INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
SAFETY EVALUATION OF CERTAIN FOOD
ADDITIVES AND CONTAMINANTS
WHO FOOD ADDITIVES SERIES: 44
Prepared by the Fifty-third meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
World Health Organization, Geneva, 2000
IPCS - International Programme on Chemical Safety
THICKENER
CURDLAN
First draft prepared by M.E. von Apeldoorn & G.J.A. Speijers
Section on Public Health, Centre for Substances and Risk Assessment,
National Institute of Public Health and the Environment, Bilthoven,
Netherlands
Explanation
Biological data
Biochemical aspects
Absorption, distribution and excretion
Mineral bioavailability in rats
Nutritional aspects
Toxicological studies
Acute toxicity
Short-term studies of toxicity
Long-term studies of toxicity and carcinogenicity
Genotoxicity
Reproductive toxicity
Special studies
Immunogenicity
Pathogenicity of Alcaligenes faecalis var. myxogenes NTK-u
strain
Cytotoxicity of Alcaligenes faecalis var. myxogenes NTK-u
strain to HeLa cells
Observations in humans
Sensitization
Studies of adverse side-effects
Comments
Evaluation
References
1. EXPLANATION
Curdlan is a linear polymer consisting of ß-(1 -> 3)-linked
glucose residues. It is derived by fermentation from the bacterium
Alcaligenes faecalis var. myxogenes. The Committee considered its
use in food as a formulation aid, processing aid, stabilizer and
thickener, or texturizer. Curdlan has not been evaluated previously by
the Committee.
Information was available on the current per-capita intake of
curdlan in Japan and from a scenario based on levels of use of the
additive and food consumption data in the United States. This
information was inadequate to assess intake completely because data on
maximum use and the distribution of intake of foodstuffs that might
contain the additive in various regions of the world were not
provided.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
2.1.1 Absorption, distribution, and excretion
Rats
Two male Sprague-Dawley rats, seven weeks old, received an oral
dose of 20 mg/kg bw of [U-14C]curdlan prepared from [U-14C]glucose
with Alcaligenes faecalis var. myxogenes strain NTK-u suspended in
water. Expired carbon dioxide, urine, and faeces were collected for 72
h and the excreted radiolabel was determined. Considerable amounts of
radiolabel were excreted in the expired carbon dioxide (Table 1;
Matsuo & Suzuoki, 1972).
In a comparison of the metabolism of curdlan and cellulose, two
groups of three male Wistar rats, six weeks of age, received
[14C]curdlan or [14C]cellulose at a dose of 20 mg/kg bw suspended in
water. Expired carbon dioxide, urine, and faeces were collected for 48
h and the excreted radiolabel was determined. After dosing with
[14C]curdlan, excretion of [14C]carbon dioxide was low during the
first 3 h but increased linearly up to 12 h and reached a plateau of
39% of the administered radiolabel. After dosing with [14C]cellulose,
33% was excreted as [14C]carbon dioxide after 24 h. In both cases,
excretion as [14C]carbon dioxide was initially delayed (an effect not
seen in studies with starch or glucose). Less than 2% of the curdlan
and cellulose was excreted in the urine within 48 h, and faecal
excretion within this period was 38% for curdlan and 54% for cellulose
(Table 2; Matsuo & Suzuoki, 1972).
When groups of three male Wistar rats aged six weeks received
tetracycline in the drinking-water at a dose of 5 mg/ml for five days
before and two days after oral dosing with [14C]curdlan, excretion as
[14C]carbon dioxide was decreased to about one-third, whereas faecal
excretion was increased by 2.7 over that in male Wistar rats receiving
[14C]curdlan only (Table 3). The results indicate that intestinal
microflora are partly responsible for the metabolism of curdlan to
carbon dioxide (Matsuo & Suzuoki, 1972).
Three male Wistar rats, five weeks old, received an oral dose of
2.3, 23, or 230 mg/kg bw [14C]curdlan suspended in water. Excretion
as [14C]carbon dioxide decreased, with an accompanying increase in
fecal excretion, as the dose was increased from 23 mg/kg bw,
indicating limited metabolism at higher doses (Matsuo & Suzuoki,
1972).
Groups of 10 male SD/Ta rats received 0, 3, 10, or 30% curdlan or
30% agar powder in their diet for 30 days. The 24-h faeces from rats
at 30% curdlan, analysed for curdlan after 14 days, contained 4.3 g.
As the average food consumption during week 2 was 27 g and the
ingested food contained 0.3 × 27 g, or 8.1 g curdlan, the recovery of
curdlan in the faeces was 54%. The faeces also contained 2.5%
water-soluble sugar. Curdlan recovered from the faeces had lost the
ability to gel on heating (Yokotani et al., 1969).
Table 1. Recovery of radiolabel after oral administration of [14C]curdlan
Collection period Recovery of radiolabel as % of administered dose
Carbon dioxide Urine Faeces Total
0-24 h 77 2.6 7.7 88
0-48 h 85 3.1 12 100
0-72 h 89 3.3 12 100
Table 2. Recovery of radiolabel after oral administration of [14C]curdlan
or [14C]cellulose
Collection period Recovery of radiolabel as % of administered dose
Carbon dioxide Urine Faeces Total
Curdlan
0-24 h 39 1.3 34 74
0-48 h 40 1.4 38 80
Cellulose
0-24 h 33 1.6 40 75
0-48 h 36 2.0 54 92
Two male Sprague-Dawley rats, seven weeks of age, received 20
mg/kg bw [14C]curdlan suspended in water intraperitoneally and were
killed 0.5, 3, 6, and 24 h later, the animals excreted extremely
little radiolabel (Table 4), indicating that most of the curdlan
remained in the body. Whole-body radioautography showed that the
radiolabel was distributed in the intestinal fluids (Matsuo & Suzuoki,
1972).
Table 3. Recovery of radiolabel after oral administration of [14C]curdlan
with and without tetracycline
Collection period Recovery of radiolabel as % of administered dose
Carbon dioxide Urine Faeces Total
0-24 h
Curdlan + tetracycline 18 0.68 63 82
Curdlan 54 2.0 24 80
0-48h
Curdlan + tetracycline 20 0.98 69 90
Curdlan 56 2.3 26 86
Table 4. Recovery of radiolabel after intraperitoneal administration
of [14C]curdlan
Collection period Recovery of radiolabel as % of administered dose
Carbon dioxide Urine Faeces Total
0-24 h 1.8 3.5 0.05 5.4
0-48 h 4.1 4.1 0.12 6.4
Humans
Four healthy male volunteers, two of whom were pretreated with an
antibiotic for three days, received 20 µCi of [14C]curdlan (location
of radiolabel not given) as a single oral dose, and samples of blood,
urine, faeces, and carbon dioxide were monitored for radiolabel for 96
h. The antibiotic treatment suppressed the bacterial flora and in one
case appeared to have totally eliminated the faecal bacteria. Although
the extent to which curdlan is metabolized in the human
gastrointestinal tract appears to reflect the action of the gut
bacteria, even the subject with no apparent bacteria at the start of
the study had very limited carbon dioxide production. Only one subject
who had not received pretreatment had detectable radiolabel in blood
after 24 h. The presence of significant amounts (1.6% of the
administered dose) in the urine of this person and of a small but
significant amount in the other man who had not received an antibiotic
(0.014% of the administered dose) is a further indication of the
bacterial metabolism of curdlan in the gastrointestinal tract. The
difference between these two men can be explained by the predominance
of enterococcus, Escherichia coli, and Bacillus species in the
stool samples of the first and essentially only E. coli in the
second; their anaerobic bacteria also differed. The large amount of
radiolabel in the expired carbon dioxide of the first man (31% of the
administered dose) and the presence of a peak in expired radiolabel in
the second also indicate the importance of the gut bacteria. The men
pretreated with antibiotics expired the least radiolabel.
The results suggest that people with a full complement of
intestinal bacteria handle curdlan differently from individuals with
few or no intestinal bacteria. The nature of the faecal bacteria may
also play a role. The main pathway for excretion appears to be the
faeces, except for a portion that appears to be fully metabolized to
carbon dioxide. The wide discrepancy in the total recovery of the
radiolabel in three of the four men is illustrated by the high
percentages of unrecovered radiolabel (43, 83, and 58% respectively).
The authors attributed this low total recovery to incomplete stool
collection. The value of this study is therefore limited (Wazeter et
al., 1975a).
2.1.2 Mineral bioavailability in rats
Groups of 20 four-week-old male Sprague-Dawley rats weighing
67-86 g received 0, 1, 5, or 15% curdlan (purity assumed to be 100%)
or 15% cellulose in their diet for eight weeks and were then killed.
The study was performed according to GLP. All rats were observed twice
a day for deaths, morbidity, and overt signs of toxicity. Detailed
clinical examinations were performed weekly. Body weights were
determined weekly and food consumption daily. The faeces of each
animal were collected daily and weighed, pooled on a weekly basis, and
stored frozen until analysis for calcium, magnesium, iron, zinc,
copper, and magnesium. At termination of the study, blood samples and
the liver were taken from each animal and frozen until analysis for
the same minerals.
No deaths or signs of overt toxicity were observed. The body
weights of animals given 15% curdlan were statistically significantly
decreased, and the food consumption in this group was slightly
decreased, whereas that of animals given 15% cellulose was
statistically significantly increased. The faecal weights of animals
at 15% curdlan or cellulose were increased, the latter being most
pronounced (no statistical analysis was used for faecal weights). The
ratio of faecal mineral excretion to consumption in animals given 15%
curdlan was statistically significantly increased for all minerals
except zinc in week 1. Thereafter, a revised procedure was used to
avoid contamination of faeces with food particles, and the ratios of
faecal mineral excretion to consumption in all treated groups were
similar or lower than control ratios for the remainder of the study.
Statistically significantly increased ratios for all of the minerals
were observed for the group given 15% cellulose throughout most of the
study, except for zinc which was statistically significantly increased
only during weeks 1 and 6. At the end of the study, the blood
concentrations of the six minerals in curdlan-treated animals were
comparable to those of controls, whereas in animals given 15%
cellulose the iron concentration in blood was statistically
significantly increased. The manganese concentration in the liver was
statistically significantly increased in animals at 5 and 15% curdlan,
while the copper concentration in the liver was statistically
significantly decreased in those at 15% curdlan. In animals given 15%
cellulose, the concentrations of all six minerals in the liver were
comparable to those of controls (Goldenthal, 1994).
2.1.3 Nutritional aspects
Groups of five male Sprague-Dawley rats, 21 days old, received
3.44 g of carbohydrate-free basal diet (52% casein, 29% cottonseed
oil, 112% salt mixture, 4.4% cellulose powder, 0.9% D,L-methionine,
0.3% choline, and 1.5% vitamin mixture) or the same diet supplemented
with 1, 2, 3, or 6.56 g of starch or of curdlan daily for eight days.
The addition of starch was reported by the authors to provide a
nutritionally complete diet. After treatment, the rats were weighed
and killed and the full and empty caecal weights were determined. The
body-weight gain of the rats increased with increasing amounts of
starch, but addition of curdlan did not cause dose-dependent growth,
indicating that it was not used as a source of carbohydrate. Curdlan
but not starch supplementation caused enlargement of the caecum and
increased contents, and rats given curdlan excreted three to five
times more faeces than controls, with a moisture content of 70%
compared with 41-50% for rats fed carbohydrate-free diet (Matsuo &
Suzuoki, 1974a).
Groups of five male Sprague-Dawley rats, 26 days old, received
3.44 g of carbohydrate-free diet supplemented with 0, 2, 2.5, 3, 3.5,
4, or 6.56 g starch or carbohydrate-free diet plus 2 g starch
supplemented with 0.5, 1, or 1.5 g of curdlan daily for seven days.
The body weights of rats given diets supplemented with starch plus
curdlan were the same as those of rats receiving only starch,
indicating that curdlan was not used as a source of carbohydrate
(Matsuo & Suzuoki, 1974a).
Groups of two to three Sprague-Dawley rats were fasted for 20 h
and then given 4-5 g/kg bw of glucose solution or curdlan in aqueous
suspension. A control group was fed ad libitum. The blood glucose
and liver glycogen contents of the fasted animals were raised when
they were given glucose but not in those given curdlan, indicating
that curdlan was not used as a source of carbohydrate (Matsuo &
Suzuoki, 1974a).
Groups of male Sprague-Dawley rats, 26 days old, received 5 g of
basal diet (44% casein, 13% sucrose, 3.3% cellulose powder, 6.6% salt
mixture, 30% corn starch, 1.3% vitamin mixture, and 1.4% cottonseed
oil) daily for five days. They were then weighed, divided into groups
of five, and continued on the basal diet or given the basal diet
supplemented with 1, 2, or 4 g of corn starch, sucrose, or curdlan
daily for seven days, after which time they were weighed and killed
and the caecum and its contents were removed for weighing. Body weight
decreased gradually in the group fed 5 g/day basal diet, whereas that
in the groups given basal diet supplemented with starch or sucrose
increased with dose; however, no dose-dependent increase in body
weight was seen with curdlan but remarkably enlarged caeca with
increased contents were observed. The net increases in body weight,
minus the weight of the caecal contents, were slightly lower in groups
supplemented with starch than in those supplemented with sucrose, but
the groups supplemented with curdlan showed a dose-dependent decrease
in net body weight. The authors concluded that curdlan is
nutritionally inert in rats (Matsuo & Suzuoki, 1974b).
2.2 Toxicological studies
2.2.1 Acute toxicity
Studies of the toxicity of single doses of curdlan are summarized
in Table 5. No abnormalities or deaths were observed in mice or rats
after oral administration. Transient diarrhoea occurred after
intraperitoneal injection, and macroscopic examination showed an
agar-like residuum of unabsorbed test material in the abdominal
cavity. Massive amounts of the compound were detected in rats, which
received larger quantities. Rats showed adhesion of curdlan to liver
and spleen, which were covered with a white, fibrin-like coating, but
the parenchymal portion of the organs showed no lesions (Aomori &
Tanida, 1968).
2.2.2 Short-term studies of toxicity
Mice
In an eight-week study of possible side-effects (not performed
according to GLP or a current guideline), groups of 10 male (body
weight, 24-34 g) and 10 female (body weight, 19-29 g) Charles River
CD-1 mice were fed curdlan in their diet at concentrations of 0, 1, 5,
10, 20, or 30%, equal to 0, 1.4, 7.1, 14, 29, and 43 g/kg bw,
respectively. Feed and water were available ad libitum. The animals
were observed daily for changes in behaviour, appearance, and
excretion. Individual body weights and the food consumption of animals
of each sex in each group were measured weekly. All animals were
examined macroscopically.
Table 5. LD50 values for curdlan
Species Sex Route LD50 Remarks
(mg/kg bw)
Mouse M & F Oral > 10 000 10% aqueous suspension
Rat M Oral > 10 000 10% aqueous suspension
Mouse M Intraperitoneal 2 750 5% suspension in saline
Mouse F Intraperitoneal 2 500 5% suspension in saline
Rat M Intraperitoneal 2 750 5% suspension in saline
From Aomori & Tanida (1968). As the origin of the test compound was not given its purity
is unknown. The tests were not performed according to GLP or a current guideline. Only a
summary report was submitted, and the numbers of treated groups per test were not given.
Six animals per group were used (no further details) and observed for seven days.
One female mouse at 30% curdlan died of undescribed causes during
the study, and one to two mice at 1, 5, and 10% and half of those at
20 and 30% had large stools. Soft stools were seen in a few mice at 20
and 30% curdlan. The body-weight gain of male mice at 30% was
decreased by 10% in comparison with male controls. The authors
reported that the total food consumption (basal diet plus curdlan)
generally increased with dose, but the Committee concluded that the
total food consumption of males at all doses was increased by > 10%
over that of controls, with no dose-response relationship, and over
that of females at 20 and 30% on the basis of mg/kg bw feed
consumption. The actual food consumption (basal diet only) did not
differ from that of the control group. At the end of treatment, the
weights of the full caeca of males at 10, 20, and 30% curdlan and of
females at 20 and 30% and the weights of the empty caeca of males and
females at 30% and of males at 10% were statistically significantly
increased. No abnormalities were observed macroscopically in any of
the mice, including the female at 30% which died during the study.
Microscopy was not performed. The NOEL was 5% curdlan in the diet on
the basis of the increase in full caecal weights and large stools at
higher doses (Wazeter et al., 1973).
Rats
Five groups of five male Sprague-Dawley Ta rats aged five weeks
and weighing 110-158 g received a basal diet to which 3, 10, or 30%
curdlan (purity not stated) had been added (providing approximately 0,
2.5, 8.5, and 30 g/kg bw) or a basal diet to which 30% agar powder had
been added (approximately 33 g/kg bw) for one month. The study was not
carried out according to GLP or current guidelines. Data on individual
animals were not submitted.
Animals at 10 and 30% curdlan and 30% agar excreted faeces that
were two to five times larger than normal, had a slight, peculiar
odour, and were brown and grey-white and poorly adhesive. The groups
given 10 and 30% curdlan had diarrhoea after day 17, whereas those
given 30% agar did not have soft or diarrhoeal stools. The faeces of
rats given 3% curdlan were slightly greyish but of normal form. No
significant differences in body-weight gain were seen in comparison
with controls. The total food consumption (basal diet plus curdlan or
agar) of animals at 3, 10, and 30% curdlan or 30% agar was 7.8, 9.8,
26, and 40% higher than that of controls. On the basis of basal diet
only, the food consumption differed from that of the control group by
4.6, -1.1, -12, and -1.6%, respectively. Food efficiency in rats given
3, 10, and 30% curdlan or 30% agar was 98, 92, 78, and 69%,
respectively, that of the control group; when calculated on the basis
of basal diet, the values were 100, 100, 112, and 98%, respectively.
The average water consumption of these groups was 100, 96, 140, and
130% that of the control group. The differential leukocyte count
showed dose-related increases in neutrophils in groups treated with
curdlan, which were significant at 10%. Occult blood was present in
the urine of four animals given 10% curdlan, two at 30% curdlan, and
one at 30% agar. No treatment-related changes were found in other
haematological (reticulocyte count, haemoglobin, haematocrit, mean
corpuscular volume, mean corpuscular haemoglobin, mean corpuscular
haemoglobin content, or leukocyte count), biochemical (serum alkaline
phosphatase, alanine aminotransferase, or aspartate aminotransferase
activity, glucose, protein, urea, or cholesterol concentration, or
Bromsulphalein retention), or urinary (colour, pH, proteins, glucose,
ketone bodies, or urobilinogen) parameters. Statistically significant,
dose-related decreases in relative kidney weights were observed in all
treated groups, including that given 30% agar. Statistically
significant decreases in relative liver weight were seen in rats given
30% curdlan or agar. The relative adrenal weights were statistically
significantly decreased with 30% agar, and the relative pituitary
weights were statistically significantly increase with 30% curdlan.
All groups given curdlan had dose-related, statistically significant
increases in the relative weights of the empty caecum, and macroscopic
examination revealed enlarged caeca in all treated groups including
those given agar. Histopathology showed no treatment-related changes.
The NOEL was 3% curdlan in the diet on the basis of large faeces and
diarrhoea at higher doses (Yokotani et al., 1969).
Groups of 10 male and 10 female Sprague-Dawley rats aged five
weeks (the males weighing 138-160 g and the females 116-130 g)
received 0, 5, 10, or 20% curdlan (purity not given; sterilized by dry
heating for 20 min at 120 °C) in their diet for three months, equal to
0, 4.4, 9, and 19 g/kg bw for males and 0, 5.5, 12, and 24 g/kg bw for
females. The study was not conducted according to GLP or current
guidelines. The general condition of the animals was observed daily,
and body weight and food consumption were recorded twice a week. Food
efficiency was calculated. At the end of the treatment period,
haemato-logical (haemoglobin, haematocrit, erythrocyte and leukocyte
counts, differential leukocyte count, and platelets) and biochemical
(serum alkaline phosphatase, alanine aminotransferase, aspartate
aminotransferase, and lactic dehydroge-nase activity, Bromsulphalein
retention, cholesterol, glucose, blood urea nitrogen, creatinine,
total bilirubin, total proteins, albumin, globulin, the albumin:
globulin ratio, and calcium) parameters were measured in all animals.
After 6 and 11.5 weeks, urine from all animals was analysed for
colour, pH, proteins, glucose, ketone bodies, occult blood, and
sedimentation rate. The absolute and relative weights of 14 organs
were determined. Macroscopy and microscopy were performed, but the
numbers of animals and groups examined were not reported.
Animals at doses > 10% in the diet had large, slightly
discoloured, soft, deformed faeces. Body-weight gain decreased with
increasing dose in males but statistically significantly only at 20%
in the diet, while the total food intake (basal diet plus curdlan) of
males at all doses and of females at 10 and 20% increased. As a
consequence, food efficiency was decreased in males at all doses and
in females at the two higher doses. A dose-dependent decrease in
platelet count was observed in males which was statistically
significant at 10 and 20% curdlan, and statistically significant
decreases in total protein and globulin concentrations were observed
in male and female animals at 10 and 20% in the diet, leading to
increased albumin:globulin ratios which were statistically significant
only at 20%. The calcium and cholesterol concentrations in males at
20% in the diet were statistically significantly decreased and serum
alkaline phosphatase activity was significantly increased in males at
10 and 20%. Decreased urinary volume was reported at 20% (data not
given in the table), and a slight increase in urinary pH in treated
males and females was seen after both 6 and 11.5 weeks, with no
dose-response relationship. The absolute carcass weight was
statistically significant increased in males at 10 and 20% curdlan in
the diet, but decreases were observed in the absolute weight of the
liver in males at 20% (statistically significant), the absolute
weights of the kidney in males at 10 and 20% (statistically
significant), the absolute and relative weights of the ovary at 20%,
and the absolute and relative weights of the pituitary in females at
all doses (absolute weight statistically significant at 20% in diet;
relative weight statistically significant at 5 and 20% in the diet).
The relative uterine weight increased with dose (statistically
significant at 20%), and the relative weights of the adrenal and
submaxillary glands were increased in males at 10 and 20%
(statistically significant). The absolute and relative weights of the
full and empty caeca increased with dose in males and females at all
doses, but the increase was more pronounced in males and was
statistically significant at 10 and 20%. The wall of the caecum did
not become thinner. Macroscopic examination showed a dose-dependent
decrease in deposition of adipose tissue in the abdominal cavity in
males at all doses and in females at 20% in the diet. The large
intestine (full) of males and females at 20% was two to three times
larger than that of controls, and moderate enlargement was seen in
some male animals at 5 and 10% curdlan and in females at 10%. No
histopathological effects of treatment were seen. The NOEL was 5% in
the diet mainly on the basis of changes in faecal size, diarrhoea, the
weights of the full and empty caeca, and enlarged large intestines
(Nakaguchi et al., 1972)
Groups of 25 male and 25 female Charles River CD rats were paired
by litter to receive 15% curdlan in the diet or basal diet only at an
amount equal to that which the paired treated rat had consumed the
previous day. The behaviour and appearance of the rats were not
affected, but males showed a statistically significantly increase in
growth throughout treatment and females showed statistically
significantly increased growth during the first two weeks only. No
other parameters were examined (Wazeter et al., 1976a).
Dogs
In a study reported as a summary and that was not performed
according to GLP or current guidelines, groups of two beagles (assumed
to consist of one male and one female per group), aged eight to nine
months, were fed coagulated or uncoagulated curdlan (purity not
achieve a final concentration of 8 or 16%. Growth was normal although
two bitches receiving uncoagulated curdlan ate only one-third to
three-quarters of their daily diet. Stool consistency and urinary
excretion remained normal, and the haematological parameters were
normal except for increased eosinophil counts in animals receiving
uncoagulated curdlan. No biochemical abnormalities or changes in organ
weights were observed. Histopathological examination showed rare
pigmented macrophages and slight to moderate glycogen depletion in the
liver and inflammatory changes in the small intestine (dose not
stated). There was no NOEL (Woodard & Imming, 1971).
In another study that was not performed acccording to GLP or
current guidelines, groups of four male and four female beagles
weighing 7.8-12 kg (males) and 7.1-11 kg (females) received 0, 1, 5,
or 15% curdlan or 40% gelled curdlan (prepared by heating a 10%
suspension of curdlan in distilled water to 90 °C for 15 min and then
cooling it to room temperature) in the diet for one year. Appearance
and behaviour were monitored daily, and body weight and food
consumption were recorded weekly. Physical examinations were performed
before treatment, at monthly intervals during the first six months,
and at 9 and 12 months. Ophthalmoscopy was carried out in all animals
before treatment and after 3, 6, and 12 months. Haematological
(haemoglobin, haematocrit, reticulocyte, leukocyte, and differential
leukocyte counts, reticulocyte sedimentation rate, and platelet
count), serum biochemical (serum alkaline phosphatase, alanine and
aspartate aminotransferases, blood urea nitrogen, glucose, total
proteins, albumin, bilirubin, cholesterol, creatinine, sodium,
potassium, chlorine, and calcium) and urinary (appearance, colour,
volume, pH, specific gravity, albumin, glucose, bilirubin, occult
blood, and sedimentation rate) parameters were measured in all animals
before treatment and after 3, 6, and 12 months. At the end of the
study, the absolute and relative weights of the liver, kidneys, heart,
spleen, brain, caecum (full and empty), adrenals, thyroid/parathyroid,
testes/ovaries, and pituitary were determined. Macroscopy and
microscopy were performed on 28 tissues from all animals.
One male at 15% curdlan died at week 37 of the study, probably
because of intercurrent infection; excessive salivation and blood in
the refuse pan were seen on the day before death. Soft stools were
seen more frequently in animals at 15% curdlan and at 40% gelled
curdlan during the first six months of the study than in other groups,
and thereafter only at 15% curdlan. During the first two months,
mucoid stools and/or blood-tinged mucoid stools were observed rarely
in animals at 1%, occasionally in those at 5%, and frequently in those
at 15% curdlan and 40% gelled curdlan. Thereafter, the incidences
decreased at 1 and 5% curdlan and at 40% gelled curdlan but not at 15%
curdlan. Males at 1, 5, and 15% curdlan and 40% gelled curdlan had
slightly (< 10%), nonsignificantly decreased growth with no
dose-response relationship. No treatment-related changes were found in
physical appearance, food consump-tion, or haematological,
biochemical, or urinary parameters. The group mean absolute and
relative weights of the full and empty caeca were statistically
significantly increased at 15% curdlan, but no other significant
changes in absolute or relative organ weights were seen (no group mean
weights given). Macroscopic examination showed petechiation and
ecchymosis of the small intestinal mucosa in one animal at 1% curdlan,
two at 15% curdlan, and five at 40% gelled curdlan; the male at 1%
curdlan and two of the five at 40% gelled curdlan also shallow mucosal
erosion. Mucosal ecchymosis of the small intestine was also observed
in one control dog. Microscopy revealed small areas of recent mucosal
bleeding in the small intestine of one animal at 1% curdlan, two at
15%, and two at 40% gelled curdlan. The NOEL was 5% curdlan on the
basis of soft stools or diarrhoea, mucoid stools and/or blood-tinged
mucoid stools, and increased full and empty caecal weights at 15%
curdlan (Wazeter et al., 1975b).
2.2.3 Long-term studies of toxicity and carcinogenicity
Mice
In a study that was not performed according to GLP or current
guidelines, groups of 100 male and 100 female Charles River CD-1 mice
weighing 17-35 g (males) and 18-29 g (females) received diets
containing 0, 1, 5, or 15% curdlan or 40% gelled curdlan (prepared as
described above) daily until the individual groups had been reduced by
approximately 80% by sacrifices and deaths during treatment. Nine
males and five females at 1% curdlan and five males and five females
from all other groups were killed at 12 months, five males and five
females were killed after 18 months, and the remainder at weeks 99-114
when the survival rate in the groups was approximately 20%. Changes in
behaviour and appearance were checked daily, and body weights and food
consumption were recorded weekly. All mice were examined
macroscopically, and approximately 20 tissues from five males and five
females per group killed after 12 and 18 months, from all mice that
died or were killed in extremis during the study, and from all
remaining mice were examined microscopically. All gross lesions
suspected of being neoplastic were also examined microscopically.
No treatment related differences in survival or body-weight gain
were found between treated and control groups. The food consumption
(grams per mouse per day) of males at 40% gelled curdlan was increased
by about 19%, whereas that of females at 15% curdlan was decreased by
about 13% (no statistical analysis was performed). No
treatment-related macroscopic or microscopic abnormalities and no
treatment-related changes in tumour incidence were observed. The NOEL
was 5% curdlan in the diet on the basis of decreased food consumption
at 15% curdlan (Wazeter et al., 1976b).
Rats
In a study not performed according to GLP or current guidelines,
groups of 60 male and 60 female Charles River CD rats weighing 64-110
g (males) and 71-120 g (females) received 0, 1, 5, or 15% curdlan or
40% gelled curdlan (prepared as described above) in their diet for two
years. Their appearance and behaviour were checked daily, and body
weight and food consumption were recorded weekly. Ophthalmoscopy was
carried out in all rats before treatment and at 3, 6, 12, 18, and 24
months. After those same times, haematological (haemoglobin,
haematocrit, erythrocyte, leukocyte, differential and platelet
counts), serum biochemical (serum alkaline phosphatase, alanine and
aspartate aminotransferases, blood urea nitrogen, glucose, total
proteins, albumin, albumin:globulin ratio, bilirubin, cholesterol,
creatinine, sodium, potassium, and chlorine), and urinary (appearance,
colour, volume, pH, specific gravity, albumin, glucose, bilirubin,
occult blood, and sedimentation rate) parameters were measured in five
male and five female rats per group. Five male and five female rats
per group were killed after 12 months; three male and three female
rats at 5 and 15% curdlan and five male and five female rats from all
other groups were killed after 18 months; and the remaining animals
were killed after two years. The absolute and relative weights of the
liver, kidneys, heart, spleen, brain, caecum (full and empty),
adrenals, thyroid/parathyroid, testes/ovaries, and pituitary were
determined in all rats, and the animals were examined macroscopically.
Approximately 25 tissues from all animals killed after 12 and 18
months and from 10 males and 10 females per group killed after 24
months were examined microscopically.
No changes in mortality, behaviour, appearance, or ophthalmic
parameters were observed. At 15% curdlan, males gained about 11% less
and females about 10% less weight than controls, although the
decreases were not significant, and males consumed about 22% less and
females about 16% less basal diet (no statistical analysis performed).
No treatment-related changes in haematological, biochemical or urinary
parameters were observed. The absolute and relative weights of the
full and empty caeca were increased in animals at 15% curdlan at all
times and were statistically significant after 18 and 24 months. No
significant treatment-related abnormalities were seen histologically.
The NOEL was 5% curdlan in the diet on the basis of decreased
body-weight gain and food consumption and increased full and empty
caecal weights at 15% in the diet (Wazeter et al., 1976c).
In a study not performed according to GLP or current guidelines,
groups of 60 male and 60 female Charles River CD rats of the F1a
generation of a multigeneration study of reproductive toxicity,
weighing 38-110 g (males) and 33-10) g (females), received 0, 1, 5, or
15% curdlan or 40% gelled curdlan (prepared as described above) in
their diet daily until weeks 124-127 when the survival rate in each
group was 20%. Appearance and behaviour were checked daily, and body
weights and food consumption were recorded weekly. After 3, 6, 12, 18,
and 24 months and before termination, ophthalmoscopy was carried out
on all rats, and haematological (haemoglobin, haematocrit,
erythrocyte, leukocyte, and differential leukocyte and platelet
counts), serum biochemical (serum alkaline phosphatase, alanine and
aspartate aminotrans-ferases, blood urea nitrogen, glucose, total
proteins, albumin, albumin:globulin ratio, bilirubin, cholesterol,
creatinine, sodium, potassium, and chlorine), and urinary (appearance,
colour, volume, pH, specific gravity, albumin, glucose, bilirubin,
occult blood, and sedimentation rate) parameters were measured in 10
male and 10 female rats in each group. Five males and five females in
each group were killed after 12 and 18 months, and the remaining rats
were killed in weeks 124-127 when the survival rate in each group was
approximately 20%. The absolute and relative weights of the liver,
kidneys, heart, spleen, brain, caecum (full and empty), adrenals,
thyroid/parathyroid, testes/ovaries, and pituitary were determined in
all rats, which were examined macroscopically. About 25 tissues from
all rats were examined microscopically.
No changes in mortality, behaviour, appearance, or ophthalmic
end-points were observed. The body weights of animals at 15% curdlan
were statistically significantly decreased; whereas those of males at
5% curdlan were statistically significantly decreased until week 65 of
treatment and then approached those of controls. The body weights of
males and occasionally of females given 40% gelled curdlan showed
statistically significant decreases until week 78 of treatment and
then became similar to those of controls. The consumption of basal
diet was decreased by 8.3% in males and 4.4% in females at 15% curdlan
and increased by 5% in males and 13% in females at 40% gelled curdlan.
Food efficiency was slightly decreased for females at 15% curdlan and
for male and female rats at 40% gelled curdlan. No treatment-related
changes in haematological or urinary parameters were seen.
Statistically significantly increased aspartate aminotransferase
activity was seen in males and females at 15% curdlan after 12 and 29
months and in animals given 40% gelled curdlan group at some
measurement times. Statistically significant increases in serum
alkaline phosphatase activity were observed in males and females at
15% curdlan or 40% gelled curdlan up to 12 months. The absolute and
relative weights of the full caecum were statistically significantly
increased in all treated groups at terminal sacrifice, and the
absolute and relative weights of the empty caecum were increased in
males at 5% curdlan, in females at 15% curdlan, and in females at 40%
gelled curdlan at this time. At the other times, increased full and,
less frequently, increased empty caecal weights were seen in one or
more groups given curdlan. Macroscopic and microscopic examination
revealed a statistically significant increased incidence of uterine
polyps in females at 15% curdlan that were killed at the end of or
during the study, with incidences of 0/450 in controls, 3/50 with 1%
curdlan, 4/51 with 5% curdlan, 7/50 with 15% curdlan, and 2/50 with
40% gelled curdlan and thus possibly related to treatment. The NOEL
was 1% curdlan on the basis of increased empty caecal weights at 5 and
15% in the diet and decreased growth and food consumption and an
increased incidence of uterine polyps at 15% curdlan (Wazeter et al.,
1976d).
2.2.4 Genotoxicity
The results of studies on the genotoxicity of curdlan are
summarized in Table 6.
2.2.5 Reproductive toxicity
Rats
In a three-generation study of reproductive toxicity and
teratogenicity that was not performed according to GLP or current
guidelines (mating periods lasted 15 days instead of three weeks as
prescribed in OECD guideline 416; the parameters examined resembled
those in OECD guideline 416 [for reproductive toxicity] and 414 [for
teratogenicity]), groups of 20 male and 40 female Charles river CD
rats, weighing 64-120 g (males) and 59-110 g (females), received 0, 1,
5, or 15% curdlan or 40% gelled curdlan (prepared as described above)
in their diet until they were 100 days of age. The F0 parents were
mated twice. After the first litter had been weaned, the number of F0
parents was reduced to 10 males and 20 females per group. The F1
parents were mated three times and the F2 parents twice. The pups
from the first mating in each generation were examined for external
abnormalities and killed and discarded after weaning (21 days of age),
although some animals from each group of the F1a generation were
selected for use in the long-term study described above. At the age of
four group from the second matings of the F0 and F1 generations (the
F1b and F2b litters) were selected to produce the next generation.
After the third mating of F1 parents, which producing the F2c
litters, 50% of the F1 females were killed on day 13 of gestation,
laparatomies were performed, and their uteri and ovaries were
examined. The remaining 50% of F1 females were killed on day 20 of
gestation and their fetuses were removed. The parental rats were
observed daily for changes in behaviour and appearance, and individual
body weights and food consumption were recorded weekly. During the
reproduction phase, fertility, litter size, numbers of male and female
pups, the viability of the newborns, the growth of the pups, and
survival of pups to weaning were observed. After weaning of the F3b
litters, the absolute and relative weights of the liver, kidneys,
heart, spleen, full and empty caeca, and testes/ovaries were
determined in five male and five female F3b pups per group and five
male and five female F2 parental rats per group, and the animals were
examined macroscopically. About 15 tissues from five male and five
female F3b pups and five male and five female F2 parental rats from
the control, 15% curdlan, and 40% gelled curdlan groups were examined
microscopically. In the evaluation of teratogenicity, the numbers of
corpora lutea, implantations, and resorptions and the number,
distribution, and location of viable and dead fetuses were counted in
F2c dams killed on day 13 or 20 of gestation. All fetuses from F2c
dams killed on day 20 of gestation were examined for external
abnormalities, sexed, and weighed. Two-thirds of the fetuses in each
litter were examined for visceral abnormalities and one-third for
skeletal abnormalities.
Table 6. Results of studies of the genotoxicity of curdlan
End-point Test object Concentration Purity Result Reference
Reverse S. typhimurium 15-5000 µg/plate Not reported Negative in presence and Dillon
mutationa TA98, TA100, as suspension in but assumed absence of S9; slight (1994)
TA1537, TA1538 sterile ultra-pure to be 86% toxicity only in absence
TA1535, water of S9 at 5000 µg/plate in
all strains except TA1538;
no precipitation
Gene tk locus in 12.5-5000 µg/ml 86% Negative in presence and Riach &
mutationb mouse lymphoma as suspension in absence of S9; toxicity at Willington
L518Y cells tissue culture 2500 and 5000 µg/ml (1994)
medium (relative growth, 69 and
31%, respectively, in
absence of S9 and 66 and
33%, respectively, in
presence of S9)
Chromosomal Chinese 625 (1250)-5000 86% Negative in presence and Leddy
aberrationc hamster µg/ml as suspension absence of S9; no toxicity; (1994)
ovary cells in tissue culture treatment time, 22 h
medium in absence of S9, 6 h in
presence of S9; harvesting
time, 24 and 48 h after
start of treatment
Micronucleus Male and 0, 500, 1000, and 86% No micronuclei and no Holmstrom
formationd female CD-1 2000e mg/kg bw overt mortality or & Innes
mice dissolved in water adverse reactions; ratio (1994)
by gavage at 24-h of polycromatic to normal
intervals; erythrocytes unchanged
bone-marrow samples
taken 24 h after
last dose
a Performed according to GLP and OECD Guideline No. 471
b Performed according to GLP and test protocol identical to OECD Guideline No. 476
c Performed according to GLP and OECD Guideline No. 473
d Performed according to GLP and OECD Guideline No. 474
e Maximum dose attainable
In the evaluation of reproductive toxicity, male parental rats of
the F0 and F1 generations at 15% curdlan and female parental rats of
the F0 generation given 40% gelled curdlan showed slightly decreased
mean growth. The mean consumption of basal diet (in grams per rat per
day) by male and female parental rats of the F0 and F1 generations
at 15% curdlan was slightly decreased. F2 parental females given 15%
curdlan or 40% gelled curdlan showed increased absolute and relative
weights of the full caecum, which were reported by the authors to be
statistically significant ( p < 0.05), but the results of the
statistical analyses were not given in the table. The Committee noted
that the absolute and relative weights of the empty caecum in females
were also increased at these doses. No macroscopic or microscopic
treatment-related changes were observed in the F2 parents examined.
Although no treatment-related changes in fertility, gestation, or
viability indices were observed, the weights of the pups in nearly all
litters in all generations at 15% curdlan were significantly decreased
during lactation, in the F1a and F1b litters at 14 and 21 days of
age; in the F2a and F3a litters at 4, 7, 10, 14, 17, and 21 days of
age; and in F3b litters at 21 days of age. Variations in the weights
of pups in the three generations at 1 and 5% curdlan and 40% gelled
curdlan were observed occasionally during lactation but were not
consistent and were considered not to be biologically relevant. The
authors suggested that the reductions in pup weight reflected the
inexperience of primiparous dams and were due to the fact that from
the age of 7-10 days the young started to consume some of their
mothers' feed. They stated that the weights of the pups at various
times post partum were within the normal range of values for rats of
this strain, but data on historical controls were not submitted. F3b
weanlings at 5 and 15% curdlan and at 40% gelled curdlan had decreased
full caecal weights, and those at 1% curdlan and 40% gelled curdlan
had increased empty caecal weights. The authors reported that these
changes were statistically significant ( p < 0.05), but the results
of the statistical analyses were not shown. No macroscopic or
microscopic treatment-related changes were observed in the F3b pups
at 15% curdlan or 40% gelled curdlan.
In the evaluation of teratogenicity, the numbers of gravid
animals, corpora lutea, implantation sites, and viable and dead
embryos on days 13 and 20 of gestation were normal. The fetuses of
dams killed on day 20 of gestation had a normal sex ratio and showed
no increased incidence of visceral or skeletal abnormalities. The mean
live fetal weights in all treated groups were statistically
significantly increased in comparison with controls, but no
dose-response relationship was seen.
The NOEL for parental animals was 5% curdlan on the basis of
decreased growth and increased full and empty caecal weights at 15%.
The NOEL for embryotoxicity was also 5% on the basis of decreased
weight gain during lactation at 15% (Wazeter et al., 1976e).
A single-generation study with two litters was perfomed to
determine whether the offspring of treated females would grow normally
if they were nursed by untreated females and whether the offspring of
untreated females would grow normally if they were nursed by treated
dams. In this study, which did not conform to GLP or any current
guideline, a control group of 40 male and 80 female Charles River CD
rats and three test groups of 20 male and 40 female rats received 0,
5, or 15% curdlan or 40% gelled curdlan (prepared as described above)
in their diet continuously throughout the study. When they were 100
days old, a period of 15 days was allowed for mating to produce an
F1a generation. The F1a litters were reduced to 10 pups per litter on
day 0. Then, 20 dams in each group nursed their own offspring through
lactation while the remaining 20 dams nursed the litters of 60 control
dams, which in turn nursed the pups of the litters of the other 20
dams in each of the three treated groups. The exchange of pups between
control and treated groups took place within 24 h post partum. After
weaning, the F1a offspring were killed and the parental females
rested for 10 days and then bred a second time to produce F1b
litters. After weaning of the F1b litters, all parental animals and
offspring were killed. The individual body weights and food
consumption of the parents were recorded weekly, and the fertility
index, length of gestation, litter size and viability, numbers of live
and dead births, and the lactation index were determined. Pups were
examined for gross abnormalities at birth and throughout lactation,
and the individual body weights and survival indices of the pups were
recorded at 0, 4, 7, 10, 14, 17, and 21 days post partum.
No changes in mortality, behaviour, or appearance were observed.
Male parents at 15% curdlan grew slightly less (< 10%) than control
males, and the food consumption of male and female parents at this
dose was lower, whereas dams at 40% gelled curdlan had increased feed
consumption. No changes in fertility or lactation indices were
observed when the F1a and F1b litters were born, and the pups showed
no gross abnormalities. The survival of F1a pups at 5 and 40% curdlan
was statistically significantly lower than in the control group, but
that of F1b pups was normal. The F1a litters of all treated dams that
nursed their own young showed statistically significant decreases in
weight gain, the greatest decrease being observed with 15% curdlan,
but F1b litters showed reduced weight gain only at 15% curdlan. When
pups born to treated dams were transferred to control dams,
statistically significant decreases in weight gain were still observed
during lactation, but the effect was reduced. The decreases in F1a
litters appeared later and for a shorter time (days 17 and 21 with 5%
curdlan, days 10 and 21 with 15% curdlan, and day 21 with 40% gelled
curdlan), whereas F1b litters at 5 and 15% curdlan showed
statistically significant decreases in weight gain on days 4 and 14 of
lactation, respectively. When control pups were nursed by dams given
15% or 40% curdlan, statistically significant decreases in weight gain
were seen in F1a control pups on days 7-21 and day 10 of lactation,
respectively. F1b control pups nursed by dams given 5 or 15% curdlan
showed statistically significant decreases in growth on days 4 and
14-17 of lactation, respectively. The authors concluded that the young
from treated dams gained weight at a normal or nearly normal rate when
nursed by control dams but had considerably reduced weights when
nursed by treated dams. They ascribed the effect on pup weight to
consumption by the pups of their mothers' diets which contained
non-nutritive material. There was no NOEL (Wazeter et al., 1976f).
In a similar study, cellulose was fed in the diet at
concentrations of 0, 5, or 15% from 80 days before mating throughout
gestation, parturition, and lactation until weaning of two litters. No
effect on the appearance, behaviour, or survival of the parents or
pups was observed, and the body weights and feed consumption of the
parents showed no significant abnormality. Pups in the F1a and F1b
litters at 15% cellulose in the diet showed decreased weight gain
during lactation, which was ascribed by the authors to consumption by
the pups of their mothers' diet (Wazeter et al., 1976g).
A further single-generation study was performed to determine
whether withdrawal of curdlan from the diet of dams during lactation
would still result in reduced weight gain in the pups. In this study,
which was not performed according to GLP or a current guideline, four
groups of 20 male and 40 female weanling Charles River CD rats
received 0, 5, or 15% curdlan or 40% gelled curdlan (prepared as
described above) in their diet. Males received the diet continuously
throughout the study after a seven-day conditioning period. Females
received the diet after a seven-day conditioning period; when they
were 100 days old they were allowed a period of 15 days for mating to
produce an F1a generation, and the test diets were fed until day 20
of gestation and removed during the lactation period. The F1a litters
were reduced to 10 pups per litter on day 4 post partum, and all pups
were killed after weaning. The dams then received the test diets again
during a 10-day rest period and during the subsequent 15-day mating
period to produce the F1b generation, until day 20 of gestation. The
F1b litters were reduced to 10 per litter at day 4 post partum, and
all animals were killed after the weaning period. The appearance,
behaviour, individual body weights, and food consumption of the
parents were recorded weekly, and the fertility index, pup survival,
litter size, viability, numbers of live and dead births, and the
lactation index were determined. Pups were examined for gross
abnormalities at birth and during lactation, and the individual body
weights of the pups were determined on days 0, 4, 7, 10, 14, 17,
and 21.
Females at 40% gelled curdlan consistently consumed more food
than other groups, probably as compensation for the 40% of
non-nutritional material in their diet. The reproductive performance
of the parents was not affected, and no adverse effects on pups were
observed. The weight gain of the pups during lactation was not
affected even though the authors reported that the pups could have
eaten their mothers' diet (basal diet) from about day 10 post partum.
The authors considered that the lack of effect on the weight gain of
the pups supported their suggestion that the reduced weight gain of
pups in the previous studies had been due to their consumption of
curdlan in their mothers' diet. The NOEL for both parental toxicity
and embryotoxicity was 15% curdlan and 40% gelled curdlan (Wazeter et
al., 1976h).
Rabbits
In a study of teratogenicity, which was not performed according
to GLP or a current guideline, groups of 15-20 pregnant Dutch Belted
rabbits received curdlan at 0, 1, 2, or 5 g/kg bw per day orally in
gelatine capsules delivered from a syringe daily during days 6-18 of
gestation An additional group received 20 g/kg bw per day gelled
curdlan (prepared by heating a 10% suspension of curdlan in distilled
water to 90 °C for 15 min, cooling it to room temperature, maintaining
it in a refrigerator until use, and then fragmenting it in a mortar)
by the same route. The doses of 5 g/kg bw per day curdlan and 20 g/kg
bw per day gelled curdlan were given in two divided doses. The control
group received two empty capsules per day. The rabbits were killed on
day 28 of gestation and their fetuses were removed. The weights of the
dams were determined on days 6, 12, 18, and 28 of gestation, and the
uteri of all dams were examined, and the numbers and locations of live
and dead fetuses, empty implantation sites, early and late
resorptions, and corpora lutea were recorded. The fetuses were weighed
and examined for external, visceral, and skeletal abnormalities.
None of the controls died, but three at 1 g/kg bw per day, one at
2 g/kg bw per day, and three at 5 g/kg bw per day curdlan and 16 at 20
g/kg bw per day gelled curdlan died, the last effect being due to
mechanical occlusion of the pharynx by the gel. Eleven resorptions
were seen with 5 g/kg bw per day curdlan compared with four in
controls, six at 1 g/kg bw per day curdlan, five at 2 g/kg bw per day
curdlan, and two at 20 g/kg bw per day gelled curdlan; however, the
numbers of females that had resorptions were similar in all groups (4,
4, 3, 2, and 2 females at 0, 1, 2, 5, and 20 g/kg bw per day (gelled)
curdlan, respectively). No teratogenic effects were observed. The NOEL
for both maternal and embryotoxicity was 5 g/kg bw per day, the
highest dose tested. Owing to the high mortality seen with 20 g/kg bw
per day gelled curdlan, no conclusion can be drawn about its maternal
toxicity, embryotoxicity, or teratogenicity (Wazeter et al., 1974).
2.2.6 Special studies
2.2.6.1 Immunogenicity
Mice
Groups of four CH3 mice were given a single intravenous injection
of 10 µg curdlan, 10 µg dextran 2000, or 0.1 mg alum-precipitated
bovine serum albumin. Although the last two compounds induced a strong
passive haemagglutination reaction, curdlan did not induce any
reactive antibodies (Arakawa et al., 1974).
Rats
Groups of five male Wistar rats, five weeks old, were fed diets
containing curdlan at 0, 0.1, 1, or 5 g/kg bw per day for 30 days. No
antibodies against curdlan were induced, as demonstrated by passive
haemagglutination and Ouchterlony precipitation tests. An
intraperitoneal injection of 10 µg of curdlan or dextran 2000
(suspended in physiological saline) to groups of five male Wistar rats
twice a week for four weeks did not induce a specific antibody
response, whereas an intravenous injection of 1 mg of
alum-precipitated bovine serum albumin elicited strong antibody
responses (Arakawa et al., 1974).
2.2.6.2 Pathogenicity of Alcaligenes faecalis var. myxogenes NTK-u
strain
Mice
Suspensions of dead cells of the curdlan-producing strain of
A. faecalis var. myxogenes NTK-u and of Xanthomonas campestris
in 5% aqueous gum arabic solution were administered orally to groups
of eight four-week-old male ICR-JCL mice at doses of 10 or 20 g/kg bw,
and the animals were observed for seven days. No deaths occurred and
no abnormalities were found at autopsy on day 8 (Yokotani, 1969).
Groups of 9-10 four-week-old male ddY-SLC mice, groups of 10
fasted male ddY-SLC mice, and groups of 9-10 suckling male and female
ddY-SLC mice aged 14-18 days (believed to be relatively susceptible to
intestinal infection) received 1010 viable organisms of A. faecalis
var. myxogenes NTK-u orally for two days or, for comparison, the
non-pathogenic strains Alcaligeneses faecalis, Escherichia coli
Umezawa, Xanthomonas campestris (which produces xanthan gum) and/or
the pathogenic strains E. coli 0-78 and Salmonella enteritidis.
The animals were autopsied after seven days of observation and the
respective strains were measured in mesenteric lymph nodes. The only
abnormal findings or deaths occurred in mice given S. enteritidis,
and that was the only strain found in mesenteric lymph nodes.
The pathogenicity of A. faecalis var. myxogenes NTK-u was
also studied after intravenous, intraperitoneal, and intracerebral
injection into four-week-old male ddY-SLC mice and compared with the
pathogenicity of the same non-pathogenic and pathogenic strains of
bacteria listed above. The LD50 values for A. faecalis var.
myxogenes NTK-u were similar to or higher than those for the other
bacterial strains (Imai, 1972).
2.2.6.3 Cytotoxicity of Alcaligenes faecalis var. myxogenes NTK-u
strain to HeLa cells
Neither the filtrate of a culture medium in which A. faecalis
var. myxogenes NTK-u had been grown, nor the bacterium or its
components was cytotoxic to HeLa cells at concentrations up to 100
mg/ml (Ito, 1971).
2.3 Observations in humans
2.3.1 Sensitization
A modified Draize 'multiple insult' patch test, which was not
performed according to GLP or a current guideline, was used on 213
healthy male prisoners (73% white, 21% black, 4% Mexican, 2% other;
average age, 35 years) who received 10 applications of curdlan (dose
not reported) as an aqueous paste on the skin under occlusion, the
test material being applied fresh every other day. A 48-h challenge
application of the test material (concentration and amount not
reported) was given 10-14 days after the last dose on a different
site, which was examined after 48, 96, and 144 h. During the induction
phase, trace irritation was seen in a few men but was not significant.
No sensitization reactions were seen in the challenge phase (Wazeter
et al., 1975c). The Committee noted that these results are of limited
value owing to deficiencies of the tests.
2.3.2 Studies of adverse side-effects
In a study of the possible adverse side-effects of curdlan,
groups of six young men received milk-shakes with or without curdlan.
Neither the subjects nor the physician conducting the study were aware
of who received curdlan. The men were given 6 g/day for five days, 35
g/day for two weeks, and then 50 g/day from day 21 until the end of
the treatment on day 28. Prior to the study, the past illnesses,
allergies, and gastrointestinal function of each subject were
determined, and before and at the end of the study each subject was
given a complete physical examination, with laboratory tests for blood
sugar, urea nitrogen, uric acid, cholesterol, calcium, total
bilirubin, alkaline phosphatase, total protein, albumin and globulin,
phosphorus, alanine aminotransferase, lactic dehydrogenase, complete
blood count, haemoglobin, haematocrit, urinary creatinine (24 h), and
an electrocardiogram. They were also questioned at weekly intervals
about any problems or adverse effects, with particular emphasis on
gastrointestinal symptoms. Body weight, blood pressure, and pulse rate
were recorded.
No evidence of toxicity was observed during or after the study.
Three of the six men receiving curdlan reported increased flatulence
and intestinal pain due to gas, and two subjects reported some
diarrhoeal movements, in one subject particularly towards the end of
the study. In contrast, another subject receiving curdlan complained
of constipation. These transient effects did not cause the withdrawal
of any subject from the study. No other effects attributable to
administration of curdlan were reported (Wazeter et al., 1975d).
4. COMMENTS
In two studies, rats given 14C-curdlan at a dose of 20 mg/kg bw
orally excreted about 80% and 40% of the administered radiolabel,
respectively, as 14C-carbon dioxide within 24 h. In these studies,
excretion in the urine represented about 3% and 1.5% of the dose and
excretion in faeces about 8% and 34%, respectively. After 48 h, 100%
and 80% were recovered from carbon dioxide, urine, and faeces
combined. When tetracycline was given concomitantly in the
drinking-water, excretion as carbon dioxide decreased by one-third,
whereas excretion in faeces was increased, indicating that intestinal
microflora may be responsible for the metabolism of this compound.
Excretion of the radiolabel as carbon dioxide also decreased with
increasing dose of curdlan, indicating more limited metabolism at
higher doses. In humans, the faeces appeared to be the main pathway
for excretion, except for a portion that was fully metabolized to
carbon dioxide. The extent of metabolism to carbon dioxide in humans
also appeared to reflect the action of intestinal bacteria: when the
bacterial microflora were suppressed by pretreatment with antibiotics,
very limited production of 14C-carbon dioxide was seen.
Curdlan given to rats at concentrations of 1, 5, or 15% in the
diet had no effect on the bioavailability of calcium, magnesium, iron,
zinc, copper, or manganese. The LD50 value in mice and rats treated
orally was > 10 000 mg/kg bw, and no abnormalities were seen at
autopsy.
In short-term and long-term studies in experimental animals, the
only effects of orally administered curdlan were soft stools and/or
laxation, reduced body-weight gain, and increased weights of full and
empty caeca due to the presence of high concentrations of
'indigestible' curdlan. In an eight-week study in mice and a four-week
study in rats given curdlan at concentrations up to 300 g/kg of diet,
the only effects were large faecal pellets, soft stools and/or
laxation, and increased weights of full and empty caeca.
In a three-month study in rats, the lowest dose of 50 g curdlan
per kg of diet was the NOEL. Growth inhibition was seen at the highest
dose of 200 g/kg of diet, even though food intake was increased. Soft
stools, enlarged large intestines when full, and increased weights of
full and empty caeca appeared to be the major effects at 100 and 200
g/kg of diet. Dose-dependent decreases in platelet counts and protein
and globulin concentrations and increased serum alkaline phosphatase
activity, absolute carcass weight, and the relative weights of the
adrenal and submaxillary glands were seen in males at 100 and 200 g/kg
of diet. In addition, males at the highest dose had decreased serum
calcium and cholesterol concentrations, and females at this dose had
decreased relative pituitary weights and increased relative uterine
weights. At necropsy, decreased deposition of adipose tissue was seen
in the abdominal cavities of females at all doses and in males at the
highest dose.
In a one-year study in dogs treated in the diet, blood-tinged,
mucoid, soft stools were seen with curdlan at 150 g/kg of diet and
with gelled curdlan at 400 g/kg of diet (containing curdlan at 100
g/kg, providing a final concentration of 40 g/kg of diet). Increased
full and empty caecal weights were observed with curdlan at a dose of
150 g/kg of feed. The petaechial haemorrhages and mucosal ecchymosis
occasionally observed in the small intestinal mucosa of dogs at all
doses were considered to be unrelated to treatment.
In a lifetime study of carcinogenicity in mice, addition of
gelled curdlan at 400 g/kg of diet or curdlan at dietary levels of up
to 150 g/kg did not cause significant abnormalities, although
decreased food consumption was seen at the highest dose of curdlan and
increased food consumption with the gelled curdlan. No changes in
tumour incidence were observed. In a two-year study in rats, the
highest dose of curdlan (150 g/kg of diet) decreased growth and food
consumption and increased the weights of full and empty caeca. The
gelled curdlan had no effect.
In another two-year study, rats were exposed in utero. The
growth of those exposed to curdlan at 150 g/kg of diet was inhibited
and they showed a slight decrease in food consumption. Increased
emptied caecal weights were seen in males given curdlan at 50 g/kg of
diet, in females given 150 g/kg of diet, and in females given gelled
curdlan at 400 g/kg of diet. Clinical chemical analyses during
treatment showed increased aspartate aminotransferase and serum
alkaline phosphatase activities in animals given the highest dose of
curdlan or gelled curdlan. Gross and microscopic examination revealed
a significantly increased incidence of benign uterine polyps in rats
exposed to curdlan at 150 g/kg of diet, with incidences of 0/50 in
controls, 3/50 in rats given curdlan at 10 g/kg of diet, 4/47 at 50
g/kg of diet, 7/50 at 150 g/kg of diet (significant), and 2/50 with
the gelled curdlan. The authors reported that benign uterine polyps
were seen infrequently in control animals; the incidences in
historical controls were not available.
In a three-generation study of reproductive toxicity in rats,
with two litters per generation, no effect was seen on fertility,
gestation, or the viability of the pups. Parents given curdlan at 150
g/kg of diet or gelled curdlan, providing 400 g/kg of diet, showed
slight growth inhibition. Food consumption was slightly decreased in
parents at the highest dose of curdlan. Furthermore, female F2
parents given the high dose of curdlan or gelled curdlan in the diet
had increased full and empty caecal weights. The weights of the pups
in nearly all litters of dams at the high dose of curdlan were
significantly decreased during lactation: in F1a and F1b litters at
14 and 21 days of age; in F2a and F3a litters at 4, 7, 10, 14, 17,
and 21 days of age; in F2b litters only at day 4 of age; and in F3b
litters only at day 21 of age. The NOEL for both the maternal and
embryonal toxicity of curdlan was 50 g/kg of diet. Although the
authors suggested that the decrease in pup weight gain during
lactation at the highest dose of curdlan was due to consumption by the
pups of their mothers' diet, it might also have been a
treatment-related effect or a combination of consumption of the
mothers' diet and an effect through the milk. In order to investigate
these suggestions, a number of single-generation studies (two litters
per generation) were performed in which the offspring of treated
females were nursed by untreated female rats and the offspring of
untreated females were nursed by treated dams. A further
single-generation study was conducted with cellulose at 50 or 150 g/kg
of diet. In these studies, curdlan or cellulose at 150 g/kg of diet
significantly decreased pup weight gain during lactation, and transfer
of pups from treated to control dams during lactation decreased this
effect. When treatment of the dams with curdlan was withdrawn during
lactation, the weights of the pups were comparable to those of control
pups at this time.
The three-generation study of reproductive toxicity included a
study of teratogenicity in the F2c litters. No embryotoxic or
teratogenic effects were observed at any dose of curdlan up to 150
g/kg of feed or with gelled curdlan at 400 g/kg. In a study of
teratogenicity in rabbits treated orally by gavage, no effects were
seen.
Curdlan was inactive in assays for gene mutation in vitro in
bacteria and in mouse lymphoma cells and did not induce chromosomal
aberrations in hamster ovary cells. It did not induce micronucleus
formation in mice in vivo.
No pathogenicity was observed in mice that received oral doses of
live or dead cells of the curdlan-producing strain, Alcaligenes
faecalis var. myxogenes NTK-u, or in mice that received
intravenous, intraperitoneal, or intracerebral injection of live
organisms. The strain was not cytotoxic to HeLa cells.
Curdlan was not immunotoxic in mice or rats. It did not induce
skin sensitization in humans, although this study was of limited
value.
In a four-week study in which six volunteers consumed up to 50 g
of curdlan daily, increased flatulence was observed. One subject who
consumed 50 g of curdlan per day had some diarrhoea. No evidence of
toxicity was seen.
5. EVALUATION
Curdlan did not induce genotoxic, carcinogenic, or teratogenic
effects or effects on reproduction. At high doses, curdlan decreased
growth and/or food consumption and increased the weights of full
and/or empty caeca. These effects are commonly observed after the
consumption of large amounts of 'indigestible' bulking materials.
The Committee noted the significant increase in the incidence of
benign uterine polyps in rats exposed to curdlan in utero at the high
dose of 150 g/kg of diet in the long-term study. The effect appeared
to be dose-related; however, uterine polyps were not observed in the
long-term study in mice or in a long-term study in rats of the same
strain and from the same laboratory that did not involve exposure in
utero. These benign growths are known to occur naturally in older
rats at incidences of 1-20%, depending on the study and strain. In
view of the lack of genotoxicity and the structure and metabolism of
curdlan, the Committee allocated a temporary ADI 'not specified'1 for
use of curdlan as a food additive, pending the provision of
information on its use and intake. Information necessary for an
assessment of the intake of curdlan, including its use, the maximum
and typical expected levels in the food categories in which curdlan is
proposed for use, and the consumption of foodstuffs that might contain
curdlan in different regions of the world, is required for evaluation
in 2001.
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1 ADI 'not specified' is a term applicable to a food component of
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See Also:
Toxicological Abbreviations