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    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 37C 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 37C 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 g of carrageenan per milliliter
    of urine. This value (12 g) 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 g 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--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)