655. Trichlorogalactosucrose (WHO Food Additives Series 24)

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 µg 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 µg equivalents/ml). Radioactivity in plasma
declined in a multi-exponential fashion; concentrations decreased
rapidly to a mean of 0.057 µg 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 µg 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 µg
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 ¹⁴_C-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 µg/plate May, 1981
TA1535, TA1537
TA1538

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

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

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

Chromosome Human periferal 8 - 200 Negative Bootman &
aberrations lymphoctes µg/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 µg/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 µg/ml and
7,500 µg/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 µg/m
rising to between 0.02 to 0.42 µg/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 µg/plate Lodge, 1980a
TA1537; TA1535; TA1538

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

Chromosome Human periferal 40 - 1000 Negative Bootman &
aberrations (1) lymphoctes µg/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 µg/plate May, 1981
TA1537, TA1538

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

Ames test (1) S.typhimurium 16 - 10,000 Negative Bootman &
TA98, TA100 µg/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
µg/plate (2)

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

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

Gene mutation Mouse lymphoma 13 - 42 µg/ml Negative Kirby et al.,
(1) frwd. mutation 1981c
L5178Y TK+/- 56 - 133 µg/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 µg/ml 1981d
L5178Y TK+/- 10 - 40 (-S9) Negative Kirby et al.,
µg/ml 1981d
53 - 127 (-S9) Positive Kirby et al.,
µg/ml (4) 1981d

Chromosome Human periferal 1.5 - 40 µg/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 µg/ml to 2-3% at 133 µg/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 µg/ml to 3% at 127 µg/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 F1_b 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
F1_a and F1_b 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 F2_a and F2_b 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 F2_a and F2_b 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
F1_a 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 F1_a 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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rat. Unpublished report from Life Science Research Ltd., Essex, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Campbell, A.H. & Johnson, A.G. (1980). TGS: Acute oral toxicity
studies in the rat. Unpublished report from Johnson & Johnson Research
Foundation, New Brunswick, New Jersey, U.S.A. Submitted to the World
Health Organization by Tate & Lyle.

Cimino, M.C. & Lebowitz, H. (1981a). Mutagenicity evaluation of
1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-D-
galacto-pyranoside (TGS) batch C327/9470/02 in the rat bone marrow
cytogenetic assay. Unpublished report from Litton Bionetics Inc.,
Kensington, MD, U.S.A. Submitted to the World Health Organization by
Tate & Lyle.

Cimino, M.C. & Lebowitz, H. (1981b). Mutagenicity evaluation of
4-chloro-galactose (4-CG), batch P3/102/15, in the rat bone marrow
cytogenetic assay. Unpublished report from Litton Bionetics Inc.,
Kensington, MD, U.S.A. Submitted to the World Health Organization by
Tate & Lyle.

Cummins, H.A., Yailup, V., Lee, P., Ashby, R., Rhenius, S.T. &
Whitney, J. (1983). 1,6-Dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-
chloro-4-deoxy-alpha-D-galactopyranoside (TGS): Eight-week dietary
toxicity study in rats. Unpublished report from Life Science Research
Ltd., Essex, U.K. Submitted to the World Health Organization by Tate &
Lyle.

Daniel, J.W. & Rhenius, S.T. (1980). 4-Chloro-4-deoxygalactose and
1,6-dichloro-1,6-dideoxyfructose: Metabolic disposition in the rat.
Unpublished report from Life Science Research Ltd., Essex, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Daniel, J.W. & Finn, J.P. (1981). Studies in male and female mice of
the neurotoxic potential of 1,6-dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside and of an
equimolar mixture of 1,6-dichloro-1,6-dideoxyfructose and
4-chloro-4-deoxygalactose. Unpublished report from Life Science
Research Ltd., Essex, U.K. Submitted to the World Health Organization
by Tate & Lyle.

Daniel, J.W. (1987). 4,1',6'-Trichloro-4,1',6'-trideoxygalacto
sucrose: Pharmacokinetics and metabolism in the rat. Unpublished
report from Life Science Research Ltd., Essex, U.K. Submitted to the
World Health Organization by Tate & Lyle.

Danks, A., Lee, P., Ashby, R., Finn, J.P., Fowler, J.S.L. &
Willoughby, C.R. (1986). An equimolar mixture of 4-chloro-4-
deoxygalactose and 1,6-dichloro-1,6-dideoxyfructose: 13-week toxicity
study in rats with in utero exposure. Unpublished report from Life
Science Research Ltd., Essex, U.K. Submitted to the World Health
Organization by Tate & Lyle.

Das, I., Pasternak, C.A. & Hems, D.A. (1979). The influence of
4,1',6'-trichloro-4,1',6'-trideoxygalactosucrose on carbohydrate
metabolism in the rat. Unpublished report from the Department of
Biochemistry, St. George's Hospital Medical School, London. Submitted
to the World Health Organization by Tate & Lyle.

Eiseman, J.C., Alsaker, R.D., Thakur, A.K., Beaudry, N., Hepner, K.E.
& Hastings, T.F. (1985). Effects of dietary TGS on cortisone excretion
and the utilization of selected minerals in the rat. Unpublished
report from Hazleton Laboratories America Inc., Vienna, VA, U.S.A.
Submitted to the World Health Organization by Tate & Lyle.

Ford, W.C.L. (1986). The effect of 4,1',6'-trichloro-4,1',6'-
trideoxygalactosucrose (TGS) and of 6,1',6'-trichloro-6,1',6'-
trideoxysucrose (TCDS) on the glycolytic activity of rat spermatozoa.
Unpublished report from the Department of Physiology and Biochemistry,
University of Reading, U.K. Submitted to the World Health Organization
by Tate & Lyle.

Goldsmith, L.A. (1985a). Twelve month oral toxicity study in dogs:
1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-
-D-galactopyranoside (TGS). Unpublished report from Hazleton
Laboratories America Inc., Vienna, VA, U.S.A. Submitted to the World
Health Organization by Tate & Lyle.

Goldsmith, L.A. (1985b). Twenty-six week oral toxicity study in dogs
with an equimolar mixture of 4-chloro-4-deoxygalactose and
1,6-dichloro-1,6-dideoxyfructose (TGS-HP) the acid-catalysed
hydrolysis products of 1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-
chloro-4-deoxy- -D-galactopyranoside (TGS). Unpublished report from
Litton Bionetics Inc., Kensington, MD, U.S.A. Submitted to the World
Health Organization by Tate & Lyle.

Haworth, S.R., Lawlor, T.E., Smith, T.K., Williams, N.A., Simmons,
R.T., Hans, L.T. & Reichard, G.L. (1981). Salmonella/mammalian-
microsome plate incorporation mutagenesis assay (1,6-dichlorofructose).
Unpublished report from EG & G Mason Research Institute, New Brunswick,
NJ, U.S.A. Submitted to the World Health Organization by Tate & Lyle.

Hawkins, D.R., Wood, S.W. & John, B.A. (1986). Intravenous-oral cross
over dog metabolism study with ¹⁴C-1,6-dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside.
Unpublished report from Huntingdon Research Centre, Huntingdon, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Hawkins, D.R., Wood, S.W. et al., (1987a). Isolation and
identification of an unknown radioactive component present in dog
urine after intravenous administration of ¹⁴C-1,6-dichloro-1,6-
dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside.
Unpublished report from Huntingdon Research Centre, Huntingdon, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Hawkins, D.R., Wood, S.W. & John, B.A. (1987b). Studies on the
metabolism of ¹⁴C-1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-
chloro-4-deoxy-alpha-D-galactopyranoside in the rabbit. Unpublished
report from Huntingdon Research Centre, Huntingdon, U.K. Submitted to
the World Health Organization by Tate & Lyle.

Hawkins, D.R., Wood, S.W. & John, B.A. (1987c). Studies of the
metabolism of ¹⁴C-1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-
chloro-4-deoxy-alpha-D-galactopyranoside in the mouse. Unpublished
report from Huntingdon Research Centre, Huntingdon, U.K. Submitted to
the World Health Organization by Tate & Lyle.

Hawkins, D.R., Wood, S.W., Waller, A.R. & Jordan, M.C. (1987d). Enzyme
induction studies of TGS and TGS-HP in the rat. Unpublished report
from Huntingdon Research Centre, Huntingdon, U.K. Submitted to the
World Health Organization by Tate & Lyle.

Hepworth, P.L. & Finn, J.P. (1981). Studies in male marmoset monkeys
of the neurotoxic potential of 1,6-dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside and of an
equimolar mixture of 1,6-dichloro-1,6-dideoxyfructose and
4-chloro-4-deoxygalactose. Unpublished report from Life Science
Research Ltd., Essex, U.K. Submitted to the World Health Organization
by Tate & Lyle.

Hughes, H.M., Curtis, C.G. & Powell, G.M. (1987a). 4-Chloro-4-deoxy-U-
¹⁴C-galactose: metabolism in the rat. Unpublished report from the
Department of Biochemistry, University College, Cardiff. Submitted to
the World Health Organization by Tate & Lyle.

Hughes, H.M., Curtis, C.G. & Powell, G.M. (1987b). 1,6-Dichloro-1,6-
dideoxyfructose: the metabolism and dechlorination in the rat.
Unpublished report from the Department of Biochemistry, University
College, Cardiff. Submitted to the World Health Organization by Tate &
Lyle.

Jagannath, D.R. & Goode, S. (1979). Mutagenicity evaluation of C327,
Batch No. P3/49-557, in the Ames Salmonella/microsome plate test.
Unpublished report from Litton Bionetics Inc., Kensington, MD, U.S.A.
Submitted to the World Health Organization by Tate & Lyle.

Jenkins, W.R. (1984). Acute toxicity of 1,6-dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside (TGS) to
Daphnia magna. Unpublished report from Aquatox Ltd., Brixham, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Kirby, P.E., Pizzarello, R.F., Cohen, A., Williams, P.E., Reichard,
G.L, Johnson, J.L., Wattam, R.E. & Clarke, J.J. (1981a). Evaluation of
test article TGS (MRI No. 630) for mutagenic potential employing the
L5178Y TK+/- mutagenesis assay. Unpublished report from EG & G Mason
Research Institute, Rockville, MD, U.S.A. Submitted to the World
Health Organization by Tate & Lyle.

Kirby, P.E., Pizzarello, R.F., Williams, P.E., Reichard, G.L., Wattam,
R.E., Johnson, J.L., Clarke, J.J., Condon, M.B. & Madden, G. (1981b).
Evaluation of test article 4-chlorogalactose (MRI No. 628) for
mutagenic potential employing the L5178Y TK+/- mutagenesis assay.
Unpublished report from EG & G Mason Research Institute, Rockville,
MD, U.S.A. Submitted to the World Health Organization by Tate & Lyle.

Kirby, P.E., Pizzarello, R.F., Brauninger, R.M., Vega, R.A. &
Reichard, G.L. (1981c). Evaluation of test article 1,6-dichlorofructose
(MRI No. 536) for mutagenic potential employing the L5178Y TK+/-
mutagenesis assay. Unpublished report from EG & G Mason Research
Institute, Rockville, MD, U.S.A. Submitted to the World Health
Organization by Tate & Lyle.

Kirby, P.E., Pizzarello, R.F., Cohen, A., Williams, P.E., Reichard,
G.L., Johnson, J.L., Clarke, J.J., Condon, M.B. & Wattam, R.E.
(1981d). Evaluation of test article 1,6-dichlorofructose (MR1 No. 629)
for mutagenic potential employing the L5178Y TK+/- mutagenesis assay.
Unpublished report from EG & G Mason Research Institute, Rockville,
MD, U.S.A. Submitted to the World Health Organization by Tate & Lyle.

Lightowler, J.E. & Gardner, J.R. (1977). 3/C327: Acute oral toxicity
to mice. Unpublished report from Life Science Research Ltd., Essex,
U.K. Submitted to the World Health Organization by Tate & Lyle.

Rhenius, S.T., Ryder, J.R. & Amyes, S.J. (1986). 1,6-Dichloro-1,6-
dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside
(TGS): 104-week combined toxicity and oncogenicity study in CD rats
with in utero exposure. Section IV: Measurement of metabolic
adaptation. Unpublished report from Life Science Research Ltd., Essex,
U.K. Submitted to the World Health Organization by Tate & Lyle.

Roberts, A., Renwick, A.G. & Sims, J. (1986). ¹⁴C-TGS: A study of the
metabolism and pharmacokinetics following oral administration to
healthy human volunteers. Unpublished report from the Clinical
Pharmacology Group, University of Southampton, England. Submitted to
the World Health Organization by Tate & Lyle.

Roberts, A., Renwick, A.G. & Sims, J. (1987). A study of the
metabolism of 1,6-dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-chloro-4-
deoxy- -D-galactopyranoside (TGS) after oral and intravenous
administration to the rat. Unpublished report from the Clinical
Pharmacology Group, University of Southampton, England. Submitted to
the World Health Organization by Tate & Lyle.

Rodgers, P.B., Jenner, M.R. & Jones, H.F. (1986). Stability of
chlorinated disaccharides to hydrolysis by microbial plant and
mammalian glycosidases. Unpublished report from Tate & Lyle Group
Research & Development, Reading, U.K. Submitted to the World Health
Organization by Tate & Lyle.

Shephard, N.W. & Rhenius, S.T. (1983). 1,6-Dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside. Absorption
and excretion in man. Unpublished report from Medical Science
Research, Beaconsfield, U.K. Submitted to the World Health
Organization by Tate & Lyle.

Shephard, N.W. (1984). A randomised double-blind study in normal
subjects to investigate the influence of 1,6-dichloro-1,6-dideoxy-ß-
D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside (TGS) on
the absorption of sucrose and the secretion of insulin. Unpublished
report from Medical Science Research, Beaconsfield, U.K. Submitted to
the World Health Organization by Tate & Lyle.

Shephard, N.W. & Kyffin, P. (1984). A tolerance study in normal
subjects of varying doses of TGS administered continuously for a
period of 13 weeks. Unpublished report from Medical Science Research,
Beaconsfield, U.K. Submitted to the World Health Organization by Tate
& Lyle.

Smyth, D.V. (1986). Sucralose: Determination of toxicity to the green
alga Selenastrum capricornutum. Unpublished report from Imperial
Chemical Industries Brixham Laboratory, Brixham, U.K. Submitted to the
World Health Organization by Tate & Lyle.

Street, J.R. (1985). Trichlorogalactosucrose (TGS): Acute toxicity to
bluegill sunfish (Lepomis macrochirus). Unpublished report from
Imperial Chemical Industries Brixham Laboratory, Brixham, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Tesh, J.M., Willoughby, C.R., Hough, A.J., Tesh, S.A. & Wilby, O.K.
(1983a). 1,6-Dichloro-1,6-dideoxy-ß-D-fructofuranosyl-4-chloro-4-
deoxy-alpha-D-galactopyranoside (TGS): Effects of oral administration
upon pregnancy in the rat. Unpublished report from Life Science
Research Ltd., Essex, U.K. Submitted to the World Health Organization
by Tate & Lyle.

Tesh, J.M., Willoughby, C.R., Tesh, S.A. & Wilby, O.K. (1983b). An
equimolar mixture of the hydrolysis products of 1,6-dichloro-1,6-
dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside
(TGS): Effects of oral administration upon pregnancy in the rat.
Unpublished report from Life Science Research Ltd., Essex, U.K.
Submitted to the World Health Organization by Tate & Lyle.

Tesh, J.M. & Willoughby, C.R. (1986a). 1,6-Dichloro-1,6-dideoxy-ß-D-
fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside (TGS): Two
generation reproductive study in rats. Unpublished report from Life
Science Research Ltd., Essex, U.K. Submitted to the World Health
Organization by Tate & Lyle.

J.M. & Willoughby, C.R. (1986b). An equimolar mixture of
4-chloro-4-deoxygalactose and 1,6-dichloro-1,6-dideoxyfructose: Two
generation reproductive study in rats. Unpublished report from Life
Science Research Ltd., Essex, U.K. Submitted to the World Health
Organization by Tate & Lyle.

Tesh, J.M., Ross, F.W. & Bailey, G.P. (1987). 1,6-Dichloro-1,6-
dideoxy-ß-D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside
(TGS): Teratology study in the rabbit. Unpublished report from Life
Science Research Ltd., Essex, U.K. Submitted to the World Health
Organization by Tate & Lyle.

Willis, C.A. (1984). The acute toxicity of 1,6-dichloro-1,6-dideoxy-ß-
D-fructofuranosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside (TGS) to
rainbow trout. Unpublished report from Aquatox Ltd., Brixham, U.K.
Submitted to the World Health Organization by Tate& Lyle.

    See Also:
       Toxicological Abbreviations