ß-CYCLODEXTRIN
First draft prepared by
Professor R. Walker
School of Biological Sciences
University of Surrey, Guildford U.K.
1. EXPLANATION
ß-Cyclodextrin is a cyclic heptamer composed of seven glucose
units joined "head-to-tail" by alpha-1,4 links. It is produced by
the action of the enzyme, cyclodextrin glycosyl transferase (CGT),
on hydrolyzed starch syrups. CGT is obtained from Bacillus
macerans, B. circulans or related strains of Bacillus.
As a result of its cyclic structure, ß-cyclodextrin has the
ability to form inclusion compounds with a range of molecules,
generally of molecular mass of less than 250. It may serve as a
carrier and stabilizer of food flavours, food colours and some
vitamins. Intake of ß-cyclodextrin from use as a food additive has
been estimated at 1-1.4 g/day. Other applications in decaffeination
of coffee/tea and in reducing the cholesterol content of eggs by
complexation followed by separation of the complex would make a much
lower contribution to intakes.
ß-Cyclodextrin has not been reviewed previously by the Joint
FAO/WHO Expert Committee on Food Additives.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1. Absorption, distribution, and excretion
ß-cyclodextrin is resistant to hydrolysis by acid (Szeftli &
Budai, 1976), alpha- and ß-amylases and yeast. It is thus not
readily digested in the upper gastrointestinal tract by gastric or
pancreatic enzymes (Jodal et al. 1984).
In a preliminary comparative metabolism study in rats, groups
each of 2 animals were given 14C-labelled alpha-cyclodextrin,
ß-cyclodextrin or gelatinised potato starch by gavage as
approximately 2.5 ml of a 2.5% aqueous solution. Urine, faeces and
expired air were collected for 17-23 hours after which residual
radioactivity was assayed in the gastro-intestinal tract (caecum and
caecal contents separately), liver, kidneys, heart, lung, spleen,
gonads and residual carcass. Animals receiving ß-cyclodextrin
metabolized the compound more slowly than starch, as indicated by
the time course of elimination of 14CO2 in expired air, but by the
end of collection the total amount of the dose excreted by this
route (48.6% and 66.8% of the dose as 14CO2 after 17 and 23 hours
respectively) was similar to that of the group given starch;
excretion of activity in the urine (3.6-5.1%) and faeces (0-5.4%),
and the residual levels in organs and carcass were also similar in
the groups given ß-cyclodextrin and starch (Andersen et al.
1963).
ß-Cyclodextrin, purity >77%, and glucose, uniformly labelled
with 14C, were administered to rats by gavage as solutions in 20%
aqueous dextran at dose levels of 13 mg/kg bw for glucose and 36 and
313 mg/kg bw for ß-cyclodextrin. In the case of glucose, blood
levels of radioactivity peaked within 10-30 minutes of dosing
whereas with the lower dose of ß-cyclodextrin, peak blood levels
were observed between 4 and 11 hours following administration. At
the lower dose level of ß-cyclodextrin, the respired radioactivity,
as a percentage of the dose, was similar to that after dosing with
glucose whereas at the higher dose a smaller percentage was
respired. In the eighth hour after the high dose (313 mg/kg bw) no
more than 3-50ppm ß-cyclodextrin was detectable in blood. It was
concluded that ß-cyclodextrin is not absorbed to a significant
extent from the stomach or small intestine of rats but that
hydrolysis to open chain dextrins/glucose occurs in the large
intestine by a combination of the action of the gut microflora and
endogenous amylases. This process may be saturated, and unabsorbed
material can be excreted in faeces at high dose levels. The oxygen
consumption of slices of rat small intestine was measured in the
presence of glucose, maltose, starch and ß-cyclodextrin as
substrates; the rate of oxygen consumption was increased by all
substrates except ß-cyclodextrin (Szejtli et al. 1980; Gerlóczy
et al. 1981; 1985).
The intestinal absorption, digestibility by the colonic
microflora, and urinary excretion of ß-cyclodextrin were studied.
Using everted sacs of rat small intestine in vitro and ligated gut
loops in vivo, absorption was shown to be slow, concentration-
dependent, not saturable and not inhibited by phloretin; this
indicates that a passive transport process is involved. Rat caecal
microflora were able to utilize ß-cyclodextrin under anaerobic
conditions in vitro, indicating that the compound may be
hydrolyzed to glucose by bacterial enzymes. It was concluded by the
authors that ß-cyclodextrin may be utilized by the rat but only
indirectly by the activity of the gut flora (Szabo et al.
1981a,b).
Fasted male Sprague-Dawley rats were given a dose of 1 500 mg
ß-cyclodextrin (about 3 235 mg/kg bw). Only small amounts (0.6-4%
of the dose) were excreted in the faeces in a 60 hour period post-
dosing and negligible amounts (0-0.9% of the dose) remained in the
gastrointestinal tract. In a separate experiment using a similar
dose, a large proportion was shown to be converted to glucose
between 3 and 8 hours after dosing (Suzuki & Sato, 1985).
In a 13 week short-term study in beagle dogs (see section
2.2.2.3) animals received ß-cyclodextrin at dietary levels of 1.25,
2.5, 5 or 10%, equal to mean daily doses of 570, 1 234, 2 479 or 4
598 mg ß-cyclodextrin/kg bw. Twenty-four-hour urine samples were
analyzed for ß-cyclodextrin at weeks 7 and 13, faeces at week 13 and
serum was collected 1, 3, and 6 hours after dosing during week 13.
The urinary concentrations of ß-cyclodextrin at week 7 were
332±76, 510±163, 1 222±491 and 4 218±1 833 mg/l in the respective
dose groups; the corresponding values at week 13 were 631±707,
467±335, 817±395 and 2 206±528 mg/l. Although, because of the daily
dietary dosing regime, it is not possible simply to express the
amount excreted as a percentage of dose, approximately 0.6-1.7% of
the mean 24-hour dose was excreted in 24-hour urine and the
percentage was not dependent on dose. In week 13, faecal
concentrations of ß-cyclodextrin (based on dry matter) in the
respective dose groups were 1.3, 1.1, 1.8 and 6.5%. Serum levels of
ß-cyclodextrin increased in a dose-dependent manner and with time
after dosing up to 6 hours. No ß-cyclodextrin was detected in the
lowest dose group and only low levels (1 mg/l increasing to 5 mg/l
in the 2.5% dose group; serum levels increased from 4 to 8 mg/l in
1 to 6 hours in the 5% dose group and from 8 mg/l to 46 mg/l in the
10% group. These results indicate that a small proportion of the
dose may be absorbed from the gastrointestinal tract in the dog
(Smith et al. 1992).
2.1.2. Biotransformation
Twenty-four out of thirty strains of Bacteroides isolated
from the human colon were able to degrade ß-cyclodextrin and utilize
it as a sole carbon source. Detailed examinations carried out on
the dextrinases from two strains, Bacteroides ovatus 3524 and
Bacteroides distasonis C18-7 indicated that cyclodextrinase
activity was predominantly cell bound and inducible in both
organisms. The products of hydrolysis of crude cyclodextrinase
preparations from these two organisms were markedly different, the
former producing only glucose whereas the latter produced a series
of maltooligomers (Antenucci & Palmer, 1984).
In vitro, ß-cyclodextrin was resistant to hydrolysis by
purified alpha-amylase from Aspergillus oryzae. On incubation for
24 h at an initial ß-cyclodextrin concentration of 15.8 mM, only 17%
was degraded; the breakdown products were glucose (12%), maltose
(2%) and maltotriose (3%) (Jodal et al. 1983).
2.2. Toxicological Studies
2.2.1. Acute toxicity studies
The results of acute toxicity studies with ß-cyclodextrin are
summarized in Table 1.
Table 1. Results of acute toxicity studies with ß-cyclodextrin.
Species Sex Route LD50 Reference
(mg/kg bw)
Mouse M oral >3 000 Sebestyén (1980)
Mouse F oral >3 000 Sebestyén (1980)
Mouse M i.p. 372 Sebestyén (1980)
Mouse F i.p. 331 Sebestyén (1980)
Mouse M s.c. 419 Sebestyén (1980)
Mouse F s.c. 412 Sebestyén (1980)
Rats M oral >5 000 Sebestyén (1980)
Rats F oral >5 000 Sebestyén (1980)
Rats M i.p. 373 Sebestyén (1980)
Rats F i.p. 356 Sebestyén (1980)
Rats M & F i.v. 788 Frank et al. (1976)
Rats M s.c. >1 000 Sebestyén (1980)
Rats F s.c. >1 000 Sebestyén (1980)
Rats M & F dermal >2 000 Sebestyén (1980)
Rats M & F inh >4.9* Busch et al. 1985
Dog M oral >5 000 Sebestyén (1980)
Dog F oral >5 000 Sebestyén (1980)
* milligrams/litre of air for 4 hours
2.2.2. Short term toxicity studies
2.2.2.1. Mice
In a repeat-dose study of very limited scope which was not
reported in detail, mature male mice (number not stated) were given
daily oral doses of 6 ml of a 1% solution of ß-cyclodextrin for 15
days. No effects were observed on body weight or relative liver
weight. ß-Cyclodextrin was detected in excreta (unclear whether
urine and/or faeces) by paper chromatography but could not be
detected in liver or gastrointestinal tract (limit of detection not
stated)(Miyazaki et al. 1979).
2.2.2.2. Rats
In a 13 week oral toxicity study in Long-Evans rats, body
weight 80-100 g at commencement, ß-cyclodextrin (purity not
specified) was administered by gavage as a suspension in aqueous 1%
methylcellulose to groups of 10 male and 10 female animals at daily
dose levels of 200, 400 or 600 mg/kg bw; controls (15 animals of
each sex) received an equal volume of 1% methylcellulose solution.
Following high mortality (4 males, 3 females) due to misdosing in
the 400 mg/kg bw group, a repeat experiment was carried out at this
dose level with a separate control group of 5 animals. Body weight
and food intake were recorded weekly and urinalysis, haematological
and clinical biochemical were performed at termination. At autopsy,
heart, lung, liver, kidneys and spleen were weighed, and examined
macroscopically and histologically together with gonads, stomach,
intestine, pancreas, adrenals and brain.
No abnormal clinical signs were observed and there were no
deaths other than 1 male in the low-dose group and 1 female in the
high-dose groups, attributed to misdosing. There were no
significant treatment-related changes in mean body weight, food
consumption, or relative organ weights at termination. No effects
due to ß-cyclodextrin were observed in haematological parameters
(Hb, haematocrit, MCH, RBC, total and differential leucocytes) or in
the clinical biochemical indices ASAT, Alk-P-ase, BUN, bilirubin or
creatinine; dose-related but non-statistically significant changes
were reported for ALAT in females (decreased) and Alk-P-ase
(decreased in males, increased in females) but these were not
considered to be of toxicological relevance. Urinalysis for colour,
pH, protein, glucose, urobilinogen, bilirubin, ketones and sediment
gave similar results between treated animals and controls except for
blood detected in the urine of some females of the low-dose group.
There were no significant, dose-related changes in the incidence of
any lesions in any of the tissues examined histologically but there
was a high background incidence of parasitic and bacterial
infection.
The power of this study was limited by the small number of
animals used and the incidence of lesions due to infection but,
within these limits, the NOEL was 600 mg/kg bw/day, the highest dose
tested (Sebestyén, 1979; Tury, Aobos-Kovacs & Somogyvari, 1979).
Groups of 17 (19 at the highest dose) male and female Sprague-
Dawley rats, 4 weeks old at commencement of the study, were given
ß-cyclodextrin by gavage in aqueous suspension at daily dose levels
of 0, 100, 400 or 1 600 mg/kg bw After 3 months administration an
interim sacrifice was made as follows: Control, 5 males & 4 females;
100 mg/kg bw/d group, 3 males & 5 females; 400 mg/kg bw/d group, 5
males & 4 females; 1600 mg/kg bw/d group, 2 males & 4 females. The
remaining animals were maintained on the same dosing regime to 6
months. Food and water intake and body weights were determined
weekly. At termination, urinalysis (pH, protein, glucose, ketone
bodies, blood, bilirubin, urobilinogen) was carried out on 5 animals
of each sex in each group; haematological (RBC, WBC, haemoglobin and
haematocrit) and serum biochemical analyses (protein,
albumin/globulin, GOT, GPT, Alk-P-ase, BUN, bilirubin, total
cholesterol and glucose) were carried out on all survivors. At
autopsy, the following organs were weighed: brain, pituitary,
thyroid, thymus, heart, lung, liver, kidneys, adrenals, spleen,
pancreas, testes or ovaries; these organs and stomach and intestinal
tract were examined histologically (haematoxylin & eosin).
A total of 18 animals died during the study as follows:
100 mg/kg bw/d, 3 males; 400 mg/kg bw/d, 2 females; and 1600 mg/kg
bw/d, 6 males and 7 females. It was claimed that these deaths were
due to misdosing, as no abnormalities were detected other than
"pneumonia-like" lung pathology. There was a small decrement of
weight gain of both sexes in the top-dose group only, otherwise
weight gain, food and water intake were similar to controls and
there were no treatment-related effects on organ weights, urinalysis
or haematological parameters. Serum biochemical indices generally
were within the normal range although some significant differences
from controls were observed, notably a dose-related increase in
alk-P-ase in males and a decrease in blood glucose in females of the
top two dose groups. Gross and histopathological examination did
not reveal any treatment-related abnormalities. If the deficit in
weight gain at the top-dose level is considered an adverse effect in
the absence of other, pathological, changes, the NOAEL for this
study is 400 mg/kg bw/day by gavage (Makita et al., 1975).
Following a two-week pilot study, a 26 week oral toxicity study
was conducted in Long Evans rats, body weight 80-100 g at
commencement, in which ß-cyclodextrin (purity not specified) was
administered by gavage as a suspension in aqueous 1% methylcellulose
to groups of 15 male and 15 female animals at daily dose levels of
200, 400 or 600 mg/kg bw; controls received an equal volume of 1%
methylcellulose solution. Additional groups of 6 animals of each
sex were similarly dosed for 6 months followed by a 2-month recovery
period prior to autopsy. Body weight and food intake were recorded
weekly and urinalysis, haematological and clinical biochemical
examinations were performed at termination. At autopsy, heart,
lung, liver, kidneys, spleen and testes were weighed; macroscopical
and histological examinations (haematoxylin, eosin, Oil red O, and
PAS) were carried out on heart, lungs, liver, kidneys, spleen,
gonads, stomach, intestine, pancreas, adrenals, lymph nodes and
thymus.
In the course of the study there were 5 deaths, not dose-
related and attributed to intercurrent disease. No adverse effects
of treatment on clinical condition were seen in any dose group.
Food consumption was reduced in all treated male groups between
weeks 18 and 24, body weight gain was reduced in males of the two
highest dose groups between weeks 6 and 21 leading to a 10% deficit
in weight gain in the top-dose group; females showed little change
in food consumption or weight gain. Statistically significant
differences were observed in some haematological and clinical
biochemical parameters but in most cases these were not
systematically related to treatment and/or were within normal
physiological ranges. Dose-related increases in blood glucose were
observed in all groups, significantly so in females of all dose
groups and in males of the top-dose group. Statistically
significant increases, seen only at the top dose level, occurred in
total bilirubin and Ca (males only), and in Cl, total protein and
albumin/globulin ratio (females only) but were considered not to be
of toxicological significance. No significant treatment-related
changes in organ weights were observed except for a dose-related
reduction in spleen weight, significant in all female treatment
groups and in males of the top-dose group. However, these changes
did not appear to associated with functional or histological
alterations in the spleen and no treatment-related histological
abnormalities were observed in heart, lung, liver, kidney, adrenals,
gastrointestinal tract, gonads, lymph nodes, pancreas or thymus; no
nerve tissue was examined histologically.
In the 2 month withdrawal period following 6 months of
treatment, the deficit in body weight in the high-dose males was
largely recovered. A significant increase in relative lymphocyte
numbers was reported in all treated males but not in females.
Changes were noted in some organ weights, notably increased relative
weights of lungs and kidneys in males of the top two dose groups, a
decreased relative spleen weight in males in the top-dose group and
decreased relative liver weights in females of the low and high (but
not intermediate) dose groups. These changes were small and
unaccompanied by obvious functional changes, and were not considered
to be of physiological significance. Blood glucose levels remained
higher than controls in all treated male groups and in the top-dose
female group but were within the normal range as were the other
clinical biochemical parameters. The reductions in spleen weights
seen in both sexes (up to 40% reduction in top dose males) were
reported not to be accompanied by histopathological changes but
individual histological details were not provided and a no-observed-
effect-level cannot be established from this study (Gergely, 1982;
Mészáros & Vetési, 1982).
A 90-day study was conducted by dietary administration of
ß-cyclodextrin to male and female Sprague-Dawley-derived OFA rats.
The test material, 99.7% pure on a dry weight basis, was
administered to groups of 20 male and 20 female animals, 6-8 weeks
old at commencement, by replacing starch in a semi-synthetic diet at
levels of O, 1.25, 2.5, 5 or 10%; a "carbohydrate control" group
received lactose at a dietary level of 10%. Body weight and food
intake were recorded weekly; water intake was monitored 3 times per
week. Ophthalmological examinations were conducted on all animals
at the start and on 10 animals from the two control groups and the
highest dose group at termination. Urine volumes and concentrations
of ß-cyclodextrin were recorded at 6 weeks and at the end of the
study. Blood and urine biochemistry, haematology and histopathology
examinations (controls and high-dose group only) were carried out at
termination. The histopathological studies included histochemical
examination (Perl's reaction) for ferric iron in liver, kidneys,
spleen and lymph nodes. Serum analyses included cholesterol,
triglycerides, glucose, urea, creatinine, total bilirubin, total
protein, albumin, GOT, GPT, Alk-P-ase, Na, K, Ca and Cl-;
urinalyses included glucose, urea, uric acid, creatinine, total
protein, Na, K and Ca. Haematological examinations included
haemoglobin, PCV, MCH, MCHC, MCV, RBC, total and differential
leucocyte counts and prothrombin time. At autopsy, relative organ
weights were determined for brain, gonads, kidneys, spleen, thymus,
caecum, heart and liver.
One male from the lowest ß-cyclodextrin dose group died in the
course of the study but there were no indications that the death was
related to treatment. There were inconsistent differences in food
consumption between groups but no significant difference between the
10% ß-cyclodextrin and the 10% lactose groups. The dose achieved in
the top-dose group was approximately 4.4 g/kg bw/day and 5.3 g/kg
bw/day for males and females respectively. No significant
differences were recorded in water consumption between treatment
groups in males but the lactose control group showed a reduced
intake in weeks 4, 5 and 6. With females there was a slight
increase in water consumption for the 5 and 10% ß-cyclodextrin
groups. No significant differences in body weight were observed in
any of the groups, except for the male lactose control group, and
there were no dose-related adverse effects on haematology, serum
biochemistry or urine composition. A small fraction of the dose of
ß-cyclodextrin was recovered in urine of animals of the top two dose
groups and represented 0.1-0.3% of the highest dose administered.
The absolute and relative filled caecal weights of the rats, of both
sexes, were increased following administration of diets containing
ß-cyclodextrin or lactose (a common feature in rats receiving poorly
absorbed and slowly digestible carbohydrates). No treatment-related
effects which were considered by the authors as indicative of a
toxic response were found from the histopathological examination and
it was concluded that ß-cyclodextrin appeared to lack toxicological
activity at the doses tested (Olivier et al. 1991).
A review of the above study was carried out by an ad hoc
Scientific Advisory Group which essentially confirmed the authors'
conclusions. In noting that in the caeca of female rats of the high
ß-cyclodextrin dose group there was a significant increase in
sub-mucosal lymphoid follicles the reviewers considered that this
was not of toxicological importance as it is commonly associated
with caecal enlargement. The increase in the presence of sinus
macrophages in the lymph nodes seen in both males and females of the
high-dose group was considered to be a physiological rather than a
toxic response to the high levels of ß-cyclodextrin in the diet.
The review group did not comment on the observation that there was a
low incidence of hepatic focal fibrosis in animals from the top-dose
group (3/20 and 1/20 in males and females respectively) which was
not seen in controls or in the group receiving lactose. The group
concluded unanimously that this subchronic study was well designed
and properly conducted (Blumenthal et al. 1990).
2.2.2.3 Dogs
The oral toxicity of ß-cyclodextrin (purity not specified) was
examined in a 24-week study in beagles. Groups of 3 males and 3
females were given daily oral doses of 0, 100, 250 or 500 mg/kg bw,
corrected for body weight changes every three weeks. The material
was administered in boluses made from egg yolk and dried breadcrumbs
before the standard diet. Food intake, clinical signs (behaviour,
pulse rate, respiratory rate) were recorded initially and at the
third, sixth, twelfth, eighteenth and twenty-fourth week of
treatment; at the same time blood samples were collected for
haematology (Hb, PCV, MCHC, WBC, differential leucocytes) and
biochemical analyses (ASAT, ALAT, Alk-P-ase, BUN, glucose,
bilirubin, total protein, inorganic P and Ca). At autopsy, the
following organs were weighed: liver, spleen, kidneys, gonads,
heart, lung and brain; in addition to these organs, histological
examinations were performed on adrenals, stomach, small and large
intestine, pancreas, spinal cord and tissues showing gross lesions.
All animals survived, but one dog from the top-dose group
developed fever, lost appetite and showed catarrhal symptoms during
the last week of the study, necessitating treatment with
anti-distemper serum and antibiotics for four days; treatment with
ß-cyclodextrin was continued during this period. Diarrhoea was
observed in some cases but did not appear to be due to
administration of ß-cyclodextrin and the condition resolved
spontaneously or after treatment with Tannocarbon and bolus
astringents for 1-2 days. No treatment-related changes in pulse or
respiratory rates were detected. There were no significant dose-
dependent changes in blood biochemical parameters, although the
values for bilirubin were very scattered, presumably due to
haemolysis, at 0 and 3 weeks. Haematological examination also did
not reveal any treatment-related changes; an increased eosinophil
count in some cases was due to parasitic infestation, confirmed at
autopsy. There were no significant changes in organ weights
although absolute and relative liver weights tended to be lower in
all treated groups, and mean relative and absolute spleen weights
were increased in all treated groups. Histopathological changes
observed were attributed to the method of sacrifice (magnesium
sulfate injection) and were not dose-related. The poor condition of
some of the animals due to parasitic infestations and the agonal
changes due to the method of sacrifice limited the sensitivity of
the study but no obvious compound-related toxicity was detected
(Haraszti, 1978; Tury et al. 1978).
In a 13-week study, groups of 2 male and 2 female beagles were
given ß-cyclodextrin in the diet at levels of 0, 1.25, 2.5, 5 or 10%
(the top-dose group received the test material at 5% of the diet for
the first week and 10% thereafter). The purity of the
ß-cyclodextrin tested was stated to be > 99.0%. The animals were
acclimatised for four weeks prior to administration of the test
compound during which time they received inoculations and
anthelminthic treatment. The condition of the animals and food
intake were recorded daily and body weight was measured weekly.
Ophthalmoscopic examinations were performed at the beginning and end
of the study; blood was collected for comprehensive haematology and
clinical biochemical analysis prior to dosing and on weeks 6 and 13;
total 24-hour urine samples were collected for analysis in weeks 7
and 13. Urine samples, and faeces collected during week 13, were
analysed for ß-cyclodextrin as were blood samples collected 1, 3 and
6 hours after feeding. At termination organ weights were recorded
for adrenals, brain, heart, kidneys, liver, lungs, pancreas,
pituitary, spleen, thymus, thyroids and gonads. An extensive range
of tissues was fixed at autopsy but histopathological examination
was limited to the alimentary tract (8 levels), kidneys, liver,
pancreas, urinary bladder and all macroscopically abnormal tissues.
The achieved group mean intakes of ß-cyclodextrin averaged over
the 13 weeks of the study were 570, 1234, 2479 and 4598 mg/kg bw/day
in the 1.25, 2.5, 5 and 10% dietary dose groups, respectively. In
the course of the study, liquid faeces were noted spasmodically in
all animals, including controls, but the incidence was higher in the
top-dose group (46.7%) than in controls (11.8%) or the 5% dose group
(15.7%) and the higher frequency was considered to be treatment-
related; there was also a significant decrement of weight gain in
the top-dose group only, not associated with reduced food intake.
No other clinical symptoms attributable to treatment were observed
and the ophthalmoscopic examinations were normal. Haematological
parameters were similar to controls except that red cell counts,
haematocrit and haemoglobin levels were statistically significantly
reduced in the top-dose group at weeks 6 and 13, and in the 5% dose
group at week 6 only; however, the changes were small and similar
trends were observed in the acclimatisation period so the
toxicological significance was considered equivocal. Reductions
were observed in the group mean serum levels of cholesterol, HDL and
ß-lipoprotein at weeks 6 and 13 for dogs in the 5% and particularly
10% dose groups; in the top-dose group, total protein, albumin,
calcium and phospholipid levels were slightly reduced at weeks 6 and
13, and sodium levels were slightly reduced in week 13 only. Other
biochemical parameters were unaffected by treatment. Urinalysis
indicated that protein levels were elevated in the 5 and 10% dose
groups at weeks 6 and 13 but there were no other notable findings
due to treatment. At autopsy, organ weights were generally
unaffected by treatment; the group mean absolute thymus weight in
the top-dose group was significantly lower than controls (p<0.5)
but similar to the lowest-dose group and there was no dose-related
trend with the mean group weight being non-significantly higher than
controls in the 5% dose group. It was concluded that treatment had
no conclusive effect on organ weights. No macroscopic abnormalities
were observed and there were no observable treatment-related changes
in the tissues examined histologically. The authors concluded that
the 2.5% dose level (equal to 1234 mg/kg bw/d) was a NOEL and that,
in the absence of any treatment-related macroscopic or microscopic
pathological findings, the other effects noted were indicative of
only a mild toxic response. However, the small number of animals
and the restricted histological examination limited the power of
this study. ß-Cyclodextrin in dose-dependent concentrations was
detected in urine, faeces and blood of these animals dosed orally
(see Biochemical Aspects above) (Smith et al. 1992).
2.2.3. Long-term toxicity/carcinogenicity studies
No information available.
2.2.4. Reproduction studies
No information available.
2.2.5. Special studies on teratogenicity
2.2.5.1. Rats
A teratogenicity study was conducted in CFY rats in which
groups of 40 mated females (30 in control group) with positive
vaginal smears were given ß-cyclodextrin by gavage in 1% methyl
cellulose suspension at dose levels of 0, 200, 400 and 600 mg/kg
bw/dy on days 7-11 post coitus. The animals were killed on day 21
and the number of implants, resorptions, live and dead fetuses,
fetal weights and rates of congenital anomalies recorded (no details
were given of the methodology of the teratological examinations).
The conception rate was poor (about 30%) giving only 11-12
pregnant animals in each group. Five animals from the top and mid-
dose groups and 1 from the low-dose group died during the study, the
deaths being attributed to bronchopneumonia due to mis-dosing.
Maternal weight gain was reduced in a dose-related manner but there
were no changes in mean number of implants (12.7 -14.1), resorptions
(1.3-7.3%) fetal viability (93-99%) or fetal weight (3.5-3.9 g).
There were 5 congenital anomalies reported out of 628 fetuses, 2 in
the low-dose group (hydronephrosis, cardiac anomaly), 1 in the mid-
dose group (cardiac anomaly) and 2 in the top-dose group (both
absence of right kidney); no anomalies were seen in the control
group. No details were given of any examination for skeletal
anomalies. The authors state that the incidence of congenital
malformations corresponded to the "spontaneous" incidence but no
anomalies were seen in controls and no historical data were
presented to support this conclusion (Jellinek et al, undated).
Following a pilot investigation at dose levels of 500 and 2 500
mg/kg bw/day a teratology study was performed in Wistar rats given
ß-cyclodextrin as a suspension in 1.25% aqueous methyl cellulose by
gavage at doses of 0, 100, 500 and 2 500 mg/kg bw/day on days 7-16
of pregnancy. The group sizes were 26-28 sperm positive females, of
which 22-25/group proved to be pregnant. The dams were sacrificed
on day 21 and the following parameters monitored: number of corpora
lutea and implantations, preimplantation loss, embryonic and late
fetal mortality, and number of viable fetuses. About 50% of the
fetuses from each litter were examined for soft tissue defects
(Wilson's technique) and the remainder were cleared, stained
(Alizarin Red or Alcian Blue/Alizarin red) and examined for skeletal
anomalies.
The doses used in this study had no effect on the clinical
condition, food consumption or weight gain of the dams and there
were no significant effects on intra-uterine mortality, viable
fetuses or on the incidence or type of congenital malformation. The
anomalies recorded in this study were regarded as sporadic and
unrelated to treatment, and had been seen previously among fetuses
from over 600 control dams. Under the conditions of this study,
there was no evidence of fetotoxicity or teratogenicity (Druga,
1985).
In a further teratogenicity study in Sprague-Dawley rats,
groups of 24-30 mated females were given ß-cyclodextrin as a
suspension in 1% aqueous methyl cellulose by gavage at doses of
0, 1 250, 2 500 and 5 000 mg/kg bw/day on days 7-16 of pregnancy.
The dams were sacrificed on day 21 and the following parameters
monitored: number of corpora lutea and implantations, live and dead
fetuses and fetal size. About 50% of the fetuses from each litter
were examined for visceral anomalies (Wilson's technique) and the
remainder were cleared, stained (Alizarin Red) and examined for
skeletal anomalies.
A slight growth retardation was observed in the highest dose
group, significant from day 8 to 21, which was associated with a
reduced food intake; otherwise there was no effect of treatment on
weight gain of the dams. No mortality which could be ascribed to
the test compound was observed but 5 animals in the top-dose group
died as a result of misdosing and oesophageal perforation, reducing
the effective number from 30 to 25. There were no statistically
significant differences between groups in any of the following:
uterine weight (full and empty), placental weight, weight of
fetuses, number of fetuses (all were alive), number of implantation
sites, resorptions and corpora lutea or sex ratio. There were no
significant effects on the incidence or type of congenital
malformation. The anomalies recorded in this study were regarded as
unrelated to treatment. Under the conditions of this study, there
was no evidence of fetotoxicity or teratogenicity at doses of up to
5 000 mg/kg bw; retardation of weight gain of the dam seen at this
dose level was not apparent at a dose of 2 500 mg/kg bw/dy (Leroy
et al. 1991).
2.2.5.2. Rabbits
Following a pilot investigation at dose levels of 250, 500 and
1 000 mg/kg bw/day a teratology study was performed in groups of
12-14 thalidomide-sensitive New Zealand white rabbits. The dams
were artificially inseminated and given ß-cyclodextrin as a
suspension in 1.25% aqueous methyl cellulose by gavage at doses of
0, 150, 300 and 600 mg/kg bw/day on days 7-19 of gestation. The
dams were sacrificed on day 28 of gestation and the following
parameters monitored: number of corpora lutea and implantations,
preimplantation loss, embryonic and late fetal mortality, and number
of viable fetuses. All of the fetuses from each litter were
examined for external and visceral abnormalities on the day of
autopsy and the fetuses then fixed, stained (Alizarin Red) and
examined for skeletal anomalies.
At the doses used, ß-cyclodextrin had no effect on the clinical
condition, food consumption or weight gain of the dams and there
were no significant effects on intra-uterine mortality, fetal size
or number of viable fetuses. The incidence of minor congenital
malformations was slightly but not significantly elevated in all
treated animals and was not dose-dependent. The anomalies recorded
in this study were regarded as sporadic and unrelated to treatment,
and under the conditions of this study, there was no evidence of
fetotoxicity or teratogenicity (Dóczy 1985).
2.2.6. Special studies on genotoxicity
The results of genotoxicity studies with ß-cyclodextrin are
summarized in Table 2.
2.2.7 Special studies on nephrotoxicity
The experimental use of ß-cyclodextrin in dialysis fluids has
been associated with a characteristic nephrosis. Groups of 4 rats
of 100-125 g body weight were given single subcutaneous injections
of ß-cyclodextrin at dose levels of 225, 450 or 900 mg/kg bw and
sacrificed 12, 24, 48 or 96 hours later. In a repeat-dose study,
similar animals were given doses of 225, 450, 675 or 900 mg/kg daily
for 1, 2, 3, 4 or 7 days and killed 24 hours after the last dose.
The kidneys were examined by light and electron microscopy. A
characteristic nephrosis was observed, manifested as a series of
alterations in the vacuolar organelles of the proximal convoluted
tubule. The changes started with an increase in apical vacuoles and
appearance of giant lysosomes followed by extensive vacuolation,
cell disintegration and amorphous mineralization. These lesions
were evident following a minimal single dose of 675 mg/kg bw and a
crude dose-response relationship was established. The earliest
manifestations, midcellular cytoplasmic vacuoles, were observable
24 hours after injection. Following repeated dosing, light
microscopic lesions were found in one rat given 225 mg/kg bw/dy for
4 days and daily injections of 450 mg/kg bw resulted in severe
nephrosis but no deaths; all the animals given repeated doses of
900 mg/kg bw/dy died within 4 days. It was concluded by the authors
that intracellular concentration of non-metabolisable ß-cyclodextrin
by the lysosomal pathway represents a "perversion" of the
physiologic function of the proximal tubule, leading to cell death
(Frank et al. 1976).
In studies of the use of ß-cyclodextrin in peritoneal
dialysates to accelerate removal of i.v. phenobarbital in adult
rats, it was observed that a number of the animals given 1.5%
ß-cyclodextrin solution in physiological phosphate i.p. at a level
of 15% of body weight died overnight. In a follow-up study, blood
urea nitrogen (BUN) was determined after administration of
ß-cyclodextrin orally as 10 ml of a 6% suspension or i.p. as a 0.75%
solution in phosphate buffer. Following fasting for 5 hours and
gavage with single or three daily doses, BUN was within the normal
range 24 hours after the last dose. Intra-peritoneal administration
of ß-cyclodextrin was accompanied by a significant increase (3-4
fold) in BUN 24 and 72 hours after administration. The BUN levels
fell after 100 hours and returned to normal (Perrin et al. 1978).
Table 2. Results of genotoxicity studies with ß-cyclodextrin.
Test system Test Object Concentration Results Reference
Host-mediated assay E.coli WP2uvr A trp-, Doses to rat of - Igali 1978
in rat S.typhimurium TA1538 0, 100 or
1 000 mg/kg bw
Chromosome aberration Long Evans rat (?)bone 0, 200, 400 or -* Czeizel 1978
marrow bw/d for 3 m. 600 mg/kg
Mouse micro-nucleus Mouse bone-marrow 100 mg/kg bw - Weill, 1988
test
Sex-linked recessive Drosophila melanogaster 1.6, 8 & 16 mM - Parádi 1987
lethal mutation
Ames test (1) S. typhimurium TA98, 0, 0.1, 0.5, 1, - Weill, 1987
TA100, TA1535, TA1537, 2 & 4 mg/plate
TA1538
HPRT mutation V79 Chinese hamster 10, 30, 100, 300 - Marzin et al. 1990
(6-thio-guanine cells & 1 000 µg/ml
resistance) (1)
In vitro chromosome Human lymphocytes 100, 300 & - Marzin et al. 1991
aberration test (1) 1 000 µg/ml
*The number of cells examined was small relative to normal guidelines
(1) With and without rat liver S9 fraction
Subcutaneous administration of ß-cyclodextrin at a daily dose
level of 450 mg/kg bw in saline on 7 consecutive days resulted in
polyuria and proteinuria, a doubling in relative kidney weight and a
decrease in the activities of succinic dehydrogenase, alkaline
phosphatase, glucose-6-phosphatase and ß-glucuronidase in proximal
convoluted tubules (Hiasa et al. 1981).
2.2.8 Special studies on skin irritancy/sensitisation
2.2.8.1 Guinea-pigs
Evaluation of the cutaneous delayed hypersensitivity of
ß-cyclodextrin was carried out in albino Dunkin-Hartley guinea-pigs
using groups of 20 animals of both sexes. Seven applications
(induction phase) and challenge were carried out using 0.4 g
ß-cyclodextrin moistened with 0.5 ml water. Macroscopic
examinations were carried out 6, 24 and 48 hours after removal of
the occlusive patches. Histopathological examinations were carried
out on 6 animals showing doubtful reactions at 6 hours. No delayed
hypersensitivity reactions were provoked by this protocol (Mercier,
1990).
2.2.8.2 Rabbits
A primary dermal irritation study was conducted in New Zealand
white rabbits by application of 0.5 g of test compound moistened
with 0.5 ml saline was applied to the shaved dorsal skin of 3
animals under occlusion for 24 hours. The mean primary irritation
score was 0.50 (minimally irritating) based on a barely perceptible
erythema after 24 hours, there was no eschar or oedema and the
treatment sites were normal by 24 hours after removal (Reagan &
Becci, 1985).
A primary dermal irritation study in albino rabbits using an
abraded skin protocol. The index of primary cutaneous irritation
which was obtained (0.01) classified ß-cyclodextrin as non-irritant
(Leroy et al. 1990).
2.2.9 Special studies on eye irritancy
In an ocular irritancy/corrosion test in albino rabbits,
ß-cyclodextrin was classified as slightly irritant (Leroy et al.
1990).
2.2.10 Special studies on tumour promotion
Subcutaneous injection of ß-cyclodextrin at a dose of 450 mg/kg
bw daily for 7 days during week 3 increased the number and size of
renal tubular cell tumours in inbred Wistar rats treated with
N-ethyl- N-hydroxyethylnitrosamine (EHEN) in the diet at a
concentration of 1 000 mg/kg in the preceding 2 weeks. After 32
weeks, the incidence of tumours was 50% in animals treated with the
EHEN alone and 100% in animals subsequently given ß-cyclodextrin.
In addition, ß-cyclodextrin promoted the development of renal
tumours in rats treated with 500 mg EHEN/kg diet, which was a sub-
threshold dose for renal tubular cell tumourigenesis (Hiasa et al.
1982).
2.2.11 Special studies on cell-membranes
The interactions between cyclodextrins and membrane
phospholipids, liposomes and human erythrocytes were studied
in vitro. ß-Cyclodextrin did not alter the differential scanning
calorimetric behaviour of phospholipids, and did not increase the
permeability of dipalmitoyl-phosphatidylcholine liposomes. No
effects on active or passive transport of 42K or 86Rb into
erythrocytes was observed at concentrations of up to 10-2mol/litre
but at 1.7x10-2mol/litre, ß-cyclodextrin caused a significant
increase in passive transport and 8-10% haemolysis (Szejtli et al.
1986).
ß-Cyclodextrin induced haemolysis of human erythrocytes
in vitro in isotonic solution with a threshold concentration of
3 mM (3 400 mg/l). Swelling of erythrocytes, associated with
release of cholesterol from the membrane, was observed at lower
concentrations, approximately 20% of the membrane cholesterol was
released from the membrane at a concentration of 2 mM (2 300 mg/l)
ß-cyclodextrin (Irie et al. 1982).
2.3 Observations in humans
2.3.1 Absorption, distribution, metabolism and
excretion
The fate of ß-cyclodextrin the human gastrointestinal tract was
studied in ileostomy subjects and in normal volunteers after
administration of 10 g in a fasting state or after 3 doses of 10 g
daily with meals. In the ileostomy subjects, the recovery of
ß-cyclodextrin in the ileal effluent was 97±10% and 91±5%
respectively. In normal subjects, in which utilization was
estimated by the breath hydrogen technique and analysis of stools,
breath hydrogen was low, and insignificant levels of ß-cyclodextrin
were detectable in faeces. It was concluded that ß-cyclodextrin is
hardly hydrolyzed or absorbed in the human small intestine but is
fermented by colonic microflora with minimal apparent hydrogen
production (Flourié et al. 1992).
2.3.2 Human tolerance studies
In three successive periods of one week, eighteen healthy
males, aged 23±2 years, were given doses of 0, 24 or 48 g
ß-cyclodextrin/day in addition to the normal diet. The volunteers
were randomly assigned to the treatment groups in what was stated to
be a placebo-controlled, double blind protocol and the test compound
was administered in a chocolate drink equally divided over three
meals. At the high dose level "to avoid too drastic influences on
bowel function" the subjects were given 24 g ß-cyclodextrin on the
first day, 36 g on the second day and 48 g on days 3-7. Tolerance
was evaluated by subjective assessment of abdominal complaints using
a questionnaire. At the end of each 7-day period, breath hydrogen
concentration was measured. One of the volunteers was withdrawn
from the study on after three days and replaced with a substitute
because of too many adverse events, resembling lactose intolerance
(abdominal cramps, nausea, diarrhoea) which were not reported before
the start of the study. It is not clear what dose this volunteer
received before withdrawal or whether the symptoms preceded
treatment.
There was a significant increase in complaints of flatulence
(p<0.05) at the higher intake level; other scores of abdominal
complaints, reported defaecation patterns and breath hydrogen were
stated not to change significantly. The authors concluded that the
dose of 24 g ß-cyclodextrin/day was well tolerated on a short term
basis (van Dokkum & van der Beek 1990).
2.3.3 Sensitization/irritation
In a repeated insult occlusive patch test in 58 subjects
(1 male and 57 females aged 21 to 68 years) ß-cyclodextrin did not
induce irritation or allergic contact dermatitis. Three subjects
showed scattered, transient, barely perceptible to mild, non-
specific patch test responses during the induction or challenge
phases of the study, none of which were irritant or allergic in
nature (Alworth et al. 1985).
3. COMMENTS
Metabolic studies in animals and humans consistently indicate
that ß-cyclodextrin is poorly hydrolyzed or absorbed in the upper
gastrointestinal tract but is largely utilized following hydrolysis
by the gut microflora in the lower gut. A small proportion of
ingested ß-cyclodextrin may be absorbed intact.
A number of acute and short-term toxicity studies were reviewed
which indicated low toxicity by the oral route, although most of
these studies used smaller numbers of animals or more limited
histological examination than would normally be appropriate for
establishing an ADI. In a well-conducted short-term toxicity study
in rats, there were no effects of toxicological significance other
than caecal enlargement and an increased number of macrophages in
intestinal lymph nodes at doses of up to 10% ß-cyclodextrin in the
diet; these effects are a common feature of poorly absorbed
polysaccharides.
In in vitro stidies, ß-cyclodextrin sequestered cholesterol
from erythrocyte membranes and caused haemolysis, but only at
concentrations much higher than those seen in the blood of dogs
given ß-cyclodextrin at a level of 10% in the diet. No effects on
mucosal cells of the gastrointestinal tract were seen in the high-
oral-dose studies. ß-Cyclodextrin was non-genotoxic in a range of
tests, and it does not have a structure likely to be associated with
such activity. Given its poor bioavailability and lack of
genotoxicity, the Committee concluded that a long-term
carcinogenicity study was not required for the evaluation of this
substance.
When administered parenterally to rats, ß-cyclodextrin was
nephrotoxic, but no renal toxicity was observed in any of the short-
term toxicity studies using oral administration. In dogs, the
urinary excretion of unchanged ß-cyclodextrin was low, even when the
compound was given at a dose level of 10% in the diet, indicating
that it is unlikely that systemic levels following oral
administration would be high enough to cause renal toxicity.
4. EVALUATION
The Committee was informed that a 1-year oral toxicity study on
ß-cyclodextrin in dogs was under way, and requested the results of
this study to complete the evaluation of this compound.
Despite its low toxicity, the Committee was concerned about the
possible sequestering effects of ß-cyclodextrin on lipophilic
nutrients and drugs. In particular, further data on the effects of
ß-cyclodextrin on the bioavailability of lipophilic nutrients are
required.
The Committee concluded that there were sufficient data to
allocate a temporary ADI of 0-6 mg/kg bw for ß-cyclodextrin, based
on a NOEL of 2.5% in the diet, equal to 1230 mg/kg bw/day in the
study in dogs and a safety factor of 200.
The results of the 1-year study in dogs and information on the
effects of ß-cyclodextrin on the bioavailability of lipophilic
nutrients are required by 1995.
As this is a novel product with a wide range of potential
applications, the Committee requested further information on the
range of possible production methods that could be used in its
manufacture.
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