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    TRICHOROGALACTOSUCROSE

    EXPLANATION

         TGS has not previously been evaluated by JECFA.
    Trichlorogalactosucrose (1,6-dichloro-1,6-dideoxy-beta-D-
    fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside) (TGS),
    is derived from sucrose by selective replacement of the three
    hydroxyl groups at positions 4, 1' and 6' by chlorine atoms which
    greatly increases sweetness. At room temperature in water TGS has
    a sweetness potency of approximately 600-650 relative to sucrose at
    a concentration of 4-5%.

         In acid solution, TGS hydrolyses slowly to its constituent
    monosaccharides, 4-chlorogalactose (4-CG) and 1,6-dichlorofructose
    (1,6-DCF). This process is influenced by temperature and pH.

    BIOLOGICAL DATA

    Biochemical aspects

    Absorption, distribution, and excretion

         After intravenous administration of 14C-TGS (20 mg/kg) to
    mice, the dose was excreted rapidly and mainly in the urine. In
    three of the four pairs of animals, a total of over 80% of the dose
    was excreted in the urine during five days, with the remaining dose
    excreted in the faeces. Overall, these data indicated that about
    10%-20% of the dose was probably excreted via the bile in the
    faeces.

         After oral administration of 14C-TGS (100 mg/kg b.w.) to mice
    the dose was excreted mainly in the faeces. Profiles of excretion
    were similar in both sexes with totals of 60%-75% of the dose
    excreted in faeces and 20%-25% excreted in urine during 5 days. The
    total excreted in urine represents a minimum for the extent of
    absorption of the dose as it does not take account of the
    proportion of the absorbed dose excreted via the bile. Less than
    0.5% of the dose was excreted in expired air.

         After oral administration of 14C-TGS to mice at dose levels
    of 1500 mg/kg b.w. and 3000 mg/kg b.w., the profiles of excretion
    in faeces and urine were very similar to those found at the
    100 mg/kg b.w. dose level (Hawkins et al., 1987c).

         Rats given a single oral dose of TGS labelled with either
    chlorine-36 (100 or 1000 mg/kg b.w.) or carbon-14 (10 or 50 mg/kg)
    excreted an average of 8% (range 3 to 22%) of the dose in the urine
    primarily over the first 24 hours and the remainder in the faeces
    within 3 to 5 days. No 14CO2 was detected in the expired air.

         When 36Cl-TGS was administered to rats by intravenous
    injection (20 mg/kg), approximately 9% of the radioactivity was
    eliminated in the faeces and 83% in the urine in 48 hours.

         The amount of radioactivity excreted in the rat bile following
    the oral administration of TGS (50 or 100 mg/kg) labelled with
    chlorine-36 ranged from 0.8 to 8.9% of the dose.

         36Cl-TGS (5 uCi, 100 mg/kg) was administered by intravenous
    injection to rats. The distribution of radioactivity 15, 30, 60 and
    360 min following the injection indicated that TGS was located
    principally in the liver, blood, kidney and small intestine at
    15 min, with some associated with intercostal and intervertebral
    cartilage. There was no evidence for uptake into the central
    nervous system at

    any time after the injection The distribution was qualitatively
    similar at 30 and 60 mins. The concentration in the individual
    organs other than the large intestine was reduced substantially 6
    hours after injection.

         36Cl-TGS (100 mg/kg 25 uCi) was administered by gastric
    intubation to groups of male and day 16 pregnant rats. Two animals
    of each sex were sacrificed 30 and 60 minutes and 2, 4, 6, 7.5, 24
    and 48 hours after dosing. Most of the radioactivity at each
    interval was located in the lumen of the small and/or large
    intestine. Maximum plasma levels in both sexes were achieved within
    one hour of dosing. The levels of radioactivity in all other
    tissues, with the exception of the liver and the kidney, were
    uniformly low.

         Radioactivity present in fetuses, ovaries, placentae, uterus
    and amniotic fluid of pregnant female rats were always below the
    plasma levels (Daniel, 1987a).

         Six lactating female rats were given 36Cl-TGS (100 mg/kg) by
    oral gavage on the 16th day after parturition. Analysis of milk
    indicated levels of radioactivity of 0.06, 0.06, 0.04, 0.13, 0.17,
    0.11 and 0.08 g equivalents/0.1 ml at 0.5, 1, 1.5, 2, 3, 4, and 24
    hours, respectively. Only the values for 2, 3 and 4 hours are
    considered significant, the remainder being at the limit of
    detection. An average of 7.5% (range 4.6 to 9.5%) of the dose was
    excreted in the urine in 48 hours of which approximately 10% was
    attributed to inorganic chloride (Daniel, 1987a).

         14C-TGS was given orally and intravenously to adult male and
    female rats (10 mg/kg b.w. and 2 mg/kg b.w., approximately 1
    uCi/mg, respectively) and urine and faeces were collected up to 4
    days after the dose.

         The total recovery of 14C for the oral dose was 97.5% (range
    92.4 - 101.4%) with most of the radioactivity (92.6; range
    87.6 - 95.4%) in the faeces.  The remainder was eliminated in the
    urine (5.29; range 4.69 - 5.94) and cage washes (1.2%).  The
    excretion of 14C was rapid with a mean of 86.2% of the dose being
    eliminated in the first 24 h.

         The total recovery of 14C for the intravenous dose was 97.4%
    (range 90.7 - 102.0%) with most of the radioactivity (76.5%; range
    67.7 - 82.2%) in the urine with the remainder occurring in the
    faeces (16.1%, range 14.5 - 17.4) and cage washes (4.8%). Similar
    to the oral dose, excretion was rapid with a mean of 89.5% of the
    dose being eliminated in the first 24 h (Roberts et al., 1987).

         The metabolism and excretion of TGS have been studied in
    non-pregnant female rabbits and pregnant rabbits after oral doses
    of the 14C-labelled compound. After single oral doses of 14C-TGS to
    non-pregnant and pregnant rabbits at a dose level of 10 mg/kg,
    radioactivity was excreted mainly in the faeces. During 24 hours
    after dosing, a mean of 16.8% of the dose was excreted in the
    faeces of non-pregnant animals, increasing to 31.8% during
    48 hours and 54.7% during 120 hours. Excretion of radioactivity
    in the faeces of pregnant rabbits was similar, with means of 27.8%,
    43.0% and 65.2% of the dose excreted by this route during 24, 48
    and 120 hours after dosing, respectively. Means of 5.3% and 4.2%
    close were excreted in the faeces of non-pregnant and pregnant
    rabbits respectively during 96-120 hours after dosing, indicating
    that excretion of radioactivity was not completed after 5 days,
    probably because of the copraphagic behaviour of rabbits. During
    24 hours, means of 8.3% and 8.6% of the dose were excreted in the
    urine of non-pregnant and pregnant rabbits, respectively. Mean
    totals of 22.3% (non-pregnant rabbits) and 21.5% (pregnant rabbits)
    of the dose was gradually excreted in the urine during 5 days after
    dosing. Radioactivity was still being excreted in the urine of
    rabbits (up to 2.9% dose) during 96-120 hours after dosing. Mean
    total recoveries of radioactivity from the urine and faeces of non-
    pregnant and pregnant rabbits after 5 days accounted for 80.3% and
    87.0% of the dose respectively. The dose not accounted for was
    presumably still to be excreted since a total of up to 8.4% of the
    dose was excreted during 96-120 hours after dosing. There were no
    notable differences in the absorption and excretion of single oral
    doses of 14C-TGS between non-pregnant and pregnant rabbits (Hawkins
    et al., 1987b).

         The pharmacokinetics and metabolism of TGS have been studied
    in the dog after oral or bolus intravenous doses of the
    14C-labelled compound. After single intravenous doses of 14C-TGS to
    dogs at a dose level of 2 mg/kg (5.8 uCi/kg) radioactivity was
    rapidly excreted mainly in the urine. Urinary excretion accounted
    for means of 29.3%, 63.9% and 74.1% of the dose during 3, 6 and 12
    hours after dosing respectively, increasing to 80.9% of the dose
    after 5 days. Faecal excretion accounted for a mean of 10.4% dose
    after 24 hours, increasing to 11.9% dose after 5 days. Plasma
    radioactivity was maximal at 5 minutes after dosing (the first time
    of sampling, 8.46 g equivalents/ml). Radioactivity in plasma
    declined in a multi-exponential fashion; concentrations decreased
    rapidly to a mean of 0.057 g equivalents/ml at 12 hours after dosing
    but thereafter declined more slowly, and were still detectable in all
    animals at 120 hours after dosing (mean, 0.013 g equivalents/ml).
    Consideration of whole-blood and plasma concentrations indicated
    that radioactivity was cleared more slowly from blood cells than
    from plasma (Hawkins et al., 1986).

         After single oral doses of 14C-TGS to dogs at a dose level of
    10 mg/kg (5.2 uCi/kg) radioactivity was excreted mainly in the
    faeces. Faecal excretion accounted for a mean of 65.9% of the dose
    during 24 hours increasing to 68.4% dose after 5 days. Excretion of
    radioactivity in the urine accounted for means of 13.8%, 22.3% and
    26.5% of the dose during 6, 12 and 24 hours after dosing
    respectively, increasing to 27.6% of the dose after 5 days.
    Concentrations of plasma radioactivity were maximal at 1 hour.
    After the time of maximal concentrations, radioactivity declined in
    an apparently multi-exponential fashion to a mean of 0.242 g
    equivalents/ml after 12 hours and after 96 hours were near or below
    the limit of accurate determination in all animals. Comparison of
    the mean areas under the plasma concentration-time curves after
    oral or intravenous doses (adjusted for dose level) indicated that
    about 35% of the oral dose was absorbed (Hawkins et al., 1986).

         Three male subjects given a single oral dose (1.11 mg/kg b.w.,
    0.3 uCi/kg) of TGS uniformly labelled with carbon-14 excreted an
    average of 13.5% of the radioactivity in urine and 82.1% in faeces
    in 5 days. No 14CO2 was detected in expired air collected during
    the initial 8 hours after dosing. Maximum levels of radioactivity
    in the blood occurred within 2-3 hours and in two of the subjects
    declined with a half-life of approximately 2.5 hours.
    Chromatographic examination of the 0-3 hours urines indicated the
    presence of only a single radioactive component (Shepard & Rhenius,
    1983).

         14C-TGS (1 mg/kg; 100 uCi > 98% pure) was given orally
    dissolved in water to 8 normal, healthy male volunteers and blood,
    urine and faeces collected for up to 5 days after the dose. The
    total recovery of 14C-activity was 92.7% (range 87.8-99.2%) with
    most of the radioactivity 78.3% (range 69.4-89.6%) in the faeces,
    and the remainder 14.4% (range 8.8-21.7%) in the urine.

         The plasma concentrations of 14C-activity reached a peak at
    about 2h after the dose, with levels of 14C equivalent to
    approximately 250 ng/ml of TGS. The plasma concentrations fell
    rapidly between 2 and 12h followed by a more gradual decrease until
    72h by which time the levels of radioactivity were near or below
    the limit of accurate determination. The mean 'effective half-life'
    calculated on the basis of a mean residence time (MRT) of 18.8h
    gives a value of 13.0h (Roberts et al., 1986).

    Biotransformation

         Profiles of radioactivity in urine and faeces of mice given a
    single intravenous dose (20 mg/kg) were examined by quantitative
    thin-layer chromatography. Unchanged 14C-TGS was the major
    radioactive component in both urine and faeces in both sexes,
    generally accounting for more than 90% of the urinary
    radioactivity, about 95% of the faecal radioactivity in males and

    about 90% of the faecal radioactivity in females. Remaining
    radioactivity was associated with several very minor components,
    including chromatographically polar material bound to the origin of
    the TLC plate and two components (A and B), chromatographically more
    polar than TGS. Component B was more notable in the urine of female
    mice.

         Profiles of radioactivity in mouse urine after oral
    administration at three dose levels (100 mg/kg, 1500 mg/kg and
    3000 mg/kg) were similar, showing that unchanged compound
    represented 80%-90% of sample radioactivity in both sexes. The same
    minor radioactive components observed after intravenous dosing
    accounted for the remaining sample radioactivity. Virtually all
    faecal radioactivity was accounted for by unchanged TGS (92%-99%).
    Small amounts of component A were detected at dose levels of 1500
    and 3000 mg/kg and component B at 3000 mg/kg only.

         The extent of metabolism was limited, generally representing,
    in total, less than 5% after oral administration and 10% after
    intravenous administration. Two metabolites were identified in both
    urine and faeces. One component had the same chromatographic
    characteristics as n minor metabolite of TGS present in human urine
    and previously identified as a glucuronic acid conjugate of TGS
    (Hawkins et al., 1987c).

         Chromatographic analysis of rat urine following oral
    administration of 14C-TGS (10 mg/kg b.w. 1 uCi/mg) revealed the
    presence of small amounts of radiolabelled metabolites in addition
    to TGS. The non-TGS material, which represented a mean of 0.5 +/-
    0.3% of the dose (in 0 - 24h urines) was resolved into two separate
    components for both males and females in an ammonia containing TLC
    solvent. The major radioactive component in urine corresponded to
    unchanged TGS.

         Chromatographic analysis of rat urine following intravenous
    administration of 14C-TGS (2 mg/kg b.w.; 1 uCi/mg) again revealed
    the presence of small amounts of radiolabelled metabolites in
    addition to TGS. The non-TGS material, which represented a mean of
    1.8 +/- 0,9% of the dose (in 0 - 24h urines) was resolved into one
    or two separate components for both males and females in an
    ammonia-containing TLC solvent.

         Chromatographic analysis of the faeces from animals given an
    intravenous dose showed that all samples contained only a single
    peak of 14C activity which corresponded to TGS (Roberts et al.,
    1987).

         A single oral dose of TGS (100 mg/kg, 10 uCi), uniformly
    labelled with carbon-14, was administered to male and female rats
    that had been maintained for 26, 52, 83 and 85-87 weeks on diet
    containing 3% TGS or  control diet. Urine and faeces were analysed 

    for radioactivity at each period, expired air was analysed for
    14C02 during week 83. Urine was analysed by thin layer
    chromatography at 26, 52, 85-87 weeks, as were faeces obtained
    during week 85-87.

         Approximately 7% of the radioactivity was excreted in the
    urine and 80% in the faeces. There was no evidence for the
    production of 14C02. Chromatography of urine at weeks 26 and 52
    revealed a single radioactive component. Examination of urine and
    faeces from weeks 85/87 indicated that 97% of the radioactivity in
    the 0-48 hour samples was TGS. Evidence for TGS absorption from the
    diet was provided by quantitative chemical analysis of TGS in urine
    from animals previously exposed to control diet or diet containing
    TGS. A single dose of radiolabelled TGS was given by gavage
    following overnight fasting and approximately 4 hours prior to
    reinstatement of control diet or diet containing non-labelled TGS.
    TGS concentrations in urine from animals receiving dietary TGS were
    greater than in controls and greater than could be inferred from
    radiochemical analysis, the increase being accounted for by TGS
    absorbed from the diet.

         It is concluded that an oral dose of TGS is eliminated
    essentially unchanged in urine and faeces. No evidence for
    metabolic adaptation to TGS in male or female rats was found in
    animals following chronic dietary treatment at 3% for more than 18
    months (Rhenius et al., 1986).

         Chromatographic analysis of radioactivity present in urine of
    pregnant and non-pregnant rabbits following oral administration of
    14C-TGS (10 mg/kg, 11 uCi/kg) indicated that 14C-TGS was mainly
    excreted unchanged and accounted for about 70-80% of total
    radioactivity in most urine samples. However, in some early samples
    (0-6 hour) unchanged 14C-TGS accounted for only about half of total
    sample radioactivity. Urinary radioactivity that was not associated
    with unchanged 14C-TGS appeared to be chromatographically more
    polar in nature and was associated with a small number of minor
    components (Hawkins et al., 1987b).

         Chromatographic analysis of radioactivity in dog urine
    indicated that unchanged TGS was the major component after either
    oral (10 mg/kg, 5.2 uCi/kg) or intravenous (2 mg/kg, 5.8 uCi/kg)
    dosing. After oral dosing, unchanged TGS accounted for 53-79% of
    sample radioactivity. The remaining radioactivity in these samples
    was apparently mainly associated with one component (Component A)
    which accounted for about 15% of 0-3-hour urine sample with
    radioactivity increasing to about 24% of 3-6 and 6-12 hour urine
    sample radioactivity. After intravenous dosing approximately 90% of
    0-3 hour urine sample radioactivity was associated with unchanged
    TGS decreasing to 54-74% and 54-68% of 3-6 and 6-12 hour urine
    sample radioactivity, respectively. Component A was the only
    notable metabolite in urine from intravenously dosed animals,
   

    accounting for 2-13% of 0-3 hour urine sample radioactivity and
    21-42% of 3-6 and 6-12 hour urine sample radioactivity. This
    component accounted for about 15-20% of the intravenous dose and
    for about 2-8% of the oral dose. Radioactivity in faeces after
    oral dosing was associated almost entirely with unchanged TGS and
    component A was not detected (Hawkins et al., 1986).

         The major urinary metabolite of TGS in the dog has been
    isolated and purified by thin-layer chromatography and high
    pressure liquid chromatography and identified, using mass
    spectrometry, as a glucuronic acid conjugate of TGS. Mass spectral
    evidence indicated that the glucuronic acid was placed on the 
    4-chloro-4-deoxygalactopyranosyl moiety of although the precise
    position of conjugation remains ambiguous. It is possible that TGS
    could be conjugated at the 2,3- or 6- carbon position (Hawkins et
    al., 1987a).

         Chromatographic analysis of radioactivity in urine from eight
    human volunteers given a single oral close of 14C-TGS (1 mg/kg;
    100uCi >98% pure) indicated the presence of radiolabelled
    metabolites in addition to TGS. The non-TGS material, which
    represented a mean of 2.6% of the dose, was resolved into two
    separate components. The major radioactive component in urine was
    shown to be TGS by its TLC characteristics and confirmed by gas
    chromatography-mass spectrometry. Chromatographic analysis of the
    faeces showed that nearly all samples contained only a single peak
    of 14C-activity corresponding to TGS. A small number of samples
    contained an additional minor peak of radioactivity which accounted
    for 1% or less of the dose in all subjects (Roberts et al.,
    1986).

         14C-TGS (10 mg/kg; 20uCi; >99% pure) was dissolved in water
    and given orally to two normal, healthy male volunteers and urine
    and faeces were collected up to five days after the dose. The total
    recovery of 14C for subjects 1 and 2, respectively, was 96.8 and
    96.4% with 84.1 and 86.8% in the faeces, and 12.7 and 9.6% in the
    urine. The excretion of 14C in urine was rapid with 11.1 and 8.3%
    of the dose being eliminated in the first 24h. Chromatographic
    analysis of urine revealed the presence of small amounts of
    radiolabelled metabolites in addition to TGS. The non-TGS material
    which represented less than 2% of the dose (0-12h, XAD-2 resin
    column chromatography concentrated urines) was resolved by thin
    layer chromatographs into two separate components for both
    subjects. The major radioactive component in urine corresponded to
    unchanged TGS. Isolation and partial purification of the unknown,
    more polar metabolite (MI) was achieved by use of XAD-2 resin and
    HPLC. Incubation of XAD-2 concentrates of 0-3 hour urine samples
    with beta-glucuronidase and beta-glucuronidase/sulphatase mixtures


    followed by thin layer chromatography, showed that the metabolite
    MI was completely hydrolysed. Sulphatase treatment alone failed to
    hydrolyse MI. These data suggest that MI is a glucuronide conjugate
    of TGS which is hydrolysed by glucuronidase (Roberts et al., 1988).

    Special studies on enzymes and other biochemical parameters

         TGS has been tested as substrate for seven microbial and plant
    glycosidases and two mammalian intestinal extracts which contain a
    range of glycosidases: alpha-galactosidase and amyloglucosidase
    from Aspergillus niger, yeast alpha-glucosidase, almond beta-
    glucosidase, beta-galactosidase from Escherichia coli, invertase
    from bakers yeast and Candida utilis, acetone extracts of porcine
    and calf-intestine. In all cases, TGS was not hydrolysed by any of
    the enzymes tested (Rodgers et al., 1986).

         TGS had no effect on the utilisation of either glucose or
    lactate when added at a concentration of 5 mM to preparations of
    rat brain, liver, kidney, diaphragm or ileum or when administered
    to rats by intraperitoneal injection at a dose of 500 mg/kg. TGS at
    different concentrations (100, 500 and 1000 mg/kg) is not
    insulinotropic in rats, introduced by intravenous, intraperitoneal
    or by gastric feeding, and is without effect on the respiratory and
    phosphorylating activities of hepatic and renal mitochondria at
    concentration of 5 and 20 mM.

         It is concluded that TGS does not exert any influence on the
    regulation of carbohydrate metabolism in the rat (Das et al.,
    1979).

         Four groups of ten male rats of CD strain had been treated for
    28 days by oral gavage as follows: Group 1 - Water;, Group 2 -
    6-chloro-6-deoxyglucose (6-CG), 24 mg/kg/day; Group 3 - TGS
    500 mg/kg/day; Group 4 6,1',6'-trichloro-6,1',6'-trideoxysucrose
    (TCDS), 100 mg/kg/day. Animals were killed on day 29 and sperm
    collected from the cauda epididymides. Similar numbers of
    spermatozoa were recovered from the rats in each group. The
    spermatozoa were incubated with 14C-glucose for 2 hours.
    Spermatozoa from Group 1 (water), Group 3 (TGS) and Group 4 (TCDS)
    produced similar amounts of 14CO2 (73.5 +/- 19.28, 79.4 +/-
    56.17 and 83.6 +/- 42.16 nmol glucose converted to CO2/108
    sperm/2h, respectively, mean +/- SD), whereas less (2.3 +/- 0.78)
    was produced by spermatozoa from Group 2 (6-CG). The concentration
    of ATP at the end of the experiment was similar in spermatozoa from
    Groups 1 (water), 3 (TGS) and 4 (TCDS) (28.8 +/- 14.79, 29.9 +/-
    21.9 and 39.9 +/- 18.62 nmol/108 spermatozoa respectively, mean
    +/-SD) but appreciably less in spermatozoa from Group 2 (6-CG)
    (4.2 +/- 4.24).

         The treatment of rats by gavage for 28 days with 1,6-dichloro-
    1,6-dideoxy-beta-D-fructofuranosyl-4-chloro-4-deoxy-D-
    galactopyranoside (TGS) at 500 mg/kg/day or with 6,1',6'-trichloro-
    6,1',6'-trideoxysucrose (TCDS) at 100 mg/kg/day had no effect upon
    the ability of their spermatozoa to oxidise glucose or on the
    concentration of ATP. This contrasted with the inhibitory effect of
    6-chloro-6-deoxyglucose at 24 mg/kg/day on both parameters (Ford,
    1986).

         TGS was administered to groups of rats (5 male and 5 female)
    by oral gavage on each of 21 consecutive days at dose levels of
    either 500 or 1500 mg/kg/day. Control animals received an
    equivalent volume of vehicle (distilled water) daily during the
    same treatment period. A positive control group was administered by
    interperitoneal injection a single dose of Aroclor 1254 five days
    before sacrifice (500 mg/kg). Twenty-four hours after the final
    dose, the rats were sacrificed and the livers immediately removed
    and weighed. Samples of the livers were taken for microsome and
    cytosolic fraction preparation. The following hepatic parameters
    were measured; microsomal and cytosolic protein, cytochrome P450,
    7-ethoxyresorufin-O-deethylase activity (cytochrome P448 mediated),
    p-nitrophenol-glucuronyl transferase activity and glutathione-S-
    transferase activity. Aroclor 1254 treatment produced statistically
    significant increases in liver weights and all the hepatic
    parameters measured. TGS treatment had no inducing effect on liver
    weight, microsomal protein, cytochrome P450, 7-ethoxyresorufin-O-
    deethylase activity, p-nitrophenol-glucuronyl transferase activity.
    Cytochrome P450 levels were reduced compared to the control group
    in female rats receiving 1500 mg/kg/day TGS although the levels
    were within the historic control range for this species and sex
    (Hawkins et al., 1987d).

    Toxicological studies

    Acute toxicity

                                                                        

    Species   Sex          Route    LD50            References
                                    (mg/kg b.w.)
                                                                        

    Mouse     Not          oral     > 16,000        Lightowler & Gardner,
              specified                             1977

    Rat       Male         oral     > 10,000        Campbell & Johnson,
                                                    1980
                                                                        

        Results of mutagenicity assays on TGS
                                                                                              

    Test system        Test object           Concentration         Results         Reference
                                             of TGS
                                                                                              

    Ames test (1)      S.typhimurium         16 - 10,000           Negative        Bootman &
                       TA98, TA100           g/plate                              May, 1981
                       TA1535, TA1537
                       TA1538

    Ames test (1)      S.typhimurium         0.5 - 1000            Negative        Jagannath &
                       TA98, TA100           g/plate                              Goode, 1979

    DNA repair         E. Coli               0.5 - 1000            Negative        Jagannath &
    test (1)           W3110 (pol A+)        g/plate                              Goode, 1979
                       p3478 (pol A-)

    Gene mutation      Mouse lymphoma        1,335 - 10,000        Positive        Kirby
    (1)                frwd. mutation        g/ml                 (2)             et al.,
                       L5178Y TK+/-                                                1981a

    Chromosome         Human periferal       8 - 200               Negative        Bootman &
    aberrations        lymphoctes            g/ml                                 Rees, 1981
    (3)

    Chromosome         Rat, os               5  2000 mg/kg        Negative        Cimino &
    aberrations        bone marrow                                                 Lebowitz,
                       (in vivo)                                                   1981a

    Micronucleus       Mouse, os             1000 - 5000           Negative        Bootman
    test               (in vivo)             mg/kg                                 et al.,
                                             24, 48, 72 h                          1986
                                                                                              

    (1)  Both with and without rat liver S-9 fraction.
    (2)  TGS at a concentration of 10,000 g/ml induced approximately a three fold increase
         in the mutant frequency of the nonactivated culture with a cell viability of 35%.
         S-9 containing cultures treated at the two highest doses (10,000 g/ml and
         7,500 g/ml) also exhibited mutant frequencies which were greater than twice
         background with cell viabilities of 18% and 35%, respectively.
    (3)  TGS was not tested with rat liver S-9 fraction.
    
    Special studies on carcinogenicity

    Mouse

         Groups of 52 male and 52 female mice of the CD-1 strain received
    TGS, continuously via the diet, at concentrations of 0, 3,000, 10,000
    and 30,000 ppm. A group of 72 male and 72 females mice received diet
    devoid of TGS and served to generate contemporaneous control data.

         No adverse effect of treatment with TGS on survival was noted.
    Survival at 78 weeks of treatment was at least 74% for each group of
    the males and 87% for each group of the females, and at termination
    after 104 weeks of treatment at least 35% in the males and 58% in the
    females. Body weight gain in both male and female mice receiving TGS
    at 30,000 ppm in diet was significantly lower than that of controls
    throughout the treatment period. Marginally lower body weight gain was
    recorded notably during the first 78 weeks of treatment for female
    mice receiving 10,000 ppm although this was not statistically
    significant. Exposure to 3,000 ppm had no influence on weight gain in
    either sex. Food consumption, after correction for the concentration
    of TGS in the diet, was marginally lower for female mice receiving
    30,000 ppm than for their controls. Efficiency of food conversion,
    after correction for the amount of TGS in the diet, tended to be
    marginally lower for mice receiving 30,000 ppm than for their
    respective controls.

         Measurement of water consumption over a 24-hour period each week,
    for the first 13 weeks, revealed that the intake for male mice
    receiving TGS at 30,000 ppm was slightly higher than that for
    controls. Water consumption for all other groups was essentially
    similar to that of their controls.

         Haematological investigation after 104 weeks of treatment
    revealed marginally lower erythrocyte counts in mice receiving TGS at
    30,000 or 10,000 ppm. Differences only achieved statistical
    significance (P < 0.05) in female mice receiving 30,000 ppm; none was
    considered to be of biological significance.

         Analysis of organ weights, using terminal body weight as the
    covariate, revealed a possible association between treatment with TGS
    and elevation of liver weight in female mice only. No difference in
    absolute liver weight, relative to controls, was observed and there
    were no treatment-related histopathological findings in the liver of
    male or female mice to corroborate the increased relative liver weight
    in female mice.

         The higher incidences of degeneration of testicular tubular
    germinal epithelium in mice treated at 30,000 and 3,000 ppm and of
    testicular tubular mineralisation at 30,000 ppm, although 
    statistically significant, were without apparent trend with dosage or

    trend in increasing severity with dosage. The intergroup differences
    noted are considered to be the result of chance distribution for this
    common but variable geriatric lesion and are not considered to be
    related to treatment.

         There was no increase in the incidence of any tumour in mice
    receiving TGS at any concentration that was considered to be
    treatment-related (Amyes et al., 1986a).

    Special study on mineral utilization

    Rats

         Dietary level of TGS 0, 10,000, 20,000, 40,000, and 80,000 ppm
    were fed to the animals for 59 days. During this time, the health
    status, body weight, food and water consumption and urine and faeces
    output were monitored daily with the exception of weeks 7 and 8.
    Mineral excretion as well as urinary free corticosterone levels were
    determined on pooled weekly samples from each animal. Mineral intake
    was calculated weekly and was based on mineral content in the diet and
    weekly food consumption for each group by sex.

         Dietary TGS had no effects on health status. There appeared to be
    a trend toward lower body weights in animals fed the higher dietary
    levels of TGS. At 9 weeks, body weights in the females fed 8% TGS in
    the diet were significantly lower than the corresponding controls.

         Water consumption was significantly increased during week 9 in
    males fed 2, 4, and 8% TGS in the diet. Food consumption was
    significantly decreased in females fed TGS at 1, 4, and 8% during the
    first week TGS was presented in the diet. Fecal output was
    significantly increased approximately 1.5-fold over control levels in
    the 4% animals during weeks 3-9 and approximately twofold over control
    levels in both sexes at the 8% level; and the dry/wet faeces ratio was
    significantly decreased in both males and females during week 3,
    confirming that the increase in faecal weight was clue to an increase
    in water content. There were no consistent dose-related effects on
    urine output, caloric efficiency, urinary free corticosterone levels,
    or the excretion of calcium, magnesium, zinc or copper. A transient
    decrease in faecal copper was seen in both males and females fed 4 and
    8% dietary TGS during the second week, and an increase only in males
    in the ninth week. A not always significant or dose-related trend
    toward decreased urinary excretion of phosphorous was observed in both
    males and females throughout the study.

         Terminal body weights for females fed 4 and 8% TGS in the diet
    were significantly lower than the control terminal body weights. Wet
    cecum weights with contents were increased significantly in both males
    and females fed 4 and 8% TGS in the diet. No other tissue weights were

    significantly different from the corresponding controls. However,
    there was a dose-related trend toward decreased thymus weight in the
    females. Kidney calcium levels were unaffected as were serum free
    corticosterone levels and white blood cell counts (Eiseman et al.,
    1985).

    Special study on neurotoxicity

    Mice

         There were no behavioural effects or any morphological changes in
    the central nervous system following the administration of TGS
    (1000 mg/kg/day) or water (negative control) to 5 male and female mice
    of the CD1 strain for 21 days.

         The administration of 6-chloro-6-deoxyglucose (6-CG) (500 mg/kg/
    day), as a positive control, induced lesions at light microscopy
    and observed by electron microscopy in particular brain nuclei and in
    the spinal cord grey matter and were observed to be a microglial
    reaction associated with vacuolation referable to astrocytic swelling
    (Daniel & Finn, 1981).

    Monkey

         Two groups of 3 male marmosets each received for 4 weeks either
    TGS or TGS hydrolysis products (TGS-HP) orally by gavage at the dosage
    of 1000 mg/kg/day: a third group received 6-chloro-6-deoxyglucose
    (6-CG) at a dosage of 500 mg/kg/day and served as a positive control.
    A similarly constituted group received the vehicle, distilled water,
    served as a negative control group.

         Two monkeys receiving 6-CG were killed after seven days of
    treatment and the remaining animal was killed in extremis on Day 28.
    All three animals had shown clinical evidence of neurotoxicity before
    sacrifice.

         Signs attributed to treatment were salivation in all treated
    groups, subdued mood and emesis shortly after dosing in marmosets
    receiving 6-CG and TGS-HP. Tremors were seen in one animal and a
    single convulsion in another receiving 6-CG.

         Food consumption of animals receiving TGS remained similar to
    that of the negative controls throughout the treatment period; the
    food consumption of animals receiving 6-CG was lower than that of the
    negative controls.

         The overall body weight gain of monkeys receiving TGS was
    unaffected by treatment; weight losses were recorded in two animals
    receiving 6-CG.

         Neurological examinations revealed clear evidence of
    neurotoxicity in marmosets receiving 6-CG. A number of reflexes were
    depressed on Day 13 in two animal receiving TGS, but were considered
    normal on Day 28. The initial depressed responses were attributed to
    the normal variations in primates.

         The macroscopic appearance of tissues of all animals at necropsy
    was unremarkable. Microscopic examination and electron microscopy
    revealed bilaterally symmetrical degenerative changes in the nuclei of
    the central nervous system only in marmosets receiving the positive
    control (6-CG) (Hepworth & Finn, 1981).

    Special study on palatability

    Rats

         Groups of 20 female rats of the CD strain received either
    control diet (Group 1) or diet containing 30,000 ppm TGS (Group 2)
    ad libitum while animals in Group 3 were pair-fed with the amount of
    diet consumed by animals in Group 2, after allowing for the TGS
    content. The amount of TGS consumed by animals in Group 2 in the diet
    was administered as an aqueous solution by gavage to animals in Group
    5 while those in Group 4 received the vehicle alone.

         Total food intake and body weight gain of animals fed diet
    containing TGS and of animals pair-fed control diet were significantly
    lower than those of animals receiving control diet ad libitum (85%
    and 82%, respectively). Rats receiving TGS by gavage consumed
    significantly more food and gained significantly more weight than
    those receiving vehicle only.

         Overall food conversion efficiency for animals fed diet
    containing TGS was similar to that for animals pair-fed control diet
    (12.1 in Group 2, 12.5 in Group 3). However, conversion efficiencies
    for both these latter groups were lower than that of animals of the
    ad libitum control group (13.4). There was no difference between the
    group receiving TGS by oral gavage and its control group (13.1 and
    13.5, respectively).

         Water intake of animals receiving TGS, either in the diet or by
    oral gavage, was higher than that of their respective control groups.

         It is concluded that the inclusion of TGS in rat diet impairs the
    palatability of the diet and that the effect on growth is a
    consequence of the reduction in the amount of food consumed (Amyes &
    Aughton, 1985).

    Special study on reproduction

    Rats

         TGS was administered continuously in the diet at concentrations
    of 3,000, 10,000 and 30,000 ppm to groups of 30 male and 30 female
    rats of the Charles River CD strain (Sprague Dawley) throughout two
    successive generations. A fourth group, serving as control, received
    basal diet without the test material.

         F0 animals were treated for 10 weeks before pairing twice in
    succession. The first pairing produced the F1A litters which were
    discarded at weaning. After the second pairing males and females from
    the F1B litters were selected to form the F1 generation and were
    treated for 10 weeks before being paired, twice in succession to
    produce F2a and F2b offspring. These offspring were discarded after
    weaning. The general condition of F0 and F1 animals was unaffected
    by treatment.

         TGS was associated with dosage-related reductions in body weight
    gain by male and female rats during maturation and with reduced weight
    gain of offspring before weaning in all treated groups. Reduced food
    intake was recorded for F0 animals, but little effect was observed in
    the second generation. A slight dosage-related increase in water
    intake was noted in the F0 females. Water intake was increased for
    both sexes principally at the higher dosage levels in the second
    generation. A number of intergroup differences in absolute and
    relative organ weight were noted.

         After covariate analysis of organ weights and body weight of F0
    animals, statistically significant effects recorded in both sexes were
    limited to increased weight of the caecum with its contents at 10,000
    and 30,000 ppm and of the empty caecum at 30,000 ppm. Full caecal
    weight was also increased for females at 3,000 ppm. Kidney weight was
    increased in males receiving 30,000 ppm and the same animals had
    slightly increased pituitary weight; females at 30,000 ppm showed
    increased ovarian weight and slightly decreased thymus weight.

         Oestrous cycles, mating performance, fertility index, gestation
    length and gestation index were unaffected by treatment. Litter size
    and offspring viability indices were similar in all groups. Dosage-
    related reductions in the initial body weight and body weight
    gain were recorded for the F1A offspring.

         Sex ratio, physical development and auditory and visual functions
    of offspring were unaffected by treatment. The general condition of
    F1 males and females was unaffected by treatment. Necropsy of F1
    adults and F2 offspring revealed no macroscopic abnormalities that
    could be attributed to treatment.

         After covariate analysis of organ weights and body weight of F1
    generation, statistically significant effects recorded in both sexes
    were limited to increased weight of the caecum with its contents at
    all treatment levels and of the empty caecum and the kidneys at
    30,000 ppm. Empty caecal weight was also increased for males at
    10,000 ppm and kidney weight was increased in females receiving
    10,000 ppm. Ovarian weight was increased at the low dose and
    intermediate dose levels (3,000 and 10,000 ppm) but not at 30,000 ppm.
    Thymus weight was reduced in high dose males (30,000 ppm) and in all
    treated groups of females but the reduction in weight did not appear
    to be dosage related in the females.

         Oestrous cycles, pre-coital interval, gestation length, gestation
    index and fertility were unaffected by treatment. There were no
    adverse effects of treatment upon litter size or upon offspring
    viability at either pregnancy.

         Sex ratio, offspring development and auditory and visual
    functions were unaffected by treatment (Tesh & Willoughby, 1986a).

    Special study on teratogenicity

    Rats

         TGS was administered by gastric intubation at dosage of 500,
    1000, or 2000 mg/kg/day, to pregnant female rats of the CD strain from
    Day 6 to Day 15, inclusive, of gestation. Control animals received the
    vehicle, distilled water, throughout the same period. All females were
    killed on Day 21 of gestation for examination of their uterine
    contents. The general condition of females was similar in all groups
    and no deaths occurred.

         Body weight gain and food intake of treated females were similar
    to controls throughout gestation. A slight increase in water intake
    during treatment was recorded at 2000 mg/kg/day, but values for the
    pre- and post-treatment periods, and for the other treated groups,
    were unaffected. The number of implantation sites, viable young, the
    extent of pre- and post-implantation losses, and foetal and placental
    weights were similar in all groups.

         At necropsy, a single abnormal foetus with multiple malformations
    of the forepaws, hindlimbs and tail was found in the highest dosage
    group (2000 mg/kg/day), but in other respects all groups were similar;
    free-hand serial sectioning and skeletal evaluation revealed no
    abnormalities that could be related to treatment with TGS. TGS at
    levels up to 2000 mg/kg/day, during organogenesis, was without adverse
    effect upon the progress or outcome of pregnancy in the rat (Tesh
    et al., 1983a).

    Rabbits

         TGS was administered by gavage to pregnant New Zealand rabbits
    (16-18 per group) during organogenesis from Day 6 to Day 19 of
    gestation inclusive at dosages of 175, 350, and 700 mg/kg/day. A
    fourth group (16 females), serving as controls, received the vehicle,
    distilled water, at the same volume-dosage during the same treatment
    period. On Day 29 of gestation, females were killed to allow
    examination of their uterine contents. Thirteen animals died or were
    killed in extremis during the course of the study, distributed as
    follows: Group 1 (negative control) 1; Group 2 (175 mg/kg/day) 4;
    Group 3 (350 mg/kg/day) 2; Group 4 (700 mg/kg/day) 6. The deaths of
    only two animals both receiving 700 mg/kg/day, were attributed to
    treatment with TGS. The remainder were either the result of accidental
    tracheal intubation or were considered not to have been related to
    treatment.

         Numbers of females which survived to term with viable young were
    13, 13, 12, 5 for the control group and the groups receiving 175, 350
    and 700 mg/kg/day, respectively. A total of 7 females receiving
    700 mg/kg/day showed evidence of gastro-intestinal tract disturbance
    during the later part of the dosing period. Three females receiving
    700 mg/kg/day died or were killed in extremis, which were not
    considered to be caused by accidental trauma, two of which
    demonstrated gastro-intestinal tract disturbance. Four of the nine
    pregnant females receiving 700 mg/kg/day aborted following periods of
    weight loss, three also exhibited signs of gastro-intestinal tract
    disturbance.

         Body weight gain and food and water intakes of females which
    survived to term with viable young were essentially unaffected by
    treatment. Females which aborted or died as a result of treatment
    exhibited marked reductions in these parameters during the late
    treatment period.

         Litter parameters were either comparable with the concurrent
    controls or were within the background control range. The mean
    post-implantation loss for the five animals treated at 700 mg/kg/day
    which survived to term with viable young was increased (18.9%)
    compared to concurrent controls (8.6%) but was still within the
    background control range (1.0-20.5%). Fetal examinations at necropsy
    and after skeletal clearing and staining revealed no evidence of any
    adverse response to treatment.

         It was concluded from this investigation that daily oral
    administration of TGS to pregnant rabbits during organogenesis at a
    dosage of 700 mg/kg/day was associated with a marked adverse maternal
    response. At lower dosages of 175 and 350 mg/kg/day no maternal
    effects or evidence of embryotoxicity or teratogenicity were recorded
    that could be attributed to treatment (Tesh et al., 1987).

    Short-term studies

    Rats

         Groups of 15 male and 15 female CD rats received TGS in the diet
    at concentrations of 10,000, 25,000 or 50,000 ppm. A similarly
    constituted group of rats received diet alone without added TGS and
    acted as a control group. These animals were killed after four weeks
    of treatment. An additional six male and six female rats were assigned
    to each of the control and highest treatment (50,000 ppm) groups and
    were treated for eight weeks. The animals were originally designated
    to investigate the reversibility of any effects of treatment with TGS,
    but were used instead for a four-week extended treatment period. There
    were no deaths. Food and water consumption of treated rats were
    essentially unaffected by the administration of TGS. Reduced body
    weight gain was recorded for male rats receiving 25,000 ppm and male
    and female rats receiving 50,000 ppm of TGS, when compared with that
    of the control animals. Food utilisation was less efficient in rats
    receiving 50,000 ppm, when compared with that of the control animals.

         Ophthalmoscopic examination during week 3 of treatment revealed
    no abnormality that could be ascribed to the administration of TGS.

         Haematological differences between treated and control rats were
    limited to a marginally lower number of lymphocytes in females
    receiving 50,000 ppm. No inter-group differences were evident in bone
    marrow smears after four or eight weeks of treatment.

         A dosage-related reduction in plasma alanine aminotransferase
    activity was recorded after four weeks of treatment in all treated
    groups of rats. This effect was not apparent after eight weeks of
    treatment. When compared with the controls, increased urinary calcium
    and magnesium excretion was recorded after three weeks of treatment in
    rats receiving 50,000 ppm of TGS. A similar trend was also apparent in
    females after seven weeks of treatment. Lower urinary volume was also
    recorded at both examinations for male rats receiving this dosage.

         A dosage-related increase in caecum weights was recorded for rats
    sacrificed after four weeks of treatment. A similar but less marked
    effect was recorded for rats killed after eight weeks. There was a
    trend towards a reduction in the weights of the spleen, thymus,
    adrenals and ovaries following treatment with 50,000 ppm TGS for
    either four or eight weeks. There were no macropathological
    observations for rats killed after four or eight weeks of treatment
    that could be associated with the administration of TGS.

         Treatment-related changes were observed microscopically in the
    caecum, liver, spleen and thymus. Goblet cell hyperplasia of the
    caecum and periacinar hepatocytic hypertrophy were present, in some of
    the males that received 25,000 or 50,000 ppm for four weeks. The
    hepatic effects were present after eight weeks in males and females
    that received 50,000 ppm.

         Lymphoid hypoplasia of the spleen and thymus was apparent after
    both four and eight weeks in males fed 50,000 ppm. The only effect in
    females was splenic lymphoid hypoplasia after eight weeks of treatment
    in those receiving 50,000 ppm.

         It was concluded that 10,000 ppm of TGS represents a minimum
    physiological effect level, 25,000 ppm represents a level of slight
    change and 50,000 ppm elicited a clear response to treatment. None of
    the observed effects was sufficiently marked, however, to cause any
    overt change in the general well-being of the animals (Cummins
    et al., 1983).

    Dogs

         Groups of four male and four female beagle dogs were fed diet
    containing 0, 0.3, 1.0, 3.0% TGS corresponding to doses of
    approximately 90, 285 and 874 mg/kg/day for twelve months. The effect
    of the test material was investigated using complete clinical
    observations; data on body weight, food consumption, body temperature,
    hematology, clinical chemistry, urinalysis, ophthalmoscopy; organ
    weight determinations and calculations; and complete gross and
    microscopic pathology.

         No well-defined treatment effects were demonstrated in this study
    by clinical laboratory analysis. There were mild lymphophenia in
    males, elevation of glucose in males, decreased serum phosphorus in
    females, and inhibition of lactate dehydrogenase in both sexes. The
    changes were generally minimal, sporadic and not dose related. The
    organ weight data together with gross, and microscopic evaluations did
    not reveal any changes attributable to the oral administration of the
    test material.

         No toxic effects were judged to be present following the oral
    consumption of TGS by beagle dogs (Goldsmith, 1985a).

    Long term studies

    Rat

         Sprague-Dawley CD rats were fed diet containing TGS at a
    concentration of 0, 3000, 10,000 and 30,000 ppm continuously for up to
    two years. These animals were derived from parents that had themselves

    received TGS at the same concentrations for four weeks prior to
    pairing and during gestation. During lactation, the highest dietary
    concentration was reduced from 30,000 ppm to 10,000 ppm.

         In the oncogenicity phase, each treated group comprised 50 male
    and 50 female rats. Two similar constituted control groups received
    diet containing no TGS.

         In the toxicity phase, 30 males and 30 female rats were assigned
    to each treated group and to one control group. After 52 weeks of
    treatment, 15 males and 15 females from each group of the toxicity
    phase were sacrificed; all surviving animals of the toxicity phase
    were sacrificed after 78 weeks of treatment. In the oncogenicity
    phase, sacrifice of surviving animals was initiated after completion
    of 104 weeks of treatment.

         Mating performance, fertility, litter size and pup viability were
    unaffected by treatment with TGS; gestation length, for animals
    receiving 10,000 or 30,000 ppm, was slightly longer than the value for
    controls; this slight difference was not considered to be of
    biological significance. The weight gain of the treated parental
    animals, prior to mating and during gestation, and of their treated
    offspring from day 14 post partum, was lower than the gain of
    control animals. The weight gain of treated animals was higher than
    that of controls during lactation. The food consumption of the treated
    animals was lower than that of their controls during the first week of
    treatment. Efficiency of food conversion for the treated parental
    animals was lower than that of the controls during the first week of
    treatment. The conversion efficiency for the treated offspring was
    lower than that for the controls from Week 15 of the toxicity and
    oncogenicity phases onwards. Treatment with TGS for two years had no
    adverse effect on survival.

         No changes in appearance or behaviour were observed that were
    associated with treatment. During the chronic toxicity and
    oncogenicity phases body weight gain for animals receiving any
    concentration of TGS was lower than that for controls (13-26%). During
    the chronic toxicity and oncogenicity phases, food consumption at any
    concentration was lower than controls (5-11%). The effect of TGS on
    food consumption, and secondarily body weight gain, was demonstrated
    to be the result of the unpalatable nature of TGS at high dietary
    concentrations to rats.

         Water consumption of treated rats was higher in a dose related
    manner than that of controls. There were no treatment-related findings
    at ophthalmoscopic examination. There were no changes in the cellular
    or chemical components of blood apart from a lower alanine
    aminotransferase activity in males receiving the highest concentration
    of TGS during and at the end of treatment (103 weeks).

         The urine volume of treated female rats tended to be lower than
    that for controls. Magnesium excretion of animals receiving the
    highest concentration of TGS tended to be higher than the excretion
    for controls during the first year of the study. This trend was
    reversed for samples collected after 77 weeks.

         The full and empty caeca, from animals receiving 30,000 ppm, were
    generally heavier than those of the controls. This and other
    intergroup differences in organ weight were not associated with any
    treatment-related histopathological findings. In particular, the
    higher caecal weights (caecal enlargement), although treatment-
    related, are considered a physiological adaptive response consistent
    with the known effect of other poorly absorbed compounds administered
    at high dietary levels.

    Analysis of covariance, using the terminal body weight as the
    covariate, revealed that the sporadic organ weight differences noted
    in animals sacrificed after 52, 78, and 104 weeks of treatment were
    not correlated with the treatment or with histopathological findings.

         There were no macroscopic abnormalities that were considered to
    be related to treatment. Changes in the incidences of non-neoplastic
    findings, minimal or slight, (renal pelvic nephrocalcinosis) in the
    kidneys of female rats of the oncogenicity phase were associated with
    treatment. A higher incidence of renal pelvic epithelial hyperplasia
    among all groups of treated females was noted. This was considered
    secondary to renal pelvic mineralisation (pelvic nephrocalcinosis),
    which was observed at a higher incidence among females that had
    received the intermediate or high dose. There was no effect on the
    kidneys of male rats. There were no neoplasms that were attributed to
    treatment (Amyes et al., 1986b).

    Observations in man

         Eight normal subjects (4 male and 4 female) first received
    ascending oral doses of TGS in doses of 0.0, 1.0, 2.5, 5.0 and
    10.0 mg/kg at 48-hour intervals. This was followed by 7 days continuous
    administration of 2.0 mg/kg daily for the first three days and
    5.0 mg/kg daily for the remaining four days.

         There were no adverse reactions or complaints throughout the
    study. No clinically significant changes were observed in temperature,
    pulse, B.P. (supine or standing), respiratory rate and general
    conditions. Detailed haematological and biochemical studies were made
    before the study, then after 2.0 mg/kg/day and 5.0 mg/kg/day during
    the second phase. There were no abnormalities in any of the parameters
    measured. E.C.G. studies throughout showed no changes from the initial
    findings. Urinary examination showed no abnormalities, and an
    assessment of 24 hour urine volumes throughout the study detected no
    effects. Blood insulin levels measured one haft hour after each dosage

    showed levels ranging from 4.038.0 mu/ml, which were within the
    fasting range. On Day 25 of the study, blood insulin levels were
    measured one half hour after 50 g standard sucrose. All showed the
    characteristic increase within the range 53-108 mu/ml (Baird et al.,
    1984).

         A single blind randomised controlled study was conducted over a
    period of 13 weeks to compare the effects in normal human volunteers
    of the high-intensity sweetener TGS with that of fructose administered
    daily. One hundred and eighteen (118) subjects were recruited of which
    108 completed the study - 77 of these received TGS (47 males, 30
    females) and 31 received fructose (17 males, 14 females).

         The total daily dose of TGS, administered as an aqueous solution,
    (50 ml) was: Weeks 1-3 inclusive: 125 mg (2  62.5 mg); Weeks 47
    inclusive: 250 mg (2  125 mg); Weeks 8-13 inclusive: 500 mg
    (2  250 mg). The maximum daily intake of TGS varied between 4.8
    and 8.0 mg/kg for males and 6.4 and 10.1 mg/kg for females.

         Fructose was administered twice daily at a constant dose of
    50 g/day dissolved in water. Three subjects receiving TGS withdrew
    from the study for reasons not related to TGS toxicity.

         There were no pathological changes on physical examination, no
    abnormal changes in E.C.G., haematology, biochemistry, or urinalysis
    in any of the subjects. Slit lamp examination before and after the
    study in 24 subjects (20 male, 4 female) showed no abnormal changes.
    Steady state measurement of TGS in the blood were made in ten subjects
    (8 male and 2 female) on five consecutive days during Week 12. Blood
    was collected on each occasion immediately before and two hours after
    the morning "dose". Pre-dose levels ranged from 0.01 to 0.16 g/m
    rising to between 0.02 to 0.42 g/ml 2 hours after dosing (Shepard and
    Kyffin, 1984).

         In this double blind crossover study, eight normal subjects were
    given TGS alone (10.0 mg/kg), sucrose alone (100 8) and a mixture of
    sucrose (100 g) and TGS (10.0 mg/kg) in random order at intervals of
    48 hours. Blood samples were taken over the three hour period after
    dosing and analysed for glucose, fructose and insulin. The levels of
    serum glucose and fructose following the administration of sucrose
    plus TGS did not differ significantly from those after sucrose alone.

         TGS was without effect upon the insulin response to sucrose.
    There was an absence of effect upon insulin levels when TGS was given
    alone.

         Under the conditions of this study TGS did not interfere with the
    absorption of sucrose and did not influence insulin secretion
    (Shepard, 1984).

    4-CHLOROGALACTOSE (4-CG) and 1,6 DICHLOROFRUCTOSE (1,6-DCF)

    BIOLOGICAL DATA

    Biochemical aspects

    Biotransformation

         The absorption, excretion and metabolism of 4-chloro-4-deoxy-
    (U-14C)-galactose was studied in 5 adult male rats (Sprague Dawley CD
    strain). After a single oral dose (5 mg/kg; 7.1 uCi/kg) 82% (range
    78.2% to 87.0%) of the administered radioactivity was excreted in the
    urine and 3.7% (range 1.9% to 8.5%) in the faeces over 5 days. The
    majority of the radioactivity was eliminated in the urine within the
    first 24 hours, predominantly in the form of 4-chloro-4-deoxy-
    (U-14C)-galactose (96%) with trace amounts (2%) of an unknown
    metabolite (Hughes et al., 1987a).

         Adult male and female rats of the Sprague-Dawley CD strain given
    a single oral dose of chlorine-36 labelled 4-chloro-4-deoxygalactose
    (36Cl-4-CG; 100 mg/kg; 2 uCi) excreted 86% of the radioactivity in
    urine and less than 4% in the faeces in 72 hours. About 4% of the dose
    was identified as inorganic chloride. A further 3% was recovered in
    urine collected over days 4 - 14 (Daniel & Rhenius, 1980).

         Adult male and female rats of the Sprague-Dawley CD strain given
    chlorine-36 labelled 1,6-dichloro-1,6- dideoxyfructose (36Cl-1,6-DCF;
    100 mg/kg; 2 uCi) excreted 80% of the radioactivity in the urine in 14
    days, approximately half of which was identified as inorganic chloride.
    The major organochlorine derivative in the urine was isolated and
    characterised by gas chromatography/mass spectrometry as a reduction
    product of 1,6-dichloro-1,6-dideoxyfructose with an identical mass
    fragmentation pattern to 1,6-dichloro-1,6-dideoxymannitol (Daniel &
    Rhenius, 1980).

         After oral administration of 14C-DCF (100 mg/kg b.w.; 1 uCi) to
    free-range male rats, 44%-55% of the radioactive dose was excreted in
    urine and 22%-25% in faeces over 5 days. After intravenous
    administration of 14C-DCF at the same dose, between 39%-55% was
    excreted in urine and 13%-20% in faeces over 5 days. Analysis of urine
    (after i.v. and intraduodenal dosing) by thin-layer chromatography
    revealed 5 major radioactive metabolites. One of these has been
    identified as 1,6-dideoxymannitol and three metabolites corresponded
    in mobility to the degradation products of 6-chlorofructose-GSH. In
    two experiments (one rat dosed i.v. and one dosed orally)
    insignificant amounts of radioactivity was detected in expired air
    collected between 0-6h after 14C-DCF administration at 100 mg/kg
    (Hughes et al., 1987b).

         When 14C-DCF (5mg/kg b.w.; 3.2-4.0 uCi) was administered either
    by intravenous or intraduodenal injection to anaesthetised rats, about
    20% of the radioactive dose was excreted in bile within 8h. Following
    the i.v. administration of 36Cl-DCF (3.4-35 mg/kg b.w.; 0.333.1 uCi)
    approximately 11% of the radioactivity was excreted in bile. Virtually
    no free inorganic 36Cl-chloride was detected in the bile and the
    metabolic profile obtained after 36Cl-DCF administration was similar
    to that obtained after 14C-DCF administration indicating that the
    major biliary metabolites of DCF contain at least one chlorine atom.
    Urine (0-6h) contained 37% of the radioactive dose, approximately 2%
    of which was inorganic 36Cl-chloride. The urinary metabolic profile
    obtained after 36Cl-administration was similar to that obtained after
    14C-DCF administration indicating that the major metabolites contain
    at least one chlorine atom. The major urinary metabolites, however,
    were chromatographically distinct from the major metabolites present
    in bile. The amount of free inorganic 36Cl-Chloride excreted in the
    bile and urine over 6h under-estimates the extent of dechlorination of
    DCF in vivo owing to the slow rate of excretion of inorganic
    chloride (Hughes et al., 1987b).

         In vitro incubations and isolated perfused liver experiments
    showed that the major biliary metabolite of DCF is a glutathione
    adduct and that glutathione-dependent dechlorination occurs in liver
    and in blood. The ability of glutathione to form a conjugate with DCF
    or 1-chlorofructose but not with 6-chlorofructose would suggest that
    DCF is dechlorinated only at position 1. NMR analysis confirms this
    mechanism and shows that the sulphur of glutathione is attached to
    carbon-1 of the sugar, i.e., 6-chlorofructose-1-glutathione. Isolated
    perfused liver experiments with the preformed glutathione adduct
    demonstrated that 6-chlorofructose-glutathione formed extrahepatically
    is not metabolised by the liver nor is it available for biliary
    excretion. The adduct excreted in the bile after DCF administration
    therefore must be formed in the liver. There is no evidence for an
    enterohepatic circulation of the adduct: experiments in which
    preformed adduct was administered orally to free-range rats showed
    that it is absorbed from the G.I. tract (at least 55%) undergoes
    metabolism by extrahepatic tissues and that the metabolites are
    excreted in urine. DCF can also be dechlorinated in vitro at pH 7.4
    by a glutathione-independent mechanism with the formation of inorganic
    chloride and 6-chlorofructose. This pathway does not occur in the
    presence of excess glutathione (Hughes et al., 1987b).

    Special studies on enzymes and other biochemical parameters

         TGS-HP (hydrolysis products 4-CG and 1,6-DCF) was administered
    to groups of rats (CD strain, 5 male and 5 female) by oral gavage on
    each of 21 consecutive days at dose levels of 25 or 75 mg/kg/day.
    Control animals (5 male and 5 female) received an equivalent volume
    of vehicle (distilled water) daily during the same treatment period.
    A positive control group (5 males and 5 females) was administered by

    interperitoneal injection a single dose of Aroclor 1254 five days
    before sacrifice (500 mg/kg; 2.5 ml/kg). Twenty-four hours (5 days for
    Aroclor 1254 group) after the final dose, the rats were sacrificed and
    the livers immediately removed and weighed. Samples of the livers were
    taken for microsome and cytosolic fraction preparation. The following
    hepatic parameters were measured; microsomal and cytosolic protein,
    cytochrome P450), p-nitrophenol-glucuronyl transferase activity and
    glutathione-S-transferase activity. Aroclor 1254 treatment produced
    statistically significant increases in liver weights and all the
    hepatic parameters measured. TGS-HP treatment had no inducing effects
    on liver weights, microsomal protein levels, cytochrome P450,
    7-ethoxyresorufin-O-deethylase activity or glutathione-S-transferase
    activity.

         p-Nitrophenol-glucuronyl transferase activity was significantly
    increased compared to control animals in both sexes receiving
    75 mg/kg/day TGS-HP, no effect was found in animals receiving
    25 mg/kg/day TGS-HP. Cytosolic protein levels were found to be raised
    when expressed as total protein per liver in females, receiving
    75 mg/kg/day TGS-HP (other female groups and all male groups were
    unaffected). These results were probably a consequence of the lower
    weight of the liver in the female control group (Hawkins et al.,
    1987d).

        Results of mutagenicity assays on 4-CG
    
                                                                                              

    Test System        Test Object           Concentration of 4-CG   Results      Reference
                                                                                              

    Ames test (1)     S.typhimurium            16 - 10,000           Negative     Bootman &
                      TA98; TA100              g/plate                           Lodge, 1980a
                      TA1537; TA1535; TA1538

    Gene mutation     Mouse lymphoma           1,335 - 10,000        Negative     Kirby et al.,
    (1)               frwd. mutation           g/ml                              1981b
                      L5178Y TK+/-

    Chromosome        Human periferal          40 - 1000             Negative     Bootman &
    aberrations (1)   lymphoctes               g/ml                              Rees, 1981

    Chromosome        Rat, os,                 5  50 mg/kg          Negative     Cimino &
    aberrations       bone marrow              5  150 mg/kg                      Lebowitz,
                      (in vivo)                5  500 mg/kg                      1981b
                                                                                              

    (1) Both with and without rat liver S-9 fraction.
    
        Results of mutagenicity assays on TGS-HP
                                                                                              

    Test System        Test Object         Concentration        Results        Reference
                                           of TGS-HP
                                                                                              

    Ames test (1)      S.typhimurium       16 - 10,000          Negative       Bootman &
                       TA98, TA100         g/plate                            May, 1981
                       TA1537, TA1538

                       TA1535              250 - 1000           Negative       Bootman &
                       TA1535              2500 - 5000          Positive       May, 1981
                                           g/plate             (2)

    Ames test (1)      S.typhimurium       16 - 10,000          Negative       Bootman &
                       TA98, TA100         g/plate                            Lodge, 1980b
                       TA1537, TA1538
                                                                                              


    Results of mutagenicity assays on 1,6-DCF
                                                                                              

    Test System        Test Object         Concentration        Results        Reference
                                           of 1,6-DCF
                                                                                              

                       TA1535              16 - 1000            Negative       Bootman &
                       TA1535              2000 - 5000          Positive       Lodge, 1980b
                                           g/plate             (2)

    Ames test (1)      S.typhimurium       60 - 6000            Negative       Haworth
                       TA98, TA100         g/plate                            et al., 1981
                       TA1537, TA1538

                       TA 1535 (-S9)       60 - 6000            Negative       Haworth
                       TA1535 (+S9)        60 - 3000            Negative       et al., 1981
                                           6000 g/plate        Positive
                                                                (2)

    Gene mutation      Mouse lymphoma      13 - 42 g/ml        Negative       Kirby et al.,
    (1)                frwd. mutation                                          1981c
                       L5178Y TK+/-        56 - 133 g/ml       Positive       Kirby et al.,
                                                                (3)            1981c
                                                                                              

                                                                                              

    Test System        Test Object         Concentration        Results        Reference
                                           of 1,6-DCF
                                                                                              

    Gene mutation      Mouse lymphoma      13 - 169 (+S9)       Negative       Kirby et al.,
                       frwd. mutation      g/ml                               1981d
                       L5178Y TK+/-        10 - 40 (-S9)        Negative       Kirby et al.,
                                           g/ml                               1981d
                                           53 - 127 (-S9)       Positive       Kirby et al.,
                                           g/ml                (4)            1981d

    Chromosome         Human periferal     1.5 - 40 g/ml       Negative       Bootman &
    aberrations (1)    lymphoctes                                              Rees, 1981

    Sex-linked         Drosophila          3 days               Negative       Bootman &
    recessive          melanogaster        0.2 - 2.0 mg/ml                     Lodge, 1981
    lethal             (in vivo)

    Chromosome         Rat, gavage,        1000 mg/kg           Negative       Bootman &
    aberrations        bone marrow                                             Whalley, 1981
                       (in vivo)           5 x 50 mg/kg         Negative
                                           5 x 150 mg/kg
                                           5 x 500 mg/kg
                                           24h interval
                                                                                              

    (1)  Both with and without rat liver S-9 fraction.
    (2)  The number of revertant colonies was increased two- to three-fold when
         1,6-DCF was incubated.
    (3)  The increase in the mutant frequency was associated with a dose-related decreased
         in the viability of the cultures (from 42-60% at 56 g/ml to 2-3% at 133 g/ml).
    (4)  The increase in the mutant frequency was associated with a dose-related decrease
         in the viability of the cultures (from 45% at 53 g/ml to 3% at 127 g/ml).
        Toxicological studies

    Special studies on carcinogenicity

    Rat

         Groups of 50 male and 50 female Sprague-Dawley rats of the CD
    strain received an approximately equimolar mixture of the hydrolysis
    products TGS-HP, continuously for 104 weeks at concentrations of 0,
    200, 600, or 2,000 ppm in the diet. No adverse effect of treatment on
    survival was observed. Survival in this study at 104 weeks of
    treatment ranged from 36-58% in the males and 36-64% in the females.

    Overall weight gain of male and female rats receiving the highest
    dietary concentration and of males of the intermediate dose group was
    significantly lower than that of their controls. Rats of either sex
    receiving TGS-HP at the highest dietary concentration consumed less
    food than rats of their respective control groups on a g/rat/week
    basis. A similar, but less pronounced, response was observed for males
    receiving the intermediate concentration and females receiving the
    intermediate or low concentration. Achieved dosages (mg/kg body
    weight/day) were 29, 85, and 275 mg/kg for males and 28, 81, and
    256 mg/kg for females during Week 1 and declined in line with changing
    body weight: food intake ratio to 5 to 59 for males and 8 to 75 for
    females at Week 104. Ophthalmic examination prior to commencement, and
    serially throughout the treatment period, revealed no evidence of
    treatment-related changes. In females, mean absolute heart, liver,
    spleen and kidney weights for rats receiving the highest dietary
    concentration were lower than their respective control values.

         In addition to the testicular differences observed attributed to
    the low value of the control group, mean absolute adrenal weight for
    males receiving the highest dietary concentration and kidney weights
    for males receiving the lowest dietary concentration were lower than
    their respective control values. Analysis of covariance with terminal
    body weight as the covariate showed significantly decreased adrenal
    gland weight in males treated at 2000 ppm and decreased spleen and
    kidney weights in males treated at 200 and 2000 ppm. Decreased heart
    and kidney weights were noted in females treated at 2000 ppm and
    testicular weight was also increased in all males receiving treatment.
    There were no macroscopic abnormalities which were considered to be
    related to treatment with TGS-HP. The incidences of a number of
    non-neoplastic findings were higher in treated groups than in control
    groups. These included pulpitis in the teeth and increased
    pigmentation of syncytial macrophages in the mesenteric lymph-nodes
    (generally graded as minimal or slight) of female rats treated at
    2000 ppm; hepatocytic clear-cell foci (generally graded as minimal or
    slight) in males and females treated at 2000 ppm; focal pneumonitis
    (generally graded as minimal or slight) in male rats treated at
    2000 pm and females treated at 2000 ppm. The pigmentation within the
    syncytial macrophages of treated female rats was similar in appearance
    to that seen in the controls and in male rats. Special histological
    demonstration techniques did not detect bile pigment but ferric iron
    (haemosiderin) and lipofuscin were present in most macrophages of the
    samples taken from control and highest dosage groups showing
    macrophages with increased pigmentation and the control group showing
    normally pigmented macrophages. Treatment with TGS-HP was not
    associated with any statistically significant increase in the
    incidence of any tumour (Amyes et al., 1986c).

    Special study on neurotoxicity

    Mice

         There were no behavioural effects or any morphological changes in
    the central nervous system following the administration of an
    equimolar mixture of 1,6-dichloro-1,6-dideoxyfructose and
    4-chloro-4-deoxygalactose TGS-HP (0, 50, 150, 500, and 1000 mg/kg/day)
    to 5 male and 5 female mice of the CD1 strain for 21 days. The
    administration of 6-chloro-6-deoxyglucose G-CG (500 mg/kg/day), as a
    positive control, induced lesions observed at light microscopy and
    examined by electron microscopy in particular brain nuclei and in the
    spinal cord grey matter and were observed to be a microglial reaction
    associated with vacuolation referable to astrocytic swelling (Daniel &
    Finn, 1981).

    Monkeys

         Groups comprising three male marmosets received for four weeks
    an equimolar mixture of 1,6-dichloro-1,6-dideoxyfructose and
    4-chloro-4-deoxygalactose (TGS-HP) orally, by gavage, at a dosage of
    1000 mg/kg/day; a third group received 6-chloro-6-deoxyglucose (6-CG)
    at a dosage of 500 mg/kg/day and served as a positive control. A
    similarly constituted group received the vehicle, distilled water, and
    served as a negative control group. Two monkeys receiving 6-CG were
    killed after seven days of treatment and the remaining animal was
    killed in extremis on Day 28. All three animals had shown clinical
    evidence of neurotoxicity before sacrifice. Signs attributed to
    treatment comprised salivation in all treated groups, subdued mood and
    emesis shortly after dosing in marmosets receiving 6-CG or TGS-HP,
    tremors, and a single convulsion in animals receiving 6-CG. Food
    consumption of animals receiving TGS-HP remained similar to that of
    the negative controls throughout the treatment period; the food
    consumption of animals receiving 6-CG was lower than that of the
    negative controls. The overall body weight gain of monkeys receiving
    TGS-HP was unaffected by treatment; weight losses were recorded in two
    animals receiving 6-CG.

         Neurological examinations revealed clear evidence of
    neurotoxicity in marmosets receiving 6-CG. One of the animals treated
    with TGS-HP exhibited a depressed segemental response prior to dosing
    and throughout the study. The same animal exhibited increased
    salivation following gavage and for these reasons the skeletal muscle
    and the salivary glands of all animals on study were evaluated
    histopathologically.

         The macroscopic appearance of all animals at necropsy was
    unremarkable. Light microscopic examination and electron microscopy
    revealed bilaterally symmetrical degenerative changes in the nuclei of
    the central nervous system only in marmosets receiving the positive
    control (6-CG) (Hepworth & Finn, 1981).

    Special studies on reproduction

    Rats

         An equimolar mixture of TGS-HP was administered continuously in
    the diet at concentrations of 0, 200, 600, or 2000 ppm to groups of 30
    male and 30 female rats of the Charles River CD strain throughout two
    successive generations. F0 animals were treated for 10 weeks before
    pairing twice in succession. After the second pairing, males and
    females from the F1b litters were selected to form the F1 generation
    and were treated for 10 weeks before being paired twice in succession.
    The general condition of F0 animals was unaffected by treatment. At
    the highest level (2000 ppm), body weight gain was significantly
    reduced in males and females during maturation and in females during
    both gestation phases, although females showed slight net weight gain
    during both lactation phases. Lesser reductions in body weight gain
    were recorded for females at 600 and 200 ppm during maturation and
    both pregnancies, whereas males were unaffected at these levels. Food
    intake was marginally reduced during maturation in males and females
    receiving 2000 ppm but was essentially unaffected at the lower
    concentrations of TGS-HP.

         Food conversion efficiency during maturation was marginally
    reduced in Group 4 males (2000 ppm) and in females in Groups 2, 3 and
    4 (200, 600 and 2000 ppm). Water intake of males was essentially
    unaffected by treatment; water intake of females was slightly reduced
    in all treated groups but there was no systematic dosage-related
    effect. Oestrous cycles, mating performance, fertility index,
    gestation length and gestation performance, fertility index, gestation
    length and gestation index were unaffected by treatment. Litter size
    and offspring viability indices were similar in all groups. Initial
    body weight of offspring was essentially similar for all groups and
    body weight gains at 200 or 600 ppm were unaffected; at 2000 ppm,
    however, body weight gain of offspring to weaning was significantly
    reduced. Sex ratio, offspring development and responses to simple
    auditory and visual stimuli were unaffected by treatment. Necropsy of
    F1a and F1b offspring and of F0 adults revealed no macroscopic
    abnormalities that were considered to be related to treatment. Organ
    weight analysis revealed a number of statistically significant
    intergroup differences but there were no differences that were
    considered to be of toxicological importance.

         Initial body weights of males and females selected to form the F1
    generation were significantly reduced at 2000 ppm and body weight gain
    in this group was significantly reduced for males and females during
    maturation and for females during both pregnancies but there was
    evidence suggestive of net increase in body weight during both
    lactation phases. At the lower treatment levels (200 and 600 ppm) body
    weight gain of females during maturation showed a slight, significant
    reduction but no other effects were seen. Food intake during

    maturation showed a dosage-related reduction for both sexes at 600 and
    2000 ppm (approximately 95% and 88%, respectively) but food conversion
    efficiency did not appear to be affected. Water intake was similar in
    all groups of animals during maturation. Oestrous cycles and
    pre-coital interval, gestation length, gestation index and parturition
    were unaffected by treatment. All animals receiving 2000 ppm were
    fertile at both matings. A number of infertile matings occurred in the
    other group and these were most evident in the control group and there
    was no indication of adverse response to treatment with TSG-HP. Litter
    size at birth was similar in all groups; offspring viability was
    normal in all treated groups and was considerably higher than in the
    concurrent control group where viability indices were low largely due
    to total loss of some litters. Initial body weight of offspring was
    unaffected by treatment but body weight gain up to weaning was
    significantly reduced for both F2a and F2b litters at 2000 ppm. Body
    weight gain of offspring at the lower treatment levels (200 and 600
    ppm) was unaffected. Sex ratio, offspring development and responses to
    simple auditory and visual stimuli were similar in all groups.
    Necropsy of F2a and F2b offspring and of F1 adults revealed no
    macroscopic abnormalities that were considered to be related to
    treatment. Organ weight analysis revealed a number of statistically
    significant intergroup differences but there were no differences that
    were considered to be of toxicological importance (Tesh & Willoughby,
    1986b).

    Special studies on teratogenicity

    Rat

         TGS-HP was administered by gastric intubation at dosages of 0,
    30, 90 or 270 mg/kg b.w., to groups of 20 pregnant female rats of the
    CD strains from day 6 to day 15 of gestation. Control animals received
    the vehicle, sterile water, throughout the same period. All females
    were killed on day 21 of gestation for examination of their uterine
    contents. The general condition of females was similar in all groups
    except for slight staining of the bedding at 270 mg/kg/day. No deaths
    occurred Maternal body weight gain and food intake were depressed
    during treatment at 270 mg/kg/day but were unaffected at the lower
    dosages. The number of implantation sites, viable young and the extent
    of pre- and post-implantation losses were similar in all groups but at
    the highest dosage (270 mg/kg/day) fetal and placental weights were
    reduced compared with values recorded in the control. Fetal
    examinations revealed a marginal increase in the incidence of the 14th
    rib at 270 mg/kg/day but, with this possible exception, there were no
    developmental abnormalities that could be related to treatment with
    TGS-HP. TGS-HP had no effects on maternal health or upon fetal
    development at dosages of up to 90 mg/kg/day (Tesh et al., 1983b).

    Acute toxicity TGS-HP

                                                                        

    Species    Sex        Route    LD50           References
                                   (mg/kg b.w.)
                                                                        

    Mouse      male &     oral     3499           Buch & Gardner, 1982a
               female

    Rat        male &     oral     1629           Buch & Gardner, 1982b
               female
                                                                        

    Short-term studies

    Rat

         Groups of twenty male and twenty female sprague Dawley rats
    received and approximately equimolar mixture of the hydrolysis
    products (TGS-HP) of TGS, continuously via the diet, for 13 weeks, at
    concentrations of 200, 600 and 2000 ppm. A similarly constituted
    control group received untreated diet. Animals were selected from the
    F1a generation, the parents of which had been exposed to the same
    concentrations of the test material for 10 weeks prior to mating and
    subsequently throughout gestation, lactation and weaning. There were
    no deaths or signs of reaction to treatment. The body weight gain of
    animals receiving 2000 ppm TGS-HP was depressed some 15% when compared
    with that of the control group while food consumption was reduced 5%
    in the females and 10% in the males. The overall efficiency of food
    conversion was reduced in females in the top dose group. There was no
    consistent effect on water consumption in the F0 and F1a generation.
    Ophtalmoscopy of the control and top dose groups during Week 12 did
    not reveal any effects of treatment. No abnormalities in the
    haematological parameters were detected during Week 12. Small, but
    statistically significant, reductions in the following serum
    constituents were observed at Week 12; alanine aminotransferase
    activity in animals in the top dose group; aspartate aminotransferase
    activity in the intermediate and top dose group females as well as the
    top dose male group and a similar trend in the intermediate male
    group, alkaline phosphatase and glucose levels in males in the top
    dose group. There were no differences in the absolute weights of the
    major organs apart from a reduction in that of the heart in the top
    dose groups and an increase in the female liver weight in the
    intermediate dose group. When adjusted for terminal body weight by
    analysis of covariance, the liver weight was higher in all treated

    females compared to the controls while a marginal increase was found
    for the kidneys in the intermediate and top dose groups. There were no
    treatment-related changes in the macroscopic or microscopic pathology
    of the tissues examined (Danks et al., 1986).

    Dog

         Groups of four male and four female beagle dogs were dosed orally
    for 26 weeks with an equimolar mixture of the hydrolysis products of
    TGS, (TGS-HP), incorporated into the basal diet at the following dose
    levels: 0, 10, 50, or 250 mg/kg b.w. The general condition and
    survival of dogs was unaffected by treatment. There were no
    treatment-related findings at ophthalmoscopic examination and there
    were no changes in the cellular or chemical components of blood. With
    the exception of a slight reduction in absolute and relative thymus
    weights in males receiving the highest dose, other organ weights were
    unaffected. There were no gross or microscopic changes attributable to
    the oral administration of TGS-HP (Goldsmith, 1985b).

    COMMENTS

         Extensive studies were carried out in animals and humans with TGS
    and with an equimolar mixture of 4-CG and 1,6-DCF which constitute the
    TGS-HP.

         With regard to the animal studies, the Committee evaluated
    pharmacokinetic, metabolic, mutagenicity, teratogenicity, reproduction,
    neurotoxicity, short-term, long-term, and carcinogenicity studies.
    TGS is poorly absorbed after oral administration in mouse, rat, dog
    and man, and is excreted essentially unchanged in human urine. Its
    half-life in man is approximately 13 hours.

         TGS is poorly absorbed after oral administration in the rat,
    (3-23%) mouse (13-26%), dog (7-36%) and man (8-22%). TGS is excreted
    essentially unchanged in urine and faeces and is not hydrolysed or
    dechlorinated. A small proportion (2%) is excreted in the urine as a
    conjugate with glucuronic acid. There is no metabolic adaptation
    following prolonged (87 weeks) dietary administration to rats. TGS
    does not appear to accumulate in man which is consistent with the
    effective half life of approximately 13 hr. There is the potential for
    the accumulation of TGS metabolites in the fetus.

         TGS is not embryotoxic or teratogenic in rats or rabbits.
    However, rabbits exhibited a marked maternal toxicity, with some
    deaths, associated with severe disturbances in gastrointestinal
    function, though only at the highest dose tested (700 mg/kg). This
    appears to be a non-specific effect caused by the sensitivity of the
    rabbit to high doses of compounds producing osmotic effects in the
    large bowel. A two-generation study in rats, each of two litters at
    dietary concentrations of 0, 3000, 10,000, and 30,000 ppm provided no
    indication of any adverse effects on mating performance, fertility,
    gestation, length, litter size, sex ratio or viability of the progeny.
    Food consumption was reduced in all treated groups as was weight gain
    during maturation in both the F0 and F1 generation.

         There was no increase in tumour incidence in the 2-year studies
    conducted in the mouse and the rat, and in a 1-year study in the dog
    at doses up to 3% in the diet. The depressed growth rate seen in the
    rat studies was shown to be due to the impalatability of diets
    containing TGS. However, depressed growth rate was also seen in the
    mouse study, and the mechanism for the effect in this species is not
    clear.

         No neurological effects were observed in mice and marmoset
    monkeys receiving TGS or TGS-HP.

         TGS is not clastogenic in vitro in peripheral human lymphocytes
    or in rat bone marrow cells in vivo and does not induce the formation
    of micronuclei in mice. It is not a bacterial mutagen either with or
    without metabolic activation. A 2 to 3 fold increase in the mutant
    frequency at the TK-locus in mouse lymphoma cells occurred only at
    cytotoxic concentrations (7.5 to 10 mg/ml).

         TGS (10 mg/kg b.w.) did not stimulate the secretion of insulin or
    influence the absorption of sucrose in normal subjects and no effects
    of clinical significance were observed in a study in normal subjects
    (500 mg/day for 13 weeks).

    Hydrolysis products

         TGS will slowly hydrolyse in acidic products with the formation
    of 4-chloro-4-deoxygalactose (4-CG) and 1,6-dichloro-1,6-dideoxy-
    fructose (1,6-DCF). Both are readily absorbed when administered
    orally to rats and while 4-CG is excreted essentially unchanged in the
    urine, DCF is converted to the corresponding alcohol and to
    6-chlorofructose-1-glutathione, with the liberation of inorganic
    chloride. 6-chlorofructose is a potential intermediate in 1,6-DCG
    metabolism.

         1,6-DCF was positive in the Ames-test, strain TA 1535, and in the
    mouse lymphoma assay. It did not produce chromosomal abnormalities in
    peripheral human lymphocytes or in rat-bone marrow cells after acute
    and sub-acute dosing. A mouse micronucleus assay was negative as was
    that in Drosophila melanogaster. There was no evidence for the
    production of dominant lethal mutations when mice were treated with an
    equimolar mixture of the hydrolysis products at dosages up to
    270 mg/kg bw/day.

         4-CG was negative in the Ames-test and in the mouse lymphoma
    assay. It did not produce chromosomal abnormalities in peripheral
    human lymphocytes or in rat-bone marrow cells after sub-acute dosing.

         There was some developmental toxicity produced by TGS-HP
    (TGS-hydrolysis products) in the rats, though this was only seen at
    doses producing maternal toxicity. A no-effect level may be
    established for maternal and developmental toxicity of 90 mg/kg b.w.
    TGS-HP.

         There were no adverse effects upon reproductive performance at
    levels up to 0.2% TGS-HP in the diet in a two-generation study in
    rats. A 26-week study in dogs revealed no adverse effects. There was
    no increase in tumour incidence in a carcinogenicity study in rats at
    up to 0.2% TGS-HP in the diet.

         A 26-week study in dogs revealed no changes apart from a slight
    reduction in the weight of the thymus in males receiving the
    hydrolysis products at a dietary concentration equivalent to 250 mg/kg
    b.w./day.

         The hydrolysis products were not carcinogenic when fed to CD rats
    at dietary concentrations of 0, 200, 600, and 2000 ppm for 104 weeks.
    Body weight was depressed in both sexes in the top dose group as was
    food consumption in the females. The incidence of hepatic clear cell
    foci, focal pneumonitis and pigmentation in the syncytial macrophages
    of the mesenteric lymph-nodes were increased in animals in the
    top-dose groups.

    EVALUATION

    Level causing no toxicological effect

         Rat: 30,000 ppm in the diet, equivalent to 1500 mg/kg b.w./day.

         Mouse: 10,000 ppm in the diet, equivalent to 1500 mg/kg b.w./day.

         Dog: 30,000 ppm in the diet, equivalent to 750 mg/kg b.w./day,

         Man: 500 mg/day (highest dose used) equivalent to 7.1 mg/kg
    b.w./day.

    Estimate of temporary acceptable daily intake for man

         0 - 3.5 mg/kg b.w.

    Further studies or information required

         1. Information on the absorption and metabolism of TGS in humans
    after prolonged oral dosing.

         2. Results of studies to ensure that TGS produces no adverse
    effects in insulin-dependent and maturity-onset diabetics.

         3. Results of further studies in rats on the elimination of TGS
    from pregnant animals and from the fetus to exclude the possibility of
    bioaccumulation.

         4. Results of a short-term rat study on 6-chlorofructose.

    

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    See Also:
       Toxicological Abbreviations