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
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
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.
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,
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 &
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
Special studies on large-bowel ulceration in various species
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).
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).
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
No untoward effects were found in gerbils which consumed a diet
containing 5% carrageenan (derived from Chondrus crispus) for six
months (Golberg, 1971).
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).
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).
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).
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.
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 &
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).
No gastrointestinal ulceration was seen in hamsters fed on a diet
containing 5% degraded carrageenan for five months (BIBRA, 1971).
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).
No abnormalities were found in gerbils consuming 3.5 g/kg/day of
degraded carrageenan for six months (Golberg, 1971).
No ulceration was seen in ferrets fed on a diet providing
1.5 g/kg/day of degraded carrageenan for four weeks (BIBRA, 1971).
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).
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).
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
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.
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
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).
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,
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).
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).
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.,
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.
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-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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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.
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,
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).
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
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
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.
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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