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    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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    ANON. (1977).  Acute oral toxicity of acetamide in female SPF Wistar
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    HOLLANDER, H., WEIGAND, W. & KRAMER, M. (1985).  Acetoacetamide-N-
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    JUNG, R. & HOLLANDER, W. (1986).  Sunett. Study of the mutagenic
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    KELLNER, H. M. & CHRIST, O. (1975b).  Pharmacokinetic studies of
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    KELLNER,  H. M. & ECKERT, H. (1983a).  Re:  Acesulfame  K. 
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    KELLNER,  H. M. & ECKERT, H. (1983b).  Re:  Acesulfame  K.
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    KREILING, R. & JUNG, R. (1988a).  Test for primary dermal irritation
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    KREILING, R. & JUNG, R. (1988b).  Test for primary eye irritation in
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    MARKERT, U. & WEIGAND, W. (1979a).  Akute Toxizitat von Acesulfam-K
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    MARKERT, U. & WEIGAND, W. (1979b).  Akute Toxizitat von Acesulfam-K
    an Goldorfen  (Leuciscus Idus f. melanotus)  Bericht  Nr. 755/79. 
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    MAYER, D. & KRAMER, F. (1980).  Studies on the effect of Acesulfame
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    MAYER, D., WEIGAND, W. & KRAMER, M. (1978a).  Acesulfame  K
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    MAYER, D., WEIGAND, W. & KRAMER, M. (1978b).  Report on
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    MAYER, D., WEIGAND, W. & KRAMER, M. (1978c).  A report on the
    experimental studies of the effect of Acesulfame K salt in causing
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    MAYER, D., WEIGAND, W. & KRAMER, M. (1978d).  Report on studies of
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    See Also:
       Toxicological Abbreviations
       Acesulfame Potassium (WHO Food Additives Series 16)
       Acesulfame potassium (WHO Food Additives Series 18)
       ACESULFAME POTASSIUM (JECFA Evaluation)