CARRAGEENAN & FURCELLARAN
Explanation
These substances have been evaluated for acceptable daily intake
by the Joint FAO/WHO Expert Committee on Food Additives in 1969 and
1974. New data on carrageenan have become available and are summarized
in the present monograph.
Native carrageenan is a mixture of highly sulfated
polygalactosides and is extracted from seaweeds. The detailed
structure varies slightly between samples depending on the source but
all samples have a sulfate/galactoside ratio of approximately one and
a molecular weight of 800 000 to one million. Native carrageenan is
used by the food and toiletry industry.
Degraded carrageenan is prepared from the extract of Eucheuma
spinosum by partial hydrolysis, using dilute HC1, followed by
purification. The sulfate/galactoside ratio is the same as in native
form but the molecular weight is only 20 000. This material is not
used by food manufacturers but is sold as an antipeptic agent on the
continent. It has, however, been used as a model compound in many of
the investigations into the mechanism of action of native carrageenan
since it produces the same lesion in guinea pigs as the native form
but much more rapidly and consistently.
Furcellaran is derived from the other varieties of seaweeds and
has not been tested as such. However, it belongs chemically to the
general range of carrageenan and carrageenan-like substances.
Degradation of carrageenan
Some researchers have noted the possibility of degradation of
native carrageenan in the gut. This possibility seems to be of limited
toxicological significance for, if native carrageenan were
sufficiently degraded in the gut to cause ulceration or tumor growth,
then the feeding studies would have shown it.
Since food grade carrageenan does not have the same effects as
degraded carrageenan, it is either not degraded, not degraded to the
same molecular weight, or not degrated in the same way.
The evidence we have so far is that it is only partly degraded,
that most of the degradation takes place in the stomach and that this
limited degradation has no effect on the gut wall. In the stomach
where pH is very low, acid hydrolysis undoubtedly does occur. In
vitro experiments with a kappa-lambda mixture in simulated gastric
juice at pH 1.2 and 37°C showed that in three hours the breakdown of
glycosidic linkages was less than 0.1% (Stancioff et al., 1975).
Ekstrom & Kuivinen (1983) recently reported a breakdown for kappa
carrageenan that was about 15 times greater. Furthermore, they
hydrolyzed for six hours at pH 1.0 - rather drastic conditions
unlikely to occur normally in the stomach. In a full stomach the pH
would be expected to be considerably higher.
There is currently no evidence that carrageenan is degraded in
the lower gut. Incubation of a carrageenan solution with the cecal
contents of rats for several hours at 37°C did not alter its
viscosity, which indicates that the microbial flora, of the rat gut at
any rate, will not break down carrageenan (Grasso et al., 1973).
Ochuba & Ven Riesen (1980) reported in vitro degradation of
carrageenan by a large number of intestinal bacteria but their results
are not valid because the carrageenan they used contained 20% reducing
sugar which would have given a positive result in their test method.
Among the bacteria they claimed would break down carrageenan were
Kelbsiella pneumonia and Escherichia coli. However, both these
species can be grown on carrageenan gel media (Epifanio et al., 1981).
If these bacteria had been able to degrade carrageenan, they would
have liquified the gel medium on which they were growing.
Pittman et al. (1976) reported breakdown of food grade
carrageenan isolated from feces of guinea pigs, rats and monkeys.
There was no indication of where the breakdown occurred. It is to be
noted that there were no intestinal lesions associated with this
breakdown, suggesting that the molecular weight attained was not as
low as that of degraded carrageenan. Indeed, Pittman's data showed
that the molecular weight was reduced to about 40 000 or 50 000
(compared to 10 - 20 000 for degraded carrageenan) irrespective of the
species of animal it was fed to.
Pittman's work also shows that degraded carrageenan is absorbed
while the high molecular weight material is not. This is further proof
that degradation in the gastrointestinal tract is limited.
BIOLOGICAL DATA
ABSORPTION
Native carrageenan
Rat
Native carrageenan, untreated or heat sterilized in milk, is
quantitatively excreted in the feces of the rat (Tomarelli et al.,
1974). Chen et al. (1981) failed to find carrageenan present in the
livers of rats fed 25 native carrageenan in the diet for one month.
Similarly, subchronic rat feeding studies in which rats were fed diets
containing 1 or 5% of carrageenan did not result in the storage of
carrageenan in the liver (Coulston et al., 1975). Pittmann et al.
(1976) fed rats diets containing 5% C. crispus carrageenan for 13
weeks and reported that carrageenan was not found in the liver. Grasso
et al. (1973) did not detect the presence of carrageenan in the small
and large intestine of rats fed 5% native carrageenan. Nicklin and
Miller (1983) reported that orally administered carrageenan of high
molecular weight could penetrate the mucosal barrier of adult animals
via transport by macrophases in Peyer's patches. Carrageenan
administration did not affect the number or distribution of these
cells. However, when antigen was administered systematically to
carrageenan fed rats the antigen specific antibody response was
suppressed. This result suggest that carrageenan may interfere with
antigen processing by macrophages and thus modify normal immune
function.
Analyzing liver samples from rats fed 25% native carrageenans
(Chondrus crispus or iridaea) in the diet for one month only the
second was found in the livers of two animals. Gamma metachromatic
reaction sites were seen in the Kupffer cells of these two rats
indicating the presence of carrageenan (Chen et al., 1981).
Monkey
Rhesus monkeys given 1% native carrageenan in drinking water for
7-11 weeks (with a subsequent 11-week recovery period) showed no
evidence of storage of carrageenan (Abraham et al., 1972). In another
study on rhesus monkeys, Mankes & Abraham (1975) found no tissue
storage of carrageenan when the monkeys were given 1% native
carrageenan in the drinking water for 10 weeks. Pittman et al. (1976)
reported that monkeys receiving daily doses of native carrageenan
(500 mg/kg) for 15 months excreted 12 µg of carrageenan per milliliter
of urine. This value (12 µg) was reported to be at the limit of
detectability of the method. Monkeys receiving 500 mg/kg, 200 mg/kg or
50 mg/kg of native carrageenan did not show evidence of storage of
carrageenan in the liver or other organs after 7.5 years of oral
administration (Abraham et al., 1983).
Guinea pig
Grasso et al. (1973) fed native carrageenan (Eucheuma spinosum)
at 5% in the diet for 21-45 days which resulted in the accumulation of
36 to 400 µg carrageenan/g of cecal or colonic tissue. The carrageenan
was contained intracellularly in macrophages.
Engster and Abraham (1976) reported that food grade carrageenans
administered as a 1% solution in drinking water for 2 weeks are not
retained in the guinea pig cecum.
Rabbit
Udall et al. (1981) reported in an abstract that carrageenan
could be detected in the liver, stomach and small intestine of new-
born rabbits orally dosed with 40 mg undegraded carrageenan.
Carrageenan was not detected in the cardiac or portal blood, 4 hours
after the treatment.
Degraded carrageenan
Rat
Rats given 5 g/kg of degraded carrageenan daily by stomach tube
over a period of 12 weeks, showed a storage of the test compound in
membrane-bound vacuoles both in hepatocytes and in mononuclear
phagocytes (Kupffer cells). The stored material was metachromatic;
acid phosphatase activity was detected at similar sites in hepatocytes
but not in Kupffer cells (Abraham & Ringwood, 1977).
Rats given 5% solution of the degraded carrageenan in the
drinking water for 3 weeks, had carrageenan material in the spleen,
liver, colon, kidney, cecum and ileum (Grasso et al., 1975).
Monkey
Rhesus monkeys were given solutions of 2% of degraded carrageenan
in the drinking water for 7-11 weeks followed by a 24 weeks period of
recovery, and 1-0.5% solutions for 14 weeks without recovery. Degraded
carrageenan was taken up by, and selectively stored in lysosomes of
the reticuloendothelial system in the liver (Abraham et al., 1972).
Degraded carrageenan was administered in a 2% solution for 10
weeks in the drinking water to 6 rhesus monkeys. Two animals were
allowed to recover for 24 weeks. Submucosal macrophages from the colon
was observed to contain lysosomes with fibrillar material
(carrageenan) even after a period of prolonged recovery (Mankes &
Abraha, 1975).
Guinea pig
The administration of degraded carrageenan as 0.5 and 0.25%
solutions in the drinking water for 4 and 12 weeks, or of 5% solution
in drinking water for 5-9 days to guinea pig resulted in carrageenan
storage in the intestine, spleen, liver and kidney (Grasso et al.,
1973, 1975). Similar results were obtained by Pittman et al. (1976).
Rabbit
Three rabbits given 1.5 g/kg degraded carrageenan by gavage daily
for 28 days had carrageenan in epithelial cells and macrophages of the
caecum and proximal colon (Grasso et al., 1973).
Pittman et al. (1976) concluded that the absorption of
carrageenan depended on its molecular weight: the higher the molecular
weight, the less material was absorbed. They estimated that molecules
of molecular weight 10 000 to 85 000 represented an upper limit of the
size of carrageenan molecules absorbed. This upper limit is likely to
be influenced by the medium in which it is administered (i.e. drinking
water or diet). Carrageenan is probably more "available" in drinking
water than it is in the diet where it is likely to be complexed to
dietary proteins. Engster & Abraham (1976) investigated the effect of
carrageenan molecular weight and carrageenan type on cecal carrageenan
accumulation. Food grade carrageenan (kappa and lambda) were not
accumulated regardless of molecular weight, whereas accumulation of
iota carrageenan occurred over a molecular weight range of 8 000 to
107 000. The apparent discrepancy between these results and the
results of Pittman et al. (1976) are not explained.
Systemic administration
Rat
Kappa, lambda and iota carrageenans were administered i.p.
(125 g/kg) to Sprague-Dawley rats. Blood samples were taken after 1.5
hours, 1, 2, 4, 7 and 14 days. Each carrageenan (but especially kappa
and lambda) caused thrombocytopenia and red-cell damage within 2 days,
This was followed by rebound thrombocytosis and persistent anaemia,
accompanied by a reticulocytosis. A two-fold increase in fibrogen was
observed at 24 or 48 h (Davidson et al., 1981).
Groups of Sprague-Dawley rats (7 male) were injected i.p. with
125 mg/kg carrageenan kappa, lambda and iota for 14 days. Kappa was
clearly nephrotoxic (increase of serum urea, creatinina levels,
N-acetyl-ß-delta-glucosaminidase and aspartate aminotransferase
activity) (Thomson & Whiting, 1981).
Mouse
Native carrageenan was injected i.p. to mice 0.5-2.5 g/animal.
The mice were killed 1-4 days after injection. Carrageenan inhibited
the immune response to T-cell-dependent antigens, while the immune
response to T-cell-independent antigens was not affected. Changes of
the spleen weight and DNA synthesis in spleen cells were seen (Antoni
et al., 1979).
Five mg potassium carrageenan given to mice in 0.5 ml as a single
i.p. injection was found to be hepatotoxic. Raised serum transaminase
activity was correlated with necrosis of the liver (Fowler et al.,
1977).
TOXICOLOGICAL STUDIES
Reproduction and teratology studies
Rat
In a three generation reproduction and teratology study groups of
Osborne-Mendel rats (40 males and 40 females for each) were fed with a
diet containing calcium carrageenan at levels of 0.5, 1.0, 2.5 or
5.0%. After weaning, all animals were fed carrageenan in their diets
for 12 weeks before mating. Carrageenan ingestion caused dose-related
and significant decrease in the weights of offspring at weaning, but
no effects were detecteds in respect of fertility, average litter
size, average number of liveborn animals, viability or survival of
offspring. Diarrhoea was marked in animals fed the two highest dose
levels (Collins et al., 1977a).
Long-term multigeneration effects of the dietary intake of
calcium carrageenan were measured in a three-generation reproduction
and teratology study in Osborne-Mendel rats. Dietary levels of 0.5,
1.0, 2.5 or 5.0% were ingested throughout the study. Developmental
effects were studied in the F2C and F3C litters. No dose-related
effect on maternal weight gain was observed. The average numbers of
corpora lutea, implantations and early or late deaths, and the average
percentage resorptions per litter showed no dose-related differences.
No specific external, skeletal or soft-tissue anomaly could be
correlated with dosage (Collins et al., 1977b).
Four groups each of 21-24 pregnant rats were fed 1%, 5% with
sodium and calcium carrageenan from day 6 of gestation through day 16.
In addition to the two dietary levels of each carrageenan, concurrent
groups received the basal (control) diet and one group each received
aspirin by stomach tube at the same levels currently used in positive
controls. Terminal sacrifice for all groups was scheduled on day 20.
The uterine contents were examined and the numbers of implants,
resorptions, live and dead fetuses recorded, as was the average weight
of the liver pups in each litter. All fetuses were examined grossly
for evidence of external abnormalities. During the mid-trimester of
gestation, the ingestion of diets containing as much as 5% of either
the sodium or calcium salt of carrageenan has no detectable effect on
maternal or fetal survival, on the rate of nidation, or on the degree
of maturation of fetuses. Under the conditions of the experiment,
neither material was a teratogen for rats (Bailey & Morgareidge,
1973).
Sprague-Dawley rats were given a diet containing 1.8, 0.9 and
0.45% calcium carrageenan. Treatments were administered 14 days prior
to mating, 1-14 days during breeding, throught gestation (22 days),
lactation (21 days) and postweaning testing (69 days) i.e. from
weaning at 21 days of age until the termination of the experiment at
90 days of age. Observations were made on reproduction and on the
physical and behavioural development of the offspring. Effects
observed in the carrageenan groups were inconsistent and not dose-
related (Vorhees et al., 1979).
Hamsters
Four groups each of 21-26 pregnant hamsters were fed 1% and 5%
with sodium and calcium carrageenan from day 6 through day 11. In
addition to the two dietary levels of each carrageenan, concurrent
groups received the basal diet and one group each received aspirin by
stomach tube at the same levels currently used in positive controls.
For all groups terminal sacrifice was scheduled on day 14.
The uterine contents were examined and the numbers of implants,
resorptions, live and dead fetuses recorded, as was the average weight
of the live pups in each litter. All fetuses were examined grossly for
evidence of external abnormalities. During the mid-trimester of
gestation, the ingestion of diets containing as much as 5% of either
sodium or calcium salt of carrageenan has no detectable effect on
either maternal or fetal survival, and on the degree of maturation of
fetuses. There was only a marginally significant reduction in the
pregnancy rate of females fed 5% of the calcium salt in the diet.
Under the conditions of the experiment, neither material was a
teratogen for hamsters (Bailey & Morgareidge, 1973).
Randomly selected syrian hamsters were intubated with sodium or
calcium native carrageenan or degraded carrageenan in distilled water
at dose levels of 10, 40, 100 or 200 mg/kg on days 6-10 of gestation.
Animals of the control groups were intubated with distilled water on
the same days. Day 0 was considered the day on which sperm were found
in the vagina. At least 21 pregnant females were examined at each dose
level of calcium and sodium carrageenan. Only eight pregnant females
were tested at each dose level of degraded carrageenan as only a
limited supply of this compound was available. The highest level of
200 mg/kg was chosen because the gelling capacity of the compounds
precluded higher concentrations. The animals were killed on day 14.
No dose-related teratogenic or foetotoxic effects occurred with any of
the three compounds tested (Collins et al., 1979).
Chick embryo
Prior to incubation, 240 chick eggs were injected in the yolk sac
0.1 mg of a sterile suspension of the 0.1% lambda-carrageenan in 0.9%
sodium chloride. For controls, 240 eggs were injected with 0.1 ml
saline solution, and 240 eggs received no treatment. After mating, for
assessing the potential embryotoxic effect of carrageenan, the
following parameters were determined; mortality rate of embryos in
which development based was arrested, retardations of development
based upon body weight and length of 3rd toe and beak and incidence of
gross malformations. Mortality of embryos in the groups injected with
carrageenan was significantly higher than in the other two groups.
Anomalies of treated groups were mainly localized in the cephalic end
(exencephaly, abnormal beak, anophtalmy, etc.). All abnormal chicks of
the group injected with carrageenan showed two or more anomalies.
Growth of newborn chicks in the carrageenan-injected group was
significantly diminished until the 4th day of age.
So lambda carrageenan, in these experimental conditions, has
teratogenic and lethal effects on chick embryo development (Rovasio &
Monis, 1980).
Furcellaran was tested for toxic and teratogenic effects to the
developing chick embryos. It was administered in water via the air
cell at pre-incubation (0 hours) and at 96 hours of incubation, and
via the yolk at 0 hours at 96 hours. Administration of furcelleran at
0 hours by both routes resulted in a line whose slope was not
significantly different from zero, while administration at 96 hours by
both routes resulted in a line with a negative slope. No LD50
estimates could be made from the regression lines (Anonymous, 1976).
Furcellaran was tested for toxic and teratogenic effects to the
developing chicken embryo under four sets of conditions. It was
administered in water as the solvent by two routes and at two stages
of embryonic development; via the albumen at pre-incubation (0 hours)
and at 96 hours of incubation, and via the yolk at 0 hours and at 96
hours using techniques that have been described previously. The route
of albumen, instead of the usual air cell, was chosen because of the
fact that the administered furcellaran solution formed globular
coagulates as soon as it was injected into the air cell, and
absorption through the embryonic membrane was not conceivable.
Furcelleran was found to be quite embryotoxic when administered
to the embryos under all conditions of the test. Probit analysis
resulted in an LD50 of 1.611 mg/egg at 0 hours and that of 1.449
mg/egg at 96 hours via the albumen. Yolk treatment at 0 hours resulted
in an LD50 of 1.089 mg/egg, while the same treatment at 96 hours
resulted in a line whose slope was not significantly different from
zero. From these results it cannot be concluded that furcelleran is
teratogenic to the chicken embryo. However, it is a note-worthy
finding that in both the albumen and yolk sac administration routes,
with the higher dose levels (5.0 mg and 1.0 mg/egg) at 0 hours there
were anomalies of the eye and maxilla which were not observed in the
solvent-treated embryos. This may warrant an additional study of the
effect of furcelleran on embryonic development (Hwang et al., 1974).
Special studies on mutagenicity
Sodium Carrageenan was tested for genetic activity in a series of
in vitro microbial assays with and without metabolic activation on
Salmonella typhimurium and Saccharomyces cerevisiae at the
concentration 0.5, 1.0, 2.0%. The test compound, sodium carrageenan,
did not demonstrate genetic activity (Brusik, 1975).
In vivo cytogenic studies
Furcellaran was administered to male rats with an average body
weight of 300-350 gr. In the acute study (single dose) and in the
subacute study (five doses) a dose of 5000 mg/kg was employed.
Metaphase chromosome spreads were prepared from the bone, marrow cells
of these animals and scored for chromosomal aberrations. Neither the
variety nor the number of these aberrations differed significantly
from the negative controls; hence, furcelleran can be considered non-
mutagenic as measured by the cytogenetic test.
Dominant lethal
Furcellaran, edible, was administered to ten male rats (400 gr)
at a dose level of 5 000 mg/kg according to acute (single dose) and
subacute (five doses) protocols. Each treated male rat was mated with
two virgin female rats each week for seven (subacute) or eight (acute)
weeks. Two weeks after mating, these female rats were sacrificed and
the fertility index, preimplantation loss and lethal effects on the
embryos were determined and compared with these same parameters
calculated from negative (saline-dosed) and positive (0.3 mg/kg TEM-
dosed) control animals.
The values calculated from these parameters from animals with
compound FDA T-52, furcelleran, edible, did not significantly vary
from those obtained from the negative controls, except for an increase
in corpora lutea during week 2 of the acute. Since the increase in
corpora lutea was not matched by an increase in the number of
implants, it is also refelcted as a preimplantation loss. No unusual
explanation for this increase in corpora lutea is apparent. TEM
caused a significant preimplantation loss and embryo resorption during
the first five weeks.
Comparing these data with the previously obtained valus for dose
levels of 715 mg/kg, 71.5 mg/kg and 7.15 mg/kg revealed no dose-
response or time trend patterns, thus indicating that furcelleran does
not include dominant lethal mutations as measured by this test.
Effect on lipid metabolism
One group of rats were fed 5% alphacel plus 15% undegraded
carrageenan. The control group was treated with 5% alphacel. After 33
weeks the data indicated that dietary carrageenan exhibited
cholesterol-lowering effect in rats, increase in feces weight and in
the concentration and daily output of fecal cholesterol and total bile
acids (Reddy et al., 1980).
Short-term studies
Degraded carrageenan
Rat
Groups of 10 male and 10 female Sprague-Dawley rats were given a
5% solution of degraded carrageenan in the drinking water for 3
months. Control rats received distilled water. The gross appearance
and histology of the ceca was normal. On rare occasions, the cecum was
slightly distended with soft, semi-fluid contents. The blood vessels
in these ceca were more prominent than usual. Small groups of
macrophages exhibited moderate acid phosphatase activity in cecal
sections (Abraham et al., 1974).
Four groups of rats were fed with basal diet (group I and II) or
were fed diet containing 10% degraded carrageenan for two weeks and
then basal diet for 28 weeks (group III and IV). Dimethylhydrazine
(DMH) was administered subcutaneously to group II and IV at a dose of
10 mg/kg once a week for 15 weeks. The animals were observed for 30
weeks and then sacrificed. Colorectal squamous metaplasia of the
mucosa was seen in all animals in group III (10% carrageenan) and IV
(carrageenan + DMH). In the same groups erosions with active
inflammatory reaction were observed in the colon. The incidence of
tumours of the small intestine was 20% in group II and 50% in group
IV. The incidence of intestinal tumors induced by DMH was higher in
animals fed on diet containing degraded carrageenan than animals fed
on basal diet (Kawaura et al., 1982).
Degraded carrageenan was administered to rats as a 5% solution in
the drinking water for 3 weeks. Other 2 groups (18 males and 18
females) received degraded carrageenan in drinking water at 0.5 and
0.25% for 12 weeks. At 5%, rats developed severe diarrhoea and gained
less weight than the controls. Macroscopically no changes were evident
in the caecum colon or rectum. Microscopically there was oedema in the
distal part of the rectum but no clear evidence of microphage
infiltration was present. Histochemically no carrageenan was
demonstrable in the small or large intestine or rectum. The rats
treated with 0.5, 0.25% degraded carrageenan appeared to be healthy
throughout the experiment and gained weight. The feces were well
formed but softer than normal (Grasso et al. 1975).
Rats (10 males group) were given 5-1% degraded carrageenan in the
drinking water for 56 days. The animals with 5% dose had diarrhoea
with watery stools that was less evident with 1% level. No ulcerations
were seen in both groups (Grasso et al., 1973).
Guinea pigs
20 male guinea pigs receiving 5% degraded carrageenan in the
drinking water for 5-9 days developed severe diarrhoea and began to
lose weight after day 1. Occult blood was detected in the faeces from
day 4 onwards and the condition of the animals deteriorated rapidly
until they were killed between days 5 and 9. At autopsy, haemorrhagic
areas were present in the wall of the caecum and proximal colon of all
animals. Histopathological investigation revealed these to be large
areas of gross submucosal oedema, haemorrhage and ulceration. The
cellular infiltrate in the lamina propria consisted mainly of
lymphocytes with few macrophages and polymorphonuclear cells.
The addition of neomycin to the diet markedly reduced the
population of polymorphonuclear cells in the ulcer but did not affect
the apparent incidence of ulcers or the time of their appearance
(Grasso et al., 1973).
10 female guinea pigs given 2% degraded carrageenan in the
drinking water for 21-45 days developed multiple pinpoin caecal and
colonic ulcerations after 3-5 weeks of treatment. Histologically, the
ulcers consisted of extensive macrophage infiltration at the base,
over which lay a thin layer of fibrin. Polymorphonuclear cells and
lymphocytes were present in appreciable numbers. The epithelium around
the ulcer was heavily infiltrated by macrophages, polymorphs and
lymphocytes. In some animals, heavy polymorphonuclear infiltration
occurred, leading to the formation of micro-abscesses close to the
ulcerated areas. The addition of neomycin (0.1%) to the diet markedly
reduced the population of polymorphonuclear cells in the ulcers but
did not affect the apparent incidence of ulcers or the time of their
appearance (Grasso et al., 1973).
20 male guinea pigs were given 1% degraded carrageenan in the
drinking water. Groups of 2-3 animals were killed every 3-4 days up to
42 days. In another experiment with the same concentration, 28 male
were treated for 3 weeks. Groups of 4 killed at week 1, 2, 3, 4, 7, 11
and 15.
Caecal and colonic ulcers were seen in animals receiving 1%
degraded carrageenan in the drinking water. The stages leading to
ulceration were observed in the animals killed sequentially.
Observation at week 2 showed an increase in the number of macrophages
in the lamina propria. The macrophages often formed dense masses and
obliterated the mucosal crypts (granulomas). They varied considerably
in size: a few were visible to the naked eye as pale raised areas.
This macrophage infiltration was present in five out of six animals
examined. The collections of macrophages were strongly acid
phosphatase-positive and ultrastructurally were shown to possess large
vacuoles lined by single membranes. Macrophage necrosis was sometimes
observed at week 3 and 4.
Multiple ulcerations were found in seven of the 12 animals
examined. They were pin-point in size when viewed macroscopically and
were invariably found microscopically to be accompanied by marked
macrophage infiltration. Loss of epithelium occurred only over areas
of these pronounced macrophage accumulations. Carrageenan was
demonstrable within the macrophages forming the base of the ulcer.
Three out of four guinea pigs killed immediately after consuming
a 1% solution of degraded carrageenan in the drinking water for 3
weeks were found to have caecal ulcerations. Histologically the
lesions were similar to those observed in our earlier studies. No
caecal or colonic ulceration was seen in animals killed 1 or 4 weeks
after the cessation of treatment, but distinct granulomas with
demonstrable carrageenan within the macrophages could be identified.
In animals killed at a later date, the histology of the caecum and
colon was indistinguishable from that in the controls and no
carrageenan could be identified in the macrophages within the lamina
propria (Grasso et al., 1973).
Ingestion of the 2% of degraded carrageenan in drinking water by
8 female guinea pigs for 2 weeks resulted in ulcerative lesions in the
caecum, and, to a lesser extent, in the colon, and in oedema of the
mesentery and hypertrophy of the mesenteric lymph nodes.
Sequential studies, conducted over a period of 7 to 14 days, of
the changes leading to ulceration, revealed focal increases in the
number of large foamy macrophages in the lamina propria between the
glands. The epithelium overlying the macrophages became attenuated;
more accumulation, death and disintegration of macrophages was
associated with loss of glands and more severe degeneration of luminal
epithelium. Ulceration occurred in these altered areas and was
followed by infiltration of heterophils. Granulomas with a central
core of large foamy macrophages were often observed in the submucosa
beneath the ulcers. After 8 days of administration of the 2% solution,
acid phosphatase activity was moderately increased, in the epitehlial
cells as well as in the macrophages of the lamina propria. By 14 days,
the time at which caecal ulcerations were predominant, a striking
increase in acid phosphatase activity was observed in the epithelium
and in the subepithelial macrophages, with formation of large dense
particulates. A significant departure from normality in the form of an
increase in the number of dense bodies (lysosomes) was noted in the
epithelial cells (Abraham et al., 1974).
14 male guinea pigs received degraded carrageenan at 5% in the
drinking water over periods from 20 to 45 days. All animals showed
loss of weight (15-25%) which became apparent after the second week.
At the end of the first week treated animals showed looseness of the
stools. Tests for the occult blood in the faeces became positive
in over half the animals by the 18th day and in all animals
carrageenan-fed by the 30th day. Ulcerative lesions in the large
intestine occurred in 12 of the 14 animals treated for 20 to 45 days.
4 animals were killed between the 20th and 25th days: 2 showed
ulceration of the caecum and proximal colon. All guinea pigs killed on
the 30th day (6) and between 35th and 45th days (4) showed ulcerative
lesions in various parts of the large intestine.
There was fatty change in the liver of 9 animals in experimental
group (Watt & Marcus, 1971).
142 male guinea pigs treated with 5% (w/v) solution of degraded
carrageenan for 30 days as the sole source of fluids. 21 or more days
after the experiment was started, all animals had gross and
microscopic evidence of disease (ulcerative lesions). None of the 10
untreated animals had any evidence of ulcerative lesions. Germfree
guinea pigs given the same solutions for comparable periods of time
did not develop caecal ulcerations (Onderdonk et al., 1981).
Seven iota fractions with intrinsic viscosities (dl/g): 0.113,
0.285, 0.685, 1.62, 4.19, 5.34 and 7.51 (molecular weights from 5 000
to 145 000); three kappa fractions: 0.177, 1.45 and 11.95 (molecular
weights 8 500, 51 500 and 314 000) and three lambda fractions: 0.503,
2.243 and 10 250 (molecular weights 20 800, 74 800 and 275 000) were
given to female guinea pigs as a 1% solution in the drinking water for
2 weeks. Six of these iota fractions (0.113 dl/g was not used) were
also fed to female guinea pigs in the diet at 2% level for 10 weeks.
When given in the drinking water, all iota fractions except those with
intrinsic viscosities (dl/g) of 0.113 and 7.51 were absorbed and
retained in acecal lamina propria and submucosal macrophages as
indicated by toluidine blue staining, by the presence of fibrillar
material in membrane-bound vacuoles, and by increased lysosomal acid
phosphatase activity. Histopathological changes in the caecum of
guinea pigs given iota fractions of intrinsic viscosities (dl/g)
0.285, 0.685, 1.62, 4.19 and 5.34 in the drinking water were
epithelial thinning, slight erosion, cellular infiltration and crypt
abscesses. Ulceration of the caecal mucosa was present in guinea pigs
given the two iota fractions of 0.685 and 1.62 dl/g in the drinking
water. The lowest (0.113 dl/g) and highest (7.51 dl/g) iota fractions,
administered in the drinking water, were neither absorbed nor stored
nor caused any histopathological effects. Iota fractions given to
guinea pigs in the diet produced no inflammatory response, erosion or
ulceration of the caecum. Caecal damage was not noted in groups of
animals given the kappa and lambda carrageenan fractions in drinking
water (Engster & Abraham, 1976).
Five male guinea pigs were given 5% degraded carrageenan in the
drinking water for 2 weeks. The carrageenan group consistently lost
weight throughout the treatment period in comparison to the control.
Animals carrageenan treated ate considerably less than the controls
(-45%). Also fluid consumption was depressed in the carrageenan
groups (-44%). One animal died on the 10th day. The group receiving
carrageenan showed a high incidence of fecal occult blood in the
caecum upon autopsy. One animal had a slight heamorrhage along the
caecum vessels. A second animal had congested vessels on the mucosal
surface. The caecum of the other animals appeared normal (Boxenbaum &
Dairman, 1977).
In another experiment 2% degraded carrageenan was dissolved in
milk and given to 3 guinea pigs over a three months period. An equal
number of control animals received milk alone. Ulcers were not
observed in the caeca of treated guinea pigs. Histological examination
revealed both the mucosa and submucosa to be normal in disposition.
Most macrophages in the lamina propria showed no metachromasia. In
contrast to the macrophages in the lamina propria, those in the
submucosa were strongly metachromatic, as were the fibroblasts and
endothelial cells. Acid phosphatase activity was not increased, and
distribution of lysosomes was similar to control animals. All the
caeca of guinea pigs receiving milk alone were normal and similar to
control animals (Abraham et al., 1974)
Monkey
6 monkeys (Macaca mulatta) male and female, were given 2%
degraded carrageenan in drinking water for 10 weeks. Equal number of
control animals were used. The monkey caecum was normal in all
respects in treated animals. Both groups had normal epithelium,
glands, lamina propria and muscularis mucosa. In the treated animals
there were numerous macrophages in the submucosal region that
contained fibrillar (carrageenan) material. Cells containing
carrageenan-like material were absent in other areas of the caecum
(Abraham et al., 1974).
5 males and 5 females received various concentrations of degraded
carrageenan in the drinking water up to week 14. 6 animals drank a 2%
solution, 2 a 1% solution and the remaining 2 a 0.5% solution. The
corresponding average daily intakes of degraded carrageenan were
calculated to be 2.9, 1.4 and 0.7 g/kg respectively. 2 monkeys from
the 2% were sacrificed after 7 and 11 weeks of exposure. The remaining
4 animals were kept on their dosage regimen for 14 week and then
allowed to recover on tap-water for a period of 20-24 weeks, when they
were killed. The 4 animals given the other dose levels were killed at
the end of the 14 week treatment period. 2 males and 2 females monkeys
were used as controls. 5 out of the 6 monkeys given 2% degraded
carrageenan did not gain weight. Almost immediately after the onset,
the consistency of the stools became loose and watery. After 2-3 weeks
occult blood was found in stool samples and melaena appeared sometimes
associated with discharge of frank blood and mucus.
Monkeys given lower levels of degraded carrageenan (1 or 0.5%)
gained weight or maintained their initial body weight. Faeces were for
the most part firm or soft, rarely loose or watery. Occult blood in
the faeces appeared 2-3 weeks after the beginning of treatment. In one
animal killed at 7 weeks (2% degraded carrageenan) the entire
intestinal tract was macroscopically normal and the microscopic
abnormalities were limited to the colon. In a monkey killed at 11
weeks more pronounced changes were seen in the caecum and colon and
their regional lymph nodes. Microscopically, slight oedema, increased
number of macrophages, two submucosal abscesses were seen. The colon
had multiple fresh mucosal haemorrhages throughout its entire length,
ulcerations and crypt abscesses.
The two monkeys that received 1% degraded carrageenan for 14
weeks had similar lesions in the lower intestinal tract. The abnormal
findings in the 2 animals given 0.5% degraded carrageenan for 14 weeks
were less conspicous. Only the female had several mucosal
haemorrhages.
In the four monkeys that had received 2% degraded carrageenan for
14 weeks followed by a 24 week recovery period, the tests for fecal
occult blood were positive for 10 weeks after the withdrawal of
carrageenan. At the autopsy the intestinal tract was grossly and
microscopically normal in both males. In one female several large deep
crypt abscesses with degenerative changes of the epithelium were found
in the colon. The colon of the 2nd female had two ulcers (Benitz et
al., 1973).
2 female squirrel monkeys were given 1.5 g/kg body weight of
degraded carrageenan by gavage daily for 28 days. Soft faeces were the
principal effect produced. The treated animals did not develop ulcers
(Grasso et al., 1973).
Rabbit
3 female rabbits received 1.5 g/kg bw. of degraded carrageenan by
gavage daily for 28 days. Faecal softening was the principal effect.
Granuloma formation was seen in the caecum and proximal colon of the 3
rabbits, but ulceration was visible macroscopically in only one
(Grasso et al., 1973).
Ferret
3 females were given 1.5 g/kg by gavage of degraded carrageenan
for 28 days. Soft stools were observed (Grasso et al., 1973).
Hamster
8 female hamsters were given 5% degraded carrageenan in the diet
for 6 months. The animals developed a frank diarrhoea with watery
stools. No ulcers were present in the gastro-intestinal tract (Grasso
et al., 1973).
Native carrageenan
Rat
A single dose of 0.5 to 50 mg native carrageenan was given to
Lewis rats by gavage. Low doses of carrageenan resulted in significant
suppression of spleen and mesenteric lymph node responses to the
phyto-hemagglutinin (PHA) while high doses showed no effect. In a
second series of experiment, 2 litters of DA rats were weaned on
distilled water containing 0, 0.1, 1 mg/ml carrageenan for 8 weeks
(daily intake of carrageenan was 2.3 and 23.1 mg respectively).
The dose of 0.1 mg/ml markedly suppressed spleen cell responses
to PHA while 1 mg/ml gave responses equivalent to the water controls
(Bash & Vago, 1980).
RE 7064 Iridaea carrageenan (25%) was incorporated into three
different basal diets: WayneR Lab Blox, a commercial rodent diet
prepared from chemically undefined sources; TD 76110, a complete high
protein synthetic rodent diet and TD 76112, identical to TD 76110
except for the deletion of vitamin E.
Rats fed RE 7064 in WayneR diet for 4 weeks lost weight and
their growth was retarded. Soft stools, decreased relative liver
weight and gross hepatic atrophy were also observed. In contrast to
these effects, rats fed RE 7064 in TD 76110 showed no adverse effects.
Reduction of dietary protein by simple dilution yields 18% protein in
WayneR diet (TD diets are not affected by dilution, their protein
value remains at 25%). It is thus suggested that protein binding by
Iridaea carrageenans reduces the dietary availability of protein below
a minimal requirement and produces hepatic atrophy (nutritional
atrophy). Rats fed vitamin E deficient TD 76112 for 3 weeks, with or
without 25% RE 7064, did not develop vitamin E deficiency. Despite
this obvious shortcoming it appears that high protein diet still
prevents hepatic atrophy, as only 1/6 rats given RE 7064 in TD 70112
developed liver atrophy.
Neomycin sulfate was incorporated at the level of 50 mg/kg of
either WayneR diet and 25% RE 7064 and fed to rats for 4 weeks. This
treatment failed to either protect or alter the atrophic response of
rats fed RE 7064. Surprisingly, rats fed neomycin alone had in their
livers numerous inflammatory foci, councilman bodies and autophagic
vacuoles. These observations have not been previously reported. As
neomycin sulfate inhibits protein absorption, the similarity of
hepatic changes in rats fed RE 7064 and those fed neomycin sulfate
support the concept of protein depletion being responsible for
initiation of hepatic atrophy. Autophagic vacuoles were present after
2 weeks of recovery but were rarely seen after 4 weeks. Further, no
evidence of fibrosis or repair was seen in liver sections. In keeping
with earlier studies, carrageenan was stored in Kupffer cells even
after 4 weeks of recovery (Mankes R.F., 1977).
Rats received for 50 days native carrageenan in the diet of 0, 15
and 25% level (10 females and 10 males each group; 25 males and 25
females the control group). It was noted on the 4th day that all the
animals receiving the test diet had begun to develop diarrhoea. The
symptoms appeared more marked at the 25% level than at the 15% level.
Males and females at the 25% level excreted bloody stools. All of the
animals receiving native carrageenan on the 8th day appeared to be
losing hair from the dorsal mid-line surfaces on the back. The
symptoms were more severe at the 25% level and in the females. On the
31st day 5 males and 5 females for each group were killed. The
carrageenan treated animals appeared smaller than the control. On the
50th day the remaining animals were sacrificed and necropsied. No
gross pathological abnormalities were noted. Many of the livers
examined had a mottled appearance, but this phenomenon occurred also
in the control rats. The rate of feed consumption for the test animals
was generally less than the controls (Anonymous, 1975).
Nutritional studies were conducted on rats fed diets containing
carrageenan that had been mixed into skim milk at a concentration
equal to that of the protein. When fed in a simulated milk powder
diet, the processed carrageenan at a dietary level of 4% had no
influence (compared to glucose or cellulose) on growth rate, diet
energy efficiency, absorption of protein, fat, calcium, blood
coagulability, utilization of protein for growth, or utilization of
iron. Gross and microscopic examination of the caecum and colon, after
6 months feeding, revealed no abnormalities (Tomarelli et al., 1974).
The effect of dietary undegraded carrageenan (Viscarin 402) on
colon carcinogenesis was studied in female inbred F344 rats. Weanling
rats were fed semipurified diets containing 0 or 15% undegraded
carrageenan. At 7 weeks of age, all animals except controls were given
azoxy-methane (AOM) s.c. at a dose rate of 8 mg/kg body weight per
week for 10 weeks or methylnitrourea (MNU) intrarectally at a dose
level of 2 mg/rat twice a week for 3 weeks. The AOM groups were
autopsied 40 weeks and MNU groups 30 weeks after the first injection.
No tumors were induced in the colon or in other organs of untreated
rats fed the control diet. One untreated rat fed the carrageenan diet
showed a colon adenoma. The animals fed the carrageenan diet and
treated with AOM or MNU had a higher incidence of colorectal tumors
(number of rats with colorectal tumors and number of tumors per
tumor-bearing rat) than did those fed the control diet and treated
similarly. The undegraded carrageenan (Viscarin 402) in the diet had
an enhancing effect in colorectal carcinogenesis in rats evoked by AOM
or MNU (Watanabe et al., 1978).
Iridaea carrageenan was given orally to groups of 15 male and 15
female rats at dietary levels of 1% or 5% for 90 to 92 days. A group
of 13 males and 17 females was given 5% alpha cellulose and served
as control. No remarkable compound related changes in behaviour,
body weight, food consumption, hematology, clinical chemistry,
urinanalysis, fecal analysis, gross pathology, histochemistry, or
electron microscopy were elicited at any dose level. However,
councilman bodies, increased frequency of monocytic accumulations and
pigmented Kupffer cells were noted in liver sections from rats fed 5%
Iridaea carrageenan. In addition, high dose rats displayed significant
decreases in lung weight, spleen weights, and brain-heart weight ratio
(only in females) after 90 days. Mid-dose rats of both sexes had only
an increased frequency of monocytic accumulations and councilman
bodies in liver sections (Abraham et al., 1979).
Guinea pig
Female guinea pigs received 5% native carrageenan in the diet for
21-45 days. The animals developed multiple pin-point caecal and
colonic ulcerations after 3-5 weeks. Histologically, the ulcers
consisted of extensive macrophage infiltration at the base, over which
lay a thin layer of fibrin. Polymorphonuclear cells and lymphocytes
were present in appreciable numbers. The epithelium around the ulcer
was heavily infiltrated by macrophages, polymorphs, and lymphocytes.
In some animals, heavy polymorphonuclear infiltration occurred,
leading to the formation of micro-abscesses close to the ulcerated
areas. The addition of neomycin to the diet markedly reduced the
population of polymorphonuclear cells in the ulcers but did not affect
the apparent incidence of ulcers or the time of their appearance
(Grasso et al., 1973).
Mouse
25% RE 7064 (Iridaea) given in the diet to mice for 4 weeks
resulted in significant decreases in body weight, growth and relative
liver weight. Unlike the rat, however, no gross hepatic atrophy was
observed. Histologic examination of the livers from these mice
revealed eosinophilic (autophagic) vacuoles, focal inflammation and
occasional necrosis of hepatocytes. Carrageenans were found both
within vacuoles of Kupffer cells and hepatocytes, and unlike the rat,
mitochondrial aberrations were observed in mouse hepatocytes (Mankes
R.F., 1977).
Monkey
3 males and 3 females Rhesus monkeys received native carrageenan
in the drinking water as a 1% solution corresponding to an average
daily intake of 1.3 g/kg.
Two animals were sacrificed after 7 and 11 weeks respectively,
the remaining four being put back on plain tap-water and allowed to
recover for 11 weeks. These animals were then used for a dose-ranging
experiment, using excalating daily doses of carrageenan ranging from
50 to 1250 mg/kg daily for up to 12 weeks. The highest dose was given
for 8 weeks. After this second period of exposure, all four monkeys
were killed. All animals gained weight and remained in good condition.
Occult blood occurred sporadically in the faeces as was the case in
controls. Only minor changes, none attributable to carrageenan
administration, were found in the intestinal tract at autopsy and on
microscopic examination. Similar findings were recorded in animals
given 1% native for 11 weeks and subsequently after a recovery period
of up to 11 weeks on tap-water, given escalating daily doses of
50-1250 mg native/kg for up to 12 weeks (Benitz et al., 1973).
Male and female infant baboons were reared from birth to 112 days
of age on infant formulas containing concentrations of 300 and
1500 mg/l of carrageenan (equivalent to 86 and 432 mg/kg/day).
For the first 14 days, the animals were fed 5 times per day; four
times for the next 14 days; 3 times for the next 56 days and 2 times
for the 28 days until 112 days of age. Carrageenan content of the
formula did not affect weight, characteristics of urine and faeces,
findings on physical examination, hematological variables, blood
chemical analyses, organ system weights, or the macroscopic and
microscopic appearance of the gastrointestinal tract (McGil et al.,
1977).
Rat
Two strains of rats (Osborne/Mandel and Sprague-DawleyL) were
given two basal diets (FDA and AMC) as well as 5% carrageenan for 9
months. 18 males and 18 females were employed in each group. The
control animals were treated with 5% alphacel in the diet. No
significant macroscopic or microscopic changes were observed in the
liver related to the administration of carrageenan. Livers were normal
in both control (alphacel) and treated rats on gross and microscopic
examination obtained from both strains of rats. No evidence of storage
of carrageenan-like material (metachromatic) was observed in the
parenchymal or Kupffer cells of the rats in all the groups examined by
light microscopy; no fibrillar material (carrageenan-like material)
was observed by electron microscopy in the livers in any of the
groups; changes were observed in the appearance of the stools (soft
and semi-fluid) at the 5% level with both strains of rats; occult
blood in the faeces was sporadic in all groups in time of appearance
and quantity (Coulston et al., 1976).
Different groups of Sprague-Dawley rats received 5-1% of the
three types of carrageenan (kappa from Chondrus crispus, lambda from
Gigartina acicularis, iota from Eucheuma spinosum) in the diet for
9 months (15 males and 15 females). The control animals were given 5%
alphacel in the diet.
5 male and 5 female rats from each group were killed at 3 months.
The complete gross examination did not result in any grossly
detectable abnormalities. No morphollogical changes were seen. Rats
receiving both 5% and 1% carrageenan in the diet over a 40 week period
grew well.
Occult fecal blood was detected in 75% of the rats given 5%
alphacel and in 25-50% of the rats fed the two levels of carrageenans.
Stools in treated rats were slightly soft. No metachromatic material
was noted in either hepatic or Kupffer cells in rat livers. No
fibrillar material (carrageenan) was present in Kupffer cells or
hepatocytes (Coulston et al., 1975).
Degraded carrageenan
Long-term studies
Rat
30 male and 30 female rats were given 10, 5, 1 and 0% of degraded
carrageenan (average m.w. 20-40 000) in the diet up to 24 months.
Grossly visible blood on the surface of the stools and soft to
semi-fluid stools were present in 10% and 5% groups.
Colorectal squamous metaplasia and tumors were observed in 10%
and 5% dose levels. Metaplasia was first observed in both groups after
6 months and final incidence was 98.3% and 88.3% respectively. The
tumors were squamous cell carcinomas, adenocarcinomas and adenomas.
Tumors were first observed in both groups after 12 months and final
incidence was 31.7% and 20% respectively.
No effect was observed with 1% degraded carrageenan treatment
(Wakabayashi et al., 1979).
20 male and 20 female Sprague-Dawley rats were given a 5% aqueous
solution of degraded carrageenan as drinking water for 15 months.
The control groups received distilled water. Colorectal squamous
metaplasia was first observed after 4 months and the final incidence
was 100%. Tumors (squamous cell carcinomas, adenocarcinomas, adenomas
and amyosarcoma) were first observed after 10 months and the final
incidence was 17.5% (Wakabayashi et al., 1978).
Groups of 10 male and 10 female rats were given either a 5%
solution of degraded carrageenan as their sole source of drinking
water or a dose of 5 g of degraded carrageenan per kg in aqueous
solution by stomach tube (once daily, 6 days per week) for 15 months.
Groups of 5 rats of each sex, given distilled water, either as
drinking fluid or by stomach tube, served as controls. Rats were
killed at intervals ranging from 1 to 15 months. At 15 months,
degraded carrageenan was withheld from the 2 remaining rats - one
female from the group that had received degraded carrageenan in
drinking water, and one male from the group that received it by
stomach tube. These rats were killed at 17 and 16.5 months,
respectively. In the second experiment, groups of 20 female rats were
given by stomach tube, daily doses of 0.5 and 5 g/kg for 6 months. A
control group of 20 female rats was given daily doses of distilled
water. Four rats were killed from each group at 4, 17 and 26 weeks.
Additional rats were killed from the 5 g/kg group at 9, 14 and 19
weeks.
In rats given 5 /kg by gavage, or 5% solution as drinking water
(6 to 10 g/kg/day) the stools were soft to semi-fluid. Occult blood
was first detected in those faeces within 3 to 7 days. After 2 to 3
weeks grossly visible blood was occasionally seen on the surface of
the stools. Administration of degraded carrageenan to rats in daily
doses of 5 to 10 g/kg resulted in squamous metaplasia of the rectal
mucosa and accompanied by accumulation of metachromatic material
(presumably carrageenan) in macrophage lysosomes. Such changes were
not observed in rats given 0.5 g/kg/day for 6 months (Fabian et al.,
1973).
Eight-week-old male Fischer 344 rats were maintained on a diet
containing 10% degraded carrageenan. Thirty-nine animals received this
ratio for two months (group 1), 42 animals for six months (group 2)
and 42 animals for nine months (group 3), respectively. Forty-six
additional animals, referred to as controls, received the same diet
without carrageenan. No mortality had occurred by 18 months following
the start of the carrageenan feeding, when all animals were
sacrificed. A 100% incidence of colorectal squamous metaplasia was
found in all treated groups. Tumors were also reported in 5/39 animals
in group 1 (3 squamous-cell carcinomas, 1 adenoma, 1 anaplastic
carcinoma), 8/42 in group 2 (6 squamous-cell carcinomas,
1 adenocarcinoma, 1 adenoma) and 17/42 in group 3 (14 squamous-cell
carcinomas, 4 adenicarcinomas). Colorectal changes were detected in
none of the control rats (Oohashi et al., 1981).
Groups of rats of 15 males and 15 females each were given 5, 1
and 0 g of degraded carrageenan/kg body weight by stomach tube for 15
months. The control group was given distilled water. In group 1
(5 g/kg) colorectal squamous metaplasia was first observed after 15
months. Metaplasia alone was observed after 14 months in group 2
(1 g/kg). Final incidence of metaplasia in groups 1 and 2 was 100% and
36.7% respectively. Colorectal tumors (adenocarcinomas and adenomas)
were first observed in group 1 after 15 months and the final incidence
was 27.6% (Wakabayashi et al. 1978).
Guinea pig
0.02 and 0.2% degraded iota carrageenan was given in drinking
water to 8 females for 12 and 10 months respectively. The gross
appearance and the histological organization of the caecum and colon
were normal. The number of macrophages within the lamina propria of
the caecum was similar to that in control animals, but on staining
with toluidine blue, a few of the macrophages from the 0.2% degraded
carrageenan animals contained metachromatic material (Abraham et al.,
1974).
Native carrageenan
Rat
Groups of 30 male and 30 female of MRC rats received native
carrageenan at dose levels of 5%, 2.5% and 0.5% in the diet for the
lifespan. A group of 100 males and 100 females served as control.
Average daily carrageenan intake was 4.022, 1.998, 0.360 g/kg
respectively. Animals occasionally develop a solf stool consistency,
particularly at the beginning. The histopathololgical alterations in
rats could not be attributed to the prolonged action of carrageenan
(Rustia et al., 1980).
Rats (Sprague-Dawley) were given two types of carrageenan
(RE 7063, an extract of Hypnea and RE 7064, an extract of Iridaea) in
the diet at levels of 1% and 5% for one year. Significant weight loss
(p = 0.05) was observed in male and female rats fed the two types of
carrageenan at both levels as compared to the alphacel control group.
Livers were normal at the 1% diet level on gross and microscopic
examinations obtained from rats receiving the two carrageenans. Gross
and microscopic examinations of livers obtained from 5% RE 7063
animals were normal in all respects except for 2/12 livers, that had
nodules. Gross observations on the liver in the group of rats
receiving 5 RE 7064 indicated a) ten rats (10/13) had decreased in
size, rough surface and vacularization of the livers and probably
related to the administration of RE 7064 at the 5% level in the diet;
b) microscopically these livers were normal in the 5% carrageenan
(RE 7064) group with the exception of 1 out of 10 where focal necrosis
was noted.
No evidence of storage of carrageenan-like material
(metachromatic) was observed in the liver cell of rats in all the
groups.
No fibrillar material (carrageenan) was observed by electron
microscopy in the livers of any of the groups. No changes were
observed in the stools at 1% level with either carrageenan. Female
rats (RE 7064 5%) showed loose stools (12/100). Male rats given either
carrageenan at the 5% level also showed loose stools. Hematest results
in all groups were sporadic in appearance and therefore not
significant. Except for gross changes in the livers in one group
(RE 7064 at 5%) no chemical-related effects were observed in rats in
this experiment (Coulston et al., 1975).
Monkey
40 Rhesus monkeys (19 male and 21 female) were given 0, 50, 200,
500 mg/kg bw. native carrageenan by gavage daily for six days a week
for the first 5 years. Thereafter for the remaining 2.5 years,
carrageenan was incorporated into diet and given to monkeys.
Loose stools, chronic intestinal disorders, poor appetite and
emaciation were seen in an apparently random distribution. Stool
consistency was decreased in a dose related trend over 7.5 years while
positive fecal occult blood findings were increased in a similar
fashion.
No statistical differences in mean survival times were detected
in any group as compared to controls. No gross or microscopic changes
were detected in tissues examined. Sporadic body weight changes from
controls were noted in a random fashion. Only females had significant
body weight depression from years 5 to 7.5, which did not appear to be
dose-related. No consistent statistically significant changes occurred
in hematology, clinical chemistry values, absolute organ weights, or
organ to body weight ratios after 7.5 years of feeding carrageenan.
Cytochemical and ultrastructural observations revealed no storage of
carrageenan-like material in livers obtained at biopsy or in other
organs obtained at necropsy from monkeys given carrageenan. No other
gross or microscopic changes in tissues occurred in a dose-response
relationship (Abraham et al., 1983).
Hamster
30 male and 30 female Syrian golden hamsters received native
carrageenan at dose levels of either 5%, 2.5% or 0.5% in the diet
daily for the lifespan. The animals did not develop neoplasm in
response to treatment at any dose levels. The histological alterations
in hamsters could not be attributed to the prolonged action of
carrageenan (Rustia et al., 1980).
OBSERVATIONS IN MAN
Six patients suffering from malignant disease of the colon were
given 5 g degraded carrageenan daily for 10 days before a colectomy
was performed. Samples of normal sections of the colon obtained at
surgery were examined for any signs of ulceration and analyzed
histochemically and chemically for the presence of degraded
carrageenan in the tissue. There were no signs of any ulceration in
these samples of gut, nor was degraded carrageenan detected by either
the histochemical or the analytical method (Grasso et al., 1973).
Comments
Carrageenan is a high molecular weight sulfated galactan derived
from a number of species of red seaweeds of the class Rhodophyceae.
The carrageenan used in food has a high molecular weight
(800 000 / 100 000) and has useful thickening and gelling properties
at concentrations as low as 0.01%.
Degraded carrageenan has a very low molecular weight
(20 000 / 30 000) and has no gelling properties whatsoever, even at
very high concentrations of 10% or more.
Most of the degradation takes place in the stomach and that
limited degradation has no effect on the gut wall. In vitro
experiments with kappa and lambda mixture showed that in three hours
the breakdown of glycosidic linkages was less than 0.1%. Carrageenan
is resistant to attack by bacteria and degradation by human intestinal
bacteria is very rare. Even though there are indications that the
microbial flora of the rat gut at any rate, will not break down
carrageenan, it has been reported breakdown of food grade carrageenan
(40 000/50 000 MW) isolated from faeces of guinea pigs, rats and
monkeys. There were no intestinal lesions associated with this
breakdown; in fact, the molecular weight attained was not as low as
that of degraded carrageenan.
In in vitro mutagenesis assays native carrageenan was not
mutagenic to S. typhimurium strains TA-1555, TA-1537 or TA-1538 or
the yeast strain S. cerevisiae D4, with or without metabolic
activation. In teratology studies with dietary concentrations up to 5%
native carrageenan given to rats and hamster resulted in no adverse
effects. In utero exposure of rat to undergraded carrageenan caused
no effects on physical or behaviour development of offspring of
parental animals fed 1.8% prior to mating during gestation and
lactation and after weaning. Injection of 0.1% native carrageenan into
the yolk sac of chicken eggs has resulted in increased lethality, a
decrease in hatching weight and a slight increase in malformations. In
a three-generation rat reproduction study a decrease in birth weight
and body weights at weaning were seen in the offspring of dams fed
1.0, 2.5 and 5.0. No effects were seen on reproductive parameters at
any dose level. Native carrageenan administered in the food to rats at
concentrations of more than 5% showed no adverse effects in studies
ranged from 30 days to 12 months. The administration of 5% carrageenan
in the diet for 21-45 days to guinea pigs caused caecal and colonic
ulcerations.
No adverse effects were seen when native carrageenan was
administered to infant baboons (432 mg/kg bw) and to Rhesus monkeys
(1300 mg/kg bw) for 112 days and 12 weeks respectively. Rats and
hamsters have been fed 5% carrageenan for their lifetimes. Survival
was not affected in either species. No statistically significant
pathological effects were seen in either species at any of the dietary
level used in the study. Rhesus monkeys have been fed 500 mg/kg/day
for 7.5 years. The only effects seen were soft stools or diarrhea and
occasional fecal occult blood which was also seen in untreated
controls. No pathologic effects attributable to the intake of
carrageenan were seen in the monkeys.
Degraded carrageenan administered in dose above 1%, whether in a
water or food vehicle, causes ulceration and metaplasia of colorectal
region of the intestinal tract in rats. The ulcerative effects
reported for the low molecular weight degraded carrageenan appear
dependent upon the animal species and the method of oral
administration. In fact, guinea pigs and rats are the most susceptible
species to ulcerative changes in the large bowel.
There were no signs of an ulceration in samples of gut, nor was
degraded carrageenan detected by either the histochemical or the
analytical method when six patients were given 5 g degraded
carrageenan daily for 10 days.
EVALUATION
Estimate of acceptable daily intake for man
Not specified.
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See Also:
Toxicological Abbreviations
Carrageenan and furcellaran (FAO Nutrition Meetings Report Series 46a)
Carrageenan and Furcellaran (WHO Food Additives Series 5)