Review of harmful gastrointestinal effects of carrageenan in animal experiments. (Research Review).

In this article I review the association between exposure to carrageenan and the occurrence of colonic ulcerations and gastrointestinal neoplasms in animal models. Although the International Agency for Research on Cancer in 1982 identified sufficient evidence for the carcinogenicity of degraded carrageenan in animals to regard it as posing a carcinogenic risk to humans, carrageenan is still used widely as a thickener, stabilizer, and texturizer in a variety of processed foods prevalent in the Western diet. I reviewed experimental data pertaining to carrageenan's effects with particular attention to the occurrence of ulcerations and neoplasms in association with exposure to carrageenan. In addition, I reviewed from established sources mechanisms for production of degraded carrageenan from undegraded or native carrageenan and data with regard to carrageenan intake. Review of these data demonstrated that exposure to undegraded as well as to degraded carrageenan was associated with the occurrence of intestinal ulcerations and neoplasms. This association may be attributed to contamination of undegraded carrageenan by components of low molecular weight, spontaneous metabolism of undegraded carrageenan by acid hydrolysis under conditions of normal digestion, or the interactions with intestinal bacteria. Although in 1972, the U.S. Food and Drug Administration considered restricting dietary carrageenan to an average molecular weight > 100,000, this resolution did not prevail, and no subsequent regulation has restricted use. Because of the acknowledged carcinogenic properties of degraded in animal models and the cancer-promoting effects of undegraded in experimental models, the widespread use of carrageenan in the Western diet should be reconsidered. Key words: carcinogenesis, carrageenan, carrageenase, diet, furcelleran (furcellaran), hydrolysis, inflammatory bowel disease, nutrition, poligeenan, promoter, sulfated polysaccharide. Environ Health Perspect 109:983-994 (2001). [Online 24 September 2001] http://ehpnet1.niehs.gov/docs/2001/109p983-994tobacman/abstract.html

During the latter half of the twentieth century, inflammatory bowel disease and gastrointestinal malignancy have been major causes of morbidity and mortality in the United States. Even with improvements in treatment and cancer screening, colorectal cancer remains the second leading cause of cancer mortality in the United States. The Western diet has been considered a possible source of inflammatory bowel disease and colorectal malignancy, and intensive efforts have been undertaken to study the impact of specific constituents of the Western diet, such as fiber and fat (1-3).

One food additive, carrageenan, has been associated with induction and promotion of intestinal neoplasms and ulcerations in numerous animal experiments; however, carrageenan remains a widely used food additive. In 1982, the International Agency for Research on Cancer (IARC) (4) designated degraded carrageenan as Group 2B, noting sufficient evidence for the carcinogenicity of degraded carrageenan in animal models to infer that "in the absence of adequate data on humans, it is reasonable, for practical purposes, to regard chemicals for which there is sufficient evidence of carcinogenicity in animals as if they presented a carcinogenic risk to humans" (p. 90). The National Research Council has noted this designation for degraded carrageenan in their 1996 monograph (5). Recognizing the impact of carrageenan in animal models, several European and British investigators have advised against the continued use of carrageenan in food (6-11). Several reports have called attention to the problems associated with carrageenan consumption (6-11).

Extracted from red seaweed, carrageenan has been used in food products for centuries and was patented as a food additive for use in the United States in the 1930s. It has been used widely as a food additive, contributing to the texture of a variety of processed foods. It has also been used as a laxative, as treatment for peptic ulcer disease, and as a component of pharmaceuticals, toothpaste, aerosol sprays, arm other products (12-15). In 1959, carrageenan was granted GRAS (Generally Regarded as Safe) status in the United States. GRAS substances are permitted to be incorporated into food products as long as good manufacturing processes are used and the substance is used only in sufficient quantity to achieve the desired effect (16,17).

In the United States, the status of carrageenan was reconsidered by the Food and Drug Administration, and an amendment to the Code of Federal Regulations for the food additive carrageenan was proposed in 1972 (18). To diminish the public's exposure to degraded carrageenan, the amendment supported inclusion of an average molecular weight for carrageenan of 100,000 and a minimum viscosity of 5 centipoises (cps) under specified conditions. However, the actual regulation was not amended, although several publications indicated that it had been modified (7,8,19-23). In 1979, the proposal to include the average molecular weight requirement of 100,000 and the associated viscosity requirement in the Code of Federal Regulations was withdrawn. It was anticipated that a new rule-making proposal on carrageenan that would comprehensively address all food safety aspects of carrageenan and its salts would be published in about a year, but this has not been forthcoming (24,25). The proposal withdrawal referred to interim specifications for food-grade carrageenan using the Food Chemical Codex; these include a viscosity stipulation, but no average molecular weight requirement (26).

In the Food Chemicals Codex and supplements, carrageenan is described with attention to specific requirements for its identification and tests of its properties, including its sulfate content, heavy metal content, solubility in water, content of acid-insoluble matter, and viscosity [a 1.5% solution is to have viscosity [is greater than or equal to] 5 cps at 75 [degrees] C] (26,27). Although the viscosity is stipulated, viscosity may not adequately protect food-grade carrageenan from contamination by the lower molecular weight degraded carrageenans that IARC has denoted as Group 2B. Because undegraded carrageenan may have molecular weight in the millions, the actual viscosities of commercial carrageenans range from about 5 to 800 cps when measured at 1.5% at 75 [degrees] C (14). Native carrageenan has molecular weights of 1.5 x [10.sup.6]-2 x [10.sup.7] (28); poligeenan or degraded carrageenan is described as having average molecular weight of 20,000-30,000 (4). The average molecular weight of poligeenan has been described elsewhere as 10,000-20,000, but extending up to 80,000 (29). Food-grade carrageenan has been described as having average molecular weight of 200,000-400,000 (29), and elsewhere as having molecular weight of 100,000-800,000 (19). Furcelleran (or furcellaran), a degraded carrageenan of molecular weight 20,000-80,000, has a sulfate content of 8-19% (12,17). No viscosity or minimum average molecular weight was designated for furcelleran in the 1972 or 1979 Federal Register documents (18,24). In the Food Chemical Codex (fourth edition), a 1.5% solution of furcelleran at 75 [degrees] C is described as having minimum viscosity of 5 cps (27).

Today, carrageenan is still included among the food additives designated GRAS in the Code of Federal Regulations. The stipulations for its use include the following: a) it is a sulfated polysaccharide, the dominant hexose units of which are galactose and anhydrogalactose; b) range of sulfate content is 20-40% on a dry-weight basis; c) the food additive is used or intended for use in the amount necessary for an emulsifier, stabilizer, or thickener in foods, except for those standardized foods that do not provide for such use; d) to assure safe use of the additive, the label and labeling of the additive shall bear the name of the additive, carrageenan. Also included are similar standards for carrageenan salts and for furcelleran and furcelleran salts (30). In 1999-2000, approved uses for carrageenan were extended to include additional incorporation into food and medicinal products, including both degraded and undegraded carrageenan in laxatives (31-33).

For use in experimental models, degraded carrageenan (poligeenan) is derived from carrageenan by acid hydrolysis, frequently by a method developed by Watt et al. (34). This method is expected to yield a degraded carrageenan of average molecular weight 20,000-30,000 (35). Experiments demonstrate that reaction conditions similar to those of normal digestion can lead to the formation of degraded carrageenan (9-11). In addition, experimental data have revealed the contamination of food-grade carrageenan by substantial amounts of degraded carrageenan (10). Also, some bacteria are known to hydrolyze carrageenan and form low molecular weight derivatives (36-40).

The sections that follow and the accompanying tables summarize many experimental observations with regard to the intestinal effects of carrageenan. In addition, I review possible mechanisms for production of degraded carrageenan from undegraded carrageenan under physiologic conditions, as well as evidence that provides a basis for the mechanism of carrageenan's effects and for the reconsideration of the safety of carrageenan in the human diet.

Characteristics of Carrageenan

Three forms of carrageenan predominate, known as kappa, iota, and lambda. All have similar D-galactose backbones (alternating [alpha]-1,3 to [beta]-1,4 linkages), but they differ in degree of sulfation, extent of branching, solubility, cation binding, and ability to form gels under different conditions. [lambda]-Carrageenan is the least branched and the least gel forming; it is readily soluble at cold temperatures, in contrast to [kappa]- or [iota]-carrageenan. Table 1 presents some of the basic characteristics of [kappa], [iota], and [lambda] carrageenan (4,12-15,20-22,31-33,41-44)

In addition to food additive uses, carrageenan has been used in cosmetics, pesticides, and pharmaceuticals, as well as in toothpaste and room deodorizers. It has been used as a treatment of ulcers and as an emulsifier in mineral oil laxatives, liquid petrolatum, and cod liver oil. However, its predominant role has been in food preparations, in which it is used across a wide variety of food groups because of its ability to substitute for fat and its ability to combine easily with milk proteins to increase solubility and improve texture. Hence, it is used in low-calorie formulations of dietetic beverages, infant formula, processed low-fat meats, whipped cream, cottage cheese, ice cream, and yogurt, as well as in other products. From its original use several centuries ago as a thickener in Irish pudding and its incorporation into blancmange, the food additive use has extended widely and cuts across both low-fat and high-fat diets. It is often combined with other gums, such as locust bean gum, to improve the texture of foods (12-14,22,41,42).

In 1977, data obtained by the survey of industry on the use of food additives produced an estimate of daily carrageenan intake of 100 mg for individuals older than 2 years. The 1971 survey of industry had indicated that formula-fed infants in the first 5 months of life had an intake of 108 mg/day (21,43). Informatics, Inc., in a report prepared for the Food and Drug Administration, cited daily carrageenan consumption of 45 mg (19); this is similar to the reported intake of 50 mg/day of carrageenan in France (45). Nicklin and Miller (20) reported intake of 0-1.5 g/day, depending on choice of diet and total food consumed. Although the Food and Nutrition Board of the National Research Council of the National Academy of Sciences of the United States in 1971 initially estimated 367 mg/day for carrageenan intake for individuals older than 2 years in the United States, this was subsequently revised to 11 mg/day. The wide range of estimates may be attributed to inconsistencies in how industry has reported carrageenan production and consumption data, variation in processed food formulations with regard to extent of incorporation of carrageenan, and changes in use of carrageenan in nonfood products. Daily individual consumption of between 50 mg/day and 100 mg/day is consistent with total consumption in the United States of 7,700 metric tons, as estimated for 1997 (46).

The content of carrageenan in several commonly consumed food products is summarized in Table 2. Because manufacturing practices vary and change over time and the food formulae are proprietary, carrageenan content is indicated by a range (12,13,47-49). The content is expressed as the percent by weight of carrageenan used in the production of the food.

Experimental Results in Animal Models

Intestinal lesions after exposure to carrageenan in animal models. Table 3 summarizes the laboratory investigations that associate exposure to carrageenan with the occurrence of intestinal lesions (50-93). Several animals were tested, including guinea pig, rat, monkey, mouse, rabbit, and ferret. The guinea pig seemed most susceptible to ulceration and the rat most susceptible to malignancy. Many studies used exposure to carrageenan in a drinking fluid, at concentrations generally of 1%. Some were feeding studies, in which carrageenan was added to a solid diet. Some studies used gastric or duodenal intubation to ensure intake at a specified level; however, this method may have affected the way that carrageenan was metabolized by gastric acid (74,82-84,91). Feeding of carrageenan with milk may also have affected study results, because carrageenan binds tightly to milk proteins (caseins), affecting its metabolism (12-15, 22,41,42,47). The degraded carrageenan used in most of the experiments had molecular weight from 20,000 to 40,000. Several major findings in relation to neoplasia and ulceration were observed in these animal studies. All of these studies observed the effects of carrageenan in comparison to appropriate control animals.

In the footnote to Table 3, several subdivisions of the table are indicated with citation of the entries from the table. The subdivisions include: a) studies in which carrageenan alone induces abnormal proliferation or malignancy, b) studies in which carrageenan alone induces intestinal ulcerations, c) studies in which carrageenan appears to be a promoter of malignancy in association with another agent, d) studies using a rat model, e) studies using a guinea pig model, f) studies using degraded carrageenan, g) studies using undegraded carrageenan, h) studies indicating uptake of carrageenan into an extraintestinal site(s), i) studies indicating intestinal breakdown of carrageenan into lower molecular weight forms, and j) studies demonstrating ulcerations in rats using degraded carrageenan. In the table, the classification of the carrageenan used in the experiments as [kappa], [lambda], or [iota] is indicated when this information is clear from the original report.

Neoplasia. Wakabayashi and associates (72) demonstrated the appearance of colonic tumors in 32% of rats fed 10% degraded carrageenan in the diet for less than 24 months. The lesions included squamous cell carcinomas, adenocarcinomas, and adenomas. With exposure to 5% degraded carrageenan in drinking water, there was a 100% incidence of colonic metaplasia after 15 months. Metastatic squamous cell carcinoma was observed in retroperitoneal lymph nodes in this experiment. In addition, macrophages that had metachromatic staining consistent with carrageenan uptake were observed in liver and spleen.

Other studies have demonstrated the development of polypoidal lesions and marked, irreversible squamous metaplasia of the rectal mucosa, the extent of which was associated with duration and concentration of carrageenan exposure (67,70). Oohashi et al. (67) observed a 100% incidence of colorectal squamous metaplasia that progressed even after degraded carrageenan intake was discontinued in rats fed 10% degraded carrageenan for 2, 6, or 9 months and sacrificed at 18 months.

Fabian et al. (84) observed adenomatous and hyperplastic polyps as well as squamous metaplasia of the anorectal region and the distal colon in rats given 5% carrageenan as a drinking fluid. Similarly, Watt and Marcus (90) observed hyperplastic mucosal changes and polypoidal lesions in rabbits given carrageenan as drinking fluid for 6-12 weeks at a concentration of 0.1-5%. Focal and severe dysplasia of the mucosal epithelium was observed in rabbits after 28 months of 1% degraded carrageenan in their drinking fluid (58).

Promotion of neoplasia. Several studies demonstrated an increased occurrence of neoplasia in relation to exposure to undegraded or degraded carrageenan and associated exposure to a known carcinogen. Experimental data with undegraded carrageenan included enhanced incidence of colonic tumors in rats treated with azoxymethane (AOM) and nitrosomethylurea (NMU), when carrageenan was added to the diet. Groups of rats received control diet; control diet with 15% carrageenan; 15% carrageenan plus 10 injections of AOM given weekly; carrageenan plus NMU; NMU alone; and AOM alone. AOM or NMU with carrageenan led to 100% incidence of tumors, versus 57% with AOM alone and 69% with NMU alone (p < 0.01). Controls had 0%, and carrageenan alone led to an incidence of 7%. In addition, when undegraded carrageenan was combined with AOM, there was a 10-fold increase in the number of tumors per rat (73). (Figure 1)

Using undegraded carrageenan as a solid gel at concentration 2.5% for 100 days, Corpet et al. (50) found that after exposure to azoxymethane, there was promotion of aberrant crypt foci by 15% (p = 0.019). Exposure of rats to 6% undegraded carrageenan in the diet for 24 weeks, with 1,2-dimethylhydrazine (1,2-DMH) injections weekly, was associated with an increase in tumors from 40% to 75% and with the more frequent occurrence of larger and proximal tumors (57).

Degraded carrageenan in the diet of rats at a 10% concentration in association with exposure to 1,2-DMH weekly for 15 weeks was associated with an increase in small intestinal tumors from 20% to 50% and in colonic tumors from 45% to 60% (64). Iatropoulos et al. (77) found that in rats given 5% degraded carrageenan in the drinking water for less than 30 weeks in association with injections of 1,2-DMH weekly, there were increases in poorly differentiated adenocarcinomas and in tumors of the ascending and transverse colon, as well as increased proliferation of cells in the deep glandular areas.

Several investigators have measured the effect of carrageenan on thymidine incorporation and colonic cell proliferation. Wilcox et al. (51) observed a 5-fold increase in thymidine kinase activity in colon cells with 5% undegraded or 5% degraded carrageenan. There was an associated 35-fold increase in proliferating cells in the upper third of crypts with degraded carrageenan and an 8-fold increase with undegraded carrageenan (51). With 5% [lambda]-undegraded carrageenan fed to rats for 4 weeks, Calvert and Reicks (55) observed a 4-fold increase in thymidine kinase activity in the distal 12 cm of the colon (p < 0.001). Fath et al. (59) observed a 2-fold increase in colonic epithelial cell proliferation, with increase in labeling indices in both proximal and distal colon and extensive increase of the proliferative compartment in the proximal colon to the upper third of the intestinal crypt, after exposure of mice to 10% degraded carrageenan in drinking water for 10 days.

Ulceration. Many studies have demonstrated significant ulceration of the cecum and/or large intestine after oral exposure to carrageenan in guinea pigs, rabbits, mice, rats, and rhesus monkeys (34,35,53,56,58,59,62,63,65,68,70,71,75, 78-80,82,83,86-93). Ulcerations arose in association with exposure to either degraded or undegraded carrageenan. Lesions occurred initially in the cecum of guinea pigs and rabbits, but could be induced in more distal parts of the colon of the guinea pig, as in an experiment in which carrageenan was introduced directly into the colon after ileotransversostomy (63). In rats, the ulcerative lesions appeared initially in distal colon and rectum (8). Undegraded and degraded carrageenan have been associated with epithelial cell loss and erosions in rats (51,65,70,87,93).

Watt et al. (34) first observed ulcerations in response to carrageenan exposure in animal models more than three decades ago. They noted that 100% of guinea pigs given 2% degraded carrageenan as liquid for 20-30 days had colonic ulcerations and that 75% of the animals > 200 ulcers (34). When guinea pigs were given 1% undegraded carrageenan as liquid for 20-30 days, 80% developed colonic ulcerations (92). The lesions were routinely produced with carrageenan concentrations of 0.1-1%, which is similar to the concentration in a variety of food products (7,12-14).

Grasso et al. (83) demonstrated pinpoint cecal and colonic ulcerations in guinea pigs and rabbits given 5% undegraded, as well as degraded, carrageenan in the diet for 3-5 weeks. Lesions were not observed in ferrets and squirrel monkeys given degraded carrageenan by gastric intubation (83). Other investigators have also observed ulcerations after exposure to either degraded or undegraded carrageenan (75,88). Engster and Abraham (75) observed ulceration of cecum in guinea pigs given ??-carrageenan of molecular weight 21,000-107,000, demonstrating ulcerations were also caused by higher molecular weight carrageenan. Cecal ulcerations were not found with exposures to [Kappa] or [lambda] carrageenan of molecular weight varying from 8,500-314,000.

Investigators have noted that carrageenan-induced ulcerations of the colon are dose dependent and related to duration of exposure (52,53,67,68,70,89,90). Kitsukawa et al. (52) observed small epithelial ulcerations in guinea pigs who received carrageenan in their drinking fluid at two days. Olsen and Paulsen (68) observed cecal lesions after 24 hr and confluent ulcerations after 7 days in guinea pigs that ingested a 5% carrageenan solution. In rats, superficial erosions were observed at the anorectal junction at 24 hr after 10% dietary carrageenan (70); these extended more proximally over time. In 5 days of feeding with a 5% carrageenan solution, Jensen et al. (62) observed as many as 111 ulcerations/[cm.sup.2] over the mucosal surface of the cecum in the guinea pig.

Benitz et al. (82) observed a dose effect when degraded carrageenan was given at concentrations of 0.5-2% in drinking fluid to rhesus monkeys for 7-14 weeks. Watt and Marcus (89) observed that in rabbits given 0.1% degraded carrageenan as drinking fluid, 60% of the animals developed ulcerations, whereas 100% of those given 1% carrageenan had ulcerations when exposed for 6-12 weeks.

Resemblance to ulcerative colitis. Several investigators have noted the resemblance between the ulcerative lesions and accompanying inflammatory changes induced by carrageenan and the clinical spectrum of ulcerative colitis (56,94-99). Since the development of the carrageenan-induced model of ulcerative disease of the colon in 1969, carrageenan exposure has been used to model ulcerative colitis and to test for response to different treatments (52,62,100,101).

Clinical features in the experimental animals exposed to carrageenan have included weight loss, anemia, diarrhea, mucous in stools, and visible or occult blood in stools. The absence of small intestinal lesions and the lack of remission and exacerbation are also characteristic features of the carrageenan model (99,102).

Onderdonk (94) discussed the similarity between the carrageenan model of colitis and ulcerative colitis in humans and considered whether animal models for inflammatory bowel disease were also models for intestinal cancer because of the increased risk of colon cancer in individuals with ulcerative colitis. He reviewed the findings from carrageenan-treated animals, including loss of haustral folds, mucosal granularity, crypt abscesses, lymphocytic infiltration, capillary congestion, pseudopolyps, and strictures. Other observations have demonstrated an apparent sequence from colitis to squamous metaplasia and then to tumors of the colorectum (67,72,102). Atypical epithelial hyperplasia in the vicinity of carrageenan-induced ulcerations resembled findings from human ulcerative colitis that provide a link to intestinal neoplasia (86,98).

Proposed mechanism of development of lesions. A common feature observed in the animal models of ulceration in association with carrageenan exposure is macrophage infiltration (35,56,63,65,68,75,76,78-81, 83,84,88,92,102-104). Fibrillar material and metachromatic staining of the macrophages were observed. Notably, the macrophage lysosomes appeared to take up the fibrillar material and to become distorted and vacuolated. It appeared that colonic ulcerations developed as a result of macrophage lysosomal disruption, with release of intracellular enzymes, subsequent macrophage lysis, and release of intracellular contents that provoked epithelial ulceration (75, 76, 79, 84, 85, 88, 105,106). In the rhesus monkey, Mankes and Abraham (76) observed vacuolated macrophages with fibrillar material when the animals were given undegraded carrageenan of molecular weight 800,000 as a 1% solution in their drinking fluid, demonstrating the occurrence of these changes after exposure to undegraded as well as to degraded carrageenan.

In an effort to clarify further the precise pathogenic changes that occurred, Marcus et al. (35) evaluated pre-ulcerative lesions after exposure of guinea pigs to degraded carrageenan for only 2-3 days. The animals received 3% drinking solution of carrageenan, with an average daily carrageenan intake of 5.8 g/kg. Early focal lesions were observed macroscopically in the cecum in only one animal with this brief exposure. However, in all test animals, a diffuse cellular infiltrate, with macrophages and polymorphonuclear leukocytes, was apparent microscopically. Inflammatory changes in the cecum and ascending colon were present in all animals, and in the distal colon and rectum in three of four animals. Metachromatic staining material was noted in the lamina propria of the colon and surface epithelial cells from cecum to rectum, as well as in colonic macrophages. The surface epithelial cells and the macrophages contained vacuoles filled with the metachromatic material, which was not found in the controls and not seen in more advanced lesions in previous studies. These early lesions suggested that the presence of degraded carrageenan within surface epithelial cells might be associated with the subsequent breakdown of the mucosa and to ulceration by a direct toxic effect on the epithelial cells (35).

Hence, a model of mechanical cellular destruction by disruption of lysosomes from carrageenan exposure arises from review of the experimental studies in animals. The observed changes in the lysosomes resemble the characteristic changes observed in some lysosomal storage diseases, in which there is accumulation of sulfated metabolites that cannot be processed further due to sulfatase enzyme deficiency (107-110). Table 4 presents a proposed mechanism of the effects of carrageenan.

Possible role of intestinal bacteria. The relationship between the intestinal microflora and the biologic activity of carrageenan has been reviewed (111,112). Investigators have examined the impact of antibiotics and alteration of the resident microbial flora on the activity of carrageenan. Grasso et al. (83) studied the impact of neomycin treatment on the development of ulcerations by carrageenan. Pretreatment against coliforms failed to attenuate the course of carrageenan-associated ulcerations (80,83). Pretreatment with metronidazole was effective in preventing carrageenan-induced colitis in another experiment, although there was no benefit in established colitis (71). Aminoglycosides administered after carrageenan exposure were associated with reduced mortality, but not with reduction in the number of colonic ulcerations (94). Hirono et al. (65) found increased ulcerations and squamous metaplasia from the anorectal junction to the distal colon in germ-free rats fed 10% carrageenan for less than 63 days.

Additional considerations about the mechanism of action of carrageenan involved the role of production of hydrogen sulfide gas from metabolism of carrageenan in the digestive tract. Because carrageenan is heavily sulfated (up to 40% by weight), bacterial sulfatases and sulfate reductases can produce hydrogen sulfide gas or H[S.sup.-] from carrageenan. Carrageenan, as well as other sulfated polysaccharides, has been shown to stimulate [H.sub.2]S production from fecal slurries (113). Sulfide has been implicated in the development of ulcerative colitis, perhaps attributable to interference with butyrate oxidation by colonic epithelial cells (114,115). Butyrate has been shown to induce intestinal cellular differentiation, suppress intestinal cell growth, and decrease expression of c-myc, among other functions in colonic epithelial cells (116-118).

No fermentation of carrageenan was reported after testing with 14 strains of intestinal bacteria. The increase in sulfide production observed arising from incubation of [kappa]-carrageenan with colonic bacteria demonstrates that intestinal metabolism of carrageenan does occur. However, data pertaining to breakdown of carrageenan by fecal organisms are limited (112,113).

Extraintestinal manifestations of carrageenan exposure. Trace amounts of undegraded carrageenan have been reported to cross the intestinal barrier, with accumulation of label in intestinal lymph nodes (61,74). Several investigators have noted uptake of carrageenan by intestinal macrophages with subsequent migration of these macrophages to lymph nodes, spleen, and liver (61,67,74,78,82,84,85).

In association with carrageenan-induced intestinal ulcerations, Delahunty et al. (56) observed an increased permeability to large molecules, such as [[sup.3]H]PEG (polyethylene glycol)-900. This finding suggested that the intestinal changes induced by carrageenan may be a factor in subsequent absorption of carrageenan or other large molecules.

Other experimental data. Because it can induce acute inflammation, carrageenan has been widely used in experimental models of inflammation, to assess activity of anti-inflammatory drugs and to study mediators of inflammation (4,61,106,119,120). Injected into an experimental site, such as the plantar surface of a rat's paw, pleural cavity, or subcutaneous air bleb, carrageenan induces an inflammatory response, with edema, migration of inflammatory cells, predominantly polymorphonuclear leukocytes, and possibly granuloma formation (61,120). Undegraded carrageenans in vitro can inhibit binding of basic fibroblast growth factor (bFGF), transforming growth factor [beta]-1, and platelet-derived growth factor but not insulin-like growth factor-1 or transforming growth factor-[alpha] (121).

Macrophage injury and destruction caused by carrageenan may be a factor in the reduced cytotoxic lymphocytic response associated with carrageenan exposure in vivo (122). In addition to depression of cell-mediated immunity, impairment of complement activity and of humoral responses have been reported. Prolongation of graft survival and potentiation of tumor growth have been attributed to the cytopathic effect on macrophages (96,123). Because of its effect on T-cells, carrageenan has been studied for its impact on viral infections with herpes simplex virus types 1 and 2 (124) and HIV-1 (125,126), as well as infections with Chlamydia trachomatis (127).

In experimental systems, undegraded carrageenan has produced destruction of several different cell types in addition to macrophages, including small intestine epithelial cell monolayers (54), androgen-dependent malignant prostatic cells (128), bFGF-dependent endothelial cell line (128), rat mammary adenocarcinoma 13762 MAT cells (129), and human mammary myoepithelial cells (130). Lysosomal inclusions and vacuolation have been observed in macrophages, intestinal epithelial cells, and myoepithelial cells exposed to carrageenan (79,85,131).

Injections of carrageenan were noted to induce sarcomas, as well as mammary tumors in animal models, in an early study (132). In other experiments, mammary and testicular tumors have been observed (69,133). Carrageenan has also been noted to have anticoagulant activity, and large systemic doses have been fatal through nephrotoxicity (4).

Mechanisms for Production of Degraded Carrageenan from Undegraded Carrageenan

Gastrointestinal metabolism of carrageenan to form smaller molecular weight components has been observed by several investigators, who reported that carrageenan of high molecular weight changed during intestinal passage, compatible with hydrolysis yielding lower molecular weight components (9,10,74,75).

Under conditions such as might occur in digestion, 17% of food-grade carrageenan degraded to molecular weight < 20,000 in 1 hr at pH 1.2 at 37 [degrees] C. At pH 1.9 for 2 hr at 37 [degrees] C, 10% of the carrageenan had molecular weight less than 20,000 (9). These data suggest that substantial fractions of lower molecular weight carrageenan are likely to arise during normal digestion.

Table 5 presents data with regard to contamination of food-grade carrageenan by lower molecular weight carrageenan. Twenty-five percent of total carrageenans in eight food-grade carrageenans were found to have molecular weight < 100,000, with 9% having molecular weight < 50,000 (9). In addition, several bacteria have been identified that are able to hydrolyze carrageenan into smaller products, including tetracarrabiose. These bacteria, including Cytophaga species and Pseudomonas carrageenovora, are of marine origin; it is unknown whether the human microbial flora can perform similar hydrolysis reactions (36-40,134).

Extent of Human Exposure to Carrageenan

Indirect evidence relating exposure to carrageenan and the occurrence of ulcerative colitis and intestinal neoplasms consists of the similar geographic distribution between higher consumption of carrageenan and higher incidence of inflammatory bowel disease and colorectal cancer. Ulcerative colitis is more common in North America, the United Kingdom, and Scandinavia; and less common in Central and Southern Europe, Asia, and Africa (135). This incidence distribution is similar to distributions for colorectal malignancy and for carrageenan consumption, providing some ecologic evidence to support a potential etiologic role of carrageenan in human disease (46,136).

The reported T[D.sup.50] (tumorigenic dose 50% = the dose rate, in milligrams per kilogram body weight per day, which will halve the probability of remaining tumorless over the life span of the exposed animal) by the Carcinogenic Potency Database for degraded carrageenan is 2,310 mg/kg body weight/day, based on rodent experiments (137,138). This extrapolates to 138.6 grams for a 60-kg individual. If the total carrageenan intake per person in the United States is about 100 mg a day (43), about 9 mg of carrageenan with molecular weight < 50,000 is likely to be ingested through contamination of food-grade carrageenan by degraded carrageenan, and at least 8 mg with molecular weight < 20,000 is likely to arise during normal digestion (simulated by exposure to pH 1.9 with pepsin for 1 hr at 37 [degrees] C). This suggests an average intake of about 10 mg/day of degraded carrageenan for an individual older than 2 years of age in the United States.

An important issue is whether 10 mg/day degraded carrageenan is safe to ingest. By the Delaney clause, no carcinogen should be permitted in food. The Food Quality Protection Act (FQPA) established a usage level for negligible risk associated with pesticide residue in food at 1 ppm (139,140). Applying this standard to the extrapolated T[D.sub.50] for degraded carrageenan for a 60-kg person, the anticipated average intake of 10 mg/day is 70-fold greater than this standard (138.6 g/[10.sup.6]/day). These calculations do not take into consideration possible exposure to furcellaran (molecular weight 20,000-80,000), or the wide range of possible intakes of carrageenan.

Conclusion

Inflammatory bowel disease and colorectal malignancy represent major sources of morbidity and mortality in the United States. A possible factor in the etiology of these pathologies is exposure to carrageenan.

Several investigators have expressed their concerns about the use of undegraded carrageenan in food products (6-10), yet no legislative protection to restrict incorporation of low molecular weight fractions has been enacted. In fact, there has been no substantive review by the Food and Drug Administration of carrageenan since the studies undertaken more than two decades ago. However, there has been increased evidence regarding the cancer-promoting activity of undegraded carrageenan and further confirmation of the carcinogenic potential of degraded carrageenan.

Evidence for the role in carcinogenesis of carrageenan appears to support a nongenotoxic model based on direct toxic effects, for carrageenan has been nonmutagenic in Salmonella mutagenicity testing and nongenotoxic by DNA repair tests (60,102). A model of cellular destruction--from disruption of lysosomes by accumulation of carrageenan by-products or by interference with normal cellular oxidation-reduction processes from sulfate metabolites--emerges from review of the experimental studies. The impact of sulfatases, of either bacterial or human origin, on the metabolism of carrageenan requires further investigation. By interference with the normal intracellular feedback mechanisms associated with arylsulfatase activity, including steroid sulfatase, the highly sulfated carrageenan may have an impact on the availability of active, unsulfated hormones, such as dehydroepiandrosterone, derived from dehydroepiandrosterone-sulfate, and estrone-1, derived from estrone-1 sulfate.

Genetic characteristics that affect sulfatase and hydrolysis reactions as well as the individual intestinal microflora may influence how carrageenan is metabolized and how its effects are manifested. These factors may determine how carrageenan is metabolized differently by different individuals, but these characteristics may not be accessible to manipulation. A basic factor that can be controlled is the intake of carrageenan, which is amenable to dietary modification or food additive regulation.

Although carrageenan is widely used as a food additive for its texture-enhancing properties, other gums, some of which are used in combination with carrageenan, such as locust bean gum, gum arabic, alginate, guar gum, or xanthan gum, potentially can be used alone or in different combinations as substitutes for carrageenan (41,46). Alternatively, higher fat composition can lead to changes in food properties that may compensate for exclusion of carrageenan. Other hydrocolloids that are used as stabilizers and thickeners have not been associated with harmful gastrointestinal effects, and it is reasonable to expect that they could replace carrageenan in many food products. Although the dietary fibers pectin and psyllium affect intestinal motility, ulcerations or neoplasms have not been induced with either these or the other water-soluble polymers used as food additives. In contrast, other highly sulfated polysaccharides, amylopectin sulfate and dextran sulfate sodium, have induced ulcerations and neoplasia, suggesting that the degree of sulfation and polysaccharide molecular weight may be critical for induction of the observed effects (102).

The major pieces of evidence that support an argument to reconsider the advisability of use of carrageenan as a GRAS food additive are:

* Degraded carrageenan is a known carcinogen in animal models

* Undegraded carrageenan is a known co-carcinogen in animal models of carcinogenesis

* In animal models, both degraded and undegraded carrageenan have been associated with development of intestinal ulcerations that resemble ulcerative colitis

* Hydrolysis such as may occur by exposure to gastric acid in the human stomach can lead to the depolymerization of undegraded carrageenan and the availability of degraded carrageenan

* Food-grade carrageenan may be contaminated with low molecular weight, degraded carrageenan that may arise during food processing

* The use of a viscosity measurement to characterize a carrageenan sample is insufficient because the presence of a small number of large molecules (and undegraded carrageenan may have molecular weight in the millions) may obscure a significant low molecular weight fraction.

The potential role of carrageenan in the development of gastrointestinal malignancy and inflammatory bowel disease requires careful reconsideration of the advisability of its continued use as a food additive.

Table 1. Characteristics of carrageenan (4,12-15,27,28,41-49).

Chemical composition  Hydrocolloid composed of [alpha]-D-1,3 and
                       [beta]-D-1,4 galactose residues that are
                       sulfated at up to 40% of the total weight.
                       Strong negative charge over normal pH range.
                       Associated with ammonium, calcium, magnesium,
                       potassium, or sodium salts.
Solubility            [lambda] is readily soluble in cold or hot
                       aqueous solution; [kappa] is soluble in hot
                       solution; treatment of aqueous solution with
                       potassium ion precipitates [kappa]-carrageenan.
Gel formation         [lambda] does not form gels; [lambda] and [iota]
                       form right-handed helices; potassium chloride
                       promotes gel formation of [kappa]; calcium ion
                       promotes gel formation of [iota].
Metabolism            Hydrolysis of glycosidic linkages at lower pH,
                       especially pH [less than or equal to] 3.0; also
                       desulfation by sulfatases.
Viscosity             Near logarithmic increase in viscosity with
                       increasing concentration. Viscosity of food-
                       grade carrageenan defined as not less than 5
                       cps at 75 [degrees] C for a 1.5% solution;
                       viscosity ranges from 5 to 800 cps for 1.5%
                       solution at 75 [degrees] C.
Source                Red algae; predominantly aqueous extraction
                       from Chondrus, Gigartina, and various Eucheuma
                       species.
Molecular weight      Discrepancies in definitions. Native carrageenan
                       reported to have average molecular weight of
                       1.5 x [10.sup.6] - 2 x [10.sup.7]; food-grade
                       carrageenan reported as 100,000-800,000 or
                       200,000-400,000. Degraded carrageenan (poli-
                       geenan) has average molecular weight of 20,000-
                       30,000; furcellaran has average molecular
                       weight 20,000-80,000.
Properties            [lambda] and [kappa] combine easily with milk
                       proteins to improve solubility and texture;
                       serve as thickening agent, emulsifier,
                       stabilizer.
Synergistic effects   With locust bean gum, increase in gel strength.
                       Other hydrocolloids may also affect gel
                       strength and cohesiveness.
Concentration in      0.005-2.0% by weight.
 food products
Major uses            Milk products, processed meats, dietetic
                       formulations, infant formula, toothpaste,
                       cosmetics, skin preparations, pesticides,
                       laxatives
Table 2. Range of content of carrageenan in some commonly
consumed foods.

                                           Percent carrageenan
Food                                         (g/100 g food)

Bakery products                                 0.01-0.1
Chocolate milk                                  0.01-0.2
Cottage cheese                                  0.02-0.05
Frosting base mix                                  3-4
Ice cream, frozen custard, sherbets, etc.       0.01-0.05
Jams and jellies                                 0.5-1.2
Liquid coffee whitener                             0.3
Pie filling                                      0.1-1.0
Pimento olive stuffing                             2.0
Processed cheese                                0.01-0.06
Processed meat or fish                           0.2-1.0
Pudding (nondairy)                               0.1-0.5
Relishes, pizza, barbecue sauces                 0.2-0.5
Yogurt                                           0.2-0.5

Because manufacturing processes vary and there can be
substitutions of one hydrocolloid for another, the content of
carrageenan for any individual product may differ from these
estimates. Unpublished manufacturers' data indicate that these
content estimates for processed cheese, frozen dairy dessert,
cottage cheese, and jams and jellies are significantly lower
than current usage (4, 13, 14,47).
Table 3. Experimental data related to intestinal effects
of dietary carrageenan exposure.

                                          Experiment
Type of
carrageenan,                                         Dose
molecular weight           Animal       %CG      (g/kg bw/day)

1. Undegraded (a,d)         Rat          10          27.4
Undegraded (a,d)            Rat         0.25          0.2
                                        2.5            4

2. Undegraded               Rat       0.5, 1.5,
[iota], >100,000                          5
(a,b)
Degraded [iota],            Rat       0.5, 1.5,
20,000 (a,b)                              5

3. Degraded [iota],      Guinea pig  2, 2.5, 5
30,000 (c)

4. Degraded,             Guinea pig      3            5.8
20,000-30,000 (c)

5. Degraded (c)          Guinea pig    1, 2, 3      2, 3, 4

6. Degraded (c)          Rat ileal                 0-1.5 g/L
                            cell
                         monolayers

7. Undegraded               Rat          5
[lambda],
300,000 (a,b)

8. Degraded                Rat,          5
[iota] (c)               Guinea pig      5

9. Undegraded               Rat          6            0.8
[kappa] (a,d)

10. Degraded               Rabbit        1
[lambda] (a,b,c)

11. Degraded                Mice         10
(a,b,c)

12. Degraded,             Cultured                1 mg/10 mL
(20,000-40,000),         rat hepa-                    or
and                      tocytes or               1 mg/100 mL
Undegraded (a)           intestinal
                          mucosal
                           cells

13. Undegraded              Rat         0.5        0.15-0.25
[kappa], [lambda],
and [iota] (e)

14. Degraded (c)         Guinea pig      5

15. Degraded (c)         Guinea pig      5

16. Degraded (a,d)          Rat          10

17. Degraded,               Rat          10           15
(20,000-40,000)
(a,b,c,g)

18. Degraded             Guinea pig      3
[kappa], [lambda],
[iota]

19. Degraded
(20,000-40,000)             Rat          10
(a,b,e)

20. Degraded (c)          Guinea          5
                            pig

21. Undegraded,             Rat,     0.5, 2.5, 5   0.36, 2, 4
(800,000)                 hamster
(largely [kappa])

22. Degraded                Rat          10
(a,b,c,g)

23. Degraded (c)          Guinea         2
                            pig

24. Degraded (c)           Guinea        5
                            pig

25. Degraded                Rat        10, 5, 1
(20,000-40,000) (a,b)                    5

                                       1 or 5

26.Undegraded               Rat          15
[lambda] (a,d)

27. [iota],                 Rat                       0.5
(8,700-145,000) (e,f)
Undegraded [kappa]/         Rat          5
[lambda],
(186,000-214,000)
[iota],                  Guinea pig      2
(5,000-145,000)
[iota],                  Guinea pig      1
(5,000-145,000)
[kappa]                  Guinea pig      1
(8,500-275,000)
[lambda]                 Guinea pig      1
(8,500-275,000)
Undegraded [kappa]/
[lambda]                   Rhesus                0.05, 0.2, 0.5
(185,000)                  monkey
[iota]                                                0.2

28.[kappa] (c)             Guinea        1
(314,000)                   pig
(51,500)
(8,500)
[lambda],
(275,000)
(74,800)
(20,800)
[iota],
(145,000)
(107,000)
(88,000)
(39,000)
(21,000)
(8,700)
(5,000)

29. Degraded               Rhesus        2
[iota] (C16),              monkey
(20,000)
Undegraded                 Rhesus        1
[kappa][lambda]            monkey
mixture,
(800,000)

30. Degraded                Rat          5
([iota], C16),                                      7.5 g,
(10,000-30,000) (a,d)                              then 5 g

31. Degraded (c,e)          Rat      0.2, 0.5, 5
                         Guinea pig  0.25, 0.5

32. Degraded             Guinea pig  2, 0.2, 0.02
([iota], C16) (c)
                         Guinea pig      2
                            Rat          5
                           Monkey        2

33. Degraded (c)         Guinea pig     2, 5        1.7-3.3

34. Undegraded              Pig                  0.05, 0.2, 0.5
[kappa],
(200,000)

35. Degraded               Rhesus      0.5-2      0.7, 1.4, 2.9
(C16, [iota]), (c)         monkey
(20,000)
Undegraded                 Rhesus        1            1.3
(largely [kappa]),         monkey
(800,000)
                                        1-3        0.05-1.25

36. Undegraded           Guinea pig      5
                           Rabbit        5
Degraded (c)             Guinea pig    1, 2, 5
                           Rabbit        2
Degraded                   Humans                  5-g dose
                           Ferret                     1.5
                          Squirrel                    1.5
                           monkey
                          Rabbit,                     1.5
                           mouse
                            Rat          1
                            Rat          5
Undegraded                  Rat          5
                          Hamster        5

37. Degraded                Rat          5           6-10
(c16, [iota]),                                      0.5-5.0
(20,000-30,000) (a,b,e)

38. Undegraded             Rhesus        1
([kappa]:[lambda]          monkey
= 70:30),
(800,000) (e)
Degraded                   Rhesus     0.5, 1, 2
(C16, [iota]),             monkey
(20,000-30,000) (e)

39. Degraded (c)           Guinea        5       [less than or
                            pig                  equal to] 2 g

40. Degraded (a,b,c,g)      Rat          5

41. Undegraded (c)         Guinea        5
                            pig
Degraded (c)               Guinea        1
                            pig

42. Degraded (a,b,c)       Rabbit     0.1, 1, 5    0.07, 0.8,
                                                     1.4

43. Degraded (c)           Guinea                     4-5
                            pig
Degraded                    Rat                   [less than
                                                  or equal
                                                  to] 16.5
Degraded,                   Rat,                  0.07-4
Undegraded                 mouse

44. Undegraded           Guinea pig      1       [less than or
                                                 equal to] 1.5

Degraded                   Guinea    [less than  [less than or
                            pig      or equal    equal to] 2
                                     to] 5

45. Degraded             Guinea pig    0.1-5
                           Rabbit
                            Rat
                           Mouse

Type of                                   Experiment
carrageenan,
molecular weight           Animal    Duration      Route

1. Undegraded (a,d)         Rat      8 days,       Jelly
                                     sacrificed
                                     at 30 days
Undegraded (a,d)            Rat      100 days      Liquid
                                     100 days      Solid gel

2. Undegraded               Rat      [less than    Diet
[iota], >100,000                     or equal to]
(a,b)                                91 days
Degraded [iota],            Rat      [less than    Diet
20,000 (a,b)                         or equal to]
                                     91 days

3. Degraded [iota],      Guinea pig  7 days        Drink
30,000 (c)

4. Degraded,             Guinea pig  2-3 days      Drink
20,000-30,000 (c)

5. Degraded (c)          Guinea pig  2 weeks       Drink

6. Degraded (c)          Rat ileal   19, 30,       Media
                            cell     54 hr
                         monolayers

7. Undegraded               Rat      4 weeks       Diet
[lambda],
300,000 (a,b)

8. Degraded                Rat,      4 months      Drink
[iota] (c)               Guinea pig  3 weeks       Drink

9. Undegraded               Rat      24 weeks      diet
[kappa] (a,d)

10. Degraded               Rabbit    8 weeks,      Drink
[lambda] (a,b,c)                     12 months,
                                     28 months

11. Degraded                Mice     10 days       Drink
(a,b,c)

12. Degraded,             Cultured   20 hr
(20,000-40,000),         rat hepa-
and                      tocytes or  2 hr
Undegraded (a)           intestinal
                          mucosal
                           cells

13. Undegraded              Rat      90 days       Drink
[kappa], [lambda],
and [iota] (e)

14. Degraded (c)         Guinea pig  5 days        Drink

15. Degraded (c)         Guinea pig  14 days       Drink

16. Degraded (a,d)          Rat      2 weeks       Diet

17. Degraded,               Rat      < 63 days     Diet
(20,000-40,000)
(a,b,c,g)

18. Degraded             Guinea pig  3 weeks       Drink
[kappa], [lambda],
[iota]

19. Degraded                         2, 6, 9       Diet
(20,000-40,000)             Rat      months
(a,b,e)                              sacrificed
                                     at 18 months

20. Degraded (c)           Guinea    [less than    Drink
                            pig      or equal
                                     to] 28 days

21. Undegraded,             Rat,     Lifetime      Diet
(800,000)                 hamster
(largely [kappa])

22. Degraded                Rat      1 day to 12   Diet
(a,b,c,g)                            weeks, some
                                     sacrificed
                                     at 27 weeks

23. Degraded (c)          Guinea     2 weeks       Drink
                            pig

24. Degraded (c)           Guinea    21 days
                            pig

25. Degraded                Rat      [less than    Diet
(20,000-40,000) (a,b)                or equal to]
                                     24 months
                                     15 months     drink
                                     15 months     Stomach tube

26.Undegraded               Rat      [less than    Diet
[lambda] (a,d)                       or equal to]
                                     40 weeks

27. [iota],                 Rat      9 months      Gavage
(8,700-145,000) (e,f)
Undegraded [kappa]/         Rat      13 weeks      Diet
[lambda],
(186,000-214,000)
[iota],                  Guinea pig  7-10 weeks    Diet
(5,000-145,000)
[iota],                  Guinea pig  2-3 weeks     Drink
(5,000-145,000)
[kappa]                  Guinea pig  2-3 weeks     Drink
(8,500-275,000)
[lambda]                 Guinea pig  2-3 weeks     Drink
(8,500-275,000)
Undegraded [kappa]/
[lambda]                   Rhesus                  Stomach tube
(185,000)                  monkey
[iota]                                             Stomach tube

28.[kappa] (c)             Guinea
(314,000)                   pig      2 weeks       Drink
(51,500)                             2 weeks       Drink
(8,500)                              2 weeks       Drink
[lambda],
(275,000)                            2 weeks       Drink
(74,800)                             2 weeks       Drink
(20,800)                             2 weeks       Drink
[iota],
(145,000)                            2 weeks       Drink
                                     10 weeks      Diet
(107,000)                            2 weeks       Drink
                                     10 weeks      Diet
(88,000)                             2 weeks       Drink

(39,000)                             2 weeks       Drink
                                     10 weeks      Diet
(21,000)                             2 weeks       Drink
                                     10 weeks      Diet
(8,700)                              2 weeks       Drink
                                     10 weeks      Diet
(5,000)                              2 weeks       Drink

29. Degraded               Rhesus    10 weeks      Drink
[iota] (C16),              monkey
(20,000)
Undegraded                 Rhesus    10 weeks      Drink
[kappa][lambda]            monkey
mixture,
(800,000)

30. Degraded                Rat      [less than    Drink,
([iota], C16),                       or equal to]  diet
(10,000-30,000) (a,d)                30 weeks

31. Degraded (c,e)          Rat      [less than    Drink
                                     or equal to]
                                     12 weeks
                         Guinea pig  [less than    Drink
                                     or equal to]
                                     4 weeks

32. Degraded             Guinea pig  12 months,    Drink
([iota], C16) (c)                    10 months
                         Guinea pig  3 months      In milk
                            Rat      3 months      Drink
                           Monkey    10 weeks      Drink

33. Degraded (c)         Guinea pig  30-44 days    Drink

34. Undegraded              Pig      83 days       Jelly
[kappa],
(200,000)

35. Degraded               Rhesus    7-14 weeks,   Drink
(C16, [iota]), (c)         monkey    then reco-
(20,000)                             very for 20-
                                     24 weeks for
                                     some before
                                     sacrificed
Undegraded                 Rhesus    7-14 weeks,   Drink
(largely [kappa]),         monkey    then 11
(800,000)                            weeks reco-
                                     very
                                     [less than    Tube
                                     or equal to]
                                     12 weeks,
                                     after reco-
                                     very

36. Undegraded           Guinea pig  1-45 days     Diet
                           Rabbit                  Diet
Degraded (c)             Guinea pig  1-45 days     Drink
                           Rabbit                  Drink
Degraded                   Humans    10 days       Diet
                           Ferret    28 days       Tube
                          Squirrel   28 days       Tube
                           monkey
                          Rabbit,    28 days       Tube
                           mouse
                            Rat      56 days       Drink
                            Rat
Undegraded                  Rat      56 days       Diet
                          Hamster    6 months      Diet

37. Degraded                Rat      [greater      Drink
(c16, [iota]),                       than or
                                     equal to] 25
                                     weeks
(20,000-30,000) (a,b,e)              1-15 months   Tube

38. Undegraded             Rhesus    7-12 weeks    Drink
([kappa]:[lambda]          monkey
= 70:30),
(800,000) (e)
Degraded                   Rhesus    7-12 weeks    Drink
(C16, [iota]),             monkey
(20,000-30,000) (e)

39. Degraded (c)           Guinea    20-45 days    Drink
                            pig

40. Degraded (a,b,c,g)      Rat      6 months      Drink

41. Undegraded (c)         Guinea    2-4 weeks     Diet
                            pig
Degraded (c)               Guinea    2-4 weeks     Drink
                            pig

42. Degraded (a,b,c)       Rabbit    6-12 weeks    Drink

43. Degraded (c)           Guinea                  Drink
                            pig
Degraded                    Rat                    Drink
Degraded,                   Rat,     28 days-      Tube
Undegraded                 mouse     6 months

44. Undegraded           Guinea pig  20-30 days    Drink
Degraded                   Guinea    20-30 days    Drink
                            pig

45. Degraded             Guinea pig  30 days-      Drink
                           Rabbit    1 year        Drink
                            Rat                    Drink
                           Mouse                   Drink

                                     Experiment        Effects
Type of
carrageenan,                         Additional        Digestive/
molecular weight           Animal    exposure          systemic

1. Undegraded (a,d)         Rat      AOM injection     Less weight
                                     (5 mg/kg ip)      gain with
                                                       2.5% CG

Undegraded (a,d)            Rat      AOM injection
                                     (20 mg/kg ip)
                                     prior to CG

2. Undegraded               Rat
[iota], >100,000
(a,b)

Degraded [iota],            Rat
20,000 (a,b)

3. Degraded [iota],      Guinea pig  Eicosapen-        2% led to FOB+
30,000 (c)                           taenoic acid      at 7 days; 5%
                                     (300 mg/kg/day)   led to 38%
                                     at 14 days        mortality

4. Degraded,             Guinea pig
20,000-30,000 (c)

5. Degraded (c)          Guinea pig

6. Degraded (c)          Rat ileal
                            cell
                         monolayers

7. Undegraded               Rat
[lambda],
300,000 (a,b)

8. Degraded                Rat,
[iota] (c)               Guinea pig

9. Undegraded               Rat      1,2-DMH
[kappa] (a,d)                        (20 mg/kg bw)
                                     SC x 16 wks

10. Degraded               Rabbit
[lambda] (a,b,c)

11. Degraded                Mice                       Bloody diarrhea
(a,b,c)

12. Degraded,             Cultured
(20,000-40,000),         rat hepa-
and                      tocytes or
Undegraded (a)           intestinal
                          mucosal
                           cells

13. Undegraded              Rat
[kappa], [lambda],
and [iota] (e)

14. Degraded (c)         Guinea pig

15. Degraded (c)         Guinea pig  With ileo-
                                     transvers-
                                     ostomy

16. Degraded (a,d)          Rat      1,2-DMH weekly
                                     injections for
                                     15 weeks (10
                                     mg/kg bw) with
                                     DCG vs. DMH
                                     alone

17. Degraded,               Rat      Germ-free vs.
(20,000-40,000)                      conventional
(a,b,c,g)                            gut flora

18. Degraded             Guinea pig                    Diarrhea after
[kappa], [lambda],                                     7 days. Mori-
[iota]                                                 bund after 9
                                                       days of [iota]
                                                       DCG.

19. Degraded                         Basal diet after
(20,000-40,000)             Rat      DCG exposure
(a,b,e)

20. Degraded (c)           Guinea
                            pig

21. Undegraded,             Rat,                       Diarrhea in
(800,000)                 hamster                      some
(largely [kappa])

22. Degraded                Rat
(a,b,c,g)

23. Degraded (c)          Guinea                       Loose stools by
                            pig                        2 weeks

24. Degraded (c)           Guinea                      17/22 died
                            pig                        by day 21

25. Degraded                Rat
(20,000-40,000) (a,b)

26.Undegraded               Rat      NMU 2 mg twice
[lambda] (a,d)                       weekly rectally
                                     for 3 weeks;
                                     AOM (8 mg/kg bw)
                                     SC for 10 weeks;
                                     UCG with NMU,
                                     UCG with AOM

27. [iota],                 Rat
(8,700-145,000) (e,f)
Undegraded [kappa]/         Rat
[lambda],
(186,000-214,000)
[iota],                  Guinea pig
(5,000-145,000)
[iota],                  Guinea pig
(5,000-145,000)
[kappa]                  Guinea pig
(8,500-275,000)
[lambda]                 Guinea pig
(8,500-275,000)
Undegraded [kappa]/
[lambda]                   Rhesus
(185,000)                  monkey
[iota]

28.[kappa] (c)             Guinea
(314,000)                   pig
(51,500)
(8,500)
[lambda],
(275,000)
(74,800)
(20,800)
[iota],
(145,000)
(107,000)
(88,000)
(39,000)
(21,000)
(8,700)
(5,000)

29. Degraded               Rhesus
[iota] (C16),              monkey
(20,000)
Undegraded                 Rhesus
[kappa][lambda]            monkey
mixture,
(800,000)

30. Degraded                Rat      1,2-DMH           Watery, bloody
([iota], C16),                       (20 mg/kg)        stools
(10,000-30,000) (a,d)                SC/wk

31. Degraded (c,e)          Rat                        Severe diarrhea
                                                       in 3 days
                                                       with 5%

                         Guinea pig                    Diarrhea

32. Degraded             Guinea pig
([iota], C16) (c)
                         Guinea pig
                            Rat
                           Monkey

33. Degraded (c)         Guinea pig  Trimethoprim/     Blood in stools
                                     Sulfame-
                                     thoxazole

34. Undegraded              Pig
[kappa],
(200,000)

35. Degraded               Rhesus                      Diarrhea,
(C16, [iota]), (c)         monkey                      hemorrhage
(20,000)
Undegraded                 Rhesus
(largely [kappa]),         monkey
(800,000)

36. Undegraded           Guinea pig  Neomycin          Diarrhea,
                           Rabbit    (0.1%) added      hemorrhage
Degraded (c)             Guinea pig
                           Rabbit
Degraded                   Humans
                           Ferret
                          Squirrel
                           monkey
                          Rabbit,
                           mouse
                            Rat                        SI diarrhea
                            Rat                        Diarrhea
Undegraded                  Rat                        SI diarrhea
                          Hamster                      Diarrhea

37. Degraded                Rat                        FOB+ by
(c16, [iota]),                                         3-7 days with
                                                       > 5 g/kg/day;
                                                       gross blood
(20,000-30,000) (a,b,e)                                by 2-3 weeks

38. Undegraded             Rhesus
([kappa]:[lambda]          monkey
= 70:30),
(800,000) (e)
Degraded                   Rhesus
(C16, [iota]),             monkey
(20,000-30,000) (e)

39. Degraded (c)           Guinea                      FOB+,
                            pig                        diarrhea
                                                       by 1 week

40. Degraded (a,b,c,g)      Rat

41. Undegraded (c)         Guinea
                            pig
Degraded (c)               Guinea
                            pig

42. Degraded (a,b,c)       Rabbit                      Diarrhea,
                                                       blood by
                                                       day 7,
                                                       weight loss

43. Degraded (c)           Guinea
                            pig
Degraded                    Rat
Degraded,                   Rat,
Undegraded                 mouse

44. Undegraded           Guinea pig                    FOB+
Degraded                   Guinea                      Diarrhea by
                            pig                        10 days,
                                                       FOB+

45. Degraded             Guinea pig                    Weight loss in
                           Rabbit                      guinea pig and
                            Rat                        rabbit, not rat
                           Mouse                       or mouse. Blood
                                                       and mucous in
                                                       stool.

Type of                                     Effects
carrageenan,
molecular weight           Animal    Histopathologic changes

1. Undegraded (a,d)         Rat
Undegraded (a,d)            Rat

2. Undegraded               Rat      Epithelial cell loss, macrophage
[iota], >100,000                     infiltration, loss of crypts,
(a,b)
Degraded [iota],            Rat
20,000 (a,b)

3. Degraded [iota],      Guinea pig  Cecal ulcerations; foamy
30,000 (c)                           macrophages; small epithelial
                                     ulcerations at 2 days.

4. Degraded,             Guinea pig  Microscopic mucosal changes from
20,000-30,000 (c)                    cecum to rectum; apparent macro-
                                     molecule absorption by colonic
                                     epithelium, macrophage infiltra-
                                     tion, macrophages with vacuoles.

5. Degraded (c)          Guinea pig  100% had cecal ulceration after
                                     3% for 4 days; crypt abscesses.

6. Degraded (c)          Rat ileal
                            cell
                         monolayers

7. Undegraded               Rat      3/8 had slight congestion
[lambda],                            and erythema of distal colon.
300,000 (a,b)

8. Degraded                Rat,      Increased permeability to
[iota] (c)               Guinea pig  ([sup.3] H) PEG-900; ulcerations
                                     in guinea pig, crypt abscesses,
                                     macrophage infiltration.

9. Undegraded               Rat
[kappa] (a,d)

10. Degraded               Rabbit    Ulcerative lesions at 8 wks; at
[lambda] (a,b,c)                     12 months had chronic
                                     inflammatory changes.

11. Degraded                Mice     Ulceration in proximal and distal
(a,b,c)                              colon, with dilatation of cecum
                                     and ascending colon.

12. Degraded,             Cultured
(20,000-40,000),         rat hepa-
and                      tocytes or
Undegraded (a)           intestinal
                          mucosal
                           cells

13. Undegraded              Rat      CG able to penetrate intestine;
[kappa], [lambda],                   CG in mesenteric lymph node,
and [iota] (e)                       and macrophages of villus and
                                     lamina propria.

14. Degraded (c)         Guinea pig  Small, superficial ulcerations
                                     over mucosal surface of
                                     cecum (1-111 ulcerations/
                                     [cm.sup.2]).

15. Degraded (c)         Guinea pig  Ulcerations in cecum and proximal
                                     colon in unoperated. Postproce-
                                     dure, crypt abscesses in rectum
                                     and ulcerations in distal colon
                                     and rectum. Macrophage infiltra-
                                     tion.

16. Degraded (a,d)          Rat

17. Degraded,               Rat      Erosions; aggregates of foamy
(20,000-40,000)                      metachromatic macrophages
(a,b,c,g)                            in submucosa and lamina
                                     propria.

18. Degraded             Guinea pig  No cecal or colonic
[kappa], [lambda],                   lesions seen.
[iota]

19. Degraded                         CG in mucosa
(20,000-40,000)             Rat      and RE system.
(a,b,e)

20. Degraded (c)           Guinea    Cecal lesions after 24 hr;
                            pig      confluent ulcerations after
                                     7 days. Macrophage infiltration.

21. Undegraded,             Rat,     No difference in
(800,000)                 hamster    ulcerations from control.
(largely [kappa])

22. Degraded                Rat      Superficial erosions
(a,b,c,g)                            at anorectal junction
                                     at 24 hr; at 2 weeks,
                                     more proximal erosions.

23. Degraded (c)          Guinea     100% with colonic ulcerations;
                            pig      75% had over 200 ulcers.

24. Degraded (c)           Guinea    All had mucosal ulcerations
                            pig      from cecum to rectum by day 14.

25. Degraded                Rat
(20,000-40,000) (a,b)

26.Undegraded               Rat
[lambda] (a,d)

27. [iota],                 Rat      Av MW of CG in liver was
(8,700-145,000) (e,f)                at 10,000; all CG in feces
Undegraded [kappa]/         Rat      had MW < 100,000.
[lambda],
(186,000-214,000)
[iota],                  Guinea pig
(5,000-145,000)
[iota],                  Guinea pig
(5,000-145,000)
[kappa]                  Guinea pig
(8,500-275,000)
[lambda]                 Guinea pig
(8,500-275,000)
Undegraded [kappa]/
[lambda]                   Rhesus
(185,000)                  monkey
[iota]

28.[kappa] (c)             Guinea    Cecal ulceration not seen with
(314,000)                   pig      [kappa] or [lambda,]; [iota]
(51,500)                             fractions of MW 21,000-107,000
(8,500)                              led to ulcerations of cecum,
[lambda],                            crypt abscesses, and epithelial
(275,000)                            thinning. [iota] fractions
(74,800)                             absorbed and seen in vacuolated
(20,800)                             macrophages. Intense lysosomal
[iota],                              enzymatic activity in macrophages
(145,000)                            of lamina propria.
(107,000)
(88,000)
(39,000)
(21,000)
(8,700)
(5,000)

29. Degraded               Rhesus    Macrophages given DCG had
[iota] (C16),              monkey    fibrillar material and
(20,000)                             vacuolations.
Undegraded                 Rhesus    Vacuolations seen with UCG.
[kappa][lambda]            monkey
mixture,
(800,000)

30. Degraded                Rat      Distal rectum transformed
([iota], C16),                       to stratified squamous by
(10,000-30,000) (a,d)                DMH with DCG.

31. Degraded (c,e)          Rat      DCG contained within
                                     macrophages of spleen,
                                     liver, kidney, small and
                                     large intestine; cecal
                         Guinea pig  and colonic ulcerations
                                     at 4 weeks.

32. Degraded             Guinea pig  2% CG in water, but not in
([iota], C16) (c)                    milk, led to cecal ulceration
                         Guinea pig  in guinea pig. DCG in macro-
                                     phages of submucosal layer in
                                     guinea pigs, rats, and monkeys.
                            Rat      No cecal ulceration seen in
                           Monkey    rats or monkeys.

33. Degraded (c)         Guinea pig  Cecum and distal colon had
                                     ulcerations, crypt abscesses;
                                     enlarged cecal or colonic
                                     lymph nodes; more extensive
                                     ulceration with 5%; fewer
                                     lesions with antibiotic.
                                     Infiltration of foamy
                                     macrophages.

34. Undegraded              Pig      Focal irregularities without
[kappa],                             ulcerations; thickened lamina
(200,000)                            propria; macrophage infiltration.

35. Degraded               Rhesus    Ulcerations of colon; hypertrophy
(C16, [iota]), (c)         monkey    of mesenteric lymph nodes and
(20,000)                             granulomas; multiple crypt
                                     abscesses; dose effect present.
Undegraded                 Rhesus    Without colonic changes.
(largely [kappa]),         monkey
(800,000)

36. Undegraded           Guinea pig  Multiple pinpoint cecal and
                           Rabbit    colonic ulcerations after 3-5
Degraded (c)             Guinea pig  weeks in guinea pig and rabbit.
                           Rabbit    Macrophages increased; inclusions
                                     and vacuoles in macrophages;
                                     granulomas seen. Neomycin did not
                                     affect incidence of ulcers or
                                     time of onset.
Degraded                   Humans    Patients had colon malignancy
                                     with colectomy planned to follow
                                     CG exposure, no ulcerations seen.
                           Ferret    No lesions seen.
                          Squirrel   No lesions seen.
                           monkey
                          Rabbit,
                           mouse     No lesions seen.
                            Rat
                            Rat
Undegraded                  Rat
                          Hamster

37. Degraded                Rat      Metachromatic material
(c16, [iota]),                       thought to be CG found in
                                     RE cells of liver, spleen,
                                     lymph nodes, macrophages
(20,000-30,000) (a,b,e)              of lamina propria and
                                     submucosa. No cecal lesions.

38. Undegraded             Rhesus    No changes in liver.
([kappa]:[lambda]          monkey
= 70:30),
(800,000) (e)
Degraded                   Rhesus    Membrane-bound vacuoles
(C16, [iota]),             monkey    with fibrillar material in RE
(20,000-30,000) (e)                  cells of liver.

39. Degraded (c)           Guinea    Multiple ulcers in cecum, colon,
                            pig      and rectum in 100% of animals
                                     by day 30.

40. Degraded (a,b,c,g)      Rat      Ulceration of cecum in 4/12,
                                     associated with stricture;
                                     marked glandular hyperplasia
                                     at ulcer margins.

41. Undegraded (c)         Guinea    Ulceration of mucosa as
                            pig      consequence of macrophage
Degraded (c)               Guinea    accumulation in lamina propria,
                            pig      then submucosa.

42. Degraded (a,b,c)       Rabbit    Ulceration of colon in
                                     100% of those fed 1%;
                                     60% of those fed 0.1%.

43. Degraded (c)           Guinea    Mucosal erosions in cecum,
                            pig      rarely into colon in guinea pig;
                                     without erosion in rat or mouse.
Degraded                    Rat
Degraded,                   Rat,
Undegraded                 mouse

44. Undegraded           Guinea pig  Multiple ulcerations of cecum;
                                     80% had ulcerations. Crypt
                                     abscesses present; macrophages,
                                     with metachromatic material.
Degraded                   Guinea    100% had ulcerations; ulceration
                            pig      extended into distal colon
                                     and rectum.

45. Degraded             Guinea pig  Hemorrhagic and ulcerative
                           Rabbit    lesions in cecum, colon, or
                            Rat      rectum in all four species;
                           Mouse     crypt abscesses present.

                                                     Effects
Type of
carrageenan,                                                   Refe-
molecular weight           Animal    Neoplastic changes        rence

1. Undegraded (a,d)         Rat      UCG jelly (10% x 8 days)   (50)
                                     did not initiate tumor.
Undegraded (a,d)            Rat      UCG solid gel promoted
                                     growth of aberrant crypt
                                     foci (+15%; p = 0.019).

2. Undegraded               Rat      5-Fold increase in         (51)
[iota], >100,000                     thymidine kinase
(a,b)                                activity in colon cells
Degraded [iota],            Rat      with 5% UCG or DCG.
20,000 (a,b)                         35-Fold increase in
                                     proliferating cells in
                                     upper third of crypt
                                     with DCG, 8-fold with
                                     UCG.

3. Degraded [iota],      Guinea pig                             (52)
30,000 (c)

4. Degraded,             Guinea pig                             (35)
20,000-30,000 (c)

5. Degraded (c)          Guinea pig                             (53)

6. Degraded (c)          Rat ileal   Retarded cell growth       (54)
                            cell     caused cell death;
                         monolayers  at 0.25g/L inhibited
                                     DNA synthesis by 20%.

7. Undegraded               Rat      4-Fold increase in         (55)
[lambda],                            thymidine kinase
300,000 (a,b)                        activity in distal
                                     12 cm of colonic
                                     mucosa.

8. Degraded                Rat,                                 (56)
[iota] (c)               Guinea pig

9. Undegraded               Rat      More tumors with           (57)
[kappa] (a,d)                        UCG than control diet
                                     (75% vs. 40%); also
                                     larger, more proximal
                                     tumors.

10. Degraded               Rabbit    At 28 months, focal        (58)
[lambda] (a,b,c)                     and severe glandular
                                     atypism; precancerous
                                     changes seen.

11. Degraded                Mice     2-Fold increase in         (59)
(a,b,c)                              colonic epithelial cell
                                     proliferation; increase
                                     in labeling indices and
                                     extension of prolifera-
                                     tive compartment to
                                     upper third of crypt.

12. Degraded,             Cultured   DCG and UCG nonmuta-       (60)
(20,000-40,000),         rat hepa-   genic in Salmonella
and                      tocytes or  mutagenicity test; DCG
Undegraded (a)           intestinal  nongenotoxic by DNA
                          mucosal    repair test.
                           cells

13. Undegraded              Rat                                 (61)
[kappa], [lambda],
and [iota] (e)

14. Degraded (c)         Guinea pig                             (62)

15. Degraded (c)         Guinea pig                             (63)

16. Degraded (a,d)          Rat      Increase in tumors         (64)
                                     of small intestine
                                     (50% vs. 25%) and
                                     colon (60% vs. 45%)
                                     with CG than occurred
                                     with DMH alone.

17. Degraded,               Rat      Squamous metaplasia        (65)
(20,000-40,000)                      from anorectal junction
(a,b,c,g)                            to distal colon.

18. Degraded             Guinea pig                             (66)
[kappa], [lambda],
[iota]

19. Degraded                         100% incidence of          (67)
(20,000-40,000)             Rat      colorectal squamous
(a,b,e)                              metaplasia that prog-
                                     ressed after DCG intake
                                     discontinued.

20. Degraded (c)           Guinea                               (68)
                            pig

21. Undegraded,             Rat,     Increased incidence        (69)
(800,000)                 hamster    of benign mammary
(largely [kappa])                    tumors and testicular
                                     neoplasms (at 2.5%
                                     level) in rats only.

22. Degraded                Rat      Squamous metaplasia        (70)
(a,b,c,g)                            of rectal mucosa at 2
                                     weeks; extended after no
                                     longer being fed CG.

23. Degraded (c)          Guinea                                (34)
                            pig

24. Degraded (c)           Guinea                               (71)
                            pig

25. Degraded                Rat      Squamous cell              (72)
(20,000-40,000) (a,b)                carcinomas,
                                     adenocarcinomas,
                                     adenomas. 32% and
                                     fed 10% diet had tumors.
                                     100% incidence of meta-
                                     plasia with 5% drink.

26.Undegraded               Rat      100% had tumors with       (73)
[lambda] (a,d)                       AOM and UCG vs. 57%
                                     with AOM alone.
                                     100% had tumors with
                                     NMU and UCG vs. 59%
                                     with NMU alone.
                                     0 tumors in control, 7%
                                     tumors with UCG alone.
                                     UCG with AOM had 10-fold
                                     increase in number of
                                     tumors per rat.

27. [iota],                 Rat                                  (74)
(8,700-145,000) (e,f)
Undegraded [kappa]/         Rat
[lambda],
(186,000-214,000)
[iota],                  Guinea pig
(5,000-145,000)
[iota],                  Guinea pig
(5,000-145,000)
[kappa]                  Guinea pig
(8,500-275,000)
[lambda]                 Guinea pig
(8,500-275,000)
Undegraded [kappa]/
[lambda]                   Rhesus
(185,000)                  monkey
[iota]

28.[kappa] (c)             Guinea                               (75)
(314,000)                   pig
(51,500)
(8,500)
[lambda],
(275,000)
(74,800)
(20,800)
[iota],
(145,000)
(107,000)
(88,000)
(39,000)
(21,000)
(8,700)
(5,000)

29. Degraded               Rhesus                               (76)
[iota] (C16),              monkey
(20,000)
Undegraded                 Rhesus
[kappa][lambda]            monkey
mixture,
(800,000)

30. Degraded                Rat      DMH with DCG-              (77)
([iota], C16),                       induced proliferation
(10,000-30,000) (a,d)                of deep glandular areas;
                                     more poorly differentia-
                                     ted adenocarcinomas;
                                     more frequently found
                                     tumors of ascending and
                                     transverse colon with
                                     DMH and DCG.

31. Degraded (c,e)          Rat                                 (78)
                         Guinea pig

32. Degraded             Guinea pig                             (79)
([iota], C16) (c)
                         Guinea pig
                            Rat
                           Monkey

33. Degraded (c)         Guinea pig                             (80)

34. Undegraded              Pig                                 (81)
[kappa],
(200,000)

35. Degraded               Rhesus                               (82)
(C16, [iota]), (c)         monkey
(20,000)

Undegraded                 Rhesus
(largely [kappa]),         monkey
(800,000)

36. Undegraded           Guinea pig                             (83)
                           Rabbit
Degraded (c)             Guinea pig
                           Rabbit
Degraded                   Humans
                           Ferret
                          Squirrel
                           monkey
                          Rabbit,
                           mouse
                            Rat
                            Rat
Undegraded                  Rat
                          Hamster

37. Degraded                Rat      Adenomatous                (84)
(c16, [iota]),                       and hyperplastic
                                     polyps in one rat.
                                     Squamous metaplasia
(20,000-30,000) (a,b,e)              of anorectal region
                                     and distal colon.

38. Undegraded             Rhesus                               (85)
([kappa]:[lambda]          monkey
= 70:30),
(800,000) (e)
Degraded                   Rhesus
(C16, [iota]),             monkey
(20,000-30,000) (e)

39. Degraded (c)           Guinea                               (86)
                            pig

40. Degraded (a,b,c,g)      Rat                                 (87)

41. Undegraded (c)         Guinea                               (88)
                            pig
Degraded (c)               Guinea
                            pig

42. Degraded (a,b,c)       Rabbit    Hyperplastic               89,
                                     mucosal changes,           90)
                                     polypoidal lesions.

43. Degraded (c)           Guinea                               (91)
                            pig
Degraded                    Rat
Degraded,                   Rat,
Undegraded                 mouse

44. Undegraded           Guinea pig                             (92)
Degraded                   Guinea
                            pig

45. Degraded             Guinea pig                             (93)
                           Rabbit
                            Rat
                           Mouse

Abbreviations: AOM, azoxymethane; bw, body weight, CG,carrageenan;
DCG, degraded carrageenan; DMH, dimethylhydrazine; FOB, fecal occult
blood; ip, intraperitoneal; NMU, nitrosomethylurea; PEG, polyethylene
glycol; SC, subcutaneous; SI, slight; tube, gastric intubation; UCG,
undegraded carrageenan.

(a) Studies are associated with neoplastic changes,
unlike studies predominantly demonstrating intestinal
ulcerations. (b) Increased proliferation or neoplasm and
carrageenan alone. (c) Ulcerations and carrageenan alone.
(d) Neoplasms in which carrageenan promoted carcinogenesis.
(e) Studies with uptake to lymph node or other site.
(f) Study demonstrating breakdown to lower molecular weight.
(g) Studies demonstrating ulcerations in rat using degraded
carrageenan.
Table 4. Proposed mechanism for effects of carrageenan
(9,10,35,67,72,75,76,79,84-86,88,98,102,105,107-110,114,115).

Site               Effect

Intestinal lumen   Ingested carrageenan can undergo acid hydrolysis
                   in stomach possible breakdown by intestinal
                   bacteria.

Intestinal         Take up degraded carrageenan, as indicated by
 epithelial cells  metachromatic staining from cecum to rectum.
                   Vacuoles observed to contain metachromatic
                   material. Epithelial cells may undergo lysis from
                   effect of lysosomal disruption producing erosions.

Inflammatory       Polymorphonuclear cells and macrophages infiltrate
 infiltrate        to site of intestinal inflammation. Macrophages
                   have metachromatic staining associated with uptake
                   of degraded carrageenan. Lysosomal vacuolation
                   occurs as well as lysosomal disruption with release
                   of intracellular enzymes from macrophage
                   destruction, leading to intestinal ulcerations.
                   Process of chronic inflammation, as with ulcerative
                   colitis.

Macrophage         Macrophages may circulate and may lead to
 circulation       extraintestinal effects related to carrageenan.
Table 5. Experimental evidence for presence of low molecular weight
carrageenan in food-grade carrageenan and production of low molecular
weight carrageenan by acid hydrolysis or by bacteria. (9,10,36-40).

Degraded carrageenan in food-grade carrageenan
 25% of total carrageenans in eight food-grade [kappa]-carrageenans
 had MW < 100,000
 9% of total carrageenan in eight food-grade [kappa]-carrageenans had
 MW < 50,000
Production of degraded carrageenan by acid hydrolysis of food-grade
carrageenan
 In simulated gastric fluid (including pepsin and HCL}, [kappa]-
 carrageenan at pH 1.2, 37 [degrees] C
  for 1 hr leads to 17% degraded carrageenan with MW < 20,000
  for 2 hr leads to 25% with MW < 20,000
 In simulated gastric fluid (including pepsin and HCL), [kappa]-
 carrageenan at pH 1.9, 37 [degrees] C
  for 1 hr leads to 8% with MW < 20,000
  for 2 hr leads to 10% with MW < 20,000
 [kappa]-carrageenan in solution at pH 1.0, 37 [degrees] C, for 6
 hours, leads to 25% with MW < 20,000
 [iota]-carrageenan in solution at pH 1.0, 37 [degrees] C, for 6 hours,
 leads to 10% with MW < 25,000
Hydrolysis of carrageenan by bacterial carrageenases
 [kappa]- and [iota]-carrageenase from cell-free supernatant from
 culture of Cytophaga genus
 [kappa]-carrageenase isolated from cell-free medium of cultured
 Pseudomonas carrageenovora
 [lambda]-carrageenase from cell-free medium of Pseudomonas
 carrageenovora cultures

MW, molecular weight.

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Joanne K. Tobacman
College of Medicine, University of Iowa, Iowa City, Iowa, USA

Address correspondence to J.K. Tobacman, Department of Internal Medicine, University of Iowa Health Care, 200 Hawkins Drive, Iowa City, Iowa 52242-1081, USA. Telephone: [phone]. Fax: [phone]. E-mail: joanne-tobacman@ uiowa.edu

Received 17 January 2001; accepted 17 March 2001.