions, with the goal of identifying patterns of hazard or risk associated with the use of dietary supplements. The consumer rightfully assumes and expects that all stakeholders interested in dietary supplement safety have done due diligence prior to a product reaching the market. A reasonable expectation of product safety standards must align with the precautionary principle used by toxicologists involved in providing risk assessments to prevent potential harm in a very dynamic, demanding, lucrative, and growing marketplace.

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OVERVIEW OF REGULATION OF DIETARY SUPPLEMENTS IN THE UNITED STATES

OVERVIEW OF REGULATION OF DIETARY SUPPLEMENTS IN THE UNITED STATES Federal Food, Drug, and Cosmetic Act Dietary supplements are governed by four major sections of the Federal Food, Drug, and Cosmetic Act (Federal FD&C Act). • Sec. 201 [21 U.S.C. • Sec. 402 [21 U.S.C. (f) and (g) • Sec. 403 [21 U.S.C. • Sec. 413 [21 U.S.C. INGREDIENTS

321] DEFINITIONS (ff) 342] ADULTERATED FOOD 343] MISBRANDED FOOD 350b] NEW DIETARY

Dietary supplements are regulated under the Federal FD&C Act (the Act), as amended by the Dietary Supplement Health and Education Act (DSHEA) of 1994, and under the Fair Packaging and Labeling Act. DSHEA established a framework for regulating the safety of dietary supplements. This gave regulatory authority to the FDA to take action if there are product safety concerns (Hobson, 2009). Implementing regulations for certain provisions of DSHEA can be found in Title 21 of the Code of Federal Regulations (21 CFR). The term dietary supplement as defined in section 201(ff) of the Federal FD&C Act (the Act) is a product (other than tobacco) intended to supplement the diet that contains one or more of certain dietary ingredients, such as a vitamin, mineral, herb, other botanicals, amino acid, or dietary substance for use by man to supplement the diet by increasing the total dietary intake, of a concentrate, metabolite, constituent, extract, or combination of the preceding ingredients. Section 201(ff) of the Act further limits the term dietary supplement to mean products that are intended for ingestion in a form described in section 411(c)(1)(B)(i) of the Act (tablet, capsule, powder, softgel, gelcap, or liquid), which are not represented as conventional food, or as the sole item of a meal or of the diet, and that are specifically labeled as dietary supplements. According to 201(ff), “[e]xcept for purposes of section 201(g), a dietary supplement shall be deemed to be a food within the meaning of this Act.” Based on case law, dietary supplements are not only regulated as food but must also be ingested, where “ingestion” means to take into the stomach and gastrointestinal tract by the oral route of administration having first passed through the mouth. The term dietary supplement does not include products that are approved drugs, certified antibiotics, licensed biologics, or products that are authorized for investigation as a new drug, antibiotic, or biologic (and for which substantial clinical investigations have been instituted and for which the existence of such investigations

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have been made public), unless the product was marketed as a dietary supplement or as a food before it was approved as a drug, antibiotic, or biologic, or, in the case of investigational products, before the public disclosure of such investigations. Dietary supplements may not contain dietary ingredients that present a significant or unreasonable risk of illness or injury as set forth in section 402(f) of the Act, or that may render the supplement injurious to health (see section 402(a)(1) of the Act). Under section 413 of the Act, a manufacturer of a dietary supplement that is or that contains a new dietary ingredient (NDI) (an ingredient that was not marketed in the United States before October 15, 1994) must submit to FDA, at least 75 days before marketing, information substantiating the manufacturer’s conclusions that use of the dietary supplement will be expected to be reasonably safe for its intended use. Alternatively, a manufacturer of a dietary supplement that is or contains an NDI may petition FDA to issue an order prescribing the conditions under which such a dietary supplement will be expected to be reasonably safe. Nevertheless, it is still the responsibility of the manufacturer to ensure that a dietary ingredient used in a dietary supplement is safe for its intended use. It is important to remember that there is no authoritative “list” of substances that were marketed in the United States as ingredients in dietary supplements or foods before October 15, 1994. Therefore, the manufacturer must determine if an ingredient may be a “new dietary ingredient” under the Act for which a notification is required. If a manufacturer does not have a basis to conclude that a notification is not required, and they introduce a product containing the ingredient into interstate commerce, they do so at the risk that it may be considered adulterated as a matter of law. In the absence of evidence that a dietary ingredient does not require a notification to be submitted for it, manufacturers may wish to make the required notification to avoid uncertainty. In the September 23, 1997 Federal Register, FDA published a final rule promulgating a procedural regulation in 21 CFR 190.6 for making the required notification. DSHEA was followed by the Dietary Supplement and Nonprescription Drug Consumer Protection Act (Public Law 109e462), which was signed into law on December 22, 2006 and became effective on December 22, 2007. This law amended the Federal FD&C Act with respect to reporting of serious adverse events related to dietary supplements and nonprescription drugs. The law has four major provisions: requiring (1) the collection of all adverse event reports by manufacturers, distributors, and retailers of dietary supplements; (2) the reporting of serious adverse event reports to the FDA; (3) firms to maintain records of reports of all adverse events from use of the dietary supplement(s) such that FDA

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is allowed to audit those records; and (4) that dietary supplement labels provide information to facilitate the reporting of serious adverse events associated with their use by consumers. Lastly, Public Law P.L. 111e353, the FDA Food Safety Modernization Act, was signed into law on January 4, 2011. The major elements of the law include: prevention-based controls, inspection, and compliance to hold industry accountable; greater tools for increased food safety; mandatory recall authority for all food products; and enhanced partnerships among food safety agencies.

Regulatory Challenges There is no requirement for the manufacturer of a dietary supplement to provide FDA with evidence of a product’s efficacy. The manufacturer also need not provide FDA with evidence of ingredient safety prior to marketing, unless the product contains an NDI that has not been part of the food supply as an article used for food or is an article of food that has been modified in a form that is constitutionally, chemically altered. FDA’s Division of Dietary Supplement Program is tasked with the challenge of regulating a progressively expanding portfolio of dietary ingredients and finished products that are marketed as dietary supplements for a wide array of uses. When DSHEA amended the act in 1994, there were approximately 4000 products on the market; as of a 2009 Nielsen market survey, FDA estimated that there were 85,000 products on the market and this number is continuing to grow globally. With such growth the agency has encountered a number of unforeseen challenges. For example, FDA has found that dietary supplements marketed for sexual enhancement, weight loss, body building, and “calming” influences, can contain active pharmaceutical ingredients, analog of approved drugs, and other compounds, such as anabolic steroids, that do not qualify as dietary ingredients. Finding and testing these supplements for hidden, undeclared ingredients and removing those products from the market are a top priority for the Agency. FDA’s regulation of dietary supplements is particularly challenging given the seemingly endless volume of products that potentially transgress current regulations and the painstaking and costly process required by the government agency in proving that a violation exists. FDA continues to see dietary supplements marketed with ingredients that have been banned (i.e., ephedrine alkaloids and kratom) as well as dietary ingredients for which a notification to support the reasonable expectation of safety as an NDI was never submitted or containing an active ingredient that is undeclared on the labeling.

After identifying one of numerous potentially violative products, FDA is responsible for analyzing the ingredients of a product to determine whether a specific product is not in compliance. FDA conducts the laboratory analysis to prove the presence or absence of a product’s ingredients that may compromise its safety. Even if the labeling of a particular dietary supplement identifies an ingredient suspected to render the product violative, the process requiring confirmation of a violation must be instituted, which is rigorous, extremely time-consuming, and expensive. When an ingredient that may render a product violative is adequately identified, FDA must then determine whether the ingredient is a dietary ingredient through an extensive search of the available scientific literature. Once it is determined whether the ingredient is a dietary ingredient, an examination of the product’s other ingredients, labeling, and other promotional material is required to determine its proper regulatory status (e.g., unapproved new drug or an adulterated dietary supplement, i.e., nondeclared NDI).

Dietary Ingredient Safety The Federal FD&C Act (the Act) prohibits the distribution of adulterated foods in interstate commerce. Under section 402(f)(1)(A) of the Act (21 U.S.C. 342(f)(1)(A)), a food is considered adulterated if it is a dietary supplement containing a dietary ingredient that presents a significant or unreasonable risk of illness or injury under the conditions of use recommended or suggested on the labeling, or if there are no stated conditions of use suggested or recommended in the labeling, under conditions of ordinary use. FDA bears the burden of establishing that the product presents a significant or unreasonable risk. Because dietary supplements are presumed to be safe, FDA’s evaluation of whether a dietary supplement presents a significant or unreasonable risk generally takes place after the product is already on the market, with the exception of products that contain certain NDIs. Whether there is suspicion of a banned ingredient, concern over combinations of dietary ingredients, or safety concerns over an NDI, the use of biomarkers is crucial in determining whether dietary ingredients are adulterated. Although this list is not all-inclusive, Table 39.1 highlights several dietary ingredients of concern; the biomarkers that should be analyzed in the ingredient for quality control and may represent a component for possible risk and the known primary target organ systems associated with exposure causing clinical symptoms associated with the adverse event that is known to occur after ingestion. In the case of NDIs, manufacturers must demonstrate

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TABLE 39.1

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Dietary Ingredients of Concern, Their Biomarkers of Toxicity, and Target Organs Affected as a Result of Their Chronic Consumption in Dietary Supplements

Ingredient of Concern in a Dietary Supplement

Biomarker of Toxicity: Toxicant/Toxin, Drug, Metabolite

Target Organ System

Aristolochia spp. and Asarum spp.

Aristolochic acid

Renal/Urinary tract, liver

Blue-green algae

Microcystin

Hepatic

Bovine Source

Prohibited cattle material (21 CFR 189.5)

Central Nervous System

Butterbur (Petasites hybridus) root

Pyrrolizidine alkaloids

Hepatic, Cardiovascular

Comfrey (Symphytum officinale L)

Pyrrolizidine alkaloids

Hepatic, Cardiovascular

DMAA (1,3edimethylamylamine)

1,3-Dimethylamylamine

Cardiovascular, Respiratory, Central and Peripheral Nervous system, Gastrointestinal

Ephedra

Ephedrine alkaloids

Cardiovascular, Central, and Peripheral Nervous System

Hydroxycut (previous formulation)

Unknown

Cardiovascular, Hepatic, Central Nervous System, Skeletal Muscle (Rhabdomyolysis), Renal

Kava (Piper methysticum)

Unknown

Hepatic

Lichen (Usnea)

Usnic acid

Hepatic

Kratom (Mitragyna speciosa)

Mitragynine, 7-hydroxymitragynine

Central Nervous and Respiratory System Depression

Rauwolfia

Reserpine

Central and Peripheral Nervous System, Cardiovascular

Red yeast rice

Lovastatin

Skeletal muscle toxicity, cardiovascular

Silver (listed as colloidal, ionic, native, alginate, protein, etc.)

Silver: Consumer advisorydrisk of argyria at levels greater than RfD: 5 mg/ kg BW/day

Dermal, Ophthalmologic (Note: All organ systems can be noticeably affected)

Desiccated thyroid glandulars

Thyroid hormone (9 CFR 310.15)

All organ systems

the process by which particular biomarkers of toxicity are absent within the process of notification to the FDA for an NDI. As part of the process of determining tolerance and safe usage of a dietary supplement, a risk assessment is performed by a qualified toxicologist experienced in nutritional product safety. Because so little information and independent, peer-reviewed research is available for many of the products coming on to the market, the toxicologist responsible for the risk assessment needs to be armed with an extensive knowledge base and resources for the interpretation of a broad range of credible, scientific, primary, peer-reviewed data, including but not limited to in vitro, in vivo, and molecular studies as well as human clinical trials, case reports, and a myriad of other scientific and nonscientific, nutritional, historical, and environmental data to support the generation of safety profiles of dietary ingredient(s) for a reasonable expectation of tolerance and safety under the conditions of their intended use.

The Risk Assessment In general, acceptable and recommended dietary daily intakes for the majority of vitamin and mineral exposures in the general population of healthy men, women, children, infants, and pregnant and/or lactating women have been well characterized. These guidelines are by no means static but are part of a dynamic process of characterization that continues to develop as methods of analyzing nutritional science, toxicology, and risk assessment develops among various subpopulations. For the purposes of this discussion on risk assessment, and the use of biomarkers of exposure and effect for the majority of newly marketed dietary supplements, the focus of this discussion will be on the role of biomarkers within the context of risk assessment of botanically derived dietary supplements. Human populations are inherently outbred. The complexity of a “general” population and individual variation is currently being developed with customized tools and models provided by global genomics, epigenomics,

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proteomics, metabolomics, and big data such as Read-Across (Berggren et al., 2015). A case in point is the investigation and discovery of the various functions and perturbations of the microbiome and the use of prebiotics, probiotics, and synbiotics as dietary supplements. Biomarkers are objective measures of biochemical pathways, systemic organ function, internal and external perturbations of environmental markers, and chemical fingerprints of mixtures, plants and a multitude of other identifiers that may be present in natural or synthetic biological systems. Targeted research is just beginning to answer questions about the multisystemic, gender, environmental, cultural, and ancestral effects that nutrition and microbes contribute to a person’s health. A single, botanical, dietary ingredient (BDI) is a complex mixture of chemical compounds. Many of the products marketed as dietary supplements have multiple BDIs in formulations that have the potential for a multitude of additive, synergistic, potentiating, and/or antagonistic properties. In addition, constituents of these products are being grown, manufactured, and transported from vendors from all over the world. The ability to provide a reasonable expectation of safe use of these constituents by assessing quality, purity, stability, identity, and consistency of product, free from contamination or adulteration, under Good Manufacturing Practices, is vitally important for the protection of the consumer for the products intended use. Assessment of biomarkers and the chemical characterization or fingerprint of chemical constituents are vitally important to the process of public safety procedures and risk comparisons between BDIs. This is very important as new products come to market that may not be not in their natural state but in a chemical or physically modified form. Although there is very little information or guidance specifically addressing the risk assessment of dietary supplements in the peer-reviewed literature, the nutritional toxicologist can follow the basic principles of risk assessment, weight of evidence, and the precautionary principle when assessing dietary supplements. There are four basic components to risk assessment: Hazard Identification, Hazard Characterization, Exposure potential, and Risk Characterization (Boobis, 2007). The following is an overview of the steps that form this process for the evaluation of dietary supplements.

in a dietary supplement is safe for its intended use prior to distribution, industry must do due diligence to identify possible hazards that may be inherent to a BDI before it is exposed to the general population. It must also establish guidelines and biomarkers of botanicals for quality control. Hazard identification forms the basis for the development of the product label. This is a diagnostic challenge for botanically based products. Before a BDI product is introduced for development, there is usually some history of use. Important features associated with an investigation of a botanical are the region or country of origin; botanical nomenclature, plant genetic identification, and chemotype; original cultural purpose for use and dosing regimen; original methods of processing a BDI such as seasonal conditions for harvesting, part of plant used, processing, extraction methods, and storage. It is within this context for comparison that the risk characterization can be performed in relation to the suggested label usage under development for a specific BDI. Because there is typically evidence of use of the BDI of interest or comparable products that have either recent or historical use, a comprehensive search of the literature for medical case reports of toxicity, clinical research, animal studies, in vitro analysis and any other pertinent mechanistic or molecular data of the BDI, or a specific chemical component of the BDI, is undertaken. Interpretation of the quality of data is highly dependent on the ability of the assessor(s) to have the experience to integrate the information to produce a toxicological narrative of risk. Experience with the principles of toxicological assessment, pathology, animal and human physiology and medicine, pharmacology, biochemistry, nutrition, and research study development and the principles of epidemiology and public health, all factor in to the creation of a robust risk assessment. In addition, it is very important to understand the origin of the data. Questions should be asked as to who owns the original study data? Who owns the laboratory or Contract Research Organization doing the study, the analysis, and the interpretation of the data? What was the initial incentive or purpose for the research project or clinical study? Was there a null hypothesis or a priori theoretical proposal or was there another motivation for conducting the study with expected outcomes and usage for the data?

Hazard Characterization Hazard Identification Hazard identification is performed to identify the range of possible intrinsic capabilities of a specific dietary supplement, its dietary ingredients (BDIs), and the characterized chemical constituents of the ingredients for the potential to cause harm. Because it is the responsibility of the manufacturer to ensure that a dietary ingredient used

Hazard characterization is based on many components including but not limited to the results of cluster analysis and correlation of multiple biomarkers associated with human clinical studies, animal studies, molecular and mechanistic analysis, toxicokinetic and toxicodynamic information, clinical pathologic and histopathologic testing and documentation of adverse events occurring within

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BIOMARKERS OF TOXICITY: DIETARY INGREDIENTS

the context of various studies. Results are compared for consistency in physiological effect across studies of similar BDIs and their chemical components. There is a cumulative need for multiple tests to assess toxicity and predictive signatures (Waters and Merrick, 2009). Conservation along with corroboration of more than one functional pathway that merges within and between various animal physiological systems is necessary to establish healthbased guidance values such as the upper limit of tolerance for various populations of consumers. Negative outcome data are critical to identifying a lack of physiological changes that have the possibility of being associated with an adverse effect. Consistent with quality research, randomized, blinded, controlled studies utilizing credible, robust, and objective biomarkers associated with comprehensive baseline serum chemistries, physical examinations, health status of the population(s) tested, exposure data, ADME, and evaluation procedures for long-term chronic effects and reversibility are warranted. Many clinical studies associated with the use of BDI’s tend to focus on the beneficial effects of a given product utilizing a wide variety of disparate, subjective outcomes. The studies tend to lack a standardized platform of objective, repeatable criteria for comparative analysis between studies. A standardized set of biomarkers indicating the presence or absence of adverse events is an integral function of robust clinical research studies and needs to be better established for research specifically relating to the use of BDI’s. Typical of most past and current studies, if adverse effects are considered by the researcher to have not occurred, there will be a short statement of "No adverse events were recorded." This is insufficient. Procedural guidelines, a priori, for how the adverse events were collected, recorded, and the criteria for evaluation are critical to the interpretation of a study and a possible adverse incident. Did these events occur in more than one individual? Were they recurring? Were there variables associated with subpopulations including race, gender, and life-stage that will help in identifying individual variation in intake and sensitivity (Boobis, 2007)? Were they self-reported or were they conducted by trained personnel capable of obtaining objective assessment? This process is very fluid. As more information surrounding the use (and misuse) of BDIs in the public sector is garnered, epidemiological data for long- and short-term exposure outcomes and postmarketing data generated by academia, government and industry, in addition to the regulatory guidelines established by the FDA for adverse event reporting, will become critical for future product development and quality control.

Exposure Assessment Risk is a function of hazard and exposure. Outside of acceptable or estimated daily intake for consumption of

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components of BDIs that may occur in a specific population’s normal diet on a local basis, global sourcing and production of BDIs in dietary supplements carries significant challenges to manufacturers and distributers. Supply-chain exposure to contaminants in the field, residual solvents after manufacture, and contamination or economically motivated adulteration of product at various stages of production can occur. Close associations with suppliers and supply chainmanagement in conjunction with standard markers for the presence or absence of toxicants are key to decreasing exposure to contamination and subsequently, risk. As an example, the raw, unprocessed butterbur plant (Petasites hybridus L.) contains pyrrolizidine alkaloids (PAs), which have been associated with hepatobiliary veno-occlusive disease and neoplasia (Danesch and Rittinghausen, 2003; Sadler et al., 2007; Burrows and Tyrl, 2013). Butterbur has been used for hundreds of years as an analgesic and antiinflammatory remedy. Currently it is being used as adjunct support for more chronic conditions such as migraine headaches, allergic rhinitis, and asthma. The pharmacologically active substances are believed to be the sesquiterpene esters, petasin, and furanopetasin (Anderson et al., 2009; Chizzola et al., 2006). During processing PAs are removed. Standard, preemptive, operating procedures and specifications need to be in place for quality analysis and control indicating the absence of PAs at critical points of production prior to distribution to the consumer to assure its absence for chronic use that may result in more severe, debilitating, and life-threatening conditions.

Risk Characterization Because of limited available data available on many dietary supplements, toxicologists find themselves quite often in the position of proving a negative, that the dietary supplement or ingredient under investigation will not be associated with any serious adverse events to support a reasonable expectation of safety. A conservative approach is always warranted in an environment where there may be competing objectives for production and release of a dietary supplement product to the market. Toxicologists, charged with completing a risk assessment on a NDI or supplement, will need to rely on the weight of evidence from known data in conjunction with the precautionary principle when evaluating product. In some cases it is quite possible and appropriate for the toxicologist to conclude that there is insufficient evidence to produce a tolerable upper limit or observable safe level for consumption of a dietary ingredient that would have a reasonable expectation of safety under the conditions of its intended use.

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BIOMARKERS OF TOXICITY: DIETARY INGREDIENTS Biomarkers Associated with the Presence of Banned Ingredients Banned Dietary Ingredients Causing NeurostimulationdEphedrine Alkaloids Dietary ingredients are banned if they present an unreasonable risk of illness or injury. In the Federal Register of February 11, 2004 (69 FR 6788), FDA interpreted and applied the “unreasonable risk” standard in a final rule declaring dietary supplements containing ephedrine alkaloids to be adulterated because they present an unreasonable risk of illness or injury. As the rule explained, “unreasonable risk” implies a riskebenefit calculation that weighs a product’s risks against its benefits under the conditions of use recommended or suggested in the product’s labeling, or if the labeling is silent, under ordinary conditions of use. The Secretary of Health and Human Services also has authority under the statute to act where a product poses an “imminent hazard” to public health. The riskebenefit calculation is also true for multiple or combinations of dietary ingredients with common mechanisms and/or modes of action. Ephedrine alkaloids are chemical stimulants that were originally found to naturally occur in some botanicals. In the 10 years prior to the published Ephedrine Final Rule of 2004, dietary supplements containing ephedrine alkaloids were labeled and marketed with claims for weight loss, energy, or enhancement of athletic performance. Today, FDA still encounters dietary supplement products containing ephedrine alkaloids in the form of raw botanicals imported from foreign suppliers intended for use in dietary supplements or destined for production of methamphetamine. Ephedrine alkaloids are members of a large family of pharmacologically active compounds known as phenylethylamines or sympathomimetics. They include pseudoephedrine (a decongestant), phenylephrine (cold tablets, nasal sprays, and hemorrhoid treatment), phenylpropanolamine (PPA) (for treatment of urinary incontinence) and amphetamine-based products (for treatment of narcolepsy and attention deficit disorder). Their mechanism of action is due to the release of catecholamines (dopamine and norepinephrine) and serotonin. Primary effects associated with toxicity are due to the activation of both a- and b-adrenergic receptors causing serious adverse effects in primarily the central nervous and cardiovascular systems. Clinical symptoms that have been associated with toxicity due to phenylethylamines are hallucination, hyperactivity, agitation, tremors, seizures, sleep disorders, vasoconstriction, arrhythmias, hypertension, platelet aggregation,

disseminated intravascular coagulopathy, hyperthermia, tachypnea, hyperinsulinemia, cardiovascular collapse, cerebrovascular hemorrhage/infarction, multiorgan failure, and death. Ephedrine alkaloids are primarily derived from raw material and extracts from the plants Ephedra sinica Stapf, Ephedra equisetina Bunge, Ephedra intermedia var. tibetica Staph, and Ephedra distachya L. (the Ephedras) (Betz and Tab, 1995; World Health Organization, 1999). Other botanical sources found to contain ephedrine alkaloids include Sida cordifolia L. and Pinellia ternate (Thunb.) Makino (Ghosal et al., 1975; Oshio et al., 1978). Ma huang, Ephedra, Chinese Ephedra, and epitonin are several names used for the botanical sources. The plants were mostly found in desert regions of China and Mongolia and were the original source of ephedrine and pseudoephedrine prior to the early 1900s. Other common names that have been used for the various plants that contain ephedrine alkaloids include sea grape, yellow horse, joint fir, popotillo, and country mallow. Although ephedrine is the primary alkaloid isolated from these plants, other closely related chemicals associated with these botanicals can also be used as biomarkers indicating contamination or adulteration of a dietary supplement. They include norephedrine, methylephedrine, norpseudoephedrine, and methylpseudoephedrine (Chen and Schmidt, 1930; Mahuang, 1987; Karch, 1996; Bruneton, 1995; Cui et al., 1991). Banned Dietary Ingredients Causing NeurosuppressiondMitragyna speciosa (Kratom) Mitragyna speciosa and extracts of its leaf are collectively referred to as kratom or by other synonyms and pseudonyms, including Nauclea speciosa, biak-biak, cratom, gratom, kakuam, katawn, kedemba, ketum, krathom, mambog, madat, maeng da, mitragynine extract, Mitragyna javanica, red vein/white vein, thang, ithang, and thom. M. speciosa or kratom is a botanical ingredient that would generally qualify as a dietary ingredient under section 201(ff) of the Federal FD&C Act (the Act) [U.S.C. 321(ff)]. The leaf and extracts are usually imported with a product code 54 as they are almost always intended as dietary supplements for ingestion. Kratom is obtained overseas from the dried leaves of a tree that grows most commonly in Thailand, Malaysia, Indonesia, Sumatra, Bali, and Vietnam. The whole leaf, crushed leaf, powder forms, and extracts are shipped directly to consumers for oral consumption or indirectly through US distributors in the United States. Dry leaves of kratom are typically ingested as a tea or the dried leaves are crushed into smaller particulates for ingestion in foods that mask kratom’s bitter taste. The powders, dry leaves, and crushed leaf forms can also be ingested by either directly placing them in blank capsules or wrapping them in bathroom tissue squares.

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After kratom is placed in bathroom tissue, the tissue is twisted at both ends to contain the material, and then placed into the mouth and swallowed. This process is termed “parachuting.” The raw botanical material is also incorporated into finished dietary supplements. M. speciosa gained use among American consumers in the belief that it will relieve pain, anxiety, and depression and treat the symptoms of opioid addiction and for its euphoric properties for recreational use. It contains two potent alkaloids, mitragynine and 7hydroxymitragynine. Both are biomarkers for M. speciosa toxicity. In low concentrations, these alkaloids produce euphoria, energy, and other stimulating effects. In higher concentrations, they are sedating and possess antinociceptive properties. Based on scientific research and modeling by the FDA, they have concluded that the compounds in Kratom have structural similarities with other opioid analgesics and exert effects on mu, delta (Thongpradichote et al., 1998), and kappa opioid receptors (Boyer et al., 2008; Dale et al., 2012), and they are now classified as opioids (FDA, 2018a). There are safety concerns regarding the dietary ingredient kratom that support detaining this ingredient and legal enforcement against any dietary supplements known to contain kratom. These products are considered adulterated under section 402(f)(1)(B) of the Act (21 U.S.C. 342(f)(1)(B)). FDA’s review of publicly available information regarding kratom concluded that there is not a reasonable assurance that the ingredient does not present a significant or unreasonable risk of injury to consumers. The scientific literature has disclosed serious concerns regarding the toxicity of kratom in multiple organ systems. Consumption of kratom can lead to respiratory depression, nervousness, agitation, aggression, sleeplessness, hallucinations, delusions, tremors, loss of libido, constipation, skin hyperpigmentation, nausea, vomiting, addiction, and severe withdrawal signs and symptoms after refraining from use. Detention and refusals of kratom imports do not rely on the provision in section 413 of the Act that deems a dietary supplement containing an NDI to be adulterated unless the manufacturer or distributor has filed an NDI notification with FDA at least 75 days before marketing (21 U.S.C. 350b).1 Instead, kratom detentions and refusals rely on the adulteration standard in section 402(f)(1)(B) of the Act, which provides that a food is adulterated if it contains an NDI for which there is inadequate information to provide a reasonable assurance that any such ingredient does not present a significant

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or unreasonable risk of illness or injury. In general, FDA would not be inclined to support the detention of bulk NDIs unless it had identified concrete safety concerns. Reports of addiction clinics dedicated to kratom abuse and emergency room visits for kratom intoxication have increased over the past 6 months. Kratom is an emerging issue of public health that should be taken seriously. From 2010 to 2015, calls to poison control centers have increased 10-fold (FDA, 2017) along with at least 36 deaths associated with its use (FDA, 2017). Based on this information the FDA has exercised its jurisdiction to seize imports and enforce voluntary destruction by companies that had imported kratom for the intent of distribution to consumers (FDA, 2017; 2018b).

Biomarkers Associated with the Presence of Endocrine Disruptors Dessicated Thyroid Glandulars Dietary supplements containing animal-derived, thyroid glandular tissue are currently marketed for weight loss, body building, nutritional support, and altering thyroid hormone levels. Marketing such a dietary supplement to explicitly or implicitly treat hypothyroidism, with respect to an individual’s thyroid stimulating hormone (TSH) or thyroid hormone level, would be a disease claim as per Title 21 CFR 101.93(g). The public health issue for using such a product, even for weight loss, is the tendency for consumers to incorrectly selfmedicate themselves to achieve weight loss by jumpstarting a perceived underactive thyroid. Dietary supplements are considered food under the Federal FD&C Act (the Act). Animal-derived thyroid tissue is declared on many dietary supplement product labels as a dietary ingredient, but it is not. In contrast to FDA-approved thyroid-containing products, which are synthetic drugs approved for defined medical conditions, at consistently defined and approved concentrations and dosages of administration, desiccated thyroid supplements are primarily derived from the thyroid glands of slaughterhouse animals. The thyroid tissue that is ingested, even if it is cooked, or frozen prior to cooking, contains variable levels of the thyroid hormones, levothyroxine (T4), and levothyronine (T3). Both forms of thyroid hormone are active after ingestion and absorption into the general circulation with the potential of resulting in multisystemic effects (Table 39.2) that can result in the clinical syndrome known as thyrotoxicosis factitia (Bouchard, 2015).

1

There is an exception to the notification requirement for dietary supplements that contain only dietary ingredients that have been present in the food supply as articles used for food without chemical alteration. 21 U.S.C. 350b(a)(1).

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Clinical Signs and Symptoms of Hyperthyroidism due to Consumption of Thyroid Glandular Ingredients Contained in Dietary Supplements (Bouchard, 2015)

Organ Systems Affected by Exogenous Thyroid Hormone Toxicity

Clinical Sign or Symptom

Ocular

Alterations in vision, photophobia, eye irritation, exophthalmos

Musculoskeletal system

Fatigue, muscle weakness, exertional intolerance, rhabdomyolysis, accelerated osteoporosis

Respiratory system

Dyspnea

Nervous system

Irritability, anxiety, behavioral changes, mental disturbance/psychosis, insomnia, eating disorders, tremor, paralysis, coma

Cardiovascular system

Palpitations, tachydysrhythmias (atrial fibrillation/flutter, extra systoles, highoutput congestive heart failure), widened pulse pressure (elevated systolic, decreased diastolic), left ventricular hypertrophy, focal myocarditis, cardiac failure, pulmonary hypertension, angina pectoris, thromboembolism, myocardial infarction, stroke, sudden death

Gastrointestinal system

Gastrointestinal disturbance (nausea, vomiting, diarrhea)

Reproductive system

Impaired fertility and fetal development

Metabolic

Hyperthermia, heat intolerance, increased diaphoresis, weight loss, hypothyroidism

Dermal

Delayed palmar desquamation

Historically, ingestion of animal-derived thyroid gland has been used by for a variety of cosmetic and medical purposes. Ancient Egyptians used them as a beauty aid. Enlarged, goiterous necks were considered fashionable in women (Bouchard, 2015). Up until the 1950s thyroid hormone from animal tissue was used to treat conditions such as hypothyroidism (Bouchard, 2015). Currently, thyroid hormone derived from animal tissue is being marketed as an aid for weight loss and as a stimulant. Its use has been fraught with adverse events. In some cases consumption has resulted in severe, clinical, multisystemic symptoms, and even sudden death (Bouchard, 2015) (Table 39.2).

Thyroid tissue as an ingredient invariably contains an unknown amount and ratio of thyroid hormones that have the potential to result in hyperthyroidism. Thus, in accordance with 9 CFR 310.15(a)2, livestock thyroid glands and laryngeal muscle tissue shall not be used for human food. Accordingly, thyroid tissue does not appear to fit within any category of “dietary ingredient” in 201(ff)(1). It is not considered a “dietary substance” because it is not part of usual food or drink for humans and even if thyroid tissue could be identified as a “dietary ingredient,” it would fall under the category of “adulterated” in accordance with Section 402(f)(1)(A) of the Federal FD&C Act (the Act) [U.S.C. 342(f)(1)(A)] because it presents a significant or unreasonable risk of illness or injury. Contamination of dietary supplements by thyroid tissue is identified by analysis of product for the presence of the biomarkers thyroxine (T4) and triiodothyronine (T3). Clinical symptoms of thyrotoxicosis factitia are not readily identifiable through blood analysis of either thyroxine (T4) and/or triiodothyronine (T3) due to the fact that even in the presence of normal or low levels, severe adverse events associated with toxic exposure can occur (Bouchard, 2015). Diagnosis of exposure of an individual from either acute or chronic consumption of excessive thyroid hormones can be identified primarily through blood analysis for the suppression of circulating TSH. Any argument to support allowing residual or trace amounts of thyroid hormone in a dietary ingredient because it is present in many common foods that we normally eat is not a valid argument. First, consumption of meat containing bovine thyroid tissue has been shown to lead to thyrotoxicosis (Hedberg et al., 1987; Kinney et al., 1988). Second, the mere fact that a substance exists in minuscule quantities in a food humans typically consume does not make that substance a “dietary” one. There are many substances found in typical human foods that people avoid eating, and which could never be termed a “dietary ingredient.” Examples of substances people may unintentionally ingest include pesticides and heavy metals. Just because they may be there in trace amounts from environmental contamination does not make them a dietary ingredient. Supplementing the diet with oral thyroid hormone from a glandular supplement is not needed or recommended in normal, euthyroid individuals. Medical literature contains numerous case reports of adverse events in individuals who ingested herbalal supplements adulterated with thyroid hormone.

2

Under 21 CFR 316.3(b)(2), active moiety means “the molecule or ion, excluding those appended portions of the molecule that cause the drug to be an ester, salt (including a salt with hydrogen or coordination bonds), or other noncovalent derivative (such as a complex, chelate, or clathrate) of the molecule, responsible for the physiological or pharmacological action of the drug substance.”

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Many describe life-threatening events. In April 2006, French health authorities reported an incident causing one death and several hospital intensive care unit admissions after taking a “slimming aid” consisting of powdered thyroid extract (Thyroid Extract, 2006). All had symptoms of thyrotoxicosis, including heart palpitations and altered consciousness. Another case series described five individuals who took Enzo-caps, which claimed to be a natural food product containing papaya, garlic, and kelp, as a weight loss adjunct. All five patients suffered from symptoms suggestive of hyperthyroidism. Each tablet was found to contain up to 112 mg of T4 and 11.1 mg of T3. It was later found to also contain a sympathomimetic agent and diuretic. For comparison, the mean dosage of levothyroxine for treatment of diagnosed, hypothyroid, adult patients is 1.7 mg per kg body weight per day, for a typical daily dose of 100e150 mg (Bouchard, 2015; Braunstein et al., 1986). Depending on age, subpopulations can have enhanced sensitivity to thyroid hormone. An average daily dosage for an elderly, adult, hypothyroid patient is 12.5e50 mg/day (Bouchard, 2015). Thyroid glandular tissue is just one example of the importance of analysis for certain biomarkers as part of a standard process of manufacturing for identification of contamination of raw materials and end-products, by even minute amounts of endocrine disrupting agents. Standard operating procedures for identification of these types of adulterants cannot be overemphasized as companies perform the due diligence necessary to protect consumers in a global environment.

Biomarkers Associated With the Presence of Carcinogens Aristolochic Acid Aristolochic acid refers to a family of nitrophenanthrene compounds found concentrated in the roots/rhizomes of plants of the family Aristolochiaceae, and in particular in the genus Aristolochia. Lower concentrations are found in the fruit and leaves. There are many other genera in the family Aristolochiaceae that can be found distributed around the world. Many are used as medicinal plants. These include species of plants in the genera Asarum, Bragantia, Stephania, Clematis, Akebia, Cocculus, Diploclisia, Saussurea, Menispermum, and Sinomenium, as well as Mu tong, Fang ji, Guang fang ji, Fang chi, Kan-Mokutsu (Japanese), and Mokutsu (Japanese). In the latter part of the 20th century reports started to appear for serious adverse events associated with renal failure and renal fibrosis occurring in individuals ingesting herbal weight loss aids. The herbal product was labeled to have contained Stephania tetrandra, a botanical

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not known to contain aristolochic acid, but was later found to be inadvertently substituted with the botanical Aristolochia fangchi. Subsequent reports confirmed that the renal lesions were related to exposure due to ingestion of products containing aristolochic acids (Vanhaelen et al., 1994; Schmeiser et al., 1996; Nortier et al., 2000; Muniz-Martinez et al., 2002). Similar diagnoses were reported elsewhere in Europe, Asia, and the United States. In July 1999, two cases of nephropathy, associated with the use of Chinese botanical preparations, were reported from the United Kingdom. Both of these patients had ingested botanical preparations for the treatment of “eczema.” Renal biopsies showed a pattern of extensive tubular loss, most prominent in the outer cortex, and severe interstitial fibrosis. These pathological features are typical of what has now come to be called “Chinese herb nephropathy” due to the toxicity associated with ingestion of aristolochic acid. In 2001, after reports of rapidly developing renal failure and urothelial carcinoma associated with the use of herbal preparations containing aristolochic acid containing dietary ingredients, manufacturers and distributors of supplements were advised to remove these products from the market. Public health statements were released, advising consumers not to consume these supplements (Schwetz, 2001). An FDA import alert (IA #54-10) called for the detention of any botanical, drug, or other products found to contain the more than the 69 types of aristolochic acids; herbs known to contain aristolochic acids; and/or herbal preparations that had been found to have been replaced by herbs containing aristolochic acids. At that time it was clear that consumption of herbal products containing aristolochic acids, both intentionally and through apparent misbranding, was associated with a rapid onset of renal interstitial fibrosis, renal failure, and an increased incidence of urothelial carcinomas. The rapid onset, severity, and irreversibility of the damage suggested that detection of any amount of aristolochic acid should be viewed as a potential health risk. The carcinogenic potential of aristolochic acid started to appear in the 1980s. In addition to renal damage, safety studies reported an increased incidence in pathologic lesions indicating aristolochic acideinduced carcinogenicity in rodents orally administered aristolochic acid (Mengs et al., 1982). The rodents developed multiple cancers that included lymphoma, as well as cancers in the kidney, bladder, stomach, and lung. Short-term studies resulted in tumors in the urinary tract in addition to the other renal pathological changes indicative of Chinese herbal nephropathy (Mengs et al., 1982; Mengs, 1987; Mengs and Stotzem, 1993). The precipitating event for aristolochic acid was an outbreak in Belgium. In 1992, a Belgian cohort of 70 patients, known to have consumed a slimming regimen

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containing a powdered extract of several Chinese herbs, was found to have interstitial renal fibrosis, with 30 individuals progressing to end-stage renal disease (Vanherweghem et al., 1993; Cosyns et al., 1994; Depierreux et al., 1994; Vanherweghem, 1998). The diagnosis was made based on an unusual and distinctive pattern of renal nephropathy and the finding of biomarkers for aristolochic acideDNA adducts in the urothelium indicating exposure and potential toxicity. The renal fibrosis occurred 12e24 months after the initial injury with an increased incidence of urothelial cancer being associated with this pattern of renal nephropathy (Hung, 2015; Debelle et al., 2008). The International Agency for Research on Cancer (IARC), an arm of the World Health Organization, would later conclude that there was sufficient evidence to determine that aristolochic acidecontaining herbs are carcinogenic in humans, thus classifying them as Group I carcinogens (IARC, 2002). To this day, reports from around the world, and especially in east Asia, continue to link rapid onset of renal failure and/or renal cancer with consumption of teas brewed from the ground root of these botanicals (Gillerot et al., 2001; Tanaka et al., 2001; Krumme et al., 2001; Cronin et al., 2002; Yang et al., 2003; Lee et al., 2004).

Pyrrolizidine AlkaloidsdSymphytum Spp. (Common Name, Comfrey) In June 2001, FDA published an Advisory to dietary supplement manufacturers to remove comfrey as an ingredient in dietary supplements. The Advisory alerted the supplement industry to the available scientific information that firmly establishes that dietary supplements that contain comfrey or any other source of PAs are adulterated under the Act. Also, the Agency stressed that manufacturers need to identify and report adverse events associated with any product that contains an ingredient that has the potential to contain PAs. The Agency cited serious adverse health effects and opined on the presence of PAs as potent hepatotoxins. Comfrey, like a number of other plants (e.g., Senecio species), contain PAs. The toxicity of PAs to humans is well documented (Huxtable and Cheeke, 1989; Winship, 1991; McDermott and Ridker, 1990; Burrows and Tyrl, 2013). FDA’s position with regard to comfrey is that the PAs present in comfrey are toxic chemicals that can lead to serious adverse health effects if taken into the body. It is clear that oral exposure is potentially hazardous. It is equally clear that PAs are harmful when allowed to enter the body through broken skin, or other nonoral routes, such as suppositories. The Federal Trade Commission has limited them to external use only.

Accordingly, FDA believes that external use of comfrey products, containing PAs, present minimal risks to consumers when such products are not used as suppositories and are not applied to broken skin. Hepatic veno-occlusive disease and neoplasia as well as pulmonary hypertension are the major documented forms of injury to humans (Burrows and Tyrl, 2013) from chronic or excessive intake of PA-containing herbals. Laboratory animal studies, epidemiological studies, and veterinary case reports suggest that the toxic effects are much broader. Animal exposure has resulted in pulmonary, kidney, and gastrointestinal pathologies including cancer (Burrows and Tyrl, 2013). Four countries, the United Kingdom, Australia, Canada, and Germany, have restricted the availability of products containing comfrey, and other countries permit use of comfrey only under a physician’s prescription. There is variability in the levels of PAs in various species of plants, thus, the concerns FDA has about a comfrey-containing dietary supplement or ingredient depends on the exact species identified. Identification of this biomarker is a crucial component for the development, manufacture, and distribution of a dietary supplement derived from plants that have the potential to containing PAs as part of the process for quality control. This is especially important in light of the potential for chronic exposure to result in hepatic lesions, including cancer, if due diligence is not adhered to by the manufacturer prior to marketing. The FDA ruling banned internal use of Symphytum officinale L. (common comfrey), Symphytum asperum Lepech (rough or prickly comfrey), and Symphytum xuplandicum Nyman (Russian comfrey), as well as any other plant/substance containing PAs. While FDA did not examine the safety of other comfrey species such as Symphytum tuberosum L. (tuberous comfrey), which is suggested to contain negligible amounts of PAs, FDA and potential manufacturers of products would rely on the presence of the PA to determine whether this species is to be permitted to be used as a dietary ingredient. Only after such sampling and analysis of this biomarker of toxicity, would they be in a position to provide an opinion on whether its use in dietary supplements would not present a significant or unreasonable risk of illness or injury under the conditions of use recommended on the label. Because the burden and responsibility for assuring that such a product is not adulterated under the Act lies with the manufacturer, and with the associated costs involved during the product life cycle, alternative species that are known to not have an association with the presence of pyrrolizidine alkaloids should take precedence rather than risking the potential for future contamination of product. Although FDA continues to voice its concern about the safety of dietary supplements containing comfrey,

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this does not mean that FDA prohibits the marketing of dietary supplements that contain comfrey. Although dietary supplements do not require premarket approval or approval from FDA before marketing and distribution, the manufacturer is responsible for determining that its products are safe under the Act. However, the Agency is unaware of conclusive scientific data that would establish that a dietary supplement containing comfrey would be safe. Therefore, for a manufacturer interested in marketing a comfrey-containing dietary supplement, it would be required to submit information in the form of a NDI notification that forms the basis by which it establishes that such a product is safe within the meaning of the Act. A manufacturer would then have to demonstrate that the comfrey-containing ingredient did not contain the toxic PA biomarker as part of the NDI notification, as well as chemistry and additional safety data to market such a product as a dietary supplement for its intended use.

BIOMARKERS OF TOXICITY: NEW DIETARY INGREDIENTS Premarket Notification of a New Dietary Ingredient The Act requires a premarket safety notification for NDIs introduced into commerce following the passage of the DSHEA of 1994. Under section 413(c) of the Act (21 U.S.C. 350b(c)), an “NDI” is a dietary ingredient that was not marketed in the United States before October 15, 1994. A dietary supplement that contains an NDI is deemed to be adulterated unless one of the following two conditions are met: (1) The dietary supplement must contain only dietary ingredients that have been present in the food supply as an article used for food in a form in which the food has not been chemically altered; or (2) there must be a history of use or other evidence of safety establishing that the dietary ingredient when used under the conditions recommended or suggested in the labeling of the dietary supplement will reasonably be expected to be safe, and the manufacturer or distributor of the dietary supplement containing the NDI must provide FDA with information, including any citation to published articles, which forms the basis on which the manufacturer or distributor has concluded that a dietary supplement containing such dietary ingredient will reasonably be expected to be safe. FDA has established requirements for premarket notification of NDIs in 21 CFR x 190.6, based on authority granted in sections 201(ff), 301, 402, 413, and 701 of the Federal FD&C Act (21 U.S.C. 321(ff), 331, 342, 350b, 371). 21 CFR 190.6(a) which states that at least 75 days before introducing or delivering for introduction into

705

interstate commerce a dietary supplement that contains an NDI that has not been present in the food supply as an article used for food in a form in which the food has not been chemically altered, the manufacturer or distributor of that supplement, or of the NDI, shall submit to the Office of Nutritional Products, Labeling and Dietary Supplements, Center for Food Safety and Applied Nutrition information including any citation to published articles that is the basis on which the manufacturer or distributor has concluded that a dietary supplement containing such dietary ingredient will reasonably be expected to be safe. That notification should include pertinent information that satisfies the regulatory requirements established under 21 CFR 190.6, documentation demonstrating clear identification of the test article, history of use, and weight of evidence for a reasonable expectation of safety. If a dietary supplement containing an NDI is subject to the notification requirement and this requirement is not met, or if there is no history of use or other evidence of safety establishing a reasonable expectation of safety, the dietary supplement is deemed to be adulterated under section 402(f)(1)(B) of the act because there is inadequate information to provide reasonable assurance that the product does not present a significant or unreasonable risk of illness or injury. Within this context, biomarkers are used at many critical points of the product life cycle to establish safety. This includes but is not limited to identification of possible contamination or adulteration of product at any point in the process for quality control; physiological effects of use of product through the evaluation of animal and human research; a comprehensive evaluation of the peer-reviewed literature concerning any evidence to substantiate use and safety of product; the establishment of labeling including cautions of use for specific subpopulations of individuals; epidemiological evaluation of use and historical and ancillary use of product.

Premarket Notification for New Dietary Supplement as Enhancers of Metabolism, Energy, and Weight Loss Usnic Acid On November 20, 2001, US FDA warned consumers to stop using LipoKinetix, a dietary supplement marketed for weight loss, because it was associated with a number of serious adverse event reports involving hepatotoxicity (Favreau et al., 2002) including acute hepatitis, irreversible liver injury, liver failure, and eventual transplant. US FDA reported on six persons who developed acute hepatitis and/or liver failure while using LipoKinetix. The injuries reported to FDA occurred in

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persons between 20 and 32 years of age, and no other cause for liver disease was identified in the cohort. In all cases, no preexisting medical condition that would predispose the consumer to liver injury was identified. Liver injury occurred fairly rapidly, between 2 weeks and 3 months, after starting LipoKinetix. Although the product contained multiple dietary ingredients such as norephedrine (PPA), caffeine, yohimbine, diiodothyronine and sodium usniate, usnic acid was the dietary ingredient suspected to have led to hepatotoxicity in the adverse event reports. Usnic acid is typically derived from botanicals of Usnea spp. (Ingo´lfsdo´ttir, 2002), but it is also present in other genera of lichens, including Alectoria, Cladonia, Evernia, Lecanora, and Ramalina. Usnic acid is a complex dibenzofuran derivative, produced naturally by certain lichen species. In recent years, usnic acid and its salt form, sodium usniate, have been marketed in the United States as an ingredient in dietary supplement products, mostly with claims and marketing to promote weight reduction, enhancement of metabolism, and inhibition of bacteria. Although lichens containing Usnea have been formulated into topical products and used as traditional medicines in other countries, FDA is unaware of any evidence that usnic acid was marketed before October 15, 1994. Similar to many NDIs on the market, FDA has not received any notification for usnic acid or usniate as an NDI for which a premarket notification pursuant to 21 U.S.C. 50b(a)(2) is a requirement. The NDI notification process, a premarket gate to ensure reasonable expectation of safety, is designed to pick up toxicological signals in the data before the dietary ingredient contained in the dietary supplement reaches the market. The notification process can only work in a premarket capacity if notifications for NDIs are submitted to FDA. Lichens have evolved an innate capacity to survive extreme environmental conditions. They excrete bioweathering and bioactive secondary metabolites (e.g., usnic acid), which provide chemical protection from invading biologicals such as viruses, bacteria, protozoa, competing fungi, algae, plants, and animal predators. Although lichens produce a vast array of secondary metabolites, representing diverse classes of chemical compounds (e.g., lactones, aromatic compounds, quinines, terpenes, diphenyl esters, dibenzofuranes, and aliphatic acids) (Fiedler et al., 1986; Huneck and Yoshimura, 1996; Huneck, 1999), the most characterized and studied is the polycyclic usnic acid (Correche et al., 1998). Usnea content varies depending on the lichen species and region. Alectoria spp. are known to contain up to 6% usnic

acid, whereas the thallus of Usnea laevis from the Venezuelan Andes contains approximately half that amount (Marcano et al., 1999). Although Usnea spp. synthesize and excrete usnic acid in response to a toxic environment, usnic acid is a useful biomarker for assessing pollution as well as toxicity in dietary supplements. Although adverse events suggest that usnic acid is the culprit for liver toxicity, we do not have a definitive mechanism for how this occurs. Subchronic and chronic toxicology studies in rodents and clinical studies in humans are absent. What is known regarding usnic acid toxicity is related to its apparent mechanism of action to uncouple oxidative phosphorylation in mitochondria (Johnson et al., 1950; Abo-Khatwa et al., 1996; Cardarelli et al., 1997). This mechanism could account for its ability to kill microorganisms (Lauterwein et al., 1995), accelerate metabolism, cause weight loss, and induce liver toxicity (Kra¨henbu¨hl, 2001; Sonko et al., 2011; Yellapu et al., 2011; Sahu et al., 2012; Liu et al., 2012; Moreira et al., 2013). In vitro analysis to screen for mitochondrial damage should be a consideration for research and development of products with similar claims. Biomarkers indicative of mitochondrial toxicity should be taken under consideration as a required step in the premarket analysis for justification of safety of any weight loss or energy products considering the possible seriousness of adverse events that might result due to “enhanced” metabolism.

BIOMARKERS OF TOXICITY: NEW DIETARY INGREDIENTS Active Moiety of a New Dietary Ingredient Red Yeast RicedMonacolin K and Statin Activity Another issue of concern is the physiological effect and regulatory status of the relevant article or active moiety2 in any marketed dietary supplement or NDI. Some NDI notifications received by the FDA concern ingredients that do not meet the statutory definition of a dietary ingredient and are therefore excluded as dietary ingredients under the U.S.C. 321(ff)(3)(B)3, section 201(ff)(3)(B) of the Federal FD&C Act (The Act) (21 U.S.C. x 321(ff)(3)(B)). Such dietary supplements may not include articles approved as a new drug or authorized for investigation as a new drug under section 505 of the Act, unless the article was marketed as a dietary supplement or food before its approval as a drug. The case law clarifying DSHEA regarding this issue involved the dietary supplement product called

The term “dietary supplement” is defined in 21 U.S.C. 321(ff). Under 21 U.S.C. x 321(ff)(3)(B), dietary supplements may not include articles approved as a new drug under 21 U.S.C. x 355 (section 505 of the Act), unless the article was marketed as a dietary supplement or food before its approval as a drug. 3

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Cholestin (red yeast rice) marketed by Pharmanex (35F. Supp. 1341, 2001). Cholestin was a “traditional” Asian product known alternatively as “red yeast rice,” “Hong Qu,” or “Red Koji.” Red yeast rice has been used both in traditional Asian cuisine and medicine. Products that are marketed as dietary supplements that contain red yeast rice, like all dietary supplements, must meet the requirements set forth in the Federal FD&C Act, as amended by the DSHEA of 1994. Red yeast rice (Monascus purpureus) contain monacolins that are fungal, secondary metabolites capable of inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase, which is involved in the synthetic pathway for cholesterol production. They are more commonly known as Statins. By definition, the presence of biomarkers for monacolins in red yeast rice would not permit its use as a dietary ingredient in a dietary supplement. The term “dietary supplement” as defined in 21 U.S.C. 321(ff) contains a number of exclusions. For example, under section 321(ff)(3)(B)(i), an article that is approved as a new drug under 21 U.S.C. 355 is excluded from being a dietary supplement unless it was marketed as a dietary supplement or as a food before such approval. It is this provision of the FD&C Act that bears directly on the legal status of Pharmanex’s product that was the subject of an FDA enforcement action on Cholestin and certain other products that contain red yeast rice, specifically sold as dietary supplements. In 1998, FDA issued an administrative proceeding stating that Pharmanex’s product, Cholestin,4 which was promoted as a dietary supplement intended to reduce cholesterol levels, was not a dietary supplement, but instead is considered an unapproved drug under the FD&C Act (Pharmanex, Inc, 1998). FDA based its decision, in part, on the fact that the red yeast rice in Cholestin was not simply red yeast rice that traditionally had been used as food. Instead, the agency concluded that Pharmanex had taken several actions in the marketing and manufacturing of Cholestin such that the relevant ingredient in the product, Monacolin K, otherwise known as lovastatin, an approved drug in the product Mevacor, was the basis for their product claims. This prohibited Cholestin from being marketed as a dietary supplement under the exclusion clause in section 321(ff)(3)(B)(i). Traditional red yeast rice does not contain lovastatin in measurable concentrations. The fact that Pharmanex took steps to artificially induce and enhance lovastatin production in its red yeast rice product and promoted its product to reduce blood

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cholesterol levels caused the product to be a drug under the FD&C Act. The FDA’s final determination was that lovastatin was not marketed as a dietary supplement prior to its approval as a new drug and Pharmanex failed to show that lovastatin was previously marketed as a dietary supplement, food, or component of a food; therefore, Cholestin did not qualify as a dietary supplement. All red yeast rice products manufactured with the purpose of increasing levels of monacolins are considered a threat to public health. Their chronic use comes with the potential for adverse events such as myopathies as well as the potential to cause damage to the kidneys with chronic usage. There is also added risk for individuals who are taking other statin medications or concurrent dietary supplements such as niacin that can potentiate the risk and incidence of adverse reactions. On February 16, 1999, the United States District Court for the District of Utah set aside the FDA’s May 20, 1998 administrative determination that Cholestin is a drug. The United States Court of Appeals for the 10th Circuit reversed the District Court’s decision on July 21, 2000 and remanded the case back to the District Court for consideration of record-based issues not reached by the lower court in its original decision. On March 30, 2001, the United States District Court for the District of Utah issued a Memorandum Decision and Order on the remaining record-based issues. The District Court affirmed the FDA’s administrative decision that Cholestin is a drug, not a dietary supplement. Taken together, the courts’ decisions in the Pharmanex litigation means that red yeast rice products containing lovastatin are unapproved new drugs in violation of the FD&C Act and may not be marketed as dietary supplements (Pharmanex, Inc. v. Shalala, 2001). The decision in the Pharmanex case does not, however, prohibit the marketing of all red yeast rice-containing products as dietary supplements. The decision only limits the marketing of products as dietary supplements if they contain substances that are excluded from the dietary supplement definition. The agency has not objected to the marketing of dietary supplements containing red yeast rice if they do not contain lovastatin or other substances that are approved drugs. The Congressional intent of the exclusion clause that is the basis of the agency’s decision on Cholestin was to protect the research development incentives for new drugs to treat serious diseases. There is no “action level” for lovastatin, or any other substance excluded under section 201(ff)(3)(B), which

4

Cholestin consists of the yeast Monascus purpureus when fermented on premium rice powder. The fermentation of the rice with this yeast, under certain conditions, produces a product that contains lovastatin, the active ingredient in the prescription cholesterol-lowering drug Mevacor.

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would trigger action by FDA. Rather, as was the case with Cholestin, FDA would look at the following: • level of a substance in a proposed dietary ingredient • consider the manufacturing, processing, and composition and determine how it compares to traditionally prepared dietary ingredients • provision for evidence of intended use or claims for the product to determine whether a given product violates the Act

BIOMARKERS OF TOXICITY: NEW DIETARY INGREDIENTS Safety Assessment of Multiple Dietary Ingredients in a Dietary Supplement As with other dietary supplements to be marketed, those containing multiple dietary ingredients, must substantiate through a history of use or other evidence of safety, that under the conditions recommended or suggested in the labeling of the dietary supplement, the combination of dietary ingredients will have a reasonable expectation of safety. As with other new ingredients, at least 75 days before being introduced or delivered for introduction into interstate commerce, the manufacturer or distributor of the dietary ingredient or dietary supplement must provide the Secretary (and by delegation, FDA) with information, including any citation to published articles, which forms the basis on which the manufacturer or distributor has concluded that a dietary supplement containing such dietary ingredients will not pose an unreasonable risk for illness or injury to the general public. This is especially true when the ingredients achieve the same effect through either different or similar mechanisms of action.

New Dietary Ingredients (Supplementation with Multiple, Combinations of Dietary Ingredients) Rauwolfia and Yohimbe Bark Extract Pharmaceutical and dietary ingredients are capable of chemical and physiological interactions that have the potential to increase the incidence of adverse events in individuals. Interactions can lead to antagonism or potentiation of drug activity and/or exacerbate underlying medical conditions that may be present with or without other diagnosed systemic disease. Because of an assumption of safe use of dietary supplements in the general, healthy population, a lack of knowledge concerning the interactions of many dietary 5

ingredients and the belief by many consumers that because many of the products are “natural” or of plant origin, that they can be assumed to be safe, selfmedication of multiple products is one of the most common precipitating circumstances surrounding adverse events and product complaints. Drug interactions can lead to changes in the activity of one or more dietary ingredients with concurrent use or a similar situation can occur when multiple dietary ingredients are placed in the same dietary supplement. This is significant if the dietary ingredients within the dietary supplements have similar pharmacological effects. For example, both Rauwolfia, containing the active ingredient, reserpine, and yohimbe bark extract, containing the a-2 adrenergic antagonist, yohimbine, can lower blood pressure. When combined in a dietary supplement, there exists the potential for an additive or synergistic response. This may go beyond permitted structureefunction claims on dietary supplement products for maintaining blood pressure in the normal range. Combination products have the potential to precipitate serious adverse events such as hypotension, dizziness, reflex tachycardia, and heart palpitations, especially in individuals who may be taking other medications with a similar mechanism of action or who have other systemic, underlying health conditions. Reserpine, an alkaloid isolated from plants in the genera Rauwolfia, was one of the first drugs developed for treatment of high blood pressure. Reserpine is an FDA-approved prescription medication for the treatment of hypertension. The prescribed, adult, label dosage for reserpine, for use as an antihypertensive agent, is 0.1e0.25 mg per individual per day.5 According to a recent study, reserpine can be found in Rauwolfia serpentina at a concentration of 0.1442% (Kumar et al., 2010). A dietary supplement product, labeled to contain 50 mg of R. serpentina in each serving (1 capsule) with directions to take two capsules per day, could potentially contain upward of 0.0721 mg reserpine (0.1442% of 50 mg) per capsule and lead to a total daily exposure level of 0.144 mg reserpine. This is in the range for an amount that would be indicated in a prescription medication for an individual with hypertension. As an FDA-approved prescription medication, reserpine contains potential risks and toxicological effects that restricts its use to uncomplicated hypertensive patients. Reserpine is classified as a pregnancy category C. When reserpine is administered parenterally it has been shown to be teratogenic in rats and to have an embryocidal effect in guinea pigs. It is unclear how reserpine affects the dietary supplement consumer who is normotensive. Furthermore, the US National

Reserpine [package insert]. Princeton (NJ): Sandoz, 2011.

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Toxicology Program has considered reserpine to be a “probable cancer-causing substance” and “reasonably anticipated to be a human carcinogen” based on sufficient evidence of carcinogenicity from studies in experimental animals (NTP, 2011). These risks are unreasonable for a dietary supplement that must have a reasonable expectation of safe use, in a healthy adult, in the general population. The package insert states that concomitant use of reserpine with other antihypertensive agents necessitates careful titration of dosage with each agent. This is because hypotension, hypothermia, central respiratory depression, and bradycardia may develop in cases of overdose of reserpine or synergistic action of reserpine with other antihypertensives. Thus, the combination of reserpine with any other antihypertensive botanical such as yohimbe bark extract can precipitate an adverse event. Their combination would necessitate a separate NDI notification to address this impact in the normotensive consumer. In contrast, the NDI Rauwolfia vomitoria (NDI 013) contains the botanical with the reserpine component removed. To receive an acknowledgment from FDA at this time, all Rauwolfia spp.ederived botanicals, for use in dietary supplements, must demonstrate that reserpine has been removed from the end-product prior to distribution to consumers. This example illustrates the risks that can result when dietary ingredients with similar pharmacological actions are combined in the same product without submitting a premarket NDI; without appropriate warnings to the consumer of toxicological effects that can potentially develop and without premarket analysis for biomarkers of physiologically active ingredients that should not be present in the final product for distribution.

New Dietary Ingredients (Supplementation with Multiple, Combinations of Dietary Ingredients)dGalantamine and Huperzine A (Cholinesterase Inhibitors) In a race to market, galantamine and huperzine A, both reversible cholinesterase inhibitors, were acknowledged dietary ingredients through NDI notification before they were investigational new drugs. Therefore, they are permitted to be used in dietary supplements under the conditions of use and consumption level as described in the “acknowledged” NDI notifications. FDA has concerns with increases in serving levels for galantamine and huperzine A whether they are used together or alone. Going forward, a manufacturer or distributor would be obliged to submit an NDI with safety evidence if they were to market the same ingredient (manufactured using the same process); if they wanted to market new products with higher serving

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levels of ingredients; if they wanted to produce dietary supplement combinations with other dietary ingredients (i.e., huperzine A combined with galantamine); or if they developed new technologies to manufacture the dietary ingredient. In other words, any change in formula and/or chemical composition would elicit a need to submit an NDI prior to distribution of a new product. The chemical structures for galantamine and huperzine A are provided in Fig. 39.1. Galantamine and huperzine A are reversible inhibitors of the enzyme acetylcholinesterase (AChE) (Taylor et al., 1996). Because their acute toxicity is due to the inhibition of AChE, measurement of AChE activity in red blood cells (whole blood analysis) is a critical biomarker for monitoring the toxicity of these dietary ingredients to adequately ensure a reasonable expectation of safety under the ordinary conditions of use for the product. AChE serves to limit the duration of the neurotransmitter acetylcholine (ACh) at muscarinic and nicotinic cholinergic receptors and therefore prevents its accumulation at synaptic junctions (Hilmas et al., 2009; Williams and Hilmas, 2010). Exposure to AChE inhibitors, both reversible and irreversible, results in symptoms indicative of widespread overstimulation. Fig. 39.2 illustrates the target organs, symptoms, and clinical signs that are associated with overstimulation. These symptoms include bronchoconstriction, increases in tracheobronchial secretion, lacrimation, increased urination, increased gastrointestinal motility, diarrhea, emesis, muscle weakness, diaphoresis, decreased heart rate, and central nervous system signs such as dizziness, blurred vision, mental confusion, respiratory depression, tremors, seizures, paralysis, and coma. Galantamine and huperzine A have a very rapid onrate and slow off-rate or release. Their kinetic profiles indicate predicted tight interactions with AChE in docking models. Best-fit docking models for both compounds involve different functional groups. Fig. 39.3 illustrates the three-dimensional docking of galantamine to both human and Torpedo californica forms of AChE. Fig. 39.4 illustrates three-dimensional docking of huperzine A to human AChE. Thus, they have been used in as prophylactic and postexposure therapeutic agents in preclinical studies to protect rodents against muscular paralysis and seizures induced by nerve agents. Safety evidence in an NDI notification would have to demonstrate that AChE inhibition was not cumulative as a result of chronic (daily) consumption. Red blood cell, muscle, and brain cholinesterase activity have all been used, primarily in in vivo and in vitro studies, to measure and evaluate peripheral and central neurotoxicity as a result of galantamine and huperzine A administration. These activities should correlate to the whole

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FIGURE 39.1 Chemical structures of galantamine, huperzine A, honokiol, lobeline, magnolol, and yohimbine. The authors would like to acknowledge Dr. Mariton Dos Santos for providing this illustration.

blood or plasma levels of galantamine or huperzine A in the animal model (Steiner et al., 2012). There are other botanicals that contain reversible cholinesterase inhibitors. An example is honokiol and magnolol, present in Magnolia spp. (Fig. 39.1). The combination of an extract of Magnolia with galantamine or huperzine A may pose significant synergistic activity. Another is Lobeline (Fig. 39.1), a nicotinic agonist that may also act synergistically to produce a cholinergic crisis if combined with galantamine, huperzine A, or any other ingredient with AChE-inhibiting activity. Appropriate safety studies should measure and evaluate AChE activity as a biomarker of toxicity when these ingredients or others are suspected of combined

nicotinic and muscarinic AChE activity prior to their use in a dietary supplement product.

CONCLUDING REMARKS AND FUTURE DIRECTIONS Dietary supplements are becoming more ubiquitous in the market and are becoming accepted in the general public as important components of many foods and beverages. Some may call them functional foods, others may call them nutraceuticals, dietary supplements, botanicals, or natural products to support health. With the

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FIGURE 39.2 Overstimulation of muscarinic and nicotinic cholinergic systems due to inhibition of acetylcholinesterase (AChE) inhibition. Some dietary ingredients inhibit AChE, resulting in overstimulation by the neurotransmitter ACh at both muscarinic and nicotinic receptors located in various organ systems throughout the body. This figure illustrates the various physiological markers of toxicity which result from cholinergic activation in the presence of AChE inhibition. The authors would like to acknowledge Alexandre Katos for providing this illustration.

FIGURE 39.3 Three-dimensional docking of galantamine into AChE from human and Torpedo californica. The most appropriate portions of human AChE are displayed. The ball display on the left-hand side is the active site tryptophan, and the balls of AChE on the righthand side represent the active site serine. Galantamine, represented in ball-and-stick configuration, is shown by atom type and has a transparent solvent accessible surface to demonstrate that it is filling a pocket. This docking was performed using Insight II software by aligning the backbones of the two AChEs.

increasing costs of health care, many people are looking for ways to lead healthier lifestyles, and whether good or bad, they are listening to broad avenues of media and marketing about the pros and cons of these products. Education of the public is important but at the same time the majority of individuals do not have the background in science and nutrition to adequately understand the complexity of what they are ingesting. Thus, it is vitally important for the medical community, industry, government regulators, academia, and media outlets to work together to make sure that only highquality products are manufactured, marketed, and distributed properly in the general marketplace. This chapter uses examples of dietary supplements and their ingredients that have been or are currently still available to consumers in a review for the use of biomarkers as they apply to various stages of the life cycle of these products. An additional goal of this chapter is to remedy the misconception that dietary supplements are not regulated. They are. It should be stated that they are regulated not as a pharmaceutical where a lack of efficacy is considered an adverse event for its intended use but

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FIGURE 39.4 Three-dimensional docking of huperzine A, a known anticholinesterase alkaloid, into human AChE. The threedimensional rendering shows a best-fit model for docking of huperzine A functional groups with the two tyrosine functional groups (Tyr 337 and Tyr 341) on human AChE. Although these tyrosines are normally not involved in GAL docking, they may play a role in huperzine A docking. For reference, the active site tryptophan of AChE is oriented in the background; the active site serine is on the right-hand side. Huperzine A is colored by atom using a space-filling ball model configuration to show it is filling a pocket, rather than the ball-andstick configuration with transparent solvent accessible surface as shown in the previous figure. The two tyrosines are in a ball-and-stick model configuration. This docking was performed using Insight II software.

are more closely aligned to the guidance’s found for historical and NDIs that may be found in food products for human consumption. Information has also been provided for how biomarkers inform worldwide regulatory decisions; the development of risk assessments for the protection of public health; and for industry to be able to manufacture and distribute dietary supplements with an appropriate weight of evidence to support an expectation of tolerance and safety for the general population of consumers for their proper, intended use.

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