TRICHLOROGALACTOSUCROSE
First draft prepared by Dr D.L. Grant,
Toxicological Evaluation Division
Health and Welfare Canada.
1. EXPLANATION
Trichlorogalactosucrose (TGS) has been evaluated previously by
the 33rd Joint FAO/WHO Expert Committee on Food Additives in 1989
(see Annex 1, reference 83). The Committee allocated a temporary
ADI of 0-3.5 mg/kg b.w. and indicated that the following further
studies or information were 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 people with insulin-dependent and maturity-
onset diabetes.
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.
Trichlorogalactose (1,6-dichloro-1,6-dideoxy-beta-D-fructo-
furanosyl-4-chloro-4-deoxy-alpha-D-galactopyranoside) (TGS), is
derived from sucrose by selective replacement of the three hydroxy
groups at positions 4',1' and 6' by chlorine atoms, which greatly
increases sweetness. At room temperature, in water, TGS is 600-650
times sweeter than 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. Under
the extreme conditions (treatment of TGS with 0.11N aqueous
hydrochloric acid at 68 °C for 72 h) used to produce sufficient
quantities of 4CG and 1,6-DCF for toxicological testing, an aqueous
solution was obtained composed of: 1,6-dichlorofructose (47.5%); 4-
chlorogalactose (49.1%); 6-chlorofructose (0.3%); 1-chlorofructose
(0.2%) and TGS (1.2%).
Since the previous evaluation, the results of additional
studies and additional information have become available. At the
present meeting, the Committee evaluated new and existing data
which, are summarized and discussed in the following addendum to the
monograph.
2. BIOLOGICAL DATA
2.1 Special study on palatability
2.1.1 Rat
Groups of 10 female Sprague-Dawley, CD strain rats had free
access to two bottles containing either tap water or TGS solution
at concentrations ranging from 20 2560 mg/ml for 32 days. Rats
showed a range of individual variability in their preference for TGS
vs. water. As a group, however, they displayed a distinct
preference for TGS over water at concentrations up to 320 mg/100 ml.
At concentrations above 640 mg/100 ml, water was preferred over the
TGS solution. There was no mortality and no signs of clinical
toxicity were observed (Amyes & Aughton, 1987).
Groups of 20 young female Sprague-Dawley rats, Crl: CDBR
strain had access to two feeding jars containing either basal diet
or a mixture of basal diet containing TGS. The starting
concentration of TGS was 50 ppm and was doubled every fourth day to
a final concentration of 3200 ppm (32 days). No statistically
significant difference in the selection of basal diet or TGS
containing diet was observed at 50 or 100 ppm. A statistically
significant preference for TGS diet was observed at 200 ppm. At the
400 ppm concentration, basal and TGS-containing diets were consumed
in similar amounts. At TGS concentration of 800 ppm or higher, a
statistically significant preference was displayed for basal diet.
The total intake of food by treated rats was similar to that of
their controls. No death, nor apparent effect on the appearance,
behavior, or body weight was reported (Amyes & Aughton, 1988).
2.2 Short-term studies
2.2.1 Rat
Groups of Sprague-Dawley rats were given TGS (dissolved in
water) by gavage at dose levels of 4000 mg/kg bw/day (15 rats/sex)
for periods of 4, 9, or 13 weeks, respectively. Corresponding
control groups (receiving water) with the same number of animals as
the treated groups were included. A significant increase of caecal
weights was observed in rats of both sexes at all dose levels.
Sporadic significant changes in some parameters of haematology and
clinical chemistry and some differences in organ weights (in some
groups at some time points) were observed. The changes were not
clearly dose-related and not considered as treatment-related. Other
investigated parameters were not adversely affected, these included:
clinical signs of toxicity, mortality, body weight gains, food and
water consumption, urinalysis, gross and histopathology (Perry et
al., 1988).
4-CHLOROGALACTOSE(4-CG) and 1,6-DICHLOROFRUCTOSE (1,6-DCF)
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution and excretion
No information available.
2.1.2 Biotransformation
The metabolic fates of 1,6-DCF and its metabolites were
evaluated and included experiments to determine the potential for
1,6-DCF to metabolize to 6-chlorofructose (6-CF).
An anaesthetized rat with bile duct and ureter cannulas was
injected intravenously with a single dose of 14C-6-chloro-6-
deoxyfructose (14C-6CF; 1.1 µCi) at a level of 1.4 mg/kg b.w. Bile
and urine samples were collected up to 5 h and analyzed for
radioactivity. The radioactivity in the blood was also determined.
About 17% of the 14C(14% in urine and 3% in bile) was detected
within 5 h. No significant amounts or radioactivity were detected
in blood. Thin layer chromatography of urine revealed 5 radioactive
components, none of which co-chromatographed with 1,6-dichloro-1,6-
dideoxymannitol (DCM) or with 6-CF.
Two anaesthetized rats with ureter cannulas were given 14C-
1,6-dichloro-1,6,dideoxyfructose (14C-1,6-DCF; 4.4 µCi)
intraduodenally at a dose level of 5 mg/kg bw. Maximum levels of
14C in blood occurred 15 min. after dosing. The level decreased
with time although radioactivity (about 12% of the dose) was still
present at 360 min. The distribution of radioactivity between RBC
and plasma was constant between 15 and 360 min. About 80% of the
14C dosage was associated with the RBC. About 50% of the 14C
dosage was eliminated in the urine within 360 min.
Three rats with cannulated bile ducts received intravenously a
single dose of 14C-1,6-dichloro-1,6-dideoxyfructose (14C-1,6-DCF)
at a level of 100 mg/kg bw and urine samples were collected for the
following 6 h. The major urinary metabolite was DCM. Other
metabolites were widely distributed over various fractions and were
not identified (Hughes et al., 1988).
2.2 Genotoxicity
2.2.1 Results of mutagenicity assays with TGS-HP
Test System Test Object Concetration Result Reference
Dominant Mouse 30-270 mg/kg Negative Bootman &
lethal gavage 5 day bw/day Whalley, 1983
assay (in vivo)
2.2.2 Results of mutagenicity assays with 1,6-DCF
Test System Test Object Concentration Results Reference
of 1,6-DCF
Micronucleus Mouse, 415-1600 mg/kg bw Negative Ivett, 1988a
assay gavage,
bone marrow
(in vivo)
Micronucleus Mouse, 1000-2500 mg/kg bw Negative Ivett, 1988b
assay gavage,
bone marrow
(in vivo)
Sister chromatid Mouse, 200-2000 mg/kg bw Negative Ivett, 1988
exchanged gavage,
bone marrow
(in vivo)
Covalent DNA Rat, gavage 21 mg/kg bw Negative Martin, 1989
binding (liver, (in vivo)
kidney, small
intestine, colon,
stomach, bone
marrow)
6-CHLOROFRUCTOSE(6-CF)
2. BIOLOGICAL DATA
No information available.
2.2 Toxicological studies
2.2.1 Special studies on neurotoxicity
2.2.2.1 Mouse
Groups of male CD-1 mice (8 mice/group) were dosed daily (by
gavage) with 6-chlorofructose (6-CF) (one of the TGS hydrolysis
products obtained by treating TGS with aqueous hydrochloric acid
[0.11N] at 68°C for 72 h) at dose levels of 240 or 480 mg/kg b.w.
day for 28 days. Mice were weighed daily and examined for hind limb
paralysis at the same time. Negative control group animals were
given water while positive controls received 6-chloroglucose (6-CG)
at a level of 480 mg/kg b.w./day. The results showed that 6-CF at
both dose levels caused dose-related hind-limb paralysis in some
treated animals (240 mg/kg bw/day: 2/8; 480 mg/kg b.w./day: 7/8)
starting between 4 to 6 days post-dosing (Ford & Waites, 1982).
2.2.3 Special studies on reproduction
2.2.3.1 Rat
Groups of mature male CD rats (3-8 rats/group) were exposed
orally to 6-CF at dose levels of 6, 12, 18, or 48 mg/kg b.w./day for
14 days. Treated male rats were mated with untreated females during
the final 7 days of dosing. Mated females were killed 10 days after
mating and live embryos, resorptions and the corpora lutea were
counted. The results showed that males exposed to 6-CF at dose
levels of 18 or 48 mg/kg b.w./day became infertile (conception rate:
control - 79%; treated groups - 0%). Three weeks after cessation of
treatment the conception rates had recovered to the level of control
groups. The lowest dose level of 6 mg/kg/b.w./day did not have any
adverse effect on the fertility of male rats (Ford & Waites, 1978a;
Ford et al., 1981; Ford & Waites, 1982).
Three groups of male Sprague Dawley rats of the CD strain (6
rats/group) were given 6-chlorofructose dissolved in water by gavage
twice daily at dose levels of 0, 3 or 9 mg/kg b.w./day for 10 weeks.
An additional group consisting of 6 males received 6-chlorofructose
twice daily at a level of 27 mg/kg b.w./day for 4 weeks. After 7
days of treatment, males were paired on a one-to-one basis with
untreated females. Each morning the females were examined for
copulation plugs and presence of spermatozoa in a vaginal smear.
Quantity and quality of spermatozoa were assessed. Females which
had not conceived during the first four days of pairing were
sacrificed and their reproductive organs examined. The pairing
procedure was repeated at weekly intervals for 10 or for 8 weeks
except for males of the 27 mg/kg b.w./day dose group which were
paired weekly for 4 weeks. Males were observed daily for signs of
clinical toxicity and mating behavior. Body weight gains were
recorded weekly. After completing the series of matings, the males
were killed, necropsied and the following organs weighed: testes,
epididymis, prostate and seminal vesicle. Testes and epididymis of
the control and 9 mg/kg b.w./day dose groups were
histopathologically examined. On days 8-10 post coitum, females
were sacrificed and the uterine horns checked for the presence of
implantation sites. For each male, mating performance, conception
rate and fertility index were calculated.
Males receiving 27 mg/kg b.w./day became infertile for the
entire treatment period (4 weeks), but regained their fertility
within the first 7 days of recovery period. Females mated by males
of the 9 mg/kg b.w./day dose group showed a reduction in the number
of implantation sites, indicating a possible decrease in male
fertility. The fertility of males of the 3 mg/kg b.w./day dose
group was not affected (Tesh, et al., 1984).
Acute toxicity TGS-HP
Species Sex Route LD50 Reference
(mg/kg bw)
Rat female oral 4450 Campbell et al.
1980
3. COMMENTS
At its present meeting the Committee reviewed new and
previously available data. Although no new data on the absorption
and metabolism of TGS in humans were received, the Committee
concluded that there was no indication that these processes would
change on prolonged oral dosing. This conclusion was drawn from the
comparative metabolic data for TGS in various species, including
humans, and the lack of evidence of toxicity in extensive animal
studies. Nevertheless, the Committee recognized that the data did
not address all possibilities, particularly the potential effects of
adaptation of the gastrointestinal microflora.
No specific studies on possible adverse effects of TGS in
people with insulin-dependent and maturity-onset diabetes had been
performed. However, the Committee decided that this concern could
be satisfactorily addressed through consideration of data which
showed that TGS had no effect on the secretion of insulin in humans
or rats, blood glucose levels or carbohydrate metabolism.
Furthermore, the Committee was aware of proposed studies invovling
both types of diabetics.
On re-assessment of the overall data on TGS, including the
metabolic data in various species, including humans and pregnant and
non-pregnant rabbits, and in the absence of any significant finding
in the two-generation reproduction study in rats, the Committee
concluded that the question on the accumulation of TGS in pregnant
animals and fetuses was satisfactorily addressed, and that there was
no evidence to suggest a difference in metabolism in pregnant and
non-pregnant animals.
The Committee reviewed additional studies relating to the
possible toxicity of the potential breakdown product of TGS,6-
chlorofructose. In a short-term study (28 days) in which 6-
chlorofructose was administered at 240 and 480 mg per kg of body
weight per day, male mice showed hind-limb paralysis. In addition,
three special studies were conducted to assess reproductive function
in rats. Administration of 6-chlorofructose at 18-48 mg per kg of
body weight per day for 7-14 days caused a loss of fertility in male
rats. In two of these studies, the no-effect-levels were 3 and 6 mg
per kg of body weight per day. The Committee noted, however, that
6-chlorofructose is only a potential breakdown product of TGS.
While a hypothetical maximum exposure of 1.15 µg per kg of body
weight per day to humans would occur if TGS were subject to extreme
conditions, such as 0.1 mol/l GC1 at 68 °C for 72 hours, the
Committee expected that exposure to 6-chlorofructose would be
virtually nil under all foreseeable storage or physiological
conditions.
The Committee received a request to reconsider the title
"trichlorogalactosucrose" it had adopted at its thirty-third meeting
(Annex 1, reference 83) and to rename it "sucralose". At that time,
the Committee established guidelines for designating titles for
specifications monographs and selected the title in accordance with
those guidelines. The Committee considered the information
submitted at the present meeting and concluded that criteria 1
(names established by international organizations) and 2 (names
established by governmental legislation) were not applicable. It
did not believe that the information submitted in relation to
criterion 3 (names established by common usage) was sufficiently
compelling to change the name, and reaffirmed its view, reached in
relation to criterion 4 (available scientific, common or trivial
names), that "trichlorogalactosucrose" was appropriate. In this
regard, the Committee noted that the name "sucralose" had been
designated as a synonym in the specifications monograph. The
existing tentative specifications were revised and the Committee
agreed to deleted the "tentative" classification.
4. EVALUATION
Finally, the Committee concluded that since the 2-year study in
rats, which included a period of exposure to TGS in utero,
represented a greater proportion of the lifetime of the species than
did the 1-year study in dogs, the former study should be used for
the purposes of setting an ADI. A safety factor of 100 was
therefore applied to the no-observed-effect level in the long-term
study in rats (1500 mg per kg of body weight per day), and an ADI of
0-15 mg per kg of body weight was allocated.
Additional immunotoxicity studies to assess the significance of
observed weight changes in the spleen and thymus and changes in
lymphocyte counts in rats were considered to be desirable. As yet,
a causal relationship between these findings and high levels of
exposure to TGS cannot be excluded.
5. REFERENCES
AMYES, S.J. & AUGHTON, P. (1987). Acceptability of aqueous
solutions of sucralose, saccharin and aspartame to female rats.
Unpublished report for Life Science Research Ltd., Suffolk, U.K.
Submitted to the World Health Organization by Tate & Lyle.
AMYES, S.J. & AUGHTON, P. (1988). An investigation of the
acceptability of rats of diet containing sucralose. Unpublished
report for Life Science Research Ltd., Suffolk, U.K. Submitted to
the World Health Organization by Tate & Lyle.
BOOTMAN, J & WHALLEY, H.E. (1983). Dominant lethal study in the
mouse after subacute treatment with an equimolar mixture of the
hydrolysis products derived from TGS. Unpublished report from the
Life Science Research Ltd., Essex, U.K. Submitted to the World
Health Organization by Tate & Lyle.
CAMPBELL, A.H., HARKINS, R.W. & LORD, G.H. (1980). 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.
FORD, W.C.L. & WAITES, G.M.H. (1978a). Chlorinated sugars: A
biochemical approach to the control of male fertility. Int. J.
Andrology, Suppl., 2, pp. 541-564.
FORD, W.C.L. & WAITES, G.M.H. (1978b). A reversible contraceptive
action of some 6-chloro-6-deoxy sugars in the male rat. J. Reprod.
Fert., 52, 153-157.
FORD, W.C.L., HARRISON, A. & WAITES, G.M.H. (1981). Effects of 6-
chloro-6-deoxysugars on glucose oxidation in rat spermatozoa. J.
Reprod. Fert., 63, 67-73.
FORD, W.C.L. & WAITES, G.M.H. (1982). Activities of various 6-
chloro-6-deoxysugars in (S)a-chlorohydrin in producing
spermatocoeles in rats and paralysis in mice and in inhibiting
glucose metabolism in bull spermatozoa in vitro. J. Reprod.
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HUGHES, H.M., CURTIS, C.G. & POWELL, G.M. (1988). 1,6-Dichloro-1,6-
dideoxyfructose: metabolism in the rat (supplementary report).
Unpublished report from University College, Cardiff, U.K. Submitted
to the World Health Organization by Tate & Lyle.
IVETT, J.L. (1988a). 1,6-Dichloro-1,6-dideoxy-D-fructose: in
vivo micronucleus assay in the mouse. Unpublished report from the
Hazleton Laboratories America, Inc., Maryland, U.S.A. Submitted to
the World Health Organization by Tate & Lyle.
IVETT, J.L. (1988b). 1,6-Dichloro-1,6-dideoxy-D-fructose: single
exposure dose selection study for the in vivo micronucleus assay
in the mouse. Unpublished report from the Hazleton Laboratories
America, Inc., Maryland, U.S.A. Submitted to the World Health
Organization by Tate & Lyle.
IVETT, J.L. (1988c). 1,6-Dichloro-1,6-dideoxy-D-fructose: in vivo
sister chromatid exchange assay in the mouse. unpublished report
from the Hazleton Laboratories America, Inc., Maryland, U.S.A.
Submitted to the World Health Organization by Tate & Lyle.
MARTIN, C.M. (1988). Study to evaluate the covalent DNA binding
potential of 1,6-dichlorofructose in organs of treated rats.
Unpublished report from the Microtest Research Limited, York, U.K.
Submitted to the World Health Organization by Tate & Lyle.
PERRY, C.J., HUDSON, P. & FINN, J.P. (1988). Sucralose: Special
investigation in the rat of the effect of administration by oral
gavage on body weight and selected organs. Unpublished report from
Inveresk Research International, Musselburgh, Scotland. Submitted
to the World Health Organization by Tate & Lyle.
TESH, J.M., ROSS, F.W., WILLOUGHBY, C.R. & SECKER, R.C. (1984). 6-
Chlorofructose: Antifertility evaluation in the male rats.
Unpublished report from the life Science Research Ltd., Suffolk,
U.K. Submitted to the World Health Organization by Tate & Lyle.
See Also:
Toxicological Abbreviations