http://informahealthcare.com/txcISSN: 1040-8444 (print), 1547-6898 (electronic)
Crit Rev Toxicol, Early Online: 1–26! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10408444.2013.861798
REVIEW ARTICLE
Food additive carrageenan: Part II: A critical review of carrageenanin vivo safety studies
Myra L. Weiner
Toxpertise, LLC, Princeton, NJ, USA
Abstract
Carrageenan (CGN) is a seaweed-derived high molecular weight (Mw) hydrocolloid, primarilyused as a stabilizer and thickener in food. The safety of CGN regarding its use in food isreviewed. Based on experimental studies in animals, ingested CGN is excreted quantitatively inthe feces. Studies have shown that CGN is not significantly degraded by low gastric pH ormicroflora in the gastrointestinal (GI) tract. Due to its Mw, structure and its stability whenbound to protein, CGN is not significantly absorbed or metabolized. CGN also does notsignificantly affect the absorption of nutrients. Subchronic and chronic feeding studies inrodents indicate that CGN at doses up to 5% in the diet does not induce any toxicologicaleffects other than soft stools or diarrhea, which are a common effect for non-digestible highmolecular weight compounds. Review of several studies from numerous species indicates thatfood grade CGN does not produce intestinal ulceration at doses up to 5% in the diet. Effects ofCGN on the immune system following parenteral administration are well known, but notrelevant to food additive uses. The majority of the studies evaluating the immunotoxicitypotential were conducted with CGN administered in drinking water or by oral gavage whereCGN exists in a random, open structured molecular conformation, particularly the lambda form;hence, it has more exposure to the intestinal mucosa than when bound to protein in food.Based on the many animal subchronic and chronic toxicity studies, CGN has not been found toaffect the immune system, as judged by lack of effects on organ histopathology, clinicalchemistry, hematology, normal health, and the lack of target organ toxicities. In these studies,animals consumed CGN at orders of magnitude above levels of CGN in the human diet:�1000 mg/kg/d in animals compared to 18–40 mg/kg/d estimated in the human diet. DietaryCGN has been shown to lack carcinogenic, tumor promoter, genotoxic, developmental, andreproductive effects in animal studies. CGN in infant formula has been shown to be safe ininfant baboons and in an epidemiology study on human infants at current use levels.
Keywords
Carrageenan, chronic toxicity, degradation,food additive, gastrointestinal tractevaluations, infant formula additive
History
Received 6 May 2013Revised 26 October 2013Accepted 30 October 2013Published online 27 January 2014
Table of Contents
Abstract ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 2
Importance of vehicle... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 3Acute toxicity studies ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 5
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 5Acute oral, dermal, and inhalation toxicity ... ... ... ... ... ... ... ... ... 5Eye irritation potential, skin irritation, and sensitization
potential ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 5Degradation, excretion, absorption, and metabolism ... ... ... ... ... ... ... 5
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 5Degradation and excretion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 5Absorption ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 6
Rat ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 6Guinea pig ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 7Primate ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 7
Special studies on nutrient absorption ... ... ... ... ... ... ... ... ... ... ... 7Metabolism ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 7
Subchronic and long-term oral toxicity studies... ... ... ... ... ... ... ... ... ... 8
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8Subchronic oral toxicity ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8
Primates ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8Rats ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 8Mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 9
Long-term oral toxicity ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 9Immune system effects ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 9
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 9Ulcerative effects ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 11
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 11Mice ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 14Rats ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 15Guinea pigs ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 15Hamsters ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 15Pigs ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 15Primates ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 15
Carcingenicity studies ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16
Genetic effects ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16
Tumor promotion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 16
Reproductive and developmental toxicity studies ... ... ... ... ... ... ... 17Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 17
Address for correspondence: Dr. Myra L. Weiner, Owner and Principal,TOXpertise, LLC, 100 Jackson Avenue, Princeton, NJ 08540, USA.E-mail: [email protected]
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Studies on special age groups ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 18Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 18Young animals and humans ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 18
Primates ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 18Humans ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 18
Epidemiology studies ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19Regulation of dietary uses ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19
Summary ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19Adults ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19
Regulatory background ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19Dietary exposure ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19
Infants ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19Regulatory background ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 19Dietary exposure ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 20
Conclusions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 21Acknowledgements ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 21Declaration of interest ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 21References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 21Appendix 1... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 24Appendix 2... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 25
Introduction
Carrageenan (CGN) has been widely used as a food additive
for decades and its safety is based on a large database of
studies. CGN is used in food processing primarily to bind
water and promote gel formation, to thicken and stabilize
structure for food products by binding protein, and to improve
palatability. CGN is used in a variety of foods, including dairy
products, water-dessert gels, meat and fish products, bever-
ages, condiments, infant formula, and pet food. In addition to
conventional foods, CGN is used in foods labeled as Kosher,
Halal, and processed foods labeled as ‘‘organic’’. CGN is
listed in the US FDA Inactive Ingredient Guide as an
excipient that is presently used in oral drug products
(capsules, syrups, and coatings), lotions, and dental pastes.
CGN is also used as an ingredient in personal care products.
Chemically, CGN is a high molecular weight sulfated
polygalactan derived from a number of species of red seaweeds
of the class Rhodophyceae. It is a polymer of the sugar,
galactose, composed of repeating galactose units that may have
sulfate groups attached. The prevalent copolymers in CGN are
designated as kappa- (k), iota- (E), and lambda (�). k-CGN is
mostly the alternating polymer of D-galactose-4-sulfate and
3,6-anhydro-D-galactose. E-CGN is similar, except that the 3,6-
anydrogalactose is sulfated at carbon 2. Between k-CGN and
E-CGN, there is a continuum of intermediate compositions
differing in degree of sulfation at carbon 2. In �-CGN, the
alternating monomeric units are mostly D-galactose-2-sulfate
(a-1,3-linked) and D-galactose-2,6-disulfate (b-1,4-linked).
The anionic ester sulfate groups on CGN bind to positively
charged groups on food proteins, imparting functionality as a
thickener and stabilizer in foods (Blakemore & Harpell, 2010).
JECFA (1999) considers the various forms of CGN (k, �, E)
derived from various species of seaweed to show no major
differences in terms of toxicology and safety in foods. Food
grade CGN is included in foods and infant formula in which
CGN is tightly bound to protein.
Detailed regulatory specifications outline the criteria for
identity, purity, functionality, composition, heavy metals, and
weight average molecular weight (Mw) of food grade CGN,
shown in Table 1 (Commission Regulation, 2012; Food and
Agriculture Organization of the United Nations, 2007; the
Food Chemicals Codex, 2013; Japan Food Additives
Association, 2009).
From a toxicological perspective, the Mw of CGN is
important for an understanding of toxicological effects. There
has been much confusion in the literature between CGN, the
high Mw polymer used as a food additive, and poligeenan,
also known as degraded CGN, a much lower Mw polymer
(10–20 kDa) generated by subjecting CGN to the extreme
conditions of acid hydrolysis at low pH (0.9–1.3) and high
temperatures (480 �C) for several hours (Bixler, 2013).
Poligeenan has been shown to produce ulceration of the
intestinal tract and gastrointestinal tumors in animals (IARC,
1983). Early literature studies used the term ‘‘degraded
CGN’’, now known as poligeenan, to describe the low Mw test
material used in toxicology studies and erroneously called this
low Mw material ‘‘CGN’’. These errors in chemical nomen-
clature have led to confusion over the toxicological properties
of CGN both in the literature and more widely on the Internet
via consumer and health advocacy groups (Bixler, 2013;
Carthew, 2002; Tobacman, 2001). Consumers are clearly
confused by the interchangeable terminology. To clarify the
two materials, ‘‘degraded CGN’’ and food additive CGN,
the US Adopted Names Council (USAN, 1988) assigned the
name ‘‘poligeenan’’ to the substance previously referred to as
‘‘degraded CGN’’. The USAN defines poligeenan as having
an average Mw 10–20 kDa. Unfortunately, the term ‘‘poligee-
nan’’ has not been substituted for the term ‘‘degraded CGN’’
on a consistent basis.
The conditions required to produce poligeenan do not exist
in vivo in the gastrointestinal (GI) tract; hence, poligeenan is
not generated from food additive CGN in humans or animals
by degradation at physiological pH and temperature or by gut
microflora. Poligeenan is not a food additive and has no
utility in food. The toxicological and purity profiles of CGN
and poligeenan differ significantly. See Appendix 1 for a
comparison of key aspects of CGN and poligeenan. In this
regard, it is important to properly identify and specify
the analytical profile and Mw of test material used in
toxicology studies.
CGN permitted for food use must meet a viscosity
specification, which has been shown to correlate well with
molecular weight. Viscosity is an easily determined property
of solutions; therefore, various regulatory agencies use
viscosity measurement as an indirect measure of molecular
weight. The viscosity test for CGN is carried out under
standard conditions using a 1.5% solution at 75 �C. Regulatory
specifications require a viscosity of not less than 5 cPs or
5 mPa s (cP or centipose is the same as mPa s or milliPascal
seconds, both units of viscosity) for all seaweed-derived food
grade CGN (Commission Regulation, 2012; Food and
Agriculture Organization of the United Nations, 2007; the
Food Chemicals Codex, 2013; Japan Food Additives
Association, 2009). This viscosity corresponds to a molecular
weight (Mw) of approximately 100–150 kiloDaltons (kDa). In
its review of CGN, the European Commission’s Scientific
Committee on Foods (SCF) acknowledged that there was no
evidence to indicate adverse effects from CGN exposure, but
2 M. L. Weiner Crit Rev Toxicol, Early Online: 1–26
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the SCF concluded that ‘‘if feasible’’ a molecular weight limit
of not 45% below 50 kDa should be introduced into the
specification’’ to minimize ‘‘the presence of any degraded
carrageenan.’’ (European Commission, 2003). Even though a
validated analytical method did not exist, Commission
Directive (2004) amended the purity criteria for CGN to
include: ‘‘Low molecular weight carrageenan (molecular
weight fraction below 50 kDa) – not more than 5%’’.
Currently, a validated analytical method is still not available.
There is no upper Mw limit for CGN.
Commercial CGN has an average Mw 200–800 kDa and
often includes different proportions of E-, k-, and �-CGN,
depending on the desired functionality characteristics (Bixler,
2013; Blakemore & Harpell, 2010). Uno et al. (2001b)
surveyed the Mw distributions of 29 samples of food-grade
refined CGNs using high-performance liquid gel permeation
chromatography (GPC). The Mw of 29 food-grade CGN
samples ranged from 453 to 652 kDa with a mean of the
average molecular weights (Mw) for all 29 samples of
530 kDa. This analysis did not detect poligeenan (Mw
of 10–20 kDa) in the CGN samples at a detection limit
of 5% (weight/weight) (Uno et al., 2001b).
CGN was reviewed by the Joint FAO/WHO Expert
Committee on Food Additives in 1974, 1999, 2002, and
2008. It is currently assigned an Acceptable Daily Intake
(ADI) of ‘‘not specified’’ and permitted for use in foods
at the level of good manufacturing practices, determined
by functionality in a given product (JECFA, 2002, 2008).
See Section on ‘‘Regulation of dietary uses’’ for more
information.
The purpose of the present review is to clarify and
summarize the toxicological properties of food grade CGN
and to distinguish it from poligeenan. In this review, the term
‘‘poligeenan’’ will be used to identify the low Mw polymer,
previously denoted as degraded CGN, which was often not
distinguished in other reviews. This review discusses the
physical and functional properties of CGN and how they
relate to toxicity, and includes references not available in
previous reviews. It also discusses all aspects of toxicological
studies; thus, significantly adding to the Cohen & Ito (2002)
review, which covered only the rodent bioassay and tumor
promotion studies and the Michel & Macfarlane (1996)
review, which covered only the GI tract studies. This review
covers the published literature on CGN, searched by NERAC
on an on-going daily basis with search terms such as CGN,
carbohydrate polymer, hydrocolloid, molecular weight deter-
mination, molecular weight profiling, and low molecular
weight fraction for toxicology endpoints. A few unpublished
studies and reports are also included: Albany Medical College
(1975, 1983), Bailey & Morgareidge (1973); FDRL (1972a,
1972b), Friedman & Douglass (1960), Mankes (1977), and
Weiner (2007). The published studies often do not specify the
purity, molecular weight and/or compliance with food addi-
tive grade specifications of a test sample of CGN. For
example, see Watt & Marcus (1969), Onderdonk & Bartlett
(1979), or Bhattacharyya et al. (2012). It is critically
important to accurately describe the Mw and purity of the
test material used in toxicology studies. Appendix 2 shows the
identity of the food additive CGN samples used as test
materials in studies quoted in this review in order of section,
including the type of CGN. k- and E-CGNs exist as both
organized (presence of helical structures and associations)
and disorganized conformations (random coil) in the presence
of water, but �-CGN exists only in the disorganized
conformation (unable to form helices) in water. The inter-
pretation of toxicology studies on CGN (k, E, or �) should
consider the possible conformation under the given experi-
mental conditions. References in Appendix 2 are shown on
the first time only that they are cited in the text. In the
following sections, results of toxicology studies will include
the type of CGN and the vehicle used for clearer, more
accurate interpretation of results.
Importance of vehicle
The vehicle employed to administer CGN is critical to an
understanding of the toxicological effects and an interpret-
ation of their meaning because the conformation of the CGN
molecule changes depending on the vehicle. Most animal
studies administered CGN in diet or in drinking water with
some studies conducted by oral gavage in water. When CGN
is dissolved in water at dilute concentration (50.1%) with no
Table 1. Molecular weights of carrageenan and related materials.
Polymer material Molecular weight (Mw): Daltons and/or viscosity specifications Reference
Food grade carrageenanCommercial carrageenan 200 000–800 000 Blakemore & Harpell (2010)Regulatory specifications Viscosity not less than 5 mPa (1.5% solution at 75 �C) Commission Regulation (2012)
Not more than 5% below 50 000 Da molecular weight CGN incommercial CGN*
Commission Directive (2004)
Viscosity not less than 5 mPa s (1.5% solution at 75 �C) Food Chemicals Codex (2013)Viscosity not less than 5 cP at 75 �C (1.5% solution) Food and Agriculture Organization of the
United Nations (2007)Viscosity not less than 5 mPa s Japan Food Additives Association (2009)
Artificially formed polymer (not for food use)Poligeenany 10 000–20 000 USAN (1988)
*Currently, there is no validated analytical method to measure the low molecular weight tail (LMT). Using the best available technology, the LMTfraction of commercial CGN is typically 1.9–12% of the total commercial carrageenan product (Weiner et al., 2007). This LMT fraction ofcommercial carrageenan is primarily due to the incomplete molecular synthesis of the seaweed plants during natural growth prior to harvesting, andthe bulk of the LMT is within the 20 000–50 000 Da range, with generally less than 10% of the LMT being lower than 20 000 Da.yCompared to the LMT fraction of commercial carrageenan detailed above, generally over 90% of poligeenan is less than 20 000 Da. Poligeenan and the
LMT are completely different with respect to molecular weight profile.
DOI: 10.3109/10408444.2013.861798 Food additive carrageenan: Part II 3
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added cations, dietary solids or protein, the polymer exists in
a disorganized random conformation (Blakemore & Harpell,
2010). Under these conditions, all the CGN molecules are
available for maximum interaction with the GI tract cells.
When CGN is dissolved in water at higher concentrations,
such as animal drinking water studies, the carragenan may be
in the ordered or disordered conformation, depending on the
type of CGN and cations present. For example, k-CGN in the
presence of potassium cations and E-CGN in the presence of
calcium ions will gel at concentrations above 0.1% in water
(Blakemore & Harpell, 2010). The cation levels necessary for
such gelation depend on the specific CGN salt. For example,
k- and E-CGN may exist as partially disorganized and partially
organized conformations, depending on the concentration and
cation balance; whereas �-CGN is always in the disorganized
or non-gelling conformation at all concentrations for all
cations (Blakemore & Harpell, 2010). The large number of
negatively charged sulfate moieties in the structure of �-CGN
precludes it from forming helices in water. This last point is
important because several studies use �-CGN in drinking
water, and it is this form that has an open molecular structure
in water when not bound to protein. In dietary studies, where
the CGN concentration is well below the protein content of
food, for example, in rodent chow and infant formula, then the
organized conformation will exist.
As shown in Figures 1 and 2, CGN has numerous
negative charges due to the sulfate groups; protein has both
negatively charged carboxyl groups and positively charged
amine groups. Below the isoelectric point of the protein, a
direct polyanion CGN and polycation protein interaction can
occur, resulting in gelation or precipitation. Also, this direct
CGN–protein bonding occurs above the isoelectric point, but
to a lesser degree as the pH is increased. However,
regardless of pH, all these CGN–protein bonds are strong
and difficult to break. Above the isoelectric point, positively
charged cations, such as calcium or other polyvalent cations,
form a bridge across the negatively charged sulfate groups
on CGN to the negatively charged carboxyl groups on the
protein, forming stable gels (Figure 1). In addition, CGN–
CGN molecules interact to form helical bridges, adding
strength to the gels. In the absence of protein, CGN forms a
loose random conformation in water solution rather than the
structured helical coil conformation formed in the presence
of protein (Figure 2) (Blakemore & Harpell, 2010). Based
on these properties, �-CGN in water will have more sulfate
groups free and exposed to the GI mucosa where irritation
or ulceration may occur. However, it has to be noted that,
even with the absence of helices, the molecular bonds
between �-CGN and protein (both direct and via cations) are
strong and difficult to break. The hypothesis that CGN
conformation has an impact on its toxicological activity is
supported by the numerous studies in this review.
Figure 2. Carrageenan reactivity with casein,a two-step process.
Carrageenan reactivity with casein,a 2-step process
Carrageenanchains
Casein micellewith kappa
casein on thesurface
Step 1.At high temperatures,carrageenan reactselectrostatically withkappa casein
Step 2.On cooling, carrageenanchains interact, creating anetwork
Coolingsol gel
Helical chain
NH2 CO-2 NH2 CO-
2 NH2 CO-2 NH2 CO-
2 NH2 CO-2
++ ++ Ca++ Ca++ Ca++Ca Ca
SO-4 SO-
4 SO-4 SO-
4 SO-4
NH+3 CO2H NH+
3 CO2H NH+3 CO2H NH+
3 CO2H NH+3 CO2H••••
••••••
••••••
•••
••••••
••••••
•••
••••••••
••••••••
••••••••
SO-4 SO-
4 SO-4 SO-
4 SO-4
SO-4 SO-
4 SO-4 SO-
4 SO-4
NH+3 CO2H NH+
3 CO2H NH+3
NH+3
NH+3CO-
2
CO-2
CO-2
Protein
Protein
CarrageenanA
CarrageenanB
CarrageenanC
ABOVE ISOELECTRIC POINT
BELOW ISOELECTRIC POINT
Protein
Figure 1. Carrageenan binding to protein. (A) Above isoelectric point:carrageenan–protein interaction – through divalent cation. (B) Aboveisoelectric point: carrageenan–partial protein interaction – direct.(C) Below isoelectric point: carrageenan–full protein interaction –direct. Figures 1 and 2 are used with kind permission of FMC Corporation.
4 M. L. Weiner Crit Rev Toxicol, Early Online: 1–26
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Acute toxicity studies
Summary
CGN is not toxic by oral, dermal, and inhalation routes of
exposure; is non-irritating to eyes and skin; and is not a skin
sensitizer.
Acute oral, dermal, and inhalation toxicity
CGN is non-toxic by the oral, dermal, or inhalation routes of
exposure in acute toxicity studies. The oral LD50 in rats,
hamsters, and mice is 45000 mg/kg (FDA, 1972c) and
confirmed in rats using E-CGN by Weiner (1991). The oral
LD50 in rabbits ranges from 2280 to 5340 mg/kg (FDA,
1972c). Acute oral LD50 levels of45000 mg/kg are considered
non-toxic by regulatory authorities, including the FDA (2000).
The dermal LD50 in rabbits is 42000 mg/kg, indicating
that E-CGN is non-toxic via the dermal route (Weiner, 1991;
Weiner et al., 1989).
CGN also has been used in human topical preparations,
such as cosmetics, toothpaste, shampoos, and shaving creams
(Tye, 1991; Weiner, 1991). No adverse effects have been
attributed to the use of CGN in these products. Data
developed for potential occupational exposure found an
inhalation LC50 of 4931 mg/m3 (4 h, maximum attainable
concentration) in the rat, indicating that E-CGN is non-toxic
via inhalation exposure (Weiner, 1991; Weiner et al, 1989).
Gravimetric analysis indicated a mass median aerodynamic
diameter of 8.8� 2.1 mm with 62% of the particles less than
10 mm (Weiner et al., 1989).
Eye irritation potential, skin irritation, and sensitization
potential
Standard animal studies for eye irritation, skin irritation, and
skin sensitization potential were conducted to provide infor-
mation for workplace exposures. I> -CGN is non-irritating to
eyes which were not washed eyes and minimally irritating to
eyes that were washed in rabbits (Weiner, 1991).
k/�-CGN is non-irritating to intact skin and minimally
irritating to abraded skin in rabbits (Weiner, 1991; Weiner
et al., 1989). In a guinea pig skin sensitization study,
k/�-CGN was found to be non-sensitizing (Weiner, 1991;
Weiner et al., 1989). Thus, CGN is not predicted to produce
skin sensitization following dermal exposure in humans.
Based on the current state of scientific knowledge,
repeated dermal exposure to CGN in human preparations
intended for topical use (sunscreens, etc.) is not predicted to
cause skin irritation or skin sensitization reactions in humans.
Degradation, excretion, absorption, and metabolism
Summary
CGN is not significantly degraded in the GI tract and is
excreted unchanged in the feces. CGN is not significantly
absorbed and is not metabolized. CGN does not affect nutrient
absorption.
Degradation and excretion
In an early study, Hawkins & Yaphe (1965) found that
89–101% of ingested food grade k/�-CGN, fed at levels of
2–20% in the diet, was excreted in rat feces, using the
resorcinol method. The resorcinol method measures the 3,6-
anhydrogalactose content as an indication of the presence of
CGN in feces or other medium. Because it relies on a
colorimetric method, the accuracy of the method depends on
the level of clean-up of the solution used to measure the CGN
content and the use of appropriate controls with no CGN to
determine any background interference (such as untreated
feces or feces with CGN intentionally added). If clean-up of
the solution is not thorough, then some CGN will remain
bound to protein in feces and not be extracted. It is important
to insure that there are either no interfering compounds to the
colorimetric measurement, or to account for interfering
compounds by validation using appropriate blanks and CGN
spike and recovery controls.
Dewar & Maddy (1970) found that 80% of the E-CGN fed
to young rats at a level of 5% in the diet was excreted in feces
within 24 h. The CGN content of feces was also estimated by
measurement of the 3,6-anhydrogalactose content using the
resorcinol method. The authors suggest that the low value for
the percent of CGN excreted (�80%) may have been due to
inaccuracy or errors in measuring the daily feed intake and in
assessing the background level of the 3,6-anhydrogalactose
content of the feces from rats on control diet (Dewar &
Maddy, 1970). Tomarelli et al. (1974) administered k/�-CGN
at a level of 5% in diet or CGN prepared in heat sterilized
milk at a level of 4% in milk, to rats and quantitatively
measured CGN excreted in the feces. Excretion of k/�-CGN,
determined by the 3,6-anhydrogalactose content of the feces,
was 98.8� 2.7% from diet-fed rats and 98.3� 2.2% in
processed milk-fed rats (Tomarelli et al., 1974). Tomarelli
et al. (1974) modified the resorcinol method for greater
accuracy by introducing a correction ‘‘blank’’ for non-
specific color formation and subtraction of the optical density
of a sample treated with resorcinol-free reagent from the
complete color reagent. With these modifications, as well as
the inclusion of feces from rats fed milk-based diets
without CGN to measure ‘‘zero’’ background, and standard-
ization of the calibration curve with the same lot of CGN used
in the experimental samples, the authors were able to
accurately quantitate CGN in the feces of treated rats
(Tomarelli et al, 1974).
Pittman et al. (1976) found that the electrophoretic
mobility of CGN (k, �, E) present in the feces from rats,
guinea pigs, or monkeys was lower than that originally
administered, indicating that some degradation may have
occurred in the GI tract or that recovery was not adequate.
Due to variations in the electrophoresis from run-
to-run, the authors were not able to estimate the reduction
of the Mw of the dosed CGNs or to quantitate it.
Nevertheless, they conclude that excretion occurs primarily
in the feces.
In more recent studies, Arakawa et al. (1986, 1988) found
that dietary k-CGN with a Mw4100 000 was not metabolized
to lower Mw material in the feces of rats and that 97–98% of
the ingested CGN dose (4 g/kg) was quantitatively excreted in
the feces. Fecal CGN had a similar Sephacryl S-300 elution
pattern to that of the CGN sample added to the treated diet,
indicating identical molecular weight profiles (Arakawa et al.,
1988). No degradation of CGN excreted in the feces was
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found. This work agrees with the earlier work of Hawkins &
Yaphe (1965) and Tomarelli et al. (1974).
Tache et al. (2000) fed 2.5% k-CGN in water as a gel to
conventional rats with normal rodent gut microflora and rats
with their gut microflora replaced by human fecal microflora.
When CGN is gelled, it has a gel-mode helical conformation,
and, thus is not absorbed from the GI tract. CGN gel is used in
food products that do not contain protein, such as water
dessert gels. The average Mw of the CGN found in feces was
the same as that administered to both conventional rats with
typical rodent microflora and rats with human fecal micro-
flora and also the same as that from rat feces with CGN added
ex vivo as an extraction test. The authors conclude that the
average Mw of CGN was not changed significantly during its
passage through the GI tract. The conclusion that the average
Mw of CGN was not significantly changed was shown when
human fecal microflora had the opportunity to act on ingested
CGN gel. Thus, CGN gel, like CGN in diet, is not degraded
in the GI tract.
Uno et al. (2001a) used GPC to separate high and low
Mw �-CGN components in feces and used ICP atomic
emission spectroscopy (ICP-AES) for direct detection of the
sulfur in feces, thus utilizing the sulfur to find the CGN
present in feces. Food grade �-CGN was included in the diet
of rats at a level of 5%. Animals were deprived of food for
one night, then given access to the treated diet for one day.
For two days after CGN intake, the rats were given access to
the untreated basal diet. Feces were collected in the same
rats at three time points: day 1 (after ingestion of CGN), day
2, and day 3 (following CGN ingestion, but on basal diet).
The Mws of the CGN sample itself and CGN found in the
feces were determined. The Mws of the CGN found in the
feces after day 1 and day 2 were nearly the same as that of
the CGN in the blended diet: 782 kDa in feces compared to
832 kDa in diet. No CGN was detected in feces excreted
on day 3. Uno et al. (2001a) concluded that dietary �-CGN
is excreted in the feces without any decomposition to lower
Mw fractions.
The work of Uno et al. (2001a) and Arakawa et al. (1988)
both used the GPC method to characterize the Mw of CGN
excreted in feces; whereas Pittman et al. (1976) used
electrophoresis for estimation of molecular weights. Uno
et al. (2001a) suggest that the conflicting results are due to
differences in these methods. In electrophoresis, the distance
of travel is determined by Mw and the charge that exists on the
molecules; hence, differences in electrophoretic mobility do
not necessarily indicate a change in Mw alone. According to
Uno et al. (2001a), the feasibility of simple electrophoresis of
high Mw polysaccharides of several hundred thousand Daltons
is questionable. In addition, Uno et al. (2001a) suggest that
the drying and storage of collected feces by Pittman et al.
(1976) may have resulted in decomposition of the CGN
excreted in the feces at the time of drying. Such drying would
strengthen CGN–protein bonds, resulting in making subse-
quent extraction of CGN more difficult. In addition, Pittman
et al. (1976) included boiling feces in water for 2 h, which
may not have completely removed the CGN because it is
strongly bound to protein in feces. Such heat treatment may
also cause some thermal degradation of the CGN, depending
on the pH of the feces.
Arakawa et al. (1988) also explain the difference in their
results from those of Pittman et al. (1976) by the different
composition of the diets used in the two studies and possible
changes in the physiochemical environment in the rat intestine
due to the diet (Arakawa et al., 1988).
In summary, studies on the excretion of food-grade CGN
in the feces indicate that CGN is excreted unchanged
without significant degradation to lower Mw fractions as
verified by GPC, ICP-AES and other methods (Arakawa
et al., 1986, 1988; Uno et al., 2001a). Excretion of CGN was
shown to be 498–100% of the ingested amount by most
researchers (Arakawa et al., 1986, 1988; Hawkins & Yaphe,
1965; Tomarelli et al., 1974; Uno et al., 2001a). JECFA
(1999, 2008) concluded that breakdown of CGN in the GI
tract is probably of limited toxicological significance since,
‘‘if native CGN were sufficiently degraded to cause
ulceration or tumor growth, this would be detected in
feeding studies’’.
Absorption
The absorption of CGN has been reviewed by Roch-Arveiller
& Giroud (1979) and Weiner (1988). Numerous studies
indicate that food grade CGN is not significantly absorbed
after oral exposure due to its high molecular weight.
Rat. Grasso et al. (1973) did not detect the presence of
CGN in the intestinal walls of rats fed 5% E-CGN, although
systemic absorption was not evaluated in this study. CGN
(k, E) was not stored in the livers of rats, following dietary
administration at levels of 1 or 5% for one year, as judged
by the absence of histochemical staining using toluidine blue
dye for the presence of metachromatic material, such as
CGN, followed by visualization by light and electron
microscopy (Albany Medical College, 1975). The toluidine
blue method for the detection of CGN was standardized for
heparin (MacIntosh, 1941) and refined for CGN for accuracy
and specificity (Roberts & Quemener, 1999). Pittman et al.
(1976) fed rats diets containing 5% k/�-CGN extracted from
C. crispus with a Mw488 000 for 13 weeks and
reported that no CGN was detected in the liver, hence not
absorbed.
Absorption of CGN was studied by Nicklin et al. (1988)
using radiolabeled 3H-E-CGN administered orally by gavage
in water to rats. Radiolabeling was achieved by incubating
CGN with galactose oxidase and tritiated borohydride. The
–CH2OH group receives tritium with one 3H tritium bonded
to the carbon atom which is stable and one 3H bonded tritium
to the oxygen atom which is labile and capable of exchanging
with body water. The majority of the radioactivity was
excreted in the feces (Nicklin et al., 1988). Some uptake of the
label into the intestinal wall, Peyer’s Patches, mesenteric
lymph node, cecal lymph node, and serum was observed.
However, the authors cautioned that due to the exchange of
the 3H tritium label with the hydrogen of body water, only
levels of radioactivity greater than those found in serum can
be considered significant. Thus, only the cecal lymph node,
Peyer’s patches, and possibly the intestinal wall exhibited the
presence of bound tritium. Autoradiographs of tissues
indicated that the bound radioactivity was apparently
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associated with the macrophages. The authors suggest that
CGN probably enters the body via the macrophages of the
Peyer’s Patches and cecal lymph nodes.
Nicklin & Miller (1984) administered 0.5% CGN (k, E, �)
in drinking water to rats for 90 d and detected the presence
of small amounts of stained material within cells of the
intestinal villi, lamina propria, and basement membrane of
lymphatics, using Alcian blue stain for CGN. This method
relies on a color shift of the dye from green to purplish
green at 520 nm in the presence of CGN (Roberts &
Quemener, 1999). The amounts of CGN were not quanti-
tated and no difference in staining was seen between the
three forms of CGN. In addition, there were no histopatho-
logical findings or abnormal effects on the GI tract and no
alteration in the immune response to orally administered
antigen in treated animals.
The effect of CGN in diet on intestinal permeability to
macromolecular markers, polyethylene glycol (PEG) 900 and
4000, was evaluated in female rats (Elsenhans & Caspary,
1989). Following 4 weeks of feeding 20% CGN (an exces-
sively high dose which could result in nutritional deficiencies
due to replacement of a significant portion of the normal diet
by a non-nutrient additive), rats showed increased cecal and
intestine weights, increased intestinal length, and decreased
excretion of PEG-900, compared to rats on fiber-free diets.
The excretion of PEG-4000 was not affected by CGN
administration. These results are difficult to interpret.
It appears that CGN at an excessively high dose did not
increase the intestinal permeability to either marker and
actually decreased the permeability to the smaller marker.
Guinea pig. Pittman et al. (1976) detected E-CGN prepared
with a Mw range of 88–107 kDa, but not CGN prepared with a
Mw of 275 kDa, in the livers of guinea pigs after dietary
administration of specific isolated molecular weight fractions
(from 88 to 275 kDa). The majority of the administered CGN
was present in the feces. Detection of CGN in the urine of
guinea pigs administered artificially prepared fractions of
CGN in drinking water was found only in Mw fractions
�21 kDa (Pittman et al., 1976).
In contrast to CGN administered in the diet, Engster &
Abraham (1976) reported that CGN (k, �, E) with number
average molecular weight or Mn4145 000 administered as a
1% solution in drinking water was not retained in the guinea
pig cecum; whereas lower Mn forms, artificially prepared
from CGN (�107 kDa), were retained in the cecal lamina
propria and submucosal macrophages.
No CGN fragments with Mws439 000 were found in the
urine following administration of 1% CGN in drinking water
for 2–3 weeks to guinea pigs (Pittman et al., 1976). In both
these cases, it can be deduced, based on the work of others
using a broad spectrum of CGN Mw fractions (Engster &
Abraham, 1976; Pittman et al., 1976; Uno et al., 2001a) that
CGNs with Mw4100 kDa are not absorbed and are excreted
in the feces.
Primate. Rhesus monkeys given 1% k/�-CGN (average Mw
800 kDa) in drinking water for 7–11 weeks (with a subsequent
24-week recovery period) showed no evidence of CGN
located in the reticuloendothelial cells or Kupffer cells of the
liver after 11 weeks or after the recovery period, using
toluidine blue to stain for polysaccharides, in particular, CGN
(Abraham et al., 1972). In another study on rhesus monkeys,
Mankes & Abraham (1975) found no tissue storage of CGN
when the monkeys were given 1% k/�-CGN in the drinking
water for 10 weeks. Pittman et al. (1976) reported that
monkeys receiving daily doses of a k/�-CGN fraction (Mw
185 kDa) (500 mg/kg) by stomach tube for 15 months
excreted 12 mg of CGN per milliliter of urine. This value
(12 mg/ml) was reported to be at the limit of detection of the
method, and confirmation by isolation of the material from
urine was not made. Monkeys receiving daily doses of 500,
200, or 50 mg/kg of k/�-CGN in food for 7.5 years of oral
administration did not show evidence of the uptake or storage
of CGN in the liver, lymph nodes, small intestine, colon, or
cecum using toluidine blue staining by electron microscopy
(Albany Medical College, 1983).
Special studies on nutrient absorption
Five percent CGN in the diet had no effect on utilization of
casein, soybean protein, or other proteins by rats (Friedman &
Douglass, 1960). No difference in protein economy of the
growing rat was observed with a diet of 0.5–5% CGN and
either a high quality protein (casein) or a low quality
vegetable protein (JECFA, 1974). Three percent CGN in the
diet has been reported to reduce the plasma cholesterol level
in chicks by 50% (Riccardi & Fahrenbach, 1965). K/�-CGN
exhibited a cholesterol-lowering effect in rats; an increase in
feces weight and in the concentration and daily output of fecal
cholesterol and total bile acids after 33 weeks of feeding
(Reddy et al., 1980). When fed in a simulated milk powdered
diet to adult rats, k/�-CGN, at a dietary level of 4%, had no
influence (compared to glucose or cellulose) on growth rate,
diet energy efficiency, absorption of protein, fat, calcium,
blood coagulability, utilization of protein for growth, or
utilization of iron (Tomarelli et al., 1974). The fecal excretion
of calcium, iron, zinc, copper, chromium, and cobalt was
slightly higher in animals fed 10% k/�-CGN for 8–147 d
compared to control animals, indicating that CGN has no
chelating powers with respect to metallic ions (Harmuth-
Hoene & Schelenz, 1980). K/�-CGN in an infant formula fed
to rats did not inhibit calcium absorption (Koo et al., 1993).
Vitamin A (300 000 IU) was administered by Kasper et al.
(1979) to 11 young human female subjects, in a formula diet
containing 20 g of CGN. Under these experimental condi-
tions, CGN promotes the assimilation of vitamin A, since the
blood level of vitamin A was significantly higher some hours
after its administration. Similar effects were observed with
bran, microcrystalline cellulose, pectin, guar gum, and locust
bean gum. The authors did not interpret these experiments,
but hypothesized that possible modifications due to peristal-
sis, viscosity, transport, and enzymatic actions may occur in
the presence of these materials. Orally administered CGN
does not significantly affect the absorption of other nutrients.
Metabolism
Dietary feeding studies have reported cecal enlargement and
decreased concentrations of cecal bacteria in the rat following
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administration of high doses (5%) of hydrocolloid fibers, such
as E-CGN, pectin, guar gum or carboxymethyl cellulose
(Mallett et al., 1984, 1985). This enlargement may be
considered a bulking effect due to the osmotic pressure that
results from large amounts of non-digestible hydrocolloid in
the feces. There was a two- to three-fold enlargement of the
cecum as well as a significant decrease in the concentration of
gut microflora bacteria and alterations in several enzyme
activities of these bacteria in vitro following E-CGN admin-
istration to rats, mice and hamsters (Mallett et al., 1985).
Rowland & Mallet (1986) reviewed the effects of dietary
hydrocolloids on gut microflora in animals and concluded
that extrapolation to humans is difficult. They found that the
effects on gut microfloral enzyme activities (nitrate reductase
and b-glucuronidase) due to dietary pectin in rats were not
observed in humans following ingestion of 18 g pectin
(Rowland & Mallet, 1986). Rowland et al. (1986) compared
five enzyme activities (azoreductase, beta-glucosidase, beta-
glucuronidase, nitrate reductase, and nitroreductase) asso-
ciated with hindgut microflora in six species of animal (rat,
mouse, hamster, guinea-pig, marmoset, and man). They found
marked differences in the cecal activities of these enzymes in
the four rodents, with no one species exhibiting consistently
higher or lower enzyme activity. None of the animal species
provided an approximation of the enzyme profile associated
with human fecal microflora. They concluded that these
results indicate that it may not be valid to extrapolate the
results of bacterial metabolic studies between closely related
species, or from animals to man (Rowland et al, 1986).
Under the most drastic artificial stomach conditions, rapid
acidification for 3 h, breakdown of food-grade k/�-CGN was
very limited, with less than 10% of the CGN having a
Mw5100 kDa and less than 3.6% having a Mw550 kDa
(Capron et al., 1996). CGN was not fermented by any of
154 different anaerobic bacterial strains isolated from
human feces (Salyers et al., 1977). CGN and other sulfated
polysaccharides did produce hydrogen sulfide gas during a 48 h
in vitro incubation with human fecal slurries. However, CGN
fermentation is thought to provide only a very limited amount
of hydrogen sulfide in the large intestine (Michel &
MacFarlane, 1996).
In summary, following oral administration, CGN is not
metabolized and is not significantly broken down to lower Mw
material in the GI tract. Most of the CGN administered in the
diet (498%) is excreted unchanged in the feces.
Subchronic and long-term oral toxicity studies
Summary
Subchronic and chronic dietary studies demonstrate that CGN
does not result in target organ toxicities. The only effects
noted are soft stools and/or diarrhea, typically seen with high
levels of dietary fibers (5% or more in the diet). One study
finding an effect of CGN on glucose utilization in mice
employed �/k-CGN in drinking water (Bhattacharyya et al.,
2012), but does not agree with other publications.
Subchronic oral toxicity
Many of the earlier toxicology evaluations done with CGN
have been subchronic rodent feeding studies (reviewed by
FDA, 1973; Weiner, 1991). These have ranged from a few
weeks to 90 d of treatment using dietary concentrations of
1–25% CGN in rodents (Abraham et al., 1985; Grasso
et al., 1973; Mankes, 1977). The majority of these studies
have used dietary concentrations of 1% or 5%. In all
studies, a dietary level of 1% fed for 90 d has caused no
adverse effects. The only finding noted was soft stools
after dosing at the 5% dietary concentration. Very high
non-physiological dietary concentrations (25%) were given
to rats for 4 weeks (Mankes, 1977). The adverse effects
noted included growth retardation, inflammatory changes
in the gut, feces coated with fresh blood, and apparent
storage of CGN in the Kupffer cells of the liver (Mankes,
1977). It must be noted that these effects were not due to
direct toxicity, but rather to the replacement of a large
portion of the diet (25%) with an inert substance with a
strong likelihood to have nutritional effects, which will
confound the interpretation of any study. OECD guidelines
452 and 453 specify the highest dose of test material
tested in dietary feeding studies should be no more than
5% to avoid nutritional deficiencies for non-nutritive
additives: ‘‘For substances administered via the diet or
drinking water it is important to ensure that the quantities
of the test substance involved do not interfere with normal
nutrition or water balance. In long-term toxicity studies
using dietary administration, the concentration of the
chemical in the feed should not normally exceed an
upper limit of 5% of the total diet, in order to avoid
nutritional imbalances’’ (OECD, 2009a, 2009b).
Primates. Adult rhesus monkeys (10/sex) were adminis-
tered 1% high Mw k-CGN in drinking water for 11 weeks,
corresponding to an average daily intake of 1.3 g/kg/d
(Benitz et al., 1973). Animals were terminated after 7 or 11
weeks of treatment or were given plain tap water and
allowed to recover for 11 weeks. Because no adverse effects
were apparent after the recovery period, the animals then
received escalating oral doses of 50–1250 mg/kg/d in water
by stomach tube for up to 12 weeks. The only effects noted
were occasional soft or watery stools in treated and recovery
group animals (Benitz et al., 1973). Gross and microscopic
examination of the GI tract found no changes related to
treatment with high Mw CGN.
Rats. Groups of Fischer 344 rats (20/sex/group) were fed
control or treated diets at levels of 0, 2.5%, or 5.0%
k-CGN for 90 d in a standard FDA and OECD guideline
subchronic study conducted under GLP Guidelines (Weiner
et al., 2007). The sample of k-CGN used had an average
Mw range of 196–257 kDa, with a relatively high percent-
age of low molecular weight tail (LMT) (a mean of 7%
550 kDa with a range of 1.9–12.0% below 50 kDa). The
Mw of the LMT was determined by an experimental
method. Clinical observations were performed daily.
Individual feed consumption/body weight measurements
were made weekly. Ophthalmic exam was conducted prior
to and after treatment. Hematology, serum chemistry, and
urinalysis evaluations were done at necropsy, as were
organ weight determinations for adrenals, brain, heart,
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kidneys, liver, ovaries, spleen, testes, and thyroid with
parathyroids. Full histopathological examination was con-
ducted on organs from animals in the control and high-
dose groups. In addition, the GI tract was evaluated in
detail using the Swiss roll technique, which permits more
extensive evaluation of the intestine than routine sections.
The results indicate no treatment-related effects on body
weight, urinalysis, hematology, or clinical chemistry par-
ameters, organ weights, or ophthalmic, macroscopic, or
microscopic findings. Even with the relatively higher
amount of low molecular weight material present in the
test material, there was no evidence of erosions, ulcer-
ations, inflammation, regeneration, hyperplasia, or any
other abnormalities of the entire GI tract (Weiner et al.,
2007). The NOAEL was at least 5.0% in the diet or
calculated k-CGN consumption of 3394� 706 mg/kg/d in
males and 3867� 647 mg/kg/d in females. The results of
the study provide evidence that it is not necessary to
characterize CGN by a food additive specification for a
LMT (less than 5% below 50 kDa Mw), as established by
the European Commission (Commission Directive 2004/45/
EC of 16 April 2004).
Mice. Bhattacharyya et al. (2012) gave C57BL/6 J male
mice (6/group) drinking water without or with �–k-CGN
(Sigma Chemical Company, St. Louis, MO, USA) at a level of
10 mg/L for 18 d to evaluate potential effects on glucose and
insulin tolerances. Glucose tolerance was performed at 12
weeks of age after the 18-d exposure to CGN. For the glucose
tolerance test, mice were fasted for 18 h prior to sampling
blood for measurement of glucose with and without an
intraperitoneal (ip) injection of dextrose (2 g/kg). For the
insulin tolerance test, mice were exposed to control or treated
drinking water for an additional 15 d (33 d total). Following a
2 h fast, insulin ip injection (0.75 U/kg) was given and blood
was sampled for glucose from time 0 min until 120 min post-
injection. There were no effects on body weight, water
consumption, activity, or clinical observations in mice with
drinking water with CGN, compared with controls. The
results indicate that ingestion of CGN in drinking water under
the experimental conditions described significantly impaired
glucose tolerance and significantly impaired the response to
an injection of insulin. There are a number of possible
explanations for these findings in this publication, which
makes conclusions difficult to interpret (see discussion in
McKim, 2014). A positive control group, such as insulin
injection to another group of animals, would have helped to
validate the findings. Since no GI tract inflammation or
histopathology was evaluated, it is not possible to determine if
the authors’ hypothesis that CGN acts via an inflammatory
mechanism is correct. The relevance of these results to human
diabetes is not clear. There are no other studies showing an
effect of CGN on glucose, either in short-term or long-term
studies, including chronic studies in rats and hamsters and a
7.5-year monkey study. CGN has not shown any effects on
hematological parameters or serum clinical chemistry par-
ameters, including blood glucose, when evaluated at the end
of the 7.5 year monkey study (Albany Medical College,
1983); a 40-week dietary study in rats (Abraham et al., 1985)
and a 90-d dietary study in rats (Weiner et al., 2007).
Thus, the results of this one study (Bhattacharyya et al., 2012)
are inconsistent with data from other toxicological studies
on CGN.
Long-term oral toxicity
Long-term administration of CGN to rodents (Abraham et al.,
1985; Rustia et al., 1980) and primates (Albany Medical
College, 1983) has resulted in essentially no adverse effects.
Rats and hamsters have been fed 0, 0.5%, 2.5%, or 5.0%
k-CGN for their lifetimes (Rustia et al., 1980). Survival was
not affected in either species. No statistically significant
pathologic effects were seen in either species at any of the
dietary levels used in the study. This study only evaluated
survival and tumor development and did not include other
parameters of chronic toxicity (Rustia et al., 1980).
Administration of 0, 1%, or 5% k/�/E-CGN in the diet for
39 weeks to two strains of rats had no effect on hematology
and clinical chemistry parameters, fecal examinations, liver
weights, and liver pathology (Abraham et al., 1985).
Rhesus monkeys have been fed 50, 200, or 500 mg/kg/d for
7.5 years; for the first 5 years k/�-CGN was administered by
oral gavage 6 d/week and for the final 2.5 years, CGN was
administered in dietary pellets. Parameters evaluated included
the following: body weight, clinical chemistry, hematology,
stools, liver and testes biopsies, organ weights (liver, kidneys,
heart, brain, adrenals, ovaries/testes, pituitary), gross nec-
ropsy, complete histopathology on all organs/tissues, histo-
chemistry with toluidine blue (small and large intestines,
cecum, liver, and lymph nodes). There were no effects on
hematological or clinical chemistry parameters, including
blood glucose. No pathologic effects attributable to the intake
of CGN were seen in the monkeys. No evidence of GI tract
ulceration or other abnormal pathology was observed. There
was no evidence of storage of CGN in the liver. The only
effects seen were soft stools or diarrhea and occasional fecal
occult blood, which were also seen in untreated controls
(Albany Medical College, 1983).
In summary, subchronic and chronic administration of
CGN in the diet to several species, in well-conducted studies,
did not result in adverse effects other than soft stools or
diarrhea. These effects are commonly seen when dietary
fibers are fed at high doses (Tungland & Meyer, 2002).
Immune system effects
Summary
Existing studies on immune system parameters were con-
ducted mainly with �-CGN in drinking water, which provide
data that are not directly applicable to dietary exposure where
CGN is tightly bound to protein. Based on the available
studies, conducted in diet, CGN has not been shown to
produce immunotoxicity.
CGN exhibits a number of biological effects following
systemic administration via intravenous (iv) or ip injection
that relate to the immune system (Nacife et al., 2004;
Thompson, 1978; Thompson & Fowler, 1981; Thompson
et al., 1979). When administered by a systemic route of
exposure (ip or iv), CGN has the capacity to induce acute and
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chronic inflammation and granuloma formation; cause the
release of vasoactive amines and other humoral mediators
(kinins, prostaglandins, and cytokines) from immune system
cells; inhibit the hemolytic activity of complement; act as an
adjuvant, and suppress specific antibody- and cell-mediated
immunity. CGN has substantial effects on the immune system
when administered systemically, as shown in the mouse or rat
footpad model. Many of these models have been utilized for
the examination of basic immune processes. However, these
models all involve systemic administration, and are not
relevant to oral exposure of the high Mw CGN in the diet
(Cohen & Ito, 2002). It is important to emphasize that the
systemic routes of exposure have been used experimentally to
further pharmacological research in the management and
treatment of inflammatory diseases or conditions, and that
this work via systemic administration is not relevant to
consumption of food that contains CGN. CGN is not absorbed
from the GI tract in significant quantities (see the section
‘‘Degradation, excretion, absorption, and metabolism’’).
Bash & Vago (1980) gave rats k/�-CGN via oral gavage
in water at doses of approximately 5–500 mg/kg/d once or
daily in drinking water for 61–85 d followed by measurements
of immunity. No consistent dose–responses were observed.
A single oral dose of CGN (1 mg) in water by gavage
significantly suppressed splenic and lymph node T-cell
responses to mitogens; whereas a single dose of 10 mg was
not suppressive. Rats maintained on 0.1 or 1.0 mg/ml
solutions of CGN in drinking water for 61 d also exhibited
significant suppression of the spleen cell mitogenic response
at the lower dose only. These doses (0.1 and 1.0 mg/ml) are
stated to be equivalent to 2.3 and 23.5 mg/day/rat,
respectively. Responses returned to normal when adherent
cells (macrophages) were removed prior to mitogenic stimu-
lation. To further define the mechanism of action, macro-
phages were harvested from rats administered drinking water
for 85 d with 0.1 or 1.0 mg/ml/d of CGN, cultured for 72 h and
added to spleen cell cultures responding to phytohemagglu-
tinin (PHA). The degree of suppression of spleen cell
responses to mitogen was roughly equivalent to that seen in
control macrophage cultures pretreated in vitro with 10 mg/ml
of the same CGN preparation (Bash & Cochran, 1980). In
both the in vivo and in vitro studies, macrophage-induced
suppression of the mitogenic response was evident at the
lower doses only.
These responses (macrophage-induced suppression of
the T-cell mitogenic response) were not seen at the higher
CGN doses. In order to explain this inverse dose–response
phenomenon, the authors speculated that suppressor
macrophages were induced at the lower dose. This effect
is curious and has not been reported by other investiga-
tors, either in vitro, or in vivo via oral or systemic routes.
The �-CGN administered in drinking water or exposed to
cells in vitro exists in the random coil conformation and,
hence, not in the structured form found in food or liquids
(see Introduction).
Nicklin & Miller (1984) examined the systemic and local
(gut-associated) immune response of rats administered E-, k-,
or �-CGN in drinking water at a level of 0.5% or the
equivalent of approximately 150–250 mg/kg/d CGN for 90 d.
Gut-associated levels of IgA and of specific agglutinating
antibody to gut bacteria were not affected by CGN treatment.
Total bile IgA levels also were not affected. The response to
injected sheep red blood cells (RBCs) was reduced in animals
on drinking water containing CGN or guar gum.
Biliary anti-sheep RBCs or anti-CGN immune responses
also were not detected (Nicklin & Miller, 1984). In contrast to
orally administered antigen, the expected anti-sheep RBC
serum antibody responses following ip injection of anti-sheep
RBCs was somewhat delayed, relative to controls, after (k, �,
E) CGN administration. This was seen in the absence of any
specific T-cell effects on graft-versus-host reactivity. The
authors concluded that T-cell immunity was unaffected.
Nicklin et al. (1988) administered 0.25% E-CGN in drinking
water to rats for 184 d. Animals on tap water or CGN-
containing water were challenged with an ip injection of 1 ml
of 5% sheep RBCs. Serum was collected once a week for
4 weeks and analyzed for anti-sheep RBC antibody activity by
the hemagglutination method. Animals treated with CGN had
a delayed and reduced antibody response, compared to
controls.
Nicklin et al. (1988) demonstrated the presence of3H-radiolabeled E-CGN in the cecal lymph node (see section
‘‘Degradation, excretion, absorption, and metabolism’’). As
in their earlier report using Alcian blue staining (Nicklin &
Miller, 1984), autoradiographic analysis of the tissues
revealed that the bound radiolabel was associated with
macrophages. The pharmacokinetic data indicate that CGN
enters the body via the Peyer’s patches and cecal lymph nodes
and may be transported via resident macrophages to mesen-
teric lymph nodes. In oral feeding studies with the unlabeled
CGN, however, it took a relatively high oral dose (0.25% in
drinking water for 184 d) before suppression of immune
responses to T-cell-dependent antigens was noted. This CGN
dose level is consistent with that of earlier oral drinking water
studies. However, unlike their earlier study (Nicklin & Miller,
1984), accurate measurements of actual CGN consumption
were not made.
The relative potency of E-CGN to act as an adjuvant
following ip versus oral administration with the antigen,
ovalbumin, was studied by Coste et al. (1989). E-CGN elicited
reaginic and IgG–ovalbumin specific antibody responses
when admixed with 1 mg antigen (ovalbumin) and adminis-
tered ip to rats at doses equivalent to 5 mg/kg. When antigen
and E-CGN were administered orally via gavage in water, no
specific immune response was initiated. These results indir-
ectly support the studies of Nicklin & Miller (1984) who
demonstrated no oral adjuvant effects of CGN on local
immunity to intestinal antigens. Responses via the ip route are
not relevant to oral exposure to food containing CGN. Coste
et al. (1989) conclude that CGN is not active as an adjuvant
when administered orally in water.
More recently, there have been studies suggesting an effect
on the immune system following oral administration of
�-CGN (Frossard et al., 2001; Tsuji et al., 2003).
Unfortunately, in most of these studies, specific information
regarding the type of administered �-CGN is not provided.
Frossard et al. (2001) showed that oral gavage administration
of �-CGN in water (0.5 g/L) with low amounts of
b-lactoglobulin (BLG), a common milk antigen, could
prevent anaphylaxis in mice subsequently sensitized to BLG
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together with the potent mucosal adjuvant cholera toxin (CT).
Tolerance was induced only when both CGN and BLG were
administered together prior to sensitization to BLG; but not
when CGN was administered alone. Since CGN and BLG
were administered simultaneously, there may have been an
effect of the CGN on the structure of BLG due to the known
binding of CGN to proteins. Similarly, Tsuji et al. (2003) also
showed a suppression of the allergic reaction when low doses
of �-CGN (0.001–0.005%) were administered to mice orally
in drinking water for six consecutive days, following
immunization by ovalbumin in alum intraperitoneally.
�-CGN suppressed both antibody production and mitogen-
induced T-cell proliferation optimally at 0.001%, compared to
higher doses of40.02%. Frossard et al. (2001) and Tsuji et al.
(2003) dosed �-CGN in water. As noted in the Introduction,
�-CGN exists only in the disorganized conformation in water,
so the relevance of these results to dietary exposure, where
CGN exists in the organized conformation, is questionable.
There is little evidence in humans suggesting an effect of
dietary exposure to food-grade CGN on the immune system.
However, there is one anecdotal report in the literature
describing a case of anaphylaxis secondary to exposure to a
barium medical imaging formulation (Tarlo et al., 1995). Based
on an allergy skin test, the Radioallergosorbent Test (RAST)
evaluation, the authors concluded that the allergic reaction
occurred due to CGN and no other substances present in the
barium medical imaging formulation. Furthermore, when the
patient was placed on what was purported to be a CGN-free
diet, the patient’s previous gastrointestinal symptoms abated.
The report indicates the use of sodium CGN in the barium
medical imaging formulation. It is widely known in the
industry that poligeenan, not CGN, was the material used in the
barium medical imaging formulation (Marinalg, 2006; JECFA,
2008). The term ‘‘poligeenan’’ was not in general use
internationally at the time of the reported incident of anaphyl-
axis. In the Tarlo et al. (1995) paper, the type of CGN used for
the skin test is not detailed. The skin prick test with the barium
medical imaging formulation was positive (10 mm mean wheal
diameter with flare). The individual ingredients of the barium
medical imaging formulation were tested separately in water at
concentrations used in the commercial imaging solution. The
only ingredient demonstrating a positive response was 0.4%
‘‘sodium CGN’’ (8 mm mean wheal diameter with flare);
however, a separate skin test with a commercial skin test
preparation of CGN gum (1:50 wt/vol) gave a borderline
response (2 mm mean wheal response) (Tarlo et al., 1995).
Moreover, there are no prior or subsequent case reports of an
allergic reaction to CGN in humans or in animals. Because
CGN is not functional in the barium medical imaging
formulation (Marinalg, 2006), the ‘‘sodium CGN’’ reported
by Tarlo et al. (1995) was certainly poligeenan.
By comparison, Mallett et al. (1985) reported that antibody
to cecal bacteria was increased in Sprague–Dawley rats fed
approximately 5% E/k-CGN in the diet for 30 d.
In summary, the majority of the studies in which
animals were exposed to CGN in drinking water or by oral
gavage in water (Bash & Vago, 1980; Coste et al, 1989;
Frossard et al., 2001; Nicklin & Miller, 1984; Nicklin et al.,
1988; Tsuji et al., 2003) show conflicting results. Several
studies showed immunosupression (Bash & Vago, 1980;
Frossard et al., 2001; Tsuji et al., 2003). The conformation of
CGN in water is different from that in food, where CGN is
bound to protein (Blakemore & Harpell, 2010) (see the
section ‘‘Introduction’’). As discussed, �-CGN exists in the
random conformation in water, and �-CGN was used in the
studies of Frossard et al. (2001) and Tsuji et al. (2003). The
studies of Bash & Vago (1980) and Nicklin et al. (1988) used
�- and E-CGNs, respectively. Therefore, the relevance of these
drinking water and gavage studies to human dietary con-
sumption is unclear. The single dietary study (Mallet et al.,
1985) conducted a very limited investigation with only one
endpoint evaluated at 5% in the diet.
The data from oral exposure studies show that small
amounts of CGN may be taken up by gut associated lymphoid
tissue and may be transported to regional lymph nodes by
macrophages following ingestion. Nevertheless, the absence of
toxicity and lack of significant amounts of the material outside
the gut in feeding studies, including the long-term primate
feeding studies (Albany Medical College, 1983), demonstrate
that food grade CGN is safe from a toxicological viewpoint. In
dietary studies in which complete organ histopathology,
clinical chemistry and hematology were evaluated, CGN did
not cause any significant treatment-related toxicity related to
the immune system (Abraham et al., 1985; Weiner et al., 2007).
Doses of CGN used in the subchronic dietary study (Weiner
et al., 2007) are orders of magnitude above levels of CGN in the
human diet: 3394–3867 mg/kg bw/d in females and males,
respectively, compared to 18–30 mg/kg/d in humans (Shah &
Huffman, 2003). The long and safe use of CGN as a food
additive also attests to a general lack of toxicity and a lack of
immunotoxicity via dietary exposure.
Ulcerative effects
Summary
Due to the known ulcerative effects of poligeenan, the
potential of CGN to cause GI tract ulceration has been
extensively studied. Food grade CGN administered in diet
does not cause ulceration on the GI tract.
Poligeenan has been reported to cause intestinal ulceration
in animals when administered in drinking water or diet (see
review by Michel & Macfarlane, 1996). The ulcerogenic
potential of CGN has been studied extensively. Studies of
CGN in diet found no evidence of intestinal ulceration in rats,
hamsters, primates, and pigs when fed at levels of 5% in the
diet or less. Exposure to very high, non-physiological levels
(415%) of CGN in the diet produced ulcerative effects
(Mankes, 1977; Watanabe et al., 1978). Benitz et al. (1973)
found that exposure to poligeenan, but not CGN, in drinking
water produced gastrointestinal ulceration in adult primates.
Newborn primates given infant formula with k/�-CGN also
showed no evidence of GI ulceration (McGill et al., 1977).
Negative studies for intestinal effects have also been reported
for the hamster (Rustia et al., 1980) and the pig (Poulsen,
1973). Conflicting results have been reported in guinea pigs
with CGN (Engster & Abraham, 1976; Grasso et al., 1973;
Watt & Marcus, 1969). Table 2 summarizes the results of
studies on poligeenan (termed degraded CGN, rather than
poligeenan) published prior to 1985 demonstrating intestinal
ulceration (Michel & Macfarlane, 1996). These findings are
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.In
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important in light of the known ulcerative effects and
gastrointestinal carcinogenicity of poligeenan (IARC, 1983)
and to show the different responses of the GI tract to
poligeenan versus CGN.
Table 2 uses the term ‘‘degraded CGN’’ for poligeenan
and shows the large number of studies reporting GI tract
ulceration with this material. As can be seen, GI tract
ulceration was reported in guinea pigs, rats, rabbits, and
monkeys, but not humans, exposed to poligeenan. Grasso
et al. (1973) report that six patients suffering from malignant
disease of the colon were given 5 g of poligeenan (called
‘‘degraded CGN’’ in the paper) daily for 10 d. Sampling of
the colon found no evidence of ulceration due to poligeenan.
Similarly, a report on 200 patients receiving a preparation of
poligeenan (Ebimar) to treat peptic ulcer in France at a dose
of 5 g/d for 6 months–2 years found no signs of ulcerative
colitis by routine monitoring and radiological examinations
(Bonfils, 1970). These reports (Bonfils, 1970; Grasso et al.,
1973) demonstrate that humans receiving 5 g/d poligeenan
orally did not develop the significant intestinal ulceration
reported in laboratory animals, particularly the guinea pig,
given poligeenan.
The ulcerogenic potential of CGN has been studied in
several species, as summarized in Table 3. The weight of the
evidence indicates that CGN does not produce intestinal
ulceration in rats, hamsters, primates, and pigs unless fed to
animals at non-physiologic levels in diet (415%). Exposure to
very high, non-physiological levels (415%) of CGN in the
diet produced ulcerative effects (Mankes, 1977; Watanabe
et al., 1978). Conflicting results have been reported in guinea
pigs (Engster & Abraham, 1976; Grasso et al., 1973; Watt &
Marcus, 1969), but, the guinea pig seems to be the most
sensitive species responding to poligeenan. Negative studies
for intestinal effects have also been reported for the hamster
(Rustia et al., 1980) and pig (Poulsen, 1973). Long-term
ingestion of CGN in the drinking water or diet has not been
shown to produce GI ulceration in adult or infant primates
(Benitz et al., 1973; McGill et al., 1977). The results for each
species are summarized and discussed in more detail in
Table 3. These findings are important in light of the known
ulcerative effects and gastrointestinal carcinogenicity of
poligeenan (IARC, 1983).
Mice
Administration of �-CGN (Sigma, stated to be pure
‘‘undegraded’’ CGN that is not food grade) to mice
(5/group) for 10 weeks at a concentration of 1% or 4% in
the drinking water was reported to induce inflammatory
responses in the colon (Donnelly et al., 2004a). Inflammation
was evaluated histologically and scored as inflammatory
score, hyperplasia score and crypt loss score. All three scores
were greater in treated animals than controls, with more
severe effects noted at 4% compared to 1% �-CGN in
drinking water. The composition of the �-CGN utilized for
these studies had an extremely high viscosity, in the range
700–800 cps, compared with the compendial (including
regulatory) viscosity specification of a minimum of 5 cps
(equivalent to �100–150 kDa). Most commercial CGNs are
Table 3. Intestinal effects of carrageenan in different species.
Species VehicleCarrageenan
dose(s)Duration:
weeksHistopathological effects:
ulceration Reference
Mouse Drinking water* 1 and 4% 10 Mild inflammation at 1%,severe inflammation at 4%
Donnelly (2004a)
Rat Processed skimmedmilk
4% 24 No microscopic changes Tomarelli et al. (1974)
Diety 2.5 and 5% 13 No microscopic effects Weiner et al. (2007)Diet 5% 104 No microscopic effects Rustia et al. (1980)Dietz 15% 40 Chronic inflammatory changes
in the large intestinesWatanabe et al. (1978)
Dietz 25% 4 Pinpoint intestinal lesions[unpublished thesis]
Mankes (1977)
Diet 1 and 5% 13 and 40 No microscopic changes Abraham et al. (1985)Guinea pig Drinking waterx 1% 2 No microscopic effects Engster & Abraham (1976)
Dietx 2% 10 No microscopic effects Engster & Abraham (1976)Diet 5% 3–7 Cecal and colonic ulcerations –
no actual data providedGrasso et al. (1973)
Hamster Diet 5% 104 No microscopic effects Rustia et al. (1980)Pig Jelly in diet 500 mg/kg/d 12 No microscopic effects Poulsen (1973)Rhesus monkey Drinking water 1% 11 No microscopic effects Benitz et al. (1973)
Drinking water 1250 mg/kg/d 8 No microscopic effects Benitz et al. (1973)Baboon Infant formula 255 and 1220 mg/L
(analyzed)16 No microscopic effects McGill et al. (1977)
CGN had a Mw4145 kDa.*The study used �-CGN from Sigma Chemical Company (not food grade CGN).yCarrageenan sample used in this study was specifically altered to contain a low molecular weight tail (LMT) of 7% (mean) carrageenan to maximize
any potential effects of the lower molecular weight fraction on the gastrointestinal tract.zDoses greater than 5% test material in the diet are considered not to be nutritionally adequate because so high a percentage of the diet is replaced by an
inert substance without nutrient or caloric value. At levels of test material in the diet45%, animals will eat more food to compensate for the lowercaloric/nutritional intake. Inadequately controlled dietary variables may result in nutritional imbalances or caloric deprivation that couldconfound interpretation of the toxicity study results (FDA, 2000). The FDA suggests that if doses in dietary studies exceed 5% in the diet,additional controls may be needed (FDA, 2000) http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDocuments/FoodIngredientsandPackaging/Redbook/ucm078345.htm.xCGN had a Mw445 kDa.
14 M. L. Weiner Crit Rev Toxicol, Early Online: 1–26
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normally within the Mw range 200–800 kDa, with a typical
range of 400–600 kDa (Blakemore & Harpell, 2010; Uno
et al., 2001b). Under the conditions of the study, administra-
tion of �-CGN at 1% in water would have resulted in a very
thick solution and at 4% in water would have been a paste-like
material. These formulations would be expected to influence
the water consumption and water availability to the animals.
Such an effect was observed (depressed water consumption in
treated animals) (Donnelly et al., 2004a). The high concen-
tration in water, abnormal consistency of the dosing solutions,
and the fact that �-CGN exists in the disorganized conform-
ation in water, likely contributed to the induction of colitis
and raise serious concerns regarding the relevance of this
study to human dietary consumption.
Rats
Ulcerative effects in rats have been reported only after
exposure to extremely high, non-physiological concentrations
of CGN in the diet. Watanabe et al. (1978) reported chronic
inflammatory changes in the large intestines of rats that
received 15% k/�-CGN in the diet for 40 weeks. Multiple
pinpoint intestinal lesions were also reported in rats following
dietary exposure to 25% CGN for one month (Mankes, 1977).
This dose level greatly exceeds the recommended limit dose
for dietary studies (OECD, 2009a,b) and can result in
confounding effects due to nutritional deficiencies because
of the large percentage of the diet (25%) containing a non-
nutritive additive. Long-term or subchronic dietary adminis-
tration of CGN at concentrations of 5% or less has not
produced ulcerogenic lesions in the rat (Abraham et al., 1985;
Tomarelli et al., 1974; Rustia et al., 1980; Weiner et al.,
2007). Lifetime administration of k-CGN in the diet at
concentrations of 0, 0.5%, 2.5%, or 5% did not produce
intestinal lesions (Rustia et al., 1980). Similarly, 4% k/�-CGN
in a processed skim milk diet fed to rats for six months did not
produce gross or microscopic changes in the cecum or colon
(Tomarelli et al., 1974). The dose administered was estimated
at 1490 mg/kg/d: 14.9 g food/day with 4% CGN is 596 mg
CGN, divided by a body weight in males of 0.4 kg.
Histopathological evaluation of the GI tract in rats following
90-d of dietary exposure to 5% k-, �-, or E-CGN did not find
any evidence of intestinal ulceration or any other lesions
(Abraham et al., 1985; Weiner et al., 2007). Abraham et al.
(1985) also found no intestinal ulceration in rats fed CGN at
up to 5% in the diet for 40 weeks.
Guinea pigs
Early studies reported intestinal ulceration following admin-
istration of 1% E-CGN (molecular weight not specified) in the
drinking water for 20–30 d (Watt & Marcus, 1969). However,
later studies showed that exposure to high Mw fractions of
CGN (Mw4145 000 Da) did not produce intestinal lesions
when given in the drinking water at a concentration of 1%
E-CGN for two weeks (Engster & Abraham, 1976). In
addition, exposure to 2% E-CGN in the diet for 10 weeks
was not associated with ulcerative effects (Engster &
Abraham, 1976). Intentionally prepared fractions of CGN
(k, �, E) with Mw 21 kDa or 39 kDa did cause cecal ulceration
in the guinea pigs, and minimal erosion was noted at Mw
107 kDa fraction (Engster & Abraham, 1976). Fractions of
E-CGN with Mw of 88 kDa or 4145 kDa did not induce any
histopathological changes to the cecum. Engster & Abraham
(1976) suggest that the low Mw fractions, produced artificially
from CGN with Mws similar to poligeenan, caused cecal
ulceration by uptake of the fraction into the lysosomes of
lamina proprial macrophages, which stimulate release of
lysosomal enzymes (acid phosphatase and b-glucuronidase),
resulting in damage to the nearby tissues. Only the low Mw
fractions similar to poligeenan were able to cause cecal
ulceration and uptake into lamina propria macrophages, as
seen by toluidine-blue staining of the cecum. Interestingly,
neither �- nor k-CGN induced ulceration in guinea pigs given
1% drinking water solutions (Engster & Abraham, 1976).
In contrast, guinea pigs fed 5% E-CGN in the diet for
21–45 d developed multiple cecal and colonic ulcerations
(Grasso et al., 1973). More severe ulceration was reported in
the same study for poligeenan, including photomicrographs of
these lesions. No photomicrographs are provided to verify the
lesions reported after administration of CGN (Grasso et al.,
1973); therefore, this study is considered inconclusive due to
lack of documentation. The guinea pig and rabbit were more
sensitive to intestinal ulcerative effects due to poligeenan
exposure in drinking water than other species examined in
this study, including rat, ferret, squirrel monkey and hamster
(Grasso et al., 1973). The positive response of CGN at a high
dietary concentration in the guinea pig could be due to the
species sensitivity noted, but is not explained further by the
authors (Grasso et al., 1973).
Interestingly, Onderdonk & Bartlett (1979) have shown
that the intestinal ulceration observed with drinking water
administration of poligeenan (5% w/w) to guinea pigs can be
eliminated by using germ-free animals, rather than conven-
tional animals. Germ-free guinea pigs repopulated with
conventional guinea pig microflora developed cecal ulcer-
ations in response to poligeenan. These authors suggest that
bacteria may be a risk factor in the intestinal ulceration seen
with poligeenan (Onderdonk & Bartlett, 1979).
Hamsters
Hamsters fed 0, 0.5%, 2.5%, or 5% k-CGN in the diet for life
(100 weeks) did not exhibit intestinal alterations beyond that
shown by age-matched control animals (Rustia et al., 1980).
Pigs
In another study, Danish Landrace pigs received k-CGN
orally at levels of 0, 50, 200, and 500 mg/kg daily for 12
weeks. No intestinal ulceration was observed (Poulsen, 1973).
Primates
Administration of CGN in the drinking water of primates
has not produced any evidence of intestinal ulceration.
Rhesus monkeys administered 1% k-CGN in the drinking
water for 7–11 weeks showed no intestinal changes (Benitz
et al., 1973). An additional four animals were given CGN
for 7–11 weeks, followed by an 11-week recovery period;
the animals were then given escalating oral doses of
50–1250 mg/kg CGN daily for 12 weeks. The highest dose
of CGN, 1250 mg/kg, was administered daily for 8 of the
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12 weeks. No intestinal ulceration was noted (Benitz et al.,
1973). Infant baboons fed 1220 mg/L k/�-CGN in infant
formula for 16 weeks showed no gastrointestinal lesions
(McGill et al., 1977).
In summary, administration of CGN in the diet to
numerous animal species (rat, mouse, pig, hamster and
monkey) did not induce intestinal ulcerations or any other
lesions when administered at a level up to 5%. Both positive
(Grasso et al, 1973) and negative effects (Engster & Abraham,
1976) on the GI tract were reported for the guinea pig, a more
sensitive species for ulcerative effects, following dietary
administration of CGN. The only report in which intestinal
ulcerations were reported was the Donnelly et al. (2004a)
study. In this study, mice given drinking water containing 4%
CGN with a high viscosity developed lesions of the intestine
which could be explained by confounding effects of dehy-
dration and the use of non-food grade test article. No such
effects were seen in numerous dietary studies or in drinking
water studies in guinea pigs and monkeys. Michel &
Macfarlane (1996) concluded that only poligeenan can
induce colorectal ulceration in animals, which is character-
istic of other sulfated polysaccharides, including amylopectin
sulfate and dextran sulfate. These polymers interact with the
gut mucosa in similar ways; and all possess sulfate,
low polymer size, and anti-peptic activity (Michel &
Macfarlane, 1996).
Carcingenicity studies
Summary
CGN is not carcinogenic based on chronic oncogenicity
studies in animals. CGN has been the subject of numerous
reviews (FDA, 1972c, 1973; Hopkins, 1981; IARC, 1983).
The International Agency for Research on Cancer (IARC)
published a review of CGN in 1983 and concluded that CGN
was not carcinogenic to experimental animals and that no
conclusion could be made regarding carcinogenicity to
humans due to the lack of epidemiological data (IARC, 1983).
k-CGN was found to lack carcinogenicity in chronic
feeding studies in rats and golden hamsters at levels up to 5%
(Rustia et al., 1980). Cohen & Ito (2002) reviewed in detail
the carcinogenicity testing of CGN and concluded that there
was no evidence of carcinogenicity of CGN in animal models.
Likewise, they concluded that there was no evidence of
tumor-promoting activity by CGN in animal models due to
flaws in the reported studies (Arakawa et al., 1986, 1988;
Corpet et al., 1997; Watanabe et al., 1978; Millet et al., 1997)
(see section ‘‘Tumor promotion’’).
Tobacman (2001, 2003) published a series of articles
questioning the interpretation of the carcinogenicity of CGNs
and other safety issues. She raises questions with respect to
carcinogenicity, tumor promotion and inflammatory and
immune effects. Tobacman (2001) often cites work on
poligeenan as if it applied to CGN. Carthew (2002) expressed
concerns with this paper in a letter to the editor. Tobacman
(2001) suggested that there is extensive breakdown of CGN
in vivo to small-molecular-weight products (poligeenan) in
the GI tract, which could be carcinogenic. As the conditions
in the human GI tract are not considered severe enough to
degrade CGN to poligeenan (see the section ‘‘Degradation,
excretion, absorption, and metabolism’’, and McKim, 2014),
these conclusions should be considered speculative. Orally
administered CGN is not digested into low molecular weight
forms; it is not absorbed or distributed systemically and shows
no evidence of toxicological effects at dose levels up to 5% in
the diet (Cohen & Ito, 2002; IARC, 1983; JECFA, 1999,
2008) (see the section ‘‘Degradation, excretion, absorption,
and metabolism’’). No evidence exists to suggest that food
grade CGN causes any carcinogenic effects in vivo.
Genetic effects
Summary
CGN is not genotoxic in in vitro and in vivo studies. Food
grade CGN has not been shown to be genotoxic following
extensive testing. The results of several in vitro mutagenesis
assays have shown that CGN was not mutagenic in the yeast
strain Saccharomyces cervisiae D4 (FDA, 1975), host
mediated assay (Sylianco et al., 1993) or the Ames test
(FDA, 1972a,b, 1975; Jackson, 1997; Mori et al., 1984;
Sylianco et al., 1993). CGN did not induce chromosomal
abnormalities in rat bone marrow cells in vitro (FDA,
1972a,b) or in an in vivo test, the mouse micronucleus
assay (Sylianco et al., 1993). Negative results were also
obtained in the dominant lethal assay in rats (FDA, 1972a,b).
Tumor promotion
Summary
CGN is not considered to be a tumor promoter. There were
some questions raised regarding possible tumor-promoting
activity of CGN in the past (JECFA, 1999). As discussed in
detail by Cohen & Ito (2002), the studies in which this
concern was raised had serious flaws and did not indicate any
evidence of tumor-promoting activity for the GI tract or any
other tissues. The concerns regarding potential tumor pro-
motion of CGN noted by JECFA (1999) were addressed by
Cohen & Ito (2002) and an ‘‘ADI not specified’’ was retained
(JECFA, 2008).
Cohen & Ito (2002) concluded that there clearly was no
evidence of tumor promoting activity when taking all these
studies into consideration (Corpet et al., 1997; Hagiwara et al,
2001; Tache et al., 2000). If one accepts the conclusions of the
investigators (Corpet et al., 1997), however, there is ques-
tionable significance to humans, since there was no tumor
promotion effect when human microflora replaced rat micro-
flora in the GI tract of rats.
More recently, Donnelly et al. (2004a–c, 2005) have
reported a series of experiments on colon crypt cell staining
when N-methyl-N-nitrosourea (MNU), a known mutagen, was
co-administered with �-CGN (stated as not food grade).
These investigators utilized persistent metallothionein over-
expression within single crypts as a biomarker of colonic
crypt stem cell mutations. It should be noted that a biomarker
for specific oncogene mutations in the stem cell fraction of
normal colonic mucosa is not available at present and the
model used does not directly address oncogene mutations
(Donnelly et al., 2004a). Although these investigators
reported an effect on DNA adduct formation and metallothio-
nein crypt-restricted immunopositive staining of colonic
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crypts in the animals administered MNU alone (62.5 mg/kg
ip), no effects on these endpoints were seen with �-CGN
alone (1% or 4% �-CGN in drinking water for up to 20 weeks)
(Donnelly et al., 2004a, 2005). The addition of �-CGN
together with the mutagen, MNU, enhanced the response
observed with the mutagen alone on the number of aberrant
crypt foci, DNA adduct formation, and the metallothionein
immunopositive staining of colonic crypts (Donnelly et al.,
2004a, 2005).
The composition of the �-CGN utilized for these studies
had a considerably higher viscosity than accepted for food-
grade CGN specifications, the viscosity probably being close
to 700–800 cps per compendia viscosity test. Under the
conditions of the study, administration of this type of CGN
would be expected to form a very thick solution at 1% in water
and a paste-like material at 4% in water. These solutions
would be expected to influence the water consumption and
water availability for the animals. Such an effect was observed
(depressed water consumption in treated animals) (Donnelly
et al., 2004a). The AIN-76A diet used in these studies is
marginal in essential nutrients and the animals may become
stressed by the marginal nutrient content and exposure to
MNU and/or �-CGN. The AIN-76A diet would also cause
much of the water consumed by these animals to be absorbed,
as animals administered this diet tend to have extremely
small, compact, dry feces. The high concentration of
extremely high Mw �-CGN in water and the expected
abnormal consistency of the test solutions, along with a
nutritionally marginal diet, may have contributed to the noted
GI ulceration and raise doubts regarding the relevance of
these studies to humans. In addition, Donnelly et al. (2004a,
2005) do not describe the preparation of MNU or indicate
whether fresh solutions were prepared daily. Fresh MNU is
important because MNU can deteriorate, leading to artifacts
of the first group dosed, compared to the last group dosed on a
given day. The ip injection of MNU would be expected to
produce peritonitis, leading to confounding effects and
responses to �-CGN. The dose of MNU employed is far in
excess of human exposure to nitroso compounds (Donnelly
et al. 2004a, 2005), so the relevance to humans is questionable
based on the MNU dose alone.
Further complicating the interpretation of the results is the
finding of inflammation of the intestinal mucosa, including
ulceration (Donnelly et al., 2004a–c, 2005). As described
above, food-grade CGN in diet does not produce ulceration
(see section ‘‘Ulcerative effects’’; Cohen & Ito, 2002; Weiner
et al., 2007; see Table 3). The reports by Donnelly et al.
(2004a–c, 2005) raise concerns regarding the material that
was administered, as well as the diet employed and other
experimental factors and certainly complicate extrapolation
from the animal model to humans. In the paper in the British
Journal of Cancer (Donnelly et al., 2005), the authors indicate
that there were no statistically significant differences in large
crypts, only in smaller crypts. Other studies have shown that
large aberrant crypt foci are likely the ones that are most
meaningful with respect to the carcinogenic process
(Papanikolaou et al., 2000). Furthermore, there was no
consistent dose–response in the results for total immunopo-
sitive patches/104 crypts with �-CGN and MNU, whether the
CGN was administered for one week, one week repeated three
times, or continuous administration during the entire 20
weeks of the experiment. Donnelly et al. (2004a, 2005)
homogenized the whole intestinal tissue for DNA adduct
evaluation, whereas only a small percentage of the total cells
in the intestine, the epithelium and mucosa, are relevant to GI
tract dietary exposure. Any effect on the mucosal epithelium
would not be observed using the whole intestinal tissue
(Cohen & Ito, 2002). Most of the DNA extracted in their
procedure would have come from the inflammatory cells in
the lamina propria, normally present. Hence, the DNA adduct
formation results are not meaningful. Given all these
difficulties in interpretation, extrapolation to a conclusion of
a tumor-promoting effect of CGN in humans is highly
unlikely and not scientifically supported.
In conclusion, based on a variety of types of assays, there
continues to be no convincing evidence of tumor-promoting
activity of the GI tract for food-grade CGN in animal models.
The FDA (2012) in a response to the Citizen’s Petition by
Dr. Joanne K. Tobacman states that the use of CGN in foods
is safe and that CGN consumed in the diet is not a carcinogen,
tumor promoter or ulcerogenic substance in animal studies.
JECFA (2008) also critically reviewed the publications
on tumor promotion and also concluded that CGN is safe
in foods.
Reproductive and developmental toxicity studies
Summary
CGN is not a developmental or reproductive toxicant in
animal studies. The reproductive effects of CGN were
evaluated in a three-generation reproduction study in rats.
Groups of rats were fed k/�-CGN at concentrations of 0.5%,
1.0%, 2.5%, and 5% prior to mating, throughout pregnancy,
lactation, and weaning for three generations. The only effects
seen were soft stools at the 5.0% and 2.5% level and decreased
body weights for the dams. Decreased birth weight and body
weights at weaning were noted in the offspring of dams fed
1%, 2.5%, and 5%. No effects were seen at the 0.5% level
(Collins et al., 1977a). No effects were seen on reproductive
parameters at any dose level. A teratologic evaluation of the
F2C and F3C generations of the reproduction study showed
no teratological effects due to treatment with CGN (Collins
et al., 1977b).
In a one-generation reproduction study in rats, CGN
caused no effects on physical or behavioral development of
offspring of parental animals fed 0.45% or 0.9% CGN prior to
mating, during gestation, and lactation and after weaning
(Vorhees et al., 1979).
CGN was studied for potential teratogenicity in four
species: mice, rats, rabbits, and hamsters by oral gavage
administration. k/�-CGN (source not specified) was admin-
istered during gestation: mice at 10–900 mg/kg on days 6–15;
rats at 40–600 mg/kg on days 6–15; rabbits at 40–600 mg/kg
on days 6–18, and hamsters at 10–900 mg/kg on days 6–10.
No treatment-related malformations of offspring were
observed in any species at any dose level (FDRL, 1972a,b).
Fetotoxicity was observed in mice at the 900 mg/kg dose by
gavage. Teratology studies were also conducted in rats and
hamsters via dietary administration at 1% and 5% k/�-CGN
during gestation days 6–15 and 6–10, respectively (Bailey &
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Morgareidge, 1973). Carrageenan was shown to lack terato-
genic effects in both species at a limit dose of 5% in the diet.
CGN has been tested in a teratology study in a non-
mammalian system. In a protocol not used to determine
teratogenicity in mammals, injection of 0.1% �-CGN into the
yolk-sac of chicken eggs has resulted in increased lethality, a
decrease in hatching weight (Rovasio & Monis, 1980) and
microscopic nervous system abnormalities (Rovasio & Monis,
1987). It is difficult to assess these results due to the lack of
concordance with other mammalian studies discussed above.
The exposure of developing chick embryos to �-CGN injected
directly into eggs in an aqueous milieu is not considered
relevant to human exposure to food grade CGN in the diet.
In the chick embryo system, the developing embryo is
exposed directly to �-CGN; whereas in mammalian studies,
CGN is not absorbed into the systemic circulation and does
not reach the embryo.
In conclusion, CGN is not considered to be a develop-
mental or reproductive toxicant or a teratogen via dietary
administration.
Studies on special age groups
Summary
Existing data on infant baboons demonstrate that CGN is safe
when fed at the maximum feasible levels in formula which
exceed levels used in infant formula. New questions raised by
JECFA (2008) regarding the use of CGN in infant formula are
currently being addressed by FMC Corporation and the
International Formula Council.
Young animals and humans
In the United States, CGN has been used for decades as a
stabilizer in infant formula. The CGN builds structure with
the casein proteins by strong binding, provides the desired
mouth-feel, and stabilizes the fat emulsion. The use level of
CGN recommended and used in casein-based infant formu-
lations for stabilization of the finished food is normally within
the range 200–400 ppm (Glicksman, 1969; Moirano, 1977;
Thomas, 1999), see Table 4.
CGN is permitted in infant formula at a level of
0.03 g/100 ml in regular milk and soy-based liquid infant
formula and at a level of 0.1 g/100 ml in hydrolyzed protein or
amino acid-based liquid infant formula (Canada Gazette,
2004). The higher use level in the hydrolyzed protein and
amino acid-based formula is necessary to adequately stabilize
and suspend the constituents, as discussed above. The higher
use rate infant formula is recommended for infants with
special needs, milk allergies, and medical conditions, as
recommended by the American Academy of Pediatrics
(2000). As noted in Table 4, a concentration of 300 ppm
CGN has the consistency of thick milk and 1000–3000 ppm
CGN has the consistency of custard. Thus, the level of CGN
that can be tested in animal infant formula feeding studies is
limited by the viscosity of high levels of CGN, which can
result in undesirable palatability.
Primates
McGill et al. (1977) fed newborn baboons infant formula with
up to 1220 mg k/�-CGN/liter formula (analyzed concentra-
tion) as their only diet for 112 d. There were no effects on
health, urine or blood parameters or any histopathological
effects on the gut tissues. Ingestion of CGN in infant formulas
by primates did not have any adverse health effects to suggest
altered intestinal permeability or immune responses (McGill
et al., 1977). The McGill et al. (1977) study provides data on
primates which are similar to humans with regard to gut
closure, and are, thus, an appropriate animal model for safety
assessment.
Humans
The potential immunosuppressive effects of CGN in human
infants were studied in a retrospective study reported by
Sherry et al. (1993, 1999). In this study, the frequency of
symptomatic upper respiratory tract infection (URI) was
studied in two populations of full-term infants in the first six
months of life: those exclusively fed infant formula
containing 0.03% k/�-CGN (n¼ 1269), and those exclu-
sively fed powdered (0% CGN) infant formula (n¼ 149).
The results of statistical analysis showed no difference
between frequencies of URIs in the two populations. In
addition, a slightly higher proportion of infants fed CGN
were illness-free for the first six months of life when
compared with infants who received powdered formula
(53.4% versus 47.6%). Thus, these results support the
conclusion that CGN-containing infant formulas do not
produce immunosuppression in infants.
In another study, groups of healthy newborn infants (0–9 d
of age) were fed either powdered (PWD) casein hydrolysate-
based formula (CHF) (95 infants) or liquid (RTF) CHF
formula (100 infants) until 112 d of age (Borsches et al.,
2002). Although the type of formula used in this study is
typically used only by special needs infants, for ethical
purposes, only healthy term infants were included in the
study. The study was a masked, randomized, parallel study
with intake and stool patterns and anthropometric measure-
ments monitored at entry and on days 14, 28, 56, 84, and 112
of the study. The liquid RTF CHF formula contained 0.1 g
k/�-CGN per 100 ml product as a stabilizer; whereas the
PWD CHF formula did not contain CGN (Personal
Communication by Ross Laboratories to author, 2007).
Stabilization of the formula with CGN at this use level is
necessary to keep the ingredients in the liquid formula
homogeneous and stable. On all measures, the infants had
similar growth, tolerance to formula, and stools in treated and
untreated groups. The use of CGN in human infants at a level
of 0.1% in formula has been demonstrated to be safe when
administered for up to 112 d (Borsches et al., 2002).
Table 4. Effect of carrageenan levels on food consistency*.
Carrageenan level in food: ppm Consistency of food
300–500 Thick chocolate milk400–800 Milk shake or eggnog700–1000 Sour cream or creamcheese1000–3000 Flan or custard
*References: Glicksman (1969), Moirano (1977), and Thomas (1999).
18 M. L. Weiner Crit Rev Toxicol, Early Online: 1–26
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Epidemiology studies
Tobacman (2001, 2002, 2003) published a hypothesis that
CGN is a cause of breast cancer. These studies are based
entirely on a comparison of gross consumption of CGN in the
United States with national breast cancer incidence rates. Their
analysis assumes an average consumption by all individuals in
the United States, an assumption that is incorrect, and has been
discussed at length regarding a variety of alternative methods
for assessing consumption (Munro & Danielewska-Nikiel,
2006). Evidence over the past decade indicates that fibers of
several kinds do not correlate with breast cancer incidence in
women despite previous claims that increased fiber ingestion
protected against breast cancer (Willett et al., 1992).
Furthermore, Tobacman and her colleagues describe a positive
correlation for several other fibers, including agar, alginate,
gum arabic, and locust bean. Three of these (agar, gum arabic,
and locust bean) have been thoroughly evaluated in carcino-
genicity assays by the National Toxicology Program and found
to be negative (Melnick et al., 1983). In summary, there is no
epidemiologic evidence for a carcinogenic, tumor promoting,
or inflammatory effect of CGN in humans.
Regulation of dietary uses
Summary
Exposure to CGN in the diet has been estimated to be below
the current numeric ADI of 0–75 mg/kg/d (SCF, 1978). The
JECFA ADI is ‘‘not specified’’ (JECFA, 2008). Exposure to
CGN in dietary foods is safe.
Adults
Regulatory background
Various regulatory agencies have completed risk assessments
and safety evaluations of the food additive CGN. The Joint
FAO/WHO Expert Committee on Food Additives (JECFA)
reaffirmed an ADI of ‘‘not specified’’ for CGN (JECFA,
2008). However, as discussed below, the ADI does not apply
to infants below 12 weeks of age (JECFA, 2008).
Other agencies have also completed risk assessments on
CGN. The European Scientific Committee on Foods estab-
lished an ADI for CGN at 0–75 mg/kg body weight/d for food
use (SCF, 1978; European Commission, 2003). CGN is
permitted in follow-on formula for infants older than six
months of age.
In general, the United States FDA permits CGN use in
foods based on the amount necessary to achieve functionality
as an emulsifier, a stabilizer, or a thickener in foods. In 1961,
an amendment to title 21 Code of Federal Regulations Part
121 listing CGN as a permitted emulsifier, stabilizer, and
thickener was published in the Federal Register (FR, 1961,
FDA, 1973) (currently listed as Part 172.620). In 1973, FDA
included ‘‘Chondrus extract (carrageenin)’’ (also known as
CGN) for use as a stabilizer to its list of substances Generally
Recognized As Safe (GRAS ID Code 9000-07-1); 21 CFR
Section 182.7255.
Dietary exposure
Data on dietary per-capita intakes in 1995 derived from
poundage data and per capita intakes in 1995 for Europe and
the USA ranged from 28 to 51 mg/d and 30 to 50 mg/d
(JECFA, 2002). The Seaweed Industry Association of
the Philippines reported similar estimates, based on sales:
44 mg/person per day for the populations of Canada and the
USA and 33 mg/person per day for European populations
(JECFA, 2002).
JECFA (2002) considers estimates derived from poundage
data to be consistent with those derived for the population of
the USA in model diets, with reported mean intakes of CGN
of 20 mg/d for consumers and 40 mg/d for consumers at the
90th percentile (derived by multiplying the mean by a factor
of 2). The intakes were derived from data on the food
consumption of individuals aged 2 years and over, available in
1976 from nutrition surveys in the USA, combined with the
results of a 2-week study by the Marketing Research
Corporation of America.
Shah & Huffman (2003) conducted a survey of the use of
CGN in foods at three large supermarket chains and two
major health food markets in the United States. They surveyed
140 North American food manufacturers to obtain recent and
accurate levels of use of CGN in a variety of food products.
They surveyed subjects from ages 417 to 485 years of age
(133 females and 65 males) in South Florida across a wide age
range and socioeconomic backgrounds to determine the use of
foods containing CGN. The results of this survey found that
the mean total CGN intake with standard deviation was
18.09� 17.96 mg/kg bw/d for males and 30.19� 31.61 mg/kg
bw/d for females, with the highest average intake reported in
people under 30 years of age (33.73� 31.61 mg/kg bw/d). An
analysis of CGN intake by body mass found that the highest
consumption on a body weight basis was found in under-
weight individuals with a body mass index of 519 kg/m2.
These individuals consumed 42.19� 25.34 mg/kg body
weight/d (Shah & Huffman, 2003). The consumption or
intake values reported by Shah & Huffman (2003) are higher
than those reported by JECFA (2002) by a factor of the
average body weight. JECFA (2008) has recommended that a
new dietary exposure evaluation be conducted, employing
specific food type and use level information.
The consumption levels of CGN reported in the literature
for the general population are well below the current ADI
in Europe (0–75 mg/kg bw/d) (European Commission, 2003)
based on the current survey information (JECFA, 2002, 2008;
Shah & Huffman, 2003).
Infants
Regulatory background
In Canada, CGN is approved at a maximum level of 0.05% in
formula as a suspension agent for calcium salts in lactose-free
infant formula, based on milk protein. Infant formula based on
isolated amino acids or protein hyrolysates or both may
contain a CGN level of up to 0.1% (Canada Gazette, 2004).
CGN is also used as a stabilizer in infant formula in the
United States. JECFA specifies that the ADI does not apply to
infants 12 weeks of age and younger: ‘‘As a general principle,
the Committee considers that the ADI is not applicable
to infants under the age of 12 weeks, in the absence of
specific data to demonstrate safety to this age group’’
(JECFA, 2008).
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The Codex Committee on Nutrition and Foods for Special
Dietary Uses (CCNFSDU) initiated a program to review and
revise the Codex Standard 72-1981, the standard for infant
formula. The purpose was to evaluate the nutrient levels in
infant formula. The Committee on Nutrition of the European
Society for Pediatric Gastroenterology, Hematology and
Nutrition (ESPGHAN) undertook the task of coordinating
the evaluation at the request of the CCNFSDU. In its
conclusions, the ESPGHAN recommended that CGN be
excluded from infant formula (Koletzko et al., 2005). The
conclusion was based on ‘‘the lack of adequate information on
possible absorption of CGN by the immature gut in the young
infants and its biological effects in infancy.’’ Koletzko et al.
(2005) mistakenly attribute the reported allergic response to a
barium imaging agent reported by Tarlo et al. (1995) to CGN;
whereas the product in this report contained poligeenan (see
section ‘‘Immune system effects’’). This error was never
corrected, but is indicated in the review by JECFA (2008).
In its review of CGN, JECFA (2008) stated that, ‘‘The
European Commission’s Scientific Committee on Food
concluded that it could not be excluded that CGN might be
absorbed by the immature gut and that absorbed material
might affect the immune system in the infant. No new data
have been published addressing this issue.’’ In addition,
JECFA concluded that based on the information available, it
is not advisable to use CGN or processed Eucheuma seaweed
in infant formulas (JECFA, 2008). JECFA considers infor-
mation on the absorption of CGN from neonates to be lacking
and data on the effects of CGN in neonates to be limited.
JECFA (2008) considered the McGill et al. (1977) study and
thought that it should have included special staining proced-
ures, such as toluidine blue stain, for GI tract mast cells,
which can be mobilized by inflammation. McGill et al. (1977)
fed infant baboons (three males and five females per dose
group) formula from birth to 112 d of age at nominal dose
levels of 0, 300, and 1500 mg/L CGN. The high dose was
equivalent to 1220 mg/L CGN analytically, the highest
feasible level without effects on formula palatability or
about 4–5 times use level (300 mg/L). Although the staining
technology and necessity to include mast cell staining were
not used in this study, nevertheless, the study did include
complete histopathology of the GI tract and found no lesions
related to CGN exposure using standard techniques and stains
(hematoxylin & eosin, periodic acid-Schiff, and Prussian blue
stains) and no other treatment-related effects or indications of
inflammation (McGill et al., 1977). This study, McGill et al.
(1977), provides data to assure the safety of CGN use in infant
formula in primates at a level of CGN equivalent to
432 mg/kg bw/d. In response to the JECFA (2008) review,
the International Formula Council, as well as, FMC
Corporation, initiated a research program on the safety of
CGN, using an appropriate physiologic model of neonatal
pigs (Personal Communication, FMC Corporation). Results of
the research program are anticipated in 2014.
Dietary exposure
The average daily exposure to CGN from liquid infant
formulas was estimated to be 47 mg/kg bw per day for milk-
and soy-based formulas (0.03% CGN) and 160 mg/kg bw per
day for hydrolysed protein- and/or amino acid-based liquid
infant formulas (0.1% CGN) (JECFA, 2008). These exposure
estimates apply to infants fed exclusively on formula. The
table below summarizes estimates of infant exposure to CGN
in formula at two different use levels in very young infants
and to 12-month old infants, as well as a comparison to the
baboons evaluated for toxicity by McGill et al. (1977). These
exposure levels agree with those calculated for infants of
different age groups, based on formula consumption and
body weight values reported in the literature (Weiner, 2007)
(Table 5).
The International Programme on Chemical Safety (IPCS,
1987) provides some guidance for the conduct of studies to
support exposure of infants. IPCS indicates that toxico-
logical studies should include animals in the same period of
life, i.e. after birth during the time of nursing; however, they
state that it is difficult to recommend precise toxicological
testing procedures until more basic research has been
undertaken (IPCS, 1987). Thus, the use of adult animal
studies via drinking water or dietary exposure are not
appropriate studies for margin of exposure and risk assess-
ment calculations for the evaluation of the infant formula
use. IPCS (1987) suggested that an expert panel develop
guidelines for testing food additives in infants and neonates.
A more recent publication by IPCS also acknowledges that
there are gaps in the testing protocols for assessment of
developmental toxicity, including the limited exposure to
neonatal animals (IPCS, 2009). Currently, there are no
guidelines for the conduct of studies for constituents of
infant formula.
The infant baboon infant formula feeding by McGill et al.
(1977) is the best available study for supporting the safety of
Table 5. Carrageenan exposure to infants from the infant formula application.
SpeciesCarrageenan informula: ppm
Carrageenan:mg/kg bw/d Method of calculation Reference
Human 300 47 Assumes 100% formula fed infants JECFA (2008)Human 300 6 Assumes 12-month old infants consuming 13.7% of
caloric intake from formulaJECFA (2008)
Human 300 30.4 Assumes 100% formula fed infants, one to sixmonths old, using reported body weights
Sherry et al. (1993, 1999) andWeiner (2007)
Human 1000 160 Assumes 100% formula fed infants JECFA (2008)Human 1000 22 Assumes 12-month old infants consuming 13.7% of
caloric intake from formulaJECFA (2008)
Baboon 1220 432 100% formula fed from birth until 117 d of age, asdescribed by McGill et al. (1977)
McGill et al. (1977) andJECFA (2008)
20 M. L. Weiner Crit Rev Toxicol, Early Online: 1–26
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CGN in infant formula. It meets several criteria for relevance
to this age group: baboons were dosed by bottle from birth
through 112 d of age. Levels of CGN were 432 mg/kg bw/d
and were at the limit of feasibility. These levels are considered
to be about five-fold higher than those ingested by human
infants. Higher concentrations of CGN in formula would have
resulted in gelling and would have changed the consistency of
the formula in a way that the baby baboons would not drink it
due to palatability differences. The highest dose tested was
well above expected infant exposure. In addition, the infant
baboon study (McGill et al., 1977) provides additional
support for the safety of CGN use in infant formula because
of the similarity of gut closure timing between non-human
primates and humans.
Conclusions
CGN has been used widely in food as an emulsifier, a
stabilizer, and a thickener for more than five decades. Food
grade CGN meets the specifications established by regulatory
authorities. Food grade CGN is a high Mw polymer (200–
800 kDa for commercial product) that is excreted unchanged
in the feces and is not metabolized or significantly absorbed
from the GI tract. CGN is acutely non-toxic by oral, dermal,
and inhalation routes. The only effects reported in animal
subchronic and chronic feeding studies at high dietary
concentrations (5%) of CGN are loose stools and diarrhea,
effects commonly seen with high levels of non-digestible
dietary fibers. Results of animal studies have shown that CGN
is not genotoxic, carcinogenic, a tumor promoter, or a
developmental or reproductive toxicant. Available studies
evaluating immune system responses have generally produced
inconsistent results because animals were dosed to CGN in
drinking water or by oral gavage in which �-CGN exists in a
random conformation, allowing greater contact with the
intestinal mucosa. Nevertheless, based on a large number of
dietary toxicity studies, CGN has not been shown to cause
adverse effects on the immune system. Such studies support
the safety of CGN in food at current regulatory levels. The
JECFA ADI is ‘‘not specified,’’ indicating a high level of
safety. Human exposure from food has been estimated at
approximately 20–40 mg/kg body weight/day or less. JECFA
identified data gaps about CGN when consumed by infants
0–12 weeks of age. These datagaps are being addressed by a
research program by FMC Corporation and the International
Formula Council. Infant exposure to CGN has been shown to
be safe at levels used in infant formula based on the infant
formula feeding study in baboons and studies in human
infants. Together, these studies indicate that no effects are
expected in human infants consuming formula containing
CGN at levels of 0.03–0.10%.
Acknowledgements
The author wishes to thank Ms Eunice Cuirle, Dr James
M. McKim, and Mr William R. Blakemore, F.R.S.C. (retired)
for their generous time in reviewing this manuscript and for
their helpful comments and valuable insights. Special thanks
to Dr Samuel M. Cohen for sharing his insights on tumor
promotion and CGN. Thanks to Ms Muriel Reva for expert
editorial and administrative assistance.
Declaration of interest
The author of this paper is identified on the cover page.
Myra Weiner is the owner and principal in TOXpertise, LLC,
a consulting firm providing advice on toxicological and risk
assessment issues to private firms. The current review was
prepared for FMC Corporation under a cost reimbursable
contract. FMC Corporation is a manufacturer of CGN and
products containing CGN. The review strategy, the review of
the literature, analyses, and conclusions reported in this paper
are the professional work product of the author. The FMC
Corporation was given the opportunity to review the paper
and offer comments on the paper. Those comments did not
alter the professional opinions of the author. The author has
not appeared in any legal proceedings related to the findings
reported in this paper. The conclusions drawn are not
necessarily those of the FMC Corporation.
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Appendix 1
Comparison of carrageenan and poligeenan.
Aspect Carrageenan Poligeenan
CAS Number 9000-07-1 53973-98-1Manufacturing To preserve functionality as a food ingredient, red
seaweed is processed under alkaline conditions(�pH 9)
To degrade the complex polysaccharide into low-molecular weight pieces, acid hydrolysis occursat pH 0.9–1.3 and high temperatures
Average molecular weight range (Mw) 200 000–800 000 Da 10 000–20 000 DaFunction Primarily stabilizer, thickener, gelling agent, with
functionality at concentrations as low as 0.01% insome systems
Dispersing agent; no gelling properties even atconcentrations as high as 10.0%
Uses Food additive/GRAS, pharmaceutical excipient,personal care products
Medical imaging; NO food uses
Toxicological profile: Not genotoxicNot acutely toxicIARC (1983): not carcinogenic in animals IARC (1983): sufficient evidence of carcinogenicity
in animalsNot teratogenic or embryotoxicNot a reproductive toxicantNot harmful to GI tract Ulceration and irritation to GI tract
US FDA food additive or GRAS Yes NoEuropean Food Safety Authority Yes NoCodex Alimentarius Commission Yes No
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Appendix 2
Identification of carrageenan test materials in cited references.*
Reference Carrageenan type Animal species Comments
Section I: acute studiesWeiner et al. (1989) Iota, food grade Rat Acute testsWeiner (1991) Kappa/lambda food grade Rat, rabbit, rat Oral gavage, dermal expos-
ure, inhalationFDRL (1972a) Not reported Rat, mouse, hamster, rabbit Acute oral
Section II: degradation, excretion, absorption, and metabolismHawkins & Yaphe (1965) Kappa/lambda Rat Diet, 27–148 dDewar & Maddy (1970) Iota Rat Diet, 10 dTomarelli et al. (1974) Kappa/lambda Rat Processed heat sterilized/
diet, 6 monthsPittman et al. (1976) Kappa/lambda, Mw 88–276 kDa
IotaIota, or kappa or lambdaKappa/lambda
RatRatGuinea pigGuinea pigMonkey
Diet, 13 weeksGavage, 9 months diet7–10 weeks drinking water2–3 weeks drinking waterGavage, 11 months
Arakawa et al. (1986) Kappa Rat Diet, 24 weeksArakawa et al. (1988) Not reported Rat Diet, 3 weeksTache et al. (2000) Kappa, Sigma Chemical Co. (Sweden,
Switzerland), Mw of 345 000Rat 0.25 and 2.5% gel in
water for 100 dUno et al. (2001a) Lambda, Mw¼�832 kDa Rat Diet, one dayGrasso et al. (1973) Iota Guinea pig; rat Drinking water, 21–45 d;
diet, 3–7 weeksAlbany Medical College (1975) Like kappa, between iota and kappa Rat Diet, one yearNicklin et al. (1988) Iota, food grade; removed polymer
530 kDaRat Gavage, one time; drinking
water, 184 dNicklin & Miller (1984) Kappa, lambda, iota, food grade Rat Drinking water for 90 dElsenhans & Caspary (1989) Not reported Rat Diet, 4 weeksEngster & Abraham (1976) Kappa, Mw 5–145 kDa
Lambda, Mw 20.8–275 kDaIota, Mw 5–145 kDa
Guinea pigGuinea pigGuinea pig
Drinking water, 2 weeksDrinking water, 2 weeksDrinking water, 2 weeks;
diet for 10 weeksAbraham et al. (1972) Kappa/lambda Mw 800 kDa Rhesus monkey Drinking water, 7–11 weeksMankes & Abraham (1975) Kappa, lambda Monkey Drinking water, 10 weeksAlbany Medical College (1983) Kappa/lambda Monkey Diet, 7.5 yearsFriedman & Douglass (1960) Not reported Rat DietRiccardi & Fahrenbach (1965) Not reported Chicken DietReddy et al. (1980) Kappa/lambda Rat Diet, 33 weeksHarmuth-Hoene & Schelenz (1980) Kappa/lambda Rat Diet, 8–147 dKoo et al. (1993) Kappa/lambda Rat Infant formula, single doseKasper et al. (1979) Not reported Human Oral liquid dietMallett et al. (1984) Iota, Sigma Chemical Co. rat Diet, 4 weeksMallett et al. (1985) Iota, kappa, Sigma Chemical Co. rat Diet, 30 dCapron et al. (1996) Kappa/lambda Not applicable In vitroSalyers et al. (1977) Not reported Anaerobic bacteria
Section III: subchronic and long-term toxicity studiesBenitz et al. (1973) Kappa Monkey Drinking water, 11 weeksMankes (1977) Kappa/lambda Rat, mouse Diet, 4 weeksWeiner et al. (2007) Kappa, Mw 196–257 kDa with a mean
of 7%550 kDaRat Diet, 90 d
Bhattacharyya et al. (2012) Lambda-kappa, Sigma Chemical Co. Mouse Drinking water, 18 d, somefor 15 d more
Abraham et al. (1985) Kappa, lambda, iota, Rat Diet, 13–39 weeksRustia et al. (1980) Kappa, food grade Rat, hamster Diet, life time (90–100
weeks)
Section IV: immune system effectsBash & Vago (1980) Kappa/lambda Rat Gavage, once and drinking
water, 8 weeksBash & Cochran (1980) Kappa/lambda In vitroCoste et al. (1989) Iota Rat Gavage, intraperitonealFrossard et al. (2001) Lambda, Sigma Chemical Co. Mouse GavageTsuji et al. (2003) Lambda, Sigma Chemical Co.,
impurities removedMouse Gavage
(continued )
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Continued
Reference Carrageenan type Animal species Comments
Tarlo et al. (1995) Sodium CGN Human Skin prick
Section V: ulcerative effectsDonnelly et al. (2004a) Lambda, Sigma Chemical Co.
not food grade statedMouse Drinking water, 10 weeks
Watanabe et al. (1978) Kappa/lambda Rat Diet, 40 weeksWatt & Marcus (1969) Iota Guinea pig Drinking water, 10–20 dPoulsen (1973) Kappa Pig Oral jelly 83 dMcGill et al. (1977) Kappa/lambda Infant baboon Infant formula, 112 d
Section VI: carcinogenicity studiesMillet et al. (1997) Kappa Rat Oral-water/gel 8 or 100 dCorpet et al. (1997) Kappa, Sigma Rat Oral-water/gel 8 or 100 d
Section VII: genetic effectsFDA (1975) Kappa/lambda In vitroSylianco et al. (1993) Not reported Mouse In vivoFDA (1972a,b) Kappa/lambda Rat In vivo, in vitroMori et al. (1984) Kappa/lambda In vitroJackson (1997) Semi-refined In vitro
Section VIII: tumor promotionTache et al. (2000) Kappa, Sigma Chemical Co.,
heated in steam at 120 �Cfor 20 min in tap water
Rat 0.25% in water or 2.5% gelfor 100 d
Donnelly et al. (2004a, 2005) Lambda, undegraded, SigmaChemical Co. (UK)
Mouse Drinking water at 1% and4% for 10 weeks (2004a)or 20 weeks (2005)
Section IX: reproductive and developmental toxicity studiesCollins et al. (1977a,b) Kappa/lambda food grade Rat Diet at 0.5–5.0% for 12
weeks before mating,through gestation for 3generations
Vorhees et al. (1979) Not reported Rat Diet at 0.45–1.8%, 2 weeksprior to mating throughgestation, lactation,weaning at 21–90 d ofage
FDRL (1972a,b) Kappa/lambda Rat, mouse, rabbit, hamster Gavage during gestationBailey & Morgareidge (1973) Kappa/lambda Rat, hamster Diet, 1% and 5% during
gestationRovasio & Monis, (1980, 1987) Lambda, food grade Chick embryo Inject in yolk sac, 0.1% in
saline
Section X: studies on special age groupsSherry et al. (1993, 1999) Kappa/lambday Babies Infant formula, 6 monthsBorsches et al. (2002) Kappa/lambday Babies Infant formula, 112 d
*References are cited from the first instance of citation and not repeated.yAlthough the seaweed source and carrageenan types are not specified, the infant formula producers know that kappa/lambda type CGN is used (FMC
Corporation, personal communication).
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