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    ALITAME

    First draft prepared by
    Dr Peter J. Abbott
    National Food Authority
    Canberra, Australia

    Explanation
    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion
         Biotransformation
         Effects on liver enzymes
    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Reproductive toxicity studies
         Special studies on neonatal survival
         Special studies on embryotoxicity/teratogenicity
         Special studies on genotoxicity
         Special studies on pharmacological effects
         Special studies on neurotoxicity and neurobehavioural effects
         Special studies on melanin binding
         Special studies on -isomer of alitame
         Observations in humans
    Comments
    Evaluation
    References

    1.  EXPLANATION

         Alitame is a dipeptide amide, composed of three chemical
    moieties: L-aspartic acid, D-alanine and 2,2,4,4- tetramethyl-
    3-thietanyl amine. The major impurities are  L--aspartyl-N-
    (2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate (2:5)
    (referred to a -isomer), formed after re-arrangement of the aspartyl
    unit; and N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide (referred
    to as alanine amide), formed after hydrolysis of the L-aspartic/
    D-alanine bond. The chemical structures of alitame (CP-54,802) and the
    6-isomer (CP-63,884) are shown in Figure 1.

         Alitame has not been previously evaluated by the Committee.

    CHEMICAL STRUCTURE 2

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

    2.1.1.1  Mice

         A group of 5 male CD-1 mice was administered by gavage a single
    dose of 50 mg/kg bw 14C-alitame in distilled water. A second group
    of 5 male CD-1 mice was fed a diet containing 14C-alitame at a
    concentration of 0.3%, equal to 350 mg/kg bw. Following the gavage
    dose, 77% of the radioactivity was recovered in urine in a 24-hour
    period. About 50% of the administered dose was excreted between 4 and
    20 h. Faecal radioactivity was not measured. Following dietary
    administration, 60% of the radioactivity was found in urine and 32% in
    faeces (Pfizer, 1986).

    2.1.1.2  Rats

         A male Long-Evans rat with a bile duct cannula was administered a
    single oral dose of 50 mg/kg bw 14C-alitame. Bile and urine were
    collected for 48 h. In a second study, 4 male and 4 female Long-Evans
    rats were administered a single oral dose of 5 mg/kg bw 14C-alitame.
    Urine and faeces were collected for 7 days and analyzed for
    radioactivity. In the single animal study, 83% of the radioactivity
    was recovered, with 14% in bile and 69% in urine over 48 h, indicating
    extensive absorption. In the group study, most of the radioactivity
    was recovered in urine (83% in males, and 95% in females) in a 24-hour
    period. Faecal radioactivity was 20% in males and 4% in females during
    24 h with little detected after that time (Pfizer, 1986).

         In a study to investigate tissue distribution in rats, a group of
    24 male and 24 female Long-Evans rats received by gavage a single dose
    of 5 mg/kg bw 14C-alitame. Animals in groups of 3 males and 3
    females were killed at 2, 6 or 24 h, and at 2, 3, 7, 14 or 29 days and
    tissue levels determined. In most tissues, the half-life of clearance
    was 3 to 6 h and the levels were below the level of detection by 7
    days. Residue levels of radioactivity in the eyes decreased much more
    slowly than in other tissues and levels of 0.08 mg/kg were detected at
    day 29 (Pfizer, 1986).

         In a study to examine the potential for transplacental transfer
    of alitame, pregnant Long-Evans rats received a single oral dose of
    1000 mg/kg bw 14C-alitame on day 13 of gestation. Four animals were
    sacrificed 6 or 24 h after treatment and maternal plasma and fetuses
    were examined for radioactivity. Radioactivity was high in the plasma
    of dams at 6 and 24 h, and high levels were also found in the fetuses
    at these times, indicating transplacental transfer of alitame
    (Pfizer, 1986).

         In a study to examine potential accumulation of alitame or its
    metabolites in the milk of lactating rats, pregnant Long-Evans rats
    were fed diet containing 1% alitame (equal to 750 mg/kg bw/day)
    beginning on the last day before birth, and were continued on this
    diet for 13 days of lactation. At the end of the lactation period,
    rats were given a single oral dose of 750 mg/kg bw 14C-alitame,
    and plasma and milk were collected 6 h later. Concentration in milk
    and plasma were the same for each animal with no indication of
    accumulation in milk. Intake for each pup, based on an average
    consumption of 3.7 g milk/day on lactation day 10. was calculated to
    be between 6 and 13 mg/kg bw/day, which is less than 2% of the
    maternal dose level (Pfizer, 1986).

         In a study to examine the toxicokinetics of alitame after i.v.
    treatment, a group of 30 male and 4 female Long-Evans rats received a
    single i.v. injection of 25 mg/kg bw. Males received non-radioactive
    alitame and were used to determine the serum concentration over a
    6-hour period. The females received 14C-alitame and were used to
    determine the excretion rate and the identity of the urinary
    metabolites. Radioactivity in serum declined with an estimated
    half-life of 13 minutes. Most of the radioactivity (87%) was found in
    urine in the 48-hour period of collection. Approximately 50% of
    urinary radioactivity was unchanged alitame, in contrast to only 1%
    after oral administration. This result was consistent with a high rate
    of hydrolysis in either the intestine or during first-pass hydrolysis
    in the liver (Pfizer, 1986).

    2.1.1.3  Dogs

         Two male beagle dogs with bile cannulas received a single dose of
    10 mg/kg bw 14C-alitame. In one dog, the alitame was injected
    directly into the stomach during cannulation surgery. This dog was
    maintained under anaesthesia during the 24-h collection of urine,
    faeces and bile. In a separate study, 4 males and 4 females received a
    single oral dose of 5 or 25 mg/kg bw 14C-alitame. Urine and faeces
    were collected at 24-hour intervals over a 7-day period and analyzed
    for radioactivity. In the first experiment, biliary excretion
    comprised only 0.2-0.3% of the total radioactivity. Urinary excretion
    was approximately 36% of the administered radioactivity in the
    anaesthetized dog, and 81% in the conscious dog. Faecal radioactivity
    was 10% of the administered radioactivity in the conscious dog. In the
    second experiment, the administered radioactivity was recovered in
    urine (94-98%) and faeces (3-4%) in the first two days (Pfizer, 1986).

         In a study to examine the toxicokinetics after i.v. treatment, a
    male beagle dog received a single i.v. injection of 10 mg/kg bw
    14C-alitame. Blood samples were taken at times up to 72 h, and urine
    and faeces were collected in three 24- hour intervals. Radioactivity

    in serum gave a biphasic decline, with half-lives of 34 minutes and
    4 h. The vast majority of the radioactivity was excreted in the first
    24 h, mainly in the urine (80%). Unchanged alitame was the major
    urinary excretion product (60%) (Pfizer, 1986).

         In a study to examine whether the site of hydrolysis of alitame
    was the intestine or the liver, a female beagle dog under anaesthesia
    had a 7-inch segment of the jejunum ligated and the mesenteric vein
    cannulated. A 3 ml solution containing 14C-alitame (4 mg/kg bw) was
    injected into the ligated jejunal segment and the mesenteric blood
    collected at 2-minute intervals and peripheral blood at 15-minute
    intervals over a 2-hour period. The mesenteric blood accounted for 4%
    of the total radioactivity over the 2-hour period. The level of
    unchanged alitame remained relatively constant and accounted for
    approximately 50% of radioactivity while the percentage of the
    hydrolysis product alanine tetramethylthietane amide (CP-57,207; see
    Figure 2) increased with time from 12% to 38% over 2 h. Unchanged
    alitame was the major substance present in the jejunum, while the
    concentration of alanine tetramethylthietane amide increased with
    time. No radioactivity was found in peripheral blood or urine. The
    results suggested that the lumen of the jejunum is the site for
    hydrolysis of alitame, and that further hydrolysis occurred during
    passage through the intestinal wall (Pfizer, 1986).

    2.1.1.4  Humans

         Four male subjects were administered an oral dose of 14C-alitame
    (50 mCi; 1 mg/kg bw). Blood samples were taken at various times up to
    48 h. Exhaled 14CO2 was measured at various times up to 8 h.
    Urine and faeces were collected over 5 days.

         Plasma radioactivity levels peaked between 8 and 18 h and
    decreased rapidly within the first 48 h, indicating slow but extensive
    absorption followed by rapid excretion. There was no significant
    increase in exhaled 14CO2 above zero. Urinary radioactivity was
    maximal between 12 and 24 h with approximately 50% of radioactivity
    excreted in the first 24 h and 90% within 5 days Faecal elimination
    accounted for 7-10% of the total dose in 3 subjects and 2% in the 4th
    subject.

         The major urinary metabolites were alanine tetramethylthietane
    amide sulfoxide (CP-58,290; see Figure 2) and alanine tetramethyl-
    thietane amide glucuronide. Minor metabolites were alanine
    tetramethylthietane amide (CP-57,207) and alanine tetramethylthietane
    amide sulfone (CP-57,254). The glucuronide was the major urinary
    metabolite in the first 24 h whereas the sulfoxide was the major
    metabolite in the 24-48 h period. Unchanged alitame constituted less
    than 1% of the total urinary radioactivity. In faeces, all of the
    radioactivity consisted of unchanged alitame (CP-54,802) and alanine

    tetramethylthietane amide (CP-57,207). In plasma, the major part of
    the radioactivity (80%) consisted of alanine tetramethylthietane amide
    (CP-57,207) and its glucuronide. In contrast to rats and dogs, the
    major urinary metabolite in humans was found to be the glucuronide of
    alanine tetramethylthietane amide (Caldwell  et al., 1985).

    2.1.2  Biotransformation

         The metabolic pathways of alitame in different animal species and
    in humans are shown in Figure 2.

    2.1.2.1  Mice

         Following a single oral dose of 50 mg/kg bw 14C-alitame or a
    diet containing 3000 mg/kg 14C-alitame, metabolites identified were
    D-alanine tetramethylthietane amide sulfoxide (CP-58,290; 64.3%),
    D-alanine tetramethylthietane amide (CP-57,207; 24.7%), and unchanged
    alitame (0.9%). The remainder metabolites (10.1%) were not identified.
    Faecal radioactivity was identified as 39.5% unchanged alitame
    (CP-54,802), 51.5% D-alanine tetramethyltbietane amide (CP-57,207) and
    4.6% was not identified (Pfizer, 1986).

    2.1.2.2  Rats

         In a study to examine metabolites in plasma, a group of
    Long-Evans rats received a single oral dose of 50 mg/kg bw
    14C-alitame. Two rats were bled serially to determine the time
    course of radioactivity and a further 3 male and 3 female rats were
    killed at 6 h and the plasma radioactivity was assayed by HPLC.
    Maximum plasma levels occurred at 4 to 6 h after administration. The
    major metabolite was alanine tetramethylthietane amide sulfoxide
    (CP-58,290; 70% of the total radioactivity), in both the free and
    acetylated forms with unchanged alitame only 1% of the total
    radioactivity (Pfizer, 1986).

         In a study to examine metabolites in urine, 4 male and 4 female
    Long-Evans rats were administered a single oral dose of 5 mg/kg bw
    14C-alitame. Urine and faeces were collected over a 24-hour period
    and analyzed by HPLC. The major urinary metabolite was alanine
    tetramethylthietane amide sulfoxide (CP-58,290; 75-80%), in both free
    and acetylated forms, with unchanged alitame only 1% of the total
    radioactivity (Pfizer, 1986).

         In a study to examine metabolites in faeces, two Long-Evans
    rats received a single oral dose of 50 mg/kg bw or 1000 mg/kg bw
    14C-alitame. Urine and faeces were collected over 24 h and analyzed
    by HPLC. The major product in the faeces was unchanged alitame
    (CP-54,802; 70% of the radioactivity).  A second metabolite was
    identified as alanine tetramethylthietane amide (CP-57,207; 23%)
    (Pfizer, 1986).

    CHEMICAL STRUCTURE 3

         In a study in lactating dams, metabolites in milk were alanine
    tetramethylthietane amide sulfoxide (CP-58,290) and its acetylated
    form (54.4%), alanine tetramethylthietane amide (CP-57,207; 31.4%),
    and unchanged alitame (1%) (Pfizer, 1986).

         In a study to examine the effect of dose on metabolic pathways,
    Long-Evans rats received a single oral dose of 10, 100 or 1000 mg/kg
    bw 14C-alitame and urine and faeces were collected at 24-hour
    intervals over 3 days. Urinary metabolites were analyzed by HPLC.
    There was a slight decrease in the percentage of administered
    radioactivity excreted after the high-dose treatment, but the
    metabolic profile was essentially unchanged (Pfizer, 1986).

         In a study to compare the metabolic fate of D-alanine as either
    free alanine or when incorporated into alitame, Long-Evans rats were
    administered 14C-D-alanine or 14C-alitame (1-14C-D-alanine
    label) by oral gavage and exhaled 14CO2 was measured. While
    14CO2 was extensively exhaled following administration of
    D-alanine, no 14CO2 was expired after administration of
    14C-alitame, demonstrating the stability of the D-alanine
    tetramethylthietane amide bond to metabolic hydrolysis (Pfizer, 1986).

    2.1.2.3  Dogs

         In a study to examine metabolites in plasma, groups of 3 male and
    3 female beagle dogs received a single oral dose of 5 or 25 mg/kg bw
    14C-alitame. Plasma samples were taken at various times and the
    metabolites in plasma identified by HPLC. In both male and female
    dogs, plasma radioactivity peaked at 2-3 h. The major metabolite was
    alanine tetra-methylthietane amide sulfoxide (CP-58,290; 80%), with
    10% in the acetylated form. Approximately 5% was unchanged alitame.
    The remainder was alanine tetramethylthietane amide (CP-57,207), its
    sulfone derivative (CP-57,254) and the acetylated forms of these
    compounds. The metabolite profile was very similar to that seen in the
    rat (Pfizer, 1986).

         In a study to examine the metabolites in urine, a group of 3 male
    and 3 female beagle dogs received a single oral dose of 14C-alitame,
    and 24-hour urine and faeces collected. Urinary metabolites were
    analyzed by HPLC. In males, the major metabolite was alanine
    tetramethylthietane amide sulfoxide (CP-58,290; 80%), including 9.5%
    as the acetylated form. Approximately 5-15% of the urinary
    radioactivity was unchanged alitame (Pfizer, 1986).

         In a study to examine metabolites in faeces, a male beagle dog
    received a single oral dose of 10 mg/kg bw 14C-alitame followed
    several weeks later by a dose of 500 mg/kg bw. A female beagle dog
    received a single oral dose of 500 mg/kg bw 14C-alitame Faecal
    samples were collected over 24 h and analyzed by HPLC. Faeces

    contained 18-40% of the administered radioactive dose over the 24 h
    period. Faecal radioactivity was a mixture of unchanged alitame
    (CP-54,802) and alanine tetramethylthietane amide (CP-57,207).
    Together these two products accounted for approximately 90% of the
    total radioactivity (Pfizer, 1986)

         In a study to examine the effect of dose on metabolic pathways,
    2 male and 2 female beagle dogs received a single oral dose of 10 or
    500 mg/kg bw 14C-alitame and urine and faeces were collected in
    24-hour intervals over 3 days. Urinary metabolites were analyzed by
    HPLC. There was a slight decrease in the percentage of administered
    radioactivity excreted after the high-dose treatment than after the
    low-dose treatment, but the metabolite profile was essentially
    unchanged (Pfizer, 1986).

    2.1.2.4  In vitro studies

         The stability of alitame in gastric juices was examined to
    clarify whether the cleavage of aspartic acid is initiated in the acid
    environment of the stomach, and whether conversion of alitame to the
    -isomer might occur under these conditions. Thus, alitame was
    incubated in simulated gastric juices at 38C to determine the degree
    of hydrolysis. Controls were water and simulated gastric juices
    (boiled). Samples were taken over 24 h and analyzed by HPLC. Alitame
    was relatively stable, with only slight evidence of degradation after
    prolonged incubation for 24 h, which was not considered to be due to
    enzyme activity. Similarly, the small increase in -isomer observed in
    both treated and control samples was not considered to be due to
    enzyme activity (Pfizer, 1986).

         The stability of alitame in rat intestinal homogenate was
    examined in order to test the capacity of the rat small intestine to
    metabolize alitame. The small intestine from male rats was flushed
    with saline and homogenized in phosphate buffer at pH 7.4. The
    homogenate was incubated with 500 mg alitame/ml at 37C and the
    reaction stopped by the addition of 0.25 tool/litre NaOH. Cleavage of
    the peptidic bond of alitame was measured by the formation of alanine
    tetramethylthietane amide (CP-57,207) and was dependent on both the
    substrate concentration and time of incubation. The percentage
    conversion to the amide ranged from 13-21% after 10 minutes of
    incubation. This demonstrated the ability of intestinal homogenate
    to cleave the peptidic bond of alitame. The reaction was also
    demonstrated to be enzyme-mediated in a second experiment in which
    it was shown that the extent of cleavage was dependent on the
    concentration of homogenate. Little evidence of further metabolic
    steps, such as sulfur oxidation or acetylation, was noted (Pfizer,
    1986).

    2.1.3  Effects on liver enzymes

    2.1.3.1  Rats

         In a study to compare the rat liver enzyme inducing properties of
    alitame with those of butylated hydroxytoluene (BHT), groups of 4 male
    rats received oral doses of either BHT (100 or 500 mg/kg bw), alitame
    (100 or 500 mg/kg bw) or phenobarbital (50 mg/kg bw) daily for 5 days.
    At 15 h following the last dose, animals were killed and livers
    removed. Liver homogenates were assayed for O-demethylase, cytochrome
    c reductase, and P-450 activity. Significant enzyme induction was
    noted with BHT, but alitame showed no effect at the same dose levels
    (Pfizer, 1986).

         In a further study to examine the liver enzyme inducing potential
    of alitame and its metabolites, groups of Sprague-Dawley rats received
    single oral doses of either alitame (up to 2000 mg/kg bw), alanine
    tetramethylthietane amide (CP-57,207) or its sulfoxide (CP-58,290; 100
    or 500 mg/kg bw), or phenobarbital (50 mg/kg bw/day) for 5 days. At
    15 h after the last dose, animals were killed and livers removed.
    Liver homogenates were prepared and assayed for O-demethylase. Slight
    induction of demethylase activity was noted with both alitame and its
    metabolites. At 500 mg/kg bw, enzyme activity was approximately twice
    the low baseline activity observed in controls with either alitame or
    its metabolites (Pfizer. 1986).

         In a recent detailed study, the hepatic enzyme-inducing activity
    of alitame was examined in rats following gavage administration (pilot
    study) and dietary administration (main study). In the pilot study,
    groups of 5 male and 5 female Sprague-Dawley rats were administered
    alitame daily by gavage for 7 days at dose levels of 0, 500 or
    1000 mg/kg bw/day. Animals were killed on day 8, the livers excised,
    sections taken for histology and electron microscopy, and the
    remainder of the liver homogenized. Positive control animals were
    given -naphthoflavone (80 mg/kg bw, i.p., 3 days), phenobarbitone
    (80 mg/kg bw, i.p., 3 days), or dexamethasone (100 mg/kg bw, i.p., 3
    days). In the main study, groups of 20 male and 20 female Sprague-
    Dawley rats were fed a diet containing alitame at concentrations of
    0, 0.3% or 1% for 4 weeks. An additional 20 males and 20 females acted
    as pair-fed controls and received the basal diet at the level of
    intake of the 1% alitame group. After 28 days, 10 males and 10 females
    from each group were sacrificed, sections of liver taken for histology
    and electron microscopy and the remainder of the liver homogenized.
    Kidney and brain weights were also recorded. The remaining 10 males
    and 10 females were withdrawn from the alitame diet and kept for a

    further 14 days before being sacrificed. In each of the above studies,
    the liver homogenate was used to determine protein content, cytochrome
    P-450 content, NADPH-cytochrome P-450-dependent reductase activity,
    ethoxyresorufin O-deethylase (EROD) activity, pentoxyresorufin
    O-depentylase (PROD) activity, erythromycin N-demethylase (ERD)
    activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis
    (SDS-PAGE).

         In the pilot study, the positive controls exhibited significant
    increases with respect to hepatic microsomal enzymes and all caused an
    increase in relative liver weight. Brain and kidney weights were
    unaltered. Alitame at 500 or 1000 mg/kg bw/day for 7 days had only
    minor effects by comparison. At 1000 mg/kg bw/day, cytochrome P-450
    levels were 140% of controls, and EROD, PROD and ERD activities
    increased 3.6-fold, 6.3-fold, and 4.7-fold, respectively. Examination
    of the microsomes by SDS-PAGE provided similar evidence of low levels
    of protein induction corresponding to the above enzymes. Light
    microscopic examination revealed hepatocellular hypertrophy at both
    dose levels, and some evidence of periportal inflammation and necrosis
    in a few sections. Electron microscopy revealed an increase in rough
    and smooth endoplasmic reticulum at both dose levels.

         In the main study, growth curves indicated a reduced body-weight
    gain in alitame-treated animals compared to controls. This effect
    was more apparent in females than in males and appeared to be
    dose-related. While the effects may not have been statistically
    significant, the curves indicated a clear biologically significant
    decrease in body-weight gain in females at both the 0.3 and 1% alitame
    dose levels. There was also an increase in relative liver weight in
    females at both dose levels which was dose-related. Relative brain and
    kidney weights were unaltered.

         The parameters used to measure hepatic enzyme induction were also
    increased in a dose-related manner compared to controls and pair-fed
    animals. At the 0.3 and 1% dose levels, cytochrome P-450 levels
    increased by 12 and 20% in females, and by 46 and 101% in males,
    respectively. The activity of EROD was increased slightly in males and
    females while there was a significant increase in the activity of PROD
    in males and females at 0.3% and 1% alitame. The activity of ERD was
    only slightly increased in males at 1% alitame. Light microscopic
    examination of the liver revealed centrilobular hypertrophy at both
    dose levels, but more marked at the higher dose. Electron microscopy
    revealed a dose-related increase in both rough and smooth endoplasmic
    reticulum. Following the 14-day withdrawal period, the liver weight
    changes were reversed, and none of the hepatic parameters was
    significantly different from controls.

         The results indicated that alitame, at both the 0.3% and 1% dose
    levels, was a weak inducer of hepatic enzymes, which was accompanied
    in females by a decrease in body-weight gain and an increase in
    relative liver weight. The pattern of enzyme changes was essentially
    similar to that caused by phenobarbitone, although quantitatively
    smaller (Caldwell  et al., 1994).

         In a supplement to the above study, the cytochrome P-450
    isoenzyme from the liver microsomes of alitame-treated rats were
    separated by SDS-polyacrylamide gel electrophoresis and further
    characterized by Western blot immunodetection. Following separation by
    electrophoresis, the proteins were transferred to nitrocellulose
    membranes and the blots either stained with antibodies to specific
    cytochrome P-450 isoenzyme or, to allow visualization of total
    protein, by staining with Amido Black solution.

         The microsomes from alitame-treated rats showed an increase in
    the band attributed to CYP2B (catalytic activity) at 52 kD which is a
    protein also shown to be induced by phenobarbitone. The band at 56 kD
    attributed to CYP1A1, shown to be induced by -naphthoflavone, was
    conspicuous by its absence. The data further confirmed that the
    hepatic changes produced by alitame were similar to those produced by
    phenobarbitone (Caldwell & Reed, 1994).

    2.1.3.2  Humans

         In a 14-day study, 8 male subjects received alitame in gelatine
    capsules as 4 daily oral doses totalling 15 mg/kg bw/day. This dose
    was calculated to be 15 times the projected human exposure level,
    assuming total sucrose replacement. Induction of microsomal enzyme
    levels was measured by the rate of elimination of aminopyrine.
    Measurements were taken before treatment, after 2 weeks of treatment,
    and at 18 days post-treatment. Both plasma and saliva were collected.
    Blood samples were taken at pre-test, after 1 and 2 weeks of
    treatment, and at 7 and 18 days post-treatment for the analysis of
    clinical chemistry and haematological parameters. On the 10th day of
    treatment, a 24-hour urine sample was collected for the analysis of
    metabolites. There was no adverse effects parameters were normal
    during the course of the study.

         The analysis of the aminopyrine elimination results indicated
    that the saliva was a good indicator of plasma levels. When the
    elimination rate constants and half-lives were compared, there was no
    significant difference in aminopyrine kinetics from the pre-dose,
    post-dose and post-withdrawal samples. Urine samples indicated the
    presence of the major metabolites, alanine tetramethylthietane amide
    (CP-57,207) and it sulfoxide (CP-57,290). Alitame was present only in
    trace amounts (Caldwell  et al., 1984).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The acute toxicity of alitame was examined in both mice and rats
    and the results are summarized in Table 1.

    Table 1.   Summary of acute toxicity studies with alitame.
                                                                        

    Species   Sex       Route     LD50           Reference
                                  (mg/kg bw)
                                                                        

    Mouse     M&F       oral      > 5000         Levinsky et al. (1983)

    Rat       M&F       oral      > 5000         Levinsky et al. (1983)
                                                                        

         Groups of 10 male and 10 female mice (Crl:COBS CD-1 (ICR) BR) and
    10 male and 10 female Sprague-Dawley (Crl:COBS CD (SD) BR) rats were
    administered alitame (purity 97.43%) by gavage at a single dose level
    of 5000 mg/kg bw. Animals were observed for clinical signs over a
    14-day period after treatment, during which food consumption and body
    weights were recorded. Animals were examined for gross pathological
    changes at day 14 after treatment.

         In mice, there were no deaths during the study and no clinical
    signs apart from an increase in soft faecal material at 2.5 h
    post-treatment. Faeces were normal by day 2 post-treatment. During the
    observation period, there was normal food consumption and steady
    body-weight gain.

         In rats, there were no deaths during the study and no clinical
    signs during the first hour post-treatment, but from then onwards all
    animals displayed an increased level of activity for 1-2 days. All
    rats were jittery and exhibited a tendency for exophthalmia, with
    females noticeably more affected than males. Females exhibited
    incoordination in their movements but were not ataxic. By 24 h, 7/10
    males were essentially asymptomatic, and by day 2, all males and
    females were asymptomatic. During the observation period, food
    consumption was normal, and there was a steady body-weight gain. There
    were no gross pathological changes on day 14 in 20/20 mice and in
    19/20 rats, with one rat having an enlarged liver (Levinsky  et al.,
    1983).

    2.2.2  Short-term toxicity studies

    2.2.2.1  Mice

         In a 2-month study, groups of 10 male and 10 female Crl:COBS
    (CD-1) (ICR) BR mice were fed a diet containing alitame (purity
    98-101%) at concentrations of 0, 1, 2, or 5%, equivalent to 0, 1500,
    3000 or 7500 mg/kg bw/day.

         Animals were observed for clinical signs, and food consumption
    and body weights were recorded throughout the study period. At the end
    of the study period, liver and kidney weights were recorded and
    tissues from the major body organs were taken for histopathology.

         There were no deaths and no clinical signs of toxicity throughout
    the study period. Body weights of the intermediate- and high-dose
    animals decreased slightly on days 1 and 2, but recovered quickly.
    There was no treatment-related effect on body weight for the remainder
    of the study. There was no change in food consumption. Both absolute
    and relative liver weights increased in a dose-related manner compared
    to controls. The increase in absolute liver weight was 20, 40 and 67%
    in males and 20, 35 and 66% in females in low-, intermediate- and
    high-dose animals, respectively. Histopathological examination of the
    liver revealed a dose-related increase in centrilobular hypertrophy.
    Electron microscopy of liver tissue indicated a dose-related increase
    in proliferation of smooth endoplasmic reticulum. No degenerative
    hepatocellular changes were noted. Effects were noted at all dose
    levels and a NOEL was not established in this study (Holmes  et al.,
    1983a).

    2.2.2.2  Rats

         In a range-finding study, groups of 5 male and 5 female Crl:COBS
    CD-1 (SD) BR rats (5/sex/dose) were fed a diet containing alitame
    (purity 98-101%) at concentrations of 0, 1, 2 or 5%, equivalent to
    500, 1000, or 2500 mg/kg bw/day for 15 days. Animals were examined for
    clinical signs of toxicity, and food and water consumption was
    monitored throughout the study period. At the end of the study period,
    blood samples were taken for haematological and clinical chemistry
    examination; tissues from the major body organs were taken for
    histopathology; tissues from liver and kidney were taken for electron
    microscopy; and tissue from liver for histochemistry. There were no
    deaths throughout the study, and no clinical signs of toxicity. A
    slight body-weight loss was noted at the 2% and 5% dose levels on day
    3 but body-weight gain was normal thereafter. There was a dose-related
    decrease in food consumption in all treated groups on days 1-2
    (especially females at the 2% dose level and in both sexes at the 5%
    dose level) possibly due to unpalatability of the diet. Water

    consumption was similarly decreased. There was a slight, but not
    significant, increase in plasma cholesterol and protein levels in all
    treated groups. Haematological parameters were normal. There was a
    dose-related increase in absolute and relative liver weights in
    males and females but no microscopic abnormalities were noted.
    Histochemistry indicated an increase in glucose-6-phosphate
    dehydrogenase and NADPH2 diaphorase in liver tissue slices. Electron
    microscopy indicated a dose-related increase in smooth endoplasmic
    reticulum in liver tissue and, in some cases, an increase in lysosomes
    and lipid vacuoles. Effects were observed at all dose levels and a
    NOEL was not established in this study (Chvedoff  et al., 1981a).

         In a 5-week study, groups of 10 male and 10 female Long-Evans
    rats were fed a diet containing alitame (purity 98.2%) at dose levels
    of 0, 10, 50 or 100 mg/kg bw/day. Animals were observed daily and body
    weights and food consumption recorded. Blood samples were taken from
    the orbital sinus prior to treatment and at the end of the study
    period, and clinical chemistry and haematological parameters examined.
    After 5 weeks, the induction of liver microsomal enzymes was measured
     in vitro and animals were examined for gross pathology. Tissues from
    the major organs were prepared for histopathology.

         There was no treatment-related effect on body-weight gain or food
    consumption. The clinical chemistry, haematological data and
    histopathology were unremarkable. It was not possible to determine
    whether there was proliferation of smooth endoplasmic reticulum
    because of the presence of excess glycogen clue to lack of fasting
    before sacrifice. Induction of 0-demethylase was determined by the
    release of  p-chlorophenol. A 30% increase in enzyme induction
    occurred at the high-dose level which was not statistically
    significant. Nevertheless, on the basis of the biological significance
    of this increase, the NOEL in this study was 50 mg/kg bw/day
    (Holmes  et al., 1983b).

         In a 3-month study, groups of CrI:COBS-CD(SD)BR strain rats
    (15/sex/dose) were fed a diet containing alitame (purity 98-101%) at
    concentrations of 0.5, 1.0 or 2.0%, equal to 380, 830 or 1500 mg/kg
    bw/day. Animals were observed throughout the study period and body
    weights, food and water consumption recorded. Blood samples were taken
    for examination of clinical chemistry and haematology parameters at
    the end of the study. Urinary parameters were also examined. At the
    end of the study, gross pathological examinations were performed and
    tissues from the major organs were taken for histopathology. A group
    of 5 males and 5 females was observed for a further 1 month following
    the treatment period.

         There were no deaths throughout the study and no clinical signs
    of toxicity. There was a dose-related decrease in body-weight gain in
    both males and females in all treated groups. The decreases at 3
    months in males/females were 4.5%/1.7%, 4.5%/3.3% and 5.3%/8.5% at
    low, intermediate and high dose. Food consumption was slightly lower
    in high-dose males, but not during the withdrawal period. Water
    consumption was normal. The clinical chemistry analysis revealed a
    significant increase in plasma cholesterol at all dose levels in males
    and females. The increase was 23% at low dose and 42% at high dose.
    Levels returned to normal during the 1-month withdrawal period. There
    was also a dose-related decrease in plasma triglyceride levels in
    males, which returned to normal levels during the withdrawal period.

         The levels of cytochrome P-450 and b5 were increased in a
    dose-related manner at the end of the treatment period. The activity
    of microsomal N-demethylase was also increased in a close-related
    manner. The changes were reversed during the withdrawal period except
    for the cytochrome P-450 levels in males. Haematological parameters
    were within the normal range.

         Relative and absolute liver weights were increased in males at
    the high-dose level, while relative liver weight was increased at all
    doses in females. The liver weights returned to normal during the
    withdrawal period.

         Histopathology of the liver revealed centrilobular hypertrophy in
    all high-dose females and in 5/10 high-dose males. Histochemical
    analysis of the livers indicated increased activity of G-6-P
    dehydrogenase, gamma-glutamyl transpeptidase and NADPH-diaphorase in
    the high-dose animals. NADPH-diaphorase activity was also increased in
    intermediate-dose females. All changes were reversible during the
    withdrawal period. Electron microscopy revealed an increased
    proliferation of smooth endoplasmic reticulum at all dose levels.
    After the withdrawal period, the livers of female rats were normal
    while those of the males still showed mild proliferation of smooth
    endoplasmic reticulum at the high-dose level. The histopathological
    and enzymatic changes were indicative of a reversible adaptive
    response. There was no evidence of focal hyperplasia or hepatocellular
    toxicity. Effects were noted at all dose levels and a NOEL was not
    established in this study (Monro  et al., 1982a).

         In a one-year study, groups of 40 male and 40 female Long-Evans
    rats were fed a diet containing alitame (purity 98-101%) at
    concentrations of 0, 0.1, 0.3, or 1.0% (equivalent to 50, 150 or
    500 mg/kg bw/day). Two other groups were fed a diet containing alitame
    adjusted to achieve a nominal intake of 10 or 30 mg/kg bw/day. At 6
    months, 20 male and 20 female animals at each of the dose levels were
    removed from treatment. Of these, half were sacrificed while the other
    half were returned to normal untreated diet. Animals were observed

    throughout the study period and body weights, food and water
    consumption recorded. Ophthalmoscopic examinations were performed in
    all animals before necropsy. Blood samples were taken at 6, 9 and 12
    months for clinical chemistry and haematology. Urinalysis parameters
    were also examined. At the end of the study period, all animals were
    examined for gross pathology, and tissues from major organs were taken
    for histopathology.

         There were no deaths throughout the study and no clinical signs
    of toxicity. Ophthalmoscopic examination at 6 months and 1 year was
    unremarkable. There were no treatment-related effects on body-weight
    gain or on clinical pathology parameters. Cytochrome P-450 levels were
    measured at 6 months and were significantly increased at the high-dose
    level only (21% in males and 33% in females). The only organ weight
    change noted was an increase in relative liver weight at the high-dose
    level at 6 months, with a 12.1% increase in males and 16.0% increase
    in females. At 12 months, liver weight increases at the high-dose
    level were 8.5% and 11.7% in males and females respectively, compared
    to controls. In animals withdrawn from treatment at 6 months, liver
    weights had returned to normal levels at 12 months. The only
    microscopic finding was a retention of glycogen in both control and
    treated groups. This change was not considered to be treatment-
    related. The NOEL in this study was 0.3% in the diet, equal to
    145 mg/kg bw/day in males and 185 mg/kg bw/day in females
    (Holmes  et al., 1985a).

         A study was conducted in Sprague-Dawley rats which included
    dietary administration during mating,  in utero exposure, exposure
    during lactation, and dietary exposure for one year post-weaning.
    Groups of 20 male and 20 female Sprague-Dawley rats were selected from
    the F1 offspring of rats treated 4 weeks prior to mating and during
    gestation and lactation (Levinsky  et al., 1992). The groups were fed
    a diet containing alitame (purity 99.3%) at concentrations of 0 (two
    control groups), 0.1, 0.3 or 1% for one year. The animals were
    observed daily for clinical signs and body weights and food
    consumption were recorded weekly. Other measurements and observations
    included ophthalmological examinations (pre-test and 6 and 12 months),
    serum chemistry, haematology and urinalysis measurements at 6 and 12
    months. Tissues were collected from all animals which died or became
    moribund during the study. At the end of the study, the animals were
    sacrificed and necropsied and tissues prepared for light microscopy.
    Organ weights for liver, kidney, adrenal glands, testes and ovaries
    were recorded.

         Twenty rats died or were sacrificed during the study. The deaths
    were distributed evenly between the groups although deaths in males
    predominated over those in females by four to one. Of the 20 early
    necropsies, 4 were due to neoplasms (1 control, 2 low-dose and 1
    intermediate-dose rats). The most common non-neoplastic cause of death
    (7/16) was described as 'unexplained death/heavy rats' and was not
    accompanied by any remarkable clinical signs.

         Body-weight measurements began at birth for this study, and at
    weaning (day 21) the mean body weights for the intermediate- and
    high-dose animals compared to the control groups were -9% and -21%,
    respectively. Following selection of animals in groups for the 1-year
    study, the body weights expressed as percent of control for the
    intermediate-dose level were -9.5% (M) and -8.8% (F), and for the
    high-dose level -16.3% (M) and -13.5% (F). Over the course of the
    study, the initial body weight differentials for males at the
    intermediate- and high-dose levels diminished by about 50%. For the
    first 80 days, all groups of males appeared to gain weight at similar
    rates apart from the control group 1. For the remaining period of the
    study, the weight gains were similar for all groups. Over the 1-year
    period, the body-weight difference for the intermediate-dose males
    disappeared and the difference for the high-dose group was 6.6% and
    11.1% compared with control groups 1 and 2, respectively.

         For females, over the course of the study, the -8.8% difference
    at the intermediate-dose level disappeared and the low-dose level
    gained weight relative to the control group 1. The females in the
    control group 1, like the males, showed a slower growth rate. The
    high-dose females, unlike the males, did not recover their initial
    body weight difference (-13.5%) and after 200 days, the body weight
    difference began to increase, reaching -20% relative to control
    group 1.

         Ophthalmoscopic examination at 6 and 12 months was unremarkable.
    There were no treatment-related changes in clinical chemistry,
    haematology or urinary parameters apart from a 40-60% decrease in
    serum triglyceride levels in the high-dose females, which appeared to
    be related to the decreased weight gain in the high-dose females. The
    high-dose males showed decreased serum triglyceride values at 6
    months, but not at 12 months.

         There were no gross pathological findings in any group. The organ
    weight changes observed were a decrease in absolute liver weight in
    the high-dose females, and an increase in relative liver, kidney and
    ovary weight as a result of the decrease in mean body weights in the
    high-dose females.

         Histopathology revealed hepatocellular hypertrophy in the
    high-dose males (13/20) and females (4/20). These changes were not
    accompanied by an increase in liver weight. Other liver changes
    observed could not be related to treatment. The NOEL in this study,
    based on body and organ weight changes, was 0.3% in the diet, equal to
    173 mg/kg bw/day in males, and 205 mg/kg bw/day in females (Fisher
     et al., 1992).

    2.2.2.3  Dogs

         In a two-week range-finding study in dogs, groups of 2 male adult
    beagle dogs were fed alitame (purity 98-101%) in the diet at
    concentrations of 1, 2 or 5%, equivalent to 250, 500 or 1250 mg/kg
    bw/day. There were no control animals. Animals were observed
    throughout the study for clinical signs of toxicity and histopatho-
    logical examinations were performed on lungs, kidney, liver and heart
    after 14 days. A section of liver was examined by electron microscopy.

         There were no deaths during the study and no clinical signs of
    toxicity. There was no treatment-related effect on body weight.
    Systolic blood pressure and heart rate were normal at all dose levels.
    Clinical chemistry parameters were normal apart from a slight increase
    in alkaline phosphatase activity at all dose levels which increased in
    the second week. Urinalysis parameters were normal. Haematological
    parameters indicated a slight decrease in haemoglobin concentration,
    RBC count and packed cell volume in treated animals, but the changes
    were within normal limits and not definitely related to treatment.
    Relative liver weights were within normal limits. Microscopic
    examination revealed an increase in cytoplasmic vacuolation in the
    liver of 5/6 animals. Electron microscopic examination revealed a
    dose-related increase in proliferation of smooth endoplasmic
    reticulum. Lipid vacuoles and lysosomes were present in the liver of
    all animals but, in the absence of control animals, it could not be
    determined whether the observed microsomal changes were treatment-
    related (Chvedoff  et al., 1981b).

         In a 3-month study, groups of beagle dogs (4/sex in the low- and
    intermediate-dose groups, and 6/sex in the control and high-dose
    group) were fed a diet containing alitame (purity 98-101%) at
    concentrations of 0, 0.5, 1, or 2%, equal to 0, 120, 220 or 490 mg/kg
    bw/day. In the control and high-dose groups, 2 animals/sex were
    transferred to normal diet for an additional month after the study
    period. Animals were examined throughout the study period for clinical
    signs of toxicity and body weight was recorded weekly. At various
    times, clinical chemistry, urinary and haematology parameters were
    measured. Tests of aminopyrine clearance were measured on days 1 and
    63. Ophthalmological examinations were performed on high-dose animals.
    Tissues were examined histopathologically at the end of 3 months.
    Liver histochemistry, electron microscopy and content of microsomal
    cytochrome P-450 and b5 were also performed.

         There were no deaths throughout the study and no clinical signs
    of toxicity. Food intake was normal although there was a slightly
    decreased weight gain in high-dose animals, particularly males.
    Cardiovascular parameters were normal. Ophthalmological examination of
    high-dose animals was unremarkable. Haematological parameters were

    within the normal range. Clinical chemistry revealed a dose-related
    increase in plasma alkaline phosphatase when measured at 1, 2 and 3
    months. The increase at the high-dose level was 400% at 3 months.
    After the recovery period, levels in the high-dose group were reduced
    but still higher than control levels.

         At the end of the 3-month treatment, the levels of cytochrome
    P-450 and b5 were increased in a dose-related manner. After the
    1-month withdrawal period, cytochrome P-450 and b5 in the high-dose
    group were reduced to near control levels. Induction of the liver
    microsomal enzyme system was also demonstrated in the dogs of the
    reversibility groups (control and high dose) following administration
    of a single i.v. dose of aminopyrine (15 mg/kg bw) on days 1 and 63 of
    alitame treatment, with blood levels measured at 0, 1, 1.5, 2, 3 and
    5 h. The plasma levels reflected the induction of a liver microsomal
    demethylase. On day 1, there was little difference between treated and
    control animals. On day 63, there was a marked difference between
    control and treated animals.

         Relative and absolute liver weights were increased in all
    treatment groups. Absolute liver weight was increased by 36% and 11%
    at high and low dose, respectively. Histopathology of the liver and
    other organs was unremarkable. Histochemical examination revealed an
    increase in G-6-P dehydrogenase activity in all treated groups except
    the low-dose females. NADPH-diaphorase activity was increased in
    high-dose animals. All levels returned to normal during the recovery
    period, except for an increased G-6-P phosphatase activity in
    high-dose males. Electron microscopy revealed an increase in
    proliferation of smooth endoplasmic reticulum in all groups. This
    increase was reversed in females but persisted in males during the
    recovery period. The liver changes appeared to be indicative of an
    adaptive response due to induction of microsome oxidative metabolism.
    Effects were observed at all dose levels and a NOEL was not
    established in this study (Monro  et al.  1982b).

         In an 18-month study, groups of 4 male and 4 female beagle dogs
    were fed a diet containing alitame (purity 98-101%) at dose levels of
    0, 10, 30, 100 or 500 mg/kg bw/day. Animals were examined throughout
    the study period for clinical signs of toxicity, and body weights were
    recorded at regular intervals. Ophthalmoscopy was performed on animals
    at 6-month intervals. Electrocardiographic tracings were performed at
    regular intervals. Clinical chemistry, urinary and haematological
    parameters were measured at regular intervals. Aminopyrine clearance
    was measured in control and intermediate dose animals at pre-test and
    during weeks 3, 5, 7, 9, 40 and 74, and in the high-dose group at 39
    and 73 weeks. Gross pathological examination was performed at 18
    months together with histopathological examination of a wide range of
    tissues. There were no treatment-related effects on survival and no

    clinical signs of toxicity. Ophthalmoscopic examinations and
    cardiovascular parameters were normal. Body weights were comparable
    between groups although body-weight gain in high-dose females was
    slightly less than control animals between weeks 40 and 70. Food
    consumption was normal in all groups. All of the clinical pathology
    parameters were normal except for an increase in serum alkaline
    phosphatase activity in high-dose males and females. The alkaline
    phosphatase rise was not uniform at high dose, with some dogs showing
    little or no change.

         Analysis of the urinary metabolites over a 2-day period at week
    10 showed that the excretion rate was proportional to the dose
    administered, indicating a dose-related systemic exposure. Measurement
    of aminopyrine clearance, as an indicator of the induction of the
    liver microsomal enzyme system, revealed a slight decrease in t1/2
    in males and females at 100 mg/kg bw/day at the 2-week period. The
    significance of this result is unclear, and may be due to the high
    t1/2 in the animals pre-dosing. In males, there appeared to be a
    gradual increase in t1/2 over the study period but again the result
    is difficult to interpret. Overall, the results at 100 mg/kg bw/day do
    not appear to indicate any enzyme induction. At 500 mg/kg bw/day,
    after 39 or 73 weeks, significant microsomal enzyme induction was
    apparent.

         The only gross pathological change observed was a moderate
    increase in absolute liver weight of 31% and 20% in high-dose males
    and females, respectively. This increase was statistically significant
    in males only. Histopathology was unremarkable in the treated animals.
    A slight increase in smooth endoplasmic reticulum was found in the
    livers of high-dose animals. The NOEL in this study was 100 mg/kg
    bw/day (Holmes  et al., 1985b).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Mice

         In a 2-year carcinogenicity study, groups of 50 male and 50
    female Crl: CD-1 (ICR) BR mice were fed a diet containing alitame
    (purity, 98-101%) at concentrations of 0 (2 groups), 0.1. 0.3 or 0.7%.
    Animals were observed throughout the study period. Body weight and
    food consumption were recorded weekly for 13 weeks and then at
    N-weekly intervals for the remainder of the study. At the end of the
    study period, animals were sacrificed and subjected to gross
    pathological examination and organs weights recorded. Tissues from all
    of the major organs were prepared for histopathological examination.
    Tumour incidence was recorded.

         There was no treatment-related effect on survival. The survival
    rate for controls, low-, intermediate-, and high-dose groups in
    males/females was 52/24%, 22/26%, 22/38%, 32/36% and 38/26%,
    respectively. There were no clinical signs of toxicity in the treated
    groups. Body-weight gain and food consumption were similar for all
    groups. Gross pathological examination revealed a significant increase
    in absolute and relative liver weight at high dose in males only.
    Absolute liver weight increased by 59% and 46% compared to the two
    control groups, while relative liver weight increased by 58% and 38%
    compared to the two control groups. Slight to mild centrilobular
    hepatocellular hypertrophy was found in 12 male animals in the
    high-dose group. At the intermediate-dose level, absolute and relative
    liver weights of males were also increased. Absolute liver weight was
    increased by 41% and 30% compared to the two control groups, while
    relative liver weight was increased by 39% and 21% compared to the two
    control groups. While the increases were not statistically
    significant, nevertheless the Committee considered them to be of
    biological significance at this dose level. No hepatocellular
    hypertrophy was apparent at the intermediate- and low- dose levels.

         There were no other histopathological changes, or any
    treatment-related increase in tumour incidence. Under the conditions
    of this assay, there was no evidence of carcinogenicity in mice
    induced by alitame in the diet. The NOEL, based on the biologically
    significant liver weight changes in males, was 0.1% in the diet, equal
    to 125 mg/kg bw/day for males and 155 mg/kg bw/day for females (Holmes
     et al., 1985c).

    2.2.3.2  Rats

         A carcinogenicity study was conducted with dietary administration
    and  in utero exposure to alitame in Long-Evans rats. Groups of 50
    male and 50 female Long-Evans rats (strain Blu:(LE)BR) were fed a diet
    containing alitame at concentrations of 0, 0.1, 0.3 or 1% for 2 years.
    Animals used in this study were taken from the F1 offspring of rats
    treated 4 weeks prior to mating and during gestation and lactation
    (Holmes  et al., 1986b). Animals were observed throughout the study
    period for clinical signs of toxicity. Body weights and food
    consumption were recorded weekly. Ophthalmoscopic examinations were
    carried out at 12, 18 and 24 months. Clinical chemistry and
    haematological parameters were measured in 10 animals/sex/dose at 6,
    12, 18 and 24 months. At the end of the study period, animals were
    sacrificed and subjected to gross pathological examination. Organ
    weights were recorded. Tissues from all the major organs were prepared
    for histopathological examination.

         There were no clinical signs of toxicity or behavioural changes
    which could be associated with treatment. Ophthalmoscopic examinations
    revealed no treatment-related effects. The survival rate at 104 weeks
    for the control, low, intermediate, and high-dose groups was for
    males/females 40/46%, 34/40%, 30/42% and 28/34%, respectively. There
    was a slight decrease in survival in treated animals compared to
    controls but this was not necessarily treatment-related.

         In the second year of the study, there was a dose-related
    difference in body weights for males between control animals and those
    in the high- and intermediate-dose groups. In females, the body weight
    of high-dose animals was lower than controls at the start of the study
    and continued to be significantly lower throughout the study.
    Intermediate-dose females also had a slightly lower body weight than
    controls which became more pronounced in the 2nd year of the study.
    There was no treatment-related effect on food consumption. These
    differences in body weights were significant only at high dose.

         There were no treatment-related changes in clinical chemistry or
    haematological parameters. Gross pathological findings were generally
    unremarkable. There was a slight non-significant increase in relative
    liver weight at the high and intermediate dose levels in both males
    and females. Microscopic examination of the tissues did not reveal any
    increase in the incidence of neoplastic lesions. In the high- dose
    group, 5 males and one female showed mild centrilobular hepatocellular
    hypertrophy. The incidence of proliferative liver lesions in females
    is shown in Table 2. The first appearance of hyperplastic nodules in
    the liver of females was at week 96. Survival at this time in control,
    low-, intermediate- and high-dose females was 64%, 56%, 50% and 50%,
    respectively. Alitame, under the conditions of this assay, showed no
    evidence of carcinogenicity in rats. The NOEL, based on body-weight
    changes in both males and females, was 0.3% in the diet, equal to
    130 mg/kg bw/day for males and 160 mg/kg bw/day for females
    (Holmes et al., 1986a).

    Table 2.   Incidence of proliferative liver lesions in female rats
               (Holmes et al., 1986a).
                                                                        

                                  Control   0.1%      0.3%      1%
                                                                        

    No. of livers examined        50        46        48        49
    Focal nodular hyperplasia     1         0         3         9
    Eosinophilic foci             4         0         3         11
                                                                        

         A re-examination of the pathology slides of the 2-year
    carcinogenicity study in Long-Evans rats (Holmes  et al., 1986a) was
    conducted on two subsequent occasions. At the first re-examination,
    all available liver slides from all groups of female rats were
    examined. The reported incidence of proliferative lesions is shown in
    Table 3. There was no evidence of hepatocellular carcinomas in females
    (Farrell, 1987).

    Table 3.   Incidence of proliferative liver lesions in female rats
               (first re-examination; Farrell, 1987)
                                                                        

                                 Control     0.1%      0.3%       1%
                                                                        

    No. of livers examined       50          49        50         50
    Hepatocellular adenoma       0           0         1          1
    Eosinophilic foci            3           3         6          12
    Hyperplasia                  0           4         3          6
    (focal and multifocal)
                                                                        

         In the second re-examination of the pathology slides, slides
    showing proliferative changes in female rats in either the original
    study or in the first re-examination were examined using a revised set
    of diagnostic criteria for hepatic proliferative lesions. The
    incidence of these lesions is shown in Table 4. There was no evidence
    of hepatocellular carcinomas in females (Hardisty  et al., 1994).

    Table 4.   Incidence of proliferative liver lesions in female rats
               (second re-examination; Hardisty et al., 1994).
                                                                        

                                  Control   0.1%      0.3%      1%
                                                                        

    No. of livers examined1       8         7         16        25
    Hepatocellular adenoma        0         0         2         12
    Hepatocellular hyperplasia    0         0         0         3
    Eosinophilic cell foci        2         3         11        18
                                                                        

    1    In this re-examination, only slides identified in the original
         report and in the first re-examination as showing proliferative
         lesions were examined.

         Because of the low survival rate at 104 weeks in the Long Evans
    rats (Holmes  et al., 1986a), a second carcinogenicity study was
    conducted with dietary administration and  in utero exposure to
    alitame in Sprague-Dawley rats. Groups of 70 male and 70 female
    Sprague-Dawley rats were selected from the F1 offspring of rats
    treated 4 weeks prior to mating and during gestation and lactation
    (Levinsky  et al., 1992). The groups were fed a diet containing
    alitame (purity 99.3%) at concentrations of 0 (two control groups),
    0.1, 0.3 or 1% for two years.

         The animals were observed daily for clinical signs, and body
    weights and food consumption were recorded weekly for the first
    6 months and then every 4 weeks thereafter. Ophthalmological
    examinations were performed pre-test and at 12 and 21 months; serum
    chemistry, haematology and urinalysis measurements were taken at 6,
    12, 18, 21 and 24 months. Tissues were collected from all animals
    which died or became moribund during the study. At the end of the
    study the animals were sacrificed and necropsied and tissues prepared
    for light microscopy. Organ weights (liver, kidney, adrenal glands,
    testes and ovaries) were recorded.

         There were no clinical signs of toxicity or behavioural changes
    which could be associated with treatment. Ophthalmoscopic examination
    revealed no treatment-related effects. Survival rates were high for
    the first 12 months (>93%) but by 18 months average survival was 65%
    for males and 75% for females. Groups reached 50% survival between 18
    and 19 months for males and between 20 and 21 months for females.
    Survival rates at 24 months in the 2 controls, low-, intermediate- and
    high-dose groups were for males/females 9/37%, 4/23%, 7/31%, 9/24% and
    26/17%, respectively.

         Body-weight measurement began at birth for this study, and at
    weaning (day 21). The mean body weights for the intermediate- and
    high-dose animals were -9% and -21%, respectively, compared to the
    control groups. Following selection of animals into groups for the
    2-year study, the body weights expressed as percent of controls at the
    low dose were -4% (M) and -2% (F), at the intermediate dose -7% (M)
    and -10% (F), and at the high dose -18% (M) and -16% (F). For males,
    over tile course of the study, the initial body weight differential
    decreased as the treatment group showed increased weight gain compared
    to the controls. Body weights peaked at approximately 15 months
    followed by a gradual decline in all groups, with the high-dose group
    showing the greatest decline. In females, over the course of the first
    few months of the study, the initial body weight differentials
    decreased particularly in the low-and intermediate-dose groups, which
    showed increased weight gain compared to controls. After 6 months,

    weight gain in the low- and intermediate-dose groups was similar to
    controls. In the high-dose group, however, there was a decrease in
    weight gain compared to the controls, and by the end of the study,
    body weight differential had increased to -32%. Food consumption may
    have been slightly lower in high-dose males, but was similar in all
    groups of females.

         There were no treatment-related changes in haematology or
    urinalysis parameters. Changes observed in clinical chemistry data
    included a decrease in serum triglyceride values (50-60%) in females
    at the intermediate-dose in the first 18 months, and in high-dose
    females throughout the study. Cholesterol was also increased in female
    rats at 6 months at the intermediate-dose level (28%), and at 6 and 12
    months at the high-dose level (67%).

         Significant organ weight changes were seen in both liver and
    kidney. In males, there was a relative increase in both kidney and
    liver weight at high dose. In females, there was an absolute decrease
    in kidney and liver weights at both intermediate and high doses, and
    a relative increase in kidney weight at high dose. These changes
    accompanied the decrease in body weight in high-dose females.
    There was also an overall trend for decreased body weight in
    intermediate-dose females which was not statistically significant.
    The relative testes weight was increased in high-dose males.

         Neoplastic changes were observed in the following organs: kidney,
    liver, pancreas, lung, heart, thymus, mesenteric node, adrenal cortex,
    thymus, parathyroid, uterus, cervix, brain, spinal cord, Zymbal's
    gland, skin, bone, skeletal muscle, abdominal cavity, and soft
    tissues. There was no evidence that the incidence of these neoplasms
    was treatment-related. In the liver, hepatocellular carcinomas were
    reported in one control male and one high-dose male. There was no
    reported incidence of hepatocellular adenomas. There were also no
    treatment-related non-neoplastic changes. Alitame, under the
    conditions of this assay, showed no evidence of carcinogenicity in
    Sprague-Dawley rats. The NOEL, based on body-weight changes, was 0.3%
    in the diet, equal to 230 mg/kg bw/day for males, and 250 mg/kg bw/day
    for females (Fisher  et al., 1993).

    2.2.4  Reproductive toxicity studies

         In a 2-generation reproductive toxicity study, groups of
    Long-Evans rats (75/sex) were administered a diet containing alitame
    (purity 98-101%) at concentrations of 0, 0.1, 0.3 or 1% for either 2
    weeks (females) or 12 weeks (males) prior to mating. Alitame-
    containing diet was continued throughout pregnancy and lactation. Of
    the F1 pups, 50 pups/sex/dose level were selected for a 24-month

    carcinogenicity study, a further 25/sex/dose level were raised to
    maturity and mated to produce the F2a litters. Approximately 3 weeks
    after weaning of the F2a litters, the F1 animals were again mated
    to produce the F2b litters. Both F2a and F2b pups were exposed to
    alitame via the milk and in the food.

         In the F0 parental group, the body weight of males was similar
    for all groups during the 12 weeks of treatment, while body weight and
    food consumption of females were slightly depressed during the 2-week
    treatment period at the high-dose level. There was no treatment-
    related effect on fertility or gestation indices, and no abortions
    were noted. There was no difference in the number of pups born alive
    between treated and control groups in the F1 litters. There was no
    treatment-related effect on day 4 pup survival rates or lactation
    indices. F1 pup weights of treated animals were similar to those of
    control animals on days I and 4 of lactation, but high-dose pups had
    significantly lower body weights on days 7, 14 and 21. However, these
    decreased body weights were not significantly different from the
    historical control data.

         In the F1 parental group, body weights were similar to control
    except at the high-dose level where there was a slight decrease in
    body-weight gain. There were no treatment-related changes in pregnancy
    rates or gestation length for either F2a or F2b litters. There was
    no difference in the number of pups born alive between treated and
    control groups in the F2a or F2b litters. Survival indices for F2a
    and F2b were normal for all groups on days 1 and 4 of lactation.
    F2a pup weight for all treated groups was similar on days 1,
    4 and 7 of lactation but a significant decrease was noted in the
    high-dose animals on day 14 and in all treated groups on day 21
    compared to controls. The body weights were still within the
    historical control data range. The F2b pups weight at the high-dose
    level was significantly lower than controls throughout lactation,
    while at the intermediate-dose level body weights were lower at days
    14 and 21, and at the low-dose level, body weights were significantly
    lower than controls on days 4 to 21. No historical control data were
    available for F2b litters.

         Gross pathological examination of the F0 males and females, the
    F1 pups, and the F2a and F2b pups revealed no treatment-related
    abnormalities. Postnatal development in F2b pups was assessed with
    regard to locomotor activity, auditory function and ophthalmoscopic
    examination. At the F2b weaning, there was a significant decrease in
    both horizontal and vertical locomotor activity in F2a male and
    female pups at the high-dose level, and a significant increase in
    horizontal (but not vertical) locomotor activity in the F1 dams at
    the high-dose level group. Pups tested for auditory function and for
    ophthalmoscopic changes revealed no treatment-related changes
    (Holmes  et al., 1986b).

         In a second 2-generation study, groups of 30 male and 30 female
    Long-Evans rats (strain Blu:(LE)BR) were administered alitame (purity
    98-101%) at concentrations of 0, 0.1, 0.3, or 1% for either 2 weeks
    (females) or 12 weeks (males) prior to mating. Alitame-containing diet
    was continued through pregnancy and lactation. Of the F1 pups,
    25/sex/dose were raised to maturity and mated to produce the F2a
    litters. Approximately 3 weeks after weaning of the F2a litters, the
    F1 animals were again mated to produce the F2b litters. Both the
    F2a and F2b pups were exposed to alitame  in utero, during lactation,
    and in the food following weaning.

         At the high-dose level, 2 additional F1 groups of 25/sex were
    exposed to alitame differently. These groups were introduced to
    address the lactation effect observed in the first study. In one of
    these groups, the F1 females were removed from treated diet at the
    birth of F2a pups. In this group, the F2a pups received no alitame
    during lactation. In the other group, the F1 females were removed
    from the treated diet 3 weeks after weaning until the birth of the
    F2a pups. In this group, the F2b pups received alitame only during
    lactation. Animals were then placed on treated diet again. Post-natal
    development of F2b pups was tested with an ophthalmoscopic
    examination, an auditory function test, and a locomotor activity test.
    Locomotor activity tests were also conducted on F0 and F1 parents,
    and on F2a and F2b pups.

         In the F0 parental animals, the body weights of males were
    similar for all groups during the 12 weeks of treatment, while there
    were decreases in body-weight gain and food consumption in females at
    the high-dose level during the 2-week treatment period. Fertility
    rates for all groups were normal. The number of live F1 pups was
    similar for all groups at day 1, but was slightly reduced at the
    high-dose level on day 4. This was explained by several large litters
    at the high-dose level which declined in the first few days. F1 pup
    body weight was similar for all groups on day 1 of lactation but was
    significantly less at the high-dose level from day 4 onward.
    Body-weight gain at high dose was similar to that of control groups
    from days 4 to 21. The mean number of pups alive at day 21 compared to
    the number of pups alive at day 4 (the lactation index) was similar
    for all groups. Gross pathological examination of the F0 males and
    females and the F1 pups revealed no treatment-related changes.

         In the F1 parental group, the body weights of all female groups
    were similar. Pregnancy rates and gestation times were normal. The
    F1 males in the treated groups had slightly lower body weights than
    controls although body-weight gain was similar for all groups. The
    numbers of F2a litter live pups were similar for control, low and
    intermediate groups. For the high-dose group, the mean number of pups
    born alive (11.54) was slightly lower than controls (12.96). However,
    the mean number of pups born alive in the other two additional

    high-dose groups were similar to controls (12.75 and 12.13). The
    4-day survival indices for the F2a pups in the control, low-,
    intermediate-and high-dose groups were 0.98, 0.98, 0.93 and 0.89,
    respectively. In the two additional high-dose groups, the 4-day
    survival indices were 0.97 and 0.95. Lactation indices were for the
    control, low-, intermediate- and high-dose groups 0.98, 1.0, 0.97 and
    0.96, respectively, and for the two additional high-dose groups 0.99
    and 0.88, respectively.

         The mean body weight of high-dose F2a pups were slightly but
    significantly lower than controls at birth and throughout lactation.
    The additional F2a pups which were exposed only during lactation had
    slightly lower body weight after the first week of lactation. The
    additional group of F2a pups which had no alitame exposure during
    lactation had body weights similar to controls at all times after day
    1 of lactation. The results indicated a slight lactation effect of
    alitame at the high-dose level, although the high-dose body weights
    were still within the historical control ranges. The F2a pups at the
    intermediate-dose level also had slightly but significantly lower body
    weights at days 14 and 21 of lactation. External examination and gross
    internal examination of the F2a pups revealed no treatment-related
    changes.

         The fertility rates and gestation times associated with the
    production of the F2b litters were normal for all groups. The mean
    number of pups born alive was comparable at the control, low- and
    intermediate-dose levels, being 12.1, 11.7 and 13.0, respectively. At
    the high-dose level, the number of live pups was slightly lower (9.8).
    Survival during lactation was similar for all groups. The mean body
    weight for the F2b high-dose level pups was significantly lower than
    controls on day 14 of lactation. External examination and gross
    internal examination revealed no treatment-related changes.

         Ophthalmoscopic examination and auditory function tests were
    normal in all groups. Locomotor activity for both parental groups and
    pups showed considerable variation and the changes could not be
    correlated with treatment.

         This second study provided supporting evidence that alitame at
    high-dose levels caused some suppression of body-weight gain during
    lactation. This was further investigated in a study to examine the
    effect of alitame in the cage bedding material on the pups body weight
    (see section 2.2.5). The NOEL, based on body-weight changes in the
    F2 pups, was 0.1% in the diet, equivalent to 100 mg/kg bw/day
    (Holmes  et al., 1986c).

         In a 1-generation study in Sprague-Dawley rats, groups of 105
    male and 105 female rats (strain Crl:CDBR VAF/Plus) were administered
    alitame (purity 99.3%) at concentrations of 0, 0.1, 0.3 or 1% for 4
    weeks prior to mating, through the cohabitation period, through the
    gestation period, and up to day 21 of lactation. The aim of this study
    was to produce an F1 generation of rats, continuously exposed to
    alitame  in utero, via the milk during lactation, and through the
    feed for use in 1-year and 2-year toxicity studies. The animals were
    observed daily for clinical signs of toxicity and body weights and
    food consumption were recorded. During cohabitation, only the body
    weights of the males were recorded. During gestation, body weights of
    females were recorded on days 0, 4, 7, 14 and 20. During lactation,
    dams and viable young were weighed on days 1, 4, 7, 14 and 21
     post partum. On day 4, all litters were culled to 8 pups, 4
    animals/sex. At day 21 (weaning), litters from the control groups, 0.1
    and 0.3% treatment groups were culled to 2 animals/sex, and the
    respective dams sacrificed. High-dose litters were not culled due to
    their lower body weights, and were allowed to remain with the
    high-dose dams until it was deemed prudent to separate them. When the
    F1 animals were 3 to 6 weeks of age, 70 pups/sex/dose were selected
    for a 2-year oncogenicity study (Fisher  et al., 1993), and 20
    pups/sex/dose level were selected for a 1-year toxicity study
    (Fisher  et al., 1992). All F0 males were terminated after the
    cohabitation period. F0 females which did not deliver by day 24 of
    gestation were terminated and their uteri and ovaries were examined.
    F0 pups not used for either study were terminated.

         Reductions in body weights and food consumption were noted in the
    high-dose F0 animals, particularly during the first 2 weeks of the
    study. This may be associated with palatability. There was no
    treatment-related effect on mating, gestation length, or body weight
    during gestation and lactation. The number of implantation sites was
    slightly lower in the high-dose group and this was reflected in a
    slight reduction in the total number of pups born alive per litter.
    There was no difference between control and treated groups with
    respect to post-implantation losses. There was no treatment-related
    effect on pup survival during the lactation period. The mean
    body-weight gains at the intermediate- and high-dose levels were
    depressed compared to controls, and at weaning the body weights of the
    intermediate- and high-dose groups were 9% and 21% lower than
    controls, respectively. There were no internal or external
    abnormalities in the pups not selected for the long-term studies
    (Levinsky  et al., 1992).

    2.2.5  Special studies on neonatal survival

         A study was conducted in Long-Evans pups and lactating dams in
    order to further investigate the apparent decrease in pup body weight
    during lactation at the high-dose level in the reproductive toxicity
    studies. In this study, dams and pups were exposed to alitame which
    was added to the bedding material rather than to the feed. It was
    argued that dams and pups are normally exposed to treated feed in this
    way because of tipping over of the feed by the dam, or the tendency to
    scratch the feed into the bedding material. In this study, 2 groups of
    8 litters were formed and all dams received normal diet. On lactation
    days 1 and 5 and every 2-4 days thereafter, one of the groups had
    125 g of feed containing 1% alitame sprinkled on the bedding. The
    control group had feed without alitame sprinkled on to the bedding.
    Body weights of dams and pups were measured throughout the lactation
    period and at one week after lactation.

         Body-weight gain in 'treated' dams was slightly lower than in
    control dams on days 1 to 4 but there was no difference in body-weight
    gain on days 4 to 21 of lactation. Body-weight gain in 'treated' pups
    was decreased from day 2 and continued to be decreased throughout
    lactation and one week after lactation. For example, the mean
    body-weight difference on days 2, 7, 14 and 21 were 0.5 g (-8.4%),
    1.6 g (-13.7%), 2.7 g (-11.3%), and 3.1 g (-7.4%), respectively. After
    weaning, the weight difference continued to increase to 5.2 g (-7.2%)
    at one week. The differences were statistically different from
    controls on days 2, 3 and 5, but not thereafter. Locomotor activity
    was measured in dam and pups, and no treatment-related effect was
    noted.

         The results suggested that bedding containing 1% alitame had a
    treatment-related effect on pup body-weight gain, although no
    mechanism for this effect was apparent. Food consumption was not
    measured in this study although it was claimed to be normal. The
    consumption of food by the dams from the bedding or from their fur was
    considered to be negligible (Holmes  et al., 1986d).

    2.2.6  Special studies on embryotoxicity/teratogenicity

    2.2.6.1  Rats

         A group of 80 Crl:COBS-CD(SD)BR strain female rats were
    inseminated and divided into 4 groups of 20 animals each. The groups
    of presumed pregnant rats were administered alitame in 0.1% methyl
    cellulose by gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day
    on days 6 to 15 post-insemination. The dams were examined throughout
    the study period and body weights recorded regularly. Animals were
    killed on day 20 post-insemination. Fetuses were examined for external
    malformations, then half were stained for skeletal anomalies and half
    were examined for soft tissue defects. The ovaries and uteri of dams
    were examined on day 20.

         The pregnancy rate was 20/20 in control and intermediate-dose
    groups and 18/20 in the low- and high-dose groups. There were no
    deaths throughout the study and no clinical signs of toxicity. A
    slight decrease in food consumption occurred on day 14 at the
    high-dose level. Body weight was depressed significantly during and
    after the treatment period at the high-dose level only. The number
    of viable litters was similar for all groups, as was the number
    of corpora lutea. There were slight decreases in the number of
    implantation sites in the intermediate- and high-dose groups (10.8 and
    10.6 respectively) compared to the control (11.8) and low-dose group
    (12.2). There were also slight decreases in the number of fetuses in
    the intermediate- (9.9) and high-dose groups (9.7) compared to the
    control (11.0) and low-dose group 111.3). The implantation rate
    decreased with increasing close (91.1, 89.8, 86.7 and 84.9%,
    respectively), and the embryotoxicity rate increased with increasing
    dose (6.4, 6.8, 7.4, and 8.4%, respectively). The sex ratio (M/F) was
    decreased at the high-dose level (74/100) compared to controls
    (110/110).

         With regard to fetal development, there was no difference between
    the fetal weight of control and treated animals. Examination of the
    fetuses did not reveal any treatment-related increase in gross,
    visceral or skeletal malformations. Among all the fetuses examined,
    only one external anomaly (cleft palate) was observed. There was no
    treatment-related change in the rate of ossification. Under the
    conditions of this study, alitame showed no evidence of teratogenicity
    in the rat. The decrease in the number of fetuses at the higher dose
    levels appeared to be a direct result of the lower level of
    implantation in these groups rather than to embryotoxicity (Perraud
     et al., 1983a).

    2.2.6.2  Rabbits

         A group of 77 New Zealand white rabbits was inseminated and
    divided into 4 groups of 19, 18, 20 and 20 animals. The groups were
    administered alitame by gavage at dose levels of 0, 100, 300 or
    1000 mg/kg bw/day on days 7 to 18 post-insemination. The does were
    examined throughout the study period and body weights recorded
    regularly. Animals were killed on day 28 post-insemination. Fetuses
    were examined for external malformations then half were stained for
    skeletal anomalies and half were examined for soft tissue defects.
    Ovaries and uteri of does were examined on day 28.

         Four animals died during the study, one at each dose level and
    one in the control group. The pregnancy rate was 16/19, 15/18, 18/20
    and 16/20 in each of the groups, respectively. Food restrictions were
    introduced on days 12 to 19 to reduce the incidence of diarrhoea.
    Body-weight loss was observed in all groups during this 8-day period.
    A significant treatment-related decrease in body-weight gain was also
    noted at the high-dose level.

         The number of viable litters was similar for all groups as was
    the number of corpora lutea. There was one abortion on day 24 in the
    high-dose group. The number of implantation sites was slightly
    decreased at the low-dose level compared to other groups.
    Embryo-mortality rate was slightly higher at the intermediate- and
    high-dose levels but was not dose-related, and not considered to be
    due to treatment.

         With regard to fetal development, fetal body weights were
    slightly but not significantly decreased (7%) at the intermediate- and
    high-dose level compared to the control group. Examination of the
    fetuses did not reveal any treatment-related increase in gross,
    skeletal or visceral abnormalities. Among all the fetuses, there was
    one with cleft palate and one with club foot in the intermediate-dose
    group, and one with edema in the control group. The only visceral
    abnormality seen was agenesis of the left kidney in one control group
    fetus. There were no major malformations observed, but there was a
    significant increase in the incidence of fetuses with supernumerary
    13th ribs at the intermediate- and high-dose levels. Under the
    conditions of this assay, alitame showed no evidence of teratogenicity
    in the rabbit (Perraud  et al., 1983b).

    2.2.7  Special studies on genotoxicity

         The results of the genotoxicity tests with alitame are summarized
    in Table 5.

    2.2.8  Special studies on pharmacological effects

         Alitame was tested for its pharmacological activity in a battery
    of systems designed to assess autonomic, gastrointestinal, renal and
    metabolic functions.

         A solution of 10-4 mol/litre alitame had no contractile or
    relaxant activity in guinea-pig ileum, rat fundus, or rat vas
    deferens, and did not interact with any of the six neurotransmitters
    used in these systems. Alitame at an oral dose level of 25 mg/kg bw
    caused no change in intestinal transit time nor in renal excretion of
    fluids or electrolytes. Alitame at an oral dose level of 25 mg/kg bw
    had no effect on fasting blood glucose levels or on the deposition of
    an oral glucose load (Pearson  et al., 1982).

        Table 5.   Summary of genotoxicity tests with alitame
                                                                                                             

    Test system                 Test organism             Concentration1            Result    Reference
                                                                                                             

    Reverse mutation in         S. typhimurium            20-10000 g/plate         -         Holden et al.,
    bacteria                    TA1535, TA 1537, TA98,    (-S9)                               (1981)
                                TA100                     10-10 000 g/plate
                                                          (+S9)

    Urinary metabolites         CD-1 mice/                50-1000 mg alitame/kg     -         Holden et al,.
    in vivo/Reverse             S. typhimurium            in mice & 0.05-0.75 ml              (1981)
    mutation in bacteria2       TA1535, TA 1537, TA98,    urine/plate
                                TA100

    Reverse mutation in         Mouse lymphoma cells      220-1655 g/ml (+S9)      -         Holden et al.,
    mammalian cells             L5178Y                                                        (1981)

    Chromosome aberration in    CD-1 mice bone            0-1000 mg/kg              -         Holden et al.,
    vivo3                       marrow cells                                                  (1981)
                                                                                                             

    Table 5.   Summary of genotoxicity tests with alitame (cont'd).
                                                                                                             

    Test system                 Test organism             Concentration1            Result    Reference
                                                                                                             

    Chromosome aberrations      Human lymphocytes         0-1000 g/ml              -         Holden et al.,
    in vitro4                                                                                 (1981)

                                Human lymphocytes         0-3000 g/ml (+/-S9)      -         Amacher et al.,
                                                                                              (1991)

    Micronucleus assay5         CD-1 mice                 0-2000 mg/kg/bw/day       -         Amacher et al.,
                                                                                              (1991)
                                                                                                             

    1     The purity of alitame tested was not provided.
    2     Urine from treated CD-1 mice (14/group) was pooled over 18 h and treated with beta-glucuronidase.
    3     Groups of 5 CD-1 mice were administered alitame orally and bone marrow cells harvested at 6, 12
          and 24 h.
    4     In the first study, there was minimal provision of methodology and reporting.  The second study
          was conducted under more recent GLP regulations.
    5     Groups of 5 male and 5 female mice were treated over three consecutive days.  Bone marrow was
          prepared 24 h after file last dose.
    
    2.2.9  Special studies on neurotoxicity and neurobehavioural
           effects

         In a neurotoxicity study, alitame was tested for its ability to
    inhibit  in vitro receptor binding in rat brain homogenate and to
    produce  in vivo neurochemical effects in rats. In the  in vitro
    binding assays performed with rat brain homogenate, alitame was tested
    for its ability to inhibit the binding of a variety of receptor
    radioligands to brain membranes. Neither alitame nor the B-isomer, at
    concentrations of 10 M, had any effect on the level of binding of
    these ligands. In the  in vivo study, groups of Sprague-Dawley rats
    (8 males) were administered an oral dose of vehicle, alitame (25 mg/kg
    bw), -isomer of alitame (6.25 mg/kg bw), or alitame (12.5 mg/kg bw)
    plus -isomer of alitame (6.25 mg/kg bw). At 2 or 4 hrs, animals were
    killed and the levels of dopamine, serotonin and their metabolites in
    the brain tissue were measured. There was no significant change in
    neurotransmitter levels after treatment (Bacopoulos, undated).

         In a study to examine the effect of alitame on the behaviour of
    rats, groups of male CD rats were administered a single oral dose of
    vehicle, alitame (25 mg/kg bw), -isomer of alitame (6.25 mg/kg bw),
    or alitame (12.5 mg/kg bw) plus -isomer of alitame (6.25 mg/kg). In a
    separate experiment, animals were administered single oral doses as
    above for 13 consecutive days. No treatment-related effect on
    horizontal or vertical locomotor activity was noted in either the
    single or multiple treatment regimes (Bacopoulos, undated).

         In a study to examine the potential neurotoxicity of alitame in
    rats following acute administration, groups of 12 male and 12 female
    Sprague-Dawley rats were given alitame at a single oral dose of
    0, 2000, 3000 or 5000 mg/kg bw. The animals were examined using a
    functional observational battery (FOB) (both qualitative and
    quantitative), a motor activity test, and an auditory startle
    habituation test prior to treatment, at 5 and 7 h post-dosing, and
    on days 1 and 7 post-dosing. At the end of the study, half the
    animals were randomly selected for perfusion for possible future
    neuropathological examination while the remaining animals were given
    a full necropsy.

         Significant decreases in body-weight gains were noted in all
    treated groups on the day following treatment (days 0 to 1), followed
    by compensatory increases on the following days (days 1 to 7). A
    decrease in food consumption on day 1 was noted in all female treated
    groups and in males of the intermediate- and high-dose groups. For the
    high-dose female group, clinical symptoms included yellow abdominal/
    urogenital fur staining, dehydration, reduced activity and pallor
    between days 1 and 4 post-treatment. Effects were noted in all
    behavioural tests for both sexes in the high-dose group and in some
    tests in the intermediate- and low-dose groups on the first day. By
    day 7, results of all parameters in all treatment groups were similar
    to the control group.

         Qualitative FOB assessments on day 1 post-treatment revealed
    significant differences between the control and high-dose group for
    both sexes. At the high-dose level, there was a significant decrease
    in pinna reflex and a decrease in rearing in the arena. There was also
    an increased incidence in ataxic gait and diarrhoea in the high-dose
    animals. Animals at the lower dose levels also showed some increase in
    minor symptoms. By day 7, there were no significant differences
    between the groups. Quantitative FOB assessments found changes in
    temperature and hind limb splay in treated groups on day 1 but by day
    7 no significant differences were detected. Changes were also noted in
    treated groups with respect to the motor activity test and the
    auditory startle habituation test on day 1, but no changes were
    evident by day 7. There were no treatment-related gross pathological
    changes found on day 7. Transitory behavioural changes were observed
    immediately after treatment and in the 24 h following treatment. By
    day 7, all animals appeared normal and there was no evidence of
    neurotoxicity (Robinson  et al., 1993a).

         In a 13-week study of the potential effects of alitame on
    behaviour and neuromorphology in rats, 12 male and 12 female
    Sprague-Dawley rats were fed a diet containing alitame at
    concentrations of 0, 0.1, 0.3 or 1%. Animals were examined throughout
    the study period for clinical signs of toxicity and body weights and
    food consumption were recorded. The animals were examined using a
    functional observational battery (FOB) (qualitative and qualitative),
    a motor activity test and an auditory startle habituation at
    pre-treatment, after 7 and 14 days of treatment and in the 5th, 9th
    and 13th weeks. Passive avoidance testing was performed in weeks 4 and
    13. At the end of the study, half the animals (randomly selected) were
    perfused for neuropathological examination. Tissues from brain, spinal
    cord, and skeletal muscle were prepared as paraffin sections and
    haematoxylin and examination. Tissues from the peripheral nervous
    system and the central nervous system were prepared in epoxy
    embedding.

         There were no mortality or adverse clinical findings related to
    the treatment. The body weights of females in the 1% treated group
    were significantly lower after 3 weeks and throughout the second and
    third months of the study. The food consumption for this group was
    also significantly decreased during the first week of the study. The
    body weights and food consumption of all the other treated groups were
    normal.

         Both the qualitative (observations and handling) and the
    quantitative FOB evaluations (grip strength, hind limb splay, and body
    temperature) were unaffected by treatment for both sexes. In the
    locomotor activity tests, significant differences at single time
    points were noted for high-dose males and females. However, these
    differences were not sustained over the course of the study and could

    not be clearly related to treatment. For auditory startle habituation
    tests, occasional significant differences in the 0.1% group were noted
    but these were not attributed to treatment as the effects were not
    sustained and there was no dose-relationship. In passive avoidance
    performance at 4 weeks there were no significant differences between
    groups. At week 13, more females in the 1% group completed the
    training trial than in the control group. However, it could not be
    concluded that this was related to treatment and was of doubtful
    neurotoxicological significance.

         There were no treatment-related gross pathological findings in
    animals not selected for perfusion or in those which underwent
    perfusion. A few animals, primarily males in all groups, had pale
    areas on the livers. For rats undergoing perfusion, gross pathological
    findings in the nervous system were limited to a few animals with dark
    areas in the meninges. Histopathological examination was only
    performed in control and high-dose animals. There were no significant
    differences in the finding in the two groups. Under the conditions of
    this study, there were no behavioural or neuromorphological changes
    observed which could be attributed to treatment with alitame (Robinson
     et al., 1993b).

         In a study to examine the potential developmental neurotoxicity
    of alitame, groups of female Sprague-Dawley rats were fed a diet
    containing alitame at concentrations of 0, 0.1, 0.3 or 1% for one week
    prior to mating, then throughout the mating and gestation periods and
    up to 10 days  post-partum. Animals were observed throughout the
    study period and body weights and food consumption recorded. All
    females were given a complete necropsy. Dams were killed on days 21-23
     post-partum.

         The F1 pups were examined for malformations, sexed and the
    number alive and dead recorded. The pups in each litter were weighed
    individually on days 1, 4, 7, 11, 14, 17 and 21. On day 4, the litters
    were culled to 8 pups, 4 of each sex. Pups were examined for pinna
    unfolding (day 2), eye opening (day 12), and air righting reflex
    (day 14). A motor activity test was conducted on day 17. Pups were
    weaned on day 21.

         The F1 adult generation was formed from at least 20 different
    litters and consisted of 80 rats/sex/group. Animals were examined for
    clinical condition and physical development and body weights and food
    consumption recorded. From each litter, 3 males and 3 females were
    randomly selected for behavioural testing (motor activity test,
    auditory startle habituation test, passive avoidance test and
    water-maze test). Young adults not selected for the adult F1
    generation behavioural testing were sacrificed and underwent gross

    pathological examination. Six animals/sex/group were perfused for
    histo-pathological examination. Tissues from brain, spinal cord, and
    skeletal muscle were prepared as paraffin sections and H and E
    stained. Tissues from the peripheral nervous system and the central
    nervous system were prepared in epoxy embedding.

         In the F0 animals, there were no deaths and there were no
    treatment-related clinical findings. The body weights of the 1%
    treated animals were significantly decreased throughout the study.
    The food consumption values for the 1% treated group were also
    significantly lower than control values during the study. Gross
    pathological examinations revealed no treatment-related effects. There
    was no treatment-related effect on oestrus cycles, pregnancy rate,
    gestation index, number of live and dead pups, sex ratio or post
    implantation loss.

         In the F0 pups, viability, survival, lactation indices,
    clinical findings and gross pathology were unaffected by treatment. In
    the 1% group, pup weight was significantly decreased on days 11, 14
    and 17 and slightly decreased on day 21. There were no treatment-
    related effects on physical or reflexological development, or on
    locomotor activity.

         In the F1 adults, there were no treatment-related effects on
    mortality or clinical findings. The body weights of males and females
    in the 1% group were significantly decreased. Food consumption was
    normal. Physical development was not affected by treatment. In the
    behavioural testing, there was no treatment-related effect on
    locomotor activity, auditory startle habituation, passive avoidance or
    water maze tests. Gross pathological and neuropathological
    examinations revealed no effects which could be attributed to alitame
    treatment. Under the conditions of this study, there were no
    behavioural or neuromorphological changes in adults or pups which
    could be attributed to treatment with alitame (Robinson  et al.,
    1993c).

    2.2.10  Special studies on melanin binding

         The potential for radiolabelled alitame or its metabolites to
    bind to melanin in the eye of rats was tested by whole body
    autoradiography and by microhistoautoradiography. Two male rats from
    the albino strain, Sprague-Dawley (Crl:CD(SD)BR) and two male rats
    from the pigmented strain, Lister Hooded (Crl:(LIS)BR) were
    administered a single oral dose of 65 mg/kg bw 14C-alitame (94%
    purity). At 2 and 24 h, one animal from each strain was killed and
    the right eye of each excised and stored at -80C for micro-
    histoautoradiography. The remaining carcass was rapidly frozen at
    -75C for whole-body autoradiography.

         Microscopic examination of the micrographs of the eyes revealed a
    large number of silver grains distributed widely throughout the layer
    of the retina, choroid and sclera in animals of both pigmented and
    non-pigmented strains at both sacrifice times. The results of the
    microscopy could not be interpreted and the data were considered to be
    artifactual.

         The results of the autoradiography revealed much higher levels of
    radioactivity in the uveal tract (choroid, ciliary body and iris) of
    the pigmented rats. Quantification of the autoradiographies revealed
    concentrations of 2.99  equivalent alitame/g tissue in the uveal
    tract of albino rats at 2 h and no detectable radioactivity at 24 h.
    In the pigmented rats, the radioactivity in the uveal tract was
    28.24/g equivalent alitame/g tissue at 2 h and slightly higher levels
    at 24 h (30.55 g/g). There was no difference in radioactivity in the
    surrounding tissues in either the albino or pigmented rat.

         The study provided evidence of binding of alitame or its
    metabolites to the melanin contained in the uveal tract of the
    pigmented rats.

         In an addendum to this report, alitame and two metabolites were
    tested  in vitro for their binding affinity to a melanin preparation
    from bovine eyeballs. Alanine tetramethylthietane amide sulfoxide,
    which was found in the urine of both rats and humans, did not bind to
    melanin under the conditions of this assay, whereas its acetylareal
    form, which was found only in the urine of rats, did bind to the
    melanin. The positive control, doxepin, produced a higher level of
    binding than found with the acetylated sulfoxide metabolite. This
    study provided some evidence that the melanin binding observed in rats
    may be associated with a metabolite formed only in the rat (Whitby
     et al., 1994).

    2.2.11  Special studies on the -isomer of alitame

    2.2.11.1  Absorption, distribution, excretion, and biotransformation

         In a study to examine the toxicokinetics of the -isomer of
    alitame, Sprague-Dawley rats (4/sex) were administered a single oral
    dose of the 14C--isomer of alitame (10 mg/kg bw in water).
    Excretion of radioactivity in urine and faeces was measured over 72 h.
    A similar study was conducted with 6 male and 3 female Long-Evans rats
    following a single oral dose of 14C--isomer of alitame (25 mg/kg
    bw). A separate group of 7 male Long-Evans rats were fitted with a
    bile cannula. These animals received an oral dose of 25 mg/kg bw
    14C--isomer, and bile and urine were collected over 48 h.

         In Sprague-Dawley rats, the bulk of the radioactivity was
    excreted within 48 h, with 24% and 65% excreted in urine and faeces,
    respectively. In Long-Evans male rats, total excretion was similar,
    with 25 and 64% excreted in urine and faeces, respectively. Excretion
    in the female rats was slightly slower. In bile-duct cannulated
    animals, 13% of the total radioactivity was in bile over 48 h
    (Pfizer, 1986).

         In a study to identify the metabolites of the -isomer of
    alitame, 2 male and 2 female Long-Evans rats were administered a
    single oral dose of 10 mg/kg bw 14C--isomer of alitame. In a second
    experiment, female Long-Evans rats received 14C--isomer of alitame
    at an oral dose of 25 mg/kg bw. In a third experiment, 3 females and 6
    males received the same dose of 14C--isomer of alitame. Urinary
    metabolites were examined by HPLC. Urinary radioactivity contained
    approximately 1% of the radioactivity as unchanged -isomer in the
    males.

         Unchanged -isomer was found in the urine of female rats and
    represented approximately 13% of the total radioactivity. Other
    metabolites which represented approximately 50% of the radioactivity
    were alanine tetramethylthietane amide sulfoxide (CP-58,290), alanine
    tetramethylthietane amide sulfone (CP-57,254) and their acetylated
    derivatives (CP-71,246 and CP-58,386). The only different metabolite
    identified was the N-acetyl derivative of the -isomer which was
    formed by acetylation of the free amine group of the aspartic acid
    moiety. The N-acetyl derivative of the -isomer comprised 9% and 31%
    of the radioactivity in the urine of male and female rats,
    respectively. In faeces, the -isomer accounted for the majority
    (70-81%) of the radioactivity (Pfizer, 1986).

         In a study to examine systemic exposure to B-alitame and N-acetyl
    beta-isomer by examination of urinary metabolites in male Long-Evans
    rats, 14C--isomer of alitame or 14C-N-acetyl --isomer was
    administered i.v. initially, then orally, at a dose level of 25 mg/kg
    bw. Urine and faeces were collected at 24-hour intervals for a 48-hour
    period. Systemic exposure was calculated from a comparison of urinary
    excretion following the oral and i.v. routes. The systemic exposure to
    -isomer following oral administration was 9% in males and 10% in
    females. The systemic exposure to -isomer plus N-acetyl--isomer was
    8% in males and 18% in females (Pfizer, 1986).

         In a study to examine systemic exposure to -isomer by
    examination of serum concentrations in Long-Evans male rats,
    14C--isomer was administered i.v. initially (25 mg/kg bw), then
    orally at a dose level of 29 mg/kg bw. Serum samples were collected
    from 3 animals at various time-points and analyzed by UV absorption
    for -isomer. Serum concentration-time AUC determinations were made
    for each route of administration. The systemic exposure to -isomer
    following oral administration was 9.6%. The elimination half-life was
    29 minutes following the i.v. dose (Pfizer, 1986).

         In a study to examine the toxicokinetics and metabolism in dogs,
    2 male and 2 female beagle dogs were administered an oral dose of
    14C--isomer of alitame followed by an unlabelled dose of 5 or
    50 mg/kg bw. Urine and faeces were collected for 96 h. The two male
    dogs excreted 35 and 39%, respectively, of the administered
    radioactivity in the urine, and the females 14 and 16%, respectively.
    Unchanged 13-isomer in urine accounted for 10-16% of the dose in all 4
    dogs. All of the urinary metabolites were identical to the ones found
    after administration of alitame, but the males had higher levels of
    metabolites than the females (Pfizer, 1986).

    2.2.11.2  Short-term toxicity studies

         In a 3-month study, groups of 15 male and 15 female mice (Strain
    Crl: COBS CD-1 (ICR) BR) were fed a diet containing the -isomer of
    alitame (CP-63,884) at dose levels of 0, 6.3, 12.5 or 25 mg/kg bw/day.
    Animals were observed throughout the study period and body weights and
    food consumption were recorded weekly. Urinary metabolites were
    analyzed at weeks 11 and 13. Gross pathological examination was
    performed on all animals and organ weights recorded. Tissues were
    taken from all of the major organs for histopathological examination.

         There was no treatment-related effect on body-weight gain
    although the average body weight of treated male animals was lower
    than in control groups. Food consumption was slightly decreased at
    high dose in males. One of the urinary metabolites identified was the
    N-acetyl derivative of the/%isomer. The amount of the isomer increased
    proportionally with the dose. There were no treatment-related effects
    on organ weights. Gross pathology was unremarkable and histopathology
    did not reveal any treatment-related changes. The NOEL in this study
    was >25 mg/kg bw/day (Stadnicki  et al., 1986a).

         In a 3-month study, groups of 15 male and 15 female Long-Evans
    rats (Strain Crl: (LE) BR) were fed a diet containing -isomer of
    alitame (CP-63884) at dose levels of 0, 6.3, 12.5 or 25 mg/kg bw/day.
    Animals were observed throughout the study period and body weights and
    food consumption data were recorded weekly. Ophthalmoscopic
    examinations were performed at pre-test and prior to necropsy. Blood
    samples were taken for examination of clinical chemistry and
    haematological parameters at pre-test and at 3, 9 and 13 weeks.
    Urinary analyses were performed at 3, 9 and 13 weeks. At the end of
    the study, gross pathological examinations were performed and tissues
    from the major organs taken for histopathology.

         There were no deaths through the study and the body-weight gains
    and food consumption were similar for treated and untreated groups.
    There were no treatment-related effects on clinical chemistry,
    haematology or urinary parameters. There were no differences between
    control and treated groups with respect to organs weights. Gross
    pathology and histopathology were unremarkable The NOEL in this study
    was > 25 mg/kg bw/day (Stadnicki  et al., 1986b).

    2.2.11.3  Special studies on embryotoxicity/teratogenicity

         A group of 88 albino female rats (strain Crl:COBS-CD (SD)BR) was
    inseminated and divided into 4 groups of 22 animals. The groups of
    presumed pregnant rats were administered the 3-isomer of alitame
    (CP-63,884) in distilled water by gavage at dose levels of 0, 25, 50
    or 100 mg/kg bw/day on days 6 to 15 post-insemination. Animals were
    killed on day 20 post-insemination. Fetuses were examined for external
    malformations, then half stained for skeletal anomalies and half
    examined for soft tissue defects.

         The pregnancy rate was 20/22 in the control and low-dose groups,
    and 19/22 in the intermediate- and high-dose groups. There were no
    deaths throughout the study, nor any clinical signs of toxicity. There
    were no treatment-related effects on body weight or food consumption
    of dams during or after the treatment period. The number of corpora
    lutea, implantation sites and viable fetuses was similar for all
    groups. With regard to fetal development, there was no difference
    between fetal weight of control and treated animals. Examination of
    the fetuses did not reveal any treatment-related increase in gross,
    skeletal or visceral malformations, nor any treatment-related effects
    on the degree of ossification. Under the conditions of this study, the
    -isomer of alitame showed no evidence of teratogenicity in rats
    (Kessedjian  et al., 1986).

    2.2.11.4  Special studies on genotoxicity

         The -isomer of alitame was tested for the induction of point
    mutations, both base-pair and frameshift, at the  his locus of
     Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100,
    with or without S9 microsomal activation. The purity of the alitame
    was not provided. Doses ranged from 20 to 10 000 g/plate, with or
    without activation. There was no increase in revertants above the
    control (water) in any of the treated plates at any dose level. The
    positive controls, 9-aminoacridine, nitrofurantoin, sodium nitrite,
    2-nitrofluorene and 2-anthramine gave appropriate responses (Ellis
     et al., 1991).

    2.3  Observations in humans

         In a 90-day double-blind tolerance study, groups of male and
    female subjects (77 treated, 80 controls) were administered either
    alitame in capsules divided over three meals at a dose level of
    10 mg/kg bw/day for 90 days, or placebo in the same manner. This dose
    was chosen because it is 10 times greater than the estimated daily
    intake if all sucrose in the diet were replaced with alitame. Subjects
    were assessed throughout the study period for side-effects (by
    interview and observation). Other tests included blood pressure,
    temperature, body weight, electrocardiogram, and ophthalmoscopic

    examination. Various clinical pathology parameters were also measured.
    Microsomal activation was estimated from the elimination kinetics of
    aminopyrine at pre-dosing and at weeks 6 and 13 of treatment. Plasma
    and urine samples were collected at various time points throughout the
    study.

         Four treated subjects and 2 control subjects discontinued from
    the study due to side effects (angioedema of the lips, maculopapular
    rash, urticaria (wheals) with erythema in treated subjects, and
    tinnitus, dry eyes and abdominal cramps in the control subjects). A
    rechallenge with alitame after 6 months produced none of the above
    symptoms, making the possibility of allergic reaction very unlikely.
    There were no treatment-related changes in blood pressure, pulse,
    temperature or body weight. Electrocardiograms and ophthalmological
    examinations were unremarkable. Haematological parameters were similar
    in treated and control subjects. Measurement of aminopyrine
    elimination rates at 6 and 13 weeks did not reveal any increase in
    hepatic microsomal enzyme activity due to alitame treatment (Gerber
     et al., 1987).

         In a 90-day double-blind tolerance study in diabetic subjects,
    groups of male and female subjects having either type I or type II
    diabetes (75 treated, 80 control) were administered either alitame in
    capsules divided over three meals at dose level of 10 mg/kg bw/day for
    90 days, or placebo in the same manner. This dose was chosen because
    it is ten times greater than the estimated daily intake if all sucrose
    in the diet were replaced with alitame. Subjects were included on the
    following criteria: diabetics of type I and type II, either sex and
    aged between 15 and 70 years with stable diabetic therapy; fasting
    blood glucose <250 mg/dL; free from investigational drugs; had not
    suffered a myocardial infarction within the previous 6 months; and had
    either no hypertension or it was controlled. Subjects were excluded on
    the following criteria: females which were pregnant or nursing; drug
    or alcohol dependence; systemic disease other than diabetes; clinical
    chemistry and haematological parameters above the upper limit of
    normal; hepatic disease; and donation of blood within 4 weeks prior to
    the study. The groups were well matched with respect to such
    parameters as age, sex distribution, weight, smoking, drinking,
    intercurrent illnesses and medication as well as diabetic status.

         Subjects were assessed at weeks 0, 1, 2, 3, 4, 6, 8, 10 and 13 of
    the study period for side effects (by interview and observation).
    Effects on diabetic control were assessed by measuring fasting
    haemoglobin A1c and 2-hour postprandial blood glucose levels at
    baseline and weeks 0, 6, 8 and 13. Physical examinations were
    conducted at screening and at week 13 of the study and included blood
    pressure, temperature, body weight and electrocardiogram. Clinical
    pathology parameters (haematology, clinical chemistry and urinary)
    were measured pre-treatment and at 13 weeks.

         At the end of the study period, 16 treated and 9 control subjects
    had discontinued from the programme and, of these, 5 treated and 2
    control subjects withdrew due to illness. No subjects discontinued due
    to side effects or abnormal laboratory parameters related to alitame
    treatment. Side-effects were observed in 29% of treated subjects and
    25% of control subjects. Gastrointestinal disturbances were the most
    common side-effect. Incidence and severity of particular side-effects
    were relatively evenly distributed between the groups. No significant
    changes were noted in either the control or treated groups with
    respect to diabetic control, clinical chemistry, haematological or
    urinary parameters. No treatment-related effects were apparent from
    the physical examinations, blood pressure and pulse rate, temperature,
    body weight or EKG recordings.

         Alitame at a dose of 10 mg/kg/bw/day for 90 days was well
    tolerated by the type I and type II diabetic patients who continued in
    this study. There was no effect of alitame at this dose level on the
    ability to control serum glucose levels by these diabetic patients
    (McMahon  et al., 1989).

         In a 1- and 2-year follow-up of the subjects in the 90-day study
    in diabetics, all but one of the treated subjects received an
    interview and complete physical examination. Subjects were interviewed
    again within 24 months post-study for all but 6 treated and 4 control
    subjects.

         A variety of intercurrent illnesses were reported during the
    follow-up period. Most of the reported illnesses related to diabetes
    and its complications. The incidence of intercurrent illnesses during
    the follow-up period was 40 treated and 36 control subjects. Two
    subjects died during the follow-up period, both treated subjects. One
    subject (type I diabetic) died during the month following the study,
    cause unknown. At completion of the study no adverse reactions had
    been noted in this subject. The second subject (type II diabetic)
    suffered a fatal heart attack resulting from hypertensive
    atherosclerotic cardiovascular disease.

         At the first follow-up (12 months), 5 treated subjects and no
    control subjects were found to have suffered myocardial infarction.
    The possibility that the results were due to chance was examined using
    available epidemiological data on the incidence of myocardial
    infarction. Data from the US National Hospital Discharge Survey (NHDS)
    were used to estimate the rate of myocardial infarctions among
    diabetics in various age groups for both sexes. These data were
    adjusted to take into account the number of myocardial infarction
    casualties which are not admitted to hospital and the actual patient
    years of observation. Based on the hospitalization data for diabetics
    who suffered myocardial infarction in 1986, the numbers of subjects
    expected to suffer a myocardial infarction in a single year were 2.9

    and 3.2 in the treated and control groups, respectively. The observed
    rates of 5% and 0%, respectively, were within the 95% tolerance
    intervals of incidence expected based on the NHDS analysis. Two years
    after the study, myocardial infarctions had been encountered by 6
    treated and 3 control subjects. The statistical analysis of these data
    at 2 years supports the null hypothesis that the incidence observed
    were due to chance. The data indicate that in the two years after the
    90-day study of alitame in diabetics, no evidence was provided that
    alitame adversely affected the health of the study groups (McMahon
     et al., 1989).

    3.  COMMENTS

         A number of toxicity studies on alitame were available, as well
    as studies on the -isomer of alitame, which is formed at low levels
    in some foods.

         In metabolism studies conducted in three animal species and in
    humans, alitame was readily absorbed from the GI tract and then
    rapidly metabolized and excreted. In mice, rats and dogs, the route of
    metabolism involved cleavage of the peptide bond yielding aspartic
    acid and D-alanine tetramethylthietane amide, the latter being further
    biotransformed by oxidation at the thietane sulfur to form the
    sulfoxide and sulfone. Acetylated derivatives of these metabolites
    were commonly found in the urine of rats and dogs whereas, in humans,
    the glucuronide derivative of D-alanine tetramethylthietane amide was
    the major urinary metabolite. In rats, initial cleavage of the peptide
    bond of alitame took place in the lumen of the jejunum. In pregnant
    rats, alitame was readily transferred transplacentally, but there was
    no evidence of active secretion in the milk. In a study in rats,
    levels of alitame residues decreased rapidly in all tissues except the
    eyes. Further studies indicated that it was bound to melanin contained
    in the uveal tract (choroid, ciliary body and iris) and suggested that
    such binding was associated with a metabolite formed only in rats. The
    Committee considered that an  in vivo study in a mammalian species
    other than the rat is desirable to clarify the significance of the
    retention of alitame in the eyes.

         The -isomer of alitame was rapidly metabolized in rats, the only
    urinary metabolite not also formed with alitame being the N-acetyl
    derivative of this isomer. There was no evidence of the formation of
    significant amounts of the -isomer of alitame in the stomach or small
    intestine of the rat.

         Acute and short-term toxicity studies indicated that the toxicity
    of alitame is low. In studies conducted in mice, rats and dogs, the
    major toxicological changes observed were associated with the liver. A
    dose-related increase in liver weight and evidence of centrilobular
    hypertrophy were seen in all of the studies in these three species,
    and these changes were accompanied by an increase in the level of
    hepatic microsomal enzymes. Short-term studies on hepatic enzyme
    induction indicated that alitame was a weak inducer of microsomal
    enzymes; the pattern of induction was qualitatively similar to that
    caused by phenobarbitone but at a lower level. The liver changes were
    indicative of an adaptive response and, in rats and dogs, withdrawal
    of treatment after a 3-month period resulted in partial or complete
    reversal of the observed changes within a month.

         Two-year carcinogenicity studies have been conducted in CD-1
    mice, Long-Evans rats and Sprague-Dawley rats. The two studies
    conducted in rats included an  in utero phase. There was no evidence
    of carcinogenicity in the study in mice.

         In rats, the first carcinogenicity study was conducted in
    Long-Evans rats at dietary levels of 0.1, 0.3 or 1% and, while the
    survival rate was greater than 50% in all groups at 22 months, poor
    survival was noted at 24 months. Pathological examination of the liver
    indicated a higher incidence of focal nodular hyperplasia and
    eosinophilic foci in females at the 1% dose level than in controls,
    according to the diagnostic criteria of Squire & Levitt (1975). Two
    subsequent independent re-examinations of these data have been
    performed using the diagnostic criteria of Maronpot  et al. (1986).
    In the first re-examination (Farrell, 1987), the higher incidence of
    eosinophilic foci in females in the high-dose group was confirmed, but
    the incidence of focal hyperplasia or hepatocellular adenomas was
    similar to that in controls. In the second re-examination (Hardisty
     et al., 1994), the higher incidence of eosinophilic foci in females
    in the high-dose group was again confirmed. A higher incidence of
    these foci was also found at the mid-dose level. Also in this
    re-examination, the nodular hyperplastic lesions noted in the original
    report were largely reclassified as hepatocellular adenomas, and an
    increased incidence of these lesions was seen at the high-dose level.
    No evidence of hepatocellular carcinomas was found in female rats in
    the original examination or in the subsequent re-examinations. The
    main difference between the first and second re-examinations was in
    the nomenclature used, rather than in the recognition that lesions
    existed. The Committee expressed concern at the change in the way that
    these liver lesions were classified, possibly as a result of the use
    of different diagnostic criteria. It considered that there was
    evidence of a higher incidence of adenomas in females in this study,
    at least at the high dose level. The adenomas were found late in the
    study and, while considered unlikely to progress to hepatocellular
    carcinomas, the data were inconclusive in this regard.

         A second carcinogenicity study, this time in Sprague-Dawley rats,
    was conducted because of the low survival rate at 24 months in the
    Long-Evans rats. In this second study, the survival rate was lower at
    both 22 and 24 months than that found in the Long-Evans rats, and
    hence the study was considered inadequate to provide further
    information regarding the potential carcinogenicity of alitame.

         The Committee considered that the available studies did not
    indicate that alitame was carcinogenic, but that they were deficient
    in certain respects and did not fully address this question. It also
    considered that further research to clarify the mechanism of the
    formation of adenomas in female Long-Evans rats was desirable.

         Genotoxicity studies both  in vitro and  in vivo did not
    provide any evidence that alitame has a mutagenic potential.
    Similarly, there was no evidence from studies conducted in rats and
    rabbits that alitame has a teratogenic potential.

         Two reproductive toxicity studies (2-generation) were conducted
    in Long-Evans rats and one reproductive toxicity study (1-generation)
    in Sprague-Dawley rats. The first reproductive toxicity study with
    Long-Evans rats produced some evidence for slightly decreased
    body-weight gain in the pups during lactation at 1% in the diet, the
    highest dose administered. A similar effect was noted in the second
    reproductive toxicity study at the same dose level, but not in the
    1-generation reproductive toxicity study with Sprague-Dawley rats. A
    study of the effects of alitame-containing diet in the bedding
    material, which could have been a source of alitame for pups due to
    spilled feed, while providing a possible explanation for the decrease
    in pup body-weight gain, did not fully address this issue. A slight
    effect of alitame on locomotor activity seen in the first reproductive
    toxicity study was not substantiated in subsequent studies. The
    Committee did not regard the changes seen in the reproductive toxicity
    studies to be of toxicological significance.

         Neurotoxicity and neurobehavioural toxicity studies using an
    observational battery of tests indicated behavioural changes only when
    very high doses were administered to rats as a single dose by gavage
    (5000 mg/kg bw). These behavioural changes were of short duration and
    animals appeared to be normal by day 7. There was no evidence of
    neurotoxicity in rats fed diets containing 1% alitame (equivalent to
    1000 mg/kg bw/day) over a 3-month period. Similarly, no changes
    indicative of neurotoxicity were observed in dams or pups when the
    same dose level was given over the reproductive period.

         In a 14-day study conducted in humans there was no evidence of
    hepatic enzyme induction at a dose level of 15 mg/kg bw/day.

         Ninety-day tolerance studies were conducted in normal and
    diabetic subjects. There was no evidence of adverse effects in
    subjects of both types during the period of the study at a dose level
    of 10 mg/kg bw/day. However, in the 1- and 2-year follow-up of the
    diabetic subjects, there was a higher incidence of myocardial
    infarction in the treatment group than in the controls, namely 5/58 in
    the treatment group and 0/71 in the controls after 1 year, and 6/53 in
    the treatment group and 3/67 in the controls after 2 years. While the
    differences at 2 years were not statistically significant, and there
    was no indication of cardiovascular effects in the animal studies, the
    Committee considered that the increased incidence of myocardial
    infarction in the treatment group has not been fully explained. It was
    advised that a further study is to be performed in diabetic subjects.

         Studies conducted with the -isomer of alitame provided no
    evidence of genotoxicity, teratogenicity or systemic toxicity in mice
    or rats at dose levels of up to 25 mg/kg bw/day.

    4.  EVALUATION

         The Committee recognized that the basis on which to consider
    establishing an ADI for a chemical such as alitame is difficult to
    specify, since the most sensitive treatment-related effects observed
    were liver enzyme changes and liver weight changes, which may be
    adaptive in nature. For alitame, these changes appeared to be closely
    linked. While the prolonged hepatomegaly observed was not accompanied
    in the long-term studies by any pathological changes indicative of
    toxicity, it is not considered desirable. On the other hand, moderate
    induction of hepatic enzymes is considered a normal adaptive process.
    In the case of alitame, significant changes in body-weight gain or
    liver weight were observed at doses above the NOELs. In the 18-month
    study in dogs, the NOEL was 100 mg/kg bw/day. In the 2-year study in
    mice, the NOEL was 0.1% in the diet, equal to 125 mg/kg bw/day. In the
    lifetime studies in rats, the NOEL was 0.3% in the diet, equal to
    130 mg/kg bw/day in Long-Evans rats and 230 mg/kg bw/day in
    Sprague-Dawley rats.

         The Committee concluded that the concerns raised by the
    deficiencies in the carcinogenicity studies in rats were of such
    significance that an ADI could not be allocated.

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    See Also:
       Toxicological Abbreviations
       Alitame (JECFA Food Additives Series 50)
       Alitame (WHO Food Additives Series 37)
       ALITAME (JECFA Evaluation)