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    Toxicological evaluation of some food
    additives including anticaking agents,
    antimicrobials, antioxidants, emulsifiers
    and thickening agents



    WHO FOOD ADDITIVES SERIES NO. 5







    The evaluations contained in this publication
    were prepared by the Joint FAO/WHO Expert
    Committee on Food Additives which met in Geneva,
    25 June - 4 July 19731

    World Health Organization
    Geneva
    1974

              

    1    Seventeenth Report of the Joint FAO/WHO Expert Committee on
    Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
    FAO Nutrition Meetings Report Series, 1974, No. 53.

    CARRAGEENAN AND FURCELLARAN

    Explanation

         These substances have been evaluated for acceptable daily intake
    by the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
    Ref. No. 20) in 1969.

         Since the previous evaluation, additional data have become
    available and are summarized and discussed in the following monograph.
    The previously published monograph has been expanded and is reproduced
    in its entirety below.

         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 industries.

         Degraded carrageenan is prepared from the extract of Eucheuma
    spinosum by partial hydrolysis, using dilute HCl, followed by
    purification. The sulfate/galactoside ratio is the same as in the
    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.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption

         Native carrageenan. Rats probably do not absorb carrageenan
    (2% to 20% in diet) but excrete it quantitatively in their faeces
    (DHSS, 1972). Guinea-pigs absorb native carrageenan in the caecum and
    proximal colon and its presence can be demonstrated in the macrophages
    of the lamina propria by staining with toluidine blue. Absorption is
    also indicated by the presence in the same sites of Perls' positive
    granules when iron-labelled native carrageenan is fed for five days.
    No absorption from the gastrointestinal tract could be demonstrated by
    these techniques in the rat and mouse (BIBRA, 1971).

         Degraded carrageenan. Two baboons excreted in the urine
    2-6 mg/kg of degraded carrageenan following a single oral or
    parenteral dose of 3 g/kg. No material could be detected in the blood

    (DHSS, 1972). Degraded carrageenan is absorbed by the caecum and
    proximal colon of the guinea-pig and rabbit where it can be
    demonstrated in the macrophages of the lamina propria by staining with
    toluidine blue or by newly developed Alcian blue technique which
    distinguishes degraded carrageenan from mucopolysaccharides. It has
    also been demonstrated in these tissues by an electrophoretic
    technique (BIBRA, 1971). Absorption is also indicated by the presence
    in the same sites in the guinea-pig and rabbit of Perls' positive
    material when iron-labelled degraded carrageenan is fed. No absorption
    from the gastrointestinal tract could be demonstrated either
    histologically or better use of iron-labelled material in the rat,
    mouse, squirrel monkey, hamster and ferret. Five patients suffering
    from carcinoma of the transverse colon were given 5 g degraded
    carrageenan each day for 10 days before partial colectomy. At
    operation specimens of colon were obtained. Histological sections from
    all five resections were stained by the toluidine blue and Alcian blue
    techniques and one specimen was analysed by the electrophoretic
    technique. No degraded carrageenan could be detected in the tissues
    either histologically or chemically (the chemical method is sensitive
    to 0.5 µg/g tissue) (BIBRA, 1971).

         Rats excrete dietary carrageenan quantitatively in the faeces
    over levels of 2% to 20% and it therefore has no direct nutritive
    value (Hawkins & Yaphe, 1965). Groups of five rats received 0.5%
    native carrageenan, or 5% degraded carrageenan for 10 days. Faecal
    excretion and weight gain of the two polymers were similar (Dewar &
    Maddy, 1970). Guinea-pigs failed to excrete in their urine up to
    158 mg orally administered undegraded carrageenan. If given
    intraduodenally to guinea-pigs with ligated pylorus, it reduces
    gastric secretion. Two adult baboons received single doses of
    240 mg/kg (more than three times the therapeutic dose in man) or
    3 g/kg of degraded carrageenan while a third baboon received both the
    high and the low dose levels with a 14-day rest period between doses.
    Only at the high level were 3-6 mg/kg excreted in the urine after
    24 hours and no carrageenans could be detected in the blood up to
    18 hours after dosing (Beattie et al., 1970). Groups of six rats were
    fed diets containing 17.4 or 34.8% carrageenan for three weeks. Weight
    gain was significantly reduced especially at higher levels. Food
    efficiency showed interference with utilization of other nutrients in
    the diet. Only 10% to 15% appeared digestible as estimated from faecal
    examination (Carey, 1958).

         Single intravenous injections of degraded carrageenan into nine
    groups of three, four or five guinea-pigs produced a dose-related
    increase in urinary polysaccharide excretion. Oral or intraduodenal
    administration of degraded carrageenan gave detectable residues in the
    urine and reduced gastric secretion in guinea-pigs with ligated
    pylorus. However, the amounts absorbed were very small (Anderson &
    Soman, 1966). Subcutaneously administered degraded carrageenan lowers

    significantly the volume of fasting gastric secretion in guinea-pigs
    and tends to reduce the total acid concentration of fasting secretion.
    When administered parenterally it has a pronounced and prolonged
    inhibitory effect on histamine - stimulated gastric secretion
    (Eagleton et al., 1968). The prevention of histamine-induced gastric
    and duodenal ulceration in the guinea-pig has been demonstrated using
    a series of undegraded and degraded carrageenans. The degree of
    antipeptic activity is probably determined by a combination of
    structural features which includes molecular size and polyanionic
    properties (Anderson & Soman, 1967; Anderson & Watt, 1959).
    Artificially produced gastric ulcers (30% acetic acid injection) in
    rats did not show any significant acceleration in healing when
    carrageenan was administered concurrently (Takagi et al., 1969).
    Inhibition of peptic activity was obtained in the Shay rat preparation
    (Levey & Sheinfeld, 1954).

         Carrageenan inhibits pepsin action by binding the substrate
    (Anderson & Watt, 1959; Piper & Fenton, 1961; Houck & Bhayana, 1960
    and Bonfils et al., 1966). Interference with pepsin activity depends
    on concentration of carrageenan and protein. It occurs only over a
    narrow range (Marquez & Garcia, 1960; Vaughan et al., 1961; Vaughan et
    al., 1962 and Bonfils et al., 1960). 5% carrageenan in the diet had no
    effect on utilization of cassein, soybean protein, or of other
    proteins by rats (Friedman & Douglass, 1960). Carrageenan complexes
    with mucoproteins in the human stomach with a consequent increase in
    viscosity and eventual precipitation (Anderson & Watt, 1959 and Marcus
    & Watt, 1965).

         No difference in protein economy of the growing rat was observed
    with a diet of 0.5 to 5% carrageenan and either a high quality protein
    (casein) or a low quality vegetable protein (USFDA, 1969).

         Subcutaneously injected carrageenan stimulated the biosynthesis
    of collagen and the formation of connective tissue (Robertson &
    Schwartz, 1953). 3% carrageenan in the diet has been reported to
    reduce the plasma cholesterol level in chicks by 50% (Riccardi &
    Fahrenbach, 1965). Carrageenan acts as an antithrombic in both human
    and dog blood plasma (Hawkins & Leonhard, 1962). The presence of
    carrageenan in infant feeds does not affect the availability of any
    added iron (Gorton et al., 1963; Gorton & Cross, 1964; Owen & Fomon,
    1963; Fomon et al., 1961).

         A seven-month infant feeding study of the nutritional value of
    Formil, an infant formula containing carrageenan (concentration not
    stated), showed normal results in feeding pattern, growth, bowel
    function, and serum calcium, phosphorus and cholesterol levels (Marine

    Colloids, Inc., 1969). It has been suggested that the presence of
    carrageenan in infant food may lead to the selective presence of
    intestinal organisms capable of forming galactose from carrageenan.
    This may be important for galactosaemic infants whose food should
    therefore be free from carageenan (Davis, 1970). In 50% of patients
    suffering from chronic peptic ulcer and treated with degraded
    carrageenan, it was noted that carrageenan was still present in
    gastric juice 7 to 10-1/2 hours after a single dose (Marcus & Watt,
    1965).

    Pharmacodynamic properties

         Carrageenan is rapidly taken up by mouse peritoneal macrophages
    in tissue culture with subsequent slow interaction between the
    engulfed carrageenan, the lysosomal enzymes and the lysosomal cell
    membrane; the enzymes eventually pass into the cytoplasm of the
    macrophages which are then destroyed. The action of silica is similar
    to carrageenan except that the cells more rapidly succumb and finally
    a fibrous response is seen because the lysosomal cell membranes are
    quickly destroyed. If no lysosome destruction occurs the foreign
    material is merely stored in the intact macrophages (Allison et al.,
    1966). Monocyte infiltration, histiocyte proliferation and macrophage
    differentiation can be induced in the rat by a single subcutaneous
    injection of carrageenan. A dual origin of macrophages has been
    observed: through migration of blood monocytes and by cellular
    multiplication in situ. The evolution of the granulomatous reaction
    was followed by the histochemical demonstration of some hydrolytic
    enzymes of lysosomes (Monis et al., 1968).

         Intravenous injection of carrageenan into normal rats lowered the
    bradykininogen levels acutely, and this effect was not prevented by
    indomethacin. It is considered improbable that bradykinin could be an
    important mediator of the inflammation caused by carrageenan (Arman &
    Nuss, 1969). Carrageenan injected intravenously lowers the arterial
    blood pressure of rats. This hypotensive effect is unaffected by
    histamine antagonists and is abolished by protease inhibitors and thus
    seems to be due to kinin release from plasma substrates (Di Rosa &
    Sorrentino, 1970).

         Tissue cultures of embryonic rat-kidney fibroblasts exposed to a
    1% solution of native or degraded carrageenan for four hours showed
    metachromatic granules when stained with toluidine blue. These
    granules persisted for 120 hours following exposure without
    cytopathological changes or suppression of growth rate (Sharratt et
    al., 1970).

    TOXICOLOGICAL STUDIES

    Special studies on large-bowel ulceration in various species

    Native carrageenan

    Mouse

         Minimal changes comprising focal cellular infiltration of the
    colonic mucosa were observed in mice treated with 1% carrageenan in
    drinking water for 30 days to one year (Marcus & Watt, 1969). However,
    no ulceration was found in the gastrointestinal tract of mice
    receiving up to 4 g/kg/day of carrageenan (derived from Eucheuma
    spinosum) for six months. Mice fed on diets containing up to 25%
    carrageenan for their life-span showed no pathological changes in the
    gastrointestinal tract (DHSS, 1972).

    Rat

         Ulceration of the large bowel followed by scarring was seen in
    rats treated with 1% carrageenan in drinking water for 30 days to one
    year (Marcus & Watt, 1969). However, no ulceration of the
    gastrointestinal tract or other untoward effects were seen in rats
    receiving dietary carrageenan (derived from Chondrus crispus and
    Gigartina mamillosa) at a level of up to 12.5 g/kg/day for two years
    (DHSS, 1972). No ulceration was found in rats fed on diet containing
    5% carrageenan (derived from Eucheuma spinosum) for eight weeks
    (BIBRA, 1971) or on diets supplying up to 4 g/kg/day for periods up to
    six months (DHSS, 1972). No abnormality was found in rats receiving
    1.3 to 1.8 g/kg/day of carrageenan (derived from Chondrus crispus)
    in drinking water for six months (Golberg, 1971).

    Guinea-pig

         When fed on a diet containing 5% native carrageenan (derived from
    Eucheuma spinosum or Chondrus crispus) they develop ulcers of the
    caecum within two to four weeks (BIBRA, 1971). After administration as
    a 1% solution (from Eucheuma spinosum) in drinking water ulceration
    occurred in eight out of 10 animals in 20-30 days (DHSS, 1972). No
    ulceration was seen, however, in guinea-pigs which received 1% native
    carrageenan (from Chondrus crispus) in drinking water for six months
    (Golberg, 1971).

    Gerbil

         No untoward effects were found in gerbils which consumed a diet
    containing 5% carrageenan (derived from Chondrus crispus) for six
    months (Golberg, 1971).

    Dog

         No untoward effects were found in four dogs which received
    1 g/kg/day of carrageenan (derived from Eucheuma cottonii and
    Chondrus crispus) for six months (DHSS, 1972).

    Pig

         No gastrointestinal ulceration was found in pigs fed for four
    weeks on diets supplying carrageenan (derived from Eucheuma
    spinosum) at a level of approximately 1.7 g/kg/day (Tournot, 1970).
    Four groups of six pigs received native carrageenan jelly for 12 weeks
    at levels of 0, 50, 200 and 500 mg/kg bw per day. Another group
    received 500 mg dry carrageenan in a powdered diet. No adverse effects
    were seen in behaviour, weight gain, haematology, blood chemistry,
    urinalysis, organ weights. No ulcerative colitis or erosions of caecum
    and colon were seen (Poulson, 1972).

    Monkey

         Six rhesus monkeys received 1% carrageenan (derived from
    Chondrus crispus) in drinking water (1.3 g/kg/day) for a period of
    seven to 11 weeks. Of two monkeys killed at 11 weeks one had minimal
    changes in the colon (minute areas of capillary hyperaemia and oedema
    of mucosa) the relationship of which to carrageenan ingestion is
    questionable. After an 11-week period without treatment the remaining
    four received, by gastric intubation, up to 1.25 g/kg/day of
    carrageenan for 84 days. No abnormalities were observed in these
    animals and at post mortem examination there were no visible changes
    in the gastrointestinal tract (Golberg, 1971).

    Degraded carrageenan

    Mouse

         Marcus & Watt (1969) reported minimal changes, consisting of
    focal cellular infiltration of the colonic mucosa in mice receiving
    0.1 to 5% degraded carrageenan in their drinking water for 30 days to
    one year. Other workers have found no ulcerative lesions in the
    gastrointestinal tract of large numbers of mice receiving up to
    4 g/kg/day for up to six months. At high dosage levels (3 and
    4 g/kg/day) Maillet (1970a) found strongly metachromatic material to
    be present in the Kupffer cells.

    Rat

         Ulcerative lesions were seen in the caecal, colonic and rectal
    mucosa of rats given a 0.1 to 5% solution of degraded carrageenan in
    drinking water for 30 days to one year. The early changes included

    pin-head sized haemorrhagic and ulcerative lesions, along with
    cellular infiltrates and some crypt abscesses. In later stages there
    was loss of mucosal folds and, in some animals, there was scarring of
    the bowel which led to the formation of constriction rings (Marcus &
    Watt, 1969).

    Guinea-pig

         Many workers have shown that degraded carrageenan causes
    ulceration of the large bowel when fed to guinea-pigs (DHSS, 1972).
    While Marcus & Watt report ulceration of the colon and rectum as well
    as the caecum, other workers have found ulceration only in the caecum
    and proximal colon (BIBRA, 1971; Golberg, 1971; Maillet, 1970).

         Two forms of ulceration have been described. In the first, the
    "acute" lesion, with onset of illness appeared in a few days. Severe
    diarrhoea and weight loss occurred within two days and in many animals
    ulceration appeared in four to six days. The ulcers were large, often
    5 mm diameter and had the appearance of stripped-off epithelium. The
    subepithelial tissues were oedematous, markedly so in some cases.
    While accumulation of macrophages was often seen in the areas of
    ulceration, this was not always so and frequently the ulcer had the
    appearance of being the result of mechanical stripping. This type of
    ulceration occurred only when there was profuse diarrhoea, that is
    when degraded carrageenan was administered as a 5% (and in some cases
    2%) solution in drinking water. It appeared that the lesion was
    related more to the osmotic diarrhoea than to carrageenan per se
    since exactly the same clinical, macroscopic and microscopic picture
    was found in guinea-pigs given equiosmolar sodium sulfate or cyclamate
    solutions in place of drinking water (BIBRA, 1971).

         The second "chronic" form of ulceration was found in animals
    receiving 1% degraded carrageenan in drinking water (or native
    carrageenan at any concentration). This ulceration occurred more
    slowly, the first signs appearing after about two weeks. The ulcers
    were pin-point in size and star-shaped. The stages of development of
    this type of lesion have been investigated (BIBRA, 1971).

    Hamster

         No gastrointestinal ulceration was seen in hamsters fed on a diet
    containing 5% degraded carrageenan for five months (BIBRA, 1971).

    Rabbit

         Three out of five rabbits receiving as little as 0.07 mg/kg/day
    of degraded carrageenan in drinking-water developed ulceration of
    the large bowel in three months. The lesions were said to occur in
    the caecum, colon and rectum. Rabbits weighing 3.5 kg were given
    1.6 g/kg/day of degraded carrageenan by gastric intubation for four

    weeks. No ulceration was found but granulomas consisting of
    macrophages were seen in the lamina propria of the caecum. No
    abnormalities occurred in the distal colon or rectum. In rabbits given
    iron-labelled degraded carrageenan macrophage accumulation occurred
    together with ulceration of the caecum but again no abnormalities were
    found other than in the caecum and proximal colon (BIBRA, 1971).

    Gerbil

         No abnormalities were found in gerbils consuming 3.5 g/kg/day of
    degraded carrageenan for six months (Golberg, 1971).

    Ferret

         No ulceration was seen in ferrets fed on a diet providing
    1.5 g/kg/day of degraded carrageenan for four weeks (BIBRA, 1971).

    Pig

         No signs of ulcerative lesions were found in the large bowel in
    10 pigs which received 1.7 g/kg/day of degraded carrageenan for four
    weeks (DHSS, 1972).

    Squirrel monkey

         No ulceration of the bowel was seen in squirrel monkeys which
    received 1.5 g/kg/day of degraded carrageenan as a 10% solution by
    gastric intubation for four weeks (BIBRA, 1971).

    Man

         Patients under treatment for peptic ulceration and ulcerative
    colitis have been treated with degraded carrageenan without ill
    effects (DHSS, 1972). In addition, in France, about 50 000 patients
    are under treatment with 5 g/day of a mixture of degraded carrageenan
    and aluminium hydroxide. It is suggested that if degraded carrageenan
    contributed to the development of ulcerative colitis it might be
    expected to show up in a population of this size under medical care.
    Although treatment has been used for 10 years there is, so far, no
    indication that French peptic ulcer patients are developing ulcerative
    colitis (Shirlaw, 1971). Administration of 100 ml 5% degraded
    carrageenan as retention enema two to three times per week for three
    weeks to several months to patients with ulcerative colitis or Crohn's
    disease improved four out of five patients as seen on X-ray and
    sigmoidoscopy (Bonfils, 1972).

    Histological appearance of ulcers

    Guinea-pig

         Ulcers involve mainly the mucosa and show features of both acute
    and subacute inflammatory infiltration as well as crypt abscesses
    (Watt & Marcus, 1969a, b). Mottet (1970) who examined their material
    considered the ulcers to be in many ways morphologically comparable to
    those of human ulcerative colitis. However, he noted that epithelial
    necrosis involved all regions of the crypt and not the depth of the
    crypt as in the human disease. Alterations were noted in the amount of
    mucus in nearby glands and lymphocytic infiltration and capillary
    congestion were prominent in the lamina propria as in ulcerative
    colitis. A typical epithelial hyperplasia was also noted.

         When neomycin was included in the diet of guinea-pigs the
    carrageenan-induced lesions had a different appearance, the infiltrate
    consisting mainly of macrophages (confirmed by their strong acid
    phosphatase activity and by their appearance under EM) with only a few
    lymphocytes, plasma cells and polymorphs. The incidence and time of
    development of lesions was similar in neomycin-treated and untreated
    animals. No crypt abscesses were seen in neomycin-treated animals. Two
    small crypt abscesses were found in carrageenan-treated animals not
    receiving the antibiotic (BIBRA, 1971). Golberg (1971) found the
    lesion corresponded to that described by Marcus & Watt, but without
    crypt abscesses or epithelial hyperplasia; no antibiotic was included
    in the animals' diet.

    Rabbit

         Ulceration of the large bowel was described by Watt & Marcus
    (1970a, b). Gross thickening of the mucosa of the caecum and colon was
    seen with small sessile polypoid formations on the summit of rugi of
    the caecum. Ulcers were much as they described in the guinea-pig but
    glandular polyps and pseudopolyps (oedematous congested mucosa with
    acute inflammatory infiltration) were prominent features. Ulceration
    which occurred in the caecum of rabbits given degraded carrageenan by
    gastric intubation together with dietary neomycin developed ulcers
    which were in all respects similar to that seen in guinea-pigs
    similarly treated. Neither polyp formation nor gross glandular
    hyperplasia was seen (BIBRA, 1971).

    Development of the ulcer in guinea-pigs

         A study was carried out in which guinea-pigs were killed and the
    whole gastrointestinal tract removed and examined macroscopically and
    histologically after periods of one to 31 days on a dietary regime
    containing 0.1% neomycin and drinking fluid containing 1% degraded
    carrageenan. Histological abnormalities were found only in the caecum

    and proximal colon where the first change was an increase in the
    number of macrophages in the lamina propria after about 14 days'
    treatment. In sections stained with toluidine blue these macrophages
    contained metachromatic material. Alcian blue stained sections showed
    that this was likely to be carrageenan. These cells were more strongly
    acid phosphatase-positive than normal macrophages and the lysosomes
    were larger. Also, at this time, lysosomes, which are normally small
    and sparse in caecal epithelial cells, became larger, more numerous
    and strongly staining. The epithelial cells most strongly acid
    phosphatase-positive were superficial to the most marked accumulations
    of macrophages. The macrophages continued to accumulate and to form
    granulomas in the lamina propria. Frequently degenerating macrophages
    were seen in these granulomas (confirmed by EM). Ulceration was found
    later in those areas where the granulomas occurred.

         The granulomas could be seen on gross examination as pale, raised
    areas under the epithelium. Occasionally these pale areas developed
    into white plaques on which large numbers of pin-point ulcers
    occurred. Histological sections were prepared which stained
    degenerating epithelium overlying a mass of degenerating macrophages
    superficial to the granuloma. The authors consider it likely that
    ulceration is a consequence of the granuloma formation which follows
    carrageenan absorption with the acute inflammatory infiltrate being
    possibly due to subsequent bacterial infection of the ulcerated
    granuloma. It did not appear that the macrophage infiltrate was a
    consequence of ulceration caused by some direct action of carrageenan
    on the gastrointestinal mucosa; direct contact for four hours with 5%
    degraded carrageenan had no untoward effect on cells of the stomach,
    intestine or large bowel in rats or guinea-pigs. The authors point out
    that the pronounced macrophage response, the limitation of the lesion
    to the caecum and the absence of epithelial hyperplasia and crypt
    abscesses (in all neomycin-treated animals and many neomycin-free
    animals) are features which differ from lesion of ulcerative colitis
    in man (BIBRA, 1971).

    Reversibility of the lesion in guinea-pigs

         Maillet (1970) found that the ulcerative lesion in the guinea-pig
    caecum was reversible in eight to 12 weeks when animals were given
    normal diet. This was confirmed by a test in which animals were
    administered 1% degraded carrageenan in drinking water for three
    weeks. All animals killed at this time had caecal ulcers. Surviving
    animals were given normal diet and killed after one, four, eight and
    12 weeks and examined. Histological sections of the caecum were
    prepared. Lesions seen were small ulcers with the base infiltrated
    chiefly with macrophages but also a few mono-nuclear cells and
    polymorphs, the margins consisting of a dense infiltrate of
    macrophages with few other cells. The majority of macrophages were

    grossly vacuolated and others contained one or more inclusions,
    probably necrotic debris. The lysosomes (acid phosphatase positive
    granules) were larger and more numerous than in untreated animals both
    in the macrophages and the overlying epithelial cells.

         No ulceration was seen in animals killed after replacement of 1%
    degraded carrageenan by drinking water. In three of four animals
    killed one and four weeks after cessation of treatment the number of
    macrophages in the lamina propria was decreased although a few
    granulomas were observed. Animals killed later were histologically
    normal. Histochemical examination of specimens from animals killed
    after one, four and eight weeks still showed considerable acid
    phosphatase activity in epithelial cells and macrophages. In all four
    animals killed 12 weeks after ceasing treatment no differences from
    controls could be found (BIBRA, 1971).

    Reproduction and teratology studies

    Mouse

         Groups each of 25-40 pregnant mice were dosed orally with either
    sodium or calcium carrageenate as a 25% suspension in anhydrous corn
    oil, from day 6 through day 15 of pregnancy. The dose levels
    administered were 0, 10, 45, 470 and 900 mg/kg/day. Animals were
    observed daily for appearance and behaviour. On day 17, dams were
    subjected to Caesarean section, and the numbers of implantation sites,
    resorption sites, and live and dead fetuses determined. Body weight of
    live pups were measured. Fetuses were examined grossly for the
    presence of external congenital abnormalities, and then one-third of
    the fetuses underwent detailed visceral examination, and the remainder
    were examined for skeletal defects. Administration of either sodium or
    calcium carrageenate resulted in an apparent increase in the number of
    resorptions and/or fetal deaths in utero. There was a dose-dependent
    decrease in the number of live pups and reduction in pup weight at
    delivery. In addition, there was a concurrent retardation in skeletal
    maturation as shown by the increased incidence of missing sternebrae
    and incomplete skull closures. All other soft tissue and skeletal
    abnormalities appeared to be within normal range (Food & Drug Research
    Lab., Inc., 1972).

    Rat

         Nine pregnant rats were fed 5% degraded carrageenan in their
    drinking water and their offspring received the same material for six

    months. Five out of 12 offspring had bronchiectasis, four out of 12
    had ulceration of the colon (Watt & Marcus, 1971; Marcus & Watt,
    1971).

         Groups each of 24-29 pregnant rats were dosed orally with either
    sodium or calcium carrageenate as a 25% suspension in corn oil, from
    day 6 to 15 of gestation. The dose levels administered were 0, 40,
    100, 240 and 600 mg/kg/day. On day 20 all dams were subjected to
    Caesarean section, and the parameters described in mouse study were
    measured. Administration of either sodium or calcium carrageenate
    resulted in an apparent increase in the number of resorption sites
    without a decrease in the number of live pups delivered. At the
    highest dose level there appeared to be a decrease in birth weight of
    the pups. There was a dose-dependent increase in missing skeletal
    sternebrae, but no other compound-related abnormalities (Food & Drug
    Research Lab., Inc., 1972).

    Hamster

         Groups each of 26-30 pregnant hamsters were dosed orally with
    either sodium or calcium carrageenate as a 25% suspension in anhydrous
    corn oil, from day 6 to 10 of gestation. The dose levels administered
    were 0, 40, 100, 240 and 600 mg/kg bw per day. At day 14 all animals
    were subjected to Caesarean section, and the parameters described in
    mouse study were measured. Administration of either sodium or calcium
    carrageenate had no significant effect on nidation or on maternal or
    fetal survival. There was some evidence of delayed skeletal maturation
    (missing or incomplete centres of ossification) which was dose-
    dependent. No other compound-related effects were observed.(Food &
    Drug Research Lab., Inc., 1972).

    Rabbit

         Groups each of 15 pregnant rabbits were dosed orally with either
    sodium or calcium carrageenate as a 25% suspension in anhydrous corn
    oil, from day 6 through day 18 of gestation. The dose levels
    administered were 10, 60, and 260 mg/kg bw per day. On day 29 all does
    were subjected to Caesarean section, and the parameters described in
    the mouse study were measured. Administration of either sodium or
    calcium carrageenate had no effect on nidation or on maternal or fetal
    survival, nor were there any abnormalities in either soft or skeletal
    tissues that were compound-related (Food & Drug Research Lab., Inc.,
    1972).

        Acute toxicity
                                                                                

                                    Dosage       Measurement
    Animal      Sex   Substance     mg/kg           (1G)*       Reference
                                                                                

    Rabbit      M     Na+ Carr.     4670-5430       LD50        Food & Drug Res.
                                                                Lab. , 1972

    Rabbit      F     Na+ Carr.     4670-5430       LD50        "    "    "    "

    Hamster     M     Na+ Carr.     7530-8470       LD50        "    "    "    "

    Hamster     F     Na+ Carr.     7530-8470       LD50        "    "    "    "

    Rat         M     Na+ Carr.     5650-6250       LD50        "    "    "    "

    Rat         F     Na+ Carr.     5650-6250       LD50        "    "    "    "

    Mouse       M     Na+ Carr.     8730-9670       LD50        "    "    "    "

    Mouse       F     Na+ Carr.     8730-9670       LD50        "    "    "    "

    Rabbit      M     Ca++ Carr.    2280-3000       LD50        "    "    "    "

    Rabbit      F     Ca++ Carr.    2280-3000       LD50        "    "    "    "

    Hamster     M     Ca++ Carr.    6180-7320       LD50        "    "    "    "

    Hamster     F     Ca++ Carr.    6180-7320       LD50        "    "    "    "

    Rat         M     Ca++ Carr.    5140-5660       LD50        "    "    "    "

    Rat         F     Ca++ Carr.    5140-5660       LD50        "    "    "    "

    Mouse       M     Ca++ Carr.    8710-9590       LD50        "    "    "    "

    Mouse       F     Ca++ Carr.    8710-9590       LD50        "    "    "    "
                                                                                

    *  Compounds administered intragastrically as a 25% suspension in anhydrous corn oil.
    
    Rat

         Ten male rats received 12 g/kg/day of degraded carrageenan for 
    three days without mortality or visible adverse effect (Bonfils,
    1959). Rats survived 10 mg carrageenan intravenously, but showed

    histopathological changes in the kidney (Morard et al., 1964). Six
    groups of five male and five female rats received 0, 3.6, 7.2, 14.4,
    21.6 or 28.8 g/kg/day carrageenan for five days. There was no
    mortality or alterations in body weight gain. All test groups
    exhibited diarrhoea with grey coloured stools during the dosing
    period. At the highest dose level two rats had cloudy eyes and one had
    blood in the urine (Rorer, 1966). A group of 10 male and 10 female
    rats received a total of 23 g/kg carrageenan over a five-day period
    with five male and five female rats as controls. There were no deaths
    during the study (Rorer, 1966).

    Guinea-pig

         Guinea-pigs survived 5-10 mg carrageenan intravenously but showed
    histopathological changes in the kidney (Morard et al., 1964).
    Intravenous carrageenan in doses of 1 mg/kg kills guinea-pigs within
    30 minutes (Anderson & Soman, 1966).

    Rabbit

         Intravenous injection of undegraded carrageenan is very toxic to
    rabbits (Anderson & Duncan, 1965). Intravenous injection of 50 mg
    carrageenan killed rabbits within 48 hours. These animals showed total
    obstruction of the glomerular capillaries by a fibrinoid-like
    substance, and extensive tubular necrosis. Rabbits survived 10 mg
    intravenously, but showed histopathologic changes in the kidney.
    (Morard et al., 1964). Undegraded carrageenan given intravenously to
    rabbits (50 mg) resulted in death within 48 hours associated with
    diffuse renal cortical necrosis and widespread capillary thromboses in
    various organs. Plasma fibrinogen disappeared and serum complement
    decreased; the effects were dose related to some extent (Morard et
    al., 1964; Anderson, 1967). In four samples of C. crispus
    carrageenans harvested from four locations, the lowest intravenous
    doses required to kill rabbits within 24 hours varied from 1 to
    20 mg/kg for lambda carrageenans to 1-5 mg/kg for kappa carrageenans
    (Duncan, 1965). The injection of lambda carrageenan induced
    collagenous growth in the rabbit cornea (Dass & McCandless, 1966).

    Dog

         Five male and one female mongrel dogs received graded amounts of
    carrageenan orally for seven days. The drug level was increased daily
    until a level of 3 g/kg/day was reached on day 3. This dose was then
    continued from day 4 to day 7. Each animal was sacrificed 24 hours
    after the last dose. Diarrhoea and occasional bloody stools were the
    only clinical findings and this occurred in two dogs on day 8. Gross
    necropsy revealed a varying degree of gastrointestinal hyperaemia and
    also an ischaemic spleen in one dog and a haemorrhagic lung in one
    dog. Body weight loss was observed in five dogs. There were some
    superficial erosions of the mucosa without extensive inflammation

    (Rorer, 1966). Three groups of one male and one female mongrel dogs
    received 750, 1500 or 3000 mg/kg carrageenan for seven days. Diarrhoea
    was observed in four dogs. Gross necropsy revealed a varying degree of
    gastrointestinal hyperaemia with extensive haemorrhages in one dog
    which also showed a gradual decrease in haematocrit during the two
    weeks drug and observation period. Histopathological examination
    revealed a consistent clubbing of the free edge of the villi combined
    with focal congestion and erosion of the lining mucosa (Rorer, 1966).
    Seven groups of two mongrel dogs (male and female) received 0, 95,
    190, 375, 750, 1500 or 3000 mg/kg carrageenan for seven days.
    Diarrhoea and loss of weight occurred in 11 of the test dogs, emesis
    occurred in one dog at 3000 mg/kg and one dog at 1500 mg/kg. Gross
    necropsy revealed a varying degree of gastrointestinal hyperaemia with
    no other significant abnormal tissue. Histopathological examination of
    the tissues revealed no evidence of drug induced lesions (Rorer,
    1966). Eight groups of two mongrel dogs (male and female) received
    0, 250, 500, 640, 1000, 1280, 2560 mg/kg carrageenan for seven days.
    All animals had diarrhoea throughout the dosing period. Gross
    necropsy revealed a varying degree of gastrointestinal congestion.
    Haematology and clinical chemistry studies were negative as was the
    histopathological examination of tissues (Rorer, 1966). Two groups of
    two mongrel dogs (male and female) were given 1500 or 3000 mg/kg
    carrageenan for seven days. Diarrhoea and melena was observed at the
    3000 mg/kg dose level. Gross necropsy revealed congestion of stomach,
    duodenum and jejunum. Histopathological examination did not reveal any
    drug induced lesions. Body weight loss was observed in all surviving
    animals (Rorer, 1966).

    Monkey

         Three groups of two rhesus monkeys received 150, 750 or
    3000 mg/kg/day carrageenan for seven days. Haematological, blood
    biochemical and urological parameters were not altered. There was no
    significant effect on body weight or organ weights. Gross necropsy
    revealed the presence of numerous petechial haemorrhages scattered
    throughout the length of the colonic mucosa in the animals given 750
    or 3000 mg/kg carrageenan. In one of the latter animals the mucosa was
    definitely haemorrhagic. Although these lesions resembled those
    produced by systemic viral infection, no evidence of septicaemia was
    found (Rorer, 1966).

    Short-term studies

    Mouse

         Mice given degraded carrageenan in their drinking water in
    concentrations of 0.1-5% daily from 30 days to one year, showed no
    significant weight loss. Some changes were seen in the mucosa of the
    caecum, colon or rectum resembling those found in ulcerative colitis

    in man. The early changes included pin-head sized haemorrhagic and
    ulcerative lesions, along with cellular infiltrates and crypt
    abscesses. In later stages there was loss of mucosal folds, and in
    some animals there was scarring of the bowel which led to formation of
    constriction rings. The changes were minimal in comparison with other
    species and comprised mainly focal cellular infiltrations of the
    colonic mucosa (Marcus & Watt, 1969). Mice were given native or
    degraded carrageenan for six months at doses ranging from 70 mg to 4
    g/kg/day. There was no evidence of any pathological changes (Maillet
    et al., 1970). Seven groups of 45 mice received 0, 0.4, 1.0, 2.0, 3.0
    or 4.0 g/kg/day of degraded carrageenan for six months. The higher
    mortality at 2, 3 and 4 g/kg was due to gavage. No change was noted in
    the duodenum mucosa of stomach, colon, small gut or caecum.
    Histological examination of the liver showed no change in the hepatic
    cells. At 3 and 4 g/kg/day the Kupffer cells showed presence of a
    strongly metachromatic material (Maillet, 1970a).

    Rat

         Groups of six male rats were fed for a 10-week period 0, 5, 10
    and 20% of a refined preparation. The animals grew well except at the
    highest test level which showed a 50% mortality (Nilson & Schaller,
    1941). A group of four male and three female rats received 1 g/kg/day
    of degraded carrageenan for 40 days without mortality or visible
    adverse effect. No lesions were apparent at necropsy and pathological
    examination revealed nothing abnormal (Bonfils, 1959). In feeding
    experiments using 10 rats there was some growth retardation, decreased
    food intake and reduced urinary nitrogen excretion at dietary levels
    over 10% carrageenan (Hawkins & Yaphe, 1965).

         Degraded carrageenan was added to the drinking water of rats in
    concentrations of 0.1-5% daily from 30 days to one year. No
    significant weight loss was observed. There were changes in the mucosa
    of the caecum, colon or rectum resembling those found in ulcerative
    colitis in man. The early changes included pin-head sized haemorrhagic
    and ulcerative lesions, along with cellular infiltrates and crypt
    abscesses. In later stages there was loss of mucosal folds and in some
    animals there was scarring of the bowel which led to formation of
    construction rings (Marcus & Watt, 1969).

         Six groups of 10 male and 10 female rats received 0, 70, 350,
    700, 1000, 2000 mg of degraded carrageenan by gavage for one month. No
    adverse effects were seen on body weight, haematology or mortality.
    Some vasodilatation was seen in the colon and caecum of the test
    groups. Another four groups of 10 male and 10 female rats received 0,
    70, 350 or 700 mg of native carrageenan for one month. No adverse
    macroscopical or microscopical effects were noted. Another 150 rats
    received 2 g/kg/day of degraded carrageenan for one, two, three or six
    months at which times at least 20 were sacrificed and examined. No

    adverse macroscopical or microscopical effects were noted. Three
    further groups acted as controls (Dubrasquet et al., 1970). Five
    groups of 10 male and 10 female rats received degraded carrageenan 0,
    1.0, 2.0, 3.0 or 3.9 g/kg/day for six months. No significant
    difference was noted between the test and control groups as regards
    mortality, weight gain, food intake, haematology, urinalysis, SGPH,
    alkaline phosphatase or organ weights (Saito & Tokunaga, 1970). Two
    groups of five male and five female rats received a 10% solution of
    degraded carrageenan as drinking water resulting in consumption of up
    to 16.5 g/kg/day for one month. Moderate congestion of the caecal
    mucosa was noted but there was no evidence of erosions (Maillet et
    al., 1970; Maillet, 1970c). Two hundred rats were given for 28 days
    native or degraded carrageenan at doses ranging from 70 mg to 4
    g/kg/day. No histopathological or other changes were noted. Some rats
    received 4 g/kg for six months without adverse effect (Maillet et al.,
    1970). Six groups of 10 male and 10 female rats received 70, 350 or
    700 mg/kg/day of native carrageenan or the same quantities of degraded
    carrageenan for one month. A seventh group acted as controls. No
    appreciable difference in weight gain was noted between the different
    groups. In the two groups on 700 mg/kg moderate vasodilatation of the
    mucosa was seen but no histological changes (Maillet, 1970b).

    Guinea-pig

         Fifty male guinea-pigs were injected daily for one to 41 days
    with 0.25 mg histamine/100 g body weight in order to induce gastric
    ulceration. 50% carrageenan in the diet did not accentuate the mucosal
    lesions (Holzmann & Schott, 1963). Four groups of 10-15 guinea-pigs
    received 0, 2 or 4 g/kg/day degraded carrageenan in their drinking
    water or by gavage. All test groups developed diarrhoea and positive
    occult blood in the faeces. The chorion was infiltrated with
    macrocytes and other mononuclear cells, and at the highest dose level
    some superficial ulceration was noted (Maillet, 1969).

         Groups of 10 guinea-pigs received for 20-30 days 0 or 1%
    undegraded carrageenan or 5% degraded carrageenan. The overall
    incidence of ulceration in the three groups were 0, 80% and 100%. In
    both experimental groups the lesions were multiple, numbering
    sometimes in the hundreds. Macroscopically, they consisted of pinhead
    sized ulcers. Some had coalesced to form larger lesions, often linear
    in shape. Histologically, the ulcerations involved mainly the mucosa
    and showed features of both acute and subacute inflammatory
    infiltration, as well as crypt abscesses (Watt & Marcus, 1969a).
    Groups of 15 guinea-pigs received for five months 0 and 1% undegraded
    carrageenan. The animals appeared healthy and gained weight
    satisfactorily. In the test group all animals developed multiple focal
    ulcers in the caecum and in four the lesions had extended distally
    beyond the caecum for about 3 cm. Microscopically the ulcers involved
    mainly the mucosa and showed subacute or chronic inflammatory reaction

    (Watt & Marcus, 1969b). Samples of the lesions in guinea-pigs noted by
    Marcus & Watt were examined by Mottet who considered them to be in
    many ways morphologically comparable to those of human ulcerative
    colitis. However, he noted that the epithelial necrosis involved all
    regions of the crypt and not principally the depth of the crypt as is
    usually seen in the human disease. Alterations were noted in the
    amount of mucus in nearby glands. Lymphocytic infiltration and
    capillary congestion were noted to be very prominent features of the
    lamina propria of the mucosa and the superficial portion of the
    submucosa, as in ulcerative colitis. A typical epithelial hyperplasia
    was noted in the vicinity of the ulceration as occurs in ulcerative
    colitis (Mottet, 1970). However, when dietary neomycin was given
    advanced lesions were not seen; there were very few polymorphs while
    the macrophage and lymphocyte responses and the degree of ulceration
    were not affected. Other processes such as osmotic effects may have
    been responsible for the early lesions noted. It was therefore
    doubtful whether the carrageenan in fact simulated ulcerative colitis
    (Sharratt et al., 1971).

         Guinea-pigs which consumed between 4 and 5 g of degraded
    carrageenan daily showed mucosal erosions, but no ulceration in their
    caeca. In rare instances these erosions extended into the colon. The
    ulcers were quite different from those of ulcerative colitis in man.
    It was noted that the drinking solutions were heavily contaminated
    with microorganisms under the conditions of the experiment (Maillet et
    al., 1970). Guinea-pigs administered 5% native carrageenan in the diet
    or 1% degraded carrageenan in drinking water for two to four weeks
    produced multiple caecal ulcerations. Sequential studies have shown
    that the lesions first develop as an accumulation of macrophages in
    the lamina propria and subsequently in the sub-mucosa leading
    to the formation of pale raised areas which can easily be seen
    macroscopically. Ulceration of the mucosa then occurs, particularly in
    these areas. The ulcers are small and superficial and affect only the
    mucosa. A mixed cellular infiltrate, consisting predominantly of
    macrophages but accompanied by polymorphs, lymphocytes, and plasma
    cells, surrounds the ulcerated area. If animals are treated with
    neomycin (0.1% neomycin sulfate in the diet) in addition to
    carrageenan, the infiltrate consists only of macrophages (Sharratt et
    al., 1970). When carrageenan (native or degraded), labelled with
    ferric iron (stable at pH 2-8) was added to the diet of guinea-pigs
    for one week, particulate Perls' positive granule appeared within the
    epithelial cells and within macrophages which normally populate the
    subepithelial layers in this species. No Perls' positive material was
    found in the ileum and colon in these animals. Administration of
    equivalent amounts of ferric chloride failed to produce any Perls'
    positive material (Clarke & Hardy, 1970).

    Rabbit

         Degraded carrageenan was added to the drinking water of rabbits
    in concentrations of 0.1% to 5% daily from 30 days to one year. Loss
    of weight was noted and in some rabbits there was blood and mucus in
    the stools. In concentrations as low as 1% there were changes in the
    mucosa of the caecum, colon or rectum resembling those found in
    ulcerative colitis in man. The early changes included pin-head sized
    haemorrhagic and ulcerative lesions, along with cellular infiltrates
    and crypt abscesses. In later stages there was loss of mucosal folds,
    and in some animals there was scarring of the bowel which led to
    formation of constriction rings (Marcus & Watt, 1969). Groups of five
    rabbits received in their drinking water 0, 0.1%, 1.0% or 5.0%
    degraded carrageenan for 12, 12, seven and six weeks respectively. In
    the group on 5% carrageenan, diarrhoea was associated with visible and
    occult blood in the faeces by the end of seven days and this
    persisted. The animals lost weight and there was severe ulceration of
    the colon. In the group on 1% carrageenan only one animal developed
    diarrhoea and occult blood was present in the faeces in all the
    animals after two weeks. There was some weight loss and all the
    animals in this group showed ulceration of the colon of moderate
    severity. In the group on 0.1% carrageenan, diarrhoea did not occur
    but occult blood in the faeces was demonstrable in three of the
    animals by the end of 10 weeks. One animal lost weight and the
    remainder gained weight. Multiple ulcers in the colon were found in
    three of the five rabbits. In the control group there was no diarrhoea
    or occult blood in the faeces at any time and no ulcerative lesions
    were seen (Watt & Marcus; 1970a, b).

    Dog

         Groups of two male and two female cross labradors received either
    0 or 1000 mg/kg/day native carrageenan in their diet over 22 weeks. No
    significant adverse effects were noted regarding general appearance,
    growth, food intake, urinalysis, faecal occult blood (negative
    throughout), haematology, organ weight or histology of all major
    organs. The intestinal tract showed no evidence of any early or healed
    ulceration (BIBRA, 1970).

    Pig

         Three groups of five male and five female pigs received 40-56 g
    daily of either degraded or native carrageenan for four weeks. A
    fourth group acted as controls. The animals were sacrificed under
    general anaesthesia. There was no sign of any ulcerative lesions in
    the areas of the colon and caecum. There was a modification of the
    intestinal flora, but without reaching pathological limits. This
    modification of the flora induced a lymphoid reaction at the level of
    the submucosa of the large intestine (Tournot & Labie, 1970).

         Groups each of six Danish Landrace pigs (equally divided by sex),
    61-75 days of age, were maintained on diets to which carrageenan was
    added as a jelly at dose levels equivalent to 50, 200 or 500 mg/kg/day
    or 500 mg/kg/day carrageenan and sucrose 1 g/kg/day. The control group
    was given diets containing a corresponding amount of sucrose. The test
    period was 83 days. No effect was observed on growth rate or on feed
    utilization. Blood and urine parameters showed no significant changes
    from control and were within normal range. At autopsy, there were no
    changes in relative organ weight. Histopathologic studies were made of
    principal organs and tissues. No ulceration or erosions were seen in
    the gastrointestinal tract. Three pigs, one in the 200 mg/kg/day
    group, and two in the 500 mg/kg/day group, showed slight changes in
    the mucous membrane of the large intestine, including a few focal
    areas with irregular surface and some shift in cellular infiltration
    pattern. Bacteriological examination of the flora of the jejunum,
    caecum, colon and ampoulla of the rectum from all animals, showed that
    treatment results in changes in intestinal flora. The total counts of
    aerobic bacteria were decreased in the colon and rectum and the number
    of lacto-bacilli was reduced in the rectum (Poulsen, 1973).

    Long-term studies

    Mouse

         Groups of five male and five female mice of two strains were
    maintained over their life span on diets containing 0.1%, 5%, 15% and
    25% carrageenan without apparent ill-effects on growth, survival,
    gross and histopathology of the gastrointestinal tract, liver and
    kidneys (Nilson & Wagner, 1959).

    Rat

         Groups of five mate and five female rats of two strains were fed
    0, 1%, 5%, 15% and 25% carrageenan for their life span. Food
    consumption increased in proportion to the increased carrageenan
    content. No effect was noted on mortality. At the 25% level there was
    evidence of hepatic cirrhosis but all other levels showed no
    histopathological abnormalities of the intestinal tract, liver or
    kidneys (Nilson & Wagner, 1959).

         Three groups of rats were injected subcutaneously with
    carrageenan alone or with a promoter. Sarcomata had developed at the
    site of injection independently of the presence of a promoter in 12
    out of 88 rats by the 650th day.

    Dog

         In the course of some pharmacological studies in dogs carrageenan
    was given to dogs for prolonged periods. No deleterious effects were
    noted and one bitch gave birth to healthy puppies which appeared to
    tolerate carrageenan without trouble (Shirlaw, G., 1971).

    OBSERVATIONS IN MAN

         In the nutritional study of tocopherol requirements an infant
    formula (Similac) containing carrageenan was fed as the basic diet for
    six months to 44 healthy premature infants (Goldbloom, 1963).

         Twenty-four patients under treatment with various anti-
    inflammatory drugs and who had gastroduodenal ulcers were prescribed
    degraded carrageenan. Haemorrhages then ceased and the radiological
    and clinical picture improved (Busson et al., 1964). Ninety-five ulcer
    patients were treated with 0.15 g aluminium hydroxide and 0.5 g
    degraded carrageenan and a further 15 ulcer patients received degraded
    carrageenan and an anticholinergic drug. The patients received 6 g
    carrageenan for the first three weeks, 3 g from the third to the sixth
    week and 1.5 g from the sixth to the eighth week. The condition of all
    these patients except for 10 improved at a similar rate to others
    treated by the classical methods (Roches, 1965). Two blind trials of
    degraded carrageenan (6 g/day) and of aluminium hydroxide showed both
    to be of similar efficacy in giving symptomatic relief to peptic ulcer
    patients. No adverse effects were noted (Evans et al., 1965). Two
    hundred peptic ulcer patients were treated with degraded carrageenan
    5 g/day. Patients were followed up for an average of two years.
    Radiological controls were performed every two to four months. No
    ulcerative colitis or other intestinal side effect was seen (Bonfils,
    1970). Ten patients suffering from ulcerative colitis were given 10 g
    a day of degraded carrageenan (twice the clinical dose) for 10 days.
    Regular sigmoidoscopic examinations showed no modification either of
    the colonic ulcerations or of the bleeding of the mucosa (Bonfils,
    1970).

         The incidence of ulcerative colitis in the Oxford area diagnosed
    per 100 000 population over the decade 1951-1960 has risen apparently
    from about five to about 10. It was concluded that part at least of
    this reflected a true increase in the incidence of the disease
    although other factors may have contributed. The disease rarely
    manifested itself in children or adolescents; the incidence in both
    sexes then rose steeply with age to a peak in early middle life,
    falling again in the sixth decade (Evans & Acheson, 1965). The
    incidence of ulcerative colitis in Norway was studied for the three
    five-year periods 1946-1960. A total of 407 hospital cases were
    reported of which 362 were typical ulcerative colitis. The average
    annual rate for the three respective five-year periods starting in
    1946 and ending in 1960 were 10.5, 17.4 and 20.3 per million
    population, thus showing a definite rise in incidence in each five-
    year period. The increase was most marked for the age-groups 0-9 and
    10-19 years. The rate of reported cases was found to vary with
    geographical areas (Gjone & Myren, 1964).

    Comments:

         The native carrageenan used as food additive has a molecular
    weight of about 1 million and the degree of dispersion must be
    specified so as to reduce the presence of low molecular weight
    fractions to a minimum. The high molecular weight material is probably
    not absorbed and not associated with the production of colonic
    lesions.

         The weight of evidence points to a lack of effect of high-
    molecular carrageenan on any of the many species studied. Hence the
    colonic lesions and other pathological changes elicited with a
    particular form of degraded carrageenan are not relevant for purposes
    of evaluation. The critical criteria distinguishing between the food
    additive and degraded forms of carrageenan may be summarized as
    follows.

    1.   Physical parameters: intrinsic viscosity, separation by agarose
         gel electrophoresis and other procedures.

    2.   Biological evidence of uptake and storage within cells of the
         reticuloendothelial system in the case of degraded carrageenan
         and lack of such evidence with food-grade carrageenan.

         While some degradation of high-molecular carrageenan does appear
    to take place during processing of acid foods at relatively high
    temperatures, the extent of such degradation seems too limited to
    elicit the formation of low-molecular products possessing ulcerogenic
    potential in animals or man.

         Furcellaran is derived from other varieties of seaweed and has
    not been tested as such. However it belongs chemically to the general
    range of carrageenan and carrageenan-like substances. The reproductive
    effects elicited in the mouse and rat by gavage of calcium carrageenan
    as a suspension in oil cannot be interpreted in relation to food
    additive use in man.

    EVALUATION

    Level causing no toxicological effect

         Rat: 150 000 ppm (15%) in the diet equivalent to 7500 mg/kg bw.

    Estimate of acceptable daily intake for man

         0-75 mg/kg bw.*

              

    *    As carrageenan or furcellaran or the sum of both.

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    See Also:
       Toxicological Abbreviations
       Carrageenan and furcellaran  (FAO Nutrition Meetings Report Series 46a)
       Carrageenan and furcellaran (WHO Food Additives Series 19)