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§ 140.100 Delegations of authority. SUPPLEMENTARY INFORMATION: approving the general-purpose food use of neotame. The Commission hereby re-delegates Table of Contents the delegations of authority made to the II. Safety Evaluation Directors of the Division of Trading and I. Introduction Markets and/or to the Division of II. Safety Evaluation of Neotame A. Chemistry and Intake Considerations Economic Analysis, and their respective A. Chemistry and Intake Considerations of Neotame of Neotame designees, in all instances as they occur Neotame is the common or usual throughout this chapter, jointly to the B. Nature and Extent of Neotame Safety Studies Database name for the chemical N-[N-(3,3- respective Directors of the Division of dimethylbutyl)-L-a-aspartyl]-L- Market Oversight and the Division of C. Toxicology/Safety Assessment of Neotame phenylalanine-1-methyl ester (CAS Reg. Clearing and Intermediary Oversight, No.165450–17–9). It is synthesized by and their respective designees. 1. Metabolism and Pharmacokinetics of Neotame reductive N-alkylation of L- Issued in Washington, DC, on July 2, 2002, a. Absorption of neotame phenylalanine-L-a-aspartyl methyl ester by the Commission. b. Elimination, distribution, and with 3,3-dimethylbutyraldehyde. Jean A. Webb, potential tissue accumulation of According to the petitioner, neotame Secretary of the Commission. neotame has a sweetening potency that is [FR Doc. 02–17179 Filed 7–8–02; 8:45 am] c. Effect of neotame on drug approximately 7,000 to 13,000 times BILLING CODE 6351–01–M metabolizing enzymes that of sucrose, depending on its food d. Metabolites of neotame application (Refs. 1 and 2). 2. Critical Toxicology Studies and The peptidyl linkage in neotame is stabilized by the N-alkyl substituent and DEPARTMENT OF HEALTH AND Issues is resistant to hydrolysis under typical HUMAN SERVICES a. A 2-generation reproduction study in the rat—neurotoxicity and use and storage conditions. Food and Drug Administration behavioral effects Additionally, the N-alkyl substituent b. Chronic (52-week) dog study— effectively prevents the common 21 CFR Part 172 toxicological significance of dipeptide cyclization reaction that elevated serum (hepatic) alkaline results in the formation of a [Docket Nos. 98F–0052 and 99F–0187] phosphatase levels diketopiperazine derivative. The data c. A 104-week mouse from stability studies submitted by the Food Additives Permitted for Direct carcinogenicity study—body weight petitioner show that the degradation of Addition to Food for Human gain decrement effect neotame in aqueous solutions is pH-, Consumption; Neotame d. A 104-week rat carcinogenicity time-, and temperature-dependent. AGENCY: Food and Drug Administration, study—body weight gain decrement Based upon data from these stability HHS. effect at all dose levels tested studies on neotame, the agency concludes that minor decomposition of ACTION: Final rule. e. Chronic (52-week) rat feeding study—body weight gain decrement neotame could occur in neotame- SUMMARY: The Food and Drug effect containing foods only when stored Administration (FDA) is amending the f. Clinical studies assessments— under conditions that are not food additive regulations to provide for human tolerance to neotame considered typical for a commercial the safe use of neotame as a D. Estimating an Acceptable Daily product (Refs. 1 and 2). nonnutritive sweetener in food. This Intake for Neotame The agency has determined the action is in response to two petitions III. Comments estimated daily intake (EDI) at the 90th filed by Monsanto Co., which IV. Conclusion percentile for neotame as a general- subsequently sold the rights to the V. Environmental Effects purpose sweetener to be 0.10 milligram petitions to the NutraSweet Co. VI. Paperwork Reduction Act of 1995 per kilogram (mg/kg) body weight per VII. References day (bw/d) for consumers of all ages DATES: This rule is effective July 9, VIII. Objections (eaters only) and 0.17 mg/kg bw/d for 2 2002. Submit objections and requests for to 5 year olds (eaters only). The a hearing by August 8, 2002. The I. Introduction corresponding mean intakes are Director of the Office of the Federal FDA published notices in the Federal 0.04 mg/kg bw/d and 0.05 mg/kg bw/d, Register approves the incorporation by Register on February 10, 1998, and respectively (Refs. 2 and 3). reference in accordance with 5 U.S.C. February 8, 1999 (63 FR 6762 and 64 FR 552(a) and 1 CFR part 51 of a certain 6100, respectively), announcing that B. Nature and Extent of Neotame Safety publication in 21 CFR 172.829, as of food additive petitions, FAP 8A4580 Studies Database July 9, 2002. and FAP 9A4643, had been filed by In support of the safety of neotame, ADDRESSES: Submit written objections Monsanto Co., Skokie, IL 60077. The the petitioner submitted, within the two and requests for a hearing to the Dockets petitions propose amending the food petitions, a combined total of 113 Management Branch (HFA–305), Food additive regulations to provide for the preclinical, clinical, and special studies, and Drug Administration, 5630 Fishers safe use of neotame as a nonnutritive plus an additional 32 exploratory and Lane, rm. 1061, Rockville, MD 20852. sweetener for tabletop use (FAP screening studies in Food Master File Submit electronic objections to http:// 8A4580) and for general-purpose use in No. 575. All pivotal preclinical studies www.fda.gov/dockets/ecomments. food (FAP 9A4643) where standards of were conducted in compliance with FOR FURTHER INFORMATION CONTACT: identity do not preclude such use. FDA’s ‘‘good laboratory practice’’ Blondell Anderson, Center for Food Subsequently, the rights to the petitions regulations in 21 CFR part 58. Safety and Applied Nutrition (HFS– were sold to the NutraSweet Co., 699 The preclinical (animal) studies 265), Food and Drug Administration, North Wheeling Rd., suite 103, Mount include short-term, subchronic, and 5100 Paint Branch Pkwy., College Park, Prospect, IL 60056. This document chronic dietary toxicity tests in the rat, MD 20740–3835, 202–418–3106. grants the petitions via a regulation mouse, and dog; multi-generation

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reproduction and developmental studies absorption of neotame approaches 100 during which dietary neotame was fed in the rat; teratology studies in the rat percent in healthy male and female at 0 (control), 100, 300, or and rabbit; and lifetime/carcinogenicity subjects when administered following 1,000 mg/kg bw/d, rats were sacrificed studies in the rat and mouse. The an overnight fast and at dose levels and in vitro assays performed on genotoxicity of neotame, its metabolites, ranging from one to five times the 90th isolated liver microsomal pellets. The and decomposition products, are also percentile EDI. Individual absorption agency concludes that, when compared evaluated in several tests using both in levels range from 68 to 126 percent (Ref. against a positive control vitro and in vivo assay systems. 5). (phenobarbital, a known enzyme Extensive metabolism and b. Elimination, distribution, and inducer), neotame does not induce P– pharmacokinetic measurements were potential tissue accumulation of 450 microsomal mixed function oxidase carried out in all animal species neotame. The agency estimates that metabolizing enzymes at any dose level studied. The clinical (human) studies approximately 40 percent of the administered during the in vivo phase tested the response/acceptance to orally systemic elimination of ingested of the study. In evaluating the effects of administered neotame in both men and neotame and metabolites occurs via the neotame on phase II metabolism, the women during short-term (e.g., acute, urine, and the remainder is eliminated agency notes that livers from rats in the single-dosing) and longer-term (e.g., up via the fecal route. In a whole-body 1,000 mg/kg bw/d treatment group show to 13 weeks, repeat-dosing) periods. radiography study in the rat, following a statistically significant depression in Pharmacokinetic (PK) measurements a gavaged dose of radiolabled neotame phase II metabolism endpoints. also were carried out in a number of and serial sacrifice at timed intervals, However, at the next lower dose of these studies (Ref. 4). post-dosing, the highest levels of 300 mg/kg bw/d, which is approximately Additionally, the petitioner provided radioactivity are associated with the 3,000 times the 90th percentile EDI for three position papers in response to intestinal tract, the liver, and the neotame for humans, there are no effects FDA questions. These position papers kidney. At final sampling, no residual on these same endpoints (Ref. 5). address: (1) The potential behavioral radioactivity is detected in peripheral d. Metabolites of neotame. The initial and neurotoxic effects of neotame, (2) tissues, with some residual activity step in the metabolism of neotame in the significance of elevated serum associated with the intestinal tract. No rats, dogs, rabbits, and humans is de- (hepatic) alkaline phosphatase activity organs or tissues, including the brain, esterification to N-[N-(3,3– in neotame-treated dogs as a measure of eye, and skin, concentrate or store dimethylbutyl)-L-a-aspartyl]-L- toxicity, and (3) body weight gain radiolabled neotame or its metabolites. phenylalanine (DMB-Asp-Phe, coded in decrement in mice ingesting neotame. Further evidence for the lack of the petition as NC–00751) by Ca++- The key aspects of these position papers accumulation of neotame at expected dependent pancreatic esterases or after are discussed, as appropriate. levels of human intake is found in the absorption by plasma esterases. De- analysis of PK parameters evaluated esterification of neotame is similar in all C. Toxicology/Safety Assessment of during a 13-week dog study. In dogs species studied, including humans, Neotame consuming dietary neotame at dose although in the rat and rabbit this 1. Metabolism and Pharmacokinetics of levels of 1,200 to 2,000 mg/kg bw/d, conversion occurs at a faster rate than in Neotame there is an indication of saturation of an the dog and human. The de-esterified elimination pathway that could lead to metabolite (NC–00751) is rapidly As a component of the toxicological possible accumulation. However, these cleared from the plasma and excreted testing program on neotame, the levels are at least 10,000 times greater via the bile duct or in urine (Ref. 5). A petitioner conducted an extensive series than the 90th percentile EDI small percentage of NC–00751 may of metabolism and PK studies. These (0.1 mg/kg bw/d) of neotame for undergo peptide-bond hydrolysis to studies were designed to assess: (1) The humans. This effect is not seen in dogs form metabolites of absorption of neotame; (2) the from the next lower treatment group dimethylbutylaspartate. The 3,3- elimination, distribution, and potential (600 mg/kg bw/d), a level approximately dimethylbutyl portion of DMB-Asp-Phe tissue accumulation of neotame; (3) the 6,000 times above the 90th percentile is then oxidized to 3,3-dimethyl-butyric effects of neotame on drug metabolizing EDI. Based on these findings, the agency acid. This is followed by conjugation enzymes; and (4) the metabolites of concludes there is no concern for with glucuronic acid or with carnitine (a neotame in rodents (rats and mice), possible accumulation of neotame or its minor pathway). dogs, rabbits, and humans. metabolites at expected human intake Methanol release results from the de- a. Absorption of neotame. In all levels (Refs. 4 and 5). esterification of neotame and occurs species studied, including humans, the c. Effect of neotame on drug more rapidly in the rat and rabbit than agency finds that the absorption of metabolizing enzymes. The rat is in the dog and human. The agency ingested neotame occurs almost entirely generally considered an appropriate concludes that at the 90th percentile in the small intestine. In the animal animal model to assess the effects of EDI for neotame, exposure to resultant studies, the absorption of neotame was xenobiotics on phase I (i.e., cytochrome methanol will be insignificant, i.e., not determined under fasting conditions P–450 or mixed-function amine oxidase more than 0.008 mg/kg bw/d. This using a dose level that was microsomal enzyme systems1) and exposure level is of no toxicological approximately 150 times greater than phase II (i.e., conjugation or concern because humans are exposed to the 90th percentile estimated daily biotransformation reactions involving much greater levels of methanol intake intake (EDI) of neotame for humans. glucuronidation, sulfation, acetylation, from their daily diets (Refs. 4 and 5). Under these conditions, the amount of or glutathione-S- transferase reactions) Based on neotame metabolism studies administered dose absorbed is reported metabolism. Following a 14-day period in the rat and dog, FDA concludes that to range from 18 to 38 percent in the rat, some intestinal microvillar peptidase 15 to 44 percent in the rabbit, and 40 to 1 Sipes, I. G. and Gandolfi, A. J., activity occurs in the gut, which results 51 percent in the dog. These studies also ‘‘Biotransformation of Toxicants,’’ chapter 4, pp. in the formation of other minor plasma 88–109, in Casarett and Doull’s Toxicology: The indicate that, when mixed with the diet, Basic Science of Poisons, 4th ed., edited by M. O. metabolites of neotame, including the absorption of neotame is reduced. In Amdur, J. Doul, and C. D. Klaassen, McGraw Hill, phenylalanine (Ref. 5). Further review the human clinical studies, the Inc., 1993. indicates that approximately 13 to 17

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percent of the total available the petitioner. During its review, the group. No effects are noted on motor phenylalanine in the ingested neotame agency determined that certain studies activity in F1 females at any dose level. is released into the plasma after were more important than others to a With regard to results from the swim- absorption; the remainder is eliminated regulatory decision on neotame. This maze tests that were conducted in F1 in feces and urine as DMB-Asp-Phe. The determination was based on the nature offspring at approximately 24 to 28 days agency has estimated the amount of of the endpoints investigated in these of age, both the petitioner and the phenylalanine presented to the body studies (i.e., reproductive and agency conclude that there is a from the ingestion of neotame. The developmental effects, long-term statistically significant increase in mean phenylalanine content of neotame is 44 exposure, chronic toxicity, carcinogenic swimming time (an indicator of reduced percent by weight. Given that the 90th potential, and human tolerance), and on performance) to the ‘‘correct’’ arm of the percentile neotame EDI for a 60 kg adult specific issues presented by these Y-maze in F1 males from the is 0.10 mg/kg bw/d or 6 mg/d, and for a studies. The critical studies and issues 1,000 mg/kg bw/d group. Specifically, 2 to 5 year old (20 kg) child is presented by the studies are: (1) The 2- this increased swim time is noted in two 0.17 mg/kg bw/d or 3.4 mg/d, the generation reproduction study in rats— of six trials in the F1 males from the estimated 90th percentile phenylalanine neurotoxicity and behavioral effects, (2) high dose group. While an increase in intake is 2.6 mg and 1.5 mg, the chronic (52-week) dog study— swim time is also noted for one of six respectively. toxicological significance of increased trials in F1 males from the The agency notes that, for healthy serum (hepatic) alkaline phosphatase 300 mg/kg bw/d dose group, this adults, the daily dietary intake of levels, (3) the 104-week mouse singular observation is not accompanied phenylalanine may range from 2.5 to 10 carcinogenicity study—body weight by any other indication of treatment- grams per person per day (g/p/d), while gain decrement effect, (4) the 104-week related behavioral changes and therefore that for a phenylketonuric (PKU) rat carcinogenicity study—body weight is not considered to be indicative of a homozygous child (20 kg) may range gain decrement effect at all dose levels biologically relevant effect. As with 2 from 0.4 to 0.6 g/p/d (Koch and Wenz ). tested, (5) the chronic (52-week) rat motor activity, there are no effects on Thus, the amount of phenylalanine from feeding study—body weight gain cognitive performance (as measured by the 90th percentile intake of neotame is decrement effect, and (6) the human swim maze times) noted in F1 female trivial compared to that from the normal clinical trials—human tolerance to offspring from any treatment group. adult diet. Even for the PKU neotame. The F1 offspring from the 2- generation reproduction study also were homozygous child, the incremental a. A 2-generation reproduction study amount of phenylalanine intake that can subjected to specific tests that measured in the rat—neurotoxicity and behavioral be expected from neotame is the development of auditory and visual effects. Reproductive performance and insignificant, i.e., equivalent to no more responses. The agency’s evaluation of fertility were assessed over two than 0.3 to 0.4 percent of the daily results on auditory startle, pupil generations in CD (cesarean derived) phenylalanine intake of the PKU closure, and visual placing show no rats fed diets containing neotame at homozygous child (Ref. 5). The agency treatment-related effects in F1 males or levels of 0 (control), 100, 300, or concludes that the potential intake of females at any level of neotame tested. 1,000 mg/kg bw/d. Each treatment group phenylalanine that may result from use The finding of statistically significant consisted of 28 males and 28 females. of neotame as a general-purpose effects on two separate behavioral tests Animals were mated, the resultant sweetener does not pose any safety (i.e., motor activity and swim maze concern (Refs. 4 and 5). offspring weaned, and the F1 generation times) in F1 males from the Based on reviews of the metabolism animals selected and allowed to mature 1,000 mg/kg bw/d dose group supports and pharmacokinetic studies on for 10 weeks and then mated. The F2 the conclusion that this dose is an effect neotame, the agency concludes that the litters were terminated, post-weaning. level. Based on the findings from the metabolism of neotame is qualitatively Under the conditions of this study, the studies of motor activity and cognitive similar across all species studied. agency concludes that neotame has no function, the agency considers the Furthermore, there is no evidence that, effects on the reproduction or fertility of 300 mg/kg bw/d dose to be a no at expected levels of intake, neotame or rats exposed to neotame at levels up to observed adverse effect level (NOAEL) its metabolites will accumulate in the 1,000 mg/kg bw/d for two generations. for these endpoints (Refs. 4 and 7). body or that ingestion of neotame will Nor are there any treatment effects on Early in its evaluation of the neotame have any adverse effect in the body on measures of physical development, e.g., safety database, the agency determined Phase I and II metabolism. The pinna unfolding, hair growth, tooth that the petitioner should provide a metabolites of neotame are well eruption, or eye opening (Refs. 4 and 6). more specific assessment addressing the characterized, and the potential intakes The 2-generation study included tests potential neurotoxicity and behavioral of metabolites, such as methanol and of motor activity and cognitive function. effects of neotame. In response to the phenylalanine, are of no toxicological General motor activity was measured in agency’s request, the petitioner consequence. Therefore, the agency’s F1 offspring by counting breaks in a pair submitted a position paper entitled review of the metabolism and of infrared light beams over a 12-hour ‘‘Neotame Does Not Cause Any pharmacokinetic studies of neotame period, while cognitive function was Behavioral or Neurotoxic Effects’’ (Ref. does not raise any safety concerns (Refs. assessed by recording swim times up to 8). This document contains summaries 4 and 5). 60 seconds maximally in six and discussions of data and information consecutive trials per animal in a water- from two principal sources. The first 2. Critical Toxicology Studies and Issues filled Y-maze (Ref. 7). While the involves several ‘‘key’’ preclinical FDA reviewed all studies and petitioner concludes there were no studies (12 in all) and 4 clinical studies supplemental information submitted by significant treatment effects on motor from the neotame studies database. The activity in F1 male and female offspring, second source of information discussed 2 Koch, R. and Wenz E. J., ‘‘Aspartame Ingestion the agency’s analyses of pertinent data in the position paper is a series of 20 by Phenylketonuric Heterozygous and Homozygous Individuals,’’ chapter 30, pp. 593–603, in show a statistically significant reduction publications that are primarily related to Physiology and Biochemistry, edited by Stegink, L. in motor activity among F1 males from aspartame. Collectively, these 20 D. and L. J. Filer, Jr., 1984. the 1,000 mg/kg bw/d neotame treatment publications provide little information

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that is relevant to the agency’s overall from the 200 mg/kg bw/d dose group liver in both sexes and at all dose levels safety assessment of neotame and are and in both sexes at the 800 mg/kg bw/d appears normal on histopathological not discussed further. dose group. Other effects noted were examination. In this 52-week dog study, With regard to the ‘‘key’’ animal statistically significant dose-related FDA establishes a no observed effect studies, the petitioner states in its increases in absolute liver weights and level (NOEL) of 60 mg/kg bw/d, based on position paper that these studies in relative liver weights (liver to brain liver effects (e.g., serum (hepatic) incorporated clinical observations/ weight ratio) in female dogs in the 200 alkaline phosphatase and relative liver testing enhancements as ‘‘effective and 800 mg/kg bw/d dose groups. There weights) as the most sensitive endpoints procedures for detecting neurotoxic was no evidence of histopathological (Refs. 4 and 10). effects.’’ During the ante mortem phase changes in the liver, brain, sciatic nerve, c. A 104-week mouse carcinogenicity of the animal studies, these and spinal cord or in other organs or study—body weight gain decrement enhancements included detailed tissues examined from neotame-treated effect. CD–1 mice were fed neotame- physical, behavioral, and clinical dogs. containing diets for 104 weeks at levels observations to detect signs of Because elevated serum ALP levels of 0 (control), 50, 400, 2,000, or neurological disorder, behavioral had also been observed in shorter 4,000 mg/kg bw/d. Based on an abnormality, physiological dysfunction, duration studies (2-week and 13-week) evaluation of the histopathological data and other signs of nervous system in dogs ingesting neotame containing from this carcinogenicity study, FDA toxicity. Post mortem enhancements diets, the agency requested that the concludes that, under the conditions of included extensive histopathological petitioner provide further clarification the study, doses of neotame up to evaluations of brain, spinal cord, and on this matter. In its response, the 4,000 mg/kg bw/d administered to male peripheral nerves. petitioner submitted a position paper and female CD–1 mice for up to 2 years FDA has reviewed thoroughly all of entitled ‘‘Increases in Serum Alkaline did not induce neoplastic lesions (Ref. the preclinical and clinical studies Phosphatase in the Dog Are Not 11). discussed in the position paper. With Associated with Target Organ Toxicity,’’ Although there was no evidence of the exception of the 2-generation rat together with several publications carcinogenicity in mice exposed to reproduction study in which related to hepatotoxicity and serum ALP neotame for 104 weeks, during the statistically significant decreases in activity (Ref. 9). In this position paper, agency’s review of other endpoints, we motor activity and statistically the petitioner reasons that the increased noted negative effects on body weight significant increases in swim times are serum ALP levels observed in neotame- gain (and thus body weight) in both observed in F1 offspring males at treated dogs are not due to a hepatotoxic sexes. In light of only small decreases in 1,000 mg/kg bw/d, the preclinical response, but to a ‘‘nonspecific, cumulative food consumption, the studies do not show behavioral or physiological response’’ to the high agency was concerned about the neurotoxic effects associated with the doses of neotame. potential toxicological significance of ingestion of neotame. FDA conducted further statistical the decrease in body weight gain. In Based on available preclinical and analyses on the liver weight parameters response to the agency’s request for clinical information from the neotame mentioned previously. Based on these further clarification on this issue, the studies database, the agency concludes analyses, the agency concludes that the petitioner submitted a position paper that there is no concern for potential means for these liver effects from the entitled ‘‘In the Mouse Carcinogenicity neurotoxic or behavioral effects in 200 and 800 mg/kg bw/d dose groups are Study With Neotame Small Changes in humans from the ingestion of neotame statistically significantly higher than the Body Weight Gain at Some Intervals in as a general-purpose sweetener in foods. means for the 0 (control), 20, and Female Mice at 50 mg/kg bw Relative to This conclusion is reinforced further by 60 mg/kg bw/d treatment groups. Controls Are Due to a Decrease in Food the NOAEL of 300 mg/kg bw/d Furthermore, there are no statistically Consumption’’ (Ref. 12). In its analysis, established for motor activity and significant differences between the 0 the petitioner states that the mouse is cognitive performance in F1 males from (control), 20, and 60 mg/kg bw/d dose not a reliable model for determining the the 2-generation reproduction study, a group means for any of the liver weight relationship between body weight gain dose level that is at least 3,000 times parameters that were evaluated. and food consumption. Reasons cited greater than the 90th percentile EDI of From the review of the data from the include the small differences in body 0.1 mg/kg bw/d (Refs. 4 and 7). 52-week dog study and the weight gain over a lifetime in mice, both b. Chronic (52-week) dog study— supplemental information submitted by in absolute terms and in proportion to toxicological significance of elevated the petitioner in its position paper, the initial body weights at the start of a serum (hepatic) alkaline phosphatase. agency concludes that the changes in study, and well-known difficulties in Beagle dogs were fed diets containing serum ALP levels are most likely due to obtaining accurate measures of food neotame at levels of 0 (control), 20, 60, a nontoxic response to the higher levels intake for mice (e.g., mice frequently 200, or 800 mg/kg bw/d over a 52-week (200 and 800 mg/kg bw/d) of spill food from their food cups and period. Detailed data were collected on administered neotame. This conclusion contaminate their food with feces and animal survival, growth, food intake, is based on the following: (1) There are urine). The petitioner reiterated its clinical chemistries, hematology, no significant effects from neotame on belief that the body weight gain urinalyses, and gross organ pathology other liver enzymes (e.g., alanine decrements noted in mice during the and histopathology. At the conclusion aminotransferase, aspartate 104-week study were due to a small but of the study, a limited number of dogs aminotransferase, gamma glutamyl consistent reduction in food from the neotame treatment groups were transferase), (2) serum albumin levels consumption which is attributable to placed on a control diet for an are not decreased in neotame-treated poor diet palatability and should not be additional 4-week ‘‘reversibility dogs (a decrease would have been an viewed as a toxicological response to period.’’ During the agency’s review of indicator of chronic liver toxicity), (3) neotame. this study, a question arose about the serum bilirubin levels are normal in In further evaluation of this body gain toxicological significance of increased both sexes at high doses of neotame (an weight decrement issue, FDA subjected serum alkaline phosphatase (ALP) levels increase would have been seen if the data on body weight, body weight (of hepatic origin) noted in female dogs cholestasis was occurring), and (4) the gain, and adjusted (for neotame content)

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food intake to extensive statistical do not fully explain the magnitude of in male rats from the 30, 100, 300, and evaluation. Using an analysis of the differences in body weight and body 1,000 mg/kg bw/d neotame treatment covariance model and pair-wise dose weight gain observed in these animals at groups, while somewhat lower than comparisons of body weights and body week 52 and thereafter up to week 104. controls, are not statistically different. weight gain, the agency notes In view of the significant body weight As noted in the 104-week statistically significant effects for the gain decrement effect observed in all carcinogenicity study, female rats in the 400, 2,000, and 4,000 mg/kg bw/d dose neotame treatment groups during the 52-week dietary study were more groups. Based on these analyses, the 104-week rat carcinogenicity study, a sensitive to body weight gain decrement agency concludes that the body weight NOEL cannot be established. Lacking a effects than males. gain decrement effect in both male and suitable explanation for this effect based FDA performed a detailed analysis of female mice in the three highest dose on decreased food intake (as argued by the results from the 52-week dietary rat groups is not accounted for by the small the petitioner), the agency considered study and concludes that this study decreases in food consumption. the body weight gain decrement effect provides an adequate basis to assess the However, in the 50 mg/kg bw/d unresolved by the 104-week rat study body weight gain decrement effect noted treatment group, the effects on body (Refs. 4 and 10). in the 104-week carcinogenicity rat weight and body weight gain are not e. Chronic (52-week) rat feeding study for four reasons. First, the range statistically different from controls. study—body weight gain decrement of neotame dose levels studied in the Based on the detailed statistical effect. In order to resolve the body 52-week study is comparable to the evaluation of data pertinent to the body weight gain decrement issue in rats, the doses tested in the 104-week study. weight gain decrement noted in the 104- agency carried out a thorough analysis Second, in each study, the female rat is week dietary carcinogenicity study in of data from a 52-week rat feeding more sensitive. Third, a parallel mice, the agency establishes a NOEL of study. This study employed a wide comparison of the 52-week study and 50 mg/kg bw/d for this endpoint (Refs. 4 range of neotame dose levels, two of the first 52 weeks of the carcinogenicity and 10). which were below the lowest dose study shows that the body weight gain d. A 104-week rat carcinogenicity tested in the 104-week rat decrement effect was of a similar order study—body weight gain decrement carcinogenicity study (as discussed in of magnitude in both studies. Fourth, effect at all dose levels tested. A 104- section II.C.2.d of this document). The the magnitude of decrease in body week rat carcinogenicity study (with an results of this analysis are presented in weight gain occurring during week in utero phase) was conducted during the following paragraphs. interval 0 to 52 in the 104-week study which neotame was fed at 0 (control), In the chronic (52-week) rat feeding does not worsen during the last half of 50, 500, or 1,000 mg/kg bw/d. Based on study (with an in utero phase) rats the study. These observations add a thorough evaluation of the received neotame at 0 (control), 10, 30, strength to the utility of the 52-week histopathological data from this 100, 300, or 1,000 mg/kg bw/d. Except dietary rat study in resolving any carcinogenicity study, FDA concludes for body weight and body weight gain, concern about the body weight gain there is no evidence of neotame-induced there were no statistically significant decrement effect and in establishing a neoplastic lesions in rats ingesting diets treatment-related effects of neotame NOEL of 30 mg/kg bw/d for this containing neotame at levels up to during this 52-week feeding study. With endpoint (Refs. 4 and 10). 1,000 mg/kg bw/d for 104 weeks (Ref. respect to both body weight and body f. Clinical studies assessments— 11). weight gain, female rats appear to be human tolerance to neotame. The During its review of the 104-week rat more sensitive than males. petitioner submitted the results of six carcinogenicity study, the agency noted In regard to body weight, at the end human clinical trials that investigated effects on body weight gain (and thus of the 52-week study, body weights in the ingestion of neotame under varied body weight) in both sexes of neotame- females from the 100, 300, and conditions, including acute-single treated rats at all dose levels tested. 1,000 mg/kg bw/d groups were exposure, acute-repeat exposure, and Statistically significant decreases in statistically significantly lower than short-term (2-week) and longer-term (13- cumulative body weight gains were those of control female rats. However, week) daily exposure. Five of these observed at various intervals throughout the body weights of females from the 10 trials employed healthy adult subjects, the study. At week interval 0 to 52, and 30 mg/kg bw/d groups were not while one trial evaluated non-insulin cumulative body weight gains were 9 to statistically different from control dependent diabetes mellitus (Type II 11 percent less and 13 to 19 percent females. Among males, only the diabetic) adult subjects. In each of these less, respectively, in neotame-treated 100 mg/kg bw/d group had statistically trials, subject tolerance to neotame male and female rats, than in control significant body weight differences from intake was determined by physical animals. Similar effects were noted at control male rats. examinations, vital signs, week intervals 0 to 78 and 0 to 104, i.e, As for cumulative body weight gains electrocardiograms, routine clinical cumulative body weight gains ranging during the 0 to 52-week interval, laboratory measurements (e.g., from 10 to 13 percent less in treated statistically significant decreases are hematology, clinical chemistries, and males and 17 to 20 percent less in noted in treated females, compared to urinalysis), and self-assessments of treated females. In reporting this controls, only from the 300 and adverse experiences. information, the petitioner suggests that 1,000 mg/kg bw/d treatment groups. The levels of neotame administered in the lower body weights and lower body While the body weight gains in females these clinical trials ranged from 1 to 15 weight gains among neotame-treated from the 100 mg/kg bw/d are lower than times the 90th percentile EDI level of rats can be attributed to reduced food in control female rats, this difference is 0.1 mg/kg bw/d or 6 mg per person per intake due to reduced palatability of the not statistically significant. Compared day (mg/p/d). diets containing neotame. with controls, there are no significant The agency concludes that in all six The agency, however, based on an differences in cumulative body weight trials there are no treatment-related analysis of the food intake data, gains in females from the two lowest effects reported for any of the concludes that the decreases in adjusted treatment groups (10 and parameters examined. Although (for neotame content) food intake among 30 mg/kg bw/d) for the 0 to 52-week headache was the most frequently noted the neotame-treated rats are small and interval. Cumulative body weight gains adverse experience, the incidence of

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headache is comparable for the treated Based on reviews of these clinical D. Estimating an Acceptable Daily and control groups and is not trials, the agency concludes that the Intake for Neotame considered to be associated with ingestion of neotame at levels up to neotame intake. Results from ancillary 1.5 mg/kg bw/d (15 times the 90th In determining an acceptable daily pharmacokinetic measurements in percentile EDI) for a period as long as intake (ADI) for a new food additive, the several of the clinical trials do not raise 13 weeks is well tolerated by healthy agency relies on a comprehensive evaluation of all relevant studies and any safety concerns. In the trial with male and female subjects. The agency information submitted by the petitioner. Type II diabetic subjects, no adverse also concludes that in the study with As the agency’s evaluation of the effects are noted in any of the subjects. Type II diabetic subjects, the intake of Under the conditions of that trial, the neotame safety studies database neotame at levels up to 1.5 mg/kg bw/d agency concludes that the ingestion of progressed, four studies with attendant does not have significant effects on neotame at levels up to 1.5 mg/kg bw/d issues emerged as having the greatest does not produce significant changes in fasting plasma glucose or insulin levels impact in reaching a safety decision; either fasting-state glucose or insulin in study subjects (Refs. 4 and 13). these studies are highlighted in table 1 levels in Type II diabetic subjects. of this document.

TABLE 1.—SUMMARY OF STUDY DATA PERTINENT TO ESTABLISHING AN ACCEPTABLE DAILY INTAKE VALUE FOR NEOTAME

Study Information Pivotal Endpoint NOEL (mg/kg bw/d) Safety FactorA ADI (mg/p/d)

2-Generation Reproduction (Rat) Motor Activity and Cognitive (300)B 1,000 18 Function in F1 Males 52-week (Dog) Serum (Hepatic) ALP Levels 60 100 36 and Relative Liver Weights in Females 104-week (Mouse) Body Weight Gain Decrement 50 100 30 in Both Sexes 52-week (Rat) Body Weight Gain Decrement 30 100 18 in Females A Safety factors typically applied by the agency in establishing an ADI based on effects from a reproductive toxicity study or from a chronic study are 1000 and 100, respectively. B The value reported is the NOAEL as discussed in Section II.C.2.a of this document.

Based on the NOAEL or NOEL section II.C.1 of this document, and disagrees with the petitioner’s use identified for the most sensitive respectively) from aspartame even of the 50 percent replacement factor in endpoint in each of the four studies, though they are structurally related. their estimation of exposure to neotame. ADI values were determined ranging Therefore, the comments’ assertions The agency conservatively assumes that from a high of 36 mg/p/d to a low of about neotame are without basis. this new sweetener will replace all 18 mg/p/d. In taking a conservative Because the comments do not provide existing uses of aspartame (Ref. 14) and approach, the agency concludes that the the agency with any information uses this estimate in its safety appropriate ADI for neotame is regarding the safety of neotame, they evaluation. 18 mg/p/d (Ref. 4). This level is three will not be discussed further. No Observed Effect Level, Body Weight, times higher than the 90th percentile Estimated Daily Intake and Body Weight Gain Effects EDI for neotame of 6 mg/p/d. Several comments objected to the One comment stated that there is no tabletop use petition on the basis that NOEL established by the 104-week rat III. Comments the petitioner’s EDI for neotame is carcinogenicity study for neotame, Thirty comments were submitted to inaccurate, implying that it is too low. because all doses show adverse effects FDA’s Dockets Management Branch in In determining an EDI, FDA makes on growth. The comment also asserted response to the filing of the two projections based on the amount of the that the data contained in this study do neotame food additive petitions (25 for additive proposed for use in particular not support the petitioner’s explanation FAP 8A4580 and 5 for FAP 9A4643). foods and on data regarding the that decreases in body weights in the The issues raised in the comments are consumption levels of these particular treated rats are due to reduced identified and grouped into the foods, commonly using the 90th palatability of the neotame-containing following subject categories. percentile as a measure of high chronic diets. In addition, the comment Aspartame exposure. The agency concludes that the indicated that the petitioner did not The majority of the comments 90th percentile EDI calculated for supply any gavage, pair-feeding, or compared neotame to aspartame. In neotame, as discussed in section II.A of dietary restriction studies to prove that these comparisons, the comments this document, accurately reflects the the body weight gain decrements are assumed that neotame produces the exposure to neotame as a general- due to palatability and not toxicity. The same metabolic breakdown products as purpose sweetener in all foods (except comment also claimed that a safe usage aspartame and thus would be for meats and poultry), including level for neotame cannot be determined responsible for the same health effects tabletop use (Ref. 2). from the safety database provided in the they allege to be associated with One comment noted that the neotame food additive petitions. aspartame, which is the subject of a food petitioner assumes that neotame will FDA agrees that a NOEL cannot be additive regulation (21 CFR 172.804). In replace 50 percent of aspartame’s established based on the 104-week rat response to these comments, FDA current applications and argued that carcinogenicity study, in view of the points out that neotame is chemically this assumption may be limited unduly body weight gain (decrement) effect. and metabolically different (see section and not sufficiently conservative. FDA The agency also notes that, while II.A of this document and Ref. 1, and agrees with the comment on this point, neotame may have had some influence

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on diet palatability, the decreases in manifestation of the target organ toxicity detected in control animals. food intake (adjusted for neotame in neotame-treated dogs. Collectively, these observations support content) among neotame-treated rats of While the agency considers the 13- the agency’s conclusion that data from both sexes in the 104-week study are too week dog study useful for obtaining the 52-week study do not show small to explain the magnitude of the preliminary toxicological information evidence of hepatic toxicity in dogs body weight gain decrement that (i.e., identification of target organs) and administered neotame (Refs. 4, 17, and occurred in rats from the neotame for determining the appropriate range of 18). treatment groups (see section II.C.2.d of doses of neotame that would be fed in Several comments asserted that this document and Refs. 4 and 10). FDA the 52-week dog study, the 52-week neotame-related liver toxicity is not disagrees, however, about the necessity study provides a stronger basis for reversible, as is implied by the for additional testing requested by the assessing the potential chronic toxicity petitioner, based primarily on the comment to resolve the body weight of neotame in the dog. Because the increases in both serum ALP levels and gain decrement issue. While the results from this longer-term study relative liver weights in the dog studies. proposed studies might address supersede those of the 13-week study The agency concludes that the mechanistic relationships between food and because all of the effects noted in reversibility of these effects is not consumption and weight gain, the the shorter-term study occurred at levels relevant to a safety decision regarding agency believes that they will not of exposure well above the NOEL chronic ingestion of neotame. While provide meaningful data to explain the established by the 52-week study, the FDA agrees, as noted in section II.C.2.b magnitude of differences in body weight agency concludes that no further of this document, that increases in and body weight gain in neotame- discussion is needed in response to serum ALP levels and relative liver treated rats from the 104-week study in issues raised in comments concerning weights occur in dogs from the 200 and view of the small decreases in food the 13-week dog study. 800 mg/kg bw/d neotame groups in the consumption noted in these animals. In Several comments asserted that 52-week study, neither of these addition, FDA believes that a safe usage elevated serum ALP levels observed in parameters is affected at the lower levels level for neotame can be established the neotame-treated dogs in the 52-week tested (20 or 60 mg/kg bw/d). By from the database provided by the dog study indicate liver toxicity. As considering serum ALP and relative petitioner. As discussed in section discussed in section II.C.2.b of this liver weights as the most sensitive II.C.2.e of this document, the results in document, FDA recognizes that in the endpoints of potential neotame toxicity, the 52-week rat dietary toxicity study 52-week dog study elevated serum ALP the agency determines for the 52-week provide a strong scientific basis to levels are observed in both sexes of dogs dog study that 60 mg/kg bw/d is an resolve concerns over the body weight from as early as 13 weeks until the end appropriate NOEL (Refs. 4, 10, and 17). gain decrement effect (Refs. 4, 10, and of the study at neotame dose levels of Liver as a Target Organ for Neotame 15). Based on the 52-week rat study and 200 and 800 mg/kg bw/d. However, the Toxicity using body weight gain decrement as agency disagrees with comments that One comment emphasized the the most sensitive endpoint for toxicity, these elevated serum ALP levels are importance of the liver in animal growth the agency is able to establish a NOEL evidence of hepatic toxicity. While an and glucose homeostasis. This comment for neotame of 30 mg/kg bw/d. From this increase in serum ALP may be an asserted, based on analyses of the NOEL, FDA derives an ADI for neotame indicator of liver toxicity, such a neotame safety studies database, that of 18 mg/p/d (see table 1 in section II.D conclusion cannot be substantiated in neotame affects growth in both rats and of this document and Ref. 4). the absence of additional corroborative dogs, and appears to affect glucose Serum Alkaline Phosphatase and Liver changes. Specifically, hepatic damage homeostasis in persons with diabetes. Toxicity may result in increased levels of other Based upon these findings, along with Several comments expressed concerns liver enzymes, such as alanine the elevated serum ALP levels in regarding potentially adverse effects of aminotransferase, aspartate neotame-treated dogs and the structure neotame based on changes observed in aminotransferase, or gamma glutamyl of neotame, the comment concluded serum ALP levels in dogs consuming transferase. None of these liver enzymes that it was important to rule out the high doses of neotame (i.e., was elevated in the neotame-treated liver as a target organ. 200 mg/kg bw/d and higher) in both 13- dogs. Also, a decrease in blood albumin In regard to the effect of neotame on week and 52-week feeding studies. levels may indicate chronic liver body weight gain in the rat, the agency Additional comments suggested that toxicity. Blood albumin levels in dogs has established a NOEL of neotame is hepatotoxic, as evidenced by from all neotame dose groups were 30 mg/kg bw/d, based on the 52-week rat effects on other endpoints, such as normal and comparable to control feeding study, as summarized in section changes in absolute and/or relative liver values. Furthermore, an elevation in II.C.2.e of this document. We discuss weight, changes in serum cholesterol serum bilirubin indicates cholestasis; our analyses of the 52-week rat feeding and triglycerides, and neotame-related serum bilirubin levels were unaffected study and our resolution of the body cholestasis. by neotame treatment. weight gain effect in more detail in Refs. The agency notes that most of these Increased cholesterol levels are 10 and 15. comments focused on effects observed another indication of altered liver In regard to the effect of neotame on in the 13-week dog study. In its review function. Plasma cholesterol and body weight and body weight gain in of the subchronic (13-week) dog study, triglyceride levels in dogs from the 52- the 52-week dog feeding study, the the agency observed the liver effects week study, although somewhat effect occurred only in male dogs and referenced in the comments (Ref. 16). variable, were well within the normal only in the highest neotame dose group Ordinarily, in the absence of a longer range for dogs and unaffected by (i.e., 800 mg/kg bw/d) during weeks 1 to duration study, the agency would have neotame treatment. Additionally, 5 and 7 to 8 (Ref. 18). At all other dose given more weight to the results of the histopathological examinations of livers levels tested (i.e., 20, 60, and 13-week dog study. However, a chronic from dogs from the neotame-treated 200 mg/kg bw/d), there were no (52-week) dog study was also submitted groups did not reveal any evidence of statistically significant effects on body in support of the safety of neotame, and necrosis, blockage of bile flow, or any weight or body weight gain in either that study provides for a more complete other abnormalities that were not sex. Furthermore, as discussed in

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section II.C.2.b of this document, the 1,000 mg/kg bw/d and 4,000 mg/kg bw/d, Because of its longer duration, the 52- agency relies on more sensitive respectively. Thus, the agency week study is more definitive than the endpoints, i.e., serum ALP levels and concludes that the hypothetical subchronic (13-week) dog study for relative liver weights, for establishing a formation of nitrosamine compounds assessing the toxicity of neotame. In the NOEL for neotame from the 52-week from neotame poses no safety concerns. 52-week dog study, decreased body dog study. Finally, the agency recognizes that weight gains were noted only at the The agency also disagrees with the one cannot absolutely rule out the liver highest dose tested (800 mg/kg bw/d) comment’s assertion that neotame as a target organ for the toxic effects of and not at any of the lower dose levels appears to affect glucose homeostasis in neotame when it is ingested at (20, 60, and 200 mg/kg bw/d). persons with diabetes. We explain our exaggerated dose levels. However, as Bile Salt Metabolism and Excretion basis for concluding that neotame does discussed in the agency’s response to One comment pointed out that not appear to affect glucose homeostasis this comment, and elsewhere in this neotame produced discolored feces in persons with diabetes later in this document, the agency concludes that at (white and gray) at the highest doses document, in the discussion entitled expected levels of dietary intake of tested (200 and 800 mg/kg bw/d) in the ‘‘Type II Diabetes Study.’’ neotame there is no concern for 52-week dog study. This comment As for changes in serum ALP levels, potential toxic effects to the liver. suggested that the change in fecal color the agency does not consider these to be Systemic Exposure/Body Weight Gain was due to neotame’s effect on bile salt a manifestation of hepatic toxicity in the One comment stated that ‘‘[t]he long- metabolism and excretion. The agency 52-week dog study. Our reasons for term studies conducted in the dog agrees that dogs from the discounting the toxicological species show definite signs of toxicity 800 mg/kg bw/d treatment group significance of the changes in serum which, through close inspection of the frequently excreted gray or white feces. ALP are discussed previously (see pharmacokinetic data generated in the However, there were only two section II.C.2.b of this document and the study and specific PK metabolism incidences of gray feces from animals in fourth subject category in section III studies, is shown to be related to the 200 mg/kg bw/d treatment group (a ‘‘Serum Alkaline Phosphatase and Liver systemic exposure of the parent female on day 322 and a male on day Toxicity’’). compound.’’ Subsequently, the 328), and no changes in appearance of The comment asserted that ‘‘[t]he comment referred to ‘‘a non-linear feces from dogs in the 20 or structure of neotame suggests that the increase in systemic exposure of the 60 mg/kg bw/d treatment groups. There metabolic formation of nitrosamines by parent compound and its metabolite was also one incident of white feces gut microflora is possible as well as over the dose range studied.’’ The observed for a female in the control formation in some food products.’’ The comment asserted that this nonlinear group on day 70 of the study. Based on agency acknowledges that a number of increase in systemic exposure to the this evidence, as well as information in nitrosamine compounds are potent parent compound and its metabolite is section II.C.2.e of this document, the hepatotoxins and hepatocarcinogens. related to decreases in body weight gain agency concludes that there is no The agency also recognizes that neotame in the dog. evidence to support a correlation contains a secondary amine that could In response, the agency notes that the between fecal color and liver toxicity in hypothetically form nitrosoneotame in analysis of PK parameters (i.e., area dogs fed neotame-containing diets the presence of a nitrosating agent. under the curve, and maximum during the 52-week study (Ref. 20). However, there is no scientific evidence concentration) discussed in the Developmental (Teratology) Studies presented in this comment to comment is based on data from the 13- One comment claimed that the dose demonstrate that the presence of week dog study, which the agency does levels of neotame tested in the definitive neotame in food leads to the formation not consider to be a long-term study as rabbit developmental (teratology) study of nitrosoneotame either through claimed in the comment. In the agency’s were too low. The agency disagrees. chemical reaction in food products or by review of this study (Ref. 16), decreased FDA’s evaluation of this study shows metabolic processes in the gut upon body weight gains were observed in that there are statistically significant ingestion (Ref. 14). Furthermore, the dogs of both sexes at dietary neotame decreases in feed consumption and petitioner addressed this issue using intakes of 600 and 2,000 mg/kg bw/d maternal body weights during the many maximizing assumptions (the 2,000 mg/kg bw/d dose level was gestation period. Thus, the highest dose concerning the formation and potency reduced on day 15 to 1,200 mg/kg bw/d in the study (500 mg/kg bw/d) was of the hypothetical nitrosoneotame. In for the remainder of the 13-week study). sufficient to achieve maternal toxicity particular, the petitioner assumed that These extremely high dose levels are (Refs. 4 and 6). In addition, FDA notes nitrosoneotame would be formed and 6,000 to 20,000 times greater than the that this study satisfies dose selection that it would be as potent a carcinogen 90th percentile EDI for neotame. At criteria recommended in the agency’s as dimethylnitrosamine. Based on this lower levels of neotame intake (i.e., 60 Redbook guidelines (Ref. 21). scenario, the petitioner concluded that and 200 mg/kg bw/d), there were no Another comment raised concern over the amounts of nitrosamine that could effects on body weight gain in either post-implantation effects of neotame be formed would be extremely small, sex. In considering the PK parameters based on a maternal toxicity range- that any hypothetical risk would be derived from blood concentration data finding study in the rabbit. Because of trivial, and that additional analyses from the dogs fed these lower levels of the study’s limitations, the agency does were not necessary. After evaluating the neotame, the agency concludes (Ref. 19) not share this concern. While a range- petitioner’s reasoning, FDA agrees with that there was no evidence of increased finding study may aid in identifying a this conclusion (Refs. 1 and 14). systemic exposure to neotame or its compound’s potential target organ Furthermore, as noted in sections metabolites. (It should be noted that PK effects, the primary objective of such a II.C.2.c and II.C.2.d of this document, measurements in the dog were study is to establish appropriate dose there is no evidence of chronic liver evaluated only in the 13-week levels to be further evaluated in a more toxicity or pre-neoplastic or neoplastic subchronic study.) definitive toxicity study. In the study in liver lesions in lifetime carcinogenicity Moreover, as mentioned in Refs. 4, 10, question, the agency notes that only six feeding studies in rats and mice and 17, a chronic (52-week) neotame animals were used in each dose group, ingesting neotame in amounts up to dog feeding study was conducted. too few for an adequate assessment of

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the developmental (teratogenic) the study followed the Redbook decreases in both feed intake and body potential of a compound (Ref. 21). In the guidelines. Additionally, the agency weight gain, at the 500 mg/kg bw/d dose definitive rabbit developmental finds no dose-dependent effects on post- level. Furthermore, there is an (teratology) study, a total of 25 mated implantation data when this study’s appropriate NOEL for these effects (Refs. females were assigned to the control and treated and concurrent control groups 6 and 21). high-dose groups, and 20 each in the are compared (Refs. 6 and 21). This comment also suggested that low- and mid-dose groups (Ref. 6). This In further response to this comment, decreases in food intake and maternal larger number of animals allows for a the agency concludes that the manner in body weight gain noted in the dams more accurate assessment of the which the comment has analyzed the from the 500 mg/kg bw/d dose group teratogenic potential of neotame in the data from the rabbit developmental were due to (tissue) accumulation of rabbit as well as increasing the study is incorrect. More specifically, the neotame. Based on a review of the PK statistical power of the study. In the comment compared control and treated data from the definitive rabbit definitive rabbit teratology study, there groups on a per-fetus, rather than on a developmental study, the agency were no significant dose-dependent, per-litter incidence basis. As recognized concludes that these data do not suggest post-implantation effects due to by authoritative sources567 the maternal that bioaccumulation of neotame or its neotame treatment. animal, not the developing organism, is metabolites would occur even at a dose One comment argued that neotame- randomly and independently assigned level of 500 mg/kg bw/d (Ref. 22). With induced effects on post-implantation to control and treatment groups during regard to a possible relationship loss, fetal size, and limb development in the gestation period. Therefore, the between (tissue) accumulation of rabbits in the teratology study may be analyses of effects should be reported as neotame and decreases in feed intake masked by the quality of the study and incidence-per-litter or as number and and maternal body weight gain, the the high background incidence of these percent of litters with particular agency finds that a mechanistic effects. The comment disagreed with the endpoints. Because the comment’s explanation is unnecessary for an petitioner’s interpretation of the data on analysis is based on inappropriate per- adequate evaluation of the study post-implantation and other fetal fetus comparisons, its conclusions are because the agency has determined an observations. In particular, the comment inherently flawed. Furthermore, the appropriate NOEL for these effects. As asserted that the petitioner’s agency finds that the comparisons noted previously in section II.C.1.b of interpretation of data was scientifically between the concurrent control and this document, based on the evaluation flawed because the petitioner made treated groups, on a percent per-litter of other neotame feeding studies in the comparisons between treatment groups basis, show no treatment-related effects rat and dog, FDA concludes that there and the concurrent control group whose on the litter incidence of any fetal is no concern for the potential incidence percentages, according to the endpoint examined in the rabbit bioaccumulation of neotame or its comment, were higher than those developmental (teratology) study (Refs. metabolites at expected human intake incidence percentages typically seen in 6 and 21). levels. historical control data. One comment focused on the Type II Diabetes Study FDA disagrees with this assessment. dosimetric and pharmacokinetic aspects One comment criticized several By using concurrent control animals, of the rabbit developmental (teratology) aspects of the Type II diabetes study. the study avoided the inherent study. The comment asserted that if a The comment stated that the design of variability that may be introduced into higher dose level, e.g., this study was not adequate to detect data analyses when historical control 1,000 mg/kg bw/d, rather than small differences resulting from data are used in place of concurrent 500 mg/kg bw/d, had been used as the neotame treatment in the parameters control data. Potential sources of top dose in this definitive study, higher examined. It cited the following variability from the use of historical systemic exposure and greater toxicity inadequacies: Limited statistical power, control data include: (1) Differences in would have occurred in the neotame- parameters measured only under the animal husbandry and animal room treated rabbits. As noted earlier with quiescent metabolic condition of environment, (2) differences in diet regard to the levels of neotame tested in extended fasting, short duration, and no compositions, (3) differences in times of this study, the agency finds that overall meal test. Despite these deficiencies, the study conduct, (4) differences in the study design and dose selection were comment recommended inclusion of the sources of nutrients in animal diets, (5) sufficient to achieve maternal toxicity. Type II diabetes study in the safety differences in skills and experience of FDA believes that it is irrelevant if evaluation, because no other studies in technicians or scientists, and (6) genetic greater toxicity were to occur at a higher the neotame safety database investigated drifts, as discussed in Haseman et al., dose level than the highest dose used in the effects of neotame on glucose 19893 and Roe, 1994.4 Therefore, the the rabbit developmental (teratology) homeostasis in patients or animals with agency concludes that, within the study. The highest dose used was diabetes. Finally, the comment definitive rabbit study, in the absence of sufficient to achieve maternal toxicity, concluded that results from the Type II compelling evidence to the contrary, it based on statistically significant diabetes study were strongly suggestive is more appropriate to compare results of a treatment-related effect of neotame between treated and concurrent control 5 FDA, ‘‘Guidelines for Developmental Toxicity on fasting glucose control. animals than to compare results Studies,’’ chapter IV.C.b, section III.D, Redbook FDA agrees that although the between treated animals and historical 2000 Toxicology Principles for the Safety of Food experimental design of the Type II Ingredients (http://www.cfsan.fda.gov/~redbook/ control data. The agency also notes that diabetes study limits its utility for redivc96.html). assessing the potential effects of 6 Tyl, R. W. and M. C. Merr, ‘‘Developmental 3 Haseman, J. K., Huff, J. E., Rao, G. N., and Eustis, Toxicity Testing-Methodology,’’ chapter 7, pp. 217, neotame on glucose homeostasis in S. I., ‘‘Sources of Variability in Rodent Handbook of Developmental Toxicology, edited by Type II diabetics, it should be included Carcinogencity,’’ Fundamental and Applied R. D. Hood, CRC Press, New York, NY, 1997. in the safety evaluation of neotame (Ref. Toxicology, vol. 12(4), pp. 793–804, 1989. 7 Kimmel, C. A. and G. I. Kimmel, ‘‘Principles of 23). Based on findings obtained during 4 Roe, F. J. C., ‘‘Historical Histopathological Developmental Toxicity Risk Assessment,’’ chapter Control Data for Laboratory Rodents: Valuable 21, pp. 671–672, Handbook of Developmental a directed clinical investigator site Treasure or Worthless Trash?’’Laboratory Animals, Toxicology, edited by R. D. Hood, CRC Press, New inspection and audit of study records at vol. 28(2), pp. 148–154, (London), 1994. York, NY, 1997. the facility responsible for this clinical

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trial, FDA concludes that the study was body weight of 20 kg is reported to this action. FDA has concluded that the well-executed, irrespective of range from 0.4 to 0.6 g/p/d or 400 to action will not have a significant impact previously noted design limitations 600 mg/p/d (Ref. 5). on the human environment, and that an (Ref. 23). Using a conservative approach (Refs. environmental impact statement is not The agency disagrees with the 4 and 5), the agency calculates that the required. The agency’s finding of no comment’s conclusion that results from amount of phenylalanine exposure significant impact and the evidence the trial with Type II diabetic subjects expected from the 90th percentile intake supporting that finding, contained in an are strongly suggestive of a treatment- (0.1 mg/kg bw/d) of neotame (Ref. 2) by environmental assessment, may be seen related effect of neotame on glucose a 60 kg adult is 2.64 mg/p/d. FDA finds in the Dockets Management Branch (see control. FDA performed a detailed this amount of exposure trivial in ADDRESSES) between 9 a.m. and 4 p.m., evaluation of the study data on fasting contrast to that expected from the Monday through Friday. glucose pharmacodynamic parameters normal adult diet. For the PKU including: (1) Area under the effect homozygous child, the additional VI. Paperwork Reduction Act of 1995 curve, (2) area under curve, (3) percent phenylalanine intake expected from the This final rule contains no collection perturbation, and (4) normal variations 90th percentile ingestion of neotame of information. Therefore, clearance by in glucose concentrations. Based on (i.e., 0.17 mg/kg bw/d) (Ref. 3) by a 20 kg the Office of Management and Budget these analyses, the agency finds that individual is 1.50 mg/p/d, an under the Paperwork Reduction Act of under the conditions of the study, there incremental amount that is equivalent to 1995 is not required. were no significant changes in these no more than 0.3 to 0.4 percent of the parameters in study subjects that are PKU homozygous child’s normal daily VII. References attributable to neotame (Ref. 23). phenylalanine intake. From these The following references have been Overall, FDA concludes that under the conservative estimates, the agency placed on display in the Dockets conditions of the Type II diabetic study, concludes that the potential intake of Management Branch (see ADDRESSES), blood glucose concentrations in Type II phenylalanine that may result from use and you may review them between 9 diabetic subjects following neotame of neotame as a general-purpose a.m. and 4 p.m., Monday through treatment (at levels ranging from 5 to 15 sweetener does not pose any safety Friday. times the 90th percentile EDI of concern (Refs. 4 and 5). 1. Memorandum from DiNovi, Division of 0.1 mg/kg bw/d) are comparable to those IV. Conclusion Product Manufacture and Use, Chemistry in the same subjects when given a Review Team, to Anderson, Division of placebo, and that any changes noted are The agency has evaluated all the data Product Policy, March 31, 1998. within the normal range of variation and and other information submitted by the 2. Memorandum from DiNovi, Division of not the result of neotame treatment (Ref. petitioner in support of the safe use of Product Manufacture and Use, Chemistry 23). neotame as a general-purpose sweetener Review Team, to Anderson, Division of Methanol and Phenylalanine Formation and concludes that there is a reasonable Product Policy, August 12, 1999; addendum Several comments expressed concern certainty that no harm will result from memorandum to the August 12, 1999, memorandum from DiNovi, Division of that harmful levels of methanol and the use of neotame as proposed. In Biotechnology and GRAS Notification phenylalanine may result from ingesting accordance with a memorandum of Review, to Anderson, Division of Petition neotame-containing foods and understanding (MOU) between the Food Review, February 28, 2002. beverages. FDA disagrees with these Safety and Inspection Service (FSIS), 3. Memorandum from DiNovi, Division of comments. Methanol release results United States Department of Product Manufacture and Use, Chemistry from the de-esterification of neotame, Agriculture, and FDA (65 FR 51758, Review Team, to Anderson, Division of which occurs more rapidly in the rat August 25, 2000), a restriction from use Product Policy, December 14, 2000. and rabbit than in the dog and human ‘‘in meat and poultry’’ appears in the 4. Memorandum from Biddle, Lin, Whiteside, Division of Health Effects (see section II.C.1.d of this document). neotame regulation. This restriction is The agency concludes that, at the 90th Evaluation, to Anderson, Division of Product required when the petitioner does not Policy, January 31, 2001; addendum percentile EDI of neotame, the resultant specify whether the food additive is memorandum to the January 31, 2001, exposure to methanol would be intended for such use. At this time, FSIS memorandum from Whiteside, Division of extremely low, approximately has not made a determination on the use Petition Review, to Anderson, Division of 0.008 mg/kg bw/d (Ref. 5). Humans are of neotame in or on meat or poultry. Petition Review, February 28, 2002. exposed to much higher levels of Therefore, FDA concludes that the food 5. Memorandum from Bleiberg, Division of methanol intake from their daily diet. additive regulations should be amended Health Effects Evaluation, to Anderson, For example, the methanol content of as set forth in this document. Division of Product Policy, January 31, 2001; fruit juices ranges from 64 mg/liter (L) In accordance with § 171.1(h) (21 CFR addendum memorandum to the January 31, in orange juice to 326 mg/L in apricot 2001, memorandum from Biddle, Division of 171.1(h)), the petitions and the Petition Review, to Anderson, Division of juice. In contrast, the methanol content documents that FDA considered and Petition Review, February 28, 2002. of neotame-sweetened carbonated relied upon in reaching its decision to 6. Memorandum from Welsh, Scientific beverages is estimated to be 1.37 mg/L. approve the petitions are available for Support Branch, to Anderson, Division of Similarly, FDA concludes that the inspection at the Center for Food Safety Product Policy, January 31, 2001. potential intake of phenylalanine from and Applied Nutrition by appointment 7. Memorandum from Mattia, Scientific the use of neotame will be extremely with the information contact person. As Support Branch, to Anderson, Division of low in comparison to that present in the provided in § 171.1(h), the agency will Product Policy, January 31, 2001; addendum daily diet. Based upon data cited by delete from the documents any memorandum to the January 31, 2001, memorandum from Biddle, Division of Koch and Wenz, 1984 (see footnote 2 in materials that are not available for section II.C.1.d of this document), the Petition Review, to Anderson, Division of public disclosure before making the Petition Review, April 12, 2002. agency notes that the daily dietary documents available for inspection. 8. Position paper from The NutraSweet Co., intake of phenylalanine for a healthy V. Environmental Effects ‘‘Neotame Does Not Cause Any Behavioral or individual may range from 2.5 to 10 g/ Neurotoxic Effects.’’ p/d. The daily intake of phenylalanine The agency has carefully considered 9. Position paper from The NutraSweet Co., for a PKU homozygous child with a the potential environmental effects of ‘‘Increases in Serum Alkaline Phosphatase in

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the Dog Are Not Associated With Target a waiver of the right to a hearing on that 20740, or at the Office of the Federal Organ Toxicity.’’ objection. Each numbered objection for Register, 800 North Capitol St. NW., 10. Memorandum from Whiteside, Division which a hearing is requested shall suite 700, Washington, DC 20001. of Health Effects Evaluation, to Anderson, include a detailed description and (1) Assay for neotame, not less than Division of Product Policy, January 21, 2001. 11. Memorandum of Conference from the analysis of the specific factual 97.0 percent and not more than 102.0 Center for Food Safety and Applied information intended to be presented in percent on a dry basis. Nutrition—Cancer Assessment Committee, support of the objection in the event (2) Free dipeptide acid (N-[N-(3,3- August 16, 2000. that a hearing is held. Failure to include dimethylbutyl)-L-a-aspartyl]-L- 12. Position paper from The NutraSweet such a description and analysis for any phenylalanine), not more than 1.5 Co., ‘‘ In the Mouse Carcinogenicity Study particular objection shall constitute a percent. with Neotame Small Changes in Body Weight waiver of the right to a hearing on the (3) Other related substances, not more Gain at Some Intervals in Female Mice at objection. Three copies of all documents than 2.0 percent. 50 mg/kg bw Relative to Controls are Due to are to be submitted and are to be (4) Lead, not more than 2.0 milligrams a Decrease in Food Consumption.’’ per kilogram. 13. Memorandum from Chen, Scientific identified with the docket number Support Branch, to Anderson, Division of found in brackets in the heading of this (5) Water, not more than 5.0 percent. Product Policy, July 19, 2000. document. Any objections received in (6) Residue on ignition, not more than 14. Memorandum from DiNovi, Division of response to the regulation may be seen 0.2 percent Product Manufacture and Use, Chemistry (7) Specific rotation, determined at 20 in the Dockets Management Branch ° ° ° Review Team, to Anderson, Division of between 9 a.m. and 4 p.m., Monday C [a]D: -40.0 to 43.4 calculated on a Product Policy, January 10, 2001. through Friday. dry basis. 15. Memorandum from Whiteside, Division (c) The food additive neotame may be of Health Effects Evaluation, to Anderson, List of Subjects in 21 CFR Part 172 safely used as a sweetening agent and Division of Product Policy, January 31, 2001. Food additives, Incorporation by flavor enhancer in foods generally, 16. Memorandum from Ikeda, Division of except in meat and poultry, in Health Effects Evaluation, to Anderson, reference, Reporting and recordkeeping Division of Product Policy, May 28, 1999. requirements. accordance with current good 17. Memorandum from Ikeda, Division of Therefore, under the Federal Food, manufacturing practice, in an amount Health Effects Evaluation, to Biddle, Division Drug, and Cosmetic Act and under not to exceed that reasonably required of Health Effects Evaluation, January 31, authority delegated to the Commissioner to accomplish the intended technical 2001. of Food and Drugs, 21 CFR part 172 is effect, in foods for which standards of 18. Memorandum from Ikeda, Division of amended as follows: identity established under section 401 Health Effects Evaluation, to Anderson, of the Federal Food, Drug, and Cosmetic Division of Product Policy, June 16, 2000; PART 172—FOOD ADDITIVES Act do not preclude such use. addendum memorandum to the June 16, (d) When neotame is used as a sugar 2000, memorandum from Whiteside, PERMITTED FOR DIRECT ADDITION Division of Petition Review, to Anderson, TO FOOD FOR HUMAN substitute tablet, L-leucine may be used Division of Petition Review, February 28, CONSUMPTION as a lubricant in the manufacture of 2002. tablets at a level not to exceed 3.5 19. Memorandum from Bleiberg, Division 1. The authority citation for 21 CFR percent of the weight of the tablet. of Health Effects Evaluation, to Anderson, part 172 continues to read as follows: (e) If the food containing the additive Division of Product Policy, February 5, 2001. Authority: 21 U.S.C. 321, 341, 342, 348, purports to be or is represented to be for 20. Memorandum from Ikeda, Division of 371, 379e. special dietary use, it shall be labeled in Health Effects Evaluation, to Anderson, 2. Section 172.829 is added to subpart compliance with part 105 of this Division of Product Policy, February 5, 2001. I to read as follows: chapter. 21. Memorandum from Shackleford, Division of Heath Effects Evaluation, to § 172.829 Neotame. Dated: July 2, 2002. Anderson, Division of Product Policy, (a) Neotame is the chemical N-[N-(3,3- Margaret M. Dotzel, February 12, 2001. dimethylbutyl)-L-a-aspartyl]-L- Associate Commissioner for Policy. 22. Memorandum from Roth, Division of [FR Doc. 02–17202 Filed 7–5–02; 10:41 am] Health Effects Evaluation, to Anderson, phenylalanine-1-methyl ester (CAS Reg. Division of Product Policy, February 28, No. 165450–17–9). BILLING CODE 4160–01–S 2001. (b) Neotame meets the following 23. Memorandum from Park, Roth, and specifications when it is tested Klontz, Division of Health Effects Evaluation, according to the methods described or DEPARTMENT OF THE TREASURY to Anderson, Division of Product Policy, referenced in the document entitled January 30, 2001. ‘‘Specifications and Analytical Methods Internal Revenue Service for Neotame’’ dated April 3, 2001, by VIII. Objections the NutraSweet Co., 699 North 26 CFR Parts 1 and 602 Any person who will be adversely Wheeling Rd., Mount Prospect, IL [TD 8997] affected by this regulation may at any 60056. The Director of the Office of the time file with the Dockets Management Federal Register has approved the RIN 1545–BA76 Branch (see ADDRESSES) written incorporation by reference of this Carryback of Consolidated Net objections by August 8, 2002. Each material in accordance with 5 U.S.C. Operating Losses To Separate Return objection shall be separately numbered, 552(a) and 1 CFR part 51. Copies are Years; Correction and each numbered objection shall available from the Office of Food specify with particularity the provisions Additive Safety (HFS–200), Center for AGENCY: Internal Revenue Service (IRS), of the regulation to which objection is Food Safety and Applied Nutrition, Treasury. made and the grounds for the objection. 5100 Paint Branch Pkwy., College Park, ACTION: Correction to temporary Each numbered objection on which a MD 20740. Copies may be examined at regulations. hearing is requested shall specifically so the Center for Food Safety and Applied state. Failure to request a hearing for Nutrition’s Library, 5100 Paint Branch SUMMARY: This document contains any particular objection shall constitute Pkwy., rm. 1C–100, College Park, MD corrections to temporary regulations

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