ACESULFAME POTASSIUM
First draft prepared by Dr R. Walker,
Professor of Food Science, Department of Biochemistry,
University of Surrey, England.
1. EXPLANATION
Acesulfame potassium (Acesulfame K) has previously been
evaluated for an Acceptable Daily Intake by the Committee at the
25th and 27th meetings (Annex 1 references 56, 62). A toxicological
monograph was prepared on the first occasion (Annex 1, 57) and a
monograph addendum was prepared by the 27th Meeting (Annex 1, 63)
when an ADI of 0-9 mg/kg b.w. was allocated based on a 2 year study
in the dog in which the no-observed-effect-level (NOEL) of 900 mg/kg
b.w. was the highest dose tested. In a two-year rat study, a
higher NOEL of 1500 mg/kg b.w. was established.
Since the previous evaluation some new data have become
available and are included in the following monograph. The
previously published monograph and monograph addendum have been
expanded and are included in their entirety. Further data on the
potential breakdown products of Acesulfame K, acetoacetamide and
acetoacetamide-N-sulfonic acid are also included.
In reviewing the new and previously published data as a whole,
the Committee also considered whether the ADI might be increased
and based on the second long-term rat study.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
2.1.1.1 Rat
Single oral doses of 10 mg 14C-Acesulfame K/kg b.w. given to
rats and dogs were rapidly absorbed. Maximum blood levels reached
were 0.75 ± 0.2 µg/ml in rats, 0.5 h after dosing, and 6.56 ± 2.08
µg/ml in dogs, 1-1.5 h after dosing. In rats, 82-100% of the dose,
and in dogs, 85-100% of the dose was excreted in the urine; in both
species, 97-100% of the total radioactivity was excreted in faeces,
and total recovery approximated 100%.
Rats given 10 consecutive daily doses of 10 mg/kg orally did
not show evidence of accumulation. Three days after dosing, the
concentration in the organs and plasma was 0.4 nMol/g in liver, and
less than 0.2 nMol/g in other tissues. Seven days after dosing,
the concentration in dogs was less than 0.2 nMol/g in all tissues
examined (Kellner & Christ, 1975a).
After intravenous administration of a single dose of 10 mg
14C-Acesulfame K/kg b.w. to rats, the radioactivity was excreted
quantitatively in urine and the plasma half-life was 0.23 h (Kellner
& Christ, 1975a).
Single oral doses of approximately 15 mg 14C-Acesulfame K/kg
b.w. were administered to male and female rats which had been
pretreated with unlabelled Acesulfame K at a level of 300 mg/kg diet
for 60 days. Control animals without pretreatment were also
similarly dosed with 14C-Acesulfame K. In all animals 95.1-98.2%
of the dose was recovered in urine and cage washings and 0.95-2.86%
in faeces. Total recoveries were 96.3-99.2%. Excretion of
radioactivity was rapid and displayed biphasic kinetics; 92.6-96.8%
of the dose was excreted in 24 hours. The half-life of the rapid
phase was 4-4.5 hours and of the slower phase (accounting for <0.5%
of the dose) was 109-257 hours. No significant differences in route
or rate of excretion were observed between sexes nor between
controls and animals pretreated with Acesulfame K for 60 days (Volz
& Eckert, 1981).
In a study of the pharmacokinetics after prior exposure to high
doses, rats of both sexes were pretreated with 1% Acesulfame K in
the diet for 7 days and then given a single oral dose of 500 mg
14C-labelled compound; control groups were included without
pretreatment. Urinary excretion was rapid, 89.7-93.8% of the dose
was excreted in the first 24 h. The elimination was biphasic with
estimated half-lives of 4 h for the rapid phase and 24-36 h for the
slower phase. No significant differences were observed between the
pretreated animals and controls. All the excreted radioactivity was
in one peak which co-chromatographed with the parent compound and
the experiment confirmed the lack of metabolism of Acesulfame K even
after repeated administration of high doses (Volz et al., 1983)
The absorption, distribution and excretion of 14C-Acesulfame K
was investigated by autoradiography in pregnant rats given a dose of
10 mg/kg b.w. on the 19th day of gestation. The kinetic behaviour
in the dam was comparable with that in non-pregnant animals. The
radioactivity in the fetuses at 0.5 h and 2 h after administration,
when maternal blood levels were at their highest, was low; the
ratio of activity in fetus and maternal blood was 1:14 and 1:3 at
these two time points. The placentae contained higher
concentrations than the fetus and the radioactivity in the amniotic
fluid did not differ from background. The distribution in the
fetus was uniform apart from somewhat higher activity in the
glandular stomach (Kellner & Eckert 1983a).
In lactating rats given a single oral dose of 14C-Acesulfame K
of about 10.6 mg/kg b.w., activity was detected in the milk with a
peak concentration occurring about 5 h after administration. The
mean milk concentration over a 48 h period was about 6.3 times that
in maternal blood. The biological half-lives were similar in milk
(5.6 h) and blood (4 h). It was estimated that about 1.6% of the
dose was eliminated in milk in the first 24 h and one-tenth of this
on the second day (Kellner & Eckert, 1983b).
Following oral administration of a single dose of 3.6-4.5 mg
14C-Acesulfame K/kg b.w. to pigs, maximum blood levels ranged
between 0.35-0.72 µg/ml between 1-2 h after dosing and fell to
undetectable levels within 48 h. Excretion occurred mainly in the
urine (Kellner & Christ, 1975b).
2.1.2 Biotransformation
The metabolism of Acesulfame K was investigated in the urine
and faeces of rats and dogs which had received single oral doses of
10 mg/kg b.w., and in the urine and bile of pigs dosed orally with 5
mg/kg b.w. The analytical methods used (thin-layer chromatography,
mass spectrometry and isotope dilution) detected only the original
substance in these samples (Volz, 1975).
Separation by TLC of urinary extracts from rats used in the
above study revealed only one peak which was identical with
Acesulfame K. No metabolites were detected in control or
Acesulfame K-pretreated animals (Volz & Eckert, 1981). Similarly,
no metabolites were detected in animals which had been pretreated
with 1% Acesulfame K for 7 days (Volz et al., 1983).
2.1.3 Effects on enzymes
In vitro studies on acetoacetamide, a possible minor
breakdown product of Acesulfame K, showed that it did not function
as a substrate for thiolase, ß-hydroxyacyl-CoA-dehydrogenase, or
ß-hydroxy-butyrate-dehydrogenase indicating that in vivo
formation of acetamide is not probable (Anon., 1980b).
Investigation of the carbonic anhydrase-inhibiting effect in
vitro showed that Acesulfame K had virtually no effect,
concentrations of 180 mg/ml being required for 50% inhibition (Vogel
& Alpermann, 1974).
2.2. Toxicological studies
2.2.1 Acute toxicity
Species Route LC50 (mg/l) Reference
Zebra Fish Water >1000 Markert & Weigand,
1979a
Zebra Fish Water ca 25001 Markert & Jung, 1988
1800-25002
Golden Orfe Water >1000 Markert & Weigand,
1979b
LD50 (mg/kg)
Rat p.o. 7430 Anon. 1973
i.p. 2240 Mayer & Weigand, 1977
1 after 48 hours
2 after 96 hours
2.2.2 Short-term studies
2.2.2.1 Rat
Four groups of 10 male and 10 female weanling Wistar-derived
rats were given diets containing 0, 1.0, 3.0, or 10% Acesulfame K
for 90 days. Body weights were recorded weekly, food intake was
determined during the first four weeks and in weeks 11 and 12. In
week 13, the animals were bled from the tip of the tail and blood
samples were examined from haemoglobin content, haematocrit, RBC and
total differential white cell counts. Pooled urine samples from
each group were collected in week 13 and examined for appearance,
pH, glucose, protein, occult blood, ketones and microscopy of the
sediment. At autopsy, blood samples were examined for SGPT, SGOT,
alkaline phosphatase, total serum protein and serum albumin. Organ
weights were recorded for heart, kidneys, liver, spleen, brain,
testes/ovaries, thymus, thyroid, adrenals and caecum (filled and
empty). Histological examination was carried out on
haematoxylin/eosin sections of the weighed organs and on lung,
salivary glands, trachea, aorta, skeletal muscle, axillary and
mesenteric lymph nodes, pancreas, bladder, prostate, epididymis,
uterus, mammary gland, oesophagus, stomach, duodenum, ileum and
colon. Food consumption of rats fed Acesulfame K at the 10% level
was depressed during the first two to three weeks and body weight
gain was markedly lowered during the first four weeks; slight
diarrhoea and increased faecal water content occurred at this dose
level. A slight increase in haemoglobin concentration was observed
in males of the top dose group only, and total serum protein was
slightly decreased in females only. Caecal enlargement was observed
in both sexes receiving 10% Acesulfame K and in females receiving
3%. The relative weights of the liver and kidneys were slightly
elevated in females of the 10% group and relative spleen weights
were slightly depressed in all dose groups. Urinalysis, serum
enzyme levels and serum albumin were not affected by the treatment,
no gross pathological changes were detected, and no dose-related
abnormalities were observed histologically (Sinkeldam, et al.,
1974).
These workers considered that the caecal enlargement was a
physiological response to the presence of osmotically-active
material in the gut and that, since liver, kidney and spleen weights
were within the normal range of the strain of rat used, and no
histological changes occurred, the no toxic effect level is
conservatively placed at 3% in the diet; this is equivalent to 1.5
g/kg/day in rats.
2.2.2.2 Dog
Four groups of four female and four male beagle dogs, initially
17-21 weeks old, were fed diets containing 0, 0.3, 1.0, or 3.0%
Acesulfame K for two years. Body weight was recorded weekly for the
first 12 weeks and at four-weekly intervals thereafter. Urinalysis,
haematological examination and clinical chemistry were performed
after 12, 26, 52, 78 and 104 weeks. Urinalysis included specific
gravity, pH, sugar, protein, occult blood, ketone and microscopic
examination of sediment; haematology comprised sedimentation rate,
clotting time, haemoglobin, PCV, RBC count, WBC count and
differential leucocyte count; clinical chemical investigations
included blood sugar, urea, SGOT, SGPT, serum alkaline phosphatase,
total serum protein and serum albumin. Liver function tests
(bromosulfophthalein clearance) and kidney function tests (phenol
red excretion) were performed on control and top dose group animals
after 26, 52 and 104 weeks. At termination, gross pathological
examinations were performed and the following organs weighed:
heart, kidneys, spleen, liver, lungs, testes/ovaries, thyroids,
adrenals and brain. Histological examinations were performed on the
weighed organs and also on the following tissues: spinal cord,
sciatic nerve, salivary glands, skeletal muscle, thoracic aorta,
skin, tonsils, axillary, superficial, cervical and mesenteric lymph
nodes, bladder, oesophagus, stomach, duodenum, jejunum, ileum,
caecum, colon, pancreas, trachea, circumanal glands, eyes,
epididymis, prostate, uterus, gall bladder, tongue and thymus. A
marrow smear (rib bone) was also examined. General appearance,
condition, behaviour and survival were not affected by the
treatment. None of the examinations performed revealed adverse
effects related to the feeding of Acesulfame K. The no-toxic effect
level was found to be higher than 3% in the diet, corresponding to
an intake of 900 mg/kg/day in dogs (Reuzel & van der Heijden, 1977).
2.2.3 Long-term/carcinogenicity studies
2.2.3.1 Mouse
Four groups of 100 male and 100 female Swiss mice were fed
diets containing 0, 0.3, 1.0, or 3.0% Acesulfame K for 80 weeks.
All survivors were sacrificed and autopsied, and weights of livers
and kidneys were recorded. All tumours and tissues showing gross
lesions suspected of being tumours and the livers of all animals
were examined microscopically (haematoxylin and eosin sections).
The feeding of Acesulfame K did not cause adverse effects on general
appearance, behaviour or survival at any of the dietary levels but
body weights were slightly decreased at the 3% dose level in both
sexes. The relative liver weight was decreased at all dose levels
in males only but there was no evidence of a dose-related response.
Deaths occurring during the course of the study were attributed to
chronic nephropathy, severe liver degeneration, respiratory
infections and lung tumours. Gross and microscopic examination
revealed a variety of tumours in both test and control animals, but
evaluation of the data on type of tumour, location and incidence did
not indicate that the test compound was carcinogenic to mice at
dietary levels up to 3% for 80 weeks (Beems & Til, 1976).
2.2.3.2 Rat
A combined chronic toxicity and carcinogenicity study was
performed on Wistar rats (CIVO strain) which were obtained from the
F1a generation in a multigeneration study (see Special studies on
reproduction, above). Four groups of 60 male and 60 female weanling
rats were given diets containing 0, 0.3, 1.0, or 3% Acesulfame K for
two years. The rats were derived from parents which had been
maintained on the same diet since weaning. Periodic observations
were made of appearance, behaviour, growth, and food intake.
Haematological examinations were carried out after 13, 26, 52, 78
and 104 weeks, clinical chemical tests were performed on blood
samples after 26, 52, and 104 weeks and urinalysis was done after
26, 52, 78, and 102 weeks. At termination, survivors were autopsied
and organ weights recorded for heart, kidneys, spleen, liver, brain,
gonads, thyroid, adrenals, and caecum (filled and empty). Tissue
samples from 20 male and 20 female rats of the control and top dose
groups only were subjected to comprehensive histological
examinations; histology on other animals was limited to liver,
spleen, adrenals, thyroid, parathyroid, pituitary and ovaries, and
to grossly visible lesions suspected of being tumours. Body weight
gain was decreased in both sexes of the top dose group during the
first 44 weeks of the study but not significantly thereafter.
Death-rates of males fed 1.0 or 3% Acesulfame K and of females fed
0.3% Acesulfame K were higher than controls but it was considered
that there was no evidence of mortality being increased by
treatment, and the mortality of control rats was low for the strain
of rat used. Interim deaths were mainly due to chronic respiratory
disease and lymphoreticular malignancies of pulmonary lymphoid
tissue. The incidence of pulmonary lymphoreticular tumours was
relatively high in both males and females of the top dose group but
only achieved statistical significance in females; there was also
some evidence that these tumours appeared rather earlier in males of
the mid and top dose groups. The results of haematological,
clinical, chemical and urinalysis investigations were essentially
normal in all dose groups. The relative weights of liver, kidneys,
caecum and adrenals were increased in both sexes of the high dose
group but the differences only reached statistical significance in
(in males) liver and empty caecal weight and (in females) kidneys
and caecal weight. Gross and histopathology did not reveal any
treatment-related effects.
In commenting on these results, the authors pointed out the
problems of inter-group comparisons in multigeneration studies where
the animals in the different dose groups are not randomised. They
stated that the increased death rate in test animals was still
within the normal range for the strain of rats used and that the
mortality in controls was lower than usual.
Pulmonary lymphoreticular tumours are a common cause of death
in the strain of rats used, with very variable incidence, and the
frequency in the test groups was within the normal range. This
study concluded that their "higher" incidences and earlier
appearance were fortuitous findings and did not suggest that
Acesulfame K possessed carcinogenic properties (Sinkeldam et al.,
1977).
A second combined chronic toxicity and carcinogenicity study
was carried out on a different rat strain with a lower incidence of
pulmonary tumours in untreated animals. Four groups of 60 male and
60 female SPF-Wistar rats received diets containing 0, 0.3, 1.0 or
3.0% Acesulfame K for 120-123 weeks. The rats used were progeny
from parents which had been maintained on the same test diets since
weaning. No adverse effects, other than decreased body weight in
the top dose group, were observed in this study. In particular,
there was no increased mortality or tumour incidence in the
treatment groups. It was concluded that Acesulfame K failed to show
carcinogenic or other effects of toxicological significance when fed
to rats at levels of up to 3.0% for 120 weeks. (Sinkeldam et al.,
1979).
Following reservations about the extent of histological
examination which had been carried out in this study, a detailed
histopathological examination was performed on all animals in the
control and top-dose groups, and in the lower (0.3% and 1%) dose
groups. It was concluded that there were no treatment-related
histopathological changes and, in particular, no evidence of an
increase in the incidence or alteration of the biological type of
the neoplasms diagnosed (Newman, 1982).
2.2.4 Reproduction studies
2.2.4.1 Rat
A multigeneration study in rats was carried out, in which males
and females received Acesulfame K at dietary levels of 0, 0.3, 1.0
and 3.0% for three successive generations, each comprising two
consecutive litters. A teratogenicity study was conducted with 15
females per group of the F2b and F3a generations. Rats from the
F3b generation were submitted to clinical and pathological
examination. Pups from the F1a litters were used for a chronic
toxicity/carcinogenicity study at the same dietary levels of
Acesulfame K as the parents (see Long-term/carcinogenicity
studies). Fertility, number of young per litter, birth weight,
growth rate and mortality during the lactation period were not
adversely affected and there were no indications of increased
mortality in utero. Growth rate was slightly decreased in the top
dose group of the F0 and F1 generations, and the mid-dose group of
the F0 generation. In the teratogenicity studies, no adverse
effects were seen in appearance, food consumption, autopsy of the
dams, organ weights, or litter data; no visceral or skeletal
abnormalities attributable to the treatment were observed.
In a four-week feeding study on rats of the F3b generation,
body weights and food efficiency were slightly decreased in males at
the highest dose level. The relative weights of the caecum were
slightly increased in both sexes of the high-dose group and in males
of the mid-dose group. Gross and microscopic examination did not
reveal any treatment-related pathological changes (Sinkeldam et
al., 1976).
In a separate study, Acesulfame K was fed to pregnant rats at
dietary levels of 0, 0.3, 1.0, or 3.0% from day 6 up to and
including day 15 of pregnancy; a positive control group received 75
000 i.u. vitamin A/rat/day during the same period. An increase in
food consumption was observed at all three dose levels of Acesulfame
K, most pronounced in the 0.3% group. Mean fetal weight showed a
slight, dose-related increase in the test groups but skeletal and
visceral examination of the fetuses revealed no teratogenic effects
attributable to the feeding of Acesulfame K. A wide range of
abnormalities was induced by teratogenic doses of vitamin A in
positive controls (Koeter, 1975).
A reproduction study was carried out in which male and female
rats were fed diets containing, 0, 0.3, 1.0, or 3.0% Acesulfame K
for 12 weeks prior to mating; the dams received the same diet
throughout pregnancy and lactation. Observations were made on the
fertility of the females, number of young per litter, sex rates,
gross abnormalities, mortality, body weight, and resorption
quotient. Growth rate was slightly decreased in parent rats of the
top dose group and in the mid-dose group females. No dose-related
effects were seen in any of the observations made on the offspring,
and there were no indications of increased mortality in utero. At
weaning, 60 animals of each sex were selected from the litters for a
two-year feeding study (see Long-term/carcinogenicity studies)
(Sinkeldam, 1976).
2.2.4.2 Rabbit
An embryotoxicity study with Acesulfame K was carried out in
which female rabbits received doses of 0, 100, 300 or 900 mg/kg bw
by gastric intubation from the seventh to the nineteenth day after
mating. On the twenty-ninth day of pregnancy, fetuses were
delivered by Caesarean section; live and dead fetuses, resorptions
and placentas were counted, weighed and examined macroscopically.
The 24-hour survival was determined by incubation and half of the
fetuses examined for skeletal abnormalities and the remaining half
for visceral changes. One dam from the 300 mg/kg group had a
premature birth. All other observations were within the range of
control values and there was no evidence of compound-related
malformations (Baeder & Horstmann, 1977).
2.2.5 Special studies on antigenicity
Acesulfame K was examined for potential antigenicity in an
active systemic anaphylaxis test in guinea pigs. Groups of 10 male
guinea pigs were challenged with Acesulfame K after sensitization
with Acesulfame K plus Freund's adjuvant. Control groups were
challenged with Acesulfame K after no prior treatment or after
treatment with adjuvant alone. Positive control groups were
challenged with bovine serum albumin. Acesulfame K showed no
antigenic effect and only the animals sensitized with BSA showed
anaphylactic reactions (Donaubauer et al., 1987).
2.2.6 Special studies on caecal enlargement
These studies were performed to investigate the possible
reversibility of caecal enlargement observed in short-term and
long-term studies. Groups of 10 juvenile female Wistar rats, body
weight approximately 115g, received Acesulfame K at dietary
concentrations of 0, 3.0 or 10.0% for treatment period of 45, 49,
and 90 days. One group from each dose level was sacrificed at the
end of the treatment periods of 45 and 90 days; in addition one
group from each dose level was sacrificed after recovery periods of
41 days' treatment, and 14, 56 and 127 days following 90 days
treatment. Food and water intake, and body weight were measured
weekly. At termination, the weights of caeca with and without
contents, as well as moisture content of the contents, were
determined. At the 10% feeding level, there was an increase in food
and water intake and a reduction in body weight gain which was
reversible during the post-treatment period. At this treatment
level, filled caecal weight relative to body weight was
approximately doubled after 45 and 90 days while at the 3% dietary
level, a significant increase of about 30% was observed after 90
days only. A significant increase in the water content of caecal
contents was observed only in the highest dose group. The changes
in the caecal weights after 90 days exposure to 3% Acesulfame K in
the diet were reversible within 14 days. After 90 days treatment at
the 10% dietary level, the water content in the caecum returned to
normal within 14 days but the filled caecal weight remained
significantly increased (by approximately 30%) even after a recovery
period of 127 days (Mayer et al., 1978b).
A similar study was performed with adult female Wistar rats,
body weight approximately 220 g. Initially, animals receiving 10%
Acesulfame K in the diet experienced anorexia followed by an
increased food consumption after two weeks. Water consumption was
increased during the treatment period in both dose groups. Filled
and empty caecal weights were increased by 80% and 33% respectively
in animals receiving 10% Acesulfame K for 45 days. Increased caecal
weights were observed in both dose groups after 91 days treatment.
Caecal water content was significantly increased in both dose groups
after 45 days but not after 91 days treatment. All the changes were
reversible in animals of both dose groups treated for 40 days
followed by a 42 day recovery period. After 91 days' treatment at
the 3% dietary level, all changes were reversible within 14 days but
filled caecal weights were still significantly increased (by about
30%) after a recovery period of 127 days (Mayer et al., 1978c).
These experiments did not reveal any significant differences between
juvenile and adult animals with regard to induction or reversibility
of caecal enlargement. Complete reversibility was demonstrated at
the 3% dietary level, but not at 10% in the diet. The authors note
that the coincidence of increased water intake with increased caecal
water content and the reversibility of both of these parameters
after withdrawal of Acesulfame K probably indicates that the changes
are of osmotic origin.
2.2.7 Special studies on dermal irritation
Acesulfame K was non-irritant in a primary dermal irritation
test in the rabbit (Kreilung & Jung 1988a).
2.2.8 Special studies in diabetic rats
Wistar rats of both sexes were rendered diabetic by a single
i.v. dose of streptozotocin (60 mg/kg) 12 days prior to the
commencement of a short-term study. Groups of 20 male and 20 female
rats were selected from the diabetic animals, only those with blood
glucose levels greater than 300 mg% being used, and received
Acesulfame K in the diet at levels of 0 (control), 0.3, 1.0 and 3.0%
for 28 days. General condition, food and water intake and body
weight gain were recorded and blood glucose levels were determined
at weekly intervals; urinalysis was performed weekly. At
termination, haematological and clinical biochemical analyses were
carried out, organ weights determined and gross and histological
examinations were performed. No differences in general condition,
behaviour and food consumption were noted between diabetic controls
and treated animals; water consumption was higher in groups fed
Acesulfame K, possibly as a result of the osmotic effect of the high
dietary load. All other observations, including histology, did not
reveal any differences due to treatment with Acesulfame K (Mayer &
Kramer, 1980).
2.2.9 Special studies on DNA binding
After pretreatment for seven days with a diet containing 3%
Acesulfame K, male rats were given a dose of 250 mg Acesulfame K
containing 14C-Acesulfame K (9.6 x 108 dpm) by oral gavage. After
eight hours the animals were killed and liver and spleen excised;
DNA and chromatin protein was isolated from these organs. No
radioactivity could be detected on any DNA sample. A low level of
activity (8-11 dpm/mg protein) was associated with chromatin protein
and this was claimed to be due to non-covalent interactions of
unchanged Acesulfame K (Sagelsdorff et al., 1981).
2.2.10 Special studies on genotoxicity
Table 1
Test System Test object Concentration Result Reference
Mouse micronucleus Male and female 2x450-4500 Negative Baeder & Horstmann,
assay mice mg/kg b.w 1977
Dominant lethal assay Male rat 1-3% diet Negative Willems, 1974
Chromosome aberration Chinese hamster 450-4500 mg/kg Negative Mayer et al., 1978a
test bone marrow b.w.
In vitro mammalian Chinese hamster 10-10000 µg/ml Negative Marquardt, 1978
gene mutation (8- V79 cells
azaguanine resistance)
Cell transformation M2 mouse fibroblast 10-10000 µg/ml Negative1 Marquardt, 1978
cells
Unscheduled DNA Primary rat 25-10000 µg/ml Negative1 Myhr & Brusick, 1982
synthesis hepatocytes
Ames test2 S. typhimurium 0-100 mg/plate Negative Gericke, 1977
TA98, TA100, TA15325,
TA1537
Ames test2 S. typhimurium 4-5000 µg/plate Negative Jung & Hollander,
TA98, TA100, TA1535, 1986
TA1537, TA1538
E. Coli WP2uvrA 4-5000 µg/plate Negative
Table 1 (contd)
Test System Test object Concentration Result Reference
Ames test2 S. typhimurium 0-100 mg/plate Negative Rohrborn, 1976
TA98, TA100 TA1535,
TA1537
1 Cytotoxicity at 5000-10 000 µg/ml
2 In the presence or absence of rat liver S9 fraction
2.2.11 Special studies on primary eye irritation
Acesulfame K was examined for primary irritation in the eye of
New Zealand white rabbits. From 1 to 72 hours after application the
treated eyes of all animals showed injection of conjunctival vessels
to a diffuse beefy red colour and there was swelling of lids. The
conjunctivae and nictating membrane were blanched. At the 1 and 24
hour examinations, the irises were reddened and there was colourless
discharge. From 48 h to 7 days some haemorrhage of the conjunctiva
and nictating membranes occurred but all signs of irritancy had
reversed by day 14 (Kreilung & Jung, 1988b).
2.2.12 Special studies of the effects on bacteria
Acesulfame K was without antibacterial activity against 12
bacterial strains in vitro, and did not show antibacterial
activity in experimental septicaemia in the mouse with
Streptococcus pyogenes A77 or Salmonella typhimurium. Long term
culture (30 daily passages) of Staphylococcus aureus and E. coli
with a range of concentrations of Acesulfame K did not affect growth
characteristics nor sensitivity towards antibiotics, ampicillin,
cephalothin, tetracycline or gentiamycin (Schrinner & Limbert,
1977).
2.2.13 Special studies on nitrosation of Acesulfame K
In vitro studies were performed to investigate whether
Acesulfame K could form N-nitroso derivatives. Nitrosation was
carried out using N2O3 in glacial acetic acid, or excess NaNO2,
at pH 3 and pH 1. An N-nitroso compound was detected in low yield
with N2O3 or with nitrite at pH 1 but not at pH 3. The yield at
pH 1 was estimated to be 0.4 x 10-3% (Eisenbrand, 1979).
Acesulfame K was incubated at 37°C with excess NaNO2 (276 mg/10 ml)
at pH 3 and pH 1 for one hour and four hours. The maximum yield of
N-nitroso derivatives was 1.4 x 10-3% after four hours at pH 1.
This was considered to represent a negligible hazard in vivo
(Eisenbrand, 1982).
2.2.14 Special studies on pharmacological aspects
2.2.14.1 In vitro
No functional changes were detected after application of 50 mg
Acesulfame K to isolated guinea-pig heart using the Langendorff
techniques; the compound did not show antiarrhythmic activity in
isolated, perfused guinea-pig heart with aconitine and
digitoxin-induced fibrillation. In the isolated ileum of
guinea-pig, Acesulfame K at a concentration of 10 mg/ml had no
neurotropic or spasmolytic effect on smooth muscle. No functional
changes were detected after application of 50 mg Acesulfame K to
isolated guinea-pig heart using the Langendorff techniques; the
compound did not show antiarrhythmic activity in isolated, perfused
guinea-pig heart with aconitine and digitoxin-induced fibrillation.
In the isolated ileum of guinea-pig, Acesulfame K at a concentration
of 10 mg/ml had no neurotropic or spasmolytic effect on smooth
muscle. Addition of Acesulfame K to dog plasma in vitro was
without effect on thrombin time, thromboplastin time or
recalcification time (Vogel & Alpermann, 1974).
2.2.14.2 Mouse
Dosages of Acesulfame K of 400 mg/kg i.p., 500 mg/kg orally, or
320 mg/kg subcutaneously, did not depress motor activity of mice
excited by Pervitin. The hexobarbital sleeping time in mice was not
changed by pretreatment with Acesulfame K at doses of 500 mg/kg per
os or 160 mg/kg subcutaneously. Metrazol-induced convulsions in
mice were not influenced by Acesulfame K at doses of 500 mg/kg per
os, 300 mg/kg i.p., or 320 mg/kg subcutaneously; anti-convulsant
activity can thus be excluded. Administration of Acesulfame K (200
mg/kg i.p.) to mice was without effect on tetrabenazine-induced
ptosis and catalepsy, thus the compound is without anti-depressant
properties. Acesulfame K (200 mg/kg i.p., 160 mg/kg s.c., or 500
mg/kg per os) was without effect on compulsive gnawing behaviour
induced by the combined application of apomorphine and imipramine,
therefore antidepressant and anticholinergic effects are unlikely.
At doses of 320 mg/kg s.c. or 500 mg/kg orally, Acesulfame K had no
analgesic effect on mice (Vogel & Alpermann, 1974).
2.2.14.3 Rat
Predosing of rats with Acesulfame K (500 mg/kg orally or 160
mg/kg s.c.) was without anti-inflammatory effect on Aerosil-induced
paw oedema, and similar doses had no antipyretic effect in rats with
yeast-induced fever. Eight daily doses of Acesulfame K (0-100
mg/kg) per os had no effect on serum cholesterol, total glycerol,
free glycerol or glucose levels; relative liver weights were
unchanged. In acute tests, Acesulfame K had no effect on blood
sugar levels in rats given 100 mg/kg orally, guinea-pigs given a
similar dose i.p., or rabbits receiving 100 or 500 mg/kg orally or
50 mg/kg i.v. Acesulfame K had no diuretic effect in rats and dogs
at oral dose levels of 50 mg/kg and 20 mg/kg respectively (Vogel &
Alpermann, 1974).
2.2.14.4 Guinea pig
Acesulfame K given intravenously to guine-pigs at a dose level
of 24 mg/kg reduced digoxin toxicity. This effect was due to the
potassium content and not to antiarrhythmic activity of the
compound. Intravenous administration of 1-5 mg Acesulfame K/kg to
anaesthetised guinea-pigs one minute before treatment with histamine
was without effect on the bronchial musculature (Vogel & Alpermann,
1974).
2.2.14.5 Dog
Cardiovascular experiments in anaesthetized dogs showed that
intravenous administration of Acesulfame K was without effect up to
a dose of 6 mg/kg; doses of 12 and 24 mg/kg caused a decrease in
contractility of the heart with a transient reduction of blood
pressure and peripheral blood flow. The changes were compensated
for in 3-5 minutes. Intraduodenal administration of Acesulfame K to
anaesthetized dogs at dose levels of 0-1000 mg/kg induced a slight
reduction of blood pressure at 500 mg/kg and this was accompanied by
a reduction of cardiac contractility of about 20% at 1 g/kg. The
effect was reversed in 50-80 minutes and other cardiovascular
parameters were unchanged. In the conscious dog, after a five day
treatment with Acesulfame K at 100 mg/kg per os daily, no change
in blood pressure or cardiac activity could be detected. Acesulfame
K at doses of 0-24 mg/kg i.v. had no antiarrhythmic effect on
anaesthetised dogs poisoned with K-strophanthin. Daily oral
administration of Acesulfame K (1 g/kg) to dogs for 14 days was
without effect on thromboplastin time, thrombin time,
recalcification time and thromboelastography of plasma samples
(Vogel & Alpermann, 1974).
2.2.15 Special studies on the possible reactions of Acesulfame K
with food constituents.
Acesulfame K (1% aqueous solutions) was heated at 100 °C with
the model food constituents, ethanol, sorbitol, glycine, alanine,
glutamic acid, phenyl alanine or n-butylamine, in acetate buffer at
pH 5. Analysis by HPLC and UV-spectrometry failed to detect any
decomposition or interaction products of Acesulfame K in any of the
model systems (Clauss, 1981).
2.2.16 Special studies on thermal degradation products of
Acesulfame K - inhalation studies.
Two groups of 5 male and 5 female rats were exposed to the
products of pyrolysis of Acesulfame K at 250 °C in an inhalation
chamber. Some disturbances in respiration (irregular/intermittent
breathing) and nasal discharge were noted during exposure but all
animals survived for an observation period of 14 days. No abnormal
signs were seen at autopsy at the end of the experiment (Hollander &
Weigand, 1986).
2.2.17 Special studies on acetoacetamide.
Acetacetamide may be formed to a very small extent during
long-term incubation of Acesulfame K in fluids of low pH. A number
of studies have been conducted on this substance.
2.2.17.1 Absorption, metabolism, distribution and excretion.
The pharmacokinetics of acetoacetamide were studied in rats
after a single oral or i.v. dose of 14C-acetoacetamide, or after 10
daily oral doses of 1 mg/kg b.w. The compound was rapidly absorbed,
maximal blood levels being achieved 0.5-1 h after oral
administration. Subsequent excretion was biphasic with half-lives
of 2.7 ± 0.3 h and 99 ± 28 h; similar excretion kinetics were
observed after i.v. dosing. After repeated dosing, some cumulation
of radioactivity in the blood was observed. Within 7 days after
administration of a single dose, rats (both sexes) excreted 90-97%
(oral dose) or 93-98% (i.v. dose) of the radioactivity in urine and
faeces, urine alone accounting for more than 90% in both cases.
About 9.3% of orally administered radioactivity was in expired air.
On repeated oral dosing, excretion of activity was uniform in urine
and faeces and amounted to 90% after 24 h following the 10th day of
administration, of which about 85% was in urine (Eckert & Kellner,
1979).
Three human volunteers were given oral doses of 50 mg doubly
isotopically labelled acetoacetamide. Absorption was rapid, peak
blood levels being achieved between 15 min and 3 h after ingestion.
Elimination was rapid, t1/2 9.7 ± 0.22 h, more than 95% being
excreted in urine and less than 1% in faeces (Eckert et al.,
1980).
The metabolism of acetoacetamide was studied after single and
repeated oral administration of different doses to hamster, rat,
rabbit, dog and man. Rapid urinary excretion occurred in all
species and TLC analysis of urine revealed the presence of 5 peaks.
Apart from the original compound, the following metabolites were
identified: ß-hydroxybutyramide, erythro- and
threo-2,3-dihydroxybutyramide. In addition, with some variation
with species, the presence of oxamic acid, malic acid amide and a
conjugate of 2,3-dihydroxybutyramide were tentatively identified
and, at least in rats, some of the dose was respired as CO2. The
rabbit was most similar to humans in the metabolism of
acetoacetamide (Volz & Fehlhaber, 1981).
2.2.17.2 Acute toxicity of acetoacetamide
The oral LD50 of acetoacetamide in female rats was greater
than 15 g/kg b.w. (Anon., 1977). No gross adverse effects other
than a slight diarrhoea were seen in one male and one female beagle
dog after administration of 5 g/kg b.w.
2.2.17.3 Short-term studies on acetoacetamide
2.2.17.3.1 Rat
In a preliminary range-finding study, groups of 5 male and 5
female Wistar rats were given acetoacetamide in the diet at
concentrations of 0 or 5% for 29 days. There was an impairment of
body weight gain in the treated group from day 8 and at termination
the deficit of weight relative to controls was 31% and 26% in
treated males and females respectively. Haematological examinations
revealed an increased leucocyte count in treated males and a
significant drop in RBC and haemoglobin levels in both sexes.
Urinalysis and clinical chemistry were essentially normal except for
a reduction in serum alkaline phosphatase. Some significant changes
were observed in relative organ weights but in view of the large
difference in body weight, the relevance of these is unclear. At
autopsy, the livers of most treated animals appeared abnormal and
some animals had enlarged thyroids. Histological examination showed
marked hepatic centrilobular fatty degeneration. The dose tested
was in excess of the no observable effect level (Mayer et al.,
1978e).
Acetoacetamide was administered to groups of 15 male and 15
female rats for 90 days in the diet at concentrations of 0, 400,
2000, 10 000 and 50 000 mg/kg. Body weight gains and feed
consumption were recorded throughout and haematological examinations
and urinalysis were carried out at termination; clinico-chemical
analyses were performed in the middle of the study and at
termination. At autopsy, major organs were weighed and histological
examinations were conducted. Behaviour and condition were
unaffected by treatment but there was a reduction in weight gain of
both sexes at the highest dose level; at the 10 000 mg/kg level
there was a transient reduction in weight gain in males only.
Erythrocyte counts and haemoglobin were depressed in both sexes at
the top dose level; the RBC was also decreased in males of the 10
000 mg/kg dose group. Clinico-chemical parameters and urinalysis
results were generally unremarkable except for a transient decrease
in serum alkaline phosphatase in males of the highest dose group.
Histology revealed fatty degeneration in livers at the top dose
level and thyroid changes (Stroma parenchymatosa) at the 10 000
and 50 000 mg/kg levels. The no observed adverse effect level was
found to be 2000 mg/kg in rat diet (Mayer et al., 1979).
2.2.17.3.2 Rabbit
Four groups of 6 male and 6 female Albino Himalayan rabbits
were given acetoacetamide in drinking water for 90 days at
concentrations of 0, 1200, 6000 or 30 000 mg/l. Behaviour and
general condition of rabbits in the low and middle dose groups
remained normal throughout but animals in the top dose group showed
apathy and body weight gain was impaired, especially in males. In
all groups food and drinking water consumption were not affected and
haematological and urinalysis parameters remained normal. Clinical
chemical analysis was similarly unremarkable except for a
significant decrease in serum alkaline phosphatase in animals of
both sexes, an increase in BUN in females and of uric acid in males
of the highest dose group only. At autopsy, organ weights were
normal except for increased thyroid and decreased testis weights
confined to the highest dose group. Histological examination
revealed marked thyroid changes, interpreted by the authors as
activation, in rabbits of the highest dose group and male rabbits in
this group showed disorders of spermatogenesis. No such effects
were seen in the low and middle dose groups and the no observed
effect level was established at 6000 mg/l in drinking water,
corresponding to a mean daily dose of about 500 mg/kg b.w.
(Hollander et al., 1981).
2.2.17.3.3 Dog
Four groups of two male and two female English beagle dogs were
given acetoacetamide at doses of 0, 100, 500 or 2500 mg/kg b.w. by
gastric intubation on 14 consecutive days. Haemoglobin, haematocrit
and RBC counts were lowered at the highest dose level in both sexes
and there was an increase in reticulocytes and Heinz bodies.
Clinical-chemical examinations showed a clear increase in total
bilirubin and creatinine in the top dose group and glucose levels
were slightly lowered relative to controls. There was an increase
in alkaline phosphatase in both sexes at the 100 mg/kg dose.
Histological examination showed that there were thyroid changes at
all dose levels and, at the highest level, there was increased iron
accumulation in hepatic Kupffer cells. It was concluded that the no
observed effect level for acetoacetamide in the dog is less than 100
mg/kg b.w. (Mayer et al., 1980).
2.2.17.4 Genotoxicity of Acetamide
Table 2
Test system Test object Concentration Result Reference
Ames test1 S. typhimurium 0-100 mg/plate Negative Gericke, 1977
TA98, TA100
TA1535, TA1537
In vitro mammalian Chinese hamster 10-10000 µg/plate Negative Marquardt, 1978
gene mutation (8- V79 cells
azaguanine
resistance)
In vitro mammalian Chinese hamster 100-1011 µg/ml Negative Müller, 1989a
gene mutation (8- V79 cells
thioguanine
resistance)
In vitro chromosome Chinese hamster 101-1011 µg/ml Negative Müller, 1989b
aberration test1 V79 cells
Cell transformation M2 mouse 10-10000 µg/ml Negative Marquardt, 1978
fibroblast cells
Unscheduled DNA Human cell line 1-1000 µg/ml Negative Müller, 1989c
synthesis A549
1 In the presence or absence of rat liver S9 fraction.
2.2.17.5 Pharmacological studies
2.2.17.5.1 Mouse
Doses up to 250 mg/kg b.w. did not induce behavioural changes
in mice. Pretreatment with acetoacetamide did not affect
hexobarbital sleeping time and was without effect on
tetrabenazine-induced ptosis in mice. Oral or sub-cutaneous
acetoacetamide had no inhibitory effect on
pentylenetetrazole-induced extensor convulsions in mice; clonic
convulsions and survival time were unaffected. Oral or s.c.
acetoacetamide at doses up to 250 mg/kg did not protect against
electric-shock-induced extensor convulsions and had no analgesic
effect in the tail-flick test in mice (Alpermann & Scholtholt,
1979).
2.2.17.5.2 Rat
Acetoacetamide had no diuretic effect in the Lipschitz test in
water-loaded rats and did not have a saluretic effect in oral doses
between 10 and 250 mg/kg b.w. per os or s.c. Acetoacetamide had
no effect on blood glucose in glucose loaded rats. At doses up to
250 mg/kg b.w. orally or s.c. acetoacetamide had no
anti-inflammatory activity (carrageenan-induced paw oedema test)
and had no antipyretic effect in rats. Acetoacetamide did not
exhibit alpha-adrenolytic activity in the isolated vas deferens of
the rat (Alpermann & Scholtholt, 1979).
2.2.17.5.3 Guinea pig
Acetoacetamide (10 mg/kg b.w. i.v.) did not antagonize
histamine-induced bronchoconstriction in guinea pigs. In vitro,
acetoacetamide did not affect contractile force or heart rate in
isolated guinea pig atrium and, at a concentration of 10-5 g/ml had
no effect on contractions in isolated guinea pig ileum induced by
barium chloride, carbachol or histamine. Acetoacetamide similarly
was without effect on carbachol-induced contractions of isolated
guinea pig trachea. (Alpermann & Scholtholt, 1979).
2.2.17.5.4 Dog
Intravenous doses of 10 or 25 mg/kg, or an intraduodenal dose
of 250 mg/kg b.w., did not induce acute changes in cardiovascular
parameters in the dog (Alpermann & Scholtholt, 1979).
2.2.18 Special studies on ß-hydroxybutyramide - mutagenicity.
ß-Hydroxybutyramide, a metabolite of acetamide in some
mammalian species, was non-mutagenic in the Ames test against four
strains of Salmonella typhimurium, with and without metabolic
activation (Engelbart, 1979).
2.2.19 Special studies on acetoacetamide-N-sulfonic acid.
Acetoacetamide-N-sulfonic acid is formed to a small extent when
Acesulfame K is incubated at low pH and is thus a potential minor
contaminant. A number of studies have been carried out on this
compound.
2.2.19.1 Absorption, distribution, metabolism and excretion.
The kinetics and metabolism of acetoacetamide-N-sulfonate,
sodium salt, were studied in male rats after single oral or i.v.
doses of 10 mg/kg b.w. Maximum blood levels occurred two hours
after oral dosing (1.33 µg equiv./ml) and subsequent elimination was
biphasic with half-lives of approx. 1 and 5 hours. After i.v.
dosing elimination was rapid and predominantly renal (69.6 ± 9.2%)
whereas after oral administration, faecal excretion predominated
(55.2 ± 6.9%). After 7 days less than 0.06% of the radioactivity
remained in the organism. Distribution studies showed higher levels
in kidneys, urinary bladder and smooth muscle than in blood; after
24 hours concentrations in the tissues were generally below 0.1 µg
equiv./g except in adrenals (0.13 µg/g), bladder (0.14 µg/g) and
retro-peritoneal fat (0.15 µg/g). Incubation with faecal microflora
in vitro resulted in complete decomposition of sodium
acetoacetamide-N-sulfonate, two products being detected, one of
which was tentatively identified as ß-hydroxybutyramide-N-sulfonic
acid. In vivo, besides the starting material, up to four
metabolites were detected but not identified in the urine of male
and female rats (Gross et al., 1987).
2.2.19.2 Toxicological studies
2.2.19.2.1 Acute toxicity studies
Species Sex Route LD50 Reference
(mg/kg b.w.)
Rat M&F oral >5000 Rupprich & Weigand,
(1984a)
Rat M&F i.v. >3150 Rupprich & Weigand,
(1984b)
2.2.19.2.2 Short-term studies
2.2.19.2.2.1 Rat
Acetoacetamide-N-sulfonic acid, sodium salt, was administered
by gavage to Wistar rats at a dose of 1000 mg/kg b.w. daily, five
days per week for 4 weeks. Behaviour, general health, food an
water consumption and weight gain were unaffected. Haematological,
clinical chemical and urinalysis examinations did not indicate any
toxic changes. No adverse effects were seen at gross autopsy nor on
histological examination. It was concluded that the no adverse
effect level was over 1000 mg/kg b.w. (Hollander et al., 1985).
Groups of 30 male and 30 female rats were given
acetoacetamide-N-sulfonic acid in the diet at levels of 0, 8000, 20
000 or 50 000 mg/kg diet for three months. Ten animals of each
sex/group were used in a recovery experiment. No changes due to
treatment were observed in the 8000 mg/kg group during the in-life
phase; the two higher dose groups showed slightly reduced body
weight gain and slightly lower serum glucose which the authors
attributed to limited carbohydrate availability due to a mild
diarrhoea in these groups. The two highest dose groups showed
slightly higher serum bilirubin and calcium values but these were
only significant in males from the 20 000 mg/kg group. In addition,
animals from the highest dose group displayed a mild anaemia.
Urinalysis showed lower pH values and higher specific gravity for
animals in the two highest dose groups and these displayed higher
kidney weights, probably reflecting the increased renal excretion of
the test compound. Animals in the two highest dose groups showed
caecal enlargement, and histological examination showed a reversible
hyperplasia of the rectal lymph follicles. In the absence of other
histological signs and in the absence of anaemia, the authors
concluded that the no adverse effect level was 20 000 mg/kg diet,
corresponding to a mean daily dose of 1863 mg acetoacetamide-N-
sulphonic acid/mg b.w. (Fuchs et al., 1986).
2.2.19.2.2.2 Dog
Four groups of two male and two female beagle dogs were given
acetoacetamide-N-sulphonic acid in the feed at concentrations of 0,
3750, 7500 or 15 000 mg/kg for two weeks. All animals survived and
there were no clinical signs with the 3750 and 7500 mg/kg groups;
administration of 15 000 mg/kg caused diarrhoea. No other reactions
or changes attributable to treatment were detected on gross or
histological examination; one female from the top dose group
displayed a meningo-encephalitis which was not attributed to
treatment (Brunk et al., 1986).
2.2.19.2.2.3 Monkey
In a preliminary tolerance study, six Cynomolgus monkeys (3
males and 3 females) were given daily oral doses of 1000 mg/kg b.w.
acetoacetamide-N-sulfonic acid for thirty days. Diarrhoea of very
slight to moderate intensity was observed from day 6 but food
consumption and body weight were not affected. The results of
electrocardiographic and ophthalmoscopic examinations were normal
and haematology, blood chemistry and urinalysis did not reveal any
treatment related abnormality. Macroscopic and histological
examinations at termination did not show any changes related to
treatment. It was concluded that the dose of 1000 mg/kg b.w. did
not elicit any systemic toxicity and, apart from the sporadic
diarrhoea, the substance was well tolerated (Read et al., 1989b).
Four groups of 4 male and 4 female cynomolgus monkeys were
given acetoacetamide-N-sulfonic acid orally at dose levels of 0,
100, 315 and 1000 mg/kg b.w. daily for 13 weeks. Diarrhoea was
observed episodically in one male from the 100 mg/kg group and
chronically in 3 females and all males from the 315 mg/kg group and
all animals in the top dose group. The onset of diarrhoea occurred
3-7 h after dosing. Body weight and food consumption were
unaffected by treatment and ophthalmoscopic examination in the final
week did not reveal any treatment-related abnormalities.
Haematological, clinical chemical and urinary parameters were not
affected by treatment and gross and histo-pathology were
unremarkable. It was concluded that, apart from the digestive tract
disturbances indicated by diarrhoea, acetoacetamide-N-sulfonic acid
was well tolerated and no systemic toxicity was observed (Read et
al., 1989a).
2.2.19.2.3 Genotoxicity of acetoacetamide-N-sulfonic acid
Table 3
Test System Test Object Concentration Result Reference
Mouse Male and female 5000 mg/kg Negative Mayer & Weigand,
micronucleus mice b.w. 1985
assay
Unscheduled DNA Human cell line 1-1000 µg/ml Negative Müller, 1988a
synthesis1 A549
In vitro Chinese hamster 0-2000 µg/ml Negative Müller, 1988b
mammalian gene V79 cells
mutation (6-
thioguanine
resistance)
In vitro Chinese hamster 0-2000 µg/ml Negative Müller, 1989d
chromosome V79 cells
aberration test1
Ames test1 S. typhimurium 4-5000 Negative Müller, 1989c
TA98, TA100, µg/plate
TA1535, TA1537,
TA1538
E.Coli WP2uvrA 4-5000 Negative
µg/plate
1 In the presence or absence of rat liver S9 fraction
2.2.19.2.4 Pharmacological aspects
2.2.19.2.4.1 In vitro
Acetoacetamide-N-sulfonic acid had no effect on the force or
rate of contraction of the isolated guinea pig right atrium, and
contractions initiated by carbachol, histamine or barium chloride in
the isolated guinea pig ileum were unaffected at a concentration of
10-5 g/ml. This compound had no relaxant effect on isolated guinea
pig trachea and was without alpha-adrenolytic effect on isolated
vas deferens of the rat (Scholtholt & Alpermann 1984).
2.2.19.2.4.2 Mouse
Acetoacetamide-N-sulfonic acid at oral or s.c. doses up to 250
mg/kg b.w. did not cause behavioural changes in mice and
pretreatment with this compound had no effect on hexobarbital
sleeping time. Acetoacetamide-N-sulfonic acid had no effect on
tetrabenazine-induced ptosis. It did not inhibit the extensor
spasms induced by pentetrazole; clonic spasms and survival time
were unaffected. Oral or s.c. doses of up to 250 mg/kg b.w. were
without protective effect against extensor spasms initiated by
electric shock and were without analgesic effect in the heat pain
test (Scholtholt & Alpermann, 1984).
2.2.19.2.4.3 Rat
Acetoacetamide-N-sulfonic acid had no diuretic effect in the
Lipschitz test in rats and oral or s.c. doses of 10 to 250 mg/kg
b.w. had no saluretic effect. The compound was without effect on
blood sugar in glucose tolerance tests. It had no anti-inflammatory
effect in the carrageenan-induced rat-paw oedema test at doses up to
250 mg/kg b.w. per os or s.c. and showed no antipyretic effect
(Scholtholt & Alpermann, 1984).
2.2.19.2.4.4 Guinea pig
Acetoacetamide-N-sulfonic acid at doses of 1-10 mg/kg b.w. i.v.
had no antagonistic effect on histamine induced broncho-constriction
in the guinea pig (Scholtholt & Alpermann, 1984).
2.2.19.2.4.5 Dog
No acute changes in cardiovascular performance parameters were
observed in dogs given acetoacetamide-N-sulphonic acid at doses of
10 or 25 mg/kg b.w. i.v. or 250mg/kg intraduodenally (Scholtholt &
Alpermann, 1984).
2.2.19.3 Human studies
Six healthy male volunteers received a dose of 50 mg/kg
acetoacetamide-N-sulfonic acid. No treatment-related adverse
reactions were reported and blood pressure and heart rate did not
show any abnormal changes. Occasional, individual borderline
changes in some haematological or clinical chemical parameters were
not considered by the authors to be of clinical significance and
most parameters were within normal ranges. It was concluded that
this single dose was safe and well tolerated (Rosenkrantz et al.,
1988).
2.2.20 Special studies on the short-term toxicity of potassium
chloride
Feeding studies in rats with potassium chloride were conducted
to elucidate the possible involvement of potassium ion on Acesulfame
K in changes observed in toxicological studies. Three groups of 20
male and 20 female weanling Wistar rats were fed for 90 days on
diets containing 0, 12 000 or 37 000 ppm (0, 1.2 or 3.7%) KCl,
equivalent to the potassium content of diets containing 0, 3, or 10%
Acesulfame K. Regular measurements were made of body weight, food
and water consumption, urinary volume and potassium content. After
the animals had been sacrificed, the caeca with and without contents
were weighed and the water contents of the caecal contents were
determined. There was a dose-related increase in water consumption
in both sexes through the study and, in the first few weeks, the
food consumption of treated animals was slightly lower than that of
controls. Body weight gains were depressed in males of both
concentration groups throughout the 90 day period but in females
statistically significant differences were obtained only up to the
twenty-ninth day. Dose-related increases in urine volumes and
urinary potassium were observed. Filled caecal weights were
increased by about 10% in males and 20% in females of the top dose
group, but these differences were not statistically significant; no
differences were observed in empty caecal weights, nor in water
content of the caecal contents. It was concluded that the potassium
content of Acesulfame K could be responsible for some adverse
effects seen in toxicological studies; in particular depressed body
weight gain (Mayer et al., 1978d).
2.3 Observations in man
Three human volunteers, body weight 70-80 kg, were given a
single oral dose of 30 mg 14C-Acesulfame K in peppermint tea.
Absorption was rapid and virtually complete, maximum blood
concentrations of 0.28 mg/ml occurring after 1 to 1-1/2 h.
Elimination occurred rapidly with a plasma half-life of 2-1/2 h.
Over 99% of the dose was excreted in urine and less than 1% in
faeces; 98% of the activity was eliminated in the first 24 h. From
the pharmocokinetic data it was calculated that repeated doses of 30
mg at 3 h intervals would increase the maximum serum levels 1.7-fold
and at 2 h intervals maximum serum levels would increase 2.4-fold
relative to a single dose (Christ & Rupp, 1976). The metabolism of
Acesulfame K was studied in serum and urine from human volunteers
following a single dose of 30 mg per individual. Only the original
substance was detected in all samples (Volz, 1976).
3. COMMENTS
The Committee reviewed further data which confirmed the
validity of the earlier long-term study in rats and the no-observed-
effect level. A review of comparative pharmacokinetic data in rats
and dogs showed that the blood levels of acesulfame potassium
reached after similar doses were higher in dogs; there was no
evidence to suggest that in relation to blood levels the dog was
more sensitive to the effects of the substance than the rat.
Pharmacokinetic studies in humans showed that oral doses of
acesulfame potassium were completely absorbed and rapidly excreted
unchanged in the urine. The half-life in the plasma was 1.5 hours,
which indicated that the period of exposure to the substance was
brief and no accumulation occurred.
Since acesulfame potassium was not metabolized in any species
tested, including humans, and further studies in rats in which
repeated doses were given did not reveal any induction of metabolism
or change in pharmacokinetic behaviour, the Committee concluded that
the rat appeared to be an appropriate model for humans.
Consequently, the Committee decided that, since the 2-year study in
rats represented a greater proportion of the lifespan of the species
than did the 2-year study in dogs and included exposure to the
substance in utero, the ADI should be based on the no-observed-
effect level in the rat, i.e., 1500 mg/kg of body weight per day.
The Committee also noted new data which indicated that acesulfame
potassium had no adverse effects in diabetic rats and was not
allergenic in an active systemic anaphylaxis test in guinea pigs.
The Committee also reviewed extensive toxicological studies on
the breakdown products, acetoacetamide and acetoacetamide- N-
sulfonic acid, which indicated that these compounds have a low
toxicity and are not mutagenic.
In view of these data and of available estimates of exposure to
acesulfame potassium, the Committee concluded that acetoacetamide-
N-sulfonic acid and acetoacetamide did not represent a health
hazard under present or foreseeable conditions of use of acesulfame
potassium.
4. EVALUATION
The previously established ADI was changed to 0-15 mg/kg b.w.
based on the long-term study in the rat.
The toxicological evaluation was carried out based on the
existing specifications which should be reviewed and revised in the
near future taking into account information on current manufacturing
purification procedures.
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