[Federal Register Volume 67, Number 131 (Tuesday, July 9, 2002)]
[Rules and Regulations]
[Pages 45300-45310]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 02-17202]
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DEPARTMENT OF HEALTH AND HUMAN SERVICES
Food and Drug Administration
21 CFR Part 172
[Docket Nos. 98F-0052 and 99F-0187]
Food Additives Permitted for Direct Addition to Food for Human
Consumption; Neotame
AGENCY: Food and Drug Administration, HHS.
ACTION: Final rule.
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SUMMARY: The Food and Drug Administration (FDA) is amending the food
additive regulations to provide for the safe use of neotame as a
nonnutritive sweetener in food. This action is in response to two
petitions filed by Monsanto Co., which subsequently sold the rights to
the petitions to the NutraSweet Co.
DATES: This rule is effective July 9, 2002. Submit objections and
requests for a hearing by August 8, 2002. The Director of the Office of
the Federal Register approves the incorporation by reference in
accordance with 5 U.S.C. 552(a) and 1 CFR part 51 of a certain
publication in 21 CFR 172.829, as of July 9, 2002.
ADDRESSES: Submit written objections and requests for a hearing to the
Dockets Management Branch (HFA-305), Food and Drug Administration, 5630
Fishers Lane, rm. 1061, Rockville, MD 20852. Submit electronic
objections to http://www.fda.gov/dockets/ecomments.
FOR FURTHER INFORMATION CONTACT: Blondell Anderson, Center for Food
Safety and Applied Nutrition (HFS-265), Food and Drug Administration,
5100 Paint Branch Pkwy., College Park, MD 20740-3835, 202-418-3106.
SUPPLEMENTARY INFORMATION:
Table of Contents
I. Introduction
II. Safety Evaluation of Neotame
A. Chemistry and Intake Considerations of Neotame
B. Nature and Extent of Neotame Safety Studies Database
C. Toxicology/Safety Assessment of Neotame
1. Metabolism and Pharmacokinetics of Neotame
a. Absorption of neotame
b. Elimination, distribution, and potential tissue accumulation
of neotame
c. Effect of neotame on drug metabolizing enzymes
d. Metabolites of neotame
2. Critical Toxicology Studies and Issues
a. A 2-generation reproduction study in the rat--neurotoxicity
and behavioral effects
b. Chronic (52-week) dog study--toxicological significance of
elevated serum (hepatic) alkaline phosphatase levels
c. A 104-week mouse carcinogenicity study--body weight gain
decrement effect
d. A 104-week rat carcinogenicity study--body weight gain
decrement effect at all dose levels tested
e. Chronic (52-week) rat feeding study--body weight gain
decrement effect
f. Clinical studies assessments--human tolerance to neotame
D. Estimating an Acceptable Daily Intake for Neotame
III. Comments
IV. Conclusion
V. Environmental Effects
VI. Paperwork Reduction Act of 1995
VII. References
VIII. Objections
I. Introduction
FDA published notices in the Federal Register on February 10, 1998,
and February 8, 1999 (63 FR 6762 and 64 FR 6100, respectively),
announcing that food additive petitions, FAP 8A4580 and FAP 9A4643, had
been filed by Monsanto Co., Skokie, IL 60077. The petitions propose
amending the food additive regulations to provide for the safe use of
neotame as a nonnutritive sweetener for tabletop use (FAP 8A4580) and
for general-purpose use in food (FAP 9A4643) where standards of
identity do not preclude such use. Subsequently, the rights to the
petitions were sold to the NutraSweet Co., 699 North Wheeling Rd.,
suite 103, Mount Prospect, IL 60056. This document grants the petitions
via a regulation approving the general-purpose food use of neotame.
II. Safety Evaluation
A. Chemistry and Intake Considerations of Neotame
Neotame is the common or usual name for the chemical N-[N-(3,3-
dimethylbutyl)-L-[agr]-aspartyl]-L-phenylalanine-1-methyl ester (CAS
Reg. No.165450-17-9). It is synthesized by reductive N-alkylation of L-
phenylalanine-L-[agr]-aspartyl methyl ester with 3,3-
dimethylbutyraldehyde. According to the petitioner, neotame has a
sweetening potency that is approximately 7,000 to 13,000 times that of
sucrose, depending on its food application (Refs. 1 and 2).
The peptidyl linkage in neotame is stabilized by the N-alkyl
substituent and is resistant to hydrolysis under typical use and
storage conditions. Additionally, the N-alkyl substituent effectively
prevents the common dipeptide cyclization reaction that results in the
formation of a diketopiperazine derivative. The data from stability
studies submitted by the petitioner show that the degradation of
neotame in aqueous solutions is pH-, time-, and temperature-dependent.
Based upon data from these stability studies on neotame, the agency
concludes that minor decomposition of neotame could occur in neotame-
containing foods only when stored under conditions that are not
considered typical for a commercial product (Refs. 1 and 2).
The agency has determined the estimated daily intake (EDI) at the
90th percentile for neotame as a general-purpose sweetener to be 0.10
milligram per kilogram (mg/kg) body weight per day (bw/d) for consumers
of all ages (eaters only) and 0.17 mg[sol]kg bw[sol]d for 2 to 5 year
olds (eaters only). The corresponding mean intakes are 0.04 mg[sol]kg
bw[sol]d and 0.05 mg[sol]kg bw[sol]d, respectively (Refs. 2 and 3).
B. Nature and Extent of Neotame Safety Studies Database
In support of the safety of neotame, the petitioner submitted,
within the two petitions, a combined total of 113 preclinical,
clinical, and special studies, plus an additional 32 exploratory and
screening studies in Food Master File No. 575. All pivotal preclinical
studies were conducted in compliance with FDA's ``good laboratory
practice'' regulations in 21 CFR part 58.
The preclinical (animal) studies include short-term, subchronic,
and chronic dietary toxicity tests in the rat, mouse, and dog; multi-
generation
[[Page 45301]]
reproduction and developmental studies in the rat; teratology studies
in the rat and rabbit; and lifetime/carcinogenicity studies in the rat
and mouse. The genotoxicity of neotame, its metabolites, and
decomposition products, are also evaluated in several tests using both
in vitro and in vivo assay systems. Extensive metabolism and
pharmacokinetic measurements were carried out in all animal species
studied. The clinical (human) studies tested the response/acceptance to
orally administered neotame in both men and women during short-term
(e.g., acute, single-dosing) and longer-term (e.g., up to 13 weeks,
repeat-dosing) periods. Pharmacokinetic (PK) measurements also were
carried out in a number of these studies (Ref. 4).
Additionally, the petitioner provided three position papers in
response to FDA questions. These position papers address: (1) The
potential behavioral and neurotoxic effects of neotame, (2) the
significance of elevated serum (hepatic) alkaline phosphatase activity
in neotame-treated dogs as a measure of toxicity, and (3) body weight
gain decrement in mice ingesting neotame. The key aspects of these
position papers are discussed, as appropriate.
C. Toxicology/Safety Assessment of Neotame
1. Metabolism and Pharmacokinetics of Neotame
As a component of the toxicological testing program on neotame, the
petitioner conducted an extensive series of metabolism and PK studies.
These studies were designed to assess: (1) The absorption of neotame;
(2) the elimination, distribution, and potential tissue accumulation of
neotame; (3) the effects of neotame on drug metabolizing enzymes; and
(4) the metabolites of neotame in rodents (rats and mice), dogs,
rabbits, and humans.
a. Absorption of neotame. In all species studied, including humans,
the agency finds that the absorption of ingested neotame occurs almost
entirely in the small intestine. In the animal studies, the absorption
of neotame was determined under fasting conditions using a dose level
that was approximately 150 times greater than the 90th percentile
estimated daily intake (EDI) of neotame for humans. Under these
conditions, the amount of administered dose absorbed is reported to
range from 18 to 38 percent in the rat, 15 to 44 percent in the rabbit,
and 40 to 51 percent in the dog. These studies also indicate that, when
mixed with the diet, the absorption of neotame is reduced. In the human
clinical studies, the absorption of neotame approaches 100 percent in
healthy male and female subjects when administered following an
overnight fast and at dose levels ranging from one to five times the
90th percentile EDI. Individual absorption levels range from 68 to 126
percent (Ref. 5).
b. Elimination, distribution, and potential tissue accumulation of
neotame. The agency estimates that approximately 40 percent of the
systemic elimination of ingested neotame and metabolites occurs via the
urine, and the remainder is eliminated via the fecal route. In a whole-
body radiography study in the rat, following a gavaged dose of
radiolabled neotame and serial sacrifice at timed intervals, post-
dosing, the highest levels of radioactivity are associated with the
intestinal tract, the liver, and the kidney. At final sampling, no
residual radioactivity is detected in peripheral tissues, with some
residual activity associated with the intestinal tract. No organs or
tissues, including the brain, eye, and skin, concentrate or store
radiolabled neotame or its metabolites.
Further evidence for the lack of accumulation of neotame at
expected levels of human intake is found in the analysis of PK
parameters evaluated during a 13-week dog study. In dogs consuming
dietary neotame at dose levels of 1,200 to 2,000 mg[sol]kg bw[sol]d,
there is an indication of saturation of an elimination pathway that
could lead to possible accumulation. However, these levels are at least
10,000 times greater than the 90th percentile EDI (0.1 mg[sol]kg
bw[sol]d) of neotame for humans. This effect is not seen in dogs from
the next lower treatment group (600 mg[sol]kg bw[sol]d), a level
approximately 6,000 times above the 90th percentile EDI. Based on these
findings, the agency concludes there is no concern for possible
accumulation of neotame or its metabolites at expected human intake
levels (Refs. 4 and 5).
c. Effect of neotame on drug metabolizing enzymes. The rat is
generally considered an appropriate animal model to assess the effects
of xenobiotics on phase I (i.e., cytochrome P-450 or mixed-function
amine oxidase microsomal enzyme systems\1\) and phase II (i.e.,
conjugation or biotransformation reactions involving glucuronidation,
sulfation, acetylation, or glutathione-S- transferase reactions)
metabolism. Following a 14-day period during which dietary neotame was
fed at 0 (control), 100, 300, or 1,000 mg[sol]kg bw[sol]d, rats were
sacrificed and in vitro assays performed on isolated liver microsomal
pellets. The agency concludes that, when compared against a positive
control (phenobarbital, a known enzyme inducer), neotame does not
induce P-450 microsomal mixed function oxidase metabolizing enzymes at
any dose level administered during the in vivo phase of the study. In
evaluating the effects of neotame on phase II metabolism, the agency
notes that livers from rats in the 1,000 mg[sol]kg bw[sol]d treatment
group show a statistically significant depression in phase II
metabolism endpoints. However, at the next lower dose of 300 mg[sol]kg
bw[sol]d, which is approximately 3,000 times the 90th percentile EDI
for neotame for humans, there are no effects on these same endpoints
(Ref. 5).
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\1\ Sipes, I. G. and Gandolfi, A. J., ``Biotransformation of
Toxicants,'' chapter 4, pp. 88-109, in Casarett and Doull's
Toxicology: The Basic Science of Poisons, 4th ed., edited by M. O.
Amdur, J. Doul, and C. D. Klaassen, McGraw Hill, Inc., 1993.
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d. Metabolites of neotame. The initial step in the metabolism of
neotame in rats, dogs, rabbits, and humans is de-esterification to N-
[N-(3,3-dimethylbutyl)-L-[agr]-aspartyl]-L-phenylalanine (DMB-Asp-Phe,
coded in the petition as NC-00751) by Ca\++\-dependent pancreatic
esterases or after absorption by plasma esterases. De-esterification of
neotame is similar in all species studied, including humans, although
in the rat and rabbit this conversion occurs at a faster rate than in
the dog and human. The de-esterified metabolite (NC-00751) is rapidly
cleared from the plasma and excreted via the bile duct or in urine
(Ref. 5). A small percentage of NC-00751 may undergo peptide-bond
hydrolysis to form metabolites of dimethylbutylaspartate. The 3,3-
dimethylbutyl portion of DMB-Asp-Phe is then oxidized to 3,3-dimethyl-
butyric acid. This is followed by conjugation with glucuronic acid or
with carnitine (a minor pathway).
Methanol release results from the de-esterification of neotame and
occurs more rapidly in the rat and rabbit than in the dog and human.
The agency concludes that at the 90th percentile EDI for neotame,
exposure to resultant methanol will be insignificant, i.e., not more
than 0.008 mg[sol]kg bw[sol]d. This exposure level is of no
toxicological concern because humans are exposed to much greater levels
of methanol intake from their daily diets (Refs. 4 and 5).
Based on neotame metabolism studies in the rat and dog, FDA
concludes that some intestinal microvillar peptidase activity occurs in
the gut, which results in the formation of other minor plasma
metabolites of neotame, including phenylalanine (Ref. 5). Further
review indicates that approximately 13 to 17
[[Page 45302]]
percent of the total available phenylalanine in the ingested neotame is
released into the plasma after absorption; the remainder is eliminated
in feces and urine as DMB-Asp-Phe. The agency has estimated the amount
of phenylalanine presented to the body from the ingestion of neotame.
The phenylalanine content of neotame is 44 percent by weight. Given
that the 90th percentile neotame EDI for a 60 kg adult is 0.10
mg[sol]kg bw[sol]d or 6 mg/d, and for a 2 to 5 year old (20 kg) child
is 0.17 mg[sol]kg bw[sol]d or 3.4 mg/d, the estimated 90th percentile
phenylalanine intake is 2.6 mg and 1.5 mg, respectively.
The agency notes that, for healthy adults, the daily dietary intake
of phenylalanine may range from 2.5 to 10 grams per person per day (g/
p/d), while that for a phenylketonuric (PKU) homozygous child (20 kg)
may range from 0.4 to 0.6 g/p/d (Koch and Wenz\2\). Thus, the amount of
phenylalanine from the 90th percentile intake of neotame is trivial
compared to that from the normal adult diet. Even for the PKU
homozygous child, the incremental amount of phenylalanine intake that
can be expected from neotame is insignificant, i.e., equivalent to no
more than 0.3 to 0.4 percent of the daily phenylalanine intake of the
PKU homozygous child (Ref. 5). The agency concludes that the potential
intake of phenylalanine that may result from use of neotame as a
general-purpose sweetener does not pose any safety concern (Refs. 4 and
5).
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\2\ Koch, R. and Wenz E. J., ``Aspartame Ingestion by
Phenylketonuric Heterozygous and Homozygous Individuals,'' chapter
30, pp. 593-603, in Physiology and Biochemistry, edited by Stegink,
L. D. and L. J. Filer, Jr., 1984.
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Based on reviews of the metabolism and pharmacokinetic studies on
neotame, the agency concludes that the metabolism of neotame is
qualitatively similar across all species studied. Furthermore, there is
no evidence that, at expected levels of intake, neotame or its
metabolites will accumulate in the body or that ingestion of neotame
will have any adverse effect in the body on Phase I and II metabolism.
The metabolites of neotame are well characterized, and the potential
intakes of metabolites, such as methanol and phenylalanine, are of no
toxicological consequence. Therefore, the agency's review of the
metabolism and pharmacokinetic studies of neotame does not raise any
safety concerns (Refs. 4 and 5).
2. Critical Toxicology Studies and Issues
FDA reviewed all studies and supplemental information submitted by
the petitioner. During its review, the agency determined that certain
studies were more important than others to a regulatory decision on
neotame. This determination was based on the nature of the endpoints
investigated in these studies (i.e., reproductive and developmental
effects, long-term exposure, chronic toxicity, carcinogenic potential,
and human tolerance), and on specific issues presented by these
studies. The critical studies and issues presented by the studies are:
(1) The 2-generation reproduction study in rats--neurotoxicity and
behavioral effects, (2) the chronic (52-week) dog study--toxicological
significance of increased serum (hepatic) alkaline phosphatase levels,
(3) the 104-week mouse carcinogenicity study--body weight gain
decrement effect, (4) the 104-week rat carcinogenicity study--body
weight gain decrement effect at all dose levels tested, (5) the chronic
(52-week) rat feeding study--body weight gain decrement effect, and (6)
the human clinical trials--human tolerance to neotame.
a. A 2-generation reproduction study in the rat--neurotoxicity and
behavioral effects. Reproductive performance and fertility were
assessed over two generations in CD (cesarean derived) rats fed diets
containing neotame at levels of 0 (control), 100, 300, or 1,000
mg[sol]kg bw[sol]d. Each treatment group consisted of 28 males and 28
females. Animals were mated, the resultant offspring weaned, and the F1
generation animals selected and allowed to mature for 10 weeks and then
mated. The F2 litters were terminated, post-weaning. Under the
conditions of this study, the agency concludes that neotame has no
effects on the reproduction or fertility of rats exposed to neotame at
levels up to 1,000 mg[sol]kg bw[sol]d for two generations. Nor are
there any treatment effects on measures of physical development, e.g.,
pinna unfolding, hair growth, tooth eruption, or eye opening (Refs. 4
and 6).
The 2-generation study included tests of motor activity and
cognitive function. General motor activity was measured in F1 offspring
by counting breaks in a pair of infrared light beams over a 12-hour
period, while cognitive function was assessed by recording swim times
up to 60 seconds maximally in six consecutive trials per animal in a
water-filled Y-maze (Ref. 7). While the petitioner concludes there were
no significant treatment effects on motor activity in F1 male and
female offspring, the agency's analyses of pertinent data show a
statistically significant reduction in motor activity among F1 males
from the 1,000 mg[sol]kg bw[sol]d neotame treatment group. No effects
are noted on motor activity in F1 females at any dose level.
With regard to results from the swim-maze tests that were conducted
in F1 offspring at approximately 24 to 28 days of age, both the
petitioner and the agency conclude that there is a statistically
significant increase in mean swimming time (an indicator of reduced
performance) to the ``correct'' arm of the Y-maze in F1 males from the
1,000 mg[sol]kg bw[sol]d group. Specifically, this increased swim time
is noted in two of six trials in the F1 males from the high dose group.
While an increase in swim time is also noted for one of six trials in
F1 males from the 300 mg[sol]kg bw[sol]d dose group, this singular
observation is not accompanied by any other indication of treatment-
related behavioral changes and therefore is not considered to be
indicative of a biologically relevant effect. As with motor activity,
there are no effects on cognitive performance (as measured by swim maze
times) noted in F1 female offspring from any treatment group.
The F1 offspring from the 2-generation reproduction study also were
subjected to specific tests that measured the development of auditory
and visual responses. The agency's evaluation of results on auditory
startle, pupil closure, and visual placing show no treatment-related
effects in F1 males or females at any level of neotame tested.
The finding of statistically significant effects on two separate
behavioral tests (i.e., motor activity and swim maze times) in F1 males
from the 1,000 mg[sol]kg bw[sol]d dose group supports the conclusion
that this dose is an effect level. Based on the findings from the
studies of motor activity and cognitive function, the agency considers
the 300 mg[sol]kg bw[sol]d dose to be a no observed adverse effect
level (NOAEL) for these endpoints (Refs. 4 and 7).
Early in its evaluation of the neotame safety database, the agency
determined that the petitioner should provide a more specific
assessment addressing the potential neurotoxicity and behavioral
effects of neotame. In response to the agency's request, the petitioner
submitted a position paper entitled ``Neotame Does Not Cause Any
Behavioral or Neurotoxic Effects'' (Ref. 8). This document contains
summaries and discussions of data and information from two principal
sources. The first involves several ``key'' preclinical studies (12 in
all) and 4 clinical studies from the neotame studies database. The
second source of information discussed in the position paper is a
series of 20 publications that are primarily related to aspartame.
Collectively, these 20 publications provide little information
[[Page 45303]]
that is relevant to the agency's overall safety assessment of neotame
and are not discussed further.
With regard to the ``key'' animal studies, the petitioner states in
its position paper that these studies incorporated clinical
observations/testing enhancements as ``effective procedures for
detecting neurotoxic effects.'' During the ante mortem phase of the
animal studies, these enhancements included detailed physical,
behavioral, and clinical observations to detect signs of neurological
disorder, behavioral abnormality, physiological dysfunction, and other
signs of nervous system toxicity. Post mortem enhancements included
extensive histopathological evaluations of brain, spinal cord, and
peripheral nerves.
FDA has reviewed thoroughly all of the preclinical and clinical
studies discussed in the position paper. With the exception of the 2-
generation rat reproduction study in which statistically significant
decreases in motor activity and statistically significant increases in
swim times are observed in F1 offspring males at 1,000 mg[sol]kg
bw[sol]d, the preclinical studies do not show behavioral or neurotoxic
effects associated with the ingestion of neotame.
Based on available preclinical and clinical information from the
neotame studies database, the agency concludes that there is no concern
for potential neurotoxic or behavioral effects in humans from the
ingestion of neotame as a general-purpose sweetener in foods. This
conclusion is reinforced further by the NOAEL of 300 mg[sol]kg bw[sol]d
established for motor activity and cognitive performance in F1 males
from the 2-generation reproduction study, a dose level that is at least
3,000 times greater than the 90th percentile EDI of 0.1 mg[sol]kg
bw[sol]d (Refs. 4 and 7).
b. Chronic (52-week) dog study--toxicological significance of
elevated serum (hepatic) alkaline phosphatase. Beagle dogs were fed
diets containing neotame at levels of 0 (control), 20, 60, 200, or 800
mg[sol]kg bw[sol]d over a 52-week period. Detailed data were collected
on animal survival, growth, food intake, clinical chemistries,
hematology, urinalyses, and gross organ pathology and histopathology.
At the conclusion of the study, a limited number of dogs from the
neotame treatment groups were placed on a control diet for an
additional 4-week ``reversibility period.'' During the agency's review
of this study, a question arose about the toxicological significance of
increased serum alkaline phosphatase (ALP) levels (of hepatic origin)
noted in female dogs from the 200 mg[sol]kg bw[sol]d dose group and in
both sexes at the 800 mg[sol]kg bw[sol]d dose group. Other effects
noted were statistically significant dose-related increases in absolute
liver weights and in relative liver weights (liver to brain weight
ratio) in female dogs in the 200 and 800 mg[sol]kg bw[sol]d dose
groups. There was no evidence of histopathological changes in the
liver, brain, sciatic nerve, and spinal cord or in other organs or
tissues examined from neotame-treated dogs.
Because elevated serum ALP levels had also been observed in shorter
duration studies (2-week and 13-week) in dogs ingesting neotame
containing diets, the agency requested that the petitioner provide
further clarification on this matter. In its response, the petitioner
submitted a position paper entitled ``Increases in Serum Alkaline
Phosphatase in the Dog Are Not Associated with Target Organ Toxicity,''
together with several publications related to hepatotoxicity and serum
ALP activity (Ref. 9). In this position paper, the petitioner reasons
that the increased serum ALP levels observed in neotame-treated dogs
are not due to a hepatotoxic response, but to a ``nonspecific,
physiological response'' to the high doses of neotame.
FDA conducted further statistical analyses on the liver weight
parameters mentioned previously. Based on these analyses, the agency
concludes that the means for these liver effects from the 200 and 800
mg[sol]kg bw[sol]d dose groups are statistically significantly higher
than the means for the 0 (control), 20, and 60 mg[sol]kg bw[sol]d
treatment groups. Furthermore, there are no statistically significant
differences between the 0 (control), 20, and 60 mg[sol]kg bw[sol]d dose
group means for any of the liver weight parameters that were evaluated.
From the review of the data from the 52-week dog study and the
supplemental information submitted by the petitioner in its position
paper, the agency concludes that the changes in serum ALP levels are
most likely due to a nontoxic response to the higher levels (200 and
800 mg[sol]kg bw[sol]d) of administered neotame. This conclusion is
based on the following: (1) There are no significant effects from
neotame on other liver enzymes (e.g., alanine aminotransferase,
aspartate aminotransferase, gamma glutamyl transferase), (2) serum
albumin levels are not decreased in neotame-treated dogs (a decrease
would have been an indicator of chronic liver toxicity), (3) serum
bilirubin levels are normal in both sexes at high doses of neotame (an
increase would have been seen if cholestasis was occurring), and (4)
the liver in both sexes and at all dose levels appears normal on
histopathological examination. In this 52-week dog study, FDA
establishes a no observed effect level (NOEL) of 60 mg[sol]kg bw[sol]d,
based on liver effects (e.g., serum (hepatic) alkaline phosphatase and
relative liver weights) as the most sensitive endpoints (Refs. 4 and
10).
c. A 104-week mouse carcinogenicity study--body weight gain
decrement effect. CD-1 mice were fed neotame-containing diets for 104
weeks at levels of 0 (control), 50, 400, 2,000, or 4,000 mg[sol]kg
bw[sol]d. Based on an evaluation of the histopathological data from
this carcinogenicity study, FDA concludes that, under the conditions of
the study, doses of neotame up to 4,000 mg[sol]kg bw[sol]d administered
to male and female CD-1 mice for up to 2 years did not induce
neoplastic lesions (Ref. 11).
Although there was no evidence of carcinogenicity in mice exposed
to neotame for 104 weeks, during the agency's review of other
endpoints, we noted negative effects on body weight gain (and thus body
weight) in both sexes. In light of only small decreases in cumulative
food consumption, the agency was concerned about the potential
toxicological significance of the decrease in body weight gain. In
response to the agency's request for further clarification on this
issue, the petitioner submitted a position paper entitled ``In the
Mouse Carcinogenicity Study With Neotame Small Changes in Body Weight
Gain at Some Intervals in Female Mice at 50 mg[sol]kg bw Relative to
Controls Are Due to a Decrease in Food Consumption'' (Ref. 12). In its
analysis, the petitioner states that the mouse is not a reliable model
for determining the relationship between body weight gain and food
consumption. Reasons cited include the small differences in body weight
gain over a lifetime in mice, both in absolute terms and in proportion
to initial body weights at the start of a study, and well-known
difficulties in obtaining accurate measures of food intake for mice
(e.g., mice frequently spill food from their food cups and contaminate
their food with feces and urine). The petitioner reiterated its belief
that the body weight gain decrements noted in mice during the 104-week
study were due to a small but consistent reduction in food consumption
which is attributable to poor diet palatability and should not be
viewed as a toxicological response to neotame.
In further evaluation of this body gain weight decrement issue, FDA
subjected the data on body weight, body weight gain, and adjusted (for
neotame content)
[[Page 45304]]
food intake to extensive statistical evaluation. Using an analysis of
covariance model and pair-wise dose comparisons of body weights and
body weight gain, the agency notes statistically significant effects
for the 400, 2,000, and 4,000 mg[sol]kg bw[sol]d dose groups. Based on
these analyses, the agency concludes that the body weight gain
decrement effect in both male and female mice in the three highest dose
groups is not accounted for by the small decreases in food consumption.
However, in the 50 mg[sol]kg bw[sol]d treatment group, the effects on
body weight and body weight gain are not statistically different from
controls. Based on the detailed statistical evaluation of data
pertinent to the body weight gain decrement noted in the 104-week
dietary carcinogenicity study in mice, the agency establishes a NOEL of
50 mg[sol]kg bw[sol]d for this endpoint (Refs. 4 and 10).
d. A 104-week rat carcinogenicity study--body weight gain decrement
effect at all dose levels tested. A 104-week rat carcinogenicity study
(with an in utero phase) was conducted during which neotame was fed at
0 (control), 50, 500, or 1,000 mg[sol]kg bw[sol]d. Based on a thorough
evaluation of the histopathological data from this carcinogenicity
study, FDA concludes there is no evidence of neotame-induced neoplastic
lesions in rats ingesting diets containing neotame at levels up to
1,000 mg[sol]kg bw[sol]d for 104 weeks (Ref. 11).
During its review of the 104-week rat carcinogenicity study, the
agency noted effects on body weight gain (and thus body weight) in both
sexes of neotame-treated rats at all dose levels tested. Statistically
significant decreases in cumulative body weight gains were observed at
various intervals throughout the study. At week interval 0 to 52,
cumulative body weight gains were 9 to 11 percent less and 13 to 19
percent less, respectively, in neotame-treated male and female rats,
than in control animals. Similar effects were noted at week intervals 0
to 78 and 0 to 104, i.e, cumulative body weight gains ranging from 10
to 13 percent less in treated males and 17 to 20 percent less in
treated females. In reporting this information, the petitioner suggests
that the lower body weights and lower body weight gains among neotame-
treated rats can be attributed to reduced food intake due to reduced
palatability of the diets containing neotame.
The agency, however, based on an analysis of the food intake data,
concludes that the decreases in adjusted (for neotame content) food
intake among the neotame-treated rats are small and do not fully
explain the magnitude of the differences in body weight and body weight
gain observed in these animals at week 52 and thereafter up to week
104. In view of the significant body weight gain decrement effect
observed in all neotame treatment groups during the 104-week rat
carcinogenicity study, a NOEL cannot be established. Lacking a suitable
explanation for this effect based on decreased food intake (as argued
by the petitioner), the agency considered the body weight gain
decrement effect unresolved by the 104-week rat study (Refs. 4 and 10).
e. Chronic (52-week) rat feeding study--body weight gain decrement
effect. In order to resolve the body weight gain decrement issue in
rats, the agency carried out a thorough analysis of data from a 52-week
rat feeding study. This study employed a wide range of neotame dose
levels, two of which were below the lowest dose tested in the 104-week
rat carcinogenicity study (as discussed in section II.C.2.d of this
document). The results of this analysis are presented in the following
paragraphs.
In the chronic (52-week) rat feeding study (with an in utero phase)
rats received neotame at 0 (control), 10, 30, 100, 300, or 1,000
mg[sol]kg bw[sol]d. Except for body weight and body weight gain, there
were no statistically significant treatment-related effects of neotame
during this 52-week feeding study. With respect to both body weight and
body weight gain, female rats appear to be more sensitive than males.
In regard to body weight, at the end of the 52-week study, body
weights in females from the 100, 300, and 1,000 mg[sol]kg bw[sol]d
groups were statistically significantly lower than those of control
female rats. However, the body weights of females from the 10 and 30
mg[sol]kg bw[sol]d groups were not statistically different from control
females. Among males, only the 100 mg[sol]kg bw[sol]d group had
statistically significant body weight differences from control male
rats.
As for cumulative body weight gains during the 0 to 52-week
interval, statistically significant decreases are noted in treated
females, compared to controls, only from the 300 and 1,000 mg[sol]kg
bw[sol]d treatment groups. While the body weight gains in females from
the 100 mg[sol]kg bw[sol]d are lower than in control female rats, this
difference is not statistically significant. Compared with controls,
there are no significant differences in cumulative body weight gains in
females from the two lowest treatment groups (10 and 30 mg[sol]kg
bw[sol]d) for the 0 to 52-week interval. Cumulative body weight gains
in male rats from the 30, 100, 300, and 1,000 mg[sol]kg bw[sol]d
neotame treatment groups, while somewhat lower than controls, are not
statistically different. As noted in the 104-week carcinogenicity
study, female rats in the 52-week dietary study were more sensitive to
body weight gain decrement effects than males.
FDA performed a detailed analysis of the results from the 52-week
dietary rat study and concludes that this study provides an adequate
basis to assess the body weight gain decrement effect noted in the 104-
week carcinogenicity rat study for four reasons. First, the range of
neotame dose levels studied in the 52-week study is comparable to the
doses tested in the 104-week study. Second, in each study, the female
rat is more sensitive. Third, a parallel comparison of the 52-week
study and the first 52 weeks of the carcinogenicity study shows that
the body weight gain decrement effect was of a similar order of
magnitude in both studies. Fourth, the magnitude of decrease in body
weight gain occurring during week interval 0 to 52 in the 104-week
study does not worsen during the last half of the study. These
observations add strength to the utility of the 52-week dietary rat
study in resolving any concern about the body weight gain decrement
effect and in establishing a NOEL of 30 mg[sol]kg bw[sol]d for this
endpoint (Refs. 4 and 10).
f. Clinical studies assessments--human tolerance to neotame. The
petitioner submitted the results of six human clinical trials that
investigated the ingestion of neotame under varied conditions,
including acute-single exposure, acute-repeat exposure, and short-term
(2-week) and longer-term (13-week) daily exposure. Five of these trials
employed healthy adult subjects, while one trial evaluated non-insulin
dependent diabetes mellitus (Type II diabetic) adult subjects. In each
of these trials, subject tolerance to neotame intake was determined by
physical examinations, vital signs, electrocardiograms, routine
clinical laboratory measurements (e.g., hematology, clinical
chemistries, and urinalysis), and self-assessments of adverse
experiences.
The levels of neotame administered in these clinical trials ranged
from 1 to 15 times the 90th percentile EDI level of 0.1 mg[sol]kg
bw[sol]d or 6 mg per person per day (mg/p/d).
The agency concludes that in all six trials there are no treatment-
related effects reported for any of the parameters examined. Although
headache was the most frequently noted adverse experience, the
incidence of
[[Page 45305]]
headache is comparable for the treated and control groups and is not
considered to be associated with neotame intake. Results from ancillary
pharmacokinetic measurements in several of the clinical trials do not
raise any safety concerns. In the trial with Type II diabetic subjects,
no adverse effects are noted in any of the subjects. Under the
conditions of that trial, the agency concludes that the ingestion of
neotame at levels up to 1.5 mg[sol]kg bw[sol]d does not produce
significant changes in either fasting-state glucose or insulin levels
in Type II diabetic subjects.
Based on reviews of these clinical trials, the agency concludes
that the ingestion of neotame at levels up to 1.5 mg[sol]kg bw[sol]d
(15 times the 90th percentile EDI) for a period as long as 13 weeks is
well tolerated by healthy male and female subjects. The agency also
concludes that in the study with Type II diabetic subjects, the intake
of neotame at levels up to 1.5 mg[sol]kg bw[sol]d does not have
significant effects on fasting plasma glucose or insulin levels in
study subjects (Refs. 4 and 13).
D. Estimating an Acceptable Daily Intake for Neotame
In determining an acceptable daily intake (ADI) for a new food
additive, the agency relies on a comprehensive evaluation of all
relevant studies and information submitted by the petitioner. As the
agency's evaluation of the neotame safety studies database progressed,
four studies with attendant issues emerged as having the greatest
impact in reaching a safety decision; these studies are highlighted in
table 1 of this document.
Table 1.--Summary of Study Data Pertinent to Establishing an Acceptable Daily Intake Value for Neotame
--------------------------------------------------------------------------------------------------------------------------------------------------------
NOEL (mg[sol]kg
Study Information Pivotal Endpoint bw[sol]d) Safety Factor\A\ ADI (mg/p/d)
--------------------------------------------------------------------------------------------------------------------------------------------------------
2-Generation Reproduction (Rat) Motor Activity and Cognitive Function (300)\B\ 1,000 18
in F1 Males
52-week (Dog) Serum (Hepatic) ALP Levels and Relative 60 100 36
Liver Weights in Females
104-week (Mouse) Body Weight Gain Decrement in Both 50 100 30
Sexes
52-week (Rat) Body Weight Gain Decrement in Females 30 100 18
--------------------------------------------------------------------------------------------------------------------------------------------------------
\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 identified for the most sensitive
endpoint in each of the four studies, ADI values were determined
ranging from a high of 36 mg[sol]p[sol]d to a low of 18 mg[sol]p[sol]d.
In taking a conservative approach, the agency concludes that the
appropriate ADI for neotame is 18 mg[sol]p[sol]d (Ref. 4). This level
is three times higher than the 90th percentile EDI for neotame of 6
mg[sol]p[sol]d.
III. Comments
Thirty comments were submitted to FDA's Dockets Management Branch
in response to the filing of the two neotame food additive petitions
(25 for FAP 8A4580 and 5 for FAP 9A4643). The issues raised in the
comments are identified and grouped into the following subject
categories.
Aspartame
The majority of the comments compared neotame to aspartame. In
these comparisons, the comments assumed that neotame produces the same
metabolic breakdown products as aspartame and thus would be responsible
for the same health effects they allege to be associated with
aspartame, which is the subject of a food additive regulation (21 CFR
172.804). In response to these comments, FDA points out that neotame is
chemically and metabolically different (see section II.A of this
document and Ref. 1, and section II.C.1 of this document, respectively)
from aspartame even though they are structurally related. Therefore,
the comments' assertions about neotame are without basis. Because the
comments do not provide the agency with any information regarding the
safety of neotame, they will not be discussed further.
Estimated Daily Intake
Several comments objected to the tabletop use petition on the basis
that the petitioner's EDI for neotame is inaccurate, implying that it
is too low. In determining an EDI, FDA makes projections based on the
amount of the additive proposed for use in particular foods and on data
regarding the consumption levels of these particular foods, commonly
using the 90th percentile as a measure of high chronic exposure. The
agency concludes that the 90th percentile EDI calculated for neotame,
as discussed in section II.A of this document, accurately reflects the
exposure to neotame as a general-purpose sweetener in all foods (except
for meats and poultry), including tabletop use (Ref. 2).
One comment noted that the petitioner assumes that neotame will
replace 50 percent of aspartame's current applications and argued that
this assumption may be limited unduly and not sufficiently
conservative. FDA agrees with the comment on this point, and disagrees
with the petitioner's use of the 50 percent replacement factor in their
estimation of exposure to neotame. The agency conservatively assumes
that this new sweetener will replace all existing uses of aspartame
(Ref. 14) and uses this estimate in its safety evaluation.
No Observed Effect Level, Body Weight, and Body Weight Gain Effects
One comment stated that there is no NOEL established by the 104-
week rat carcinogenicity study for neotame, because all doses show
adverse effects on growth. The comment also asserted that the data
contained in this study do not support the petitioner's explanation
that decreases in body weights in the treated rats are due to reduced
palatability of the neotame-containing diets. In addition, the comment
indicated that the petitioner did not supply any gavage, pair-feeding,
or dietary restriction studies to prove that the body weight gain
decrements are due to palatability and not toxicity. The comment also
claimed that a safe usage level for neotame cannot be determined from
the safety database provided in the neotame food additive petitions.
FDA agrees that a NOEL cannot be established based on the 104-week
rat carcinogenicity study, in view of the body weight gain (decrement)
effect. The agency also notes that, while neotame may have had some
influence
[[Page 45306]]
on diet palatability, the decreases in food intake (adjusted for
neotame content) among neotame-treated rats of both sexes in the 104-
week study are too small to explain the magnitude of the body weight
gain decrement that occurred in rats from the neotame treatment groups
(see section II.C.2.d of this document and Refs. 4 and 10). FDA
disagrees, however, about the necessity for additional testing
requested by the comment to resolve the body weight gain decrement
issue. While the proposed studies might address mechanistic
relationships between food consumption and weight gain, the agency
believes that they will not provide meaningful data to explain the
magnitude of differences in body weight and body weight gain in
neotame-treated rats from the 104-week study in view of the small
decreases in food consumption noted in these animals. In addition, FDA
believes that a safe usage level for neotame can be established from
the database provided by the petitioner. As discussed in section
II.C.2.e of this document, the results in the 52-week rat dietary
toxicity study provide a strong scientific basis to resolve concerns
over the body weight gain decrement effect (Refs. 4, 10, and 15). Based
on the 52-week rat study and using body weight gain decrement as the
most sensitive endpoint for toxicity, the agency is able to establish a
NOEL for neotame of 30 mg[sol]kg bw[sol]d. From this NOEL, FDA derives
an ADI for neotame of 18 mg[sol]p[sol]d (see table 1 in section II.D of
this document and Ref. 4).
Serum Alkaline Phosphatase and Liver Toxicity
Several comments expressed concerns regarding potentially adverse
effects of neotame based on changes observed in serum ALP levels in
dogs consuming high doses of neotame (i.e., 200 mg[sol]kg bw[sol]d and
higher) in both 13-week and 52-week feeding studies. Additional
comments suggested that neotame is hepatotoxic, as evidenced by effects
on other endpoints, such as changes in absolute and/or relative liver
weight, changes in serum cholesterol and triglycerides, and neotame-
related cholestasis.
The agency notes that most of these comments focused on effects
observed in the 13-week dog study. In its review of the subchronic (13-
week) dog study, the agency observed the liver effects referenced in
the comments (Ref. 16). Ordinarily, in the absence of a longer duration
study, the agency would have given more weight to the results of the
13-week dog study. However, a chronic (52-week) dog study was also
submitted in support of the safety of neotame, and that study provides
for a more complete manifestation of the target organ toxicity in
neotame-treated dogs.
While the agency considers the 13-week dog study useful for
obtaining preliminary toxicological information (i.e., identification
of target organs) and for determining the appropriate range of doses of
neotame that would be fed in the 52-week dog study, the 52-week study
provides a stronger basis for assessing the potential chronic toxicity
of neotame in the dog. Because the results from this longer-term study
supersede those of the 13-week study and because all of the effects
noted in the shorter-term study occurred at levels of exposure well
above the NOEL established by the 52-week study, the agency concludes
that no further discussion is needed in response to issues raised in
comments concerning the 13-week dog study.
Several comments asserted that elevated serum ALP levels observed
in the neotame-treated dogs in the 52-week dog study indicate liver
toxicity. As discussed in section II.C.2.b of this document, FDA
recognizes that in the 52-week dog study elevated serum ALP levels are
observed in both sexes of dogs from as early as 13 weeks until the end
of the study at neotame dose levels of 200 and 800 mg[sol]kg bw[sol]d.
However, the agency disagrees with comments that these elevated serum
ALP levels are evidence of hepatic toxicity. While an increase in serum
ALP may be an indicator of liver toxicity, such a conclusion cannot be
substantiated in the absence of additional corroborative changes.
Specifically, hepatic damage may result in increased levels of other
liver enzymes, such as alanine aminotransferase, aspartate
aminotransferase, or gamma glutamyl transferase. None of these liver
enzymes was elevated in the neotame-treated dogs. Also, a decrease in
blood albumin levels may indicate chronic liver toxicity. Blood albumin
levels in dogs from all neotame dose groups were normal and comparable
to control values. Furthermore, an elevation in serum bilirubin
indicates cholestasis; serum bilirubin levels were unaffected by
neotame treatment.
Increased cholesterol levels are another indication of altered
liver function. Plasma cholesterol and triglyceride levels in dogs from
the 52-week study, although somewhat variable, were well within the
normal range for dogs and unaffected by neotame treatment.
Additionally, histopathological examinations of livers from dogs from
the neotame-treated groups did not reveal any evidence of necrosis,
blockage of bile flow, or any other abnormalities that were not
detected in control animals. Collectively, these observations support
the agency's conclusion that data from the 52-week study do not show
evidence of hepatic toxicity in dogs administered neotame (Refs. 4, 17,
and 18).
Several comments asserted that neotame-related liver toxicity is
not reversible, as is implied by the petitioner, based primarily on the
increases in both serum ALP levels and relative liver weights in the
dog studies. The agency concludes that the reversibility of these
effects is not relevant to a safety decision regarding chronic
ingestion of neotame. While FDA agrees, as noted in section II.C.2.b of
this document, that increases in serum ALP levels and relative liver
weights occur in dogs from the 200 and 800 mg[sol]kg bw[sol]d neotame
groups in the 52-week study, neither of these parameters is affected at
the lower levels tested (20 or 60 mg[sol]kg bw[sol]d). By considering
serum ALP and relative liver weights as the most sensitive endpoints of
potential neotame toxicity, the agency determines for the 52-week dog
study that 60 mg[sol]kg bw[sol]d is an appropriate NOEL (Refs. 4, 10,
and 17).
Liver as a Target Organ for Neotame Toxicity
One comment emphasized the importance of the liver in animal growth
and glucose homeostasis. This comment asserted, based on analyses of
the neotame safety studies database, that neotame affects growth in
both rats and dogs, and appears to affect glucose homeostasis in
persons with diabetes. Based upon these findings, along with the
elevated serum ALP levels in neotame-treated dogs and the structure of
neotame, the comment concluded that it was important to rule out the
liver as a target organ.
In regard to the effect of neotame on body weight gain in the rat,
the agency has established a NOEL of 30 mg[sol]kg bw[sol]d, based on
the 52-week rat feeding study, as summarized in section II.C.2.e of
this document. We discuss our analyses of the 52-week rat feeding study
and our resolution of the body weight gain effect in more detail in
Refs. 10 and 15.
In regard to the effect of neotame on body weight and body weight
gain in the 52-week dog feeding study, the effect occurred only in male
dogs and only in the highest neotame dose group (i.e., 800 mg[sol]kg
bw[sol]d) during weeks 1 to 5 and 7 to 8 (Ref. 18). At all other dose
levels tested (i.e., 20, 60, and 200 mg[sol]kg bw[sol]d), there were no
statistically significant effects on body weight or body weight gain in
either sex. Furthermore, as discussed in
[[Page 45307]]
section II.C.2.b of this document, the agency relies on more sensitive
endpoints, i.e., serum ALP levels and relative liver weights, for
establishing a NOEL for neotame from the 52-week dog study.
The agency also disagrees with the comment's assertion that neotame
appears to affect glucose homeostasis in persons with diabetes. We
explain our basis for concluding that neotame does not appear to affect
glucose homeostasis in persons with diabetes later in this document, in
the discussion entitled ``Type II Diabetes Study.''
As for changes in serum ALP levels, the agency does not consider
these to be a manifestation of hepatic toxicity in the 52-week dog
study. Our reasons for discounting the toxicological significance of
the changes in serum ALP are discussed previously (see section II.C.2.b
of this document and the fourth subject category in section III ``Serum
Alkaline Phosphatase and Liver Toxicity'').
The comment asserted that ``[t]he structure of neotame suggests
that the metabolic formation of nitrosamines by gut microflora is
possible as well as formation in some food products.'' The agency
acknowledges that a number of nitrosamine compounds are potent
hepatotoxins and hepatocarcinogens. The agency also recognizes that
neotame contains a secondary amine that could hypothetically form
nitrosoneotame in the presence of a nitrosating agent. However, there
is no scientific evidence presented in this comment to demonstrate that
the presence of neotame in food leads to the formation of
nitrosoneotame either through chemical reaction in food products or by
metabolic processes in the gut upon ingestion (Ref. 14). Furthermore,
the petitioner addressed this issue using many maximizing assumptions
concerning the formation and potency of the hypothetical
nitrosoneotame. In particular, the petitioner assumed that
nitrosoneotame would be formed and that it would be as potent a
carcinogen as dimethylnitrosamine. Based on this scenario, the
petitioner concluded that the amounts of nitrosamine that could be
formed would be extremely small, that any hypothetical risk would be
trivial, and that additional analyses were not necessary. After
evaluating the petitioner's reasoning, FDA agrees with this conclusion
(Refs. 1 and 14). Furthermore, as noted in sections II.C.2.c and
II.C.2.d of this document, there is no evidence of chronic liver
toxicity or pre-neoplastic or neoplastic liver lesions in lifetime
carcinogenicity feeding studies in rats and mice ingesting neotame in
amounts up to 1,000 mg[sol]kg bw[sol]d and 4,000 mg[sol]kg bw[sol]d,
respectively. Thus, the agency concludes that the hypothetical
formation of nitrosamine compounds from neotame poses no safety
concerns.
Finally, the agency recognizes that one cannot absolutely rule out
the liver as a target organ for the toxic effects of neotame when it is
ingested at exaggerated dose levels. However, as discussed in the
agency's response to this comment, and elsewhere in this document, the
agency concludes that at expected levels of dietary intake of neotame
there is no concern for potential toxic effects to the liver.
Systemic Exposure/Body Weight Gain
One comment stated that ``[t]he long-term studies conducted in the
dog species show definite signs of toxicity which, through close
inspection of the pharmacokinetic data generated in the study and
specific PK metabolism studies, is shown to be related to systemic
exposure of the parent compound.'' Subsequently, the comment referred
to ``a non-linear increase in systemic exposure of the parent compound
and its metabolite over the dose range studied.'' The comment asserted
that this nonlinear increase in systemic exposure to the parent
compound and its metabolite is related to decreases in body weight gain
in the dog.
In response, the agency notes that the analysis of PK parameters
(i.e., area under the curve, and maximum concentration) discussed in
the comment is based on data from the 13-week dog study, which the
agency does not consider to be a long-term study as claimed in the
comment. In the agency's review of this study (Ref. 16), decreased body
weight gains were observed in dogs of both sexes at dietary neotame
intakes of 600 and 2,000 mg[sol]kg bw[sol]d (the 2,000 mg[sol]kg
bw[sol]d dose level was reduced on day 15 to 1,200 mg[sol]kg bw[sol]d
for the remainder of the 13-week study). These extremely high dose
levels are 6,000 to 20,000 times greater than the 90th percentile EDI
for neotame. At lower levels of neotame intake (i.e., 60 and 200
mg[sol]kg bw[sol]d), there were no effects on body weight gain in
either sex. In considering the PK parameters derived from blood
concentration data from the dogs fed these lower levels of neotame, the
agency concludes (Ref. 19) that there was no evidence of increased
systemic exposure to neotame or its metabolites. (It should be noted
that PK measurements in the dog were evaluated only in the 13-week
subchronic study.)
Moreover, as mentioned in Refs. 4, 10, and 17, a chronic (52-week)
neotame dog feeding study was conducted. Because of its longer
duration, the 52-week study is more definitive than the subchronic (13-
week) dog study for assessing the toxicity of neotame. In the 52-week
dog study, decreased body weight gains were noted only at the highest
dose tested (800 mg[sol]kg bw[sol]d) and not at any of the lower dose
levels (20, 60, and 200 mg[sol]kg bw[sol]d).
Bile Salt Metabolism and Excretion
One comment pointed out that neotame produced discolored feces
(white and gray) at the highest doses tested (200 and 800 mg[sol]kg
bw[sol]d) in the 52-week dog study. This comment suggested that the
change in fecal color was due to neotame's effect on bile salt
metabolism and excretion. The agency agrees that dogs from the 800
mg[sol]kg bw[sol]d treatment group frequently excreted gray or white
feces. However, there were only two incidences of gray feces from
animals in the 200 mg[sol]kg bw[sol]d treatment group (a female on day
322 and a male on day 328), and no changes in appearance of feces from
dogs in the 20 or 60 mg[sol]kg bw[sol]d treatment groups. There was
also one incident of white feces observed for a female in the control
group on day 70 of the study. Based on this evidence, as well as
information in section II.C.2.e of this document, the agency concludes
that there is no evidence to support a correlation between fecal color
and liver toxicity in dogs fed neotame-containing diets during the 52-
week study (Ref. 20).
Developmental (Teratology) Studies
One comment claimed that the dose levels of neotame tested in the
definitive rabbit developmental (teratology) study were too low. The
agency disagrees. FDA's evaluation of this study shows that there are
statistically significant decreases in feed consumption and maternal
body weights during the gestation period. Thus, the highest dose in the
study (500 mg[sol]kg bw[sol]d) was sufficient to achieve maternal
toxicity (Refs. 4 and 6). In addition, FDA notes that this study
satisfies dose selection criteria recommended in the agency's Redbook
guidelines (Ref. 21).
Another comment raised concern over post-implantation effects of
neotame based on a maternal toxicity range-finding study in the rabbit.
Because of the study's limitations, the agency does not share this
concern. While a range-finding study may aid in identifying a
compound's potential target organ effects, the primary objective of
such a study is to establish appropriate dose levels to be further
evaluated in a more definitive toxicity study. In the study in
question, the agency notes that only six animals were used in each dose
group, too few for an adequate assessment of
[[Page 45308]]
the developmental (teratogenic) potential of a compound (Ref. 21). In
the definitive rabbit developmental (teratology) study, a total of 25
mated females were assigned to the control and high-dose groups, and 20
each in the low- and mid-dose groups (Ref. 6). This larger number of
animals allows for a more accurate assessment of the teratogenic
potential of neotame in the rabbit as well as increasing the
statistical power of the study. In the definitive rabbit teratology
study, there were no significant dose-dependent, post-implantation
effects due to neotame treatment.
One comment argued that neotame-induced effects on post-
implantation loss, fetal size, and limb development in rabbits in the
teratology study may be masked by the quality of the study and the high
background incidence of these effects. The comment disagreed with the
petitioner's interpretation of the data on post-implantation and other
fetal observations. In particular, the comment asserted that the
petitioner's interpretation of data was scientifically flawed because
the petitioner made comparisons between treatment groups and the
concurrent control group whose incidence percentages, according to the
comment, were higher than those incidence percentages typically seen in
historical control data.
FDA disagrees with this assessment. By using concurrent control
animals, the study avoided the inherent variability that may be
introduced into data analyses when historical control data are used in
place of concurrent control data. Potential sources of variability from
the use of historical control data include: (1) Differences in animal
husbandry and animal room environment, (2) differences in diet
compositions, (3) differences in times of study conduct, (4)
differences in the sources of nutrients in animal diets, (5)
differences in skills and experience of technicians or scientists, and
(6) genetic drifts, as discussed in Haseman et al., 1989\3\ and Roe,
1994.\4\ Therefore, the agency concludes that, within the definitive
rabbit study, in the absence of compelling evidence to the contrary, it
is more appropriate to compare results between treated and concurrent
control animals than to compare results between treated animals and
historical control data. The agency also notes that the study followed
the Redbook guidelines. Additionally, the agency finds no dose-
dependent effects on post-implantation data when this study's treated
and concurrent control groups are compared (Refs. 6 and 21).
---------------------------------------------------------------------------
\3\ Haseman, J. K., Huff, J. E., Rao, G. N., and Eustis, S. I.,
``Sources of Variability in Rodent Carcinogencity,'' Fundamental and
Applied Toxicology, vol. 12(4), pp. 793-804, 1989.
\4\ Roe, F. J. C., ``Historical Histopathological Control Data
for Laboratory Rodents: Valuable Treasure or Worthless
Trash?''Laboratory Animals, vol. 28(2), pp. 148-154, (London), 1994.
---------------------------------------------------------------------------
In further response to this comment, the agency concludes that the
manner in which the comment has analyzed the data from the rabbit
developmental study is incorrect. More specifically, the comment
compared control and treated groups on a per-fetus, rather than on a
per-litter incidence basis. As recognized by authoritative sources\5\
\6\ \7\ the maternal animal, not the developing organism, is randomly
and independently assigned to control and treatment groups during the
gestation period. Therefore, the analyses of effects should be reported
as incidence-per-litter or as number and percent of litters with
particular endpoints. Because the comment's analysis is based on
inappropriate per-fetus comparisons, its conclusions are inherently
flawed. Furthermore, the agency finds that the comparisons between the
concurrent control and treated groups, on a percent per-litter basis,
show no treatment-related effects on the litter incidence of any fetal
endpoint examined in the rabbit developmental (teratology) study (Refs.
6 and 21).
---------------------------------------------------------------------------
\5\ FDA, ``Guidelines for Developmental Toxicity Studies,''
chapter IV.C.b, section III.D, Redbook 2000 Toxicology Principles
for the Safety of Food Ingredients (http://www.cfsan.fda.gov/
[tilde]redbook/redivc96.html).
\6\ Tyl, R. W. and M. C. Merr, ``Developmental Toxicity Testing-
Methodology,'' chapter 7, pp. 217, Handbook of Developmental
Toxicology, edited by R. D. Hood, CRC Press, New York, NY, 1997.
\7\ Kimmel, C. A. and G. I. Kimmel, ``Principles of
Developmental Toxicity Risk Assessment,'' chapter 21, pp. 671-672,
Handbook of Developmental Toxicology, edited by R. D. Hood, CRC
Press, New York, NY, 1997.
---------------------------------------------------------------------------
One comment focused on the dosimetric and pharmacokinetic aspects
of the rabbit developmental (teratology) study. The comment asserted
that if a higher dose level, e.g., 1,000 mg[sol]kg bw[sol]d, rather
than 500 mg[sol]kg bw[sol]d, had been used as the top dose in this
definitive study, higher systemic exposure and greater toxicity would
have occurred in the neotame-treated rabbits. As noted earlier with
regard to the levels of neotame tested in this study, the agency finds
that overall study design and dose selection were sufficient to achieve
maternal toxicity. FDA believes that it is irrelevant if greater
toxicity were to occur at a higher dose level than the highest dose
used in the rabbit developmental (teratology) study. The highest dose
used was sufficient to achieve maternal toxicity, based on
statistically significant decreases in both feed intake and body weight
gain, at the 500 mg[sol]kg bw[sol]d dose level. Furthermore, there is
an appropriate NOEL for these effects (Refs. 6 and 21).
This comment also suggested that decreases in food intake and
maternal body weight gain noted in the dams from the 500 mg[sol]kg
bw[sol]d dose group were due to (tissue) accumulation of neotame. Based
on a review of the PK data from the definitive rabbit developmental
study, the agency concludes that these data do not suggest that
bioaccumulation of neotame or its metabolites would occur even at a
dose level of 500 mg[sol]kg bw[sol]d (Ref. 22). With regard to a
possible relationship between (tissue) accumulation of neotame and
decreases in feed intake and maternal body weight gain, the agency
finds that a mechanistic explanation is unnecessary for an adequate
evaluation of the study because the agency has determined an
appropriate NOEL for these effects. As noted previously in section
II.C.1.b of this document, based on the evaluation of other neotame
feeding studies in the rat and dog, FDA concludes that there is no
concern for the potential bioaccumulation of neotame or its metabolites
at expected human intake levels.
Type II Diabetes Study
One comment criticized several aspects of the Type II diabetes
study. The comment stated that the design of this study was not
adequate to detect small differences resulting from neotame treatment
in the parameters examined. It cited the following inadequacies:
Limited statistical power, parameters measured only under the quiescent
metabolic condition of extended fasting, short duration, and no meal
test. Despite these deficiencies, the comment recommended inclusion of
the Type II diabetes study in the safety evaluation, because no other
studies in the neotame safety database investigated the effects of
neotame on glucose homeostasis in patients or animals with diabetes.
Finally, the comment concluded that results from the Type II diabetes
study were strongly suggestive of a treatment-related effect of neotame
on fasting glucose control.
FDA agrees that although the experimental design of the Type II
diabetes study limits its utility for assessing the potential effects
of neotame on glucose homeostasis in Type II diabetics, it should be
included in the safety evaluation of neotame (Ref. 23). Based on
findings obtained during a directed clinical investigator site
inspection and audit of study records at the facility responsible for
this clinical
[[Page 45309]]
trial, FDA concludes that the study was well-executed, irrespective of
previously noted design limitations (Ref. 23).
The agency disagrees with the comment's conclusion that results
from the trial with Type II diabetic subjects are strongly suggestive
of a treatment-related effect of neotame on glucose control. FDA
performed a detailed evaluation of the study data on fasting glucose
pharmacodynamic parameters including: (1) Area under the effect curve,
(2) area under curve, (3) percent perturbation, and (4) normal
variations in glucose concentrations. Based on these analyses, the
agency finds that under the conditions of the study, there were no
significant changes in these parameters in study subjects that are
attributable to neotame (Ref. 23). Overall, FDA concludes that under
the conditions of the Type II diabetic study, blood glucose
concentrations in Type II diabetic subjects following neotame treatment
(at levels ranging from 5 to 15 times the 90th percentile EDI of 0.1
mg[sol]kg bw[sol]d) are comparable to those in the same subjects when
given a placebo, and that any changes noted are within the normal range
of variation and not the result of neotame treatment (Ref. 23).
Methanol and Phenylalanine Formation
Several comments expressed concern that harmful levels of methanol
and phenylalanine may result from ingesting neotame-containing foods
and beverages. FDA disagrees with these comments. Methanol release
results from the de-esterification of neotame, which occurs more
rapidly in the rat and rabbit than in the dog and human (see section
II.C.1.d of this document). The agency concludes that, at the 90th
percentile EDI of neotame, the resultant exposure to methanol would be
extremely low, approximately 0.008 mg[sol]kg bw[sol]d (Ref. 5). Humans
are exposed to much higher levels of methanol intake from their daily
diet. For example, the methanol content of fruit juices ranges from 64
mg/liter (L) in orange juice to 326 mg/L in apricot juice. In contrast,
the methanol content of neotame-sweetened carbonated beverages is
estimated to be 1.37 mg/L.
Similarly, FDA concludes that the potential intake of phenylalanine
from the use of neotame will be extremely low in comparison to that
present in the daily diet. Based upon data cited by Koch and Wenz, 1984
(see footnote 2 in section II.C.1.d of this document), the agency notes
that the daily dietary intake of phenylalanine for a healthy individual
may range from 2.5 to 10 g/p/d. The daily intake of phenylalanine for a
PKU homozygous child with a body weight of 20 kg is reported to range
from 0.4 to 0.6 g/p/d or 400 to 600 mg[sol]p[sol]d (Ref. 5).
Using a conservative approach (Refs. 4 and 5), the agency
calculates that the amount of phenylalanine exposure expected from the
90th percentile intake (0.1 mg[sol]kg bw[sol]d) of neotame (Ref. 2) by
a 60 kg adult is 2.64 mg[sol]p[sol]d. FDA finds this amount of exposure
trivial in contrast to that expected from the normal adult diet. For
the PKU homozygous child, the additional phenylalanine intake expected
from the 90th percentile ingestion of neotame (i.e., 0.17 mg[sol]kg
bw[sol]d) (Ref. 3) by a 20 kg individual is 1.50 mg[sol]p[sol]d, an
incremental amount that is equivalent to no more than 0.3 to 0.4
percent of the PKU homozygous child's normal daily phenylalanine
intake. From these conservative estimates, the agency concludes that
the potential intake of phenylalanine that may result from use of
neotame as a general-purpose sweetener does not pose any safety concern
(Refs. 4 and 5).
IV. Conclusion
The agency has evaluated all the data and other information
submitted by the petitioner in support of the safe use of neotame as a
general-purpose sweetener and concludes that there is a reasonable
certainty that no harm will result from the use of neotame as proposed.
In accordance with a memorandum of understanding (MOU) between the Food
Safety and Inspection Service (FSIS), United States Department of
Agriculture, and FDA (65 FR 51758, August 25, 2000), a restriction from
use ``in meat and poultry'' appears in the neotame regulation. This
restriction is required when the petitioner does not specify whether
the food additive is intended for such use. At this time, FSIS has not
made a determination on the use of neotame in or on meat or poultry.
Therefore, FDA concludes that the food additive regulations should be
amended as set forth in this document.
In accordance with Sec. 171.1(h) (21 CFR 171.1(h)), the petitions
and the documents that FDA considered and relied upon in reaching its
decision to approve the petitions are available for inspection at the
Center for Food Safety and Applied Nutrition by appointment with the
information contact person. As provided in Sec. 171.1(h), the agency
will delete from the documents any materials that are not available for
public disclosure before making the documents available for inspection.
V. Environmental Effects
The agency has carefully considered the potential environmental
effects of this action. FDA has concluded that the action will not have
a significant impact on the human environment, and that an
environmental impact statement is not required. The agency's finding of
no significant impact and the evidence supporting that finding,
contained in an environmental assessment, may be seen in the Dockets
Management Branch (see ADDRESSES) between 9 a.m. and 4 p.m., Monday
through Friday.
VI. Paperwork Reduction Act of 1995
This final rule contains no collection of information. Therefore,
clearance by the Office of Management and Budget under the Paperwork
Reduction Act of 1995 is not required.
VII. References
The following references have been placed on display in the Dockets
Management Branch (see ADDRESSES), and you may review them between 9
a.m. and 4 p.m., Monday through Friday.
1. Memorandum from DiNovi, Division of Product Manufacture and
Use, Chemistry Review Team, to Anderson, Division of Product Policy,
March 31, 1998.
2. Memorandum from DiNovi, Division of Product Manufacture and
Use, Chemistry Review Team, to Anderson, Division of Product Policy,
August 12, 1999; addendum memorandum to the August 12, 1999,
memorandum from DiNovi, Division of Biotechnology and GRAS
Notification Review, to Anderson, Division of Petition Review,
February 28, 2002.
3. Memorandum from DiNovi, Division of Product Manufacture and
Use, Chemistry Review Team, to Anderson, Division of Product Policy,
December 14, 2000.
4. Memorandum from Biddle, Lin, Whiteside, Division of Health
Effects Evaluation, to Anderson, Division of Product Policy, January
31, 2001; addendum memorandum to the January 31, 2001, memorandum
from Whiteside, Division of Petition Review, to Anderson, Division
of Petition Review, February 28, 2002.
5. Memorandum from Bleiberg, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, January 31,
2001; addendum memorandum to the January 31, 2001, memorandum from
Biddle, Division of Petition Review, to Anderson, Division of
Petition Review, February 28, 2002.
6. Memorandum from Welsh, Scientific Support Branch, to
Anderson, Division of Product Policy, January 31, 2001.
7. Memorandum from Mattia, Scientific Support Branch, to
Anderson, Division of Product Policy, January 31, 2001; addendum
memorandum to the January 31, 2001, memorandum from Biddle, Division
of Petition Review, to Anderson, Division of Petition Review, April
12, 2002.
8. Position paper from The NutraSweet Co., ``Neotame Does Not
Cause Any Behavioral or Neurotoxic Effects.''
9. Position paper from The NutraSweet Co., ``Increases in Serum
Alkaline Phosphatase in
[[Page 45310]]
the Dog Are Not Associated With Target Organ Toxicity.''
10. Memorandum from Whiteside, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, January 21,
2001.
11. Memorandum of Conference from the Center for Food Safety and
Applied Nutrition--Cancer Assessment Committee, August 16, 2000.
12. Position paper from The NutraSweet Co., `` In the Mouse
Carcinogenicity Study with Neotame Small Changes in Body Weight Gain
at Some Intervals in Female Mice at 50 mg[sol]kg bw Relative to
Controls are Due to a Decrease in Food Consumption.''
13. Memorandum from Chen, Scientific Support Branch, to
Anderson, Division of Product Policy, July 19, 2000.
14. Memorandum from DiNovi, Division of Product Manufacture and
Use, Chemistry Review Team, to Anderson, Division of Product Policy,
January 10, 2001.
15. Memorandum from Whiteside, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, January 31,
2001.
16. Memorandum from Ikeda, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, May 28, 1999.
17. Memorandum from Ikeda, Division of Health Effects
Evaluation, to Biddle, Division of Health Effects Evaluation,
January 31, 2001.
18. Memorandum from Ikeda, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, June 16, 2000;
addendum memorandum to the June 16, 2000, memorandum from Whiteside,
Division of Petition Review, to Anderson, Division of Petition
Review, February 28, 2002.
19. Memorandum from Bleiberg, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, February 5,
2001.
20. Memorandum from Ikeda, Division of Health Effects
Evaluation, to Anderson, Division of Product Policy, February 5,
2001.
21. Memorandum from Shackleford, Division of Heath Effects
Evaluation, to Anderson, Division of Product Policy, February 12,
2001.
22. Memorandum from Roth, Division of Health Effects Evaluation,
to Anderson, Division of Product Policy, February 28, 2001.
23. Memorandum from Park, Roth, and Klontz, Division of Health
Effects Evaluation, to Anderson, Division of Product Policy, January
30, 2001.
VIII. Objections
Any person who will be adversely affected by this regulation may at
any time file with the Dockets Management Branch (see ADDRESSES)
written objections by August 8, 2002. Each objection shall be
separately numbered, and each numbered objection shall specify with
particularity the provisions of the regulation to which objection is
made and the grounds for the objection. Each numbered objection on
which a hearing is requested shall specifically so state. Failure to
request a hearing for any particular objection shall constitute a
waiver of the right to a hearing on that objection. Each numbered
objection for which a hearing is requested shall include a detailed
description and analysis of the specific factual information intended
to be presented in support of the objection in the event that a hearing
is held. Failure to include such a description and analysis for any
particular objection shall constitute a waiver of the right to a
hearing on the objection. Three copies of all documents are to be
submitted and are to be identified with the docket number found in
brackets in the heading of this document. Any objections received in
response to the regulation may be seen in the Dockets Management Branch
between 9 a.m. and 4 p.m., Monday through Friday.
List of Subjects in 21 CFR Part 172
Food additives, Incorporation by reference, Reporting and
recordkeeping requirements.
Therefore, under the Federal Food, Drug, and Cosmetic Act and under
authority delegated to the Commissioner of Food and Drugs, 21 CFR part
172 is amended as follows:
PART 172--FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR
HUMAN CONSUMPTION
1. The authority citation for 21 CFR part 172 continues to read as
follows:
Authority: 21 U.S.C. 321, 341, 342, 348, 371, 379e.
2. Section 172.829 is added to subpart I to read as follows:
Sec. 172.829 Neotame.
(a) Neotame is the chemical N-[N-(3,3-dimethylbutyl)-L-[agr]-
aspartyl]-L-phenylalanine-1-methyl ester (CAS Reg. No. 165450-17-9).
(b) Neotame meets the following specifications when it is tested
according to the methods described or referenced in the document
entitled ``Specifications and Analytical Methods for Neotame'' dated
April 3, 2001, by the NutraSweet Co., 699 North Wheeling Rd., Mount
Prospect, IL 60056. The Director of the Office of the Federal Register
has approved the incorporation by reference of this material in
accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies are available
from the Office of Food Additive Safety (HFS-200), Center for Food
Safety and Applied Nutrition, 5100 Paint Branch Pkwy., College Park, MD
20740. Copies may be examined at the Center for Food Safety and Applied
Nutrition's Library, 5100 Paint Branch Pkwy., rm. 1C-100, College Park,
MD 20740, or at the Office of the Federal Register, 800 North Capitol
St. NW., suite 700, Washington, DC 20001.
(1) Assay for neotame, not less than 97.0 percent and not more than
102.0 percent on a dry basis.
(2) Free dipeptide acid (N-[N-(3,3-dimethylbutyl)-L-[agr]-
aspartyl]-L-phenylalanine), not more than 1.5 percent.
(3) Other related substances, not more than 2.0 percent.
(4) Lead, not more than 2.0 milligrams per kilogram.
(5) Water, not more than 5.0 percent.
(6) Residue on ignition, not more than 0.2 percent
(7) Specific rotation, determined at 20 deg.C [[agr]]D:
-40.0 deg. to 43.4 deg. calculated on a dry basis.
(c) The food additive neotame may be safely used as a sweetening
agent and flavor enhancer in foods generally, except in meat and
poultry, in accordance with current good manufacturing practice, in an
amount not to exceed that reasonably required to accomplish the
intended technical effect, in foods for which standards of identity
established under section 401 of the Federal Food, Drug, and Cosmetic
Act do not preclude such use.
(d) When neotame is used as a sugar substitute tablet, L-leucine
may be used as a lubricant in the manufacture of tablets at a level not
to exceed 3.5 percent of the weight of the tablet.
(e) If the food containing the additive purports to be or is
represented to be for special dietary use, it shall be labeled in
compliance with part 105 of this chapter.
Dated: July 2, 2002.
Margaret M. Dotzel,
Associate Commissioner for Policy.
[FR Doc. 02-17202 Filed 7-5-02; 10:41 am]
BILLING CODE 4160-01-S