5
Testing Ingredients with Preclinical Studies

ABSTRACT

Preclinical studies are a vital first step to assess the safety and quality of ingredients new to infant formulas. They must be performed before an ingredient can be considered for clinical studies in humans in order to determine the potential toxicity of the ingredient, its metabolites, and its matrix. Guidelines for these studies to assess the safety of infant formulas must be based on considerations of the diversity of potential new ingredients and the ingredients’ source and matrix. In the United States, the Food and Drug Administration’s (FDA) Redbook provides comprehensive guidelines for conducting preclinical studies to test the safety of food and color additives, but it often does not take the many special needs and vulnerabilities of infants into consideration. In Canada, there are no comprehensive guidelines for conducting preclinical studies, and decisions are made on a case-by-case basis using internationally accepted guidelines.

The committee recommends implementing a flexible, two-level assessment approach to help guide the expert panel’s decisions on the appropriate preclinical studies (including preanimal tests; absorption, distribution, metabolism, and excretion studies; toxicity studies; and neurological studies) to assess the safety of ingredients new to infant formulas. Level 1 assessments include standard measures for each organ system required for all new ingredients (e.g., commonly used screening tests of cell and organ composition and function). Level 2 assessments include in-depth measures of organ systems that would be used to explicate equivocal level 1 findings or specific theoretical concerns not typically addressed by level 1 tests.

In addition to following established guidelines, a distinct set of procedures using appropriate cellular and animal models at relevant developmental stages should be included in studies to assess safety. The most commonly used animal models for general toxicological studies are the rat and mouse, but they are of limited use for developmental studies involving ingredients new to infant formulas because of the



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5 Testing Ingredients with Preclinical Studies ABSTRACT Preclinical studies are a vital first step to assess the safety and quality of ingredi- ents new to infant formulas. They must be performed before an ingredient can be considered for clinical studies in humans in order to determine the potential toxicity of the ingredient, its metabolites, and its matrix. Guidelines for these studies to as- sess the safety of infant formulas must be based on considerations of the diversity of potential new ingredients and the ingredients’ source and matrix. In the United States, the Food and Drug Administration’s (FDA) Redbook provides comprehen- sive guidelines for conducting preclinical studies to test the safety of food and color additives, but it often does not take the many special needs and vulnerabilities of infants into consideration. In Canada, there are no comprehensive guidelines for conducting preclinical studies, and decisions are made on a case-by-case basis using internationally accepted guidelines. The committee recommends implementing a flexible, two-level assessment ap- proach to help guide the expert panel’s decisions on the appropriate preclinical stud- ies (including preanimal tests; absorption, distribution, metabolism, and excretion studies; toxicity studies; and neurological studies) to assess the safety of ingredients new to infant formulas. Level 1 assessments include standard measures for each organ system required for all new ingredients (e.g., commonly used screening tests of cell and organ composition and function). Level 2 assessments include in-depth mea- sures of organ systems that would be used to explicate equivocal level 1 findings or specific theoretical concerns not typically addressed by level 1 tests. In addition to following established guidelines, a distinct set of procedures using appropriate cellular and animal models at relevant developmental stages should be included in studies to assess safety. The most commonly used animal models for general toxicological studies are the rat and mouse, but they are of limited use for developmental studies involving ingredients new to infant formulas because of the 70

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71 TESTING INGREDIENTS WITH PRECLINICAL STUDIES difficulty of feeding formulas to a preweanling rodent. The nonhuman primate and the piglet are more amenable for these types of studies. INTRODUCTION This chapter describes the importance and the unique aspects of conducting preclinical studies to assess the safety of infant formulas. Current regulatory guidelines for preclinical studies are described, and a two-level assessment process is proposed. The committee’s recommended two-level process is a flexible approach that can accommodate a variety of potential ingredients. For example, safety assessment might not be as intense for an ingredi- ent that is not absorbed systemically. The manufacturer, in consultation with an expert panel, determines the types of tests conducted. The extent of this investigation will be determined on the basis of previous experience with the ingredient in other populations and on theoretical concerns based on the putative biological effects of the ingredient, its metabo- lite, and its matrix. Finally, the committee recommends the following preclinical tests de- scribed in detail below: 1. preanimal tests (including structure, stability, and solubility tests of the ingredients; genetic tests; and cellular studies) 2. absorption, distribution, metabolism, and excretion studies 3. toxicity studies (including acute, subchronic, chronic, developmental, and organ studies) 4. neurological studies Neurological studies are described in a section separate from other toxicity studies because the scope of work defined for this project placed special emphasis on the potential effect of ingredients on the rapidly developing infant brain. THE IMPORTANCE OF APPROPRIATE PRECLINICAL STUDIES Preclinical studies must be performed before an ingredient can be considered for clinical studies in humans in order to determine the potential toxicity of the ingredient and its metabolites and their effects in the matrix. Preclinical studies allow researchers to expose cell cultures and experimental animals to doses of ingredients not normally encountered in human consumption. Results of such assessments are used to determine the threshold of toxicity for the given ingredient (i.e., the margin of safety). Guidelines for preclinical studies to assess the safety of infant formulas must be based on considerations of: • The diversity of the potential new ingredients. The new ingredients will possess different chemical characteristics, nutritional contributions, pharmacological activities, and physiological activities. The ingredients may be a conventional synthetic or an extracted single component, a plant extract, or a complex mixture. The ingredients may also be derived from novel sources or processes (e.g., products of fermentation or biotechnology). Such diversity requires preclinical guidelines that are clear but not overly prescriptive be- cause of the disparity in the issues that each class of ingredient may represent. Nevertheless, given the vulnerability of the population to receive the ingredient (infants), it is incumbent upon the manufacturer, in consultation with the expert panel, to be overly conscientious in considering potential safety issues.

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72 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS • The ingredient’s source and matrix. The approach to evaluate ingredients new to infant formulas should be driven by the class of the functional substances and by the full characterization of the ingredient in the matrix of the infant formulas. In other words, the general approach is to deal with the targeted ingredient, as well as the nontargeted com- pounds, such as metabolites of the targeted ingredient, the vehicle required for delivery of the targeted ingredients, and impurities introduced in the manufacturing process. CURRENT REGULATORY GUIDELINES FOR PRECLINICAL STUDIES In considering how to construct guidelines for preclinical studies of ingredients new to infant formulas, the committee drew on existing guidelines for the testing of food ingredients and infant formulas. In the United States these are included in Sections 409 and 412 of the Federal Food Drug and Cosmetic (FD&C) Act (21 U.S.C. §301) and in the FDA Redbook (OFAS, 2001, 2003). The committee suggests that the risks of adding ingredients to sole nutrient sources, such as infant formulas, are not adequately ad- dressed; however some aspects of the more stringent guidelines applied to new ingredi- ents as articulated in the Redbook are appropriate for some, if not all, ingredients new to infant formulas. Sections 409 and 412 of the Federal Food, Drug and Cosmetic Act There are no explicit requirements for preclinical testing of infant formulas specified under Section 409 of the FD&C Act. The section stipulates that a petition to establish safety of a food additive shall contain “all relevant data bearing on the physical or other technical effect such additive is intended to produce. . .” (21 U.S.C. §301), but it does not dictate a specific type of preclinical study. Under Section 412, which applies to infant formulas, a formula shall be deemed to be adulterated if it does not meet the quality factor requirements prescribed by the Secretary of Health under Subsection (b)(1). Subsection (b)(1) then states, “The secretary shall by regulation establish requirements for quality factors for infant formulas to the extent possible consistent with current scientific knowledge, including quality factor require- ments for the nutrients required by subsection (i).” Currently, only protein quality is named as a quality factor in FDA regulations but there are no specific requirements to be met regarding quality factors. Subsection (i) specifies levels of certain nutrients (e.g., pro- tein, fat, and specific vitamins) that are required to be met. There is no other requirement for any specific preclinical studies. FDA Redbook The FDA Redbook II and Redbook 2000 (OFAS, 2001, 2003) provide comprehensive guidelines for conducting preclinical studies to test the safety of food and color additives. Chapters IV and V in the Redbook II and 2000 describe: • general guidelines for designing, conducting, and reporting results of toxicity studies, • special considerations in toxicity studies (e.g., pathology, statistics), and • guidelines for specific toxicity studies (e.g., genetic, acute, subchronic, chronic, im- munotoxicity, and neurotoxicity). These guidelines, however, do not consider the unique characteristics of having infants as consumers.

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73 TESTING INGREDIENTS WITH PRECLINICAL STUDIES Canada’s Food and Drug Regulations There are no explicit requirements for preclinical testing of infant formulas specified under Canada’s Food and Drug Regulations in Division 16 (Food Additives), Division 25 (Infant Formulas), or Division 28 (Novel Foods) (Canada, 2001). Divisions 16 and 28 require that a notification be submitted to Health Canada that includes information used to establish the safety of a food additive or a novel food. Health Canada refers manufacturers to internationally accepted guidelines for preclinical testing or asks to be consulted because decisions are made on a case-by-case basis. OVERVIEW OF RECOMMENDED LEVELS OF ASSESSMENT RECOMMENDATION: A hierarchy of two levels of preclinical assessment, using tech- niques from cellular-molecular studies through whole-animal studies, should be imple- mented to assess the safety of ingredients new to infant formulas on developing organ systems: • Level 1 assessments. These are suggested standard measures for each organ sys- tem (e.g., gastrointestinal, blood, kidney, immune, endocrine, and brain) and are re- quired for any new ingredient. • Level 2 assessments. In-depth measures of organ systems or functions that would be performed to explain abnormalities found in level 1 assessments and specific theoreti- cal concerns not typically addressed by level 1 tests. These are suggested measures to assess any new ingredient that primarily interacts with an organ system, has a metabo- lite that interacts with an organ system, or stimulates or changes the synthesis of factors (e.g., hormones, cytokines, immunoglobulins, endotoxin) that interact with an organ system. Figure 5-1 describes the overall flow of proposed preclinical studies, from preanimal tests (Boxes 8 and 4) to toxicity tests (Box 4) to neurological tests (Box 7). Figure 5-2 describes the two assessment levels that should be considered when conducting preclinical studies, and it refers to tables in the text for specific measurements that could be conducted. CONDUCTING PREANIMAL TESTS Structure, Stability, and Solubility Chemical and physical characterization is warranted if the targeted and nontargeted ingredients to be added to infant formulas are not well known and if there is no adequate literature about them to determine their safety. Thus the complete chemical structure and functional groups and the purity and stability of the intended and nontargeted ingredients present in the matrix must be determined using well-established physical methods (see Figure 5-1, Box 8). These methods include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and thin layer chromatography (TLC). LC-MS is a particularly versatile technique. It can be used to assess the structure, molecular mass, and purity of most classes of compounds because derivatization is not necessary. The stability to temperature and ultraviolet light and the solubility properties of the ingredients should be established by standard methodology. The percentage of the unidentifiable mate- rials should be stated in the notification or petition to the regulatory agency. The kind of

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74 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS PROPOSED PRECLINICAL ASSESSM ENT 1 New ingredient proposed for infant formula 2 Are the active component or the impurities well known ? AND Yes Is adequate literature available to determine their safety? Sidebar A: Chemical and Physical Characterization 3 Structure, Stability, and Solubility - High performance liquid chromatography, Initiate preclinical studies to evaluate toxicity No liquid chromatography-mass spectrometry, and and neurological safety (See Figure 5-2) thin layer chromatography. - The stability to temperature, ultraviolet light as well as the solubility properties of the ingredient. 8 - The percentage of the unidentifiable materials in ingredient. Chemical and Physical Characterization - The kind of so lvents, suspending agents, (See Sidebar A) emulsifiers, or other materials that will be used in administering the ingredient whether in in vitro or animal studies. - Ingredient should bestored under condit ions 9 4 that maintain its stability, quality, and purity until the subsequent studies are complete. Are chemical and Toxicity Assessment Yes - These studies should be repeated with the physical purity assured? (See Sidebar B) ingredient in the solution or matrix that would be fed to the human infants. Sidebar B: Toxicity Assessment 1. Genetic tests 2. Cellular studies 3. Animal toxicity studies - Acute,subchr onic, and chro nic toxicity - Developmental toxicity - Absorption, distribution, metabolism,,and excretion 4. Organ level studies - Gastrointestinal tract No - Hepatic - Renal - Hematology - Immune - Endocr ine - Neurolo gic 5 Any positive test for toxicity or concern for Yes safety? No 6 10 7 DISCONTINUE DISCONTINUE Neurological Safety Assessment PROCESS (See Figure 5-2) PROCESS FIGURE 5-1 Proposed preclinical assessment algorithm. = a state or condition, = a decision point, = an action, sidebar = an elaboration of recommendation or statement. solvents, suspending agents, emulsifiers, or other material that will be used in administering the new ingredients during testing, whether using in vitro or animal studies, should be disclosed. The targeted ingredient should then be stored under conditions that maintain its stability, quality, and purity. The stability and solubility studies should be performed with the ingredient both in the solution and in the matrix that would be fed to human infants.

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75 TESTING INGREDIENTS WITH PRECLINICAL STUDIES P PROPOSED LEVELS OF PRECLINICAL ASSESSMENT 1 Initiate preclinical studies to evaluate toxicity and neurological safety 2 Any evidence of abnormalities based on previous human data, other preclinical studies, or on Yes theoretical plausible perturbation of a metabolic pathway? No Sidebar A: Level 1 Assessment 7 Standard measures of genetic tests, cellular studies, animal toxicity studies, Lev el 1 Assessment (See Sidebar A) major organ systems, and neurological preclinical screening measures. (See Tables 5-1 through 5-10) Sidebar B: Level 2 Assessment Detailed measures of genetic tests, 8 3 Any evidence of cellular studies, animal toxicity studies, Lev el 2 Assessment adverse Yes major organ systems, and neurological (See Sidebar B) effect/event? preclinical measures. (See Tables 5-1 through 5-10) No 4 Any evidence of adverse effect/event or concern Yes for safety? No 6 9 5 Continue to clinical Re-evaluate results before DISCONTINUE studies considering clinical trials PROCESS FIGURE 5-2 Proposed levels of preclinical assessment algorithm. = a state or condition, = a decision point, = an action, sidebar = an elaboration of recommendation or statement.

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76 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS Genetic Tests Numerous genetic studies, such as those provided in the Redbook, are available, and the committee recommends evaluating all ingredients, their metabolites, or their secondary effectors for their ability, if any, to cause molecular changes in the deoxyribonucleic acid (DNA) or to cause structural changes in the chromosomes of cells (see Figure 5-1, Box 4). These changes may include forward and reverse mutations, point mutations, deletion muta- tions, chromosomal aberrations, micronuclei deletions, polymorphisms, DNA strand breaks, or unscheduled DNA synthesis. In vitro assessments use microorganisms and cells from multicellular animals; examples are listed in Table 5-1. Cellular Studies It is often most efficient to perform in vitro studies of metabolism before whole-animal (oral dosing) studies to provide information about future in vivo studies and estimate dosages to be used in preclinical animal studies. In vitro work and pharmacokinetic model- ing can be used to predict the potential toxicity and in vivo kinetics of the ingredients and the matrix. The in vitro studies can also provide initial evidence of the level of clinical safety or risk. Because in vitro studies are generally less complex than whole-animal studies, elucida- tion of an ingredient’s metabolic pathway and toxicity characteristics may be facilitated. Different doses of the solubilized ingredient can be incubated with either confluent or preconfluent cells cultured in 10-cm dishes or 96-wells plates to establish uptake and toxic- ity levels of both the ingredient and its metabolic byproducts. Time-course and dose-response experiments should be conducted to check the growth characteristics of the cells and the toxicity of the ingredient. The production of metabolites can also be followed upon adding TABLE 5-1 Examples of Potential in vitro Tests to Assess Genetic Toxicity Test Name Function of Test Reference Ames test Microsome reverse mutation Aeschbacher et al., 1983 Mouse lymphoma Genetic forward mutations McGregor et al., 1987, 1988a, thymidine kinase gene 1988b; Myhr and Caspary, mutation assay 1988; Myhr et al., 1990 Mammalian erythrocyte Micronuclei deletions, chromosomal Schlegel and MacGregor, 1982; micronucleus test aberrations Schlegel et al., 1986 Polymerase chain reaction Changes in gene expression and Innis, 1990 deoxyribonucleic (DNA) sequence, polymorphisms, point mutations DNA microarray a Identifies genes that are up or down Cohen et al., 2002; Daniel, 2002; regulated DeRisi and Iyer, 1999; Guengerich, 2001; Lee et al., 2000; Moreno-Aliaga et al., 2001; Perou et al., 1999; Wang, 2000; Williams, 1999 Proteonomics a Identifies proteins that are altered Anderson and Anderson, 1998; after exposure to the ingredient Bogyo and Hurley, 2003; Govorun and Archakov, 2002; Hochstrasser, 1998; Jungblut et al., 1999; MacBeath, 2002 Bioinformaticsa ? Vihinen, 2001 aThese techniques are relatively new and are not standardized for clinical use.

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77 TESTING INGREDIENTS WITH PRECLINICAL STUDIES the ingredient directly to the cellular extracts. In this case, different concentrations of the solubilized material are incubated with the cellular extract, and the production of metabo- lites as a function time is determined by analytical methods (e.g., HPLC, LC-MS, TLC). Radioactive and stable isotope studies are useful in tracing nutrient uptake by cells and intracellular metabolic pathways. The in vitro systems can also be used to measure binding adduct and conjugate forma- tion, transport across membranes, enzyme activity and concentration, enzyme substrate specificity, and other specific objectives. In addition to providing information on the effects of the ingredient and its metabolites on target receptors, cell-culture studies can also indicate subsequent downstream effects. In the future, DNA microarray technologies may be comple- mented by the capabilities of proteomics and metabolomics to assess the expression of expected and unexpected proteins and metabolites after ingredient exposure to the cells. However, at this point, these techniques are relatively new and are not standardized for clinical use. The principal problems that these advances confront lie in assessing the func- tional significance of the results of the analyses. Numerous algorithms in dealing with such analyses are available (Lander, 1999; Lee et al., 2000; Soukas et al., 2000) and should be consulted. CONDUCTING ANIMAL TOXICITY STUDIES Several toxicity studies must be performed in animals to ensure the safety of ingredients new to infant formulas (see Figure 5-1, Box 4). This section reviews the following issues: • considerations when choosing animal models, • general considerations when conducting animal studies, and • considerations when conducting the following specific animal toxicity studies: – acute, subchronic, and chronic toxicity studies, – developmental toxicity studies, – absorption, distribution, metabolism, and excretion studies, and – organ-level studies. RECOMMENDATION: In addition to established guidelines and regulations (e.g., the Redbook and regulations under Sections 409 and 412 of the FD&C Act), a distinct set of procedures using appropriate animal models at relevant developmental stages should be included in studies to assess safety. Choosing Animal Models Animal models are used as a tool in initial toxicology studies before human clinical trials are conducted. The end points of animal studies include: • repeat dose toxicity (target organ evaluation), • carcinogenicity (mutagenicity screen), • developmental toxicity, • reproductive toxicity, and • neurotoxicity. Animal models should be chosen appropriately, and it is especially important to look at the comparative development between the animal model and humans and to choose the

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78 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS model that can be developmentally extrapolated to humans. For example, the animal model chosen must have digestive and metabolic similarities to the human infant and must be at the same point in development as the human infant who consumes formulas. Investigators should also ensure that the animal model and the reason for the studies have been justified such that there is an understanding of what can and cannot be extrapolated and how the preclinical data generated will be used to interpret the safety profile. All of the following questions must be answered before selecting animal models and moving ahead with a submission to the regulatory agency: • What are the appropriate species? • What are their ages? • Should juvenile animals be assessed, or is age irrelevant for the animal models being considered? • What safety factors are needed with these models, and what are going to be the relevant scientific disciplines? At least two animal models should be selected, keeping in mind that the more animal models used, the better (OFAS, 2001, 2003), because converging lines of biological proof of cause and effect can be established. There are several additional factors to consider when conducting animal studies: • The intended ingredient should be added to the animal’s diet during the time in development that corresponds to when the average human infant will consume it. • The bioavailability of the ingredient in the human infant must be known. • The study must be designed to prevent differences in pharmacokinetics, handling of the ingredient, and dietary imbalance from competing ingredients. The most commonly used animal models for general toxicological studies are the rat and mouse. However the rat and the mouse are of limited use for developmental studies involv- ing ingredients new to infant formulas because of the difficulty of feeding formulas to a preweanling rodent. The nonhuman primate and the piglet are more amenable for these types of studies because they readily accept infant formulas as a nutrient source. The advan- tages and disadvantages of using each of these animal models is discussed below and sum- marized in Table 5-12. Nonhuman Primate Nonhuman primates are the closest analog to humans. Their diet is similar to humans and they can be fed infant formulas and followed developmentally. Humans and nonhuman primates are also comparable at a neural systems level. The animals display a wide array of sophisticated cognitive and motor behaviors (for review, see Bachevalier, 2001; Golub and Gershwin, 1984; Overman and Bachevalier, 2001). The nonhuman primate’s neurodevelop- mental trajectory is well described, with about a 4:1 ratio in relative time of development (e.g., 4 months of human brain development is equal to 1 month of development in the nonhuman primate). Nonhuman primates can be more thoroughly assessed than humans through catheters in the blood stream, direct cerebrospinal fluid sampling, deep implanted electrodes, and re- peated neuroimaging. These approaches allow a greater correlation of form and function changes.

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79 TESTING INGREDIENTS WITH PRECLINICAL STUDIES Nonhuman primates have made substantial contributions to human nutrition studies, including assessment of the neurobiological effects of long-chain polyunsaturated fatty-acid (LC-PUFA) deficiency (Neuringer et al., 1986; Reisbick et al., 1994), iron deficiency (Kriete et al., 1995), and zinc deficiency (Golub et al., 1985). This model could be used to study changes in general behavior and speed of neural processing in response to the addition of ingredients with neurological effects, such as LC-PUFAs. Furthermore, neuroimaging with magnetic resonance imaging (MRI) may reveal differences in myelination or regional brain volumes. The main disadvantages of using nonhuman primates are that they are very expensive to maintain, and most researchers are reluctant to sacrifice the animals to obtain brain tissue for a single analysis. Piglet Piglets are comparable in size to neonatal humans and metabolize fatty acids like hu- mans do. Piglets can be raised on a relatively high-fat infant formulas and are an excellent choice for studies of hypoxia, endocrinology, and lipid metabolism. Piglets can be sacrificed and, thus, tissue can be obtained for analysis. Pig models have been used extensively to study the role of added LC-PUFAs on the developing brain (Arbuckle et al., 1994; Craig-Schmidt et al., 1996; Hrboticky et al., 1990). These studies generally show that infant formulas enriched with LC-PUFAs and fed to neonatal pigs result in increased incorporation of these fatty acids into neuronal membranes and myelin. However piglets are difficult to work with in behavioral/learning paradigms, and neurodevelopmental form-function relationships are difficult to assess. Thus the inabil- ity to test these animals functionally to determine if the fatty acid incorporation results in beneficial or toxic effects is a significant drawback. Rat The rat is the most versatile model for toxicological testing for the following reasons: • There is a wealth of information available on the rat model, including the nutrient effects on biochemistry, cell biology, neurophysiology, anatomy, and behavior. • The developmental trajectory of the rat brain has been extensively compared with the human brain. For example, a postnatal day-7 rat has the structural maturity of a 34-week premature human, a postnatal day-12 animal is similar to a term human (Rice and Barone, 2000), and a postnatal day-28 rat approximates a human toddler. • The rat’s short reproductive cycle (21 days gestation) allows for rapid cycle studies and assessment of generational effects. • Rats are excellent learners on basic cognitive (medial temporal lobe) tasks. Learning paradigms in rats have been correlated with human behavioral tests, allowing extrapolation of behavioral data between species (for review, see Overman and Bachevalier, 2001). However the rat also has several limitations. Rats normally consume low-fat diets (ap- proximately 5 percent fat), while human infants consume 45 to 55 percent of their calories as fat. Therefore the rat is a poor model for assessing the effects of altering or supplementing fats (including LC-PUFAs). Studies of LC-PUFA supplementation in the developing rat pup in- volved supplementing the mother and thus enriching the composition of rat milk (Haubner et

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80 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS al., 2002; Pasquier et al., 2001). The matrix and source of these LC-PUFAs delivered to the rat pup were therefore different than the matrix and source of LC-PUFAs added to formulas and fed directly to the pup. Although a feeding system for artificial rat-milk substitute (formula) has been devised, it is invasive (i.e., it involves gastrostomy placement), it is difficult to maintain long term, and it is likely nonphysiological in nature. Rats have been used extensively in brain efficacy and toxicity studies involving LC- PUFAs (Delion et al., 1994; Vazquez et al., 1994; Wainwright et al., 1997). For example, the GRAS Notification by Martek for the addition of LC-PUFAs to infant formulas lists mul- tiple toxicity protocols involving rats (Hahn, 2000). Since it is difficult to feed infant formu- las to a preweanling rat, the developmental tenets of timing, dose, and duration cannot be addressed. The literature cited in Martek’s notification provides no mention of neuro- toxicological effects in either the developing or the mature rat. An appropriate approach to study the impact of LC-PUFAs on development would be to assess the animals for evidence of adequate synaptogenesis, myelin biochemistry, and myelin quantity. More specific probes for the effects of these ingredients on regulation of myelina- tion (e.g., microtubule associated protein-2 or synaptophysin messenger ribonucleic acid) would represent targeted approaches to potential physiological processes likely to be af- fected by LC-PUFAs. In some instances, such as when rats may possess different tissue metabolism and cortical function compared with humans, the rat model may be inappropriately extrapolated to humans. For example, the newborn human has relatively more cortical activity than the rat, so nutrient effects on cortical structures may be underestimated in the rat model. In contrast, the rat has a large and metabolically active hippocampus at birth relative to the human. Thus nutrient perturbations, such as iron deficiency, protein-energy malnutrition, or hypoglycemia, which profoundly affect the rat’s hippocampus, may overestimate the effect in humans. Mouse The mouse is an excellent model because of the ability to manipulate its genetics and to link alterations in genotype to metabolic phenotypes. The mouse genome sequence is almost complete, so it provides a major resource to link gene expression to disease. For example, the ability to manipulate the genome to alter the uptake and processing of nutrients provides valuable information on mechanistic insight. A particularly powerful emerging technology is the conditional knock-out or knock-in model where nutrient dependent genes can be altered in specific regions of the brain at specific times of development (Lee et al., 1999). Mice have been widely used in LC-PUFA research with respect to regulation of genes of lipid metabolism. In these studies it has been demonstrated that LC-PUFAs are potent inhibitors of lipogenic gene expression (Clarke, 2001; Ntambi, 1999; Shimomura et al., 1998). The main disadvantages of the mouse model are: • The mouse, like the rat, consumes a low-fat diet and cannot be fed infant formulas, making developmental effects difficult to assess. • The mouse has different tissue metabolism compared with humans, particularly with respect to adipose tissue and liver lipid metabolism. • The mouse has a limited neurobehavioral repertoire compared with the rat, making it more difficult to detect subtle neurocognitive effects after nutrient manipulations.

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87 TESTING INGREDIENTS WITH PRECLINICAL STUDIES TABLE 5-8 Immunology Assessment: Examples of Tests in Level 1 and Level 2 Assessmenta Level Level 1 T-/B-cell quantitation and function (immunological analysis of B- and T-lymphocytes and T-lymphocytes subsets [Th+Ts or CD4 and CD8]) Thymus, spleen, bone marrow, lymph nodes, tissue histology Electrophoresis (e.g., for changes in levels of γ-globulin fractions [IgG, IgM, IgA, IgE]) Total serum complement and components of complement (e.g., C3 from CH50 determinations) Levels of prostoglandin E2, balance of LTB4 and LTB 5 Immunochemical assays of γ-interferons and serum autoantibodies (antinuclear, antimitochondrial, antiparietal antibodies of B-lymphocytes) In vitro assays of activity of natural killer cells Mitogenic stimulation assay of B- and T-lymphocytes Macrophage activity assays Stem cell assays In vitro assays to assess allergenicity (source of the protein, amino acid sequence homology analysis, physicochemical properties) Level 2 Microarray/proteonomics Special histology stains Stimulation tests of immune function using the T-dependent or independent antigens or human vaccines Cell-mediated immune reactivity and host-resistance assays NOTE: The petitioner (or manufacturer), in consultation with the expert panel, determines which tests are re- quired based on a thorough analysis of the potential effects of the new ingredient. aTh = T helper cells, Ts = T suppressor cells, CD4 = cell differentiation antigen 4, CD8 = cell differentiation antigen 8, IgG = immunoglobulin G, IgM = immunoglobulin M, IgA = immunoglobulin A, IgE = immunoglobulin E, LTB4 = leukotriene B4, LTB5 = leukotriene B5. ment of human allergic disease are not currently available. The committee believes that it is of critical importance that animal models for allergenicity assessment in humans be devel- oped. Until such models are validated, the assessment of allergic potential should be ap- proached in vitro on a molecular level (e.g., utilizing sequence homologies, comparative digestive stabilities, functional antigenic determinants, and B- and/or T-cell epitope charac- teristics), and the nature of the demonstrated immune response to the known allergic prod- ucts should be determined. Endocrine Function Growth abnormalities of the test animal is an important early indication of a possible effect of a new ingredient on the endocrine system. Because endocrine effects may not be immediately apparent in growth changes, nor in other metabolic functions, some screening tests are indicated. Table 5-9 provides several examples of the types of tests that could be used in level 1 and level 2 assessments of endocrine function. CONDUCTING NEUROLOGICAL TESTS Background As explained in detail in Chapter 6, there are important reasons to include neurological tests in safety assessments of new ingredients for infant formulas, including the sensitivity of

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88 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS TABLE 5-9 Endocrine Assessment: Examples of Tests in Level 1 and Level 2 Assessmenta Level Level 1 Hormone levels (e.g., T4, TSH, LH, FSH, GH, CCK, NPY, cortisol, leptin), blood sugar, insulin Organ weight/histology (e.g., adrenals, ovaries, testes, pancreas, thyroid) Level 2 Microarray/proteonomics Provocative endocrine tests (e.g., cosyntropin stimulation) Special histology stains NOTE: The petitioner (or manufacturer), in consultation with the expert panel, determines which tests are re- quired based on a thorough analysis of the potential effects of the new ingredient. aT4 = thyroxine, TSH = thyrotropin, LH = luteinizing hormone, FSH = follicle-stimulating hormone, GH = growth hormone, CCK = cholecystokinin, NPY=neuropeptide Y. growth and development to toxic substances and the long-term predictive value of behav- ioral measures. Therefore the scope of work defined for this project placed special emphasis on the potential effects of ingredients to be added to infant formulas on the rapidly develop- ing infant brain. This section discusses the preclinical assessment tools and models that can be utilized to assess ingredients with either positive or negative neurological effects. The approach is decidedly developmental because the effects of added ingredients (or any envi- ronmental stressor) in the developing animal are highly dependent on the timing, dose, and duration of ingredient exposure (Kretchmer et al., 1996). Traditionally, two metrics—composition and performance—are applied as infant for- mula manufacturers continue to refine their products to match the gold standard of breast- feeding (Benson and Masor, 1994; MacLean and Benson, 1989). With respect to the latter, the impact of infant formulas on neurodevelopment has come to the forefront. It is widely accepted that breastfed infants demonstrate more advanced neurodevelopment (Lucas et al., 1998; Morrow-Tlucak et al., 1988; Mortensen et al., 2002; Wang and Wu, 1996). One potential explanation is that ingredients found in human milk, but not in infant formulas (e.g., LC-PUFAs, nucleotides, growth factors, and oligosaccharides), may be responsible (DeLucchi et al., 1987; Innis, 1992; MacLean and Benson, 1989). As these compounds are identified and added to infant formulas, valid and stringent assessments of their impacts on the developing brain should be undertaken. Nutrients and growth factors regulate brain development during prenatal and postnatal life. Late fetal and early neonatal life is a period of rapid brain growth and development in most mammals, including humans (Rice and Barone, 2000; for review, see Dobbing, 1990). There is a wealth of information on the critical events that take place in the brain’s develop- ment in the early neonatal period. At this stage of development, cell migration is complete, but myelination, synaptogenesis, dendritic arborization and pruning, and apoptosis are highly active. The rapidly growing brain is more vulnerable to restriction or loss of nutrients when compared with the more mature, less actively growing brain in later childhood and adulthood. Arguably, the rapidly developing brain is also more amenable to repair following nutritional perturbations and may be more highly influenced by nutrient supplements (Kretchmer et al., 1996). Researchers have argued that the persistence of neurological ab- normalities after repletion of a nutrient deficiency acquired early in life provides evidence that early neurological vulnerability outweighs potential central nervous system (CNS) plas- ticity in the developing human (Lozoff et al., 2000; Rao, 2000). Nutrients may have variable effects on the developing brain. Nutrient deficiencies may produce negative effects or may have no effect at all depending on the stage of brain

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89 TESTING INGREDIENTS WITH PRECLINICAL STUDIES development (for review, see Georgieff and Rao, 2001). Similarly, nutrient overabundance or supplementation may produce positive effects, negative effects, or no effects, and the magnitude of the effects can be regionally different. For example, iron deficiency in the young rat brain can cause severe energy and structural deficits in the hippocampus (de Ungria et al., 2000; Rao et al., 1999) and can cause abnormalities in dopamine metabo- lism without energy deficits in the striatum (Erikson et al., 2000). At an older age, the same degree of iron deficiency causes no structural deficits. Thus any nutrient’s effect on the developing brain will be based on the timing, the dose, and the duration of the exposure. All nutrients are important for neuronal cell growth and development, but manipulation of some nutrients appears to cause more effects than others. Protein, energy, iron, zinc, selenium, iodine, folate, vitamin A, choline, and LC-PUFAs are nutritional components that influence early brain development with measurable clinical effects in humans and animal models (Georgieff and Rao, 2001). A nutrient that promotes normal brain development at one time and concentration may be toxic at another time or concentration. Many nutrients, such as iron, are regulated within a very narrow range because deficiency and toxicity have profound effects in brain development. The potential effects of nutrients on brain development will determine the types of preclinical testing that are needed. Effects can be neuroanatomical, that is, affecting neu- ronal cell division and cell growth (e.g., size, complexity, synaptogenesis, dendritic arboriza- tion). Furthermore, nutrients can affect developing non-neuronal cells, such as oligodendro- cytes, astrocytes, and microglia, in turn influencing myelination, other nutrient delivery, and cell trafficking. Nutrients can affect neurochemistry through alterations of neurotransmitter and receptor expression. They can affect neurophysiology and neurometabolism with subse- quent alterations of signal propagation. The fundamental question that must be answered in the assessment of nutrient effects on neurodevelopment is whether nutritionally induced alterations in brain development result in changes in brain function, loosely defined as behavior. Specific considerations include: • Is this effect transient (e.g., only during the time when the nutrient is supplemented or deficient) or are there long-term gains or losses beyond the time of deficiency? • How close is the linkage for each nutrient and each time period of development? • What accounts for the individual variability in behavioral outcome of nutritional- based alterations in brain development? • Does a lack of behavioral effect in spite of a measurable biochemical effect imply plasticity or alternate circuit development? • Are there genetic polymorphisms from a change in nutrient status that confer greater or lesser risk or benefit to the host? Many of these questions can be answered experimentally before a nutrient is added to formulas, but a multilevel, integrated approach is required because of the limitations of human brain analysis (Nelson et al., 2002). The multiple levels of approach are shown in Table 5-10. The integrated approach (see Table 5-11) chosen should be complementary and obey the laws of timing, dose, and duration (Kretchmer et al., 1996). The animal models must be developmentally appropriate with respect to timing of brain events and must coincide with the likely time of nutrient supplementation or deficiency in the human. In other words, for brain-behavior associations to be relevant, the time at which a given nutrient alters brain developmental processes in the animal model should coincide with the time that the nutrient deficit or supplement occurs in the infant population.

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90 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS TABLE 5-10 Neurological Assessment: Examples of Tests in Level 1 and Level 2 Level Assessment Level 1 Neuronal/glial cell culture Histological examination General behavior Level 2 Electrophysiology (implanted/surface) Magnetic resonance imaging Magnetic resonance spectroscopy Neurotransmitter concentrations/receptor expression Targeted behavior assessments (e.g., learning paradigms) NOTE: The petitioner (or manufacturer), in consultation with the expert panel, determines which tests are re- quired based on a thorough analysis of the potential effects of the new ingredient. The nonhuman primate, pig, rat, and mouse are the traditional models for neurological research. Table 5-12 and the information provided earlier in this chapter describe the advantages and disadvantages to using each model for neurological research. Preclinical studies should assess the neurological safety of the nutrients by measuring the physiological and pharmacological levels of the nutrients. The goal is to match structural and biochemical alterations to changes in function (e.g., behavior). It must be recognized, however, that there can be a dissociation between biochemical and cellular changes in the brain and neurodevelopment (defined as behavior). Environmental effects on the developing brain have been historically divided into effects on the motor and nonmotor systems. The motor system is far better understood than the nonmotor system because it is more discretely localized in the CNS and has a longer history of investigation. Principles learned from the motor system serve as a model for the current approaches to experimentation in the nonmotor system. The nonmotor system includes cognitive and noncognitive behaviors. Within cognition, a number of behaviors can be assessed, including memory and emotion. Memory is further divided into declarative or recognition memory and implicit or procedural memory (Squire, 1992). The neural circuits that subserve these memory systems are quite distinct and are differentially vulnerable to nutrient insults. The difference in ontogeny of the memory systems has been extensively reviewed by Nelson (1995). It should be noted that many of the neural systems have mixed components. For ex- ample, there are connections between structures underlying recognition memory (e.g., hip- pocampus) and emotion (e.g., amygdala). Thus, for example, it may be difficult to know from behavioral testing alone whether the enhanced ability to perform a recognition memory task following a nutritional supplement is due to direct effects on the hippocampus or through attentional and motivational effects mediated by the amygdala. Certain structures, such as the cerebellum and basal ganglia, are involved in both motor and cognitive events. TABLE 5-11 Integrated Approach to the Assessment of Neurodevelopment Neurological Function System of Assessment Animal behavior (fit to human) Nonhuman primate, rat Biochemistry (neurochemistry) Human, animal model Brain structure Human, animal model Human behavior Human Physiology, cellular/molecular Animal model, cell culture

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91 TESTING INGREDIENTS WITH PRECLINICAL STUDIES TABLE 5-12 Summary of Animal Models Used in Preclinical Studies Animal Advantages Disadvantages Chicken Immunology No relevance to human nutrition, genetics Dog Metabolism, immunology, organs Behavior, genetics Hamster Lipid metabolism Immunology, genetics Mouse Genetics, molecular analysis, mechanisms, Learning paradigms, organs developmental Nonhuman primate Functional/behavioral relationship to Basic biochemistry, histopathology, humans, similar diet as humans, genetics; expense and ethical immunological studies, dermatological concerns for neural tests studies, renal function, kidney biopsy, invasive assessment Pig Comparable size to neonatal humans, Learning paradigms, poor model lipid biochemistry, organs for genetics Rabbit Biochemistry, immunology No relevance to human metabolism Rat Cellular, molecular analysis, behavior, Higher-level learning, genetics, organs, physiological studies; brain developmental development is similar to human infant The optimal use of an integrated methods approach should allow assessment of nutrient effects on behavior, neuroanatomy, neurochemistry, and form-function relationships (e.g., time-locked combinations of methods, such as functional MRI). It is important to have assessments that tap similar brain areas across species (from human to rodent), allowing extrapolation of CNS effects at the tissue level (rodents) to species where tissue is unavail- able (humans, nonhuman primates). Table 5-13 provides examples of the cross-referencing of behavioral tasks across three species. Neurological Assessment Techniques Overall there must be a systematic approach to this important aspect of safety effects of ingredients added to infant formulas. Preclinical assessment must include whole-animal work with specific attention to regional brain effects. Here, neuroimaging through his- tochemistry at autopsy and through MRI in the living animal provides information. Neuro- chemistry experiments should target plausible biological mechanisms, particularly as to how nutrients might affect neurotransmission. Electrophysiology, using either deep-implanted electrodes or scalp-surface electrodes, provides information about neuronal population func- tions (Bachevalier, 2001; Fuster and Alexander, 1971) and can be cross-referenced to the assessment of humans conducted by evoked or event-related potentials (see Chapter 6). The following sections describe various techniques for using preclinical studies to assess the effect of ingredients new to infant formulas on the developing brain. As with the non- TABLE 5-13 Examples of Cross-Species Developmental-Neural Assessment Tools Measure Infant Monkey Rat A-not-B X X Black-white discrimination X X X Delayed nonmatch to sample test X X X Maze X X Paired comparison X X X Object discrimination X X X

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92 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS neurological organs, the committee proposes a two-level hierarchy with respect to applica- tion of these techniques in the preclinical studies. Level 1 assessments are required of all proposed ingredients and are designed to survey the neurological system of the developing brain. Level 2 assessments are performed if the ingredient to be added could theoretically affect brain development through a biologically plausible mechanism or if level 1 assess- ments have previously demonstrated a safety concern for the brain (Table 5-10). Neuronal Genetics/Mutagenicity In the future, evaluations should include neuronal and glial-cell culture work to assess the effect of the added ingredient on genomics, metabolomics, and proteomics with the purpose of evaluating mutagenicity and further functional consequences. Genomic tech- niques (e.g., DNA microarray analysis) are new and promising techniques that, once refined, will likely have great utility in assessing whether there are inductions of desirable or undesir- able genes after exposure to an ingredient. These methodologies are still limited because of difficulties in interpreting the functionality of observed changes in gene expression. The parallel fields of proteomics and metabolomics complement the genomics methods by as- sessing the production of expected and unexpected proteins after nutrient exposures. Fur- thermore, cell culture work can inform about nutrient effects on target receptors (for the nutrient or its metabolite) (Alcantara et al., 1994) and the subsequent downstream effect on signaling cascades in the neurons (Gietzen and Magrum, 2001; Magrum et al., 1999). An excellent overview of this type of approach can be found in the work of Gietzen (2000) on imbalanced amino acid diet effects on feeding behavior as mediated through the neurons of the anterior piriform cortex. Gietzen’s work serves as a model system for assessing nutrient effects on signaling cascades in the relevant neuronal systems. As noted earlier in this chapter, virtually every new ingredient has the potential to advertently or inadvertently alter basic cellular processes. Therefore neuronal-cell culture techniques should be considered level 1 assessments. Neuronal- and glial-cell culture techniques were not reported in the GRAS Notification for LC-PUFAs. These techniques may have identified unwanted effects of these ingredients on gene expression through microarray screening analysis. The effect of these added ingredi- ents on the signaling cascade involved in long-term potentiation could have been assessed in hippocampal CA1 cell recordings. Neuroanatomy There are a number of techniques that assess neuroanatomy, ranging from direct histo- logical assessment by light, confocal, and electron microscopy to neuroimaging (e.g., MRI). Histological assessment allows a direct view of the brain and should be used as a level 1 tool to assess cytological effects of all proposed ingredients. The approach is used in the pig and rodent models and can visualize general neuronal structure (e.g., myelin, synapses, dendritic arborization, neuronal number), effector proteins (e.g., transporters, receptors), apoptosis/ cell death, and regulatory elements (e.g., iron regulatory proteins). The limitation of the approach is that it only shows structure, and alterations of function may not always follow structural changes. MRI can be used in the developing human and all typical animal models to assess nutrient effects on total brain volume, regional volumes, myelination, and the visualization of some nutrients (for review, see Casey et al., 2001). In spite of great technical advances, this method is generally insensitive and assesses only relatively large differences in protein-

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93 TESTING INGREDIENTS WITH PRECLINICAL STUDIES energy or fatty acid status. Therefore it has poor predictive value for future subtle disability of neurological development in humans. At this time, neuroimaging should be categorized as a level 2 assessment. Neurochemistry Neurochemistry can be assessed either directly in the tissue once it is harvested, in the tissue of the living brain through microdialysis catheters (Chen et al., 1995), or in vivo through MRI spectroscopy (Tkác et al., 2003). The goal is to assess the potential adverse ˇ effect of ingredients on neurotransmitters and intermediate metabolism. The advent of MRI spectroscopy allows for the in vivo, real time, repeatable assessment of the chemistry of the developing brain. The technique uses proton, phosphorous, or 13-carbon tracer nuclear magnetic resonance, and it can be used in humans and in animals as small as mice. Its main limitation is the inability to target compounds prospectively; the compounds that can be assessed are dependent on the major peaks that are visualized on the spectra. If the nutri- tional effect is on compounds that are in a concentration that is too low to result in a major peak, the technique is not useful. This technique is innovative and as yet not standardized. Therefore it should be considered a level 2 assessment in the evaluation of ingredients likely to affect neurochemistry. The measurement of nutrient effects on neurotransmitter systems is an important part of nutritional research on brain development. The response of neurotransmitters to changes in nutrient availability can be directly measured in the rodent (Chen et al., 1995) and in neuronal cell culture. It is important with the glutamine system that cell cultures contain both neurons and glia since there is an important shuttle of glutamate and glutamine that takes place. It is also important to assess compensatory feedback mechanisms when transmitter concentrations are affected by nutrients. In response to increased transmitter release, there can be alterations in receptor number and affinity and in presynaptic reuptake mechanisms. The effect of iron deficiency on dopamine provides an excellent example (Chen et al., 1995). The assessment of neurotransmitter systems will be a level 2 assessment for most ingredients. However many compounds being considered for addition to infant formulas (e.g., choline) are likely to have a significant impact on these systems and will require this type of assessment. Neurophysiology Nutrients have effects on the electrophysiology of neuronal populations. A fundamental question that must be addressed in any nutrient-additive experiment is the plausible biologi- cal mechanism by which neuronal output is changed, which could result in a behavioral change. Examples include changes in calcium gating through alpha-amino-3-hydroxy-5- methylisoxazole-4-proprionic acid and N-methyl-D-aspartate receptors in brain areas in- volved in learning, which could alter long-term potentiation and learning behavior (Magrum et al., 1999). If nutrient supplements have a putative biological effect on myelination, there should be demonstrable effects on the speed of processing of myelinated circuits (Birch et al., 1992; Uauy-Dagach and Mena, 1995). Indirect effects must be considered as well. For example, steroids have significant effects on hippocampal anatomy, physiology, and gene expression (for review, see Sapolsky, 1994). Therefore if a nutrient supplement presents a stress to the developing organism with increased output of corticosteroids or estrogens, secondary effects of these steroids on the hippocampus must be considered. Electrophysi- ological testing will typically be warranted as a level 2 assessment when there is a reasonable concern that a new ingredient will affect neuronal function.

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94 INFANT FORMULA: EVALUATING THE SAFETY OF NEW INGREDIENTS Behavior Ultimately the preclinical assessment must shed light on any positive or negative effects on behavior since it is the summation of the efferent expression of brain activity. An assessment of general behavior of the animal model should be performed with any added ingredient. This is part of standard animal care during experiments. Particular attention should be paid to changes in typical behaviors, such as activity level, feeding, and comfort seeking. In the case of suspected specific regional or neurotransmitter effects, a more com- prehensive second level evaluation targeting the potential neuropathology is required. Ex- amples include specific motor, cognitive, and behavioral paradigms that map on the brain systems at risk. With any behavioral assessment, cross-referenced behavioral assessments in the motor and cognitive domain are important during the period of nutrient delivery to assess acute effects. Just as importantly, down-stream, long-term effects must also be evalu- ated to assess whether the added nutrient has permanent or transient effects and whether there are any “sleeper” effects. SUMMARY Preclinical studies are a vital first step to assess the safety and quality of ingredients new to infant formulas. Regulatory guidelines for preclinical studies must be based on consider- ations of the diversity of the potential new ingredients and the ingredients’ source and matrix. In the United States, the FDA Redbook provides comprehensive guidelines for conducting preclinical studies to test the safety of food and color additives, but it often does not take the many special needs and vulnerabilities of infants into consideration. The committee recommends a two-level preclinical approach with respect to assessing the safety of ingredients new to infant formulas. The approach should take into consider- ation model systems that are appropriate for the developing infant. All proposed new ingredients require assessments using level 1 techniques (Tables 5-1 through 5-10) at the discretion of the expert panel. Any proposed new ingredient with expected effects on struc- ture or function, either on a theoretical basis or as a result of level 1 tests, will require appropriately targeted level 2 evaluations (Tables 5-1 through 5-10). REFERENCES Aeschbacher HU, Finot PA, Wolleb U. 1983. Interactions of histidine-containing test substances and extraction methods with Ames mutagenicity test. Mutat Res 113:103–116. Alcantara O, Obeid L, Hannun Y, Ponka P, Boldt DH. 1994. Regulation of protein kinase C (PKC) expression by iron: Effect of different iron compounds on PKC-beta and PKC-alpha gene expression and role of the 5'- flanking region of the PKC-beta gene in the response to ferric transferrin. Blood 84:3510–3517. Anderson NL, Anderson NG. 1998. Proteome and proteomics: New technologies, new concepts and new words. Electrophoresis 19:1853–1861. Arbuckle LD, MacKinnon MJ, Innis SM. 1994. Formula 18:2(n-6) and 18:3(n-3) content and ratio influence long- chain polyunsaturated fatty acids in the developing piglet liver and central nervous system. J Nutr 124:289– 298. Bachevalier J. 2001. Neural bases of memory development: Insights from neuropsychological studies in non- human primates. In: Nelson CA, Luciana M, eds. Handbook of Developmental Cognitive Neuroscience. Cambridge, MA: MIT Press. Pp. 365–379. Benson JD, Masor ML. 1994. Infant formula development: Past, present and future. Endocr Regul 28:9–16. Birch DG, Birch EE, Hoffman DR, Uauy RD. 1992. Retinal development in very-low-birth-weight infants fed diets differing in omega-3 fatty acids. Invest Ophthalmol Vis Sci 33:2365–2376. Bogyo M, Hurley JH. 2003. Proteomics and genomics. Curr Opin Chem Biol 7:2–4. Canada. 2001. Departmental Consolidation of the Food and Drugs Act and the Food and Drug Regulations. (Food and Drugs Act). Ottawa: Minister of Public Works and Government Services Canada.

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