ASPARTAME
Explanation
Aspartame was first evaluated by JECFA in 1975 (WHO, 1975).
At that time a special problem was posed by the presence of the
conversion product, 5-benzyl-3,6-dioxo-2-piperazine (diketopiperazine,
DKP) and no ADI for man was allocated. It was again considered by
JECFA in 1976 and its consideration was deferred in view of the
incompleteness of the information available (WHO, 1976). In 1977
JECFA had evidence that the problem with diketopiperazine was of no
significance and concluded that the safety of aspartame had been
adequately demonstrated; the Committee was prepared to establish an
ADI for man, but because of the assertion that the data base from
which the conclusions were drawn required validation the Committee
deferred its decision pending an assurance that the toxicological data
were valid (WHO, 1978). In 1979 JECFA was presented with evidence of
validation of the toxicological data and accepted the validation;
however, the Committee did not have sufficient time to reassess the
data on aspartame which were evaluated by the previous meeting (WHO,
1980).
The present monograph contains summaries of data examined by
JECFA in 1975, 1976, 1977 and 1980.
BIOLOGICAL DATA
BIOCHEMICAL STUDIES
Aspartame (10 mg) was incubated with pepsin (0.4 mg) in a KCl-HCl
buffer pH 1.0 at 37°C for 15 min. Pepsin showed neither esterase nor
peptidase activity when aspartame was the substrate. Aspartame was not
hydrolysed when incubated with dog gastric juice. Methyl 14C-labelled
aspartame was administered to rats whose stomach had been ligated
at the pylorus. Examination of the stomach contents 4 h after
administration of the test compound, showed no hydrolyses of the
methyl group and essentially complete recovery of the administered
dose. Incubation of Methyl 14C-labelled aspartame with fresh rat
plasma resulted in demethylation of the aspartame (Anonymous, 1972a).
Rat
Male rats (300 g) were dosed orally with 0.5 ml of an aqueous
solution of phenylalanine 14C-aspartame (10 mg/ml). Blood samples
were taken 1, 2 and 4 h after administration. Afterwards the animals
were sacrificed and GI tract removed and divided into stomach, small
intestine and colon. About 50% of the administered dose was absorbed
within 4 h, and 28-30% remained in the GI tract, mainly in the colon.
Chromatographic separation of the plasma radioactivity showed that
most of the radioactivity was probably associated with proteins,
peptides and amino acid conjugates. In another study rats were
administered a single dose of phenylalanine 14C-aspartame, and urine
samples collected for up to 17 days post-dosing. Less than 5% of the
administered radiolabel was excreted in the urine during this period,
with most being excreted within the first two days post-dosing
(Anonymous, 1972a).
In another study the metabolism of methyl 14C-aspartame,
phenylalanine 14C-aspartame, and aspartyl 14C-aspartame was studied
and compared with that of methanol 14C, phenylalanine 14C and
aspartic acid 14C. In these studies male albino rats (Charles River
strain), ca 250-300 g, were dosed orally with a single dose of the
test compound, at a level equivalent to 20-30 mg/kg. The CO2 in the
expired air was collected for a period 8 h post-dosing. Urine and
faeces were collected separately. Rats used for plasma studies weighed
300-400 g; indwelling carotid catheters had been inserted 2 days
before dosing. The methyl group of aspartame was metabolized in a
manner similar to that of methanol, with major portion of the
radiolabel appearing in the expired CO2, (53-73%)/(48-64%),
with traces in the urine (2-7.5%)/(1.4-2.4%), and in the faeces
(0.5-1%)/(0.02-0.09%) for methanol/aspartame respectively. The plasma
concentration of the radiolabel was similar for methanol and the
aspartame. Comparison of the metabolism of phenylalanine and aspartame
showed that the amount of label converted to CO2, and excreted into
the urine from the amino acid was greater than that of the aspartame
(13-24%)/(7-15%) for CO2, and (2.65-4.62%)/(0.7-4.3%) for urine, for
phenylalanine/aspartame respectively. Only trace amounts of radiolabel
were present in the faeces in one study. However, in a subsequent
study, with phenylalanine-14C-aspartame the level of radiolabel
recovered in the faeces ranged from 3-48%. Forty-seven to fifty-three
per cent. radiolabel from the phenyl-alanine 14C and 2.0-17% from the
aspartame was incorporated into the carcass. Total recovery of 14C in
these studies ranged from 14 to 85%. A direct comparison of aspartic
acid 14C, aspartyl 14C-aspartame is not possible because of different
times of collection of CO2. However, 68% of the radiolabel from the
aspartame was excreted as CO2 in a 48 h period (Anonymous, 1972a).
Groups of six adult male rats (S-D derived Charles River
CD-strain) were dosed by gavage with a 3:1 mixture of aspartame/DKP at
a dose level of 27 mg/kg bw for five days. On day 4, one group of rats
was cannulated to permit the removal of blood for plasma studies. On
day 6, following an overnight fast, the rats were dosed with the same
dose of 14C-aspartame/DKP mixture previously used. Blood samples were
taken from the cannulated rats at 0.5, 1, 2, 3, 4, 6 and 24 h post-
dosing. The other group of rats was used for collection of expired
14CO2 (7 h) and measurement of 14C excreted in urine and faeces (24,
48, 72 and 96 h post-dosing). Five rats respired maximum amounts of
14CO2 in 1 h, the sixth peaked at 30 min. The cumulative 14CO2
expired ranged from 12 to 23% of the administered dose. Two to four
per cent. of the total administered 14C dose was excreted in the
urine, with maximum excretion within the first 24 h. Total faecal
excretion of 14C ranged from 1.6 to 3.1% of the total dose. Peak
blood levels of 14C were attained in 3 to 4 h and showed little
change during the course of the study. The maximum level was estimated
as 0.7% of the administered dose. No aspartame or DKP was detected in
the plasma, at 0.5 h post-dosing. At subsequent time intervals through
24 h the major 14C components in hydrolysed serum protein were
present as phenylalanine or its metabolite tyrosine (Anonymous,
1972b).
Mice
Six young adult male HAM/ICR-derived (Charles River CD-1 strain)
outbred albino mice, were given by gavage aspartame at a dose level of
20 mg/kg bw daily for six days. The mice were fasted overnight and
then administered a single oral dose of 14C-labelled aspartame. CO2
from expired air was collected up to 7 h post-dosing, and urine and
faeces collected at 24 h intervals up to 96 h post-dosing. At
termination of the study the animals were sacrificed and autopsied.
Eleven to twenty-six per cent. of the administered dose of radiolabel
was expired as CO2, during the 7 h period, with 4.5-8.18% being
excreted within the first 30 min. Only small amounts of 14C were
excreted in the urine (less than 1-3.7%), with peak excretion during
the first 7 or 24 h. The cumulative faecal excretion was 4.4-7.49% of
the administered dose. For the study of plasma levels a group of 14
male mice were dosed with aspartame and then 14C-labelled aspartame
as described in the previous study. Two mice were sacrificed 0.5, 1,
2, 3, 4, 6 and 24 h post-dosing, and plasma samples prepared. Levels
of 14C in the plasma showed little change during the 0.5 and 24 h
period, but were slightly higher at the 3 h period. Chromatographic
separation of the 14C in the serum showed that the 14C was
incorporated into slowly migrating plasma components. Acid hyrolysis
of the plasma gave rise to peaks containing tyrosine and phenylalanine
(Anonymous, 1972b).
Male Charles River rats were treated for two weeks with a
standard diet or with diets containing 0.85% L-phenylalanine or 1.5%
aspartame. At the end of the test period rats from each group were
sacrificed at 08.00 (day 1), 12.00, 16.00, 20.00, 24.00, 04.00 (day 2)
and 08.00 h. Hepatic phenylalanine hydroxylase activity was decreased
during the 24 h period, and there was no change in the circadian
rhythm of the hydroxylase. Plasma phenylalanine and tyrosine levels
were directly related to the hepatic phenylalanine hydroxylase
concentration (Anonymous, 1974a).
Rabbit
Eight young adult female rabbits (New Zealand White) were given
by gavage 20 mg/kg/day aspartame for five days, and then after an
overnight fast a single dose of 14C-labelled aspartame. Four of the
rabbits were used to measure plasma 14C levels and metabolites,
samples of blood being taken from the ear vein at 0.5, 1, 2, 3, 4, 6
and 24 h post-dosing. The remaining four rabbits were used to measure
14CO2, urinary 14C and faecal 14C. Cumulative 14CO2 production
during 7 h post-dosing period ranged from 2.9 to 10.4% of the
administered dose, with large variations between individual animals.
Cumulative urinary 14C during the 96 h post-dosing period ranged from
3.5 to 6.0% with maximum excretion occurring in the first 7-24 h
period. The cumulative faecal excretion ranged from 2.6 to 5.9% of the
dose. Plasma concentration of 14C was low, reaching a maximum 3 h
post-dosing and then remaining relatively constant throughout the
study. Chromatographic separation of the 14C in the plasma indicated
a single component in the unhydrolysed plasma, and the presence of
14C phenylalanine and tyrosine in the acid hydrolysed plasma
(Anonymous, 1972b).
Male New Zealand White Leunberg strain rabbits, which had been
administered orally 20 mg/kg aspartame/day for five days were dosed
with a single dose of 14C-phenylalanine aspartame (20 mg/kg). Blood
samples were taken at 0.5 and 3 h post-dosing. Chromatographic
separation of the 14C-labelled products in the plasma shows that the
radiolabel was rapidly incorporated into protein, and by three hours
all the 14C was associated with this fraction. The 14C in the protein
was associated with phenylalanine and tyrosine (Anonymous, 1974a).
Mature New Zealand female rabbits on day 6 of pregnancy were
given a diet containing 6% aspartame. On days 6 and 9 of gestation
blood samples were taken. On day 16 of gestation animals were
anaesthetized and four foetuses (two from each uterine horn) removed,
then the mothers and remaining foetuses sacrificed. On day 20 the
remaining animals were autopsied in the same manner. All samples were
analysed for phenylalanine and tyrosine. Since it was not possible to
collect blood from the 16-day-old foetuses, homogenates of the whole
foetus were prepared. Maternal plasma phenylalanine and tyrosine were
significantly elevated in the early treatment period reaching peak
levels at day 9 of gestation (three days after commencement of
treatment). The subsequent values returned to normal. The ratio of
foetal:maternal plasma phenylalanine was 1.58 in control rabbits and
1.40 in treated animals. For plasma tyrosine the ratio was 2.29 in
controls and 2.06 in the treated animals. The aromatic amino acid
level of the amniotic fluid was consistently higher in treated animals
than in controls (Anonymous, 1974a).
Dog
Groups each of four female beagle dogs (5.4-7.9 kg) were
pre-treated for five days by oral intubation with 0.068 mmol/kg
aspartame or L-phenylalanine. On day 6, a dose of 0.068 mmol 14C-
phenylalanine aspartame was administered to four dogs via the
saphenous vein, and 0.068 mmol 14C-phenylalanine to the other four
dogs by gavage. 14CO2 elimination was measured for the period 7 h
post-dosing and urinary and faecal 14C excretion for the 96 h post-
dosing period. Plasma 14C levels were determined at 15 min intervals
during the first hour post-dosing, then hourly up to 3 h post-dosing,
and then daily for 15 days post-dosing. Phenylalanine was oxidized
more rapidly to CO2 than aspartame, the maximum level of elimination
of 14CO2 occurring within 60 min in the case of phenylalanine and
90 min in the case of aspartame. Urinary and faecal excretion of 14C
was low and similar for phenylalanine and aspartame: for urine
5.32 ± 0.43/2.63 ± 0.3 and for faeces 5.38 ± 9.2/6.17 ± 0.77, for
phenylalanine/aspartame respectively. Most of the 14C in the urine
and faeces from the phenylalanine treated animals was excreted in
the first 48 h, whereas in the case of aspartame the low level of
excretion occurred during the 96 h test period. Plasma 14C levels
were similar for phenylalanine and aspartame treated animals, with
peak values occurring at 5 h post-treatment. The plasma 14C half-time
for phenylalanine was 10.8 days, and for aspartame 12.1 days.
Fractionation of plasma into low and high molecular weight 14C-
containing compounds showed that the low molecular weight component
was being converted to a high molecular weight component.
Chromatographic studies of the low molecular weight 14C plasma
component from aspartame treated animals showed that during the first
hour post-dosing the radiolabel was associated mainly with
phenylalanine and its metabolite tyrosine (Anonymous, 1972c).
Monkey
Four young female rhesus monkeys (2-3 kg) were administered
orally either 14C-methanol, 14C-L-phenylalanine, methyl 14C-
aspartame or phenylalanine 14C-aspartame at a dose level equivalent
to 0.068 mmol. 14CO2 elimination and 14C excretion in urine and
faeces was measured for a 8 h post-dosing period in monkeys treated
with the 14C-methyl-labelled compounds, and 24 h for the 14C-
phenylalanine compound. Plasma 14C determinations were made up to
72 h post-dosing. The cumulative 14CO2 excretion expressed as a
percentage of total administered dose was 72-88% and 60-75% for
methanol and aspartame (methyl 14C label) treated animals and
9.00-21.70% and 13.17-22.60% for the phenylalanine and aspartame
(phenylalanine 14C label). Cumulative 14C excretion in the urine was
3.17% and 1.57% for methanol and aspartame (methyl 14C label) treated
animals. For phenylalanine and aspartame (phenylalanine 14C label)
treated animals the respective cumulative 14C excretion in the urine
was 1.3-4.6%, and 1.5-3.7%, and for faeces 0.6-8.8% and 0.5-3.3%.
Plasma 14C levels following administration of methanol or aspartame
(14C-methyl label) was low. The loss of 14C was slow. In the case of
phenylalanine and aspartame (14C-phenylalanine) treated animals, 14C
plasma levels reached a maximum at 5 h and showed a slow loss during
the next 75 h of the test (Anonymous, 1972a).
In another study four female rhesus monkeys (5-7 kg bw) were
dosed via the saphenous vein, with a single dose of either
14C-L-aspartic acid or 14C-aspartyl aspartame at a dose level
equivalent to 0.068 mmol/kg. 14CO2 excretion was measured for 12 h
and urinary and faecal 14C excretion for 72 h post-dosing and plasma
14C for 24 h post-dosing. Peak 14CO2 excretion occurred within 1 h
post-dosing. The cumulative mean 14CO2 excretion during the 12 h
period was 77% and 67% of the dose of the aspartame and aspartic acid,
respectively. Faecal excretion accounted for less than 2% of the total
dose for both compounds. Urinary excretion was low, 2.1 and 3.8% of
the doses of aspartame and aspartic acid respectively. There was
considerable individual variation in the plasma 14C levels. Maximum
levels were observed 1 h post-dosing, followed by a biphasic loss of
14C. Chromatographic separation of 14C from the plasma of aspartame
or aspartic acid treated animals (5 h post-dosing) showed similar
radioactive peaks. Analysis of acid hydrolysates of serum showed that
the major 14C component was aspartic acid (Anonymous, 1972b).
Groups each of three female rhesus monkeys between 4.2 and 6.4 kg
were fed daily for 10 days, 0, 15 or 60 mg/kg aspartame in peanut
butter. On day 11, a catheter was passed into the saphenous vein to
permit withdrawal of blood samples, as well as the administration of
L-phenylalanine-u-C14 (0.068 mmol/kg). After administration of the
C14-phenylalanine blood samples were collected via the catheter for
up to 7 h post-dosing and then by venous puncture for up to 10 days
post-dosing. CO2 was collected for 7 h post-dosing. Rates of
excretion of 14CO2 were nearly identical for animals pre-dosed with
0 or 60 mg/kg aspartame for 10 days. Slightly less 14CO2 was expired
from animals receiving 15 mg/kg aspartame. Plasma 14C levels
declined during the first 30 min then peaked at 3 h. The plasma
14C-phenylalanine disappearance in the three groups was similar. The
appearance and disappearance of free plasma 14C-tyrosine was similar
in all groups. The plasma 14C levels in all groups showed a similar
pattern over the 10 day study. However, the levels of 14C in plasma
of the group not previously exposed to aspartame was slightly lower
than that of the test groups. Three hours post-dosing more than 90% of
the 14C in the plasma of all groups was incorporated into protein
(Anonymous, 1972b).
Infant Macaque monkeys (species Macaca mulatta, M.
fascicularis and M. arctoides) were fasted 3-4 h, lightly
tranquillized and dosed via stomach tube with 2 g/kg bw of aspartame
(APM) or 2 g/kg aspartame plus 1 g/kg monosodium glutamate (MSG).
Control infant monkeys received either no fluid or water. Blood
samples were obtained from the umbilical vein (four-day-old or younger
monkeys) or saphenous vein (older infants) at 0, 20, 40, 60, 90, 120,
180 and 240 min post-dosing and analysed for amino acid content. At
the termination of the last measurement the monkeys were sacrificed
and the hypothalami examined by light and electron microscopy. Peak
plasma levels of aspartate in the 14 infant monkeys dosed with APM or
APM plus MSG occurred at 60 min (23 ± 33 µmol/dl versus 0.69 ± 0.43
µmol/dl for controls). Ninety minutes after aspartame loading, peak
plasma phenylalanine levels reached 95 ± 59 µmol/dl versus 5.93 ± 2.81
µmol/dl control values. The monkeys metabolized the amino acids
somewhat more rapidly than the human. No signs of pyknotic nuclei,
neuronal degeneration or dendritic swelling were noted in the
hypothalami of treated infant monkeys (Reynolds et al., 1979a).
A developmental study was performed on newborn Macaca arctoides
reared for nine months on infant formula diets containing 1, 2 or
3 g/kg bw of APM. Control monkeys received only infant formula or
formula plus 1.65 g/kg bw of phenylalanine per day. There were four
monkeys in each group and formula and water were available ad lib.
All animals were monitored for formula and water intake, weight and
growth. Blood samples were taken from the saphenous veins of all
monkeys at 2, 4, 6, 8 and 9 months on study after a 4 h fast. The
samples were analysed for serum electrolytes, osmolality, CBC and
glucose as well as plasma amino acid content. Urine was analysed
periodically for pH, occult blood, protein, glucose, ketones,
bilirubin and phenylketones. Once a month the following developmental
milestones were assessed: extent of teething; ability to vocalize;
alertness; tractability; and general behaviour. Socialization
with laboratory technicians was allowed to occur freely.
Electroencephalograms were performed at the time of first dosing and
at four and nine months on study. After removal of the monkeys from
the experimental diets, eight animals were continued on regular
formula for an additional month and given EEGs. Some infants were
tested at quarterly intervals after cessation of the experimental
diet.
While no significant differences were noted in any groups with
respect to formula and water intake over the course of the study, when
a milk to water intake ratio was calculated the 1 g/kg APM group had a
ratio significantly different (p <.05) from the other groups at five
months on study. The 3 g/kg APM and 1.65 g/kg phenylalanine groups had
the highest ratio of water to formula consumption. All treatment and
control groups had similar growth rates. Blood indices, urinalyses and
EEGs were within normal limits for all monkeys during and after the
study. No significant differences in plasma aspartate or glutamate
levels were seen between animal groups. Fasting plasma phenylalanine
levels are given below:
Plasma phenylalanine levels
Treatment and dose µmol/dl ± s.d. Reference
1 g/kg bw APM 8.88 ± 5.15
2 g/kg bw APM 28.7 ± 48
3 g/kg bw APM 66.2 ± 83.3 Reynolds et al., 1979b
1.65 g/kg bw PHE 54.4 ± 65
None 5.49 ± 1.49
No behavioural deficits in treated versus control, untreated
monkeys were found.
TOXICOLOGICAL STUDIES
Special studies
Reproduction/teratological studies
Chicken embryo
Four hundred selected fertile eggs (White Leghorn) were randomly
distributed into eight groups of 50 eggs each. Three control groups
were assigned; one such group was untreated, one received only the
thrust of the hypodermic needle, and one received a 0.05 ml injection
of distilled water. The remaining five groups of 50 eggs each received
one of the following agents: aspartame, 0.25 mg/egg; aspartame,
0.5 mg/egg; calcium cyclamate, 0.5 mg/egg; calcium cyclamate,
2.5 mg/egg; or sucrose, 0.5 mg/egg. All compounds were dissolved in
sufficient distilled water so the total dose was contained in 0.05 ml.
A vertical injection technique directly through the air cell
enabled the injection of 0.05 ml of the prescribed solution directly
into the yolk sac. The shell puncture was sealed and all eggs were
candled daily beginning on day 6 post-injection. On day 19, all eggs
with live embryos were transferred to hatching trays and returned to
the incubator. On day 21, all hatched chicks were examined closely for
gross signs of malformations. Additionally, all dead embryos observed
during the candling procedure were examined grossly for signs of
abnormality. On day 23, all surviving chicks were sacrificed and a
complete gross necropsy was performed.
Mortality rates were similar in the untreated control and the
needle-thrust control groups (40%), in the vehicle control and the
sucrose groups (60%) and in both aspartame treated groups and both
calcium cyclamate treated groups (80%). No morphological abnormalities
were observed in the embryos or hatched chicks of both aspartame
treated groups, the sucrose group, or the intact or needle-thrust
control groups. One malformed embryo was found in each of the vehicle
control and cyclamate treated groups; due to the frequency (single
incident) and the nature of the abnormalities little biological
significance was attached, however, to these findings. No
abnormalities were observed in any of the cyclamate treated hatched
chicks (Anonymous, 1970a).
Rat
Low and high dose groups of 14 male and 30 female rats (Charles
River Caesarean derived) received 2 or 4 g/kg/day of aspartame
administered in the diet throughout the pre-mating, gestation and
lactation period, and intragastrically during the mating period. A
concurrent control group of 14 males, 48 females received basal diet
or vehicle, as appropriate. For mating purposes, 12 males from each
group were randomly subdivided into 36 units and mated with three
females. This mating design (CC, LL, HH; CL, CH, LC, HC) enables
comparison within each mating unit as well as between treatment levels
and facilitates precise identification of any affected parent. Sires
were sacrificed after completion of the mating period. Fifty per cent.
of the dams from each dose level were sacrificed on day 13 of
gestation; ovaries, uterus and uterine contents were examined. The
remaining dams proceeded through natural delivery and lactation. The
progeny were thoroughly examined at birth and periodically thereafter
for evidence of maldevelopment. Ophthalmoscopic examinations were
performed on all weaned pups. Pups were sacrificed at weaning (21 days
old) or shortly thereafter. Compound ingestion closely approximated
the indicated dosages, except during the latter half of lactation,
when it increased to 2.7 and 5.9 g/kg/day. Aspartame had no effect on
parental survival rate, mating performance, fertility, or paternal
body weight gain. Paternal food consumption was significantly
depressed during approximately one-half of the pre-mating period.
Maternal food consumption was unremarkable during the pre-mating and
lactation periods, but showed a variable, statistically significant
reduction at the low dose level only during much of gestation.
Maternal body weight was unremarkable during the pre-mating, gestation
and lactation periods, but exhibited a variable, statistically
significant reduction at the high dose level only during
mid-gestation. Hysterotomy, litter examination, and the neonatal
examination data were all unremarkable. Evaluation of the reproductive
performance of the neonates (F2 generation study) was also normal
(Schroeder et al., 1972).
A two-generation reproduction study was performed in the rat to
evaluate general reproductive performance in P1 and P2 generations
continuously ingesting aspartame. Dosage levels of approximately 2 and
4 g/kg/day were employed throughout the study, with a concurrent
control group of equal size receiving the basal diet only. Rats were
randomly distributed into three groups of 12 males and 24 females
each; this constituted the first parental generation. Each group
received the appropriate diet for nine weeks prior to mating, then
throughout the mating phase and the interval prior to sacrifice. Each
litter produced was arbitrarily reduced to a maximum of 10 pups within
24 h of birth. Thirty males and 60 females from this F1 group were
utilized as the P2 generation for continuation of the study.
Following a nine week pre-mating treatment period the P2 animals were
mated to produce F2A litters. All F2A litters were reduced to a
maximum of eight pups within 24 h of birth. Five F2A litters per group
were utilized for a separate neonatal clinical pathology study (Entry
E-9); the remaining 15 litters per group were utilized for producing
F2A weaning data. All pups were sacrificed at 21 days of age and
gross necropsies performed on approximately 35% of the pups from 10
litters per group. Full sets of wet tissues were preserved. All
remaining weanlings and all P2 generation males and females were
sacrificed and discarded. Mean food consumption body weight, survival,
physical appearance and behaviour of the parental generations were
comparable between control and both treated groups. Indices of
fertility, gestation, live birth, litter size at weaning, as well as
appearance, behaviour, physical examination data and gross necropsy
data from weanling animals, were also comparable between control and
both treated groups. Growth among the low dose level pups compared
well to the controls during both reproduction phases. The only
evidence of a treatment-related effect was a statistically significant
reduction in the body weight of weanling rats of both generations
(F1A and F2A) in the high dose group (Anonymous, 1971).
Female rats were housed in groups of three plus a male of proven
fertility. Twenty-four mated females were assigned to each of three
groups, receiving 0, 2 or 4 g/kg/day of aspartame in the diet from
gestation days 6 through 15. On gestation day 20 each female was
sacrificed and the ovaries, uterus and uterine contents examined.
Foetuses were examined externally and preserved intact for subsequent
examination for soft tissue abnormalities or for skeletal anomalies.
Forty-seven litters (589 term foetuses) from treated females were
examined. Maternal survival, body weight, and necropsy findings were
comparable between control and treated groups. Food consumption was
unremarkable at the low dose level, but significantly decreased in the
high dose group throughout the treatment period (gestation days 6-15).
The mean number of resorption sites, foetuses per pregnant female, and
viable foetuses was similar in both treatment groups. Likewise, foetal
sex distribution, body weight and length, and crown-rump distance were
unremarkable at all dose levels. In short, no evidence of treatment-
related anatomical alterations was observed. Thus, continuous dietary
administration of aspartame to the primigravid rat, employing dosages
up to 4 g/kg/day during the sixth to fifteenth day of gestation,
exerts neither embryotoxic nor teratogenic effects in the developing
foetus, nor does it affect the maternal rat adversely (Schroeder &
McConnell, 1970).
Three groups of 24 pregnant rats each received 0, 2.5 or
4.4 g/kg/day of aspartame administered in the diet from gestation
days 14 through 21 (parturition), and 0, 3.6 or 6.8 g/kg/day from
postpartum days 1 to 21 (weaning). Twenty-one litters (246 pups) from
the control group level, 20 litters (236 pups) from the low dose group
level, and 22 litters (289 pups) from the high dose group level were
available and received physical examinations at birth. Maternal food
consumption, behaviour, morbidity, and mortality were comparable
between control and both treated groups. Maternal body weights were
also comparable between groups during gestation, but body weight gain
during lactation was significantly depressed in the high dose group.
Duration of gestation, litter size and live birth indices and physical
examination data at birth were unremarkable at both treatment levels.
Pup body weights at birth were significantly reduced at the high dose
level; at weaning, body weights of males were unremarkable but females
weighed significantly less at both dose levels. Weanling pups survival
was significantly depressed at the high dose level. Weanling physical
examination data revealed that the low dose pups were unremarkable but
the 3% (5/164) of the high dose pups exhibited incompletely opened
eyelids and 1% (2/164; a single litter) exhibited grossly observable
lens opacities (Schroeder et al., 1973e).
In another study aspartame and DKP in a 3:1 ratio was fed to
rats. Females were housed in pairs with a male of proven fertility.
Thirty mated females were assigned to each of four groups, receiving
0, 1, 2 or 3 g/kg/day of the 3:1 mixture in the diet from the sixth
through the fourteenth day of gestation. On gestation day 19 each
female was sacrificed and the ovaries, uterus and uterine contents
examined. Foetuses were examined externally and preserved intact for
subsequent examination; approximately 33% of each litter was examined
for soft-tissue abnormalities and the remaining 67% for skeletal
anomalies. Eighty-two litters (1026 term foetuses) from treated
females were examined. Maternal survival, body weight and food
consumption were comparable between control and treated groups. The
mean number of implantation sites, resorption sites, number of
foetuses per pregnant female, live foetuses, or dead foetuses were
similar in all treated groups. Foetal sex distribution, body weight
and crown-rump distance exhibited no biologically significant
alterations at any dosage level, nor was there any treatment-related
anatomical alterations (Schroeder et al., 1972).
Rabbit
A total of six studies have been performed on the New Zealand
White rabbit; five studies involved the administration of aspartame
only while the sixth study involved administration of a 3:1 (w/w)
mixture of aspartame:DKP. In all studies pooled sperm specimens were
employed in the artificial insemination procedure.
Food consumption was recorded daily and animals weighed
periodically. All animals were sacrificed at term, partial necropsies
performed and foetuses examined externally. Approximately one-half of
the foetuses from each litter were processed for soft tissue
examination. Viscera were removed and examined from the remaining
animals and the carcasses processed for skeletal examination.
Three Segment II rabbit studies involving intragastric
administration were carried out. In each study an aqueous suspension
of the test material was administered in equal quantities twice daily
at 3-5 h intervals, employing a vehicle of 1% aqueous Tween 80 (v/v).
Treatment was carried out on days 6-18 of gestation. Survival rates
were frequently suboptimal, resulting both from technical difficulties
encountered in intubating the rabbit and from intercurrent pulmonary
infections. In the initial study a mixture of aspartame and DKP (3:1
ratio) was administered at daily dosage levels of 1, 2 or 3 g/kg/day
of the mixture. The concurrent control group was fed ad lib. In the
other studies aspartame was administered at a single dose level of 2
g/kg/day and the control group was pair-fed with those treated females
consuming the lowest amount of food. There was a non-dose related
decrease in the maternal survival rate of treated animals in one
study. It is unlikely this was treatment related, but rather reflects
intercurrent disease and technical difficulties with the compound
administration procedure.
The cumulative mean conception rate was slightly lower in the
treated animals (70%) than in the concurrent control groups (85%).
Much of this disparity can be traced to one study, which exhibited a
reduced ovulation rate especially notable in the treated group.
Cumulative mean rates of survival to term, abortion and premature
delivery were similar between control and treated groups. The mean
number of non-viable foetuses per litter was reduced in the treated
groups. Other parameters evaluated were essentially comparable between
control and treated animals.
The foetal examination data reveal that the incidence of major
foetal malformations was lower in the aspartame treated rabbits than
in the controls, but was similar to the incidence in a historical
control group. The incidence of anomalies in the concurrent control
group of one study was unusually high, however, and this is reflected
in the increased incidence observed in the compiled concurrent control
data. The total number of litters and foetuses examined from aspartame
treated animals (63 and 486, respectively) closely approximates the
number in the historical control group (79 and 573, respectively) and
is notably larger than the concurrent control group (34 and 263,
respectively). Roughly similar fractions of each litter were examined
by the two techniques employed (soft tissue examination by the
Wilson's procedure, and skeletal examination of the maxillary bones,
vomer irregularities, and cleft palate); one foetus exhibited
underdeveloped frontal skull bones, a partial cleft palate, and
eyelids fully opened at delivery; one foetus showed absence of the
maxillary and mandibular bones, two missing cervical vertebrae and two
split thoracic vertebral centra; and six foetuses exhibited
microstomia and cleft palate and/or syn- or oligo-dactyly. Thus
aspartame or aspartame/DKP mixture was neither embryotoxic nor
teratogenic to the albino rabbit foetus when administered
intragastrically at the doses studied during the mid-portion of
gestation. It also lacked notable adverse effects on the maternal
animal. The remaining three teratology studies on the rabbit involved
administration of specially pelleted rabbit diet containing 3.28% or
6.08% aspartame. These pellets were prepared commercially (Teklad,
Inc., Monmouth, Ill.). Chemical and microbiological analyses of the
specially pelleted diets were performed at periodic intervals
throughout the study. The rabbits received the test diet from days
6-18 of gestation. The ingested mean dosage of aspartame was 1.1 and
1.9 g/kg/day.
All rabbits were sacrificed at term and the uterine contents
examined. Parameters evaluated in this study included maternal
survival rates, conception rates, body weights and hysterotomy
findings; litter size and viability; foetal size, sex distribution and
morphological development.
Maternal survival, conception, premature delivery rate and body
weight data during gestation were comparable between the control and
treated groups.
Mean food consumption during the treatment period was similar for
the two treatment groups.
In utero litter size and resorption data were comparable
between the control and both treated groups. Similarly, foetal sex
distribution, body weight and crown-rump distance data were comparable
between these same groups.
External, soft tissue and skeletal examinations of the 181
control, 151 low and 146 high dose foetuses recovered at term
sacrifice were generally unremarkable. Major malformations were
observed, in one foetus each from the control, low and high dose
groups. Minor malformations were observed in an additional four high
dose foetuses from two litters. A minor foetal malformation designated
as separated eyelids was observed in three of five foetuses from one
high dose litter. This malformation was not observed in the concurrent
control group nor has it been observed historically in this
laboratory. Its significance is not clear. No major compound-related
embryotoxic or teratogenic effects were observed (Anonymous, 1973a and
b, 1974b; Schroeder & McConnell, 1973; Schroeder et al., 1973f and g).
Mutagenicity studies
Microbial systems
Aspartame at doses of 10-5000 µg/plate was tested in the Ames
Salmonella/microsome mutagenicity screening assay in strains
TA-1535, TA-1537, TA-1538, TA-98 and TA-100, with and without S-9
activation. The assay was repeated once using DMSO as the solvent and
negative control both times. No toxicity to the organisms was seen
even at the highest dose tested, but the compound precipitated on the
agar plates at 10 000 µg. No aspartame induced increase in HIS- to
HIS+ reversions was seen either in the presence or absence of the
activating system. The positive control mutagens (sodium azide,
9-aminoacridine, 2-nitrofluorine and 2-anthramine) did induce
significant numbers of reversions in the tester strains (Simmon &
Shan, 1978).
Dominant lethal
Aspartame was administered orally as a 10% suspension at 2 g/kg
to 15 male rats (Charles River CD strain) in two divided dosages two
hours apart on a single day. The vehicle was 1% Tween 80 in distilled
water. The aspartame used contained 0.2% diketopiperazine (DKP). The
negative control was Tween 80 as a 1% solution (v/v) and was given to
15 males by the same route and dosage regimen as the aspartame. The
positive control methylmethane sulfonate (MMS) was given to five males
i.p., as a 0.7% suspension (w/v) in corn oil. The dosage was 25 mg/kg.
Two females per week were mated to each male for eight weeks and
autopsied 14 days after positive indications of mating. Fertility was
reduced in the fourth week in males treated with aspartame. The
fertility in all other time periods was normal. Females mated in the
third and eighth week to aspartame treated males had a reduced number
of corpora lutea, and significantly greater number of corpora lutea,
respectively. The number of corpora lutea for all other time periods
was normal. The number of implantations per female was unaffected by
aspartame as well as by the positive control MMS.
The positive control (MMS) produced a statistically significantly
greater number of foetal deaths in weeks 2, 3 and 4. MMS also produced
a statistically significant increase in the number of foetal
deaths/pregnant female in week 6. Females mated to aspartame treated
males in week 3 showed a statistically significant decrease in the
number of live embryos, but the decrease is a result of fewer
ovulations and as such is not considered important. Females mated to
males in weeks 2, 3 and 4 showed a statistically significant decrease
in the number of live embryos which indicates that the positive
control was eliciting strong mutagenic response. This study was
repeated with rats using MMS (positive control) at a dose level
equivalent to 50 mg/kg. The aspartame contained 0.75% DKP. The
fertility was significantly reduced in the fourth week in males
treated with the positive control. All other time periods showed
normal fertility. The number of corpora lutea was significantly
reduced in weeks 2, 3 and 4 in females mated to positive control-
treated males. There was no significant fluctuations in this parameter
at any other time period. There was a significant decrease in the
number of implantations in the fourth week in females mated to
aspartame treated males. This parameter was significantly reduced in
weeks 2, 3, 4 and 5 in females mated to positive control-treated
males. All other time periods were unaffected. Females mated to
positive control-treated males showed a significant increase in this
parameter in weeks 1, 2, 3 and 5. All other time periods were
unaffected. Females mated to aspartame treated males in week 4 showed
a significant decrease in the number of viable foetal swellings. This
effect appears spurious. Females mated to positive control treated
males in weeks 1, 2, 3, 4 and 5 showed significant decreases in this
parameter (Schroeder et al., 1973a).
In vivo cytogenetic studies
Holtzman strain rats were divided into eight groups of 10 rats
each and received either no treatment, positive control triethylene-
melamine, 0.5 mg/kg i.p./day if only positive control, days 1-5
cyclohexylamine 10 mg/kg/day or 50 mg/kg/day or SC-18862 days 1-5,
0.4 g/kg/day, 0.8 g/kg/day, 1.2 g/kg/day or 1.6 g/kg/day by gastric
intubation for five days. Chromosomal aberrations in bone marrow and
spermatogonial cells were evaluated. The positive control,
triethylene-melamine produced a statistically significant increase
in the number of cells with chromosomal aberrations. Neither
cyclohexylamine nor aspartame had any significant cytogenetic effects
(Anonymous, 1970b, 1972k).
In another study aspartame (10% w/v) suspended in 10% Tween 80
was administered intragastrically in three equally divided dosages
every three hours to Purina Caesarian derived strain male albino rats.
Dosages were 0.5, 1.0, 2.0 and 4.0 g/kg/day for five days. The
positive control was triethylenemelamine 0.5 mg/kg. It was
administered i.p., as a suspension in 1% Tween 80 in distilled water,
on the final day of treatment for the other groups. The vehicle
control received 40 ml/kg/day. Chromosomal aberrations in bone marrow
were evaluated. All groups except the positive control lost weight
during the five day treatment. The negative controls consumed less
food than any other group. The positive control produced a
"statistically significant" increase in cells with aberrations. No
significant increases were observed utilizing aspartame (Anonymous,
1970a).
Host mediated assay
Aspartame was administered orally to male rats (Purina Caesarean
derived) approximately 12 weeks old, at four dose levels: 0.25, 0.50,
1.0 and 2.0 g/kg/day in three equally divided doses for five days;
the vehicle control was given at a level of 40 ml/kg/day. DMNA was
administered i.p. at a level of 100.00 mg/kg on day 5 only.
S. typhimurium G-46 was injected i.p. on day 5, 30 min after
administration of the test compounds. The rats were sacrificed 3 h
after S. typhimurium injection; their peritoneal cavities were
aseptically exposed and washed with 2.0 ml sterile saline. As much
fluid as possible was removed from the cavity. Peritoneal washings
were diluted and plated in accordance with generally accepted
procedures. A total of 6 × 108 CFU of S. typhimurium G-46 was
injected into each rat. Recoveries from the peritoneal cavity were
extremely low - approximately 1/10-1/1000 of the original inoculum -
indicating that the bacteria were being rapidly killed. A number of
the recoveries, 0.08, 0.10, and 0.12, and 0.16 × 107 CFU/ml of
exudate translate to counts of 5, 7 and 10 colonies/three plates at
the 105 dilution, respectively. Mutant cell values of 1.6, 3.3 and
5.0 CFU/ml of exudate translate to counts of 1, 2 and 3 colonies/three
plates, respectively. As a consequence of the low cell recoveries,
only seven of 10 rats were usable from the 0.25 and 0.50 g/kg/day
groups and 5 of 10 from the 2.0 g/kg/day group.
The mean mutation frequencies (MF) were:
Negative control: 4.39 ± 4.47 × 10-7
Positive control: 35.97 ± 22.18 × 10-7
0.5 g/kg/day: 2.48 ± 1.78 × 10-7
1.0 g/kg/day: 6.40 ± 3.67 × 10-7
2.0 g/kg/day: 2.93 ± 4.82 × 10-7
4.0 g/kg/day: 10.96 ± 6.48 × 10-7
(Anonymous, 1972e and l).
Carcinogenicity studies (see also long-term studies)
Groups each of 200 female 60-90-day-old Swiss albino mice were
used for a urinary bladder tumorigenicity study by the intravescical
pellet implant technique. Pellets of 20-22 mg of purified cholesterol
(80%) and aspartame (20%, 4.0-4.4 mg) were prepared and surgically
placed into the urinary bladder. The negative control group was
exposed to pellets of cholesterol and the positive control group to
pellets of cholesterol and the 8-methyl ether of zanthurenic acid. The
study was for 56 weeks. Parameters measured included morbidity,
mortality, motor and behavioural activity, growth, general external
features and digital palpation of protruding tissue masses. All
animals dying during the experiment, or at the termination of the
study were subjected to necropsy and histopathological inspection of
the bladder. No bladder neoplasia were observed in animals dying or
killed prior to 175 days of the study. The following incidence of
bladder neoplasia was recorded in mice surviving 175 days or more:
negative control 17/155, aspartame 13/123 and positive control 40/111
(Bryan, 1974a).
Neurological effect studies
Mice A/JAX-ICR hybrids between six and 10 days of age of both
sexes, were administered by gastric intubation, aspartame as a 10%
aqueous solution at dose levels equivalent to 0.25, 0.5, 1.0, 1.5 and
2 mg/g. Three hours post-dosing the mice were killed and the brains
prepared for microscopy. At 2 mg/g and 1.5 mg/g lesions were observed
in midline structures, namely the hypothalamic arcuate nucleus, the
subfornical organ and the area of the postrema. Less damage was
observed at the 1.0 mg/g level, and at 0.5 and 0.25 mg/g no neuronal
lesions were observed (Lemkey-Johnson et al., 1977).
Special studies on DKP (5-benzyl-3,6-dioxo-2-piperazine acetic acid)
BIOCHEMICAL STUDIES
Metabolism
All studies were carried out with 14C-labelled DKP prepared from
14C phenylalanine aspartame.
Rat
14C DKP was incubated with rat plasma. There was no significant
effect. Male rats (300 g) were dosed orally with 0.5 ml of an aqueous
solution of 14C DKP (10 mg/animal). Blood samples were taken at 2,
3 and 4 h post-dosing. Four hours post-dosing the animals were
sacrificed and the GI tract removed and divided into stomach, small
intestine and colon. The major part of the administered dose of 14C
was present in the colon (40-50%), and stomach (8-18%). Less than 5%
being present in the small intestine. Only trace amounts of 14C were
detected in the plasma (less than 0.1% of the administered dose).
Separation of the plasma 14C by chromatographic techniques indicated
the presence of many components (Anonymous, 1972a).
In another study fasted rats were administered a single dose of
14C DKP (10 mg/animal), and tissue distribution of 14C determined 2,
4, and 6 h post-dosing. The tissues studied were lung, spleen, kidney,
skeletal muscle, brain, heart, plasma, stomach, small intestine and
colon. The contents of the stomach, small intestine and colon were
also sampled. Only trace amounts of 14C were present in the tissues;
lungs and spleen contained little radiolabel. The colon, plasma, liver
and small intestine contained small but significant amounts of 14C.
Analysis of the stomach, colon and small intestine for 14C compounds,
indicated significant amounts of aspartyl-phenylalanine in the stomach
contents, significant amounts of unchanged DKP, aspartyl-phenylalanine
and tyrosine were found in the contents of the colon, and
phenylalanine methyl ester was present in the intestinal contents.
Plasma radiolabel at 2 h or more presumably contained 14C
phenylalanine-containing protein. Liver 14C consisted of
phenylalanine and a phenylalanine-containing protein (Anonymous,
1972a).
In another study male rats (Charles River strain) were dosed
orally with 14C DKP at a level equivalent to 10 mg/animal. Urine was
collected for 18 days, and plasma samples for four days, post-dosing.
About 25% of the dose was excreted in the first 24 h, with smaller
amounts during the rest of the test period (cumulate excretion was
less than 30% of the administered dose). Plasma 14C levels were low,
but there was a possible biphasic response (Anonymous, 1972a).
In another study 300 g male rats were subjected to bile duct
cannulation and were given 10 mg of 14C DKP intragastrically. Bile
was collected for 48 h. About 1% of the administered 14C was excreted
in the bile in this period (Anonymous 1972a).
In another study germ-free rats (Charles River, CD strain) were
removed from their germ-free environment and administered a single
oral dose of 20 mg 14C DKP. Urine and faeces were collected for 48 h
post-dosing. The metabolic profile (urinary) was quite different from
that of neomycin or control animals. 89% of the 14C in the urinary
extracts was unchanged SAIB. However, after keeping the rats for 30
days in a regular animal room environment, administration of 14C DKP
gave rise to metabolic urinary products similar to those of control
animals (Anonymous, 1974a).
DKP was incubated under aerobic or anaerobic conditions with
faecal suspensions prepared from the faeces of male Charles River
rats. No bacterial degradation occurred (Anonymous, 1974a).
Male Charles River rats that had previously been dosed with
neomycin sulfate, 25 mg/day for five days, received by oral intubation
20 mg/kg 14C DKP. Urine and faeces were collected for 48 h. Unchanged
DKP and two metabolites were identified in urine, hippuric acid and
benzoic acid. Quantitatively and qualitatively, the amount of these
compounds present in urine was similar to that observed in rats that
had not been treated with neomycin (Anonymous, 1974a).
Rabbit
Eight young adult female New Zealand white rabbits were dosed by
gavage, daily, with DKP (17 mg/kg bw) then following an overnight fast
with a dose of 14C DKP. Four rabbits were used to measure 14C levels
and plasma metabolites, blood samples being taken at 0.5, 1, 2, 3, 4,
6 and 24 h post-dosing. The other four rabbits were used to measure
14CO2 and urinary and faecal excretion of 14C. CO2 was collected up
to 7 h post-dosing, and urine and faeces up to 96 h post-dosing.
At the termination of the study all animals were sacrificed and
autopsied. 14CO2 expiration was extremely low. The total cumulative
14CO2 collected was 0.2-0.65% of the administered 14C. Plasma 14C
levels were extremely low, 0.1-0.2% of the administered 14C dose. No
characterization of the 14C content of the plasma was possible. 5-33%
of the 14C was excreted in the urine in 96 h, with a maximum
occurring in the 24-48 h period. Chromatographic separation of
extracts of the urine indicated the presence of a single unidentified
peak. The rabbits excreted about 19% of the 14C in the faeces in
96 h, with the maximum levels occurring in the first 24 h post-dosing.
Methanol extracts of the faeces showed a steady decrease of the major
14C peak with time and appearance of other peaks. 14C DKP was
identified in the faeces but the other metabolites have not been
characterized (Anonymous, 1972b).
Monkey
Four female rhesus monkeys (4-6 kg) were given by intubation
0.068 mmol 14C DKP. CO2 was collected for 12 h post-dosing. Plasma
14C and excretion of 14C in the urine and faeces, was determined
during the 120 h post-dosing period. About 1% of the administered dose
was excreted as CO2. 48% of the administered 14C was excreted in
the urine, with 30% being excreted in the first 24 h. 36% of the
administered DKP was excreted in the faeces, the majority being
excreted in the first 48 h. The 14C level in plasma was low, the peak
level at 12 h was 1.61% dose/litre plasma. The 14C exhibited a
biphasic response. One peak occurred at 2 h, another at 12 post-
dosing. 26% of the extractable urinary radioactivity was identified as
unchanged DKP and 58% as the major metabolite phenylacetylglutamine.
Methanol extracts of the 14C-labelled material in the faeces
contained 13% phenylalanine and 62% unchanged DKP. The other 14C
materials were not identified (Anonymous, 1972a). In a later study
phenylacetic acid was identified as a metabolite of DKP in the rhesus
monkey (Anonymous, 1974a).
In another study two female rhesus monkeys were infused via the
saphenous vein with 14C DKP at a dose level equivalent to 0.068
mmol/kg. 14C plasma levels were determined up to 12 h post-dosing.
Urine was collected at one hour intervals, for up to 8 h post-dosing,
by removing urine collected in bladder and then rinsing with 10 ml
saline. Urinary collection was continued in a normal fasting for a
further 64 h. Faeces were collected for 14C assay for a 72 h period.
14C peaked in the plasma in less than a minute, and then showed a
very rapid decrease. There were 3 phases of elimination of 14C from
the plasma, namely, at t1/2 of 0.042 h, 0.40 h and 9 h. Approximately
100% of the administered 14C was recovered in the urine with most of
the radiolabel being excreted in the first 3 h. Chromatographic
examination of the 14C in the serum and urine indicated that most of
the 14C (approximately 97%) was present in unchanged DKP (Anonymous,
1972a).
Man
Three male subjects (age 25-55 years) were fasted overnight prior
to administration by mouth 14C DKP (97 mg in 120 ml water). Blood and
urine samples were taken prior to dosing and then at various intervals
up to 72 h post-dosing,. Maximum 14C levels were observed in the
plasma 1 h post-dosing, with a second peak being observed 12 h post-
dosing. It was estimated the plasma level of 14C DKP at 1 h was
70 µg/l. The plasma 14C disappearance half life of the first phase
was about 2 h and the second phase 30 h. 14C was present in the urine
within 4 h post-dosing. At the end of three days no additional 14C
was excreted in the urine. A mean of 48.3% of the administered 14C
was excreted during this period. Analysis of the 14C compounds
present in the pooled 3-day samples of urine showed that about 12% of
the extractable 14C was in the form of unchanged DKP, 29% was in the
form of phenylacetylglutamine. In addition there were two other
unidentified 14C compounds (Anonymous, 1972a). In a later study
phenylacetylglutamine was identified as major urinary metabolite of
DKP in man (Anonymous 1974a).
Acute toxicity
LD50 Reference
Animal Route (mg/kg bw)
Rat Oral >5000 Andress et al., 1973a
i.p. >1562 Andress et al., 1973a
Mouse Oral >5000 Andress et al., 1973a
Rabbit Oral >5000 Andress et al., 1973a
Short-term studies
Mouse
Groups of 10 male mice received 0 and 1000 mg/kg/day of DKP
intragastrically for two weeks. All animals survived. No significant
differences were noted between control and test animals in terms of
body weight, food consumption, physical appearance and behaviour.
Haematology showed a decrease in total wbc due apparently to a marked
decrease in polymorphonuclears and blood chemistry showed a
significant decrease in glucose and non-significant decrease in BUN
and bilirubin. Aside from a significantly increased weight of the
seminal vesicles no other effects were noted upon organ weights nor
was there any increase noted grossly or microscopically in the
incidence of severity of lesions seen in treated as compared to
control animals (Rao et al., 1971a).
Rat
Groups of 5 male and 5 female rats received 0 and 1000 mg/kg/day
of DKP intragastrically for 2 weeks. No deaths or adverse physical or
behavioural effects were noted. No effects were seen in the urinalysis
findings. Transient depression of weight gain and food consumption was
seen in treated females at one week. Haematology showed only a small
percentage increase in polymorphonuclear leucocytes and decrease in
lymphoocytes. A non-statistically significant lowering of serum BUN,
SGPT, bilirubin and small but significant lowering of K+ was seen in
the treated groups. Decreased male heart and increased prostate
weights were noted at autopsy. No treated related evidence of
microscopic tissue lesions were noted (Rao et al., 1971b).
Groups of 5 male and 5 female rats received dietary levels of 0,
1000, 2000, 4000 and 6000 mg/kg/day of DKP for 5 weeks. Each dietary
group was run in duplicate. No mortality occurred. No dose-related
variations in body weights of food consumption were observed. Terminal
body weights and food consumption were decreased at the highest dose
level, this effect being statistically significant for the females. No
adverse physical or behavioural effects were noted in the treated
animals nor were compound related eye lesions apparent in the
ophthalmoscopic examination made at termination. Haematology and
plasma chemistry did not indicate any consistent treatment related
variations. No clear dose-related effects were noted in terms of
organ weights aside from decreased male, heart weight. This was
statistically significant only at the high dose level but was dosage
related except for the 1000 mg/kg/day level. Other gross and
microscopic pathology observations were not indicative of compound
related effects (Rao et al., 1972a).
Long-term studies
Rat
Groups of 6 male and 6 female rats received dietary levels of
750, 1500 and 3000 mg/kg/day of DKP for 115 weeks. A group of 12 male
and 12 female rats served as controls. Each group was replicated 6
times. No effects were reported as being seen in terms of physical
appearance and behaviour, nor was there evidence that the compound
produced any effects in terms of survival. A consistent pattern of
dosage-related decreased weight gain was seen in both sexes. These
were reported to be statistically significant as follows:
Weeks of decreased body weight gain
Dosage
(mg/kg/day) Males Females
3000 2-100 16-termination
1500 24-64 27-84
750 24-68 Not significant
A fairly consistent, statistically significant pattern of
increased food consumption was noted for high level males. Increased
food consumption for the high level females was seen only during the
second experimental year. For the lower feeding levels food intakes
which differed significantly from control values were sporadic. No
evidence of effect was seen in terms of haematology. Clinical
chemistry findings were similarly without evidence of compound effect
aside from an apparent statistically significant decrease in serum
cholesterol seen to persist in the high level groups. Urinalysis
findings were not remarkable aside from a significant drop in pH seen
persistently in the high level females and sporadically in other
groups. Ophthalmological findings also failed to disclose any evidence
of compound related changes, on autopsy, findings with respect to
organ weights were not remarkable. Gross and microscopic pathology as
reported did not indicate the presence of tumorigenic or non-
tumorigenic changes which would be attributable to the administration
of DKP except for a dosage related increase in uterine polyps whose
numbers were significantly increased over controls for both the
intermediate and high dosage level groups. This observation will
require further clarification (Rao et al., 1974).
Mouse
Groups of 36 male and 36 female mice received dietary levels of
250, 500 and 1000 mg/kg/day of DKP for 110 weeks. A group of 72 male
and 72 female rats served as controls. No evidence of compound induced
effect was reported as being evident in terms of appearance and
behaviour as well as weight gain and food consumption or eye lesions.
Blood counts were not remarkable. Clinical chemistry results were not
suggestive of compound related effect. At sacrifice thyroid weight and
ratio of thyroid weight to body weight for the intermediate and high
dose level females were significantly elevated over control values.
Gross and microscopic pathology reported as not suggestive of
tumorigenic or non-tumorigenic changes which might be attributable to
the feeding of DKP (Anonymous, 1974c).
Reproduction studies
Rat
Low, medium and high dose groups of rats (Charles River CD
strain) 14 males and 28 females received 0.45, 0.9 or 1.8 g/kg/day of
DKP administered in the diet throughout the premating, gestation, and
lactation periods, and intragastrically during the mating period. A
concurrent control group of 14 males and 60 females received either
basal diet or the diluent. For mating purposes, the rats were randomly
subdivided into 48 mating units each containing one male and three
females. This male-female cross-mating design is the same as that
described in the aspartame studies. Sires are sacrificed after
completion of the mating period. 50% of the dams from each group are
sacrificed on gestation day 14; ovaries, uterus, and uterine contents
are examined. The remaining dams proceed through natural delivery
and lactation. The progeny are thoroughly examined at birth
and periodically thereafter for evidence of maldevelopment.
Ophthalmoscopic examinations are included. Pups are sacrificed at
weaning (21 days old) or shortly thereafter. DKP had no effect on
parental survival rate, food consumption, mating performance,
fertility, or on paternal body weight gain. Maternal body weights were
unremarkable at low and medium dose levels, but significantly
depressed during mid-gestation (day 14) and lactation (days 14 and 21)
in the high dose animals. Hysterotomy, litter examination, and
neonatal data were all unremarkable, excepting a slight but
significant decrease in mean viable litter size in the high dose group
(Schroeder et al., 1973c).
Four groups of 20 pregnant rats (Charles River CD strain) each
received 0, 0.7, 1.3 or 2.5 g/kg/day of DKP administered in the diet
from gestation day 14 through postpartum day 21 (weaning). Following
physical examination at birth, each litter was arbitrarily reduced to
a maximum of 8 pups. Maternal food consumption, body weight gain,
behaviour, morbidity, and mortality were comparable between control
and all treated groups. Likewise, duration of gestation, litter size
and live birth indices, and weanling pup survival, body weight gain,
and physical examination data (including ophthalmoscopic exams) were
normal in all treatment groups. No treatment-related anatomical
abnormalities were observed (Schroeder et al., 1973c).
Teratological studies in the rat (Charles River CD strain)
Diketopiperazine (DKP)
Female rats were housed in groups of four with a male of proven
fertility. Twenty-four mated females were assigned to each of four
groups, receiving 0, 1, 2, or 4 g/kg/day of DKP in the diet from the
sixth through the fifteenth day of gestation. On day 20 each female
was sacrificed and the ovaries, uterus, and uterine contents examined.
Foetuses were examined externally and preserved intact for subsequent
examination for soft tissue abnormalities or for skeletal anomalies.
Fifty-seven litters (711 term foetuses) from treated females were
examined. Maternal survival, conception, body weight, and food
consumption were comparable between control and treated groups. Mean
number of resorption sites was unremarkable at the low and medium dose
levels, and significantly decreased at the high dose level. The mean
number of foetuses per pregnant female was likewise unremarkable at
the low and medium dose levels, and significantly increased at the
high dose level. Foetal sex distribution, body weight, and crown-rump
distance were unremarkable at all dose levels. No evidence of
treatment-related anatomical alterations was observed (Schroeder et
al., 1973d).
Teratological studies in the rabbit
Four groups comprised of 21 artificially inseminated females of
the New Zealand white strain received aqueous suspension of DKP by
gastric intubation at dosages of 0.5, 1, or 2 g/kg/day from the sixth
day of gestation through day 18. Controls received diluent only. Body
weights were recorded periodically (7 intervals) and food consumption
was measured daily. All animals were sacrificed at term (gestation day
28 or 29). Partial necropsies were performed, and foetuses examined
externally. Approximately one-half the foetuses from each litter were
processed for soft tissue examination. Viscera from the remainder were
removed and examined. The carcasses were processed for skeletal
examination. Approximately 40-50 foetuses were processed from each
group except the high dose from which only 4-5 foetuses were available
for each type of examination. Survival rates were sub-optimal but
comparable in the control, low, and medium dose groups, with 20-30%
mortality per group. At the high dose level 90% of the rabbits died.
Pulmonary aspiration of compound suspension and gastric perforation,
resulting from technical difficulties encountered in intubating the
animals, clearly contributed notably to the mortality rate in all
groups. However, in the high dose group marked anorexia and weight
loss occurred secondary to pyloric obstruction by a gastric concretion
composed ostensibly of DKP intermixed with rabbit hair. Data on the
mean number of implantation sites, resorption sites, viable and
non-viable foetuses, and foetal length and weight were unremarkable
for control, low, medium dose groups. There were insufficient data
from the high dose group to permit a proper evaluation. External,
visceral, and skeletal examinations of foetuses from 10-14 litters per
group (except the high dose group) showed no treatment-related effects
(Anonymous, 1972f).
Mutagenicity studies
Dominant lethal study
DKP was administered intragastrically as a freshly prepared 5%
suspension w/v in a 1% solution of Tween-80 (v/v) in distilled water
to 15 male rats (Charles River CD strain). Two equally divided dosages
were administered two hours apart on a single day. The negative
control was Tween-80, as a 1% solution (v/v) and was administered to
15 males by the same route and dosage regimen as the DKP. The positive
control methylmethane sulfonate (mms) was given to 10 males i.p., as
an 0.7% suspension (w/v) in corn oil. The dosage was 50 mg/ml. Two
females per week were mated to each male for 8 weeks and autopsied 14
days after positive indications of mating. Males treated with the
positive control had statistically significant decreases in fertility
in the fourth week. There was no evidence that DKP affected the
fertility of male rats. There were significant decreases in the number
of corpora lutea in females mated to positive control-treated animals,
in the second, third and fourth weeks. Females mated to DKP treated
males showed a decrease in the number of corpora lutea in the seventh
week. The number of implantations in females mated to positive
control-treated males was significantly decreased in the second,
third, fourth and fifth week. Females mated to DKP treated males in
the fifth week showed a significant decrease in the number of
implantations. The results in all other time periods were normal. The
number of foetal deaths was significantly increased in females mated
to positive control-treated males in weeks 1, 2, 3 and 5. Females
mated to DKP-treated males were unaffected. The number of viable
foetal swellings was significantly decreased in females mated to
positive control-treated males in weeks 1, 2, 3, 4 and 5. Females
mated to DKP treated males in week 5 showed a significant reduction in
the number of viable foetal swellings (Schroeder et al., 1973b).
In vivo cytogenetics
DKP was administered intragastrically to four groups of 10 male
Purina Caesarian derived albino rats for five consecutive days. The
dosages were 0.25, 0.5, 1.0 and 2.0 g/kg/day given in three equally
divided daily dosages. The negative control received the Tween-80-
water vehicle (1% Tween-80 in distilled water). The positive control
received a single i.p. dose of triethylene melamine. Chromosomal
aberrations in bone marrow were evaluated. The mean body weight was
reduced in all groups, but more significantly at the very high dosage
level (2.0 g/kg/day). Food consumption was also reduced in the groups
receiving five days of treatment. The mean percentage of cells with
aberrations was significantly higher in the positive control-treated
groups. There were increases above control level in other DKP treated
groups, but these apparently were not evaluated statistically
(Anonymous, 1972g).
Host mediated assay
DKP was administered orally to male rats (Purina Caesarian
derived) approximately 12 weeks old at 4 dose levels; 0.25, 0.50, 1.0
and 2.0 g/kg/day in 3 equally divided doses for 5 days. The vehicle
control was given at a level of 40 ml/kg/day. DMNA was administered
i.p. at a level of 100.00 mg/kg on day 5 only. S. typhimurium G-46
was injected i.p. on day 5, 30 min after administration of the test
compounds.
The rats were sacrificed 3 hours after S. typhimurium
injection; their peritoneal cavities were aseptically exposed and
washed with 2.0 ml sterile saline. As much fluid as possible was
removed from the cavity. Peritoneal washings were diluted and plated
in accordance with generally accepted procedures.
A total of 16.0 × 108 CFU of S. typhimurium G-46 was injected
into each rat. Recoveries from the peritoneal cavity were extremely
low, approximately 1/3-1/100 of the original inoculum.
The mean mutation frequencies (MF) were:
Negative control: 1.26 0.64 × 10-7
Positive control: 23.36 10.58 × 10-7
0.25 g/kg/day 2.90 6.33 × 10-7
0.50 g/kg/day 0.83 0.84 × 10-7
1.0 g/kg/day 1.34 1.21 × 10-7
2.0 g/kg/day 2.19 1.88 × 10-7
(Anonymous, 1972e)
Special studies
Urinary bladder tumorigenicity study in the mouse by the intravesical
pellet implant technique (DKP)
Groups each of 200 female 60-90 day-old Swiss albino mice were
used. Pellets of 20-22 mg of purified cholesterol (80%) and DKP (20%,
4.0-4.4 mg) were prepared and surgically placed into the urinary
bladder. The negative control group was exposed to pellets of
cholesterol, and the positive control group to pellets of cholesterol
and the 8-methyl ether of xanthurenic acid. The study was for 56
weeks. Parameters measured included morbidity, mortality, motor and
behavioural activity, growth, general external features and digital
palpation of protruding tissue masses. All animals dying during the
experiment, or at the termination of the study were subject to
necropsy, and histopathological inspection of the bladder. No bladder
neoplasma was observed in animals dying to 175 days of the study. The
following incidence of bladder neoplasia was recorded in mice
surviving 175 days or more: control 17/155 (10.6%), DKP 17/125 (13.6%)
and positive control 40/111 (36.0%) (Bryan, 1974b).
Special studies on the possible nitrosation of DKP
Reaction of DKP and sodium nitrite in vitro
1250 mg of piperidine and 25 mg of Na nitrite were dissolved in
distilled water; pH was adjusted to 4 and volume to 10 ml. Duplicate
flasks were capped and incubated at 37°C with constant shaking. At
0.5, 1, 2 and 4 hours, 2 ml samples were transferred to tubes
containing 3 ml of ether with extraction for 15 min. The ether phase
was used for GLC detection of N-nitroso-piperidine. In duplicate
vials, 10 mg of DKP-14C-phenylalanine and 3 mg of Na nitrite were
dissolved in 0.25 M phthalic acid buffer, pH 4. The volume was
adjusted to 10 ml, the vials gassed with N2 and capped. Control vials
were prepared containing DKP but no Na nitrite. Vials were incubated
with shaking at 37°C and at 0.5, 1, 2 and 4 hours, samples (were taken
and analysed for the presence of nitroso compounds by use of multiple
sampling procedures) and the use of two different thin chromatographic
systems. Nitrosopiperidine formation occurred by reaction of
piperidine with sodium nitrite (3-6 mg or 0.2 to 0.5% of the possible
yield). DKP did not react with nitrite to form compounds that could be
detected by the methods used (Anonymous, 1972a).
A study of the possible reaction of DKP with aqueous nitrous acid
A 0.1% solution of DKP, containing 0.13% (5 molar excess) Na
nitrite was prepared in 0.1 N HCl. The mixture was allowed to stand at
room temperature for 1.5 hours. Excess nitrous acid, formed by the
interaction of HCl and Na nitrite was distilled off from the mixture
at 30 under vacuum. Following distillation, a 25 ml aliquot of the
solution was taken and to it 0.062 mg of N-ethyl, N-nitrosourethane
was added. The UV spectrum of this solution was taken against a blank
of 0.1% DKP in 0.1 N HCl. An aliquot of the solution was subjected to
UV spectral analysis (against a blank of 0.1% DKP in 0.1 N HCl) prior
to addition of N-ethyl, N-nitrosourethane. A 0.158% solution of
NaNO2 in 0.1 N HCl was also distilled and the UV spectrum taken
against 0.1 N HCl. At 245 nm, the DKP showed no increase in absorbance
as compared to the NaNO2 solution alone, suggesting that DKP had not
been nitrosated to form N-Nitroso-DKP (Anonymous, 1972h).
Reaction of DKP and Na Nitrite in vivo
Non-fasted male Charles River rats (340-360 g) were anaesthetized
with Na pentobarbital. The stomach was ligated at the cardiac and
pyloric junctions. At the pyloric junction, an incision was made
through which coarse food particles were manually expressed from the
stomach and through which a polyethylene cannula was inserted. 25 mg
of Na nitrite in 0.5 ml water followed by 5 mg 14C DKP in phthalic
acid buffer were administered. The cannula was removed, the ligature
tightened, and stomach returned to abdominal cavity. Controls received
either piperidine, HCl or Na nitrite alone with saline. Two rats were
used for each experiment. After 60 min, the animals were sacrificed
with ether, gastric contents removed, and stomach rinsed with saline
and the contents were assayed for the presence of n-nitroso compounds.
In vivo nitrosation of piperidine was demonstrated and only the
stomachs of rats given piperidine and nitrite. No reaction products of
sodium nitrite and DKP were detected by the assay methods used
(radiochromatography by several solvent systems) (Anonymous, 1972a).
Special studies with aspartame and DPK
Enzyme induction studies with aspartame and DPK
Male albino rats (Charles River strain) of 80-100 g weights were
pretreated with either saline (1 mg/kg), phenobarbital (60 mg/kg), or
aspartame (3.5 g/kg) for 4 days. Saline and phenobarbital were
administered once daily by i.p. injection. Aspartame was given twice
daily as an aqueous suspension. On the day following the last
pre-treatment day, the animals were challenged with either
hexobarbital (100 mg/kg, i.p.) or zoxazolamine (60 mg/kg, i.p.). The
duration of hexobarbitol sleeping time or zoxazolamine paralysis time
was determined, using loss and return of the righting reflex as end
points. In vitro studies were carried out on liver preparations
derived from the livers of rat following the last pretreatment day, to
measure the effect of aspartame administration on hepatic amino-N-
demethylase, p-nitrosoamisole, O-demethylase, zoxazolamine hydroxylase
and hexobarbital oxidase activity. Aspartame administration to the
rats had no effect on the in vivo and in vitro systems studied
(Anonymous, 1972a). Similar results were observed when DPK (2.0 g/kg)
was used in the diet (Anonymous, 1972a).
Effect of dietary aspartame and phenylalanine on hepatic
phenylalanine hydrolase activity in the rat
Groups each of 8 male Charles River strain rats (170 g) had free
access to one of 4 diets and water. Diets used were: (1) powdered
Rockland diet, (2) powdered diet plus 0.15% aspartame, (3) powdered
diet plus 1.5% aspartame and powdered diets and 0.85% phenylalanine
(equimolar with 1.5% aspartame). Animals were weighed at 2-day
intervals and food consumption recorded every second day. Treatment
period varied from 1 to 8 weeks. The animals were sacrificed and blood
samples taken for phenylalanine analysis. A liver preparation
consisting of the supernatant from homogenates centrifuged 45 min ×
16 000 g, was used for the enzyme assay. Feeding diets of 0.85%
phenylalanine or 1.5% aspartame for one or more weeks resulted in
decreases in phenylalanine hydroxylase activity and increases in
plasma phenylalanine levels. The hydroxylase activities were more
sensitive to the presence of the dietary aspartame and phenylalanine
than were the plasma phenylalanine level (Anonymous, 1972i).
Gastrointestinal system
Appetite inhibition studies in rats
Ten male adult Charles River rats were trained to eat during a
two-hour period each day for 4 consecutive days. Tap water was allowed
throughout the experiment. On day 5, 1 h prior to feeding, rats were
dosed intragastrically with 200 mg/kg of aspartame, DKP (an aspartame
breakdown product), or vehicle (30% propylene glycol) only. One group
received no treatment. One hour later, all except the control group,
which was fasted, were allowed food ad lib. for 2 h. The body weight
was taken before compound administration and 24 h after feeding.
Neither aspartame or DKP had any effect on food consumption or weight
loss, indicating no effect on appetite (Anonymous, 1972j).
Effects on gastric secretion in rats
Groups each of 6 male Charles River strain rats (175-225 g) were
fasted for 48 h. The animals were anaesthetized with ether and
subjected to pyloric ligation. Aspartame, DKP, or distilled water
(control) were administered in 1 ml of water intragastrically
(250 mg/kg). Five hours later, the stomachs were removed, contents
measured, and centrifuged. Acid concentration and proteolytic activity
of the gastric juice was measured. Neither aspartame nor DKP had any
marked effect on gastric juice volume, acidity or proteolytic activity
(Anonymous, 1972j).
Pepsin inhibition in vitro
Bovine pepsin was incubated with bovine haemoglobin in the
presence of aspartame or DKP at 143 µg/ml. There was no inhibition of
pepsin activity (Anonymous, 1972j).
Pancreatic lipase inhibition in vitro
Emulsified triglyceride (olive oil) containing 0.4 mg/ml of
pancreatic lipase was incubated for 2 h in the presence or absence of
aspartame or DKP at 1.25 mg/ml. There was no inhibition of lipase
activity (Anonymous, 1972j).
Effects on gastric ulceration in rats
Male Charles River rats (200-250 g) were anaesthetized with ether
and stomach ligated at the pyloric junction. The animals were given
50 mg of aspartame or DKP or vehicle only. 17-1/2 hours later, the
stomachs were removed and examined microscopically. The numbers of
ulcers in the non-secretory portion of the stomach were counted
according to size to obtain the Z score. Six rats were in each
treatment group. A known anti-ulcer agent was used as a positive
control. Neither aspartame nor DKP significantly increased or
decreased the severity of gastric ulceration (Anonymous, 1972j).
Cardiovascular system
Effects on blood pressure in anaesthetized dogs following intravenous
administration
Two mongrel dogs were anaesthetized with sodium pentobarbital and
the blood pressure monitored continuously from a femoral artery.
Aspartame or DKP was dissolved in propylene glycol and injected at
concentrations of 0.1, 1.0, and 5.0 mg/kg. Aspartame had no effect on
blood pressure at 0.1 or 1.0 mg/kg. At 5 mg/kg, one dog (of 2) had a
slightly lowered blood pressure temporarily. DKP had no effects at any
concentration (Anonymous, 1972j).
Effects on blood pressure and heart rates following oral
administration of aspartame or DKP in unanaesthetized normotensive
dogs
Blood pressure was recorded via a surgically implanted aortic
cannula. Following surgery, the animals were isolated from noise.
Systolic, diastolic, and mean arterial pressures were recorded. Heart
rate was derived from the blood pressure tracing. Blood pressure and
heart rate were determined at 5 min intervals during a 30 min control
period prior to compound administration. Aspartame, DKP, and placebo
were administered in capsule form at levels of 100 and 200 mg/kg. Two
dogs of each sex were in each treatment series. Measurements were made
at 10, 20, 30, 40, 50, 60, 120, 180, 240 min and 24 h after treatment.
Neither aspartame nor DKP showed any consistent effects on blood
pressure or heart rates (Rozek, 1972).
Inhibition of the pressor response to angiotensin in rats
Adult male Charles River rats were anaesthetized by i.p.
injection of sodium pentobarbital. Cardiovascular reflexes were
blocked with atropine and pentolinium subcutaneous injections. A
femoral vein was cannulated for injections, a femoral artery for
blood pressure measurements. Five consecutive doses of 0.01 µg of
angiotensin were given intravenously at 3 min intervals. Three minutes
after the last injection, Aspartame or DKP was injected at 10 mg/kg.
Following 15 min, the angiotensin protocol was repeated. The mean
pressor responses were compared before and after test compound
administration. A known antihypertensive agent was used as a positive
control. Neither aspartame nor DKP had any effect on the pressor
response to angiotensin in rats (Anonymous, 1972j).
Antiarrhythmic activity using the isolated rabbit heart
The ability of aspartame and DKP at concentrations of 10, 20, and
40 mg/l to affect aconitine-induced ventricular arrhythmia in the
isolated rabbit heart was determined. No significant effects of
aspartame or DKP were observed (Anonymous, 1972j).
Effects on blood coagulation in vitro
Saline solutions of aspartame or DKP were added to freshly drawn
rabbit blood and the mixture incubated at 37°C. The coagulation time
was compared to controls (no treatment) and coagulation time in the
presence of heparin. DKP or aspartame had no effects on coagulation
time (Anonymous, 1972j).
Central nervous system
General observable effects in mice
Groups of 4 mice each were injected i.p. or s.c. with aspartame
or DKP at dose levels of 0, 5, 20, 40, 80, and 320 mg/kg. Observations
and tests were carried out just prior to compound administration and
an 0.5, 1, 2, 3, and 4 h treatment. The spontaneous elicited behaviour
was rated for each mouse and tests for locomotor ataxia were carried
out by observing the behaviour of the mouse for 30 sec when placed on
a horizontal rod. Neither aspartame nor DKP caused excitation or
depression at any of the doses used. Very slight ataxia was observed
for aspartame at i.p. doses of 20, 40, 80 and 320 mg/kg and
subcutaneous doses of 40, 80 and 320 mg/kg. Similar observations were
reported for DKP following i.p. doses of 5-320 mg/kg and subcutaneous
doses of 40 and 320 mg/kg (Anonymous, 1972j).
Antidepressant activity in mice
Aspartame and DKP were checked for antidepressant activity by
determining their abilities in antagonizing the drooping of the upper
eyelid (ptosis) caused by the administration of RO-4-1284. Ten mice
were used in each group. Mice received 0, 25, or 200 mg/kg of
aspartame or DKP intragastrically. One hour later, RO-4-1284 was
administered i.p. at 20 mg/kg. The abilities of the two compounds to
antagonize the eyelid drooping caused by RO-4-1284 were rated.
Positive controls with known antidepressants were run. Aspartame or
DKP had essentially no antidepressant effects (Anonymous, 1972j).
Effects on hexobarbital hypnosis in mice
Groups of 16 male HAM/ICR mice (18-25 g) were administered saline
(control), or DKP or aspartame at dosage levels of 250, 500, or
1000 mg/kg intragastrically. Thirty minutes later, hexobarbital was
administered i.p. at 100 mg/kg. Sleeptime was defined as the time from
the loss of righting reflex until a 2-time spontaneous righting in a
15 sec interval. Aspartame did not show any effect at the doses
studied. DKP caused a significant increase in sleeping time at the
highest dose level test (1000 mg/kg) (Anonymous, 1972j).
Effect on motor coordination in mice
To groups of male HAM/ICR mice (20-30 g), aspartame or DKP was
administered intragastrically at 0, 50, 100 or 200 mg/kg. Diazepam
(positive control) was administered at 10 mg/kg. Mice were scored
after 2-1/2 hours on the ability to stay on a rotating rod (4.5 rpm)
for 1 min or longer. Neither aspartame nor DKP produced motor
incoordination. Diazepam was significantly active in this test
(Anonymous, 1972j).
Anticonvulsant activity in mice
Groups of male HAM/ICR mice (20-30 g) were administered aspartame
or DKP intragastrically at 0, 50, 100, or 200 mg/kg. 2-1/2 hours
later, the animals were exposed to a current of 50 milliamperes
delivered by corneal electrodes. Anticonvulsant effects are judged by
protections from the hindlimb extension component of the seizure.
Diphenylhydantoin was used as a positive control. Neither aspartame
nor DKP had any anticonvulsant effects at any dosage level. Groups of
the same strain of mice were given aspartame or DKP intragastrically
at dosage levels of 0, 100, or 200 mg/kg. Trimethadione and diazepam
were used for positive controls. 1-1/2 hours later, 35 mg/kg of
metrazol was administered i.p. Anticonvulsant activity was judged by
abolition of seizures. Neither aspartame nor DKP had any effect
(Anonymous, 1972j).
Analgesic activity in mice
Groups of 10 male HAM/ICR mice (18-25 g) were administered
aspartame or DKP intragastrically at levels of 0, 50, and 100 mg/kg.
The reaction time of each mouse to lick a foot or jump was measured
at 60, 40, and 20 min before, and 30, 60, 90, and 120 min after
administration of the test compound. The mice were scored on foot
licking or jumping when placed on a hot plate at 55°C, as compared
with controls (Anonymous, 1972j).
Adult male HAM/ICR mice (18-25 g) were administered aspartame or
DKP at levels of 0, 50, or 100 mg/kg intragastrically. A pressure-
standardized artery clip was placed one inch from the base of the
tail. Response to the clip was measured as compared to controls.
Neither aspartame nor DKP showed any analgesic effects (Anonymous,
1972j).
Central anticholinergic activity in mice
Groups of 10 adult male HAM/ICR mice were given i.p. or oral
doses of aspartame or DKP at levels of 0, 20, or 200 mg/kg. 20 min
later, the mice received 20 mg/kg i.p. doses of tremorine. The mice
were placed on a rotating rod 10 min after tremorine treatment and
scored on the ability to remain on the rod for 2 min as compared to
controls. Neither compound showed any effect (Anonymous, 1972j).
Effects on behaviour in rats
Naive male Fischer rats (90 days of age) were treated
intragastrically with saline or 50, 100 or 200 mg/kg of aspartame,
DKP or L-phenylalanine; 30 min later, rats were placed in a
two-compartment shuttle box equipped with electrified grid floor and
insulated from noise. A 5 sec conditioned stimulus (a tone and a
light) preceded a 0.2 milliampere footshock delivered via the grid.
The shock was terminated in 30 sec if the rat did not respond. If the
rat moved to the other chamber, the shock was avoided and the response
scored as an avoidance response. A movement to the other chamber
during the shock was scored as an escape response. If no response
was made, it was recorded as a failure response. 15 sec intervals were
given before another conditioned stimulus was given. If a shuttle
response was made during this rest interval, the shock and conditioned
stimulus were applied until the rat returned to the other side. Each
rat received 100 trials. 12 rats per dose were tested. Neither
aspartame nor L-phenylalanine had any significant effect at any dose
tested. DKP had a significant effect on the number of foot avoidances
at 50 and 200 mg/kg dose. However this effect may not be significant
since it was not dose related nor was it accompanied by a significant
increase in intertrial interval responses (Potts, 1973).
Miscellaneous pharmacological activity
Diuretic activity in rats
Six groups of 4 male Sprague-Dawley rats (188-228 g) were
administered saline or aspartame or DKP at a level of 100 mg/kg. The
rats had been maintained on a normal commercial diet, with water
ad lib. Food was withdrawn 18 h before, and water withdrawn during
the 5 h test period. Urine was collected for 5 h, and voiding induced
by bladder palpation. The volume of urine, and Na and K contents of
urine were measured. Neither aspartame or DKP showed any diuretic
activity (Anonymous, 1972j).
Effects on blood glucose in rats
Adult male Charles River rats (180-230 g) were fasted for 24 h.
Blood samples were obtained via the tail veins and blood analysed
for glucose by a published procedure. DKP or aspartame was
administered intragastrically at 100 mg/kg. Serum samples were then
obtained at 2 and 4 h post-treatment, and analysed for glucose
content. Neither aspartame nor DKP had any effect on the blood glucose
level (Anonymous, 1972j).
Effects on body weight gain and blood cholesterol in
hypercholesterolaemic rats
Adult male Charles River rats (200-250 g) were made hyper-
cholesterolaemic by receiving 0.02% of propylthiouracil in their
drinking water. Groups of 8 rats each were treated intragastrically
daily for 9 days with 0, 5, 10, 30 or 200 mg/kg of aspartame or DKP in
30% propylene glycol. On the tenth day the rats were anaesthetized
with ether, and blood samples withdrawn from the abdominal aorta.
Serum samples were analysed for cholesterol by a published procedure.
Body weights were taken on the first and tenth day of the test period.
Neither aspartame nor DKP had any significant effect on body weight
gain or blood serum cholesterol (Anonymous, 1972j).
Anti-acetylcholine activity in vitro
Segments of rabbit ileum were cleansed of adipose tissue and
suspended in Tyrode's solution. Muscle movement was recorded on a
physiograph. Maximum muscle contraction was recorded as that produced
by adding acetylcholine at a final concentration of 5 µg/ml in the
bath. Aspartame, DKP, or atropine sulfate was then added until the
contraction had been reduced by 50% of the maximum value. Each
concentration of test compound was tested over a 7 min interval with
at least 8 min intervals between tests. Concentrations of aspartame
and DKP one thousand times those used for atropine sulfate failed to
reduce the contraction by 50% indicating that anticholinergic
activity, if present at all, is less than 0.001% that of atropine
(Anonymous, 1972j).
Antihistamine activity in vitro
Segments of guinea-pig ileum were suspended in Tyrode's solution
and muscle movement recorded on a physiograph. Maximal muscle
contraction was induced by histamine diphosphate at a concentration of
50 µg/ml of bath solution. Test compounds, including the known
antihistamine agent diphenhydramine-HBr, were introduced to determine
what concentration induced a 50% relaxation of maximal contraction.
Each concentration of test compound was tested for 7 min with 8 min
periods between tests during which the bath solution was changed. Both
aspartame and DKP at a PD concentration of 4.6 failed to relax the
tissue by 50% indicating that both compounds are essentially devoid of
antihistamine activity (Anonymous, 1972j).
Autonomic ganglionic blockade effects in cats
Aspartame and DKP were tested for their effects on the superior
cervical ganglia of cats. Each cat was anaesthetized with Na
pentobarbital and (1) the right superior sympathetic nerve was
exteriorized and sectioned caudal to the superior cervical ganglion;
(2) the left common carotid artery was cannulated to monitor blood
pressure; (3) a femoral vein was cannulated for test compound
injections. The rostial stump of the cervical sympathetic nerve was
stimulated electrically and the sustained contraction of the
ipsilateral nictitating membrane was recorded on a physiograph.
Aspartame and DKP were given at a single intravenous dose of 6.4 mg/kg
in saline. Tetraethylammonium bromide was used as a positive control.
Blocking agents decrease the nictitating membrane contraction. Neither
aspartame nor DKP were effective as autonomic ganglionic blocking
agents (Anonymous, 1972j).
Effect of short-term dietary administration of DKP and aspartame on
serum levels of glucose, insulin, triglycerides, free fatty acids,
and cholesterol in rats
Male and female Charles River rats (150-200 g) were placed in
metabolism cages and fed a commercial diet containing 0, 0.2% or 2% of
aspartame or DKP. Ten rats of each sex were used at the 2 treatment
levels for both compounds; controls received commercial diet only. A
paired-feeding technique was used in which a rat of the same sex and
similar weight was given one more gram of food than that eaten by its
pair-fed rat. Body weight, food and water consumption were measured
daily. After 7 days, the rats were sacrificed by decapitation and the
serum obtained after clotting by centrifugation. The serum was
analysed for insulin (by radioimmunoassay, with rat insulin as a
standard), for fatty acids, triglycerides and cholesterol by standard
methods. Serum glucose was determined with the Beckman Glucose
Analyser. For treated rats the mean daily consumption of aspartame
was, for females, low dose/high dose 0.152/1.48 g/kg, and for males,
low dose/high dose 0.2/2.01 g/kg. Ingestion of these levels of
aspartame for one week did not significantly affect the parameters
measured. For DKP the daily dose ingested was females, low/high,
0.153/1.57 g/kg and for males, low/high, 0.197/1.88. Consumption of
the levels of DKP for one week had no significant effect on the
parameters studied (Saunders, 1972).
Endocrinological studies
Hormonal properties
Estrogenic activity
Twenty-one day-old female mice (8-10 per group), maintained
on an estrogen free diet, were orally administered aspartame or DKP
(dissolved in corn oil) at a daily total dose of 1.35 mg for 3 days.
Controls received corn oil. A positive control group received estrone
subcutaneously at doses of 0.1 or 0.3 µg. On the day after the last
injection, the mice were sacrificed and the uteri weighed. Aspartame
and DKP did not stimulate uterine weights in contrast to the marked
stimulation at both dose levels of estrone (Nutting, 1972).
Estrogen antagonism
The test was similar to that used to study estrogen activity
except that all groups were treated simultaneously with a total dose
of 0.3 µg, estrone. A positive effect consists of a limitation of
uterine growth induced by estrone. Aspartame and DKP were given orally
in total doses of 450 and 1350 µg (10-20 mice/dose). Untreated
controls (38 mice) received corn oil. Progesterone in total doses of
50, 100 and 200 µg, s.c., was used as a positive control for estrogen
antagonistic activity. Neither aspartame nor DKP significantly
antagonized uterine growth induced by estrone. In contrast,
progesterone, injected s.c., significantly decreased uterine weight
at all doses (Nutting, 1972).
Progesterone-like activity
Immature female rabbits were primed for 6 days with daily s.c.
doses of estradiol-17-Beta. After priming, aspartame or DKP in corn
oil or corn oil alone was administered bucally for 5 days. Test
compounds were given at 300 mg/day. The positive control,
progesterone, was injected s.c. at doses of 0.02, 0.05 or 0.1 mg/day.
On the day after the last injection, the rabbits were sacrificed and
a segment of uterus was examined histologically and rated as to
the degree of arborization of the endometrial glands (glandular
proliferation is a progestational effect). Increased uterine carbonic
anhydrase activity has been correlated with glandular proliferation.
Accordingly, a uterine segment was also analysed for carbonic
anhydrase activity. Neither aspartame nor DKP caused an increase in
the degree of glandular arborization or the concentration of carbonic
anhydrase in the uterus. In comparison progesterone at a dose of
0.05 mg/day or greater significantly increased both measures (Nutting,
1972).
Progesterone antagonism
The test was similar to that for progesterone-like activity
except that all groups received a daily dose of 0.1 mg progesterone in
addition to the test compounds. Aspartame (4 rabbits) and DKP (4
rabbits) were given at a dose of 300 mg/day. Controls (15 rabbits)
received vehicle alone. The positive control, estrone, was given s.c.
at a dose of 0.001 (4 rabbits) and 0.002 (12 rabbits) mg/day. A
decrease of the stimulated endometrial glandular proliferation
observed in animals treated with 0.1 mg progesterone alone was used as
one index of progesterone-antagonistic activity. After 5 daily doses
(buccal administration) of 300 mg/day, neither aspartame nor DKP
altered the glandular arborization induced by progesterone. Aspartame
also failed to alter the carbonic anhydrase activity induced by
progesterone. However, SC-19192 at 300 mg/day did reduce the
concentration of carbonic anhydrase by 59% of control. The positive
control, estrone, at a dose of 0.002 mg/day reduced both the degree of
arborization and the concentration of carbonic anhydrase. Accordingly,
in this test aspartame does not display progesterone-antagonistic
activity. DKP does, however, exhibit some progesterone antagonism at a
buccal dose of 300 mg/day for 5 days (Nutting, 1972).
Androgenic-myotrophic activity
White male rats were castrated at 22-24 days of age. After 19-21
days recovery, test compounds in oil were given daily for 7 days
(orally). Aspartame and DKP were given at doses of 50 (8 rats each)
and 350 mg (6 rats) (total dose). Controls (20 rats) received oil
alone. The positive control, methyl testosterone, was given orally at
total doses of 10 and 60 mg (8 rats each). On the day after the last
injection, the rats were sacrificed. Increases in the weights of the
seminal vesicles and ventral prostate gland compared to oil control
were used as a measure of androgenicity. An increase in levator ani
muscle weight compared to oil controls served as an index of
myotrophic activity. Methyl testosterone at a total dose of 10 or
60 mg significantly increased the weights of the seminal vesicles,
prostate gland, and levator ani muscles. Neither aspartame nor DKP
significantly altered any of these parameters. Thus, they did not show
either androgenic or myotrophic activity in this test (Nutting, 1972).
Androgen antagonism
The test was similar to that for androgen-myotrophic activity
except that all groups were treated simultaneous to the test compounds
with testosterone propionate, intramuscularly, at a total dose of
0.5 mg. Aspartame and DKP were given orally doses of 50 (15 rats) and
350 mg (7 rats each) daily for 7 days. Controls (20 rats) received
oil. A decrease in weight of the seminal vesicles and ventral prostate
glands compared to a group treated with 0.5 testosterone propionate
alone was used as a measure of inhibition of the response of
testosterone. A decrease in the weight of the levator ani muscle,
compared to a group treated with testosterone alone, was used as an
index of catabolic activity. No positive control was employed.
Although neither test compound given orally at a total dose of 50 or
350 mg/day produced a statistically significant change in the weights
of the tissues examined, both aspartame and DKP at 350 mg/day did
reduce the weight of the seminal vesicles by about 20% (Nutting,
1972).
Glucocorticoil activity
The test employed the fact that in fasted, adrenalectomized rats
the ability to store glycogen in the liver is impaired but that
glucocorticoids reverse this. Adrenalectomized adult male rats,
maintained on saline fluid and a high protein diet were fasted for
24 h, 3 days after surgery. After fasting, test compounds were
administered orally in oil in 4 equal doses at about 2 h intervals.
Aspartame and DKP were given at a total dose of 45 mg (9 rats each).
Controls (9 rats) received oil. Positive controls received cortisone
acetate, s.c., at a total dose of 0.5 or 0.2 mg (10 rats each). The
rats were sacrificed 6-8 h after the first injection and livers were
analysed for total glycogen. An increase in liver glycogen above
controls was the index of neoglycogenic or glucocorticoid activity.
This activity was demonstrated in the animals receiving 0.5 mg (total
dose) cortisone. Aspartame and DKP did not demonstrate glucocorticoid
activity in this test (Nutting, 1972).
General physiological effects
Effects on fertility (implantation)
Rats, post-ovulatory
Sexually mature female rats were mated and treated for seven
days, 60 mg/day aspartame or DKP (five rats each) (orally) beginning
on the day sperm appeared in the vagina (conception). Controls (10
rats) received vehicle (oil). A positive control, oestrone, was given
s.c. at 2 or 4 µg/day (10 rats each). On the 15th day post coitum, the
animals were sacrificed. Aspartame and DKP failed to decrease the
number of rats with normal implantation sites (Nutting, 1972).
Hamsters, post-ovulatory
Sexually mature female hamsters were mated and treated for five
days with 30 mg/day aspartame or DKP (orally; five hamsters each)
beginning on the day sperm were found in the vagina. Controls (15
hamsters) received vehicle (oil) alone. The positive control,
oestrone, was given s.c. at 10 or 20 µg/day (15 hamsters). Animals
were sacrificed six days post coitum. The corpora lutea and
implantations were counted and their condition noted. Oestrone
markedly attenuated the 1% of implantation, increased the 1% of
abnormal corpora lutea and significantly decreased the implantation
rate considering all sites with an ED50 of 12.4 µg/day. Aspartame had
no effect on any of the parameters. DKP did slightly reduce the 1%
normal implantation sites and slightly increased the % abnormal
corpora lutea but had no effect on the implantation rate value
(Nutting, 1972).
Inhibition of pituitary gonadotrophin (GTH)
The test is based on the phenomenon of stimulation of pituitary
GTH secretion in response to unilateral ovariectomy and subsequent
hypertrophy of the intact ovary. Seventy-eight-day-old female rats
were unilaterally ovariectomized. Oral administration of aspartame and
DKP at 60 mg/day or buccal administration of aspartame at 10 and
2 mg/day was carried out for 14 days beginning on the day of surgery
(9-19 rats per group). Controls (10 rats) received vehicle (oil)
orally. A positive control, northynodrel, was given s.c. at 20, 50 or
100 µg/day (29-30 rats per dose). On the day following the last
treatment, the rats were sacrificed and the remaining ovary weighed.
Norethynodral inhibited the compensatory ovarian hypertrophy at doses
of 20 µg/day or greater. None of the groups treated with aspartame or
DKP exhibited a decrease in ovarian weight; pituitary GHT secretion
was unaffected (Nutting, 1972).
Anti-inflammatory activity
Foot oedema test
An inflammatory reaction (oedema of the hind feet measured a
volume displacement) was induced in intact male rats (about 120 g) by
injecting 0.1 mg of 1% carrageenan under the plantar surface of the
hind feet. Aspartame or DKP in saline were administered orally to
eight rats each at a total dose of 36 mg one hour before injection
of the carrageenan. Controls received saline (63 rats). Volume
displacements were measured five hours after carrageenan injection.
The positive control, hydrocortisone, administered orally, reduced the
carrageenan-induced oedema at a dose of 10 mg/rat. Neither aspartame
nor DKP administered as a single oral dose of 36 mg reduced the oedema
inflammatory response to carrageenan (Nutting, 1972).
Cotton wad granuloma formation
In response to subcutaneous implantation of cotton,
adrenalectomized rats develop granulomas surrounding the cotton
pellets. Hydrocortisone prevents this. Adrenalectomized male rats
(200 g) were given four subcutaneous cotton pellet implantation and on
the following day, aspartame was administered orally at 20 and
65 mg/day (six rats each) and SC-19192 was given at 32 and 65 mg/day
10 and 21 rats, respectively). Controls (57 rats) received saline
alone. The positive control, hydrocortisone, was administered s.c. at
1 mg/day. Hydrocortisone significantly reduced the granuloma
formation. Aspartame at 20 and 65 mg/day had no effect, neither did
DKP at 32 mg/day. However, at 65 mg/day SC-19192 did produce a
significant, but small inhibition of granuloma formation (Nutting,
1972).
Chronic polyarthritis
An inflammatory response resembling rheumatoid arthritis
(assessed in rats on the basis of ankle volume) was induced in male
rats (150 g). Aspartame or DKP in saline were administered, orally, at
60 mg/day for 19 days (11-12 rats each). Negative controls (18 rats)
received saline alone and positive controls (11-12 rats per dose)
received hydrocortisone i.g. at 5.1 or 2 mg/day. Rats were sacrificed
and volume displacement of rear ankle joints were determined 24 hours
after the last injection. Hydrocortisone at all doses significantly
decreased the ankle volume, whereas aspartame and DKP at 60 mg/day had
no effect (i.e. no anti-inflammatory activity) (Nutting, 1972).
Immunosuppressive activity
In the Jerne Plaque Test the number of specific antibody
(haemolysin) producing cells in the spleens of mice sensitized to
sheep erythrocytes (SRBCs) is measured. Immunosuppressive agents
inhibit a sensitization injection of SRBCs. In the test system the
number of in vitro "plaques" formed is proportional to the number of
haemolysin producing spleen cells. Male mice (five to seven weeks old)
were injected i.p. with SRBCs in saline. SC-18862 and SC-19192 in
saline were administered once daily, orally for four days (beginning
the same day as the SRBCs injection) to groups of 6-10 mice. Dose
levels of 50, 125, 250, 500 and 750 mg/kg/day were employed. Several
different replications were performed. Additionally, several
experiments included administering phenylalanine and aspartic acid at
these same daily dose levels. In each case, 24 hours after the final
injection, the mice were sacrificed and plaque formation determined.
In one set of experiments aspartame, DKP, phenylalanine and aspartic
acid (oral) inhibited plaque formation (i.e., inhibited the immune
response) at a dose of 500 mg/kg/day. Only SC-18862 was active at a
lower (250 mg/kg/day) or at a higher (750 mg/kg/day) dose. Replicates
of this experiment failed to confirm this activity (Nutting, 1972).
Acute toxicity
LD50
Animal Route (mg/kg/bw) Reference
Rat Oral >5 000 Andress et al., 1973b
i.p. >2 033 Andress et al., 1973b
Mouse Oral >5 000 Andress et al., 1973b
i.p. >1 000 Andress et al., 1973b
Rabbit Oral >5 000 Andress et al., 1973b
Short-term studies
Mouse
Groups of five male and five female mice received 0, 3, 5 and
13 mg/kg/day of aspartame in their diets for four weeks. No compound
related differences between control and test groups in terms of
physical, motor or behavioural effects were seen, nor were effects
evident in terms of body weight or food consumption. On autopsy the
mucosa of the stomach, duodenum, and jejunum of the high dose level
animals were found to be coated with a clear moderately viscous fluid
(Rao et al., 1972b).
Rat
Groups of 10 male and 10 female rats received 0, 5 and
1250 mg/kg/day of aspartame in their diets for a two month period. No
compound related effects were noted in terms of gross appearance,
behaviour, gross eye appearance, mean body weights, food consumption
and urinalysis. Haematological findings were within normal limits.
Blood levels of glucose found were indicative of a dose related
increase, while a dose related decrease of serum albumin and SGOT was
noted particularly in the male rats. Gross and microscopic pathology
did not reveal evidence of compound related lesions (Anonymous,
1969a).
Groups of five male and female rats received 0, 2, 4 and
10 mg/kg/day of aspartame in their diets for a four-week period. No
adverse behavioural or physical effects were noted in treated animals.
A decrease in body weight noted in the high dose level animals was not
statistically significant. At necropsy no treatment related gross
alterations were noted other than a heavy coat of clear viscous
material on the mucosa of the stomach, duodenum and jejunum of the
high level rats (Rao et al., 1972c).
Groups of five male and five female rats received a basal diet or
basal diet with aspartame (100:9 w/w) or basal diet with phenylalanine
(100:5 w/w) for nine weeks. Both aspartame and phenylalanine fed
groups showed similar reductions in growth (11%) and food consumption
(20%). Aspartame treated males showed significantly lower SGPT and
plasma Ca++ and Cl- values. No treatment related effects were seen
upon haematology findings, urinalysis, organ weights and gross and
microscopic pathology (Hemm et al., 1972).
Dog
Groups of two male and three female beagle dogs received orally
5 mg/kg or 125 mg/kg of aspartame daily by capsule for eight weeks.
Two male and two female beagles served as controls. No treatment
related changes were seen in body weights, food consumption,
haematology, biochemistry, urinalysis, ophthalmoscopy. Small, dose-
related increases in testicular weight were noted. Increases in organ
to body weight ratios noted for heart, kidney and adrenal in both test
groups were not dosage related. Gross autopsy and microscopic
evaluation of tissues did not reveal any evidence of compound related
effect (Anonymous, 1969b).
Groups of five males and five female beagle dogs received 0,
1000, 2000 and 4000 mg/kg/day of aspartame (DKP content less than 1%)
incorporated into 200 g of powdered basal diet for 106 weeks. At all
levels of aspartame fed, growth was depressed. Cataracts seen in one
intermediate and one high-level dog (litter mate) were considered to
be congenital based upon appearance of a cataract in one of two pups
resulting from a remating of the parents. Consistent and statistically
lowering of haemoglobin, haematocrit, and total red blood cells was
noted in the male dogs at the high dose level. To a lesser frequency
and degree it was noted in the intermediate dogs. This was not seen in
the low dosage group. There were sporadic treatment-related changes in
some clinical chemistry parameters, but no consistent trends in time
or dose relationships. A large and statistically significant decrease
in BSP values was seen in the male intermediate and high dose level
males at 78 and 106 weeks, but no changes were seen in other liver
function tests. Gross and microscopic pathological findings were not
indicative of compound related changes (Rao et al., 1972d). A detailed
histopathological study was made of the brains of two dogs on the high
dose level. No neoplastic alternations were seen (Kommineni, 1973).
Monkey
Seven newborn rhesus monkeys were divided into three dosage
groups. Aspartame was administered dissolved in a commercial milk
preparation, first using a nursing bottle, later from a cup.
Concentrations of aspartame were increased incrementally so as to
approximate intended dosages. Treatment groups were as follows:
Dosage Age at Total days
mg/kg/day Sex start (days) on treatment
1 000 M 6 210
1 000 F 3 204
3 000 M 3 360
3 000 M 3 362
3 000 F 2 363
4 000-6 000 M 9 357
4 000-6 000 M 1 279
Data from the experimental animals was compared to historical control
data derived from 14 monkeys. Aspartame intake calculated for the low
and intermediate dosage levels was within 5% of that planned. For the
high level, intake was calculated as 1210 and 5330 mg/kg/day, mean for
the entire study being 3600 mg/kg/day. Group mean intake of the
diketopiperazine (DKP) conversion product of aspartame over the entire
study was estimated as being 4.84, 15.07 and 18.12 mg/kg/day
respectively for low, intermediate and high dose groups. Body weights
of one of the two low dose group animals, two of the three
intermediate dose group animals and one of the two high dose group
animals were below normal limits based upon the historical controls.
Growth rates however were comparable to the controls. While there was
a decrease in the total volume of liquid formula ingested in all
groups this was severe only in one of the two high treatment level
monkeys. All animals in the intermediate and high dosage groups
exhibited convulsions of the grand mal type observed for the first
time following 218 days of treatment. These were described as similar
to those induced by feeding L-phenylalanine to infant monkeys and were
ascribed to the L-phenylalanine moiety of aspartame. One of the high
dose level animals (calculated intake 1210 mg/kg/day) died after 279
days on study, cause of death not determined. No haematological
evidence of effect was noted. Clinical chemistry findings were
similarly without evidence of effect aside from serum phenylalanine
and tyrosine values for the intermediate and high dose level animals
in which the values found were similar to those found in animals fed
2000-2500 mg/kg/day of L-phenylalanine. Urinalyses were similarly not
indicative of other than the consistent presence of phenylketone in
the two higher dosage groups. No final gross or microscopic evaluation
was made, the study being abruptly terminated due to the
investigator's death (Rao et al., 1972e).
Long-term studies
Mouse
Groups of 36 male and 36 female mice received 1000, 2000 and
4000 mg/kg/day of aspartame in their diets for 110 weeks. A group of
72 males and 72 females served as controls. No effects were reported
in terms of appearance, behaviour or survival. Ophthalmological
findings were also without evidence of effect. Mean body weights of
treated animals were not markedly different from controls; food
consumption however was decreased with increased dosage. Haematology
and clinical chemistry findings though showing sporadic incidence of
statistically significant differences between test and control animals
gave no indication in trend or dose-relationship of being compound
related. At termination scattered incidences of increased organ
weights or organ to body weight ratios were noted. Among these were
thyroid, heart, and prostate. Distribution and incidence were not
indicative of compound relationship. Gross and microscopic pathology
reported did not indicate the presence of tumorigenic or non-
tumorigenic changes which would be attributed to the administration of
aspartame (Anonymous, 1974d).
Rat
Groups of 40 male and 40 female rats received 1000, 2000, 4000 or
8000 mg/kg/day of aspartame in their diets for 104 weeks. A group of
60 male and 60 female rats served as controls. No evidence of compound
related effects were noted in terms of physical appearance. While no
effects were noted upon growth and food consumption at the two lower
dose levels, these parameters were slightly decreased at the
4000 mg/kg/day and markedly decreased at the 8000 mg/kg/day level. Two
year survival, poor for all groups in both sexes and particularly for
the male control group was attributed to spontaneous disease. However,
survival for females of the 4000 and 8000 mg/kg/day groups was lower
than that of the control groups, significantly so at the 8000
mg/kg/day level where survival was 54% of the control level. No
evidence of compound related changes was noted in terms of
haematology, blood chemistry or eye examination findings. Increased
red and white blood cells were seen persistently in the urine of the
rats tested at the highest dietary level. Gross and microscopic
pathology findings were in general not treatment or dosage related
(McConnell, 1973; Anonymous, 1973c). However, astrocytomas were seen
in all treated animals none being noted in the controls. Incidence of
brain tumours found were as follows:
Feeding level
Tumours Control
1 000 2 000 4 000 8 000
Astrocytomas 0 4 1 4 1
Oligodendroglioma 0 0 0 1 0
(Hazleton Labs., 1973)
Groups of 40 male and 40 female rats received 2000 and 4000 mg/kg/day
of aspartame in their diets for 104 weeks. A group of 60 male and 60
female rats served as controls. All animals were selected from the
F1A litter of a multi-generation study in which the parents had been
exposed to corresponding dietary levels of aspartame for 60 days prior
to mating. No compound related effects were noted in terms of
appearance, behaviour, results of ophthalmological findings or
survival. Decreased weight gain and food consumption was noted for the
higher dosage level animals. Sporadic, statistically significant
variations were noted in both haematology and blood chemistry
findings; there was not however any clear trend of dose or treatment
relationship. Heart to body weight ratios were significantly decreased
for males of both treatment groups and liver weights were increased
for both female treatment groups. Grossly inconsistent treatment
related differences were noted. Histopathologically several
statistically significant alterations were noted for several organs.
These were increased stomach ulceration and gastritis in high level
females, increased incidence of liver hyperplastic nodules at both
feeding levels in females, increased incidence of nodular hyperplasia
of the adrenal cortex. Incidence of brain tumours found may be
tabulated as follows:
Feeding level
Tumour Control
2 000 4 000
Astrocytoma 4 3 1
Meningoma 0 0 1
(Anonymous, 1974e)
A detailed histopathological review of the brains, liver and pituitary
glands of control rats, and rats fed aspartame was carried out. The
incidence of intracranial neoplasm did not appear to predominate in
any treated group nor did any particular type of neoplasm. The
intracranial neoplasms appeared in both the untreated control and
treated rats. Hyperplastic nodules from the liver were considered to
be non-neoplastic and were not treatment related. Chromophobe adenomas
occurred frequently in test and control animals. It was concluded that
the neoplastic alterations in the rats were not treatment related and
within "normal" limits (Kommineni, 1974). A detailed statistical
analysis of the incidence of the hyperplastic nodules of the liver
showed that for animals sacrificed at week 104, there was no
significant increase for test animals over control animals (Springer,
1974).
Hamsters
Basic groups each consisting of five male and five female
hamsters were fed aspartame containing less than 1% DKP at dietary
levels of 1000, 2000, 4000 and 12 000 mg/kg/day for 46 weeks. Groups
of 10 male and 10 female hamsters served as controls. Each group was
replicated seven times. Somewhat erratic decreases in body weights
were seen in the high dosage level males. Transient food consumption
decreases were noted for both sexes of the high feeding level group.
No unequivocal compound related changes in physical, behavioural signs
or mortality were noted for this study. However, there was a spread of
an unidentified infection considered to be "wet tail" in both control
and treated animals considered to be responsible for the high
mortality rate (50%). It was because of this that the study was
terminated at 46 weeks. No consistent treatment or dose-related
effects were noted upon haematology, clinical chemistry and
haematology. Gross pathology was not delineated by groups, but
indicated that gross lesions were "uniformly recorded both in control
and all treated groups and were attributed to the unidentified
infection in the colony". Neoplastic changes were not reported.
Microscopic lesions reported appeared to be scattered throughout the
groups with no apparent treatment relationship (Rao et al., 1972b).
OBSERVATIONS IN MAN
Normal adults
1. Short-term tolerance
Thirty-one normal men and 38 normal women (aged 21-45) were the
subjects of this study. The study design was double blind, with the
subjects randomly assigned to receive aspartame or matching placebo
capsules. The aspartame dose was increased from 0.6 g/day during the
first week to a final level of 8.1 g during the 6th week of the test.
The total amount of aspartame consumed by each individual during the
study was 160.7 g. The following laboratory tests were done one week
before each subject entered the study, and then again at weeks 4 and 6
of the test: complete blood count, complete urinalysis, partial
thromboplastin, prothrombin time, BUN, thyroxine, bilirubin (direct
and indirect), SGOT, alkaline phosphatase, uric acid, creatinine,
cholesterol (total), triglycerides. Serum insulin and glucose levels
were determined after a four-hour fast, and again after dosing the
subject with 100 g glucose orally, during weeks 3 and 6 and follow-up
week 7. Plasma phenylalanine and tyrosine values were determined twice
a week, on blood samples following a four-hour fast. Phenylalanine
tests of urine were done during weeks 3, 5 and 7. Methanol
determinations were done on samples of serum and urine during weeks 4
and 6. General physical examinations with special eye studies were
also performed. No significant difference between the aspartame and
the placebo groups were reported for any of the tests (Langlois,
1972).
2. Long-term tolerance
The subjects used in this study consisted of individuals who had
participated in the short-term study, and who had agreed to continue
in the study for an additional 21 weeks, and subjects who would follow
the same study design and fulfil the same criteria for admission. A
total of 76 persons (30 male, 37 female) were involved in the study.
The daily intake of aspartame during the 21 weeks was 1.8 g/day. The
laboratory tests described in the short-term study were carried out
initially and at weeks 6, 12, 20 and 21 of the study. Serum insulin
and serum glucose levels, plasma tyrosine and phenylalanine levels
were measured at the commencement of the study and weeks 12, 16, 20
and 21. Urine was tested for phenylpyruvic acid prior to and at weeks
1, 10, 20 and 21 of the study. Clinically significant differences
between those taking aspartame and the placebo group were not shown
for either sex in the results of the tests performed (Frey, 1972a).
Obese individuals
The study group consisted of 95 men and women aged 21-70, whose
weight exceeded more than 20% the mean normal weight for height, sex,
body frame, and age as taken from the Metropolitan Life Insurance Co.
Weight Tables (Four Steps to Weight Control, New York, Metropolitan
Life Insurance Co., 1969). The study design and dose level of
aspartame was the same as that described for the short-term study with
normal adults. The laboratory tests were carried out over the same
period. In addition to the tests previously described, weight and
blood pressure were monitored throughout the study. Eighty-four of the
subjects completed the test (44 on aspartame, 40 on placebo). Sixty-
nine individuals who completed the first six weeks of the study,
continued taking 1.8 g of aspartame/day for an additional 21 weeks. In
addition, another 36 individuals took aspartame for 21 weeks only. The
regime for laboratory tests was the same as that described for the
long-term normal studies. No significant differences were reported
between the aspartame and placebo groups (Hoffman et al., 1972;
Hoffman & Romano, 1973).
Normal children and adolescents
Five age-groups were studied of age ranges, 2-3(13), 4-6(22),
7-9(22), 10-12(24), 13-20(45). The figures in parenthesis represent
number of individuals in the study. The study design was double blind
with the subjects randomly assigned to receive foods containing
aspartame or sucrose. The mean daily intake of aspartame ranged from
39.5 to 58.1 mg/kg bw, for the 13 weeks of the study. The following
laboratory determinations were made initially and during weeks 7 and
13 on all subjects aged two through 12 years: plasma phenylalanine,
plasma tyrosine, complete blood count, partial thromboplastin,
prothrombin time, creatinine, bilirubin (direct, indirect and total),
SGOT, urinalysis complete, urine test for phenylpyruvic acid. The
following additional tests were done on subjects aged 13 through 20
years; serum thyroxine, fasting glucose, alkaline phosphatase, uric
acid, cholesterol (total and esters), triglycerides and BUN. No
significant clinical differences were reported between the sucrose and
aspartame groups (Frey, 1972b).
Adult PKU heterozygotes
The subjects of this study were the natural parents of
phenylketonuric children. Sixty-five men and women (non-obese) between
the ages of 21 and 45 were studied. The design of the study and the
dose schedule was the same as that described for normal adults. In
addition to the tests described, electroencephalograms were taken on a
number of subjects before and after the study. Short-term tolerance
studies were conducted for six weeks, 52 of the subjects continued the
study, for a total period of 21 weeks. The design and dose schedule of
this portion of the study is the same as that described for normal
adults (long-term tolerance). There were no significant biochemical or
physical changes during the course of the study in either the
aspartame or placebo groups. There was no evidence that the
phenylalanine content of aspartame caused any disturbance of the
apparently normal phenylalanine metabolism (Koch et al., 1972; 1973).
Tolerance of loading doses by normal adolescents
A 12-year-old male and 15-year-old female were subjects of this
study. The two subjects were given a loading dose of 34 µg/kg bw
aspartame in orange juice on one occasion and two weeks later they
were given the molecular equivalent amount of L-phenylalanine
(19 µg/kg) in orange juice. A standardized diet was prescribed for
each of the three 24-hour periods before the aspartame and the
phenylalanine loads and for each of the three, 24-hour periods
following each load. Urine was collected for three successive
three-hour periods before each load, and then at 8, 16, 24-hour
post-loading. Urine was analysed by chromatography for amino acids
(phenylalanine and tyrosine), phenolic acids, phenylpyruvic acid and
phenylacetylglutamine and methanol. Serum levels of phenylalanine and
tyrosine were determined before the loading dose and at 1, 2, 4, 8,
24, 48 and 72-hour post-loading. All parameters studied remained
normal during the period of the test (Koch & Shaw, 1973).
Tolerance of loading doses by phenylketonuric (PKU) homozygous children
Two PKU homozygous boys, each approximately 14 years old were
selected for this study. One was on a liberalized Lofenalac diet with
an allowable phenylalanine dietary intake of 70 mg/kg/day. The other
was on a well-controlled Lofenalac diet (PA intake 17 mg/kg/day). The
two subjects were given a loading dose of 34 µg/kg bw aspartame in
orange juice and two weeks later were given a molecular equivalent
amount of L-phenylalanine (19 µg/kg) in orange juice. A standardized
diet consistent with the subjects' clinical state was prescribed three
days prior to and three days post-loading. Although one subject (1) on
the liberalized diet was well within permitted phenylalanine range
during the loading study (dietary intake 2539 mg, added PA from
loading 689 mg), the other subject (2) for exceeding his dietary
limitations (dietary intake 965 mg, added PA from loading, 1072 mg).
Urine samples were collected at eight-hour intervals on the day prior
to loading, then at eight-hour intervals for the first day post-
loading, and then early morning samples on subsequent days 2 and 3.
Urines were analysed for amino acids (phenylalanine and tyrosine),
phenolic acids, phenylpyruvic acid and phenylacetylglutamine. Serum
levels of phenylalanine and tyrosine were determined prior to and at
1, 2, 4, 8, 24, 48 and 72-hour post-loading. One patient (1) at the
time of the study was excreting large quantities of phenylalanine so
any slight increase may not have been observed. The other subject (2)
showed a slight increase in urinary excretion of phenylalanine and its
metabolites. Analysis of serum did not show any significant increase
in phenylalanine or tyrosine levels (Koch, 1972).
Tolerance of aspartame by diabetic subjects
Seventy-seven subjects (27 male, 50 female) aged 21-70 who were
dependent on insulin for control of diabetes were studied. Prior to
the study there was a complete physical examination and the following
laboratory tests were carried out: complete blood count (CBC), partial
thromboplastin time, prothrombin time, BUN, creatinine, T4, bilirubin
(it was required that these values be within normal range), in
addition the following tests were also carried out: SGOT, SGPT, LDH,
alkaline phosphatase, glucose (fasting blood sugar), uric acid,
cholesterol (total and esters), triglycerides. The study was double
blind with subjects randomly assigned to receive aspartame or matching
placebo capsules. The participants continued on their normal diets.
The dose level of aspartame was 1.8 g/day. There was no evidence that
either aspartame or placebo resulted in consistent changes in fasting
blood sugar or any other parameters measured in this study (Bleicher &
Stern, 1973).
Effects of aspartame loading on plasma and erythrocyte free amino
acids in normal adult subjects
Twelve normal healthy subjects (six male and six female) were
fasted overnight and administered either aspartame at 34 mg/kg bw or
an equivalent amount of aspartic acid (13 mg/kg), dissolved in orange
juice. The subjects received nothing by mouth for eight hours
following the load, except for 240 ml of water at four and six hour
post-dosing. Normal meals were allowed after that point. Plasma and
erythrocyte amino acid levels were measured at 0, 0.25, 0.5, 0.75, 1,
1.5, 2, 3, 4, 8 and 24 hours. The 24-hour sample was taken after an
eight-hour fast. No significant changes were noted in plasma
aspartate, asparagine or glutamin levels with either treatment. Plasma
glutamate, alanine and proline levels increased, for both treatments.
Plasma phenylalanine levels increased from fasting to normal
postprandial levels. A small increase in the plasma tyrosine level was
noted in the aspartame group. All other amino acid in the plasma
decreased or remained unchanged. Erythrocyte glutamate, aspartate and
asparagine levels were unchanged, after either treatment. Erythrocyte
phenylalanine and tyrosine, alanine and proline levels were similar to
those in the plasma. All other amino acids were unchanged or slightly
elevated (Stegink et al., 1977a).
In another series of tests six normal subjects (three male and
three female) were administered aspartame in successive studies at
dose levels equivalent to 100, 150 or 200 mg/kg bw. The subjects were
fasted eight hours prior to dosing, and eight hours post dosing (with
water allowed). Blood samples were taken at 0, 0.25, 0.5, 0.75, 1,
1.5, 2, 3, 4, 5, 6, 7, 8 and 24-hour post-dosing for determination of
plasma and erythrocyte amino acids. The 100 mg/kg dose of aspartame
caused no increase in plasma aspartate levels. At the 150 mg/kg bw
dose level, small increases in plasma aspartate and glutamate were
noted. Increases in plasma phenylalanine and tyrosine also occurred.
At the highest level tested (200 mg/kg) there was a small increase in
plasma aspartate and glutamate levels. Plasma phenylalanine levels
increased to a mean of 48.7 ± 15 µmol/dl after dosing and decreased
rapidly to normal levels (Ca 7 µmol/dl). Plasma tyrosine levels
increased to a mean of 13.6 ± 12.8 µmol/dl (Ca 4 µmol/dl normal
level). Erythrocyte levels of glutamate and aspartate were unchanged,
while phenylalanine and tyrosine increased to levels similar to those
in plasma (Stegink et al., 1977a; 1979d; 1979c).
In another study six healthy women with well-established
lactation were administered either aspartame or lactose at 50 mg/kg
bw. The order of administration was randomized in a crossover design,
with an interval of at least two weeks between each segment of the
study for each subject. Subjects were fasted eight hours prior to
administration of the test compound and four hours after. Plasma and
erythrocyte amino acid levels were measured at 0, 0.25, 0.5, 0.75, 1,
1.5, 2, 3 and 4 hours post-dosing. Breastmilk samples were collected
for amino acid analysis at 0, 1, 2, 3, 4, 8, 12 and 24 hours
post-dosing. There was no significant effect on plasma aspartate,
asparagine glutamine levels. Plasma glutamate and tyrosine levels were
slightly increased after aspartame ingested, but were still within
normal postprandial range. Plasma phenylalanine was also increased
after aspartame ingestion but not after lactose ingestion. The level
was higher than that generally observed postprandial. Plasma proline
and alanine were increased after both aspartame and lactose ingestion.
No significant effects were observed on other amino acids. Erythrocyte
phenylalanine and tyrosine increased after aspartame ingestion, as did
proline and alanine, but the increases were less than that in plasma.
No significant differences were observed for the other amino acids.
There was a small increase in tyrosine, phenylalanine and aspartate
levels in breastmilk after aspartame loading as compared to lactose
loading, e.g,, breastmilk phenylalanine increased from 0.5 µmol/dl to
2.2 µmol/dl, and aspartate increased from 2.2 µmol/dl to about
4.5 µmol/dl in the four-hour period post-dosing. At eight hours
post-dosing the levels were in the normal postprandial range (Stegink
et al., 1977a; 1979d; 1979e).
Plasma methanol was determined in individuals administered
aspartame at 100, 150 and 200 mg/kg bw. The peak levels of methanol
were (in mg%) 1.27 ± 0.48 (100 mg/kg group), 2.14 ± 0.35 (150 mg/kg
group) and 2.58 ± 0.78 (200 mg/kg group). No formate was detected in
the blood or urine of subjects receiving aspartame at 200 mg/kg bw
(Stegink et al., 1977a; 1979d; 1979e).
Four female subjects known to be heterozygous for phenylketonuria
(PKU) were administered aspartame at a dose level equivalent to
34 mg/kg bw. The subjects were fed eight hours prior to administration
of the test substances, and then for another eight hours post-dosing
with water being allowed. Blood samples were taken at 0, 0.25, 0.5,
0.75, 1, 1.5, 2, 3, 4 and 8 hour post-dosing. Plasma aspartate levels
were similar to those in normal subjects. Plasma phenylalanine levels
in the PKU individuals were higher than that in normal subjects
(16 µmol/dl compared to 12 µmol/dl normal). No significant differences
in plasma tyrosine were noted. Erythrocyte levels of amino acids were
similar to those in normal subjects (Stegink et al., 1977a; 1979d;
1979e).
Three male subjects (aged 25-55 years) were fasted overnight
prior to administration by mouth of phenylalanine 14C aspartame
(500 mg in 120 ml water). Four-hour post-dosing normal diet was
allowed. Blood and urine samples were taken prior to dosing and then
at various intervals post-dosing up to 64 hours for blood and 48 hours
for urine. The 14C levels in plasma rose rapidly during the first 15
minutes and reached a maximum four to eight hours post-dosing. The
initial disappearance rate of 14C from plasma was estimated to be
47.5 hours. Four hours after administration of the test substance, the
major 14C plasma radioactivity was associated with high molecular
weight substances (87%), with trace amounts being present as
phenylalanine, tyrosine, diketopiperazine and aspartylphenylalanine.
Less than 1% of the administered 14C was excreted in urine during the
test period (Anonymous, 1972a).
The effects of aspartame ingestion on blood amino acids in normal
adults and one-year-old infants were examined in individuals given
single 34, 50 or 100 mg/kg bw oral doses (Stegink et al., 1977c;
1979d; 1979e).
Plasma concentrations of aspartate were unchanged from fasting
levels in both infants and adults at all ingestion levels, indicative
of rapid aspartate metabolism. Phenylalanine levels in plasma peaked
at 45-90 minutes after dosing. The increase was dose but not age
related. The data are summarized below:
Peak plasma phenylalanine levels (µmol/dl)
Dose
Subject
0 34 mg/kg 50 mg/kg 100 mg/kg
Infants 6.1 ± 1.2 9.7 ± 2.7 11.5 ± 3.1 22.5 ± 11.6
Adults 5.7 ± 1.2 11.1 ± 2.5 16.2 ± 4.6 20.2 ± 6.8
Six normal adult subjects (three male, three female) were examined for
glutamate and aspartate plasma levels up to six hours after ingesting
a 1 g protein/kg bw meal or an identical meal containing MSG or APM at
a dose of 34 mg/kg bw (Stegink et al., 1977b; 1979d; 1979e).
Plasma glutamate and aspartate levels did not differ
significantly between groups. In an identical study using MSG at 150
mg/kg bw and aspartame at 23 mg/kg bw, plasma glutamate plus aspartate
levels did not differ significantly between the subject groups fed the
MSG alone or MSG plus aspartame (Stegink et al., 1979c; 1979e).
In a similar study, six male and three female subjects were given
a meal consisting of a clear soup and a beverage containing either no
added compound, MSG at a dose of 50 mg/kg bw, or MSG at 50 mg/kg bw
plus aspartame at 34 mg/kg bw. Plasma aspartate and glutamate were
measured for up to four hours postprandially. Plasma aspartate levels
were unchanged following ingestion of the soup-beverage meal without
added MSG or aspartame. The MSG dose produced a significant increase
in plasma aspartate values 15-30 minutes after loading. Addition of
both APM and MSG produced a small but statistically significant
(p = 0.01) increase in plasma aspartate levels above those noted from
MSG alone 30-60 minutes after loading. The addition of APM alone did
not significantly increase the mean peak values of plasma glutamate
plus aspartate (Stegink et al., 1979b; 1979e).
Six known MSG sensitive individuals (susceptible to "Chinese
Restaurant Syndrome") were given 300 ml of cold orange juice
containing either sucrose (1 g/kg bw) or aspartame (34 mg/kg bw) in a
crossover design study (Searle Labs, 1979b). No symptoms were reported
by any of the subjects and plasma aspartate levels were similar after
aspartame and sucrose loading. Plasma glutamate levels remained within
normal fasting levels (Stegink et al., 1979a; 1979e).
Multiple doses of 34, 100 or 200 mg/kg bw of aspartame were used
to investigate the average steady state levels of plasma phenylalanine
after repeated doses of the compound. The following table summarizes
the T 1/2, Ke and K1 from this study:
Plasma phenylalanine half-life, Ke and K1
Aspartame dose Half-life (T 1/2) Ka* K1**
(mg/kg bw) (hours) (hour -1) (hour -1)
34 1.65 0.420 3.47
100 1.7 0.408 2.72
200 1.7 0.408 1.26
* Ke = first rate constant for the disappearance of phenylalanine
from the plasma.
** K1= first order rate constant for input of phenylalanine in
plasma.
The following table summarizes the average steady state plasma
phenylalanine concentrations:
Plasma phenylalanine (µmol/dl)
Time interval between doses
(hours) Aspartame dose (mg/kg bw)
34 100 200
1 12 67 156
2 6 34 78
3 4 22 52
4 3 17 39
8 - 8 19
(Stegink et al., 1979e)
Adult PKU heterozygotes
Five female PKU heterozygotes were given a single 100 mg/kg bw
oral dose of aspartame in cold orange juice. As shown in the table
below, plasma aspartate levels were not affected. However, plasma
phenylalanine levels were elevated.
Plasma aspartate and phenylalanine levels µmol/dl)
Time (h)
0 0.5 1.0 1.5 2.0 3.0 4.0 Reference
0.5 0.8 0.5 0.6 0.4 0.5 0.4
ASP ± ± ± ± ± ± ±
0.3 0.6 0.3 0.5 0.3 0.3 0.3 Stegink et
al., 1979f
7.0 36 37 42 32 24 17
PHE ± ± ± ± ± ± ±
0.7 7.6 8.0 2.3 5.3 3.2 2.7
Plasma and erythrocyte amino acid levels were measured in eight
female subjects known to be PKU heterozygotes and 12 normal subjects
(six males and six females) after a single oral dose of aspartame at
34 mg/kg bw. No changes in either plasma or erythrocyte aspartate
levels were noted up to eight hours after dosing in either group. In
the normal subjects, plasma phenylalanine levels increased from
fasting values (5.66 ± 1.21 µM/100 ml) to normal post-prandial
levels (11.11 ± 2.49 µM/100 ml) and returned to close to baseline
levels at eight hours after dosing. In the PKU heterozygotes, mean
peak plasma phenylalanine levels were 16.03 ± 2.25 µM/100 ml and the
concentration curve was broader over time. However, maximum plasma
phenylalanine levels in this group were only slightly above
postprandial values in the normal human infant and adult. Erythrocyte
phenylalanine levels showed similar but smaller patterns of change
(Stegink et al., 1978).
Comments
An extensive array of toxicological studies have been carried out
with aspartame (APM and its breakdown product DKP).
Metabolic studies with APM in a variety of species suggest that
aspartame is hydrolysed to its constituent amino acids prior to
absorption from the GI tract, and its subsequent metabolism resembles
that of phenylalanine, aspartic acid and methanol. The possible
effects of subsequent increases in serum phenylalanine in neonates has
been demonstrated in the 52-week study in the infant Rhesus monkey
which showed that with APM at dose levels of 3 and 4-6 g/kg grand mal
seizures were observed at 218 days of treatment. The effect was not
observed at the low doses (ca 1 g/kg). Thereafter, sporadic
convulsions occurred inconsistently at various times. The seizures
occurred most frequently during physical handling of the animals and
were of the grand mal type similar to those induced by feeding
L-phenylalanine to infant monkeys.
In an early study in which infant monkeys were dosed with
aspartame, the monkeys from the two highest aspartame dose groups (3.0
and 3.7 g/kg) underwent seizures. In another study none of these
effects were observed in the two highest aspartame dose groups (2.0
and 2.7 g/kg). It is important to note that in the early study monkeys
were fed the total intake of milk formula in an eight-hour period,
while in the second study they were provided 24-hour access to
aspartame-in-milk formula. This would result in a less pronounced
peaking of phenylalanine blood levels in the monkeys of the later
study. It seems that in the early study ad libitum watering was not
allowed while in the second study it was. There is also evidence that
normal food intake and growth were impaired in the early study. These
methodological variations may explain the differences in the results
of the two studies, i.e. seizuring versus no seizuring. It appears
that, if proper nutrition, hydration and growth are maintained, even
extremely high intakes of aspartame (1-3 g/kg/day) do not produce
untoward effects in neonatal monkeys.
Phenylketonuria (PKU) and resulting brain damage and mental
retardation can be experimentally induced in the monkey by feeding
large doses of phenylalanine (PA) beginning shortly after birth. The
large dosing results in suppression of phenylalanine hydroxylase
activity in the liver, the enzyme required for conversion of PA to
tyrosine. The mid and high doses (ca 3 and 4 g/kg/day) of APM resulted
in a significant increase in serum PA and tyrosine levels, while the
low dose (ca 1 g/mg/day) showed no appreciable change in serum PA and
tyrosine levels.
A comparison of the serum PA and tyrosine levels of positive
control monkeys fed 2-2.5 g PA/kg/day with the serum PA levels from
the animals dosed with 3 or 4 g/kg/day APM shows that the PA levels
were similar. PA and APM at these dose levels cause similar effects in
neonate monkeys. It is reasonable to conclude that the effect is due
to the PA moiety of the aspartame, and that this problem related to
neonate infants at an age where susceptibility to phenylalanine and
the effects of PKU are critical. Long-term feeding studies with rats,
mice and dogs, as well as a bladder implant study with mice, provide
adequate data to assess the carcinogenic potential of APM as well as
other long-term effects.
In the two-year feeding studies in rats, effects at the highest
dose levels (4 and 8 g/kg bw/day) fed included decreased growth,
kidney weight increases, decreased thyroid weight, seminal vesicle
atrophy, changes in the pancreas (fibrosis, mild atrophy and nodular
hyperplasia) and gastritis and stomach ulceration. These effects were
not observed in animals on the lower dose levels (1 and 2 g/kg/day).
Neonate rats (two weeks old) which had been exposed to APM
in utero showed subtle changes in the kidney (minimal to slight
hypertrophy and vacuolation of nuclei in cells of tubules in the inner
cortex). However, in the long-term feeding study in which rats were
exposed in utero to aspartame, these effects were not observed in
the mature animals, nor did they affect the well being of the animals.
Both control and test animals showed an incidence of brain tumours,
nodular hyperplasia of the adrenal cortex, and liver hyperplastic
nodules. The brain lesions (tumours) seen in some test animals of the
first two-year rat study were investigated by a detailed pathology
evaluation of brain sections of the second, lifetime study. These same
brain lesions were seen in untreated controls as well as in test
animals in the second study. The fact that these lesions were not seen
in control animals of the first two-year study could be attributed to
biological variation and not treatment related. The statistically
significant increase of adrenal nodular hyperplasia in survivor males
of the second lifetime rat study was not borne out in non-survivor
males, in females, nor in all groups of the first two-year rat study.
The gastritis and stomach ulceration increase in female rats at high
test levels of the second lifetime rat study, although possibly
related to compound, was not seen consistently in all groups nor in
any test group of the first two-year rat study.
The incidence of hyperplastic nodules in the livers of animals of
the two-year rat study was subjected to a detailed statistical
analysis; there did not appear to be a significant increase of these
lesions in test animals over controls.
In the case of the 104-week mouse study, a detailed pathological
study did not indicate the presence of compound-related tumorigenic or
non-tumorigenic changes.
In the two-year feeding study with dogs, at the highest dose
level fed (4 g/kg/day) effects observed included decreased
haemoglobin, haematocrit and total red blood cells. BSP values were
also decreased. The effects were not noted at lower dietary levels.
Gross microscopic pathological findings did not indicate any compound-
related effect. A detailed study of the brain of test dogs did not
show any neoplastic changes.
The 56-week urinary bladder impact study in the mouse showed no
significant differences in the incidence of neoplasms in the urinary
bladder of negative control (cholesterol) and test groups (cholesterol
plus APM).
The general lack of carcinogenic response in these test systems
provides an adequate data base for the non-carcinogenicity of APM.
Studies of the carcinogenic potential of DKP include a long-term
study in the rat and mouse, as well as a bladder implant study in the
mouse. In the rat study there was a significant decrease in weight
gain at the highest dose levels. At autopsy the only compound-related
effect was a significant increase in the incidence of uterine polyps.
The significance of this effect was carefully assessed. The lesions
were considered to be non-neoplastic benign proliferations that are
known to occur spontaneously in aging rats. In the mouse study the
only compound-related effect was an increase in absolute relative
thyroid/body weight. However, there were no pathological changes. The
56-week urinary bladder implant study in the mouse showed no
significant differences in the incidence of neoplasm in the urinary
bladder of negative control (cholesterol) and test groups (cholesterol
plus DKP).
Metabolic studies with DKP in a number of animal species indicate
that some DKP may be observed unchanged, or as its metabolite,
phenylacetyl-glutamine. Other metabolic products have not been
identified. Detailed investigations on the possible nitrosation of
DKP show that n'-nitroso compounds were not formed in vitro or
in vivo.
A two-generation rat reproduction study with aspartame showed
that the only effect observed at the highest dose level tested
(4 g/kg bw) was that the body weights of F1A and F2A weanlings were
significantly reduced. This effect was not observed in the lower dose
levels (2 g/kg bw). All other parameters were normal. A single
generation reproduction study with DKP at dose levels up to
1.8 g/kg/day showed no compound-related effects. Teratological studies
with the rat and rabbit with either AMP or AMP/DKP (3:1) mixture of
DKP at dose levels up to 4 g/kg/day, administered either by gavage or
in the diet (rabbit) or in the diet (rat), showed no significant
compound-related effects. Mutagenicity studies, which included host-
mediated assay, dominant lethal and cytogenetic studies with AMP or
DKP, did not indicate that either AMP or DKP are mutagenic.
AMP and DKP were subjected to a large nurser of biological tests
to screen for any possible pharmacological, endocrinological or
behavioural effects. A positive effect was only observed in one test,
namely, that high levels of DKP exhibit some progesterone antagonism.
Aspartame was tested in human subjects of various population
types - normal adults, "healthy" obese adults, normal children and
adolescents, PKU heterozygotes (natural parents of PKU children), PKU
and normal adolescents, and insulin and non-insulin dependent
diabetics. Various doses of aspartame and various treatment periods
(from acute, large dose loading to prolonged reasonable dietary dosing
up to 90 days) were used. Comparisons were made with phenylalanine,
with sucrose controls, and normal diet controls (double-blind
studies). The data indicated no significant toxicological problems
within limitations of the studies.
Studies on aspartame loading of normal individuals showed that
this resulted in only minor changes in the levels of serum or
erythrocyte amino acids. At the highest dose studied, the peak levels
of phenylalanine were within the range tolerated in phenylketonurics
subjected to high doses of aspartame who, however, showed somewhat
higher levels of phenylalanine than normal subjects. The elevations
were transient, and well below levels that could be expected to
produce toxic effects. Loading with aspartame did not result in
significant levels of methanol in the blood, or formate in blood or
urine. Additional studies have been carried out on the effect of
aspartame ingestion on blood amino acids in infants, normal adults and
female PKU heterozygotes. The results of these studies confirm the
previous observations. Aspartame loading has also been shown not to
increase plasma glutamate levels in MSG-sensitive individuals.
EVALUATION
Aspartame:
Estimated level causing no toxicological effect in the rat
4 g/kg bw
Estimate of acceptable daily intake for man
40 mg/kg bw
Diketo piperazine:
Estimated level causing no toxicological effect in the rat
750 mg/kg bw
Estimate of acceptable daily intake for man
7.5 mg/kg bw
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potential in the rabbit. Unpublished report from Hazleton
Laboratories, Inc., submitted to the World Health Organization by
G. D. Searle & Co., Skokie, Ill., USA, 1974b
Anonymous. SC-19192: 110-week toxicity study in the mouse. Unpublished
report from Hazleton Laboratories, Inc., submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1974c
Anonymous. SC-18862: 104-week toxicity study in the mouse. Unpublished
report from Hazleton Laboratories, Inc., submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1974d
Anonymous. SC-18862: Lifetime toxicity study in the rat. Unpublished
report from Hazleton Laboratories, Inc,, submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1974e
Bleicher, S. J. & Stern, S. B. Tolerance of Aspartame by diabetic
subjects. Unpublished report from Tulane University School of
Medicine, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1973
Bryan, G. T. SC-18862: A 56 week urinary bladder tumorigenicity study
in the mouse by the intravesical pellet implant technique.
Unpublished report from University of Wisconsin School for Health
Sciences, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1974a
Bryan, G. T. SC-19192: A 56 week urinary bladder tumorigenicity study
in the mouse by the intravesical pellet implant technique.
Unpublished report from University of Wisconsin School for Health
Sciences, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1974b
Frey, G. H. Long-term tolerance of Aspartame by normal adults.
Unpublished report from Hill Top Research, Inc., submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972a
Frey, G. H. Long-term tolerance of Aspartame by normal children and
adolescents. Unpublished report from Hill Top Research, Inc.,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1972b
Hemm, R. D., Rao, K. S., Martinez, T. B., Calhoun, D. W. & Mayer,
J. E. SC-18862: Nine week oral toxicity study in the rat.
Unpublished report from Department of Pathology-Toxicology of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Hoffman, R. S. & Romano, F. Long term tolerance of Aspartame by obese
adults. Unpublished report from Staten Island Hospital, submitted
to the World Health Organization by G. D. Searle & Co., Skokie,
Ill., USA, 1973
Hoffman, R. S., McGinn, T. & Romano, F. Short term tolerance of
Aspartame by obese adults. Unpublished report from Staten Island
Hospital, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1972
Koch, R. Tolerance of loading doses of Aspartame by phenylketonuric
(PKU) homozygous children. Unpublished report from Children's
Hospital of Los Angeles, submitted to the World Health
Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972
Koch, R. & Shaw, K. Tolerance of loading doses of Aspartame by normal
adolescents. Unpublished report from the Children's Hospital of
Los Angeles, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1973
Koch, R., Peterson, R. M. & Scriver, C. R. Short term tolerance of
Aspartame by adult PKU heterozygotes. Unpublished report from the
Children's Hospital of Los Angeles, submitted to the World Health
Organization by G. D. Searle & Co., Skokie, Ill., USA, 1972
Koch, R., Peterson, R. M. & Wolfinger, H. L. Long term tolerance of
Aspartame by adult PKU heterozygotes. Unpublished report from the
Children's Hospital of Los Angeles, submitted to the World Health
Organization by G. D. Searle & Co., Skokie, Ill., USA, 1973
Kommineni, C. Personal communication to C. J. Kokoski of the United
States Food and Drug Administration, submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1973
Kommineni, C. Personal communication to C. J. Kokoski of the United
States Food and Drug Administration, submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1974
Langlois, K. J. Short term tolerance of Aspartame by normal adults.
Unpublished report from Hill Top Research., Inc., submitted to
the World Health Organization by G. D. Searle & Co., Skokie,
Ill., USA, 1972
Lemkey-Johnston, N., Reynolds, W. A. & Kulikowski, H. Damage in the
neonatal mouse brain following ingestion of Aspartame.
Unpublished report from the Illinois State Institute for
Developmental Disabilities, and the Department of Anatomy,
University of Illinois College of Medicine, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1977
McConnell, R. G. SC-18862: Two-year toxicity study in the rat.
Unpublished report from Hazleton Laboratories, Inc., submitted to
the World Health Organization by G. D. Searle & Co., Skokie,
Ill., USA, 1973
Nutting, E. F. SC-18862: A sweetening agent. Endocrine studies.
Unpublished report from Department of Biological Research of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Potts, W. J. SC-18862: A sweetening agent. Pharmacological studies.
Unpublished report from Department of Biological Research of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Rao, K. S., Martinez, T. B., Hemm, R. D. & McConnell, R. G. SC-19192:
Two week oral toxicity study in the mouse. Unpublished report
from Department of Pathology-Toxicology of Searle Laboratories,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1971a
Rao, K. S., Mauro, J. & McConnell, R. G. SC-19192: Two week oral
toxicity study in the rat. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1971b
Rao, K. S. Staunton, C. & McConnell, R. G. SC-19192: Five-week oral
toxicity study in the rat. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972a
Rao, K. S. Martinez, T. B. & McConnell, R. G. SC-18862: Four week oral
tolerance study in the mouse. Unpublished report from Department
of Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972b
Rao, K. S. Martinez, T. B. & McConnell, R. G. SC-18862: Four week oral
tolerance study in the rat. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972c
Rao, K. S. Mauro, J. & McConnell, R. G. SC-18862: 106 week oral
toxicity study in the dog. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972d
Rao, K. S. Mauro, J. & McConnell, R. G. SC-18862: 46 week oral
toxicity study in the hamster. Unpublished report from Department
of Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1972f
Rao, K. S. McConnell, R. G. & Waisman, H. A. SC-18862: 52 week oral
toxicity study in the infant monkey. Unpublished report from
Department of Pathology-Toxicology of Searle Laboratories,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1972e
Rao, K. S., Stejskal, R. & McConnell, R. G. SC-19192: 115 week oral
tumorigenicity study in the rat. Unpublished report from
Department of Pathology-Toxicology of Searle Laboratories,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1974
Reynolds, W. A., Stegink, L. D., Filer, L. J., jr & Renn, E. Aspartame
administration to the infant monkey: Hypothalamic morphology and
blood amino acid levels. Unpublished report from the Department
of Anatomy of the University of Illinois at the Medical Center,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1979a (Also submitted for publication to
Anat. Rec., 1980)
Reynolds, W. A., Bauman, A. F., Stegink, L. D., Renn, E. & Filer, L.
J., jr. Developmental assessment of infant macaques receiving
dietary Aspartame or phenylalanine. Unpublished report from the
Department of Anatomy of the University of Illinois at the
Medical Center, submitted to the World Health Organization by G.
D. Searle & Co., Skokie, Ill., USA, 1979b
Rozek, L. F. SC-18862: A sweetening agent. Pharmacological studies.
Unpublished report from Department of Biological Research of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Saunders, R. N. SC-18862: A sweetening agent. Pharmacological studies.
Unpublished report from Department of Biological Research of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Schroeder, R. E. & McConnell, R. G. Evaluation of embryotoxic and
teratogenic potential in the rat. Unpublished report from
Department of Pathology-Toxicology of Searle Laboratories,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1970
Schroeder, R. E. & McConnell, R. G. SC-18862: An evaluation of the
embryotoxic and teratogenic potential in the rabbit. Unpublished
report from Department of Pathology-Toxicology of Searle
Laboratories, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1973
Schroeder, R. E., Rao, K. S. & McConnell, R. G. Toxicological
evaluation of SC-18862: Evaluation of reproductive performance.
Unpublished report from Department of Pathology-Toxicology of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1972
Schroeder, R. E., Rao, K. S., McConnell, R. G. & Sammeta, K. SC-18862:
An evaluation of the mutagenic potential in the rat employing
dominant lethal assay. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1973a
Schroeder, R. E., Mitchell, A., Rao, K. S., McConnell, R. G. &
Sammeta, K. SC-19192: An evaluation of the mutagenic potential in
the rat employing the dominant lethal assay. Unpublished report
from Department of Pathology-Toxicology of Searle Laboratories,
submitted to the World Health Organization by G. D. Searle & Co.,
Skokie, Ill., USA, 1973b
Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
SC-19192: Evaluation of reproductive performance in the rat.
Unpublished report from Department of Pathology-Toxicology of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1973c
Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
SC-19192: An evaluation of the embryotoxic and teratogenic
potential in the rat. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1973d
Schroeder, R. E., Mitchell, A., Rao, K. S. & McConnell, R. G.
SC-18862: An evaluation of the embryotoxic and teratogenic
potential in the rabbit. Unpublished report from Department of
Pathology-Toxicology of Searle Laboratories, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1973g
Schroeder, R. E., Rao, K. S. & McConnell, R. G. SC-18862: A study of
the pregnant and lactating rat and of her offspring. Unpublished
report from Department of Pathology-Toxicology of Searle
Laboratories, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1973e
Schroeder, R. E., Rao, K. S. & McConnell, R.G. SC-18862: An evaluation
of the embryotoxic and teratogenic potential in the rabbit.
Unpublished report from Department of Pathology-Toxicology of
Searle Laboratories, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1973f
Simmon, V. F. & Shan, H. G. An evaluation of the mutagenic potential
of SC-18862 employing Ames Salmonella/microsome assay; S.A.
1385. Unpublished report from SRI International, submitted to the
World Health Organization by G. D. Searle & Co., Skokie, Ill.,
USA, 1978
Springer, J. Personal communication to C. J. Kokoski of the United
States Food and Drug Administration, submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1974
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
loading upon plasma and erythrocyte free amino acid levels in
normal adult subjects. Unpublished report from the University of
Iowa College of Medicine, submitted to the World Health
Organization by G. D. Searle & Co., Skokie, Ill., USA, 1977a
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
Aspartame and MSG ingested as a meal component. Unpublished
report from the University of Iowa College of Medicine, submitted
to the World Health Organization by G. D. Searle & Co., Skokie,
Ill., USA, 1977b
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
upon plasma and erythrocyte free amino acid levels and blood
methanol levels in normal one-year-old children. Unpublished
report from the University of Iowa College of Medicine, submitted
to the World Health Organization by G. D. Searle & Co., Skokie,
Ill., USA, 1977c
Stegink, L. D., Filer, L. J., jr, Baker, G. L. & McConnell, J. E.
Effect of Aspartame loading at 100 mg per kg body weight upon
plasma and erythrocyte levels of free amino acids in normal
subjects and subjects presumed to be heterozygous for
phenylketonuria. Unpublished report from the University of Iowa
College of Medicine, submitted to the World Health Organization
by G. D. Searle & Co., Skokie, Ill., USA, 1978
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Effect of Aspartame
loading in subjects who report symptoms of Chinese restaurant
syndrome after glutamate ingestion. Unpublished report from the
University of Iowa College of Medicine, submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1979a
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
Aspartame and monosodium glutamate when ingested together as part
of a soup-beverage meal. Unpublished report from the University
of Iowa College of Medicine, submitted to the World Health
Organization by G. D. Searle & Co., Skokie, Ill., USA, 1979b
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Metabolic studies of
Aspartame and monosodium glutamate ingested as components of a
hamburger-milk shake meal system in normal adult subjects.
Unpublished report from the University of Iowa College of
Medicine, submitted to the World Health Organization by G. D.
Searle & Co., Skokie, Ill., USA, 1979c
Stegink, L. D., Filer, L. J., jr, Baker, G. L., Brummel, M. C. &
Tephly, T. R. Aspartame metabolism in human subjects. In:
Guggenheim, B. Health and sugar substitutes. Basle, Munich,
Paris, London, New York, Sydney, S. Karger, pp. 160-165, 1979d
Stegink, L. D., Filer, L. J., jr & Baker, G. L. Personal communication
to I. C. Winter of G. D. Searle & Co., submitted to the World
Health Organization by G. D. Searle & Co., Skokie, Ill., USA,
1979e
Stegink, L. D., Filer, L. J., jr, Baker, G. L. & McConnell, J. E.
Effect of Aspartame loading upon plasma and erythrocyte amino
acid levels in phenylketonuric heterozygotes and normal subjects.
J. Nutr., 109: 708-717, 1979f
See Also:
Toxicological Abbreviations
Aspartame (WHO Food Additives Series 16)
ASPARTAME (JECFA Evaluation)