Critical Reviews in Toxicology

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/itxc20

Evidence and hypotheses on adverse effects

of the food additives carrageenan (E 407)/
processed Eucheuma seaweed (E 407a) and
carboxymethylcellulose (E 466) on the intestines: a
scoping review

Mirlinda Tahiri, Celine Johnsrud & Inger-Lise Steffensen

To cite this article: Mirlinda Tahiri, Celine Johnsrud & Inger-Lise Steffensen (2023) Evidence and
hypotheses on adverse effects of the food additives carrageenan (E 407)/processed Eucheuma
seaweed (E 407a) and carboxymethylcellulose (E 466) on the intestines: a scoping review,
Critical Reviews in Toxicology, 53:9, 521-571, DOI: 10.1080/10408444.2023.2270574

To link to this article: https://doi.org/10.1080/10408444.2023.2270574

© 2023 Norwegian Institute of Public Health.

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CRITICAL REVIEWS IN TOXICOLOGY
2023, VOL. 53, NO. 9, 521–571
https://doi.org/10.1080/10408444.2023.2270574

REVIEW ARTICLE

Evidence and hypotheses on adverse effects of the food additives carrageenan

(E 407)/processed Eucheuma seaweed (E 407a) and carboxymethylcellulose
(E 466) on the intestines: a scoping review
Mirlinda Tahiri# , Celine Johnsrud# and Inger-Lise Steffensen
Department of Food Safety, Division of Climate and Environmental Health, Norwegian Institute of Public Health, Oslo, Norway

ABSTRACT ARTICLE HISTORY

This scoping review provides an overview of publications reporting adverse effects on the intestines of Received 13 June 2023
the food additives carrageenan (CGN) (E 407)/processed Eucheuma seaweed (PES) (E 407a) and carboxy­ Revised 2 October 2023
methylcellulose (CMC) (E 466). It includes evidence from human, experimental mammal and in vitro Accepted 2 October 2023
research publications, and other evidence. The databases Medline, Embase, Scopus, Web of Science Core
KEYWORDS
Collection, Cochrane Database of Systematic Reviews and Epistemonikos were searched without time lim­ Autoimmune effects; barrier
its, in addition to grey literature. The publications retrieved were screened against predefined criteria. function; carboxymethylcel­
From two literature searches, 2572 records were screened, of which 224 records were included, as well lulose; carrageenan; colitis;
as 38 records from grey literature, making a total of 262 included publications, 196 on CGN and 101 on Crohn’s disease; food
CMC. These publications were coded and analyzed in Eppi-Reviewer and data gaps presented in inter­ additive; inflammation;
active maps. For CGN, five, 69 and 33 research publications on humans, experimental mammals and inflammatory bowel
in vitro experiments were found, further separated as degraded or native (non-degraded) CGN. For CMC, disease; intestinal
three human, 20 animal and 14 in vitro research publications were obtained. The most studied adverse microbiome; irritable bowel
effects on the intestines were for both additives inflammation, the gut microbiome, including fermenta­ syndrome; permeability;
processed eucheuma
tion, intestinal permeability, and cancer and metabolic effects, and immune effects for CGN. Further stud­ seaweed; scoping review;
ies should focus on native CGN, in the form and molecular weight used as food additive. For both ulcerative colitis
additives, randomized controlled trials of sufficient power and with realistic dietary exposure levels of sin­
gle additives, performed in persons of all ages, including potentially vulnerable groups, are needed.

Abbreviation: ACF: aberrant crypt foci; ADI: acceptable daily intake; ADME: absorption, distribution,
metabolism and excretion; AIEC: adherent-invasive E. coli; AIM: Australian Inflammatory Bowel Disease
Microbiome study; AOM: azoxymethane; APC: adenomatous polyposis coli; BCFA: branch chain fatty
acids; Bcl/BCL: B-cell lymphoma/leukemia; bw: body weight; CD: Crohn’s disease; CGN: carrageenan;
CMC: carboxymethylcellulose, DP: degree of polymerization; EC: European Commission; EEN: exclusive
enteral nutrition; EFSA: European Food Safety Authority; EGR-1: early growth response gene 1; ER: Eppi-
Reviewer; EU: European Union; FC: food category; GAG: glycosaminoglycan; HTML: hypertext markup
language; IAP: intestinal alkaline phosphatase; IARC: International Agency for Research on Cancer; IBD:
inflammatory bowel disease; IBS: irritable bowel syndrome; Ig: immunoglobulin; IL: interleukin; IOIBD:
International Organization for the Study of Inflammatory Bowel Disease; JECFA: Joint FAO/WHO Expert
Committee on Food Additives; KCO: j-CGN oligosaccharides; LPS: lipopolysaccharide; MPL: maximum
permitted level; MPO: myeloperoxidase; M-SHIME: mucosal simulator of the human intestinal microbial
ecosystem; MW: molecular weight; NIPH: Norwegian Institute of Public Health; NOAEL: no observed
adverse effect level; PBM: peripheral blood monocytes; PES: processed Eucheuma seaweed; PGN: poli­
geenan; QS: quantum satis; RCT: randomized controlled trial; ROS: reactive oxygen species; SCF:
Scientific Committee on Food; SCFA: short-chain fatty acids; SD: standard deviation; TLR: Toll-like recep­
tor; TNF: tumor necrosis factor; UC: ulcerative colitis

Table of contents
1. Introduction ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 522 1.4. Objectives ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 524
1.1. Regulation of food additives ... ... ... ... ... ... ... ... ... ... 522 2. Methodology ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 524
1.2. Carrageenan (CGN) (E 407)/processed Eucheuma sea­ 2.1. Systematic literature searches ... ... ... ... ... ... ... ... ... 524
weed (PES) (E 407a) ... ... ... ... ... ... ... ... ... ... ... ... ... ... 523 2.2. Grey literature ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 525
1.3. Carboxymethylcellulose (CMC) (E 466) ... ... ... ... ... 524 2.3. Selection of evidence ... ... ... ... ... ... ... ... ... ... ... ... ... 525

CONTACT Inger-Lise Steffensen inger-lisekarin.steffensen@fhi.no Department of Food Safety, Division of Climate and Environmental Health, Norwegian
Institute of Public Health, P.O. Box 222 Skøyen, NO-0213 Oslo, Norway.
#These authors contributed equally to this work.
� 2023 Norwegian Institute of Public Health. Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted
Manuscript in a repository by the author(s) or with their consent.
522 M. TAHIRI ET AL.
2.4. Data extraction and analyses ... ... ... ... ... ... ... ... ... 525
2.5. Presentation of the results ... ... ... ... ... ... ... ... ... ... ... 526
3. Results ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 526
3.1. Number of relevant publications obtained ... ... ... 526
3.2. Publications on carrageenan (CGN) (E 407)/processed
Eucheuma seaweed (PES) (E 407a) ... ... ... ... ... ... ... ... 528
3.2.1. Human studies ... ... ... ... ... ... ... ... ... ... ... ... ... 529
3.2.1.1. Human studies of degraded CGN ... ... ... ... 529
3.2.1.2. Human studies of native CGN ... ... ... ... ... 529
3.2.2. Animal studies ... ... ... ... ... ... ... ... ... ... ... ... ... 530
3.2.2.1. Animal studies of degraded CGN ... ... ... ... 530
3.2.2.2. Animal studies of native CGN ... ... ... ... ... ... 531
3.2.3. In vitro studies ... ... ... ... ... ... ... ... ... ... ... ... ... 534
3.2.3.1. In vitro studies on degraded CGN ... ... ... ... 534
3.2.3.2. In vitro studies on native CGN ... ... ... ... ... 535
3.2.4. Hypotheses on adverse effects of CGN ... ... 537
3.2.5. Data relevant for exposure ... ... ... ... ... ... ... ... 537
3.2.6. Absorption, distribution, metabolism and excre­
tion (ADME) studies ... ... ... ... ... ... ... ... ... ... ... ... 539
3.2.7. Risk assessment opinions ... ... ... ... ... ... ... ... ... 539
3.2.8. Non-systematic (narrative) reviews, commenta­
ries/editorials/letters and conference abstracts 539
3.3. Publications on carboxymethylcellulose (CMC) (E
466) ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 541
3.3.1. Human studies ... ... ... ... ... ... ... ... ... ... ... ... ... 541
3.3.2. Animal studies ... ... ... ... ... ... ... ... ... ... ... ... ... 542
3.3.3. In vitro studies ... ... ... ... ... ... ... ... ... ... ... ... ... 543
3.3.4. Hypotheses on adverse effects of CMC ... ... 545
3.3.5. Data relevant for exposure ... ... ... ... ... ... ... ... 545
3.3.6. Toxicokinetics and ADME studies ... ... ... ... ... 545
3.3.7. Risk assessment opinions ... ... ... ... ... ... ... ... ... 545
3.3.8. Non-systematic (narrative) reviews, commenta­
ries/editorials/letters and conference abstracts 546
3.4. Evidence presented as interactive research maps 548
4. Discussion ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 548
4.1. Methodological aspects ... ... ... ... ... ... ... ... ... ... ... ... 548
4.2. Number of various categories of publications on CGN
and CMC ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 548
4.3. Main findings on effects of CGN relevant for human
intestinal health ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 549
4.3.1. Degraded versus non-degraded (native)
CGN ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 550
4.4. Main findings on effects of CMC relevant for human
intestinal health ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 551
4.5. Strengths and limitations of this scoping review ... 552
4.6. Evidence gaps and implications of the study findings
for further research ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 552
5. Conclusions ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
6. Recommendations for further work on adverse effects
of CGN and CMC ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
Acknowledgments ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
Declaration of interest ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
Supplemental material ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
ORCID ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
References ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 553
Appendices ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 561
Appendix A. Preferred Reporting Items for Systematic
reviews and Meta-Analyses extension for Scoping
Reviews (PRISMA-ScR) Checklist filled in for this scoping
review ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 561
Appendix B. First literature search ... ... ... ... ... ... ... ... ... ... 563
Appendix C. Repeated literature search ... ... ... ... ... ... ... 565
Appendic D. Publications excluded at full-text level with
reasons ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 568
Appendix E. Eppi-Reviewer Code book ... ... ... ... ... ... ... 571
1. Introduction
Despite the existing regulations aimed to secure the safety of
additives in our food, among the general public there are per­
sons who worries about health risks from intake of food addi­
tives. The topic of potential adverse effects of food additives
also appears intermittently in the media. In this scoping review,
literature published on adverse effects on the intestines of two
commonly used food additives, carrageenan (CGN) and carbox­
ymethylcellulose (CMC), have been compiled and presented, in
order to get an overview of this issue.
1.1. Regulation of food additives
According to Regulation (EC) No 1333/2008 (2008b), a food
additive is defined as “any substance not normally consumed
as a food in itself and not normally used as a characteristic
ingredient of food, whether or not it has nutritive value, the
intentional addition of which to food for a technological pur­
pose in the manufacture, processing, preparation, treatment,
packaging, transport or storage of such food results, or may
be reasonably expected to result, in it or its by-products
becoming directly or indirectly a component of such
foods.” Only food additives included in the Community list in
Annex II of this regulation may be placed on the market as
such and used in foods under the conditions of use specified
therein, listed in the food categories (FC) to which they may
be added.
At present, 406 food additives are present in the
European Union (EU) database of food additives (Regulation
(EC) No 1333/2008 2008a). All food additives are identified by
an E number and food additives are included in the ingredi­
ent lists of foods in which they are used. Product labels must
identify both the function of the additive in the finished food
and the specific substance used by referring either to its E
number or name. Food additives are grouped into 27 func­
tional groups, of which those that are used to maintain prod­
uct consistency comprise a heterogeneous group of more
than 80 food additives. They include emulsifiers, which make
it possible to mix ingredients that are not naturally miscible,
for instance water and fat in mayonnaise, gelling agents,
thickeners and stabilizers.
In Europe, food additives on the market are authorized by
the EU in accordance with Annexes II and III to Regulation
 CRITICAL REVIEWS IN TOXICOLOGY 523

(EC) No 1333/2008 (2008b) on food additives, after risk

assessments by the European Food Safety Authority (EFSA).
At the international level, the Joint FAO/WHO Expert
Committee on Food Additives (JECFA) is responsible for eval­
uating the safety of food additives. Thus, food additives
should not, on the basis of the scientific evidence available,
pose a safety concern to the health of the consumer at the
level of use proposed.

1.2. Carrageenan (CGN) (E 407)/processed Eucheuma

seaweed (PES) (E 407a)
One controversial food additive is carrageenan (CGN) (E 407),
which has been used as thickener, gelling agent, stabilizer,
emulsifier and glazing agent (EFSA 2018a). It is obtained
from seaweeds of the families Gigartinaceae, Solieriaceae,
Hypneaceae and Furcellariaceae of the class Rhodophyceae
(red seaweeds), by extraction with water or dilute aqueous
alkali (Commission Regulation (EU) No. 231/2012 (2012); EFSA
2018a). Over time, the sources for the production of CGN have
changed as well as some of the algae names. As summarized
by EFSA (2018a), the industrial sources of CGN are mainly the
species Eucheuma cottonii, E. spinosum, Chondrus crispus, C.
ocellatus, Hypnea musciformis and several Gigartina, Iridaea and
Furcellaria species, which are harvested either in nature or pro­
duced by seaweed farming.
The closely related food additive processed Eucheuma sea­
weed (PES) (E 407a) has structural similarities with CGN (E 407)
(EFSA 2018a). The industrial sources of E 407a are strains of the
red seaweeds E. cottonii and E. spinosum, from which the addi­
tive is obtained by aqueous alkaline treatment at high tempera­
ture (Commission Regulation (EU) No. 231/2012 (2012); EFSA
2018a). Processed Eucheuma seaweed (E 407a) is distinguished
from CGN (E 407) by its higher content of cellulosic matter
(15%) and because the manufacturing does not include solubil­
isation and precipitation during processing (EFSA 2018a). The
terms carrageenan (CGN) and processed Eucheuma seaweed
(PES) are restricted in use only for the non-hydrolysed or other­
wise chemically degraded polymers.
US FDA (2023a) has listed that CGN and its ammonium,
calcium, potassium or sodium salts may be safely used
when it is the refined hydrocolloid prepared by aqueous
extraction from the following species of the families
Gigartinaceae and Solieriaceae of the class Rodophyceae:
Chondrus crispus, C. ocellatus, Eucheuma cottonii, E. spino­
sum, Gigartina acicularis, G. pistillata, G. radula and G. stel­
lata, and if it is a sulfated polysaccharide with dominant
hexose units of which are galactose and anhydrogalactose
with 20–40% sulfate content on a dry-weight basis.
Furthermore, they inform that the food additive is used or
intended for use in the amount necessary for an emulsifier,
stabilizer or thickener in foods, except for those standar­
dized foods that do not provide for such use.
The chemical name of CGN (E 407) is sulfate esters of poly­
galactose (sulfated polygalactans), and CGN consists mainly
of the potassium, sodium, magnesium and calcium sulfate
esters of galactose and 3,6-anhydrogalactose polysaccharides
(Commission Regulation (EU) No. 231/2012 (2012); EFSA
Figure 1. Structural formulas of j-, i- and k-CGN. The names j-, i- and k-CGN
do not reflect definitive chemical structures but only general differences in the
composition and degree of sulfation at specific locations in the polymer (EFSA
2018a, reused with permission from EFSA).
2018a). These hexoses are alternately linked a-1,3 and b-1,4 in
the copolymer. The three major prevalent polysaccharides in
CGN are designated k-, i-, k-CGN (Figure 1) depending on the
number and position of sulfate groups on the disaccharide
repeating unit; k-CGN with one sulfate group at C4 of the b-D-
galactose, i-CGN with one sulfate group at C4 of the b-D-galactose
and one sulfate group at C2 of the a-3,6-anhydro-D-galactose, and
k-CGN with one sulfate group at C2 of the b-D-galactose and
two sulfate groups at C2 and C6 of the a-D-galactose (Marburger
2003; Dr€ager et al. 2011; EFSA 2018a). In the processed
Eucheuma seaweed (E 407a), the main polysaccharide is j-CGN
when produced from E. cottonii and primarily i-CGN when pro­
duced from E. spinosum (EFSA 2018a). The CAS number for
native CGN (E 407) is 9000-07-1, for j-CGN 11114-20-8, for i-CGN
9062-07-1 and for k-CGN 9064-57-7 (EFSA 2018a). Degraded
CGN, often called poligeenan (PGN), has CAS number 53973-98-
1. No CAS number had been assigned to processed Eucheuma
seaweed (E 407a).
The molecular weight (MW) of CGN depends on the
extraction method used (Weiner 1991). Industrial food grade
(native) CGN is extracted with water and has average MW of
200–400 kDa. The MW is not specified for E 407 and E 407a
in the EU regulation, however, according to industry, E 407 is
defined as having a weight-average MW of 200–800 kDa
(EFSA 2018a). The low MW CGN (MW fraction below 50 kDa)
should be <5% of the total composition of commercial products
E 407 and E 407a (Commission Regulation (EU) No. 231/2012
524 M. TAHIRI ET AL.
(2012)). Degraded CGN, such as PGN, C16 and Ebimar, are not
authorized as food additives in EU. PGN is prepared from i-CGN
by acid hydrolysis at low pH (0.9–1.3) and high temperature
(>80� C) for several hours and has a MW of 10–20 kDa (EFSA
2018a). C16 is artificially formed from i-CGN by acid hydrolysis
with 0.1 M sulfuric acid at 60 � C for 1.5 h (EFSA 2018a) with MW
of about 20 kDa (Benitz et al. 1973). Ebimar is a commercial
degraded CGN, mostly k-CGN, with MW of about 20 kDa (Evans
et al. 1965).
Maximum permitted levels (MPL) of CGN (E 407) and PES (E
407a) have been defined in Annex II of Regulation (EC) No
1333/2008 (2008b) with amendments. They are authorized
food additives in the EU at quantum satis (QS) levels in many
foods, or in some foods with specific MPLs. In food safety regu­
lations in the EU, QS is a catch-all restriction for food ingre­
dients, especially food additives, which are harmless enough to
have no specific quantity restriction. E 407 is used for example
in ice cream, salad dressing, jam and marmalade, fruit or vege­
table spreads, cheese, sauces and processed meats, and E 407a
is used in meat preparation (EFSA 2018a). The group accept­
able daily intake (ADI) for CGN and PES is 75 mg/kg body
weight (bw) per day (EFSA 2018a).
1.3. Carboxymethylcellulose (CMC) (E 466)
Another controversial food additive is carboxymethylcellulose
(CMC) (E 466), also called sodium carboxy methyl cellulose or
cellulose gum, used as emulsifier, stabilizer, binder, thickener
and gelling agent in foods. It is the partial sodium salt of a
carboxymethyl ether of cellulose (Figure 2), where the cellu­
lose is obtained from natural strains of fibrous plant material
(Commission Regulation (EU) No. 231/2012 (2012)). It has CAS
no. 9004-32-4. E 466 is authorized as a food additive in the
EU at QS in most food categories (EFSA 2018b). Typical foods
and beverages that contain CMC are for instance ice cream,
salad dressings, cheese, bakery wares and various processed
foods. A numerical ADI value for the non-modified and modi­
fied celluloses, including E 466, was found not to be needed
because of no safety concern at the reported uses and use
levels (EFSA 2018b).
US FDA (2023b) has defined sodium carboxymethylcellulose
as the sodium salt of CMC not less than 99.5% on a dry-weight
basis, with maximum substitution of 0.95 carboxymethyl
groups per anhydroglucose unit and with a minimum viscosity
of 25 centipoises for 2% by weight aqueous solution at 25 � C.
Figure 2. The chemical structure of CMC, in which R ¼ H or CH2COOH (EFSA
2020, reused with permission from EFSA).
This substance is generally recognized as safe (GRAS) when
used in accordance with good manufacturing practice.
1.4. Objectives
In this work, an extensive scoping review on adverse effects on the
intestines of the food additives CGN (E 407)/PES (E 407a) and CMC
(E 466) has been conducted. The study population was the general
healthy population of both sexes and all age groups, as well as
patients with intestinal diseases. Regarding concept and context,
the adverse effects were limited to three main hypotheses: inflam­
mation, including effects on barrier dysfunction or permeability of
the intestines, autoimmune effects or effects on the intestinal
microbiome, without further limitations in geography, publication
year or other factors. The data on adverse health effects were
obtained from the three usual streams of such information; human
studies, experimental animal studies and in vitro studies. The objec­
tives of this scoping review were to obtain an overview of the
available evidence and to identify knowledge gaps for further
research on and risk assessments of these food additives.
2. Methodology
As part of planning of this scoping review, a protocol was
made but not published, however, all the same information
is included in this section. This scoping review was guided by
the framework for scoping reviews originally proposed by
Arksey and O’Malley (2005) with later enhancements and
updates of the methodology (Levac et al. 2010; Daudt et al.
2013; Munn et al. 2018; Peters et al. 2021a, 2021b; Page et al.
2021a, 2021b). In addition, the latest version of guidance and
checklist specific for scoping reviews (Tricco et al. 2018)
together with guidance for systematic reviews (Page et al.
2021a, 2021b) were consulted (Appendix A, PRISMA-ScR
checklist filled in for this scoping reviews).
2.1. Systematic literature searches
First, a pilot search was performed in December 2021 in the
database Medline (OVID) to get a quick overview of the topic
of interest (not shown). Further, an extensive peer-reviewed
literature search in several databases (Appendix B, First litera­
ture search) was performed in order to obtain publications
on the three lines of evidence; human studies, experimental
animal studies and in vitro studies.
The text words contained in the titles and abstracts of relevant
articles in the pilot search and the MeSH and other index terms
describing the articles were used to develop a full search strategy
in collaboration with the library staff at the Norwegian Institute of
Public Health (NIPH). The search strategy, including all identified
key words and index terms, was adapted for each of the included
six databases, Medline (OVID), Embase (OVID), Scopus (Elsevier
B.V.), Web of Science Core Collection [SCI-EXPANDED & SSCI]
(Clarivate), Cochrane Database of Systematic Reviews [CDSR,
CENTRAL] (Wiley) and Epistemonikos (Epistemonikos foundation)
by a research librarian, before peer-review by a second librarian.
The searches were run on 31.01.22, except for the Cochrane
Database, which was run on 28.01.22. No restrictions were
included in the searches related to publication date to obtain as

much information as possible. Also, no restrictions were used for
type of publications in order to be as comprehensive as possible.
Reviews, both systematic and narrative, were included.
Conference abstracts were included, representing new data, pos­
sibly not yet published as full-text articles. Additionally, com­
mentaries, editorial articles, letters to the editor etc. were
included to get an understanding of the reasons for the contro­
versy around these food additives. However, in this review more
emphasis was put on the original research publications (human,
experimental animals and in vitro studies). The searches were
limited to the languages English, Norwegian, Danish or Swedish.
Duplicates were removed in EndNote v. 20 (Clarivate, AZ, USA),
before the search outcome was uploaded in the Eppi-Reviewer
(ER) software (version 4.13.0.2) (Evidence for Policy and Practice
Information and Co-ordinating Centre (EPPI-Centre), Institute of
Education, University of London, UK) for further analyses.
A repeated literature search in the same databases under the
same conditions as the first search was performed by the NIPH
library to obtain additional newer publications on December 7
and 8, 2022 (Appendix C, Repeated literature search).
2.2. Grey literature
Relevant risk assessment reports were searched for in October
2022 at the web sites of EFSA, JECFA, National Institutes of
Health (NIH, USA), European Medicines Agency (EMA), the
Federal Institute for Risk Assessment (BfR, Germany), French
National Institute for Industrial Environment and Risks (INERIS),
French Agency for Food, Environmental and Occupational
Health and Safety (ANSES), National Institute for Public Health
and the Environment (RIVM, the Netherlands) and the EU
Commission Joint Research Centre (JRC).
Master and doctoral theses were searched for in PROSPERO
(International prospective register of systematic reviews),
Cristin (Current Research Information System in Norway), Oria
(a search engine for search in the Norwegian academic libra­
ries’ resources) and System for Information on Grey Literature
in Europe (SIGLE) in November-December 2022.
Grey literature was also searched in December 2022 in
Google Scholar using the search terms (carrageenan OR carbox­
ymethylcellulose) AND intestin�. However, since this search
returned approximately 19000 and 18500 publications, respect­
ively, many of which were already obtained by the library data­
base searches, were books not available unless purchased or
were irrelevant to the scope of this work, only the first 100 hits
for each food additive were scrutinized for inclusion. Many of
the publications were found in several sources, i.e. in Google
Scholar and in references list of already included publications.
2.3. Selection of evidence
The obtained publications were screened on two levels using
predefined inclusion and exclusion criteria (Table 1). Titles
and abstracts were screened independently by two reviewers
for assessment against the inclusion criteria in ER. Thereafter,
publications that full-filled the inclusion criteria were read in
full-text by two independent reviewers. Conflicts between
CRITICAL REVIEWS IN TOXICOLOGY 525
the two reviewers were resolved by discussions or by a third
reviewer, on both stages of the selection process.
The inclusion criteria comprised adverse effects on the intes­
tines of the food additives CGN (E 407)/PES (E 407a) and CMC
(E 466). From the pilot search, three main hypotheses on
adverse effects on the intestines of these two food additives
appeared to be prevalent and search terms to cover these
were therefore included in the main search strategy: 1) effects
on barrier dysfunction or permeability of the intestines, often
including inflammation, 2) autoimmune effects or 3) effects on
the intestinal microbiome. Studies mainly exploring positive
health effects of varying substances or treatments were
screened when they included a group only given CGN or CMC.
Studies on adverse effects on the general healthy human popu­
lation of both sexes and all age groups without any geograph­
ical limitations, or in experimental mammals, were included.
Publications which studied diseases of the intestinal tract, such
as inflammatory bowel disease (IBD) or irritable bowel syn­
drome (IBS) in patients or animal models, were included. IBD is
an immune-mediated, chronic inflammatory disease that affects
the gastrointestinal tract and is a common term for Crohn’s dis­
ease (CD) and ulcerative colitis (UC) (Park et al. 2020).
The results of the literature searches and the study inclusion/
exclusion process are presented in the PRISMA-ScR flow dia­
gram (Figure 3), including the reasons for exclusion at the full-
text level (see also Appendix D, Publications excluded at
full-text level with reasons). The pdfs of the included full-text
publications were obtained directly from the internet or ordered
from the NIPH library and uploaded into ER. No authors were
contacted for additional information about their publications.
2.4. Data extraction and analyses
The categories of information in the studies relevant to full-
fill the study objectives were extracted from the included
full-text publications and sorted into standardized categories,
called codes in ER, using frequency and cross-table functions
in this software. The codes are presented in Appendix E,
Eppi-Reviewer Code book. Two authors categorized each
publication independently of each other. Disagreements were
resolved by discussion.
The data included, i.e. the codes, were the food additive,
CGN (E 407)/PES (E 407a) or CMC (E 466). The food additives
were further coded in sub-categories, for CGN; j-, ί- or
k-CGN, unspecified CGN, degraded CGN (including PGN, C16,
Ebimar), modified CGN, and for CMC; non-modified CMC or
modified CMC (Appendix E, Eppi-Reviewer Code book). This
definition of modified CMC is used in a more general sense
and is not consistent with the group “chemically modified
celluloses” to which E 466 belongs, as defined by EFSA
(2018b). Other codes were publication type (human, animal,
in vitro or other types of studies, conference abstracts),
human study design (systematic review, randomized
controlled trial (RCT), non-randomized controlled study, pro­
spective/retrospective cohort study, case-control study, cross-
sectional study, case report, incidence/prevalence study),
experimental animal study (species), in vitro studies (human
or animal cells or microbiota, or other in vitro studies), other
526 M. TAHIRI ET AL.
Table 1. Inclusion and exclusion criteria used for selection of publications obtained from the literature searches.
Inclusion criteria
All forms of the food additives Carragenan (E 407)/Processed Eucheuma
seaweed (E 407a) or Carboxymethylcellulose (CMC) (E 466)
Human studies (all study designs),Experimental animal studies (all mammalian
species), In vitro studies, Other studies
Healthy general population, of both sexes and all age groups, Patients with
intestinal diseases (Inflammatory bowel disease (IBD), Irritable bowel
syndrome (IBS),Other gastrointestinal diseases)
Oral administration of the food additives to humans or animals
Hypotheses on adverse effects on the intestines; barrier dysfunction or
permeability of the intestines including inflammation, Autoimmune effects,
Effects on the intestinal microbiome, Other hypotheses
All years of publication
types of studies (toxicokinetics, non-systematic reviews, com­
mentaries/editorials/letters, studies with data relevant for
exposure and risk assessments), human study population
(general healthy population (children, adolescents, adults)) or
patients (with IBD, IBS, other diseases)) and publication year.
Lastly, formulated hypotheses, suggested mechanisms,
explanations for an effect or similar statements in the publi­
cations for adverse effects on the intestines were noted for
each publication. They were first grouped in three main
hypotheses (effects on barrier dysfunction or permeability of
the intestines, including inflammation, autoimmune effects,
effects on the intestinal microbiome), but were later divided
in more groups since other hypotheses were also mentioned
in the included publications (as shown in Figures 8 and 11).
Several publications contained more than one type of data,
such as from both experimental animal and in vitro studies.
For a few publications in addition to the conference
abstracts, the coding was based only on the title, abstract
and content list (for books), since full-text was not available.
2.5. Presentation of the results
This scoping review presents the various categories of infor­
mation from the included publications relevant for the
research questions in text, tables and figures. The figures
were made in Excel from the data extracted into various cat­
egories by using frequency and cross-table functions in ER. In
addition, two interactive research maps were made with
EPPI-Mapper included in the ER software. These maps were
made by exporting the data extracted in ER as a JSON data
file (https://www.json.org/json-en.html).
3. Results
3.1. Number of relevant publications obtained
From the first main literature search, 2318 publications were
obtained from all six databases, which after removal of dupli­
cates in EndNote was reduced to 1165. After import into ER, two
additional duplicates were observed, reducing the number of
publications obtained to 1151 that were screened at the level of
title and abstract according to the inclusion and exclusion criteria
(Table 1). These publications were handled in the ER software as
Exclusion criteria
Other food additives
All other animals than mammals
Patients with diseases outside the gastrointestinal tract
Non-oral administration, for instance by injection or intrarectally
Adverse effects on other organs than the intestines, or only positive effects
shown in Figure 3. Of these, 253 publications were considered
potentially relevant and were assessed in the full-text version
according to the same inclusion and exclusion criteria. In the fol­
low-up second literature search, 254 publications were initially
obtained, which after removal of duplicates in EndNote and ER
gave 107 publications, increasing the total to 1258 publications
that were screened at the level of title and abstract. From the
publications obtained from the second literature search, 29 were
further screened at the full-text level, increasing the total number
at this step to 282 publications. After full-text screening, 210 and
14 publications from the first and second literature search,
respectively, in total 224, were retained. Thirdly, from the exam­
ination of title/abstract and full-text in the same step of grey lit­
erature from many sources, six risk assessments from EFSA, one
risk assessment from the International Agency for Research on
Cancer (IARC), four publications from NIH and 27 publications
from Google Scholar, in total 38, were included after examination
(Figure 3). From all this literature, in total 262 publications were
finally included in this scoping review and were coded in ER for
data extraction. Of these, 196 were on CGN and 101 on CMC.
The distribution of all types of publications per five-year-
periods is shown for CGN and CMC in Figure 4, showing that
research interest in CGN has been ongoing since 1961,
whereas CMC publications are mostly from the last decade,
when also CGN interest has increased substantially.
The reasons for exclusion at full-text level of 59 publications
are listed in Appendix D. Publications excluded at full-text level
with reasons. Studies that only looked for positive health
effects of these food additives were outside the scope of this
review, or did not have a control group to compare the food
additives with, were excluded. During the screening process, it
became apparent that in many studies, CGN was injected in
animal paws or lung tissues to induce inflammation in order to
test natural substances or drugs that could reduce or eliminate
inflammation. Although this non-oral administration route was
not relevant for food additives, it was apparent from these
studies that CGN could induce inflammation in the skin or
lungs in various animal models. In other publications, the food
additives were administered intrarectally. Thus, these studies
using non-oral administration routes were outside our inclusion
criteria and the publications were excluded. Another large
group that was excluded were studies where CMC was used as
an adhesive under operative procedures in the intestines or
 CRITICAL REVIEWS IN TOXICOLOGY 527

Figure 3. PRISMA flow chart of literature identification, selection and inclusion for the scoping review, guided by Tricco et al. (2018), Page et al. (2021a, 2021b).

other organs, or was used as a component of new delivery sys­
tems for drugs. Additional publications that were excluded for
not meeting the inclusion criteria studied effects of these food
additives on other parts of the body than the intestines or
examined effects of other treatments, substances or bacteria
together with CGN or CMC, or were in nano form.
In the following, the results will be described for each
food additive separately, and for the various categories of
publications separately if not stated otherwise; original
research publications (humans, experimental animals,
in vitro), toxicokinetics and absorption, distribution, metabol­
ism and excretion (ADME) publications, non-systematic
528 M. TAHIRI ET AL.
Figure 4. The number of all types of publications studying CGN or CMC per 5-year-periods.
(narrative) reviews, commentaries/editorials/letters, confer­
ence abstracts, data relevant for exposure (from all types of
publications) and risk assessment opinions. For all codes,
there is often overlap between alternatives in the categories,
since many publications report more than one type of food
additive, type of publication, such as both animal and in vitro
studies, more than one hypothesis for adverse effects and so
on. When stated in the publications, the MW of the CGN
types was included in the text.
3.2. Publications on carrageenan (CGN) (E 407)/
processed Eucheuma seaweed (PES) (E 407a)
Since most of the publications on CGN from the literature
searches did not specify the EU food additive numbers (E 407
or E 407a), the publications have not been divided accord­
ingly, but described together. Although the main objective of
this scoping review was to elucidate the potential adverse
effects of the food additive E 407/E 407a, containing native,
non-degraded CGN, it become apparent from an early look at
the literature that even with native, non-degraded CGN there
were numerous publications reporting adverse effects, and
therefore, both categories, degraded and native CGN, were
included for comparison in this review. Data were obtained on
the three most prevalent polysaccharides of CGN (j-CGN, ί-
CGN and k-CGN), in addition to degraded CGN (sometimes
called poligeenan (PGN), C16 or Ebimar) and CGN modified in
various ways. In the following text, “degraded” is native writ­
ten when it is clear from the publication that a degraded CGN
(with lower MW than CGN) has been used. If “degraded” is
not stated in the text and there are no further specifications
to indicate that CGN was degraded, the results are assumed
to be from native CGN (i.e. not degraded). Studies in which
the type of native CGN used were not reported were catego­
rized as unspecified CGN. Overlap among these categories was
often the case in the publications.
No time limits were used for the literature searches to
obtain as many as possible of the relevant publications. The
number of research publications studying degraded CGN
showed a peak around 1971–1995 and a higher peak around
2006–2023 (Figure 5). The first period represents publications
using CGN to induce inflammation in experimental animal
models, in which various anti-inflammatory substances and
treatments could be tested. Publications on native CGN are
also present from those early years but a higher number is
published more recently, around 2011–2023.
The number of research publications studying the various
types of CGN are shown in Figure 6. Somewhat more publi­
cations were using j- or k-CGN than i-CGN. A lot of the pub­
lications stated that they used CGN, without specifying what
form of CGN they studied. An even higher number of publi­
cations was found for degraded CGN, sometimes studied
together with native CGN in the same publications. No
research publications used modified CGN. However, in a
review, it was described that red seaweed CGNs may be
chemically modified with one or more substitutions in the
same molecule in different ratios, resulting in new types of
CGNs called “hybrid CGNs” (Cian et al. 2015). In addition, an
abstract described investigations on refined food grade
j-CGN with 10% Naþ or 10% Ca2þ, which affect the gel for­
mation of CGN (Farag et al. 2018).
The distribution of various types of publications on CGN
(original research publications on human studies, experimen­
tal animal studies and in vitro studies, as well as some other
types of studies) is shown in Figure 7. Only five studies
reported research on humans (excluding publications on
exposure), in patients with intestinal diseases. The highest
number of publications was on various species of experimen­
tal animals (69), with in vitro studies as the second largest
category (33), studying microbiota from humans or animals
ex vivo-in vitro or describing mechanistic data in human or
animal primary cells or cell lines. Twenty publications
included some data on exposure or intake of CGN or its pres­
ence in foods, three publications gave information on ADME
and two risk assessment opinions of CGN were available. The
main results of the original research publications on humans,
animals and in vitro are described briefly in the following.

Figure 5. The number of research publications studying degraded or native CGN per 5-year-periods.
Figure 6. The number of research publications studying the various types of CGN as specified in the publications. Modified CGN was only mentioned in one review
publication and one abstract.
3.2.1. Human studies
Only five publications reported human studies on adverse
effects of CGN on the intestines, two using degraded CGN
and three using native CGN, all in adult patients. No publica­
tions were found on adverse effects of degraded or native
CGN on healthy adults, or on children or adolescents.
3.2.1.1. Human studies of degraded CGN. Two older studies
on adult patients without control groups were included using
degraded CGN. In the first study, 40 patients of both genders
with gastrointestinal ulcerations were given a degraded
crude extract of CGN, mostly k-CGN (Ebimar, MW of about
20 kDa) (Evans et al. 1965). One patient had mild diarrhea
and three complained of constipation. One patient needed
surgery for continuing severe pain and vomiting after two
months. In the second study, six patients who suffered from
malignant disease of the colon were given degraded CGN
(Grasso et al. 1973). No signs of any ulceration in the samples
CRITICAL REVIEWS IN TOXICOLOGY 529
of normal gut were seen and degraded CGN was not
detected in the colon by histochemistry or other methods.
3.2.1.2. Human studies of native CGN. One relevant small
randomized double-blind, placebo-controlled, multicenter,
clinical trial was found (Bhattacharyya et al. 2017). They exam­
ined whether patients of both sex (>18 years) diagnosed with
UC in remission would have a longer interval to relapse if they
followed a diet without CGN vs. after oral exposure to equal
parts of 100–200 mg food grade j-, i- and k-CGN in gelatin
capsules. The study continued until relapse or one year of par­
ticipation. Of the 12 patients completing the study, three of
five who received CGN relapsed, whereas none of the seven
patients receiving placebo relapsed (p ¼ 0.046). In the CGN-
exposed group only, interleukin (IL)-6 and fecal calprotectin
levels were increased. This clinical trial was included in the
only systematic review found, on dietary interventions for
induction and maintenance of remission in IBD by Limketkai
530 M. TAHIRI ET AL.
Figure 7. The distribution of various types of publications on CGN (degraded and native). The human studies (without studies of exposure), experimental animal
studies and in vitro studies are original research publications. Exposure studies comprise all types of publications that contain information on levels in food, expos­
ure etc., including from review publications and conference abstracts. In addition, publications on CGN containing information on ADME and risk assessments were
included.
et al. (2019). These authors calculated a risk ratio with 95% con­
fidence interval (CI) of 0.5 (0.15, 1.64) for the above-mentioned
clinical trial and it was given very low score for certainty of the
evidence (GRADE) and a high risk of bias.
In an older clinical study without control group, k-CGN pow­
der was given as treatment to 22 patients with peptic ulcers (17
men and 5 women) (Heineken 1961). The only adverse side-
effect reported from CGN was one case of dizziness.
3.2.2. Animal studies
Among the included publications reporting adverse effects on
the intestines of CGN, in total 69 were experimental animal
studies. The numbers of publications on each animal species
studied were rats (25), mice (18), guinea pigs (20), rabbits (7),
hamsters (3), ferrets (1), pigs (2) and monkeys (3). Among the
animal studies, a large proportion of the studies (23) was per­
formed with unspecified native CGN, whereas the various types
of CGN were more similarly represented, with 25, 15 and 19
publications for j-, i- and k-CGN, respectively. The number of
publications on experimental animals that had used degraded
CGN was 38, whereas a somewhat lower number (31) had
studied native (non-degraded CGN). Five publications studied
both degraded and native CGN in animals (Watt and Marcus
1969; Benitz et al. 1973; Grasso et al. 1973; Mankes and
Abraham 1975; Pintauro and Gilbert 1990).
3.2.2.1. Animal studies of degraded CGN. Nine publications
reported studies on rats using degraded CGN. The endpoints
studied were ulceration and mucosal injuries (Grasso et al.
1973; Benard et al. 2010), diarrhea and uptake in tissues
(Grasso et al. 1975), role of intestinal bacterial flora (Hirono
et al. 1981), intestinal permeability (Delahunty et al. 1987)
and colitis, followed by squamous metaplasia and finally
adenomas, adenocarcinomas, squamous cell papillomas and
squamous cell carcinomas in the colorectum, as well as
presence of CGN in macrophages in the lamina propria
mucosa and submucosa of the colorectum (Ishioka et al.
1987).
In a model for IBD, rats were sensitized by a s.c. injection
of degraded k-CGN and Freund’s complete adjuvant followed
by oral administration of the same CGN solution in drinking
water, where the degraded k-CGN caused significant small
intestinal injury shown by ulceration, abnormal villous pat­
tern, inflammation, microgranulomas and crypt abscesses,
resembling human IBD (Moyana and Lalonde 1990; Moyana
et al. 1994). Prior sensitization aggravated the effects of CGN.
Further, a study suggested that oxygen free radicals from
intestinal macrophages played a role in this intestinal injury
(Moyana and Lalonde 1991).
Three publications reported studies on mice using degraded
CGN, inducing bloody diarrhea, pericryptal inflammation and
marked dilatation of the caecum and ascending colon (Fath
et al. 1984). Combined treatment of the low MW (�4.5 kDa)
j-CGN oligosaccharides (KCO) with KCO-degrading bacteria
(Bacteroides xylanisolvens and E. coli) led to greater pro-inflam­
matory effects in the colon and rectum of mice than either KCO
or bacteria alone (Yin et al. 2021). Of several strains of mice,
DBA/2J was the most sensitive to colitis induced by degraded
k-CGN (MW 30 kDa) (Hata et al. 2006). However, b-catenin-accu­
mulated crypts, putative precancerous lesions in colon, were
not increased after initiation with azoxymethane (AOM), indicat­
ing lack of a promotor effect of degraded k-CGN in mice.
All the 20 publications reporting studies on guinea pigs
were from 2005 or earlier, and used degraded CGN, of which
the three following publications also studied native CGN. In
both guinea pigs given 1% undegraded CGN or 5% degraded
CGN in drinking water for 3–4 weeks, ulcerative lesions in the
caecum and colon, and fecal occult blood, were observed,
with more extensive damage and higher incidence of ulcer­
ation in the guinea pigs given degraded CGN (Watt and
Marcus 1969). Both 5% solutions of degraded and native

CGN in drinking water produced ulceration in the caecum
and colon of guinea pigs, with more serious effects in those
given degraded CGN, such as severe diarrhea, blood in the
feces, submucosal oedema, haemorrhage and ulceration
(Grasso et al. 1973). Guinea pigs were administered 0.2%
undegraded j-CGN, 0.2% undegraded i-CGN, 1% degraded
j-CGN or 1% degraded i-CGN in the drinking water to inves­
tigate drug-metabolizing enzyme activities relevant for cancer
promotion (Pintauro and Gilbert 1990). The undegraded
j-CGN and undegraded i-CGN increased small intestinal cyto­
chrome P-450 levels and benzo[a]pyrene hydroxylase activ­
ities, whereas the degraded j-CGN and i-CGN did not.
Most of the remaining publications studying guinea pigs
also reported effects of degraded CGN (0.5–5% in drinking
water) for from two-three days up to six weeks on colitis and
ulcerations in the intestines, sometimes with occult blood in
the feces, diarrhea or decreased body weight (Watt and
Marcus 1971; Van der Waaij et al. 1974; Grasso et al. 1975;
Boxenbaum and Dairman 1977; Onderdonk et al. 1977; Olsen
and Poulsen 1980; Onderdonk et al. 1981; Jensen et al. 1984;
Onderdonk et al. 1984; Langman et al. 1985; Marcus et al. 1989;
Kitsukawa et al. 1992 (using MW 30 kDa); Marcus et al. 1992;
Fujita and Sakurai 1995; Xiong et al. 2005). The inflammatory
response was often associated with infiltration of macrophages
or mononuclear phagocytes in the intestinal mucosa.
The majority of the studies did not specify the form of
degraded CGN used. However, in guinea pigs given either
degraded j-, i- or k-CGN in drinking water for three weeks, the
order of potency was i- > k- > j-CGN for decreasing effect on
body weight gain (Norris et al. 1981). When the effects on col­
itis of degraded Eucheuma spinosum (almost entirely i- and nu-
CGN) and degraded Eucheuma cottonii (almost entirely j- and
mu-CGN) given in drinking water to guinea pigs were com­
pared, degraded E. spinosum CGN induced mild to moderate
colitis, while E. cottonii consistently induced severe colitis in
colon and rectum (Langman et al. 1985).
Some publications on guinea pigs also studied the influence
of intestinal microflora. UC was significantly reduced by elimin­
ation of the family of Enterobacteriaceae (Van der Waaij et al.
1974). There were shifts in the major microbiota populations
during development of the ulcerative lesions in guinea pigs
(Onderdonk et al. 1977). Two pools of feces each containing 10
bacterial strains were obtained from conventional guinea pigs
administered a solution of degraded CGN, which caused ulcer­
ations in their ceca and large intestines (Onderdonk et al.
1981). When these samples were given by orogastric intub­
ation to germfree guinea pigs all these animals also developed
such ulcerations, whereas control animals did not. Further
studies showed that certain strains of Bacteroides vulgatus
were capable of inducing immune enhancement of UC
induced by degraded CGN (Onderdonk et al. 1984). Immune
animals which received B. vulgatus from a patient with inflam­
matory bowel disease, but not from a clinically normal person,
had more intestinal lesions than non-immune animals.
Guinea pigs treated with degraded i-CGN excreted more
PEG-900 than controls, suggesting increased intestinal perme­
ability (Delahunty et al. 1987).
All the seven publications reporting studies on rabbits
were from 1996 or earlier. Most of the publications studied
CRITICAL REVIEWS IN TOXICOLOGY 531
inflammation and ulceration caused by degraded CGN (0.3–
5%) in drinking water or by gastric intubation for 4–9 weeks
or even up to 7 months (Grasso et al. 1973; Aoki 1978;
Al-Suhail et al. 1984a; Al-Suhail et al. 1984b; Kitano et al.
1996 (MW 30 kDa)). Ulcerations resembled those seen in
guinea pigs and in patients with UC, and CGN was present
within macrophages. A focal, high-grade dysplasia was seen
in the colonic mucosa of rabbits sensitized to degraded CGN
by an injection and exposed to degraded CGN in drinking
water for 28 months (Kitano et al. 1986). A similar rabbit
experiment with k-CGN (30–33 kDa) suggested impairment of
the immunoglobulin (Ig)A-regulated local immune response
and an abnormality in the differentiation of immunoglobulin-
secreting cells (Matsumoto et al. 1988).
One publication on hamsters given degraded CGN in the
diet for six months and ferrets given degraded CGN by gas­
tric intubation for 28 days did not observe ulceration and
other adverse changes that were observed in guinea pigs
and rabbits (Grasso et al. 1973).
One publication on pigs administered degraded CGN (20–
30 kDa) in the drinking water found no effects on stool consist­
ency or levels of IL-1b, IL-8, IL-6, IL-10 or tumor necrosis factor-
(TNF)-a (Munyaka et al. 2016). However, CGN decreased
bacterial species richness (a-diversity) and shifted community
composition. At the phylum level, an increase in Proteobacteria
and Deferribacteres, and a decrease in Firmicutes,
Actinobacteria, Bacteroidetes and Tenericutes, were observed.
One publication on two squirrel monkeys given 1.5%
degraded CGN by gastric intubation for 28 days revealed no
ulceration and other adverse changes (Grasso et al. 1973). In
another publication, Rhesus monkeys (Macaca mulatta) were
administered degraded CGN (C16, MW 20 kDa) or native,
largely j-CGN, in drinking water for 7–14 weeks (Benitz et al.
1973). The monkeys given 0.5 (n ¼ 2) or 1% (n ¼ 2) C16 solution
gained weight, but in those on 2% C16 (about 2.9 g/kg bw per
day, n ¼ 6) weight loss was considerable, and they lost blood
from the intestinal tract and developed anaemia. Pathological
changes in the colon ranged from shallow mucosal erosions to
multiple crypt abscesses. The severity of these effects was
dose-dependent, but some reversal was indicated when
allowed to recover for 20–24 weeks. The six monkeys given 1%
native CGN (about l–3 g/kg bw per day) all gained weight and
remained in good condition. Thus, a marked difference was
observed between the effects of degraded (C16) and native
CGN in monkeys. Another study on Rhesus monkeys compared
effects of C16, 2% degraded i-CGN (MW ca. 20 kDa), adminis­
tered to six monkeys in drinking water for 10 weeks, and 1%
undegraded j- and k-CGN mixture (MW ca. 800 kDa) adminis­
tered to six monkeys in drinking water for 10 weeks (Mankes
and Abraham 1975). Degraded CGN, but not native CGN, was
detected in lysosomes in submucosal macrophages, altering
the functional state of the lysosomes. Necrosis and leucocyte
infiltration were also observed in the macrophages.
3.2.2.2. Animal studies of native CGN. Seventeen publica­
tions reported studies on rats using native CGN. No ulcer­
ation or other adverse changes were observed in rats given
5% native CGN in the diet for 56 days, as found also for
532 M. TAHIRI ET AL.
degraded CGN (Grasso et al. 1973). In rats given 50 g/kg of
i-CGN for 50 days, increased weight of the cecal contents and
decreased cecal bacterial population, as well as decreased
rate of reduction of several nitro-containing compounds,
were observed (Rowland et al. 1983). In a model of IBD, rats
given k-CGN in drinking water gradually developed intestinal
lesions, which were morphologically similar to those in
human UC, and the proliferative response of splenocytes to
known mitogens was significantly diminished (Pricolo et al.
1996). In rats which orally consumed 1% j-CGN-containing
PES (407a) for four months, inflammation developed in the
small intestine, which was associated with a strong overex­
pression of the DNA repair enzyme O-6-methylguanine-DNA
methyltransferase (MGMT) in enterocytes and in stroma,
mainly on the surface of villi (Tkachenko et al. 2018b). In the
same model, the CGN-induced inflammation was accompa­
nied by higher levels of TNF-a, IL-1b and heat shock protein-
90a upregulation in the intestinal mucosa (Tkachenko et al.
2020). In rats exposed to PES in drinking water (140 mg/kg
bw per day) for two weeks, altered small and large intestinal
morphology, infiltration of lamina propria in the small intes­
tine with macrophages, higher systemic levels of inflamma­
tion markers (C-reactive protein and middle molecules),
changes in the lipid order of the phospholipid bilayer in the
cell membranes of leukocytes, as well as activation of apop­
tosis in these cells, were demonstrated (Pogozhykh et al.
2021). In the same rat model, low-grade inflammation, as
indicated by increased T lymphocytes and macrophages in
the colonic lamina propria, was induced (Onishchenko et al.
2022). Among the endothelial-mesenchymal transition (EMT)
markers, fascin and vimentin were overexpressed in both
stromal and epithelial cells, while E-cadherin was upregulated
in the stroma and downregulated in epithelia.
In rats administered CGN in drinking water for four weeks,
CGN reduced the protective properties of mucin, accelerated
death of epithelial cells and led to interstitial inflammation in
the small intestine, together with decreased activity of poly
(ADP-ribose) polymerase (PARP), increased activity of apop­
tosis signal-regulating kinase 1 (ASK-1) and high percentage
of DNA fragmentation in the small intestine (Gubina-Vakyulyk
et al. 2015). Increased death of epithelial cells was accompa­
nied by activation of proliferation, but not sufficient for com­
plete regeneration of the epithelial cover of villi. Similarly
treated rats with gastroenterocolitis showed activation of
apoptosis in the intestinal epithelium, with changes in the
polar regions of the enterocyte membranes, but not in more
hydrophobic regions (Tkachenko et al. 2018a).
In rats given 5, 20 or 50 g i-CGN per kg diet (0.5, 2 and 5%)
for 30 days, all doses caused similar cecal enlargement and
decrease in concentration of bacteria per gram of cecal con­
tent (Mallett et al. 1985). Rats fed the highest dose showed a
significant decrease in enterobacteria, staphylococci, strepto­
cocci and total facultative anaerobes as well as in lactobacilli
and the total microscopic count. Proportions of Enterobacteria
and lactobacilli were relatively increased, while the proportion
of staphylococci was decreased. There were no differences in
degree of cecal enlargement and decrease in cecal bacterial
numbers and concentrations between j-, i- or k-CGN.
Intestinal permeability was determined indirectly by orally
administration to rats of a poorly absorbed dye, phenol red, and
measuring its recovery in feces and urine (Shiau and Chang
1986). CGN type I (80% j-CGN, 20% k-CGN) or CGN type II
(i-CGN) were fed as 5 and 15% of the diet for 31 days. Both CGN
increased permeability dose-dependently, with some differen­
ces, with adaptation apparently taken place at four weeks. The
urinary excretion 96 h after administration in drinking water of
two polyethylene glycol markers was measured in rats after
feeding CGN type II (20%) for 4 weeks (Elsenhans and Caspary
1989). The ratio of PEG 4000 to PEG 900 in the urine increased
after CGN feeding, showing that CGN could influence the intes­
tinal permeability of larger molecules.
The promoter effect of native CGN (Viscarin 402) upon colon
carcinogenesis induced by AOM or methylnitrosourea (MNU)
was studied in rats (Watanabe et al. 1978). CGN enhanced the
incidence of colonic tumors in the AOM- and MNU-treated rats.
No tumors were found with control diet, but one of 15 rats
(7%) had a colonic adenoma after CGN diet without carcino­
gen. To study carcinogenic effects of native CGN (Gelcarin,
largely composed of j-components and also k-CGN (MW ca.
800 kDa), doses of 0.5, 2.5 or 5% (average daily intake 360,
1998 and 4022 mg/kg, respectively) were given in the diet to
rats for life (Rustia et al. 1980). A trend toward increased inci­
dence of benign mammary tumors in females and testicular
neoplasms in males occurred at the 2.5% CGN dose, however,
no statistically significant increases in tumor incidences were
seen. The tumors of the gastrointestinal tracts of the rats were
one leiomyoma of the intestine and three squamous cell papil­
lomas of the forestomach with the middle CGN dose. In con­
clusion, native CGN had no carcinogenic effects on rats
throughout their life spans. The initiating and promoting
effects of native j-CGN (MW 345 kDa, type I) were studied in
conventional rats and germ-free rats gavaged with human
Bacteroides sp. and human fecal samples from healthy children
given CGN-containing desserts (Tach�e et al. 2000). The initiat­
ing effect of j-CGN was studied by scoring preneoplastic aber­
rant crypt foci (ACF) in the colon of rats given j-CGN (10% gel
and 0.25% liquid in drinking water) for eight days. j-CGN alone
did not initiate ACF. The promoting effect of j-CGN was
studied by comparing the multiplicity of ACF (number of aber­
rant crypts/focus) in rats receiving drinking water (controls),
liquid j-CGN (0.25% in water) or j-CGN gel (2.5% in water) for
100 days, after tumor initiation with AOM. In conventional rats,
2.5% j-CGN gel promoted growth of ACF, whereas 0.25%
liquid j-CGN did not. In rats with human microflora, no pro­
moting effect of 2.5% j-CGN gel was observed. This indicated
that rat microflora, but not human gut microflora, might be
involved in colonic tumor promotion by j-CGN.
The effects of i-CGN were studied in rats given diets sup­
plemented with 5% of the seaweed Sarconema filiforme for
eight weeks (du Preez et al. 2020). The rats had lower,
although not significant, food intake and body weight with S.
filiforme. The S. filiforme supplementation also modulated gut
microbiota without changing the Firmicutes to Bacteroidetes
ratio. In rats fed a high-fat diet and injected with streptozoto­
cin to induce diabetes, supplementation with 270 mg/kg
unspecified CGN did not affect glycemic control-related
parameters: fasting blood glucose, glycosylated serum

proteins, oral glucose tolerance test, insulin and the homeo­
stasis model of assessment of insulin resistance (HOMA-IR))
(Nie et al. 2021). However, CGN increased serum glucagon-
like peptide (GLP)-1, decreased leptin and increased some
unsaturated fatty acids in diabetic rats.
Fifteen publications reported studies on mice using native
CGN. Mice given 5, 20 or 50 g of i-CGN per kg diet for 30 days
showed cecal enlargement and decreased concentration of
bacteria per gram of cecal content (Mallett et al. 1985). Two %
CGN in drinking water for 4 weeks induced a marked diffuse
inflammatory response in the colon, associated with infiltration
of leucocytes and macrophages, and a two-fold increase in
prostaglandins in mice (Zijlstra et al. 1992).
Both systemic and intestinal inflammatory responses of
undegraded j-, k-CGN in drinking water (total CGN intake ca.
11.5 mg/30 g mouse) for 30 days were examined in B-cell lymph­
oma/leukemia (Bcl10) wild-type, heterozygous and null mice
(Bhattacharyya et al. 2013). Adverse effects in caecum and intes­
tines were seen in the wild-type mouse, but not in Bcl10 null
mice, consistent with reduced inflammation in the absence of
Bcl10. Fecal calprotectin and circulating keratinocyte chemo­
kine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-jB-induc­
ing kinase (NIK) and phospho(Ser36)-IjBa in the colonic
epithelial cells were significantly lower in the CGN-treated Bcl10
null mice than in controls. IL10-deficient mice exposed to CGN
in a germ-free environment showed an increase in activation of
the canonical pathway of NF-jB (RelA) activation, but without
increase in RelB or phospho-Bcl10. In mice given undegraded
CGN in drinking water for four weeks (Bhattacharyya et al.
2014b), CGN decreased arylsulfatase B activity in colonic epithe­
lial cells, with subsequent effects on C4S, galectin-3, Sp1 and
Wnt9A, which may have significant effects on Wnt-initiated sig­
naling and related cellular processes.
Mice were treated with j-CGN in doses of 1.7, 8.3 and
41.7 mg/kg bw by for 14 days prior to the administration of
2,4,6-trinitrobenzene sulfonic acid (TNBS) to test effects of
CGN on TNBS-induced inflammation (Wu et al. 2016a). j-CGN
pretreatment increased body weight loss, mortality rate and
colonic inflammation, associated with oxidative stress and
activation of the Toll-like receptor (TLR)4-NF-jB and MAPK/
ERK1/2 pathway. In a similar study, j-CGN administration
prior to induction of colitis by oxazolone showed increased
body weight loss, decreased survival and aggravated colonic
inflammation (Wu et al. 2016b). Secretion of IL-4, IL-10, TNF-a
and IL-6 also increased after j-CGN exposure, associated with
an activation of the TLR4-NF-jB pathway, a decreased ratio
of regulatory T cells and the induction of Th2-dependent
immune responses.
In another inflammation model, mice given j-CGN (1.7, 8.3
and 41.7 mg/kg diet, average MW 1000 kDa) and then later
were inoculated with Citrobacter freundii bacteria, the dose-
dependent decreases in body weight and survival were higher
with CGN than without, indicating that CGN aggravated the
bacteria-induced gut inflammation (Wu et al. 2017; Wu et al.
2021). Mice with high dietary k-CGN consumption showed
altered colonic microbiota composition that resulted in deg­
radation of the colonic mucus layer, increased fecal LPS level
and decreased short-chain fatty acids (SCFA), affecting integrity
of the intestinal barrier. These effects could be reproduced in
CRITICAL REVIEWS IN TOXICOLOGY 533
germ-free mice by fecal transplantation from mice given the
high k-CGN dose, but not in germ-free mice fed the high
k-CGN dose. A similar study with j-CGN (average MW 198 kDa)
in mice did not cause significant inflammatory symptoms, but
it reduced SCFA and decreased thickness of the mucus layer by
altering microbiota composition (Wu et al. 2022).
Administration of the pathogenic bacterium Citrobacter roden­
tium further aggravated the inflammation and mucosal dam­
age in the presence of j-CGN. Mucus layer degradation and
altered SCFA levels could be reproduced by fecal transplant­
ation from j-CGN-fed mice, but not from germ-free j-CGN-fed
mice. The results suggested that j-CGN may not be directly
inflammatory but creates an environment that favors inflam­
mation by perturbation of gut microbiota composition and
metabolism, and facilitates expansion of pathogens.
The diet appears to modulate the effects of CGN. In mice
exposed to 0.02% j-CGN together with dairy cream, no
adverse outcomes were seen on lipid metabolism, intestinal
permeability or liver damage, but a higher expression of
endoplasmic reticulum (ER) stress genes in the colon was
observed (Milard et al. 2018). j-CGN (average MW
640.80 ± 11.53 kDa) reduced colon length and induced more
serious deepening of the crypts in mice on a high-sucrose
diet (Gao et al. 2022). j-CGN on a high-sucrose/high-salt diet
induced more serious reduction in goblet cells and increased
permeability of the intestines, and reduced anti-inflammatory
bacteria and increased harmful bacteria, associated with
decrease of anti-inflammatory colonic metabolites.
CGN may affect metabolic parameters. Mi et al. (2020)
found that a 45% fat diet with 0.5% j-CGN (average MW
365 kDa) and normal drinking water increased the general
disease activity index (DAI), myeloperoxidase (MPO) activity, a
marker for infiltration of inflammatory cells, and mRNA
expression of TLR4 in colon of mice, whereas a 45% fat diet
with 5% j-CGN, but no j-CGN in drinking water, had no
such effects. No signs of colitis were observed on a 10% fat
diet regardless of the mode of vehicle used for j-CGN (diet
or drinking water), except that 5% j-CGN in 10% fat diet and
normal drinking water increased the DAI. Moreover, the
j-CGN-induced colitis on high-fat diet was correlated with
increases of the harmful bacteria Alistipes finegoldii and
Bacteroides acidifaciens, whereas the anti-inflammatory bac­
terium Akkermansia muciniphila was increased in the group
given 10% fat diet with 0.5% j-CGN in normal drinking
water. Hence, the inflammatory property of CGN is influenced
greatly by its intake form via modification of host intestinal
microecology. In a further study, in obese mice given 5%
j-CGN for 6 weeks, genes involved in the inflammatory path­
ways or tight junction protein encoding were not signifi­
cantly dysregulated by j-CGN (Zhang et al. 2021). However,
j-CGN decreased expression of genes for adipocytokines,
lipogenesis, lipid absorption and transport, and increased
expression of genes for of adipolysis and oxidation. The
food-grade j-CGN was not absorbed or significantly
degraded in the digestive tract of the obese mice. In another
study on mice fed a 60% fat diet, 0.2% or 1% j-CGN signifi­
cantly reduced body weight gain, fat mass, adipocyte size,
fasting blood glucose and blood lipids (Wang et al. 2021).
j-CGN improved high-fat diet-induced obesity by the AMPK
534 M. TAHIRI ET AL.
and PPARc signaling pathway. The level of TNF-a and LPS
in the low dose CGN group, but not in the high dose
group, was significantly lower than that in the high-fat diet
group. The abundance of Firmicutes, Proteobacteria,
Lachnospiraceae and Desulfovibrionaceae was positively cor­
related with body weight, fasting blood glucose, serum low
density lipoprotein cholesterol (LDL-c), total cholesterol (TC)
and triglyceride levels, whereas Bacteroidetes were negatively
correlated with LDL-c and TC levels, while positively with
fasting blood glucose.
Comparable activities of colitis were induced by j-, i- and
k-CGN given in drinking water for six weeks to mice (Shang
et al. 2017). All three forms of CGN decreased the anti-inflamma­
tory bacterium Akkermansia muciniphila in the gut microbiota.
Three publications on guinea pigs compared native and
undegraded CGN (Watt and Marcus 1969; Grasso et al. 1973;
Pintauro and Gilbert 1990) and are described above in
Section 3.2.2.1. There were no publications studying native
CGN in rabbits or in ferrets.
There were two publications on hamsters using native
CGN. Hamsters given 5, 20 or 50 g of i-CGN per kg diet for
30 days showed cecal enlargement and decreased concentra­
tion of bacteria per gram of cecal content (Mallett et al.
1985). To study carcinogenic effects of native CGN (Gelcarin),
largely composed of j- and also k-CGN (MW ca. 800 000),
doses of 0.5, 2.5 or 5% in the diet (average daily intake 370,
2162 and 3719 mg/kg, respectively) were given to hamsters
for life (Rustia et al. 1980). The tumors of the gastrointestinal
tracts of the hamsters were one squamous cell papilloma in
each treated group, which were not significantly different
from controls. Thus, native CGN had no carcinogenic effects
on hamsters exposed throughout their life spans.
One publication studied pigs administered native CGN
(average MW 200 kDa) orally in doses of 50, 200 and 500 mg/
kg bw per day for 12 weeks (Poulsen 1973). No effects of
CGN were seen on behavior, body weight gain, feed utiliza­
tion, haematology, blood chemistry, urine analysis or organ
weights. No UC or erosions of the mucous membrane of
caecum and colon were observed. However, CGN decreased
the total counts of aerobic bacteria in the colon and rectum,
and the number of Lactobacilli in the rectum.
Two publications compared native and degraded CGN in
Rhesus monkeys (Benitz et al. 1973; Mankes and Abraham
1975) and are described above in Section 3.2.2.1.
3.2.3. In vitro studies
In total, 33 publications with in vitro experiments reported
adverse effects on the intestines of CGN. Among the in vitro
studies, 8 studies were performed with unspecified native
CGN, whereas there were 19, 10 and 16 publications with j-,
i- and k-CGN, respectively. The numbers of publications on
in vitro studies that had used degraded CGN were 14,
whereas 26 had studied native (non-degraded CGN). Seven
publications studied both degraded and native CGN in vitro.
Six publications were studying microbiota from human
feces (three on degraded, three on native CGN), whereas the
majority (21) of the publications reported studies on human
primary cells or cell lines (three on degraded, 12 on native,
seven on both types of CGN), some also including non-
human mammalian cells. Four publications studied mamma­
lian fecal microbiota (two on degraded, two on native CGN)
and eight publications used mammalian primary cells or cell
lines (three on degraded, five on native CGN).
3.2.3.1. In vitro studies on degraded CGN. Seven in vitro
studies were using degraded CGN only. In addition, seven
publications included data on both degraded and native
CGN, described in this and/or the section on native CGN.
When fermentation of CGN, i.e. breakdown of carbohy­
drates by human fecal microbiota that cannot be digested by
the body’s enzymes, was screened in an in vitro batch fermen­
tation system, seven of eight human colonic microbiota fecal
samples showed that high MW j-CGN (450 or 100 kDa)
remained undegraded, whereas low MW j-CGN, i.e. j-CGN oli­
gosaccharides (KCO, �4.5 kDa), were degraded (Yin et al.
2021). Sulfate groups were not removed during degradation.
The concentrations of propionic and butyric acids were signifi­
cantly increased, and pH decreased, after KCO fermentation.
Bacteroides xylanisolvens and Escherichia coli isolates from the
fecal samples appeared to degrade KCO synergistically, with B.
xylanisolvens being the primary degrading bacteria. Studies of
in vitro fermentation by human gut microbiota of two j-CGN
oligosaccharides (MW <3000 kDa with different degree of
polymerization (DP)) obtained from simulated gastric diges­
tion, called KO3 and KO6, showed that both were readily
degraded (Sun et al. 2019). Both promoted pro-inflammatory
bacteria Prevotella, while inhibited anti-inflammatory bacteria
Bacteroides and Parabacteroides. The KO3 with larger DP
improved SCFA production and strongly increased the growth
of Bifidobacterium and Lactobacillius, whereas KO6 with smaller
DP reduced SCFA production and strongly increased the abun­
dance of mucin-producing bacteria Prevotellaceae. Another
study also showed that j-CGN oligosaccharides were degraded
by Bacteroides xylanisolvens and E. coli isolated from human
gut microbiota (Li et al. 2017).
Human peripheral blood monocytes (PBM) and human THP-
1 monocytic cells were cultured in the presence of either 10 or
40 kDa degraded CGN prepared from native i-CGN in vitro
(Benard et al. 2010). Degraded CGN inhibited THP-1 cell prolif­
eration and arrested the cells in G1 phase. Degraded CGN also
increased Intercellular Adhesion Molecule 1 (ICAM-1) expres­
sion in both PBM and THP-1 cells, with the strongest effect
seen with the 40 kDa degraded CGN, and stimulated monocyte
aggregation. Degraded CGN also stimulated expression and
secretion of TNF-a in both cell types. All these effects involved
NF-kB activation and indicated that degraded CGN led to an
inflammatory phenotype of the monocytes.
Associated with inflammatory effect on human colon
cancer HT29 cells, supernatants (50, 100 and 200 ml/ml) from
fermentation of j-CGN oligosaccharides KO3 and KO6
promoted secretions of IL-1b, TNF-a, secretory IgA and
mucin2 in a dose-dependent way, indicating that smaller
j-CGN oligosaccharides might have stronger influence on
inflammation (Sun et al. 2019).
Native k-CGN activated the Wnt/b-catenin signaling path­
way, shown by increased expression of Wnt9A, suppressed

Dickkopf 3 and RHOU genes, and increased accumulation of
b-catenin, and suppressed the expression and secretion of
bone morphogenetic protein-4 (BMP4) in human NCM460
cells (Bhattacharyya et al. 2007). This suggested a role of
k-CGN in malignant transformation in the human intestine.
Native and degraded j-CGN had similar reducing effect on
BMP4 and both had lower effect on BMP4 reduction than
native k-CGN.
In NCM460 cells and primary human colonic epithelial
cells that were exposed to low levels (1–10 mg/L) of unde­
graded high MW CGN for 1–8 days, increased cell death,
reduced cell proliferation and cell cycle arrest were reported
(Bhattacharyya et al. 2008a). After 6–8 days of CGN exposure,
the percentage of cells reentering G0–G1 decreased and the
percentages of cells in S and G2-M phases increased.
Increases in activated p53, p21 and p15 followed CGN expos­
ure, consistent with CGN-induced cell cycle arrest. However,
the antiproliferative response, as decline in BrdU incorpor­
ation, occurred after exposure to both undegraded j-, i- and
k-CGN as well as degraded j-CGN. No evidence of apoptosis
was seen, but the data was consistent with necrosis.
Shorter, degraded j-CGNs induced higher IL-8 levels than
longer disaccharides (Bhattacharyya et al. 2010c). Hydrolysis
of different forms of high MW CGN (j-, i-, k-CGN) by the
enzyme a-1!(3,6)-galactosidase reduced increases in IL-8
and BCL10 in NCM460 cells, but a-1!6-galactosidase,
b-1!4-galactosidase and b-1!3,6-galactosidase had no
effect. In contrast, specific j-CGNases or i-CGNases, which
hydrolyze b-1,4-galactosidic bonds, increased IL-8 and BCL10,
because of increased exposure of the immunogenic a-1!3-
galactosidic epitope of CGN to TLR4. Thus, the CGN-induced
innate immune response in NCM460 cells was modified by
enzymes that hydrolyze distinct galactosidic bonds.
Food grade j-, i- and k-CGN were mixed with milk (whey),
soy or egg protein isolates, and tested in a semi-dynamic
in vitro digestion model, which showed varying levels of dis­
turbance of gastric digestive proteolysis and a significant
decrease in pepsin activity (Fahoum et al. 2017). Further, in
human Caco-2 cells, samples of physiologically digested CGN
affected the epithelial barrier function, including redistribution
of the tight-junction protein Zo-1, changes in cellular F-actin
architecture and increased monolayer permeability of macro­
molecules. Moreover, CGN also increased the pro-inflammatory
IL-8 receptor CXC Motif Chemokine Receptor 1 (CXCR1).
Degraded k-CGN with MW of 10–40 kDa induced inflam­
mation via both NF-jB and activation protein (AP)-1, and
increased the inflammatory effects of LPS though AP-1 activa­
tion in the human THP-1-derived macrophage cell line and
the murine RAW264.7 macrophage cell line (Chen et al.
2014). Degraded k-CGN was more effective than native
k-CGN in the activation of macrophages to secrete TNF-a.
Degraded and undegraded CGN of several varieties were
compared in several human cell lines (Ariffin et al. 2014).
Food grade j-CGN (FGKC), dried sheet j-CGN (DKC), commer­
cial grade j-CGN (CGKC), food grade i-CGN (FGIC) and com­
mercial grade i-CGN (CGIC) were dissolved in hydrochloric
acid and water to prepare degraded and undegraded CGN,
respectively, with concentration range of 62.5–2000.0 lg/ml.
FGKC and DKC were PES (E 407a). Degraded FGKC, DKC and
CRITICAL REVIEWS IN TOXICOLOGY 535
CGKC reached the concentration inhibiting 50% of cell viabil­
ity (IC50) in 24, 48 and 72 h in Caco-2 (human epithelial colo­
rectal adenocarcinoma), FHs 74 Int (normal human small
intestinal), HepG2 (human hepatocellular carcinoma) and
Fa2N-4 (immortalized human hepatocyte) cell lines. Degraded
FGIC and CGIC were only toxic in Fa2N-4 cells. Apoptosis was
demonstrated in degraded j-CGN treated Caco-2, FHs 74 Int,
HepG2 and Fa2N-4 cells. Chromatin condensation and
nuclear fragmentation were seen in Caco-2 and HepG2 cell,
whereas DNA ladder was only found in HepG2 cells.
Degraded j-CGN also inactivated PCNA, Ki-67 and survivin
genes in HepG2. On the other hand, cells treated with unde­
graded FGKC, DKC, CGKC, FGIC and CGIC showed no cyto­
toxic effect after the same analyses as with degraded CGN.
PGN (MW <20 kDa) at concentrations of 1 and 10 mg/ml
showed a 3.5- and 7-fold induction of IL-8 in human HepG2
cells in the absence of serum protein, but no induction of IL-
8 was observed when serum protein was present (McKim
et al. 2016).
Partially hydrolysed (degraded) CGN (up to 1.5 mg/ml)
retarded growth and caused cell death in the rat ileum epi­
thelial cell line IEC18, as well as inhibited DNA synthesis, dis­
rupted cellular junctions and subsequently caused cell
membrane injury (up to 5 mg/ml) (Ling et al. 1988).
Pools of feces containing ten bacterial strains obtained
from conventional guinea pigs administered 5% degraded
CGN caused cytotoxic responses in Vero cells (African green
monkey kidney cells) and WI-38 cells (human lung fibro­
blasts), increased presence of long-chain fatty acids (LCFA) in
the cell culture supernatants and increased chemotactic activ­
ity (Onderdonk et al. 1981).
j-CGN oligosaccharides (KCO) (MW 1-7 kDa) were obtained
by enzymatic hydrolysis (Han et al. 2019). Culture medium was
supplemented with KCO and fermented by pig fecal microbiota
in vitro. KCO increased the concentration of a total of seven
SCFAs (only butyric acid significantly). KCO had no obvious
effects on fecal microbiota structure after the fermentation.
However, the bacterial populations at the phylum level showed
a mild increase in the abundance of Bacteroidetes in KCO cul­
ture and a total of 50 taxa displayed significant differences vs.
controls. KCO increased the abundance of butyric acid produc­
ing bacteria, such as Coprococcus_1, Ruminococcaceae and
Roseburia, and opportunistic pathogenic bacteria, including
Peptococcus, Clostridium and Bacteroides.
3.2.3.2. In vitro studies on native CGN. The majority of the
in vitro studies had only used native (undegraded) CGN (19).
In addition, seven publications compared degraded and
native CGN, described also in Section 3.2.3.1. CGN (j-, i- and
k-) had strong detrimental effect on human microbiota, using
feces obtained from a healthy person maintained ex vivo in
the dynamic stable MiniBioReactor Array model under anaer­
obic conditions (Naimi et al. 2021). CGN altered microbiota
density and composition, and induced bioactive levels of
flagellin in human embryonic kidney HEK cells with this
donor. In a study of in vitro batch fecal fermentation using
samples from 13 young healthy donors (seven women and
six men), j-CGN had no effects on production of any SCFA or
536 M. TAHIRI ET AL.
branch chain fatty acids (BCFA), but increased the abundance
of Escherichia/Shigella and inhibited the growth of f
Bifidobacterium in the human gut microbiome (Gerasimidis
et al. 2020). Effects on CGN in fecal samples from 20 healthy
persons and 10 UC patients (4 men and 6 women, adults)
were studied by Gibson et al. (1991). Fermentation of CGN
stimulated sulfide production in the colitis patients, and
increased growth and activities of sulfate-reducing bacteria,
which were higher in the healthy controls.
Studies in the human colonocyte cell line NCM460 cells and
human colonic epithelial cell line HT-29 showed that k-CGN
increased expression of colonic Wnt9A through Sp-1-mediated
transcriptional effects involving arylsulfatase B, chondroitin 4-
sulfate and galectin-3 (Bhattacharyya et al. 2014b).
Type IV k-CGN, essentially a pure k form, increased Bcl10,
IjBa phosphorylation, total and nuclear NF-jB and secretion
of IL-8 in the NCM460 cell line (1 mg/ml) and ex vivo normal
human colon epithelial tissues (10 mg/ml), implicating this sig­
naling pathway in intestinal inflammation induced by CGN
(Borthakur et al. 2007). When comparing IL-8 secretion
between the native j-, i- and k-CGNs (0.1, 1 and 10 mg/ml) in
NCM460 cells, the response was highest for k-, next for j-
and then i-CGN. Further, in NCM460 and HT-29 cell lines, it
was shown that NF-jB binding to the BCL10 promoter
involved the components RelA and RelB, and could lead to
prolonged activation of the k-CGN-induced inflammation by
a transcriptional mechanism involving a NF-jB-BCL10 loop
(Borthakur et al. 2012).
k-CGN increased production of reactive oxygen species
(ROS) in NCM460 cells and affected the Bcl10-mediated path­
way, which involves the innate immune response, and was
responsible for the majority of the effects of CGN in the
inflammatory pathway leading to IL-8 (Bhattacharyya et al.
2008b). Both the ROS and Bcl10 pathways increased NF-jB
nuclear translocation and IL-8 secretion. The Bcl10-NFjB-IL-8
inflammatory pathway affected by CGN in NCM460 cells was
mediated by TLR4, indicating that CGN-induced inflammation
in human colonocytes proceeds via innate immunity
(Bhattacharyya et al. 2008c). IL-8 secretion in response to j-,
i- and k-CGN after treatment with carrageenase and galacto­
sidase enzymes in NCM460 cells was compared and it was
found that k-CGN produced a higher IL-8 response than
j-CGN or i-CGN (Bhattacharyya et al. 2010c). Another study
suggested that k-CGN drives both the NCM460 and mouse
embryonic fibroblast (MEF) cell lines stimulated with TNF-a, a
potent proinflammatory cytokine, towards inflammation
rather than towards apoptotic cell death (Bhattacharyya et al.
2010b). Lastly, in NCM460 cells, an increase in nuclear RelA
following exposure to k-CGN was partially inhibited by
exogenous IL-10 (the canonical pathway), whereas the
increase in nuclear RelB was unaffected by exogenous IL-10
(the non-canonical pathway) (Bhattacharyya et al. 2013). In
human HT-29 cells, j-CGN did not stimulate IL-8 alone, but
increased LPS-induced IL-8 secretion via the Bcl10-NFjB path­
way and acted in synergy with LPS to increase the expression
of MD-2 and CD14 (Wu et al. 2017).
A CGN food grade blend, separate j-, k- or i-CGN, or a CGN
non-gelling mix of j- and k-CGN were reported not to bind to
and activate TLR4 signaling or reduce the LPS response when
co-administered, in a HEK293 (immortalized human embryonic
kidney cells)-TLR4 reporter cell-line model (McKim et al. 2015).
The various forms of CGN were reported to bind tightly to
serum proteins. These results were different from results of
other publications, and it should be noted that the work was
funded by FMC Corporation, a producer of CGN.
Several publications studied effects of CGN on the intes­
tinal barrier and its permeability. Choi et al. (2012) studied
effects of CGN on the pro-inflammatory transcription factors
NF-jB and early growth response gene 1 (EGR-1), evaluated
in terms of human intestinal epithelial barrier integrity using
the human cell lines HT-29, HCT-8 and Caco-2. Both pro-
inflammatory transcription factors were elevated by CGN, but
only NF-jB activation was shown to be involved in the induc­
tion of IL-8. The integrity of the in vitro epithelial cell mono­
layer under the CGN exposure was maintained by both
activated NF-jB and EGR-1. Suppression of NF-jB or EGR-1
aggravated barrier disruption by CGN, which was associated
with reduced gene expression of tight junction component
zonula occludens-1 (Zo-1) and its irregular localization in the
epithelial monolayer. Further, CGN also upregulated the
expression of the macrophage inhibitory cytokine 1 (MIC-1)
that promoted epithelial cell apoptosis. The counterbalance
between MIC-1 and the pro-survival protein activating tran­
scription factor 3 (ATF3) appeared critical for deciding the
fate of enterocytes under CGN exposure, when studied in
human HCT-8 and HCT-116 intestinal epithelial cells, CMT-93
mouse colon cancer cells and primary mouse intestinal epi­
thelial cells (Choi et al. 2014).
In an in vitro co-culture system of human intestinal epithe­
lial Caco-2 cells and human macrophage-like THP-1 cells,
j-CGN resulted in apoptosis, reduced transepithelial electrical
resistance of the Caco-2 cell monolayer and increased secre­
tion of TNF-a, IL-1b or IL-6, depending on CGN concentration
(Jiang et al. 2013).
CGN’s physiochemical properties, such as zeta-potential,
may affect gastric proteolysis, potentially leading to reduced
accessibility of proteins, peptides and amino acids in the
intestines (Fahoum et al. 2017). Different commercial CGN
samples varied in their zeta-potential, arising from their dif­
ferent degree of sulfation, throughout a range of pH values
as well as with varying CaCl2 concentrations Throughout the
tested pH range, j-CGN was the least negatively charged,
i-CGN was intermediately charged, while k-CGN was the
most negatively charged of the CGN samples at typical
luminal concentrations of CaCl2.
j-, k- or i-CGN exposure reduced the activity of several
sulfatases and increased N-acetylgalactosamine-containing
glycosaminoglycan (GAG) content and their disaccharide
composition in several human and mammalian intestinal and
mammary epithelial cell lines (Yang et al. 2012). The results
suggested that CGN inhibition of sulfatase activity led to re-
distribution of the cellular GAG composition with increase in
di-sulfated chondroitin sulfate, with potential consequences
for cell structure and function.
In a standard Caco-2 absorption model, CGN was reported
not to affect permeability or cytotoxicity (McKim et al. 2016).
Further, in the two human intestinal cell lines HT-29 and
HCT-8, CGN did not induce IL-8, IL-6 or monocyte

chemoattractant protein-1 (MCP-1) (CCL2) or produced cellu­
lar toxicity. The TLR4 agonist LPS produced weak induction
of IL-8 in HT-29 cells and no induction in HCT-8 cells. No
effect on oxidative stress was observed in HT-29 cells with
j-CGN. In the human liver cell line HepG2, k-CGN had no
effect on the expression of IL-8, IL-6 or MCP-1. These results
were different from results of other publications, and it
should be noted that the work was funded in part by FMC
Corporation, a producer of CGN, and the International Food
Additives Council (IFAC), a global association representing
manufacturers and end-users of food ingredients, including
food additives.
Among the publications on non-human microbiota,
Harmuth-Hoene and Schwerdtfeger (1979) found that in rat
feces, dry matter digestibility and apparent protein digestibil­
ity, as well as nitrogen retention and trypsin inhibition, were
decreased with 10% CGN in the diet during eight days. These
effects were attributed to the partial breakdown by intestinal
microbiota and/or their ability to partially inhibit the activity
of proteolytic enzymes in the gastrointestinal tract.
In an ex vivo-in vitro experiment, the proliferative responses
of splenocytes from rats given 2% k-CGN in drinking water to
the mitogens concanavalin A (Con A, to stimulate T cells), lipo­
polysaccharide (LPS, to stimulate B cells) and pokeweed mito­
gen (PWM, to stimulate both T and B cells) were significantly
diminished compared with splenocytes from control rats
(Pricolo et al. 1996). The proliferative responses were fully
restored with a specific inhibitor of nitric oxide synthase,
involving nitric oxide in the immunosuppression.
In rats given a fibre-free purified diet supplemented with
5, 20 or 50 g of i-CGN per kg diet for 30 days, all doses
caused reduced activities of azoreductase, b-glucosidase,
b-glucuronidase, nitrate reductase and total nitroreductase
per gram of cecal content in vitro (Mallett et al. 1985). Mice
treated similarly also had decreased activities of b-glucuroni­
dase and total nitroreductase, whereas hamsters had
increased activities of b-glucuronidase and nitrate reductase,
but decreased activity of azoreductase and total nitroreduc­
tase per gram of cecal content.
k-CGN affected the Bcl10-NFjB-IL-8 inflammatory pathway
via TLR4, since binding of k-CGN to TLR4 was markedly
reduced in RAW 264.7 mouse macrophages after exposure
with TLR4 blocking antibody (HTA-125), whereas binding to
10ScNCr/23 mouse macrophages, which are deficient in the
genetic locus for TLR4, was absent (Bhattacharyya et al.
2008c).
The four publications comparing native and degraded
CGN are described in Section 3.2.3.1. (Bhattacharyya et al.
2007; Bhattacharyya et al. 2008a; Ariffin et al. 2014; Chen
et al. 2014). Three other publications also included data on
both degraded and native CGN, and are present in both sec­
tions (Bhattacharyya et al. 2010c; McKim et al. (2016);
Fahoum et al. 2017).
3.2.4. Hypotheses on adverse effects of CGN
Many publications examined more than one specific hypoth­
esis or had several objectives for their work, and many publi­
cations examined the same hypotheses or effects, thus, a
CRITICAL REVIEWS IN TOXICOLOGY 537
complete list would be very repetitive. The number of
research publications studying degraded or native CGN in
relation to various hypotheses on adverse effects is shown in
Figure 8. Most of the hypotheses were studied both with
degraded and native CGN, with some differences. The num­
ber of studies on inflammation was higher with degraded
(43) than native CGN (35), reflecting the use of degraded
CGN as an experimental inflammation model. The number of
studies of native CGN was higher than of degraded CGN for
most of the other endpoints, especially permeability of the
intestinal barrier (10 vs. 6), effects on the gut microbiome (16
vs. 11), metabolic effects, such as obesity and metabolic syn­
drome (9 vs. 3), and effects on cell proliferation, apoptosis or
cell cycle (9 vs. 3), respectively, i.e. reflecting the more wide-
spread interest in native CGN in relation to different health
effects.
3.2.5. Data relevant for exposure
Twenty publications had some information relevant for
exposure to food grade CGN. In reviews, CGN (E 407) was
reported in 4% of all food products in the food supply in EU
(Cox et al. 2021) and the range of content of CGN in some
commonly consumed food groups was reported to vary from
0.01 g to 4 g per 100 g food (Tobacman 2001).
The estimated intake of CGN has increased over time. In
reviews by David et al. (2018) and Liao et al. (2021), it was
reported that in a FDA report (1972), the daily CGN con­
sumption in USA was reported to be 45 mg per day, an
industrial survey on the use of food additives (1977) esti­
mated CGN intake of 100 mg per day for individuals
>2 years, and the CGN intake was estimated to rise to
2.5 mg per day in 1989 and further up to 7.7 g per day (0–
1 mg/kg bw) in South Florida in 2003. In other reviews, the
estimated range of intake of CGN reported for the general
population was 18–42 mg/kg bw per day and for infants
given formula 6–160 mg/kg bw per day (Weiner 2014) and
the average daily intake of CGN in a Western diet (USA) was
estimated to be 20 to 200 mg/day (Martino et al. 2017). In
an abstract, it was reported that CGN is consumed at levels
of about 250 mg/day in the average diet (Bhattacharyya and
Tobacman 2012).
In a review, it was reported that CGN was consumed by
77.5% of the consumers among 106 489 adults in the French
NutriNet-Sant�e cohort study (2009–2020) (Chazelas et al.
2021). Mean intake per person per day was approximately
46 mg per day. The mean ± standard deviation (SD) intake
was 0.71 ± 1.07 mg/kg bw per day and the 95th percentile
intake was 2.70 mg/kg bw per day.
CGN or PES were not considered to be used in combin­
ation in the same foods and their exposure could therefore
be assessed independently (EFSA 2018a). Several different
exposure scenarios were used in the calculations of their
exposure by EFSA. The mean maximum level exposure to
CGN and PES was estimated based on the maximum
reported use levels provided by industry, since CGN and PES
are authorized as QS in almost all FC, and ranged from
21.8 mg/kg bw per day in the infants to 581.4 mg/kg bw per
day in toddlers (EFSA 2018a). The 95th percentile of exposure
538 M. TAHIRI ET AL.
Figure 8. Hypotheses on adverse effects on the intestines of degraded and native CGN in the research publications.
to CGN and PES ranged from 58.1 mg/kg bw per day in the
elderly to 999.1 mg/kg bw per day in children. The Panel
further concluded that in the refined brand-loyal exposure
scenario the exposure estimates exceeded, by up to about
10-fold, the temporary ADI of 75 mg/kg bw per day at the
95th percentile for all population groups and at the mean for
all population groups except for infants and the elderly,
which may be a safety concern (EFSA 2018a). In a reply to a
comment, David et al. (2019) presented a figure of total lev­
els of estimated exposure to CGN from foods in various age
groups based on an EFSA survey of European countries
(referring to Supplementary material in EFSA 2018a) where
the exposures in mg/kg bw per day were exceeding the ADI
for toddlers, other children and adolescents.
Average exposure to unspecified CGN in children of both
sex (n ¼ 138, mean age 14.2 ± 2.8 years) with CD was quanti­
fied from three 24-h recall days over two years obtained by
telephone (Lee et al. 2018). A total of 1325 unique foods
were recorded and 5% of the foods contained CGN.
Mean ± SD frequencies of exposures per day were 0.58 ± 0.63
(range 0–3.67) of CGN. The frequency of exposure to CGN in
20 patients with CD using 7-day food diaries analyzed for
habitual intake was 0.09 ± 0.15 (mean ± SD) per day (Sandall
et al. 2020).
In an abstract, the exposures to selected food additives
per day in children with IBD and the association with IBD
relapse were reported (Ho et al. 2021). In this single-centre
observational cohort study, children 8–18 years of age with
CD or UC in remission were enrolled. Detailed 24-h dietary
recalls were collected at baseline, between 3–6, 7–10, 11–
14 months and at time of relapse. A food list was generated,
and a database of product ingredient lists was evaluated to
quantify the frequency of dietary exposure to 10 food addi­
tives, including CGN (and CMC). In total, 22 participants with
IBD (15 CD, 7 UC) were enrolled, of which six later dropped
out. Mean age was 12.8 ± 3.5 years, with average disease dur­
ation 2.2 ± 1.9 years. Disease relapse occurred in 5/16 (31%)
of participants; 4 with CD and 1 with UC. There were no stat­
istical differences between number of exposures per day or
intake of food additives between the remission and relapse
groups.
An abstract reported emulsifier exposure using baseline
dietary data from adults and children with IBD enrolled in
the Australian Inflammatory Bowel Disease Microbiome (AIM)
study, a multicentre, prospective, longitudinal cohort study
(Reveley et al. 2022). Baseline dietary intake was measured
using a 3-day prospective food diary. Food products were
evaluated for the presence of six emulsifiers, including CGN
(and CMC), using ingredient lists. Complete data were avail­
able for 370 participants (102 with CD, 75 with UC and 193
healthy controls; 337 adults and 33 children). There was no
difference in emulsifier exposure between those with UC or
CD, and healthy controls. Mean exposure was 2.7 occasions
per day (range 0–6.0) for those with UC, 2.8 occasions (range
0–10.7) for those with CD and 2.4 occasions for healthy con­
trols (range 0–7.7). Children with IBD had significantly higher
exposure to emulsifiers than adults (p < 0.01). Daily occasions
of exposure (range) to CGN in patients with IBD were 0.4 (0–
1.7) in children (n ¼ 21) and 0.2 (0–1.7) in adults (n ¼ 156).
Three abstracts reported on previous and current con­
sumption of nine food additives including CGN (and CMC),
determined in 156 participants (92 with CD, 21 first-degree
relatives of patients with CD (FDR), 21 non-related healthy
household members of patients with CD (HHM) and 22
healthy controls (HC)) (Zhang et al. 2022a, 2022b, 2022c). No
significant differences in food additive consumption were
observed among the CD, FDR, HHM and HC groups in the
first two abstracts, whereas the third abstract reported that
while the FDR of CD patients consumed less food additives,
no significant differences were observed between the CD,
HHM, and HC groups.
One publication on use of emulsifiers in processed foods
mentioned CGN and exposure, but without providing any
new quantitative data on exposure to CGN (Laster et al.
2019).
Sixty-one exclusive enteral nutrition (EEN) formulas used
to induce remission in active CD contained 56 food additives
(median per formula was 11) (Logan et al. 2020). CGN
(unspecified) was present in 21.8% of the formulas.

The International Organization for the Study of
Inflammatory Bowel Disease (IOIBD) has recommended that
“it may be prudent to reduce intake of processed foods con­
taining CGN” (with evidence level very low, agreement
92.3%) in both UC and CD, based on data from epidemi­
ology, animal models and one very small RCT (Levine et al.
2020).
3.2.6. Absorption, distribution, metabolism and excretion
(ADME) studies
Three publications contained some information about CGN
and ADME. In a review article, it was summarized that CGN
was not significantly degraded in the gastrointestinal tract
and was excreted unchanged in the feces (Weiner 2014).
CGN was not significantly absorbed, was not metabolized
and did not affect nutrient absorption.
The ADME data on CGN was sufficient to show that high
MW CGN (MW of ca. 800 kDa) was not absorbed intact, while
low MW CGN (<88 kDa) was found in tissues of animals after
oral administration (EFSA 2018a). No data were available on
ADME of processed Eucheuma seaweed, but EFSA did not
expect it to behave differently from CGN, based on their
chemical structure. The conditions (duration, pH, tempera­
ture) for potential hydrolysis of CGN in the gastrointestinal
tract were considered less extreme than the mild acidic con­
ditions which resulted in the formation of chemically
degraded CGN. The theoretical possibility that more limited
degradation of CGN could occur under conditions representa­
tive of the in vivo situation remains uncertain although this
had not been observed to any significant extent in the avail­
able in vivo ADME studies. It was also noted that the tested
CGN differed in MW distribution and EFSA considered that
existing results did not allow for evaluation of kinetic differ­
ences between the various types of native CGN (j-, i- and k-)
and their corresponding low MW fractions.
However, in an abstract, it was reported that E. coli ST871
in feces from healthy men could efficiently metabolize
degraded j-CGN in vitro and the promoter effect of
degraded j-CGN on colon tumorigenesis included the E. coli
ST871-mediated metabolite pyrocatechol (Wang et al. 2022).
3.2.7. Risk assessment opinions
Two risk assessments of CGN were available. EFSA Panel on
Food Additives and Nutrient Sources added to Food (ANS)
reevaluated CGN (E 407) and PES (E 407a) as food additives
(EFSA 2018a). The no observed adverse effect level (NOAEL)
was 3400–3900 mg/kg bw per day, the highest dose tested,
in a subchronic toxicity rat study performed with CGN nearly
in compliance with the EU specification for E 407. No adverse
effects were detected in chronic toxicity studies with CGN in
rats up to 7500 mg/kg bw per day (the highest dose tested).
The NOAEL values of sodium and calcium CGNs for prenatal
developmental dietary toxicity studies were the highest doses
tested, 3000 and 5000 mg/kg bw per day for rats and ham­
ster, respectively. The safety of PES was considered suffi­
ciently covered by the toxicological evaluation of CGN. They
concluded that there was no concern regarding
CRITICAL REVIEWS IN TOXICOLOGY 539
carcinogenicity of CGN, and no concern for genotoxicity of
CGN and PES. However, EFSA concluded that the existing
group ADI for the two food additives E 407 and E 407a of
75 mg/kg bw per day should be considered temporary
because of the remaining uncertainties regarding chemistry
(type and MW), exposure assessment, and biological and toxi­
cological data, awaiting a new risk assessment with new data
within five years.
In this risk assessment (EFSA 2018a), they evaluated both
human studies, animal studies and in vitro studies, of which
many are included in this scoping review. In addition, 48
documentations were submitted to EFSA from industry and
other institutions not publicly available.
Previous risk assessments of these food additives (CGN,
and also CMC) from JECFA, and the Scientific Committee on
Food (SCF), the main committee providing the European
Commission with scientific advice on food safety before
being replaced by EFSA, are cited and referenced in the EFSA
opinion summarized above for CGN, and below for CMC, and
are not described here.
Native (undegraded) CGN has been classified by the IARC
as Group 3, i.e. unclassifiable with respect to carcinogenicity
in humans, whereas degraded CGN (PGN) is classified as
Group 2B, a possible human carcinogen, based on animal
data (IARC 1987).
3.2.8. Non-systematic (narrative) reviews, commentaries/
editorials/letters and conference abstracts
No other scoping review/scoping study or systematic review
on effects of CGN on the intestines in line with our objectives
was detected among the published literature. However, 54
non-systematic review publications on CGN with varying
scope and perspectives, published between 1973 and 2023,
were included. Most of these review publications (36) were
only on CGN (Watt and Marcus 1973; Watt and Marcus 1975;
Anver and Cohen 1976; MacPherson and Pfeiffer 1976; Watt
and Marcus 1981; Onderdonk 1985a, 1985b; Strober 1985;
Whittaker and Freeman 1988; Nicklin and Miller 1989;
Yoshida and Murata 1990; Weiner 1991; Kim and Berstad,
1992; Elson et al. 1995; Michel and Macfarlane 1996; Wirtz
and Neurath 2000; Tobacman 2001; Cohen and Ito 2002; Hibi
et al. 2002; Tobacman 2003; Kanneganti et al. 2011; Neurath
2012; Goyal et al. 2014; McKim 2014; Weiner 2014; Cian et al.
2015; Wells et al. 2017; David et al. 2018; Shang et al. 2018;
Dey 2019; McKim et al. 2019; Gkikas et al. 2020; Gotteland
et al. 2020; Liao et al. 2021; Liu et al. 2021; Li et al. 2023).
They were concerned with health effects and safety of CGN
and were often reviews on animal models. Many reviews
were on effects of CGN on IBD, UC and colitis, some on col­
itis-associated carcinogenesis, microbiota or immunological
effects, others mainly reviewed the use of CGN as pharma­
ceuticals, nutritional or functional food sources or prebiotics.
Of the included reviews on CGN, 18 publications also
included CMC (Liu et al. 2015; Martino et al. 2017; Paula Neto
et al. 2017; Kikut et al. 2018; Halmos et al. 2019; Laster et al.
2019; Marion-Letellier et al. 2019; Vo et al. 2019; Zino €cker
2019; Bancil et al. 2021; Borsani et al. 2021; Cox et al. 2021;
Sasson et al. 2021; Tan and Nie 2021; De Siena et al. 2022;
540 M. TAHIRI ET AL.
Levine et al. 2022; Liu et al. 2022; Kuang et al. 2023) and
reviewed effects on intestinal inflammation and disease,
microbiota, intestinal permeability, obesity and metabolic
dysregulation, or allergy.
Fourteen publications on CGN in the category commenta­
ries/editorials/letters were published in the period 1969–
2022. One of the main issues in these publications (as well as
in original research publications) seems to be the differences
between native and degraded CGN. A distinction is made
between the food grade CGN of high MW and degraded
CGN, which are not the same substances as commercial food
grade CGN (Marcus and Watt 1969; Tobacman 2002a, 2002b;
Bixler 2017; David et al. 2019; Weiner and McKim 2019).
Another issue is whether the research from experimental ani­
mal models (and in vitro model systems) is representative of
effects of CGN in humans being exposed to CGN as a food
additive or as medication (Bonfils 1970; Marcus and Watt
1970; Mottet 1972). Other emphasize conclusions on evalua­
tions of safety of CGN already performed by health author­
ities (Carthew 2002; Kirsch 2002). Three were on adverse
effects on the intestines of several food additives, also includ­
ing CMC (Zino €cker and Lindseth 2019; Cox and Sandall 2021;
Wellens et al. 2022).
The 23 conference abstracts on CGN found in the
searches, published 1984–2022, were mostly reporting studies
on unspecified CGN, either degraded or native, and mostly in
animals or in vitro. The effects of CGN in these abstracts were
often reported also as full publications (Shiau and Chang
1984; Madh�ere et al. 1994; Axelsson and Midtvedt 1997;
Sabljic et al. 2002; Bhattacharyya et al. 2010a; Bhattacharyya
and Tobacman 2011; Bhattacharyya and Tobacman 2012;
Bhattacharyya et al. 2014a; Fahoum et al. 2015; Jimenez
Loayza et al. 2019; Kang et al. 2022; Wang et al. 2022). In
addition, four abstracts were on both CGN and CMC
(Schooth et al. 2020; Zhang et al. 2022a, 2022b, 2022c).
However, seven abstracts reporting on human studies
were not obtained as full publications. In Cochrane Library, a
randomized controlled clinical trial was listed (Cochrane
Central Register of Controlled Trials 2021). The inclusion crite­
ria were UC, clinical remission verified by normal fecal calpro­
tectin 2, age 18-64 years, with no biological medications or
systemic cortisone usage for relapse. The intervention was
CGN 2-2.5 g/day vs. a corresponding dose of oat fibre, start­
ing with a 7-day run-in period (low CGN diet, instructed by a
dietician), then either a CGN period or an oat fibre (control)
period for 7 days, followed by at least 14 days of wash-out,
then the other treatment (either CGN or oat fibre), altogether
35 days. The primary outcome studied was colitis activity
measured using the Simple Clinical Colitis Assessment Index
(SCCAI) at baseline and the 7th day of both treatment peri­
ods. Secondary outcomes were gastrointestinal symptoms,
laboratory tests using blood, urine and stool samples at base­
line and at the end (7th day) of both treatment periods
measuring fecal calprotectin, hs-CRP, S-FABP-2 (intestinal per­
meability marker), U-creatinine, U-Albumin, F-albumin, F-IgG
and F-intestinal alkaline phosphatase (IAP). Macronutrient
and fibre intake will be measured using a 3-day food diary at
the end of both treatment periods. No results were reported.
Two abstracts on a randomized, double-blind, placebo-
controlled, cross-over trial (registered as NCT02629705) were
published by Wagner et al. (2018a, 2018b). Healthy males
(n ¼ 20) were randomly allocated to 14 days of CGN (250 mg
twice daily) or matching placebo. Participants had a mean (±
SD) age of 27.6 ± 4.8 years and a BMI of 24.4 ± 2.6 kg/m2. After
a wash-out period of 30 ± 7 days, they received the other
compound. At the end of each treatment phase, participants
underwent an oral glucose tolerance test (OGTT), a hyperin­
sulinemic-euglycemic clamp with labeled glucose (ISIclamp),
whole-body magnetic resonance (MR)-tomography and 1H-
MR-spectroscopy, blood immune cell phenotyping and a lac­
tulose-mannitol test for investigating intestinal permeability.
Intestinal permeability increased after CGN vs. placebo
(p ¼ 0.03). Whole-body insulin sensitivity index (ISI), both
ISIclamp and OGTT, did not differ between CGN and placebo.
No changes of hepatic fat content, body fat mass, fat distri­
bution, liver transaminases or inflammatory cytokines were
observed.
In addition, an abstract not covered by a full publication
reported results of modified CGNs. The effects of j-CGN gels
modified with Naþ and Ca2 were studied in neonatal rats,
which received a daily oral dose of pure refined food grade
j-CGN (500 mg/kg bw per day), j-CGN with 10% Naþ, j-CGN
with 10% Ca2þ or no j-CGN as control group, for 14 weeks
(Farag et al. 2018). All groups showed normal growth without
differences in body weight or feed consumption, and no mor­
tality or pathological symptoms were observed. Serum bio­
chemical parameters showed increase (p < 0.05) in total
cholesterol with j-CGN þ Ca compared with controls, whereas
no differences were observed in total glycerides, total lipids,
high or low density lipoprotein cholesterol, liver enzymes,
urea, creatinine or haematological parameters between any
groups. Serum antioxidant biomarkers in rats treated with
j-CGN þ Ca showed decreased (p < 0.05) superoxide dismu­
tase (SOD) and glutathione reductase (GR) activities vs. con­
trols, but the other two groups did not. Rats treated with
j-CGN showed increase (p < 0.05) in malondialdehyde (MDA),
whereas no differences were detected between j-CGN þ Na,
j-CGN þ Ca and controls. Catalase activity (CA) was not
affected in any groups. In contrast to serum, levels of liver anti­
oxidants showed no differences for SOD, GR, MDA or CA
between any groups. Histological investigation showed minor
changes in rat colon mucosa of j-CGN and j-CGN þ Na, such
as focal destruction of crypts of Lieberkuhn, where some were
replaced by cellular infiltration, loss of colonic crypt integrity
seen as loss of vacuolated cytoplasm, presence of dense small
nuclei and lymphocytic aggregation in the submucosa.
One abstract reported that E 407a promoted ROS gener­
ation by leukocytes and induced intestinal inflammation asso­
ciated with macrophage and T-lymphocyte infiltration after
two weeks of daily oral exposure to 140 mg/kg bw in rats
(Tkachenko et al. 2021).
Two abstracts reporting exposure to food additives,
including CGN, per day in children with IBD and the associ­
ation with IBD relapse (Ho et al. 2021) and exposure using
baseline dietary data from adults and children with IBD
(Reveley et al. 2022) are described in Section 3.2.5.
 CRITICAL REVIEWS IN TOXICOLOGY 541

Figure 9. The number of research publications studying non-modified or modified CMC per 5-year periods.

Figure 10. Distribution of various types of publications on CMC. The human studies (without studies of exposure), experimental animal studies, in vitro studies and
other types of studies (one toxicokinetic study), are original research publications. Exposure studies comprise all types of publications that contain information on
levels in food, exposure etc., including from review publications and conference abstracts. In addition, publications on CMC containing information on toxicokinetics
and ADME, and risk assessments, were included.

3.3. Publications on carboxymethylcellulose (CMC)
(E 466)
Few publications on CMC were found until the period 1991–
2023 and the number increased especially after 2016
(Figure 9). Only two publications were investigating modified
CMC, which are described in more detail below (B€ar et al.
1995, see Section 3.3.6.) and Andreev-Andrievskiy et al. 2021,
see Section 3.3.3).
The distribution of various types of original research publi­
cations on CMC (human studies, experimental animal studies
and in vitro studies) is shown in Figure 10. Only three human
studies were available. As for CGN, the highest number of
publications was on various experimental animals (20), with
in vitro studies as the second largest category (14), studying
microbiota from humans or animals ex vivo-in vitro or
describing mechanistic data in human or animal primary cells
or cell lines. Seventeen publications of various types con­
tained some information relevant for exposure to CMC, two
had some information on toxicokinetics and ADME, and five
risk assessment opinions by EFSA on CMC were found. The
main results of the original research publications on humans,
animals and in vitro are described briefly in the following.
3.3.1. Human studies
Only three publications contained original research in
humans relevant for effects of CMC. A randomized, double-
blind, controlled feeding study included healthy adults (18–
60 years) of both sexes kept first on an emulsifier-free diet at
home for three days and thereafter divided into two groups
and given either a CMC-free diet (n ¼ 9) or a CMC-rich diet
542 M. TAHIRI ET AL.
(15 g/day of CMC) (n ¼ 7) for 11 days (Chassaing et al. 2022a).
In the CMC group, an increased disturbance of the intestinal
microbiota with reduced bacteria diversity, increased stomach
discomfort, and reduction of SCFA and free amino acids in
feces, were observed. An increased amount of bacteria in the
normally sterile inner mucus layer of the intestine was found
in two of the test subjects, indicating intestinal inflammation.
According to the authors, the amount of CMC used was
somewhat higher than the CMC intake for most persons, but
that those who consume a lot of processed food would have
an intake of CMC comparable to in this study.
In a study of five healthy male volunteers (age range 24–
58 years) given 15 g CMC daily for 23 days after a 7-day con­
trol period, no adverse effects were noted either on plasma
biochemistry, haematology, urinalysis, glucose tolerance,
serum cholesterol, triglyceride and phospholipids, or breath
hydrogen and breath methane concentrations (Anderson
et al. 1986). In addition, some inconsistent changes were
observed on intestinal transit time, fecal wet and dry weights,
fecal bile acids, fecal fat and excretion of neutral sterols,
which were considered physiological and not adverse.
One case report ascribed anaphylaxis to CMC in a 14-year-
old girl (Ohnishi et al. 2019). She had four episodes of ana­
phylaxis and the common ingredient in the foods eaten in all
episodes (pizza, cheeseburger, ice lolly and a half-frozen bev­
erage) was CMC. She tested positive for CMC in skin prick
tests with a CMC solution and CMC-containing foods, and
did not react to a CMC-free ice lolly from the same manufac­
turer as eaten before. No other publications were found on
adverse effects of CMC in children or adolescents.
3.3.2. Animal studies
The 20 included publications on adverse effects of CMC on
the intestines in experimental animals were mostly on mice
(13) and/or rats (7), as well as one study on pigs.
CMC in drinking water was administered for 12 weeks to
wild-type mice and to two strains, IL-10−/− and TLR5−/−, that
are sensitive to effects on microbiota composition and
inflammation (Chassaing et al. 2015). CMC induced low-grade
inflammation and obesity/metabolic syndrome in wild-type
mice and induced profound colitis in the sensitive strains.
CMC-induced metabolic syndrome was associated with
microbiota encroachment, changes in species composition
and increased pro-inflammatory potential. Further, use of
germ-free mice and fecal transplants indicated that these
changes in microbiota were necessary and sufficient for both
low-grade inflammation and metabolic syndrome. In another
study, when the CMC-treated M-SHIME human microbiota
(see Section 3.3.3) was transferred to germ-free mice, many
of the host and microbial alterations observed in mice dir­
ectly treated with CMC were observed (Chassaing et al.
2017a). In a third study, gnotobiotic wild-type and IL-10-/-
mice were colonized with adherent-invasive E. coli (AIEC),
associated with Crohn’s disease, and subsequently adminis­
tered CMC in drinking water for 12 weeks (Viennois et al.
2020). AIEC colonization of germfree and altered Schaedler
flora (ASF) mice resulted in chronic intestinal inflammation
and dysregulations of metabolism after CMC exposure. In
IL-10-/- mice, AIEC mono-colonization resulted in severe intes­
tinal inflammation in response to CMC. Transcriptomic ana­
lysis revealed that CMC directly induced expression of genes
that mediate AIEC virulence and promotion of inflammation.
Effects of CMC on ex-germ-free IL10-/- mice colonized by
pooled fecal transplant from three patients with active IBD
were investigated by Rousta et al. (2021). In mice which
received CMC in drinking water for four weeks, CMC
increased fecal lipocalin 2 (Lcn-2) levels, histological inflam­
matory scores and inflammatory cytokine gene expression in
colon. CMC did not affect bacterial composition but signifi­
cantly decreased Caudoviricetes (bacteriophages). Thus, CMC
promoted aggressive inflammation without changing bacter­
ial composition. Twenty to 27-week-old IL-10 gene-deficient
mice received either CMC or water orally for 3 weeks
(Swidsinski et al. 2009). CMC-treated IL-10 gene-deficient
mice demonstrated a massive bacterial overgrowth, disten­
tion of spaces between villi filled with bacteria, adherence of
bacteria to the mucosa and migration of bacteria to the bot­
tom of the crypts of Lieberkuhn. Leukocytes migrated into
the intestinal lumen in four of the seven CMC-exposed mice.
The changes were similar to those observed in CD in humans
and were absent in control animals.
As opposed to other studies, in IL-10-/- mice which
develop spontaneous colitis, exposure to CMC by daily gav­
age for 14 days caused reduction of blood in the feces, feces
consistency score, histopathological injury score, colon
weight/length ratio and activity of MPO vs. controls (Ung
et al. 2010). However, CMC did not decrease the levels of the
inflammatory cytokines interferon (IFN)-c or IL-17 in colon or
caecum.
Exposure of mice to CMC in drinking water for 12 weeks
induced chronic intestinal inflammation, increased adiposity
and caused sex-dependent changes in gut microbiota com­
position (Holder et al. 2019). Additionally, CMC treatment
altered anxiety-like behaviours in males and reduced social
behaviour in females, followed by changed expression of
neuropeptides associated with regulation of feeding as well
as social and anxiety-related behaviour.
Exposure of mice to CMC in drinking water for 15 weeks
caused inflammation, reduced mucus thickness and increased
the intestinal permeability measured by increased passage of
the permeability tracer fluorescein isothiocyanate (FITC)-dex­
tran into serum (Wu et al. (2020).
When mice received oral CMC (0.45 ml of a 0.05% solu­
tion) daily for 5 days, two of 11 mice died vs. none of ten in
the controls, mean body weight was reduced by 3.6% and
level of C-reactive protein was increased (Nabarawi 2014).
CMC in drinking water for 12 weeks administered to adult
mice increased body weight and adipose tissue but did not
affect fasting blood glucose (Sandall et al. 2020). CMC also
decreased colon length, a proxy marker for intestinal inflam­
mation, compared to controls.
Increased risk of colorectal cancer associated with IBD
coined the term “colitis-associated cancer” and the concept
that inflammation promotes cancer (Viennois et al. 2017). A
colitis-associated cancer model was obtained by treating
mice with the colon carcinogen AOM and dextran sulfate
sodium (DSS) to induce inflammation. CMC in drinking water

for ca. 14 weeks exacerbated tumor development (both num­
ber of tumors and tumor area) and altered the microbiota
metagenome, characterized by increased levels of LPS and
flagellin. In a later study, the effect of CMC on cancer initi­
ation and progression in a genetic model of intestinal adeno­
mas using mice with the multiple intestinal neoplasia (Min)
mutation in the tumor suppressor gene adenomatous polyp­
osis coli (Apc), ApcMin/þ mice, was investigated (Viennois and
Chassaing 2021). CMC in drinking water for 15 weeks
increased small intestinal tumor development, apparently
independent of chronic intestinal inflammation, but it was
associated with CMC’s effects on proliferation in the intestinal
epithelium and intestinal microbiota composition.
Long-term exposure of rats and mice to dietary levels of
10 or 100 g/kg of CMC (Edifas B) for 104 and 100 weeks,
respectively, had little adverse influence on the general
health and survival of the animals (McElligott and Hurst
1968). The haematological parameters in the rats were also
unaffected. The rats appeared to compensate for the lower
nutritional value of the diet by increased food consumption,
but in the later stages of the experiment retardation of
growth was apparent. Histological examination revealed no
intestinal abnormality or evidence of the passage of Edifas B
through the intestinal wall, and there was no evidence that
the substance was carcinogenic, in either species.
The protective mucus layer in the intestines acts as a bar­
rier that protects the underlying epithelial layer against
microorganisms and chemicals. When rat intestines were dir­
ectly exposed to 1% CMC for 30 min., mucus pore size
decreased, which resulted in significantly slower E. coli speed
and passive diffusion rates of nanoparticles through mucus
(Lock et al. 2018).These results indicated that acute exposure
to CMC may impact barrier and structural properties of intes­
tinal mucus, affect interactions between intestinal lumen con­
tents, microbes and underlying tissue, which may contribute
to development of intestinal inflammation. Three concentra­
tions of CMC (0.1%, 0.2% and 0.5%) could all increase absorp­
tion of a drug with low intestinal permeability, shown by
intestinal perfusion in rats (Valizadeh et al. 2015).
Some older publications studied effects of CMC on various
physiological parameters of the intestines and feces in rats,
and reported effects on cell proliferation, tissue hypertrophy
- mostly caused by the increased bulk of feces and diarrhea
(Wyatt et al. 1988; Toyoda et al. 1994; Ju�skiewicz and
Zdun �czyk 2004).
In addition, a study on toxicokinetics in rats by B€ar et al.
(1995) is described under Section 3.3.6.
In weaned piglets given CMC in the diet for 13 days, aer­
obe bacteria were transiently higher at day 7 but coliform
counts remained unchanged and b-haemolytic E. coli were
virtually absent (Lalle�s et al. 2006). This indicated that CMC
had pro-absorptive effects on the small intestine, possibly
due to the absence of pathogenic E. coli.
3.3.3. In vitro studies
In total, 14 publications on in vitro experiments on CMC were
included, using fecal microbiota from humans (8) or animals
(rat and pig) (2), or cell lines from humans (4) or primary cells
CRITICAL REVIEWS IN TOXICOLOGY 543
from animals (rat and mouse) (3). All except one in vitro pub­
lication (Andreev-Andrievskiy et al. 2021) studied non-modi­
fied CMC.
Effects of Kagocel, a synthetic CMC derivative copolymer­
ized with gossypol, were studied in murine Peyer’s patches
lymphocytes from the small intestine of mice (Andreev-
Andrievskiy et al. 2021). The lymphocytes were stimulated
with concanavalin A (Con A), or with Con A and TLR3 ligand
poly I:C to mimic viral infection. After 24 h of stimulation, the
cells were treated with Kagocel. Expression of genes involved
in the inflammatory response, antiviral defense, lymphocyte
survival and proliferation (C1qa, C2, C3, Ccl21a, Il11, Il1b,
Il23a, Il5, Ltb4r2, Alox15, Pla2g4a, Ptger1, Mapkapk5, Hras,
Ifna1, Tlr2, Mrc1 and Mx2) was upregulated in Kagocel-
treated lymphocytes. Transcription factors (CEBPs, IRF, NFjB,
RXR, Stat, Tead4 and ZSCAN) and master regulators (cIAP,
CIKS, dock9, MEKK1, FXR, IKK, IRAK, TRAF, dsRNA:TLR3:TRIF)
were also identified. The changes in gene expression pattern
and the outcome of bioinformatics analysis suggested that
pattern recognition receptors, TLRs and dectin-1, were the
key mediators of Kagocel’s effects on the immune system,
possibly involving the interferon autocrine loop. Kagocel
upregulated genes of various components of the innate
immune defense system.
Three publications reported studies of CMC and intestinal
permeability. Concentrations of 0.02%, 0.1%, 0.5% and 1%
CMC, claimed to be commonly used in the food industry, did
not affect intestinal permeability studied ex vivo in the
Ussing diffusion chamber system using small intestinal frag­
ments from rats and in the human colorectal adenocarcin­
oma-derived cell line Caco-2, or the stimulation of the
human dendritic cell-like myeloid leukemia-derived MUTZ-3
cell line (Oscarsson et al. 2020). Testing of CMC in mucus-pro­
ducing rat intestinal cell cultures, human Caco-2 cells and
mucus-producing human colon HT29-MTX cells in a mono­
layer showed that CMC decreased mucus pore size and
reduced diffusion of various particles through the mucus
(Lock et al. 2018). CMC also resulted in partial removal of the
mucus layer, making it thinner with fewer globular mucus
aggregates, which may contribute to exposure of the epithe­
lium to intestinal contents and increase bacterial transloca­
tion, resulting in intestinal inflammation. Lastly, exposure of
AIEC to CMC in vitro increased the motility and ability to
adhere to human intestine-407 epithelial cells, but invasion
ability was not affected (Viennois et al. 2020). Transcriptomic
analysis revealed that CMC directly induced expression of
clusters of genes that mediate AIEC virulence in a dose-
dependent way and promoted inflammation.
Four publications studied changes by CMC on the micro­
biota. In an ex vivo-in vitro study on feces from pigs, dietary
CMC administered to growing pigs resulted in a distinctive
bacterial community (Metzler-Zebeli et al. 2010). CMC
affected the taxonomic composition and metabolic features
of the fecal microbiota, as shown by increases in total bac­
teria, Bacteroides-Prevotella-Porphyromonas and Clostridium
cluster XIVa, and Enterobacteriaceae and increased prevalen­
ces of E. coli virulence factors in feces.
The mucosal simulator of the human intestinal microbial
ecosystem (M-SHIME) is a dynamic in vitro model which
544 M. TAHIRI ET AL.
Figure 11. Hypotheses on adverse effects of CMC in the research publications.
simulates the lumen- and mucus-associated human intestinal
microbial ecosystem. It maintains a complex stable human
microbiota in the absence of a host. In this system, CMC
increased the pro-inflammatory potential of the human fecal
microbiota, demonstrated by increased levels of bioactive
flagellin (Chassaing et al. 2017a). In another model system,
CMC had adverse effect on human microbiota composition
when using feces obtained from a healthy person and main­
tained ex vivo in the MiniBioReactor Array model, by directly
changing the microbiota composition and gene expression
(Naimi et al. 2021). CMC had only modest effects on levels of
flagellin and LPS measured in human embryonic kidney HEK
cells with this donor.
Seven publications reported on fermentation of CMC
in vitro. CMC was barely fermented, as shown by no changes
in MW, and showed similar total SCFA production as the con­
trols when tested with feces from three healthy adult donors
(one man and two women) (He et al. 2022). However, CMC
increased the relative abundance of the Firmicutes phylum.
At the operational taxonomic unit (OTU) level, CMC could
apparently favour the growth of certain bacterial families, but
which one(s) changed with the donor.
Using fecal microbiota from 10 adult humans (four women
and six men), 0.005%, 0.05% and 0.5% CMC did not affect
the structure of the cell population and even increased total
cell count at higher concentrations, although the fraction of
living cells was unaffected by all CMC concentrations
(Miclotte et al. 2020). CMC had small impact on microbial
composition in all three concentrations and had no effect on
the general microbial metabolic activity, including SCFA pro­
duction. The effects of CMC on several parameters, including
flagellin levels, varied strongly depending on the microbiota
donor.
In a study of in vitro batch fecal fermentation, CMC
showed no or minimal effects on the broad composition and
fermentation capacity of the fecal microbiome, had no effects
on the production of any SCFA or BCFA, and had no
significant effects on the growth of broad bacterial popula­
tions, when using fecal samples from 13 young healthy
donors (seven women and six men) (Gerasimidis et al. 2020).
In a study of in vitro fermentation of CMC using fecal sam­
ples from 10 healthy adult donors (5 of each sex), CMC
increased acetic acid, butyric acid and propionic acid, as well
as a total of six SCFA (Chen et al. 2020). With CMC, signifi­
cantly lower Shannon diversity index than with controls was
observed, indicating lower microbial diversity. After CMC
exposure, there was a higher relative abundance of
Fusobacteria, Bacteroides and Proteabacteria, and a relative
decrease in Firmicutes, at the phyla level. There were clear
spatial separation and clustering of the fermentation bacteria
from the CMC-treated vs. control groups. Unique microbiota
changes after CMC treatment were increased abundance of
Escherichia-Shigella and Lachnoclostridium, and decreased
abundance of Bifidobacterium, Streptococcus, Fecalibacterium
and Ruminococcus_torques_group.
In a study on fibre composition of CMC, the majority of
CMC was soluble fibre and it was estimated that about 55%
of the glucose residues in CMC had at least one carboxy­
methyl group substitution and therefore would not be avail­
able for fermentation by gut bacteria (Bliss et al. 2013).
Further, in vitro incubation was studied with feces from 13
adult persons (nine women and four men) with fecal incon­
tinence and adverse gastrointestinal symptoms, before and
after consumption of CMC given in muffins and juice twice
daily for 20–21 days, or a placebo without CMC. The results
indicated that prior consumption of a specific fibre did not
increase its degradation by fecal bacteria.
In vitro fermentation of CMC with feces from three healthy
adult male donors resulted in formation of the SCFAs acetate,
butyrate and propionate during 24 h (Bourquin et al. 1993).
In vitro fermentation with slurries of cecal contents from
rats fed a fibre-free diet prepared under anaerobic conditions
showed that CMC was poorly fermented, and thus, not sig­
nificantly degraded (Wyatt et al. 1988).

3.3.4. Hypotheses on adverse effects of CMC
The number of research publications studying CMC in rela­
tion to various hypotheses on adverse effects is shown in
Figure 11. Effects on the gut microbiome as well as inflam­
mation are studied in the highest number of publications,
with 19 and 17 publications, respectively. Metabolic effects
(11) and fermentation (10) are the next most common topics,
followed by intestinal physiology, permeability of the intes­
tinal barrier and cancer, with 5, 5 and 4 publications,
respectively.
3.3.5. Data relevant for exposure
Seventeen publications had some information relevant for
exposure to CMC. In a review, celluloses, including CMC,
were reported to be present in 2% of all food products in
the food supply in EU (Cox et al. 2021).
Mean dietary exposure to CMC in the population aged
>2 years in USA was estimated to be 30, 27, 25 and 24 mg/
kg bw per day in the time periods 1999–2002 and 2003–
2010, both in the NHANES study, and 1999–2002 and 2003–
2010, both in the NTP NET-NID study, respectively (Shah
et al. 2017). The 90th percentile dietary exposure to CMC was
estimated to be 70, 60, 53 and 53 mg/kg bw per day and the
percentages of users of CMC were 95, 100, 100 and 100, in
the same time periods and studies, respectively. This study
was also reported in review publications by Laster et al.
(2019), Laudisi et al. (2019) and Cox et al. (2021).
In EFSA (2018b), total exposure was only indicated for all
the ten evaluated microcrystalline, powdered and modified
celluloses together, being around 660–900 mg/kg bw per
day, not for E 466 alone.
The incidence and prevalence of IBD are increasing globally,
both in Western countries and Asia, although thought to be
lower in Asia, whereas prevalences in Europe are as high as
those in USA, according to a review by Park et al. (2020). These
increases are also seen in children. The increases in IBD were
hypothesized to be caused by numerous environmental factors,
including dietary factors, such as CMC, and nutritional factors.
Average exposure to CMC in children of both sexes (n ¼ 138,
mean age 14.2 ± 2.8 years) with CD was quantified using data
from three 24-h recall days over two years obtained by tele­
phone (Lee et al. 2018). A total of 1325 unique foods were
recorded and 0.4% of the foods contained CMC. Mean ± SD fre­
quencies of exposures to CMC per day were 0.05 ± 0.13 (range
0–0.67). The frequency of exposure to CMC (mean ± SD) in 20
patients with CD using 7-day food diaries analyzed for habitual
intake was 0.14 ± 0.30 per day (Sandall et al. 2020).
In an abstract, the exposures to selected food additives,
including CMC (and CGN), per day in children with IBD and
the association with IBD relapse were reported (Ho et al.
2021) (see Section 3.2.5.). An abstract reported emulsifier
exposure using baseline dietary data from adults and chil­
dren with IBD enrolled in the Australian AIM study, a multi­
center, prospective, longitudinal cohort study (Reveley et al.
2022) (see Section 3.2.5.). It was reported that daily occasions
of exposure (range) to CMC in patients with IBD were 0.3 (0–
3.0) in children (n ¼ 21) and 0.1 (0–1.7) in adults (n ¼ 156).
Three abstracts determined previous and current
CRITICAL REVIEWS IN TOXICOLOGY 545
consumption of nine food additives, including CMC (and
CGN), in patients with CD, their first-degree relatives, their
non-related healthy household members and 22 healthy con­
trols (Zhang et al. 2022a, 2022b, 2022c) (see Section 3.2.5.).
Exposure was mentioned, but without providing quantita­
tive data on CMC, in a review by Martino et al. (2017) and in
an exposure publication by Chazelas et al. (2021).
CMC was present in 12.7% of sixty-one EEN formulas used
to induce remission in active CD (Logan et al. 2020).
The International Organization for the Study of
Inflammatory Bowel Disease (IOIBD) has recommended that
“it may be prudent to limit intake of CMC” (with evidence
level very low, agreement 92.3%) in both UC and CD, based
on data from epidemiology, animal models and one very
small RCT (Levine et al. 2020).
3.3.6. Toxicokinetics and ADME studies
E 466 was not absorbed intact, not fermented and was
excreted intact via feces (>90%) in rats, rabbits and humans
(EFSA 2018b, 2020).
Only one study included both modified, i.e. partially
hydrolysed CMC, depolymerized with a cellulase-containing
enzyme preparation from Trichoderma longhibrachiatum
(CMC-ENZ), and non-modified CMC, studying toxicokinetics of
the two CMC variants (B€ar et al. 1995). Five % of each CMC
type were administered in the diet to rats for two weeks, fol­
lowed by a single gavage of 14C-CMC-ENZ or 14C-CMC
(500 mg/kg bw). About 95% of the label was excreted in
feces, <2% in urine, <1% with CO2 and a small fraction was
retained in the body (CMC, 0.58%; CMC-ENZ, 0.75%). Tissue
retention of 14C was highest in the liver after both treat­
ments. Only about 49% and 65% of fecal 14C was extracted
with water in the 14C-CMC- and 14C-CMC-ENZ-dosed rats,
respectively. Gel permeation chromatography of the dosing
solutions and the fecal extracts revealed that CMC was depo­
lymerized during intestinal passage, whereas CMC-ENZ was
excreted nearly unchanged. Consequently, the MW distribu­
tion of the 14C-CMC and 14C-CMC-ENZ fecal excretion prod­
ucts was similar. The authors concluded that there was no
toxicologically relevant difference between the disposition of
CMC and CMC-ENZ in rats.
3.3.7. Risk assessment opinions
Five relevant risk assessments of CMC had been performed by
EFSA. Sodium CMC (E 466) was evaluated together with other
cellulose food additives in the group “chemically modified
celluloses” by the ANS Panel (EFSA 2018b), based on read-
across between all the celluloses because of their structural,
physicochemical and biological similarities. Note that modified
CMC in this and other EFSA opinions does not have the same
meaning as in other publications (B€ar et al. 1995), and, thus,
CMC was not coded as modified for the EFSA opinions in ER.
The acute toxicity of celluloses was low and there was no gen­
otoxic or carcinogenic concern. Short-term and subchronic
dietary toxicity studies at levels up to 10% in the feed did not
indicate specific adverse effects. In chronic toxicity studies, the
NOAEL values ranged up to 9000 mg/kg bw per day of
546 M. TAHIRI ET AL.
celluloses. Adverse effects on reproductive performance or
developmental effects were not observed with celluloses at
doses greater than 1000 mg/kg bw by gavage (often the high­
est dose tested). EFSA concluded that there was no need for a
numerical ADI value and that there would be no safety concern
at the reported uses and use levels for the evaluated celluloses,
including for E 466.
The EFSA Panel on Additives and Products or Substances
used in Animal Feed (FEEDAP) assessed sodium CMC as a
feed additive and concluded that it was safe for all animal
species and that the use of this substance in animal nutrition
was of no concern for consumer safety (EFSA 2020). Likewise,
the EFSA Panel on Food Contact Materials, Enzymes,
Flavorings and Processing Aids (CEF) assessed (sodium) CMC
and concluded that it did not raise a safety concern for the
consumer when used as a component of a mixture in active
food contact materials intended used as moisture and liquid
absorbers in the packaging of perishable foods to extend
their shelf-life (EFSA 2012, 2018c).
As a follow-up of the assessment by the ANS Panel in
2018 (EFSA 2018b), the Panel on Food Additives and
Flavorings (FAF) assessed the safety of E 466 for its uses as a
food additive in food for infants below 16 weeks of age
belonging to FC 13.1.5.1 (Dietary foods for infants for special
medical purposes and special formulae for infants) and
addressed the issues previously identified when used in food
for the general population, including the safety assessment
for FC 13.1.5.1 and FC 13.1.5.2 (Dietary foods for babies and
young children for special medical purposes) (EFSA 2022).
The business operators declared that E 466 was not used in
food for infants below 16 weeks of age and in FC 13.1.5.1.
Due to the lack of data, an assessment was not performed
for this FC and age group. The business operators did not
provide data to support the uses of E 466 in FC 13.1.5.2. Due
to the almost unchanged database compared to in the previ­
ous evaluation, the FAF Panel confirmed the previous conclu­
sion that the available data was still not sufficient for an
adequate assessment of the safety of use of E 466 in infants
and young children consuming foods belonging to the FC
13.1.5.2.
3.3.8. Non-systematic (narrative) reviews, commentaries/
editorials/letters and conference abstracts
No other scoping review/scoping study or systematic review
on effects of CMC on the intestines in line with our objec­
tives was detected among the published literature. In total,
26 narrative reviews on CMC (all non-modified) were
included. The reviews on CMC were published more recently,
between 2015 and 2023, compared with many of those on
CGN. Eight narrative reviews on CMC were included, review­
ing effects on inflammatory bowel diseases, the microbiome
or CMC as an obesogenic chemical (Glade and Meguid 2016;
Viennois and Chassaing 2018; Laudisi et al. 2019; Partridge
et al. 2019; Goens and Micic 2020; Park et al. 2020; Rinninella
et al. 2020; Heindel et al. 2022). In addition, 18 publications,
also including CGN, reviewed effects of CMC on intestinal
inflammation and disease, microbiota, intestinal permeability,
obesity and metabolic dysregulation, or allergy (Liu et al.
2015; Martino et al. 2017; Paula Neto et al. 2017; Kikut et al.
2018; Halmos et al. 2019; Laster et al. 2019; Marion-Letellier
et al. 2019; Vo et al. 2019; Zino €cker 2019; Bancil et al. 2021;
Borsani et al. 2021; Cox et al. 2021; Sasson et al. 2021; Tan
and Nie 2021; De Siena et al. 2022; Levine et al. 2022; Liu
et al. 2022; Kuang et al. 2023).
The nine commentaries/editorials/letters on CMC pub­
lished 2015–2022 all discussed non-modified CMC. Two of
them (Bordon 2015; Cani 2015) concerned the published
study on CMC by Chassaing et al. (2015) and two concerned
the publication by Chassaing et al. (2022a); Wellens et al.
(2022) and the answer by Chassaing et al. (2022b). One dis­
cussed CMC in relation to IBD in Australia (Niewiadomski
2018), whereas four were on adverse effects on the intestines
of several food additives and also mentioned CGN (Zino €cker
2019; Zino €cker and Lindseth 2019; Cox and Sandall 2021;
Wellens et al. 2022).
All the 24 conference abstracts on CMC published 2005–
2022 were reporting studies on non-modified CMC, mostly in
animals or in vitro. Two abstracts (Cochrane Central Register
of Controlled Trials 2018; Chassaing et al. 2020) on a random­
ized double-blind, controlled feeding experiment with CMC
had later been published in full (Chassaing et al. 2022a).
Fourteen abstracts were on one or more effects of CMC on
intestinal inflammation, microbiome and metabolic changes,
intestinal permeability, mucin-producing cells, or obesity,
metabolic syndrome and thermogenesis (Bowen-Yacyshyn
et al. 2005; Ung et al. 2009; Yacyshyn et al. 2011; Chassaing
and Gewirtz 2013; Chassaing and Gewirtz 2014; Chassaing
et al. 2016; Chassaing et al. 2017b; Chassaing and Gewirtz
2017a; Chassaing and Gewirtz 2017b; Behr et al. 2018; Xu
et al. 2019; Zheng et al. 2019; Zheng et al. 2020; Zangara
et al. 2021). In addition, four abstracts included both CMC
and CGN (Schooth et al. 2020; Zhang et al. 2022a, 2022b,
2022c).
Four additional abstracts reported human data not yet
presented in full publications. The exposures to selected food
additives per day in children with IBD and the association
with IBD relapse were reported (Ho et al. 2021). The results
in this abstract were described for ten food additives
together, including CMC (and CGN) (see Section 3.2.5.).
In an abstract by Kang et al. (2021), it was examined in vitro
how CMC affected CD mucosa-associated microbiota (CD-
MAM) in microbial cultures with cryopreserved biopsies from
five CD patients. These cultures were inoculated in medium
with 0.1% CMC (a concentration found in food) or without
additive (control). The addition of CMC appeared to have only
modest effects on the CD-MAM communities from 4 of 5 CD
patients. However, in one person, CMC resulted in large expan­
sions in the relative abundance of Bacteroides, Proteus,
Morganella, the family Enterobacteriaceae and Enterococcus,
with a concordant reduction in the Streptococcus lineages.
Emulsifier exposure was examined using baseline dietary
data from adults and children with IBD enrolled in the
Australian AIM study, a multicenter, prospective, longitudinal
cohort study, and compared with exposure of healthy con­
trols (Reveley et al. 2022). The results in this abstract were
described for six food additives together, including CMC (and
CGN) (see Section 3.2.5.).
 CRITICAL REVIEWS IN TOXICOLOGY 547

Figure 12. Image of the interactive research map showing various types of CGN and CMC in the columns, type of publications in the rows and hypotheses on the
adverse effects on the intestines as bubbles. The size of the bubbles indicates the number of studies in each square. From this map, available and missing data in
the various categories can be easily seen. The map can be downloaded from Supplementary materials Figure 1 be used interactively.

Figure 13. Image of the interactive research map showing various types of CGN and CMC in the columns, human populations (healthy general population or
patients) in the rows and hypotheses on the adverse effects on the intestines as bubbles. The size of the bubbles indicates the number of studies in each square.
From this map, available and missing data in the various categories can be easily seen. The map can be downloaded from Supplementary materials Figure 2 to be
used interactively.

An abstract reported generation of a gnotobiotic human organoids were microinjected with a live mixture of four
intestinal organoid microcosm system (HIOMS) as an in vitro functionally distinctive bacteria species, Akkermansia mucini­
model of the human gut microbiome (Xu et al. 2020). The phila, Bacteroides acidifaciens, B. cellulosilyticus and B.
548 M. TAHIRI ET AL.
intestinalis, supplied with CMC and co-cultured for 48 h. A
stable colonization of A. muciniphila and B. intestinalis was
demonstrated and their abundance was not affected by CMC.
However, CMC increased a-fucosidase activity by 360%.
3.4. Evidence presented as interactive research maps
The included evidence was presented as interactive research
maps, which give a visual overview of where in this area
there is quite a lot, some or non-existent scientific know­
ledge. The first interactive research map on evidence found
in the 262 included publications (Figure 12) is showing vari­
ous types of CGN and CMC in the columns, type of publica­
tions in the rows and hypotheses on the adverse effects on
the intestines as bubbles. The size of the bubbles indicates
the number of studies in each square.
The second interactive research map (Figure 13) is show­
ing various types of CGN and CMC in the columns, human
populations (healthy general population or patients) in the
rows and hypotheses on the adverse effects on the intestines
as bubbles.
From these maps, available and missing data in the vari­
ous categories can be easily seen. The research gaps that can
be identified from the maps indicate where future research
may be useful to carry out in relation to human (healthy per­
sons or patients), animal and in vitro studies (publication
types), and which potential adverse effects in the gut should
be studied in more detail resulting from the consumption of
CGN and CMC.
The interactive research maps are available as HTML files
and can be downloaded by clicking on the links below and
displayed in any browser, without the need for login. To use
the maps interactively, one can click anywhere in a column,
row or bubble and access all studies that address the particu­
lar food additive, publication type etc.
Hypotheses of adverse effects on the intestines caused by various
types of CGN and CMC in all the included 262 publications,
stratified by publication types.
Hypotheses of adverse effects on the intestines caused by various
types of CGN and CMC in human studies (healthy general population
or patients).
4. Discussion
4.1. Methodological aspects
Summaries of results from individual studies and across simi­
lar studies are usually not included in scoping reviews (Munn
et al. 2018). However, to get an overview of the existing
knowledge of the adverse effects of CGN and CMC, short
summaries of the individual publications were included. This
was considered particularly necessary for CGN, since some
commentaries indicated that adverse effects were only
caused by degraded CGN and not by native CGN. Therefore,
summaries were made for comparison of effects between
native and degraded CGN, as discussed in Section 4.3.1.
Many of the traditional, narrative review publications
reported data on both CGN and CMC, as well as on several
other food additives. In addition to more general information
about these food additives, they gave relevant information
about the concerns for adverse health effects, although some
mainly focused on positive effects. The commentaries, editor­
ial articles and letters also gave insight into the controversies
regarding their potential adverse health effects. A summary
of some arguments for or against the likelihood that CGN
may have adverse effects can be found in Liao et al. (2021).
The conference abstracts were included to pick up newer
research indicating adverse effects, however, the reported
results were most often covered also by full publications. As
opposed to in this scoping review, a future full systematic
review on adverse effects of these food additives would
include only the primary research publications and available
systematic reviews.
4.2. Number of various categories of publications on
CGN and CMC
In total, 2572 publications from two literature searches were
screened against predefined inclusion/exclusion criteria, and
224 of them were included in this review. In addition, 38
publications from grey literature sources were included, mak­
ing a total of 262 investigated publications. Of these, 196
and 101 were on CGN and CMC, respectively. The potential
adverse effects of CGN or CMC on the intestines have been
studied mainly in individual research publications, mostly
performed in experimental animals or in vitro. For CGN, five,
69 and 33 studies on humans, experimental animals and
in vitro experiments were found, of which two and three, 38
and 31, and 14 and 26 publications studied degraded and
native (undegraded) CGN, respectively, in the three publica­
tion categories. For CMC, three human studies, 20 experimen­
tal animal studies and 14 in vitro studies were obtained. Only
one systematic review was found, which was relevant for
native CGN in humans, although a few were listed as
planned studies (scored as abstracts) in the Cochrane data­
base, but not yet published. Publications (of all types) having
information relevant for exposure were 20 and 17 for food
grade CGN and CMC, respectively. Publications with data on
toxicokinetics and/or ADME were three and two, and risk
assessment opinions were two and five, for CGN and CMC,
respectively.
High numbers of non-systematic (narrative) review articles
were obtained, 54 on CGN and 26 on CMC, reflecting the
ongoing controversy about potential adverse effects of these
and other food additives, as well as reviewing their use as
pharmaceuticals, nutritional or functional food sources or pre­
biotics. Many of these reviews discussed food additives in
general and did not focus specifically on CGN or CMC.
Insight into the controversial issues was also gained from the
commentaries/editorials/letters, 14 on CGN and nine on CMC.
Conferences abstracts, 23 on CGN and 24 on CMC, were
included in order to obtained more recent hypotheses,
however, most of these were already available as full
publications.
The included publications presented several hypotheses
on how the two food additives may be associated with

adverse effects on the intestines, often studying effects
related to several hypotheses in the same publications, and
most often, several publications had been studying the same
hypotheses and effects.
No other scoping review/scoping study or systematic
review on effects of CGN and CMC on the intestines in line
with our objectives was detected by search in Medline up to
June 12, 2023.
4.3. Main findings on effects of CGN relevant for human
intestinal health
Effects on inflammation and effects on the microbiome,
including fermentation, were the most studied endpoints
both with degraded and native CGN in the research publica­
tions across the three evidence streams, whereas permeabil­
ity of the intestinal barrier was somewhat less studied (Figure
8), obviously reflecting the search terms used in the literature
searches. Higher number of publications had studied inflam­
mation with degraded CGN, because of its use as a model
for inflammation, and somewhat higher number had studied
the microbiome with native CGN. Effects on the immune sys­
tem and cancer were also studied with both variants of CGN.
Among the research publications, only five human studies
of CGN and effects on the intestines were included, all on
adult patients with gastrointestinal conditions, three on
native CGN and two on degraded CGN. In the randomized
double-blind, placebo-controlled, clinical trial by
Bhattacharyya et al. (2017), including 12 UC patients in remis­
sion given 100 mg food grade j-, i- and k-CGN or placebo in
gelatin capsules, three of five who received CGN relapsed,
whereas none of the seven patients receiving placebo
relapsed. However, limited weight can be put on this study
because of the low number of participants. When it was
included in a systematic review on dietary interventions of
remission in IBD, Limketkai et al. (2019) calculated a risk ratio
with 95% CI of 0.5 (0.15, 1.64) for this study and a very low
score for certainty of the evidence (GRADE) and high risk of
bias. In the other three human studies in patients with peptic
ulcers or malignant disease, which were quite old and with­
out control groups, few and mild adverse effects (dizziness,
mild diarrhea, constipation, but no ulceration) were reported
either with native or degraded CGN (Heineken 1961; Evans
et al. 1965; Grasso et al. 1973). No research publications were
found on adverse effects of degraded or native CGN on
healthy adults or on children or adolescents.
In addition, of the three publications on human studies
that were available only as abstracts, two reported results on
a randomized, double-blind, placebo-controlled, cross-over
trial (Wagner et al. 2018a, 2018b), in which 20 healthy males
were given 250 mg CGN or placebo twice daily for 14 days.
Intestinal permeability increased after CGN vs. placebo
(p ¼ 0.03), whereas whole-body insulin sensitivity index (ISI),
ISIclamp and OGTT did not differ between the groups. No
changes of hepatic fat content, body fat mass, fat distribu­
tion, liver transaminases or inflammatory cytokines were
observed.
CRITICAL REVIEWS IN TOXICOLOGY 549
Current understanding of the pathogenesis of IBD sug­
gests an interaction between genetic susceptibility of the
immune system to changes in microbiota, likely due to envir­
onmental exposures that trigger a dysregulated immune
response (Niewiadomski 2018). The rapid rise in incidence of
IBD over the last few decades also strongly points to the
environment playing a significant role, which may include
diet in general or food additives specifically. Dietary interven­
tions in patients with IBD are difficult to conclude upon with
certainty, since there are many different factors that contrib­
ute to the symptoms, and the impact of a complex and vary­
ing diet in general is difficult to evaluate. Participants in
studies on food additive-free diets most often abstain from
foods with many different additives, making it difficult to
pin-point any adverse effects to one specific food additive.
Among the 69 publications with studies of CGN in experi­
mental animals, 38 and 31 publications investigated effects
of degraded and native CGN, respectively. Several species of
experimental animals were used in both groups of studies, in
decreasing numbers, rats, guinea pigs, mice, rabbits, ham­
sters, monkeys, pigs and ferrets. Most of the publications did
not give a clear reason for the choice of animal model, how­
ever, the large use of guinea pigs and rabbits was mainly
from earlier years when animal models of inflammation with
degraded CGN were established and investigated for testing
of anti-inflammatory drugs or other treatments. The three
publications using monkeys, likely being most similar to
humans, were all from the 1970s. In two of the studies,
adverse effects (intestinal mucosa ulceration, and effects on
lysosomes and macrophages, respectively) were observed in
Rhesus monkeys given 2% degraded CGN (C16), but not
when given native CGN (Benitz et al. 1973; Mankes and
Abraham 1975), whereas a study on two squirrel monkeys
given 1.5% degraded CGN for 28 days did not find any
adverse effects (Grasso et al. 1973).
A large variety of doses and exposure times were used in
the animal studies. Concentrations of degraded CGN in diet
or drinking water used were from 0.2% to 10% with exposure
from 1–2 weeks up to 28 months - the longest studying ulcer­
ations in rabbit (Kitano et al. 1986), whereas native CGN was
used in concentrations of 0.02% to 20% with exposure from
one week up to lifetime - the longest studying cancer in rats
and hamsters (Rustia et al. 1980). Compared to the exposure
from foods consumed by humans, these highest concentra­
tions may not be relevant for studies of effects on human
intestinal health of food additives.
Some studies described a model for IBD where rats were
sensitized by a s.c. injection of a solution of degraded CGN
and Freund’s complete adjuvant followed by oral administra­
tion of the same CGN solution in drinking water (Moyana
and Lalonde 1990, 1991). Although not directly relevant for
humans exposed to CGN in food, prior sensitization aggra­
vated the ulceration and inflammation in the intestines. Thus,
the studies showed that the immune system is involved in
these effects. In another inflammation model, mice were
given CGN and then later were inoculated with Citrobacter
freundii or Citrobacter rodentium bacteria (Wu et al. 2017,
2021, 2022). The results indicated that CGN aggravated the
bacteria-induced gut inflammation, which may be relevant
550 M. TAHIRI ET AL.
for humans having bacterial infections in their gastrointes­
tinal tract. As well as when exposed together with patho­
genic microorganisms, synergistic effects of CGN on
inflammation were observed when it was given before other
inflammatory chemical agents such as trinitrobenzene sul­
fonic acid (Wu et al. 2016a) and oxazolone (Wu et al. 2016b).
The order of potency was i- > k- > j-CGN for decreasing
effect on body weight gain in guinea pigs given 3% of either
degraded j-, i- and k-CGN in drinking water for three weeks,
although the mean daily CGN consumption per group was in
the reverse order, indicating that potency was not related to
intake concentration (Norris et al. 1981). Comparable activ­
ities of colitis shown by histological score and TNF-a serum
levels were induced by native j-, i- and k-CGN given in
drinking water for six weeks to mice, and all three decreased
the anti-inflammatory gut bacterium Akkermansia muciniphila,
although they showed some other differences on the micro­
biome (Shang et al. 2017). Regarding mechanistic differences
between the three main forms of CGN, when comparing IL-8
secretion between the native j-, i- and k-CGNs in human
cells in vitro, the response was highest for k-, next for j- and
then i-CGN (Borthakur et al. 2007). There is at present not
sufficient evidence to understand potential differences in
adverse effects between the various CGN forms.
In total, 33 publications with in vitro experiments reported
adverse effects on the intestines of CGN, of which 14 and 26
studies had used degraded and native CGN, respectively.
Human colonic microbiota samples from feces studied in an
in vitro batch fermentation system showed that high MW
j-CGN (450 or 100 kDa) remained undegraded, whereas low
MW j-CGN, i.e. KCO (�4.5 kDa) were degraded (Yin et al.
2021). In vitro fermentation by human gut microbiota of two
j-CGN oligosaccharides (MW <3000 kDa with different DP),
called KO3 and KO6, obtained from simulated gastric diges­
tion, were both readily degraded (Sun et al. 2019). A third
study also showed that j-KCO were degraded by Bacteroides
xylanisolvens and E. coli isolated from human gut microbiota
(Li et al. 2017). Thus, at least in vitro, human fecal bacteria
appear to be able to degrade CGN oligosaccharides to even
lower MW, but apparently not the high MW CGN which is
used in food additives.
Some in vitro studies found that native CGN activated the
Wnt/b-catenin signaling pathway in human cells, suggesting
a role in malignant transformation in the intestine
(Bhattacharyya et al. 2007; Bhattacharyya et al. 2014b).
However, when native CGN (Gelcarin, largely composed of j-
and k-CGN, with MW of approximately 800 kDa), in doses of
0.5, 2.5 or 5% given in the diet to rats and hamsters for life,
no significant increase in tumor incidence was seen (Rustia
et al. 1980). The potential risk of intestinal cancer in humans
from a low intake of CGN as food additive needs updated
studies, both regarding promoter and initiating effects.
Regarding molecular mechanisms for the adverse effects
of CGN, k-CGN induced inflammation via activation of NF-jB
and AP-1 pathways in macrophages and enhanced LPS-
induced TNF-a secretion through AP-1 (Chen et al. 2014). In
this study, degraded k-CGN was much stronger than native
k-CGN in the activation of macrophages to secrete TNF-a.
Native k-CGN increased Bcl10, IjBa phosphorylation, total
and nuclear NF-jB, and secretion of IL-8 in human cell lines,
implicating this signaling pathway in intestinal inflammation
induced by CGN (Borthakur et al. 2007). NF-jB binding to the
BCL10 promoter involved the components RelA and RelB,
and could lead to prolonged activation of the k-CGN-induced
inflammation by a transcriptional mechanism involving a
NF-jB-BCL10 loop (Borthakur et al. 2012). j-CGN did not
stimulate IL-8 alone, but increased LPS-induced IL-8 secretion
via the Bcl10-NFjB pathway and acted in synergy with LPS
to increase the expression of MD-2 and CD14 (Wu et al.
2017). Thus, these signaling pathways seems to be important
for the effects of CGN, although one publication did not sup­
port this view (McKim et al. 2015).
4.3.1. Degraded versus non-degraded (native) CGN
Degraded forms of CGN, including PGN, are not approved as
food additives in the EU, nor are they used for this purpose
in other countries such as USA (EFSA 2018a). Thus, the
degraded CGN may be regarded as not relevant for the dis­
cussion on food additives and human intestinal health, since
as such they are not indicative of the potential adverse
effects of CGN used in regulated food additives today. It was
argued in the literature obtained that the lack of understand­
ing of the difference between non-degraded and degraded
CGN, and the misuse of CGN terms, may have contributed to
the confusion and misinterpretation of the results in many of
the studies, leading to the worries about adverse health
effects in the general population and also among scientists
(David et al. 2018, 2019; Weiner and McKim 2019). Likewise,
it could be argued that degraded CGN and PGN should not
have been included in this scoping review. However, the dis­
tinction between CGN of different MW was not clear to the
authors before this work started. Furthermore, it become
apparent from an early look at the literature that even with
native, non-degraded CGN there were numerous publications
reporting apparently adverse effects. The degraded forms of
CGN were therefore included in this review for comparison
with native CGN and to get a more complete overview of
the controversial issues.
Thus, one of the major controversial issues in this field
seems to be differences in health risk from degraded CGN vs.
native CGN. A distinction is made between the food grade
forms of CGN of high MW (MW 200–800 kDa) used in the
food additives E 407/E 407a (EFSA 2018a), which are claimed
not to be absorbed from the intestinal tract nor being
degraded during transit, and degraded CGN such as PGN,
C16 and Ebimar (MW 10–20 kDa). The degraded CGN forms
are made by low pH (<2.0) and high temperature (>80 � C),
and are often used to induce inflammation and ulcerations in
the intestinal tract of rats, guinea pigs and rabbits as experi­
mental models of inflammation in general or human IBD.
These CGN forms are not the same substances as commercial
food grade CGN. The low MW of degraded CGN forms
reduces the strength of protein binding, which allows
absorption from the intestines (McKim et al. 2019). However,
in many of the publications, the MW of the CGN (and the
form of CGN) was not specified.

The next issue is whether the presence of low MW com­
ponents in high MW food grade CGN or possible generation
of low MW CGN following food processing or gastric hydroly­
sis of native CGN may cause adverse effects in humans. Trace
amounts of native CGN have been shown to cross the intes­
tinal barrier and are demonstrated within intestinal lamina
propria cells and macrophages, and are absorbed via Peyer’s
patches and cecal lymph nodes in animals (Mankes and
Abraham 1975; Ishioka et al. 1987; Nicklin and Miller 1989).
Based on the ADME data available, EFSA (2018a) concluded
that CGN with high MW, for example j-/k-CGN with MW of
about 800 kDa studied in Rhesus monkeys (Mankes and
Abraham 1975), was not absorbed, while low MW CGN
(<88 kDa) was found in tissues (liver, intestines) and in urine
of rats and guinea pigs after oral administration.
Based on the data provided to EFSA (2018a) after a call
for data, the overlap between the low MW-average MW tail
of food-grade CGN and high MW-average MW tail of PGN is
expected to increase with the decrease of the weight-average
MW of the CGN. An example was given: for CGN with the
weight-average MW of about 770 kDa, the overlap would
encompass from ca. 30 kDa up to ca. 200 KDa molecules.
Thus, although for a commercially available CGN with a
weight-average MW of 200 kDa, the preparation would
encompass a larger fraction with a MW <50 kDa. EFSA con­
cluded that it could not be excluded that commercial CGN
preparations may contain considerable amounts of low MW
molecules when used as food additive. Therefore, with
respect to the definition of E 407 and E 407a in the
Commission Regulation (EU) No. 231/2012 (2012), the weight-
average MW range should be specified in a narrow way
avoiding a significant overlap with the MW range of PGN.
Furthermore, there is an urgent need to develop a validated
method to quantify the low MW CGN with the limit of 5%
(EFSA 2018a). Overall, EFSA considered that the information
available suggested that the ulceration and bleeding in the
intestines reported in some studies having tested native CGN
could be associated with the presence of low MW CGN (EFSA
2018a).
Regarding risk of cancer, this distinction in MW of CGN is
made by IARC, since native (undegraded) CGN has been clas­
sified as Group 3, i.e. unclassifiable with respect to carcino­
genicity in humans, whereas degraded CGN (PGN) is
classified as Group 2B, a possible human carcinogen, based
on animal data (IARC 1987).
Information about the stability of high MW CGN in food
when exposed to various temperature and pH conditions
under the authorized food use is also limited and contradict­
ory (EFSA 2018a).
4.4. Main findings on effects of CMC relevant for human
intestinal health
The three types of research publications reported effects on
intestinal microbiota, including studies of fermentation of
CMC, inflammatory potential, metabolic effects, effects on
intestinal physiology, permeability of the intestinal barrier
and cancer (Figure 11).
CRITICAL REVIEWS IN TOXICOLOGY 551
Only three human studies on CMC were found, of which a
recent study (Chassaing et al. 2022a) indicated that CMC in
high, but realistic doses, disturbed the intestinal microbiota
and led to inflammation. However, the number of subjects in
this study was low (in total 16) and they were exposed only
for 11 days, thus, such a study needs to be repeated with
higher numbers of participants exposed for longer time. In
an older study (Anderson et al. 1986) with five healthy men
given 15 g CMC daily for 23 days, only some inconsistent
changes in fecal physiology were observed. One other study
(Ohnishi et al. 2019) reported that CMC appeared to have
caused anaphylaxis in a young girl, although this appears to
be a low risk.
Of the 20 experimental animal studies on CMC, most of
them used continuous doses of 1–2% CMC in drinking water
or other oral exposure in two to 15 weeks, see for instance
Viennois et al. (2020) and Rousta et al. (2021). If this is to be
transferable to humans, it means that all the food consumed
daily contains this concentration of CMC, which is likely an
overestimation. However, a high intake of CMC may be pos­
sible in some individuals depending on their dietary habits,
such as if eating a lot of processed foods. Also of relevance,
it is not known whether some persons are more susceptible
to adverse effects of CMC than others. The sensitivity to CMC
may possibly be species-specific, so that typical laboratory
animals such as rodents may react differently to CMC than
humans.
As inflammation is implicated in intestinal cancer, some
studies tested CMC in various animal models of cancer pro­
motion. One % CMC in drinking water for ca. 14 weeks exa­
cerbated both number of tumors and tumor area after
initiation of mice with the colon carcinogen AOM and expos­
ure to DSS to induce inflammation (Viennois et al. 2017). In
another cancer model, using ApcMin/þ mice, having a muta­
tion (Min, for multiple intestinal neoplasia) in the tumor sup­
pressor gene Apc, involved in the Wnt/b-catenin signaling
pathway of colorectal cancer also in humans (Washington
and Zemper 2019), 1% CMC in drinking water for 15 weeks
increased small intestinal tumor development, apparently
independent of chronic intestinal inflammation, but associ­
ated with CMC’s effects on proliferation in the intestinal epi­
thelium and intestinal microbiota composition (Viennois and
Chassaing 2021). Thus, there are indications that CMC may
act as a tumor promoter in the colon, at least at these doses
given in drinking water.
The literature search resulted in 14 in vitro studies on
CMC, most of which used human microbiota or cells. Of the
six in vitro publications that reported on fermentation of
CMC using human donors, four found no effects such as
changes in MW or increased formation of SCFA (Bliss et al.
2013; Gerasimidis et al. 2020; Miclotte et al. 2020; He et al.
2022), whereas two studies reported increased individual or
total SCFA (Bourquin et al. 1993; Chen et al. 2020). Also, for
changes in the microbiota composition, the results varied
between the publications. Overall, it appeared that the result­
ing effects may vary strongly depending on the feces micro­
biota donor, possibly indicating individual susceptibility in
the population.
552 M. TAHIRI ET AL.
4.5. Strengths and limitations of this scoping review
This scoping review provides an overview of type of research
published on adverse effects on the intestines of the food
additives CGN/PES (E 407/E 407a) and CMC (E 466). The
strength of this scoping review is that it is based on exten­
sive literature searches comprising six library databases as
well as grey literature, without any time limit. The publica­
tions have been selected in a systematic way, to provide a
transparent overview of the current literature available about
potential adverse effects of CGN and CMC on the intestines.
It also included data from three evidence streams; human
data, experimental animal data and in vitro data. As such, it
is a good starting point for performing a systematic review
of adverse effects of these food additives. In addition, miss­
ing data within this research area are identified and illus­
trated in the interactive research maps (Figures 12 and 13),
which can be used for planning of further research employ­
ing studies in humans or animals on presence of such effects
and in vitro studies for the mechanisms behind the effects.
A limitation of this scoping review is that quality assess­
ments of the included studies and evaluation of risk of bias of
the overall body of publications were not performed.
However, the objective of this work was not to give a final
answer to whether these food additives constitute a risk to
intestinal health or not. In general, the aim of scoping reviews
is to provide extensive and transparent overviews of know­
ledge on a specific topic, in which the literature is systematic­
ally identified, selected and presented in text and visually,
rather than performing a qualitative or quantitative synthesis
of the data in order to assess the causality, or size and severity
of potential adverse effects of a stressor. Thus, the quality
assessment step is regarded as not strictly necessary for scop­
ing reviews as opposed to in systematic reviews of causal rela­
tionship between, as for this topic, the food additives CGN
and CMC, and their adverse effects on the intestines (Munn
et al. 2018; Peters et al. 2021b; Pollock et al. 2022).
Not all the data categorized from the publications fitted
one ER code precisely because of overlap within the publica­
tions of more than one study design, several streams of data,
several animal species etc. Thus, the coding system in ER
used to categorize the publications may not be entirely con­
sistent and exclusive, although the aim of each category
(code) was to be as specific as possible. This mixture of types
of data within the publications means that the total numbers
of publications in a category are often larger than what
would be expected from merely addition of numbers.
This scoping review is limited to effects on the intestines,
not other organs. The literature searches did not include all
languages. Thus, although extensive, this scoping review is
not considered a complete overview of publications on
adverse effects of CGN and CMC.
4.6. Evidence gaps and implications of the study
findings for further research
Two interactive research maps showing various types of CGN
and CMC in the columns, type of publications or human pop­
ulations in the rows, and hypotheses on the adverse effects
on the intestines as bubbles are illustrated in Figures 12 and
13. These maps can be used interactively via the links in
Section 3.4. The size of the bubbles indicates the number of
studies in each square, and thus, available and missing data
in the various categories can be easily seen. The references
to the publications constituting this map can be downloaded
directly from the map.
In a review, David et al. (2019) highlighted three unre­
solved information gaps for CGN. These were 1) insufficient
information on the current levels of public exposure to CGN,
2) a better understanding of the link between CGN’s physico­
chemical properties, its impact on digestive proteolysis and
the effects on the microbiome and inflammation, and 3) how
the digestion of CGN in the intestines may differentially affect
susceptible subjects in the population, such as children or
elderly persons, or patients with intestinal diseases.
The evidence analyzed in this scoping review suggests
that CGN, both degraded and native, and CMC, may cause
adverse effects on intestinal health, predominantly based on
studies in experimental animals and in vitro studies. These
adverse effects include decreased barrier function and/or
increased permeability of the intestines, leading to influx of
bacteria or chemicals, causing inflammation, often concomi­
tantly with potentially adverse changes in the intestinal
microbiota. However, since very few such publications were
available on human subjects, more research is needed in this
area before one can draw any firm conclusions on the effects
on humans. Also, the risk of cancer from food additives act­
ing as tumor promoters in animal models is more uncertain
in humans. There is a lack of human studies on the healthy
general population, especially children and adolescents, on
whom very few publications were found, and better data are
also needed for patients with various intestinal disease condi­
tions. The human studies should preferably be RCT as this is
defined as the gold standard for human studies, and of lon­
ger duration to examine the effects of long-term exposure to
the food additives. Studies using CGN or CMC as a dietary
supplement compared to a placebo supplement are probably
not comparable with the real-life dietary exposure to addi­
tives in foods. The results may be more realistic by compar­
ing a diet with or without a specific additive.
Older studies finding adverse effects, especially inflamma­
tion, from degraded CGN are not representative of the CGN
authorized for use as a food additive (E 407/E 407a) today.
However, many publications conclude that adverse effects
are also found in studies on native CGN. Compared to the
exposure from foodstuffs consumed by humans, the highest
concentrations of CGN or CMC used in the experimental ani­
mal studies may not be relevant for studies of effects on
human intestinal health. Both CGN and CMC are allowed at
QS in most food categories, however, there is a lack of data
on actual use levels in each food category, as well as
updated information on consumption of each category of
food for all age groups in the population.
Knowledge on the real-life exposure to the food additives
in all population groups is of importance for their risk assess­
ment, as for all other chemical substances. Dietary studies
are difficult to perform, and it must be taken into account
that the intake of food additives will vary from individual to

individual, over time and across national borders. National
food surveys should include data on concentrations of vari­
ous food additives present in the consumed foods, as well as
intake of the relevant foods. A problem is that such national
surveys are performed rather rarely, and thus, may not be
representative of new food products on the market and the
food additives therein, for example such as the recently
increased number of highly processed vegan food products
made to replace meat products.
Another issue is whether the research from experimental ani­
mals and in vitro model systems is representative of effects of CGN
or CMC in humans when being exposed to them as food addi­
tives. Especially, it is questionable whether exposure to CGN or
CMC in drinking water used in many animal experiments as the
sole source of liquid is representative of dietary exposure to addi­
tives in humans from varying types of foods. Food has a more
complex matrix where the additives may be bound to proteins,
affecting their absorption differently from water (EFSA 2018a).
5. Conclusions
Of the two food additives investigated in this scoping review,
CGN appears to be the one for which further research is most
urgently needed. Based on the information obtained, it appears
to be reasonably established that degraded CGN (with low
MW), including the artificially manufactured products such as
PGN, C16 or Ebimar, is absorbed and may be present in various
animal tissues. Degraded CGN has been extensively used as an
experimental animal model of inflammation. Thus, degraded
CGN appears clearly adverse in experimental animals, causing
colitis and possibly cancer, and likely may do so also in humans,
depending on the actual exposure (administration route, matrix
and dose). The role of native, non-degraded CGN is less clear,
however, a substantial number of publications reported adverse
effects on the intestines also of this form of CGN in experimen­
tal animals, supported by plausible mechanistic data from
in vitro studies. Also for CMC, the available animal studies indi­
cate that it may have potentially adverse effects on the intes­
tines, supported by mechanistic data from in vitro studies.
However, the relevance of these studies for human intake of
CMC as a food additive is unclear. For both food additives, their
role in reducing the intestinal barrier and increasing permeabil­
ity of the intestines to chemicals or bacteria, and their impact
on the gut microbiota, needs further clarification.
6. Recommendations for further work on adverse
effects of CGN and CMC
For further research of potential adverse health effects of CGN,
we recommend to focus the studies on the forms of CGN used
as food additives, i.e. food-grade CGN, according to Commission
Regulation (EU) No. 231/2012 (2012), and to characterize and
specify exactly which form(s) of CGN being present and their
levels, including the distribution of MW within the product.
For both CGN and CMC, new human studies, preferably
RCT, should have sufficient power, realistic dietary intake lev­
els, performed in healthy persons of all age groups, and also
in potentially vulnerable population groups such as young
CRITICAL REVIEWS IN TOXICOLOGY 553
children, pregnant women and elderly persons, as well as in
relevant patient groups, such as those with IBS, IBD, CD and
UC. One additive instead of several should be studied at the
time in order to be able to conclude on adversity of a spe­
cific additive. Good quality, longer-term animal studies with
dietary exposure to human relevant doses would also still be
useful, and the choice of animal model should be justified.
Future research should be performed in a transparent and
systematic way, with the hypothesis under scrutiny stated up
front, to gain trust from the public. In a future systematic
review on these food additives, quality assessments of the
included studies should be performed to better interpret and
understand their impact on the overall evidence. Future risk
assessments of CGN and CMC should include studies now
available on permeability and disturbance of barrier proper­
ties of the intestines and effects on the microbiome, repre­
senting relatively more recently occurring hypotheses on
potentially adverse effects than inflammation.
Acknowledgments
The authors would like to thank Ragnhild Agathe Tornes, the NIPH
library, for performing the literature searches. Håkon Valen and Gunn
Elisabeth Vist at NIPH, and Zak Ghouze at University College London, are
thanked for good advice and help with EPPI-Reviewer. The authors grate­
fully acknowledge the comments of the Editor and the external
reviewers selected by the Editor who were anonymous to the authors.
Declaration of interest
This research did not receive any grants from funding agencies in the public,
commercial or not-for-profit sectors, and the work was funded only by regu­
lar employment and engagements of the authors at NIPH. The motivation
for this work was questions to NIPH from the public about the safety of
these food additives. The authors report no conflicts of interests. The authors
have not participated in and do not anticipate participation in any legal,
regulatory or advocacy proceedings related to the contents of the paper.
Supplemental material
Supplementary data for this article can be accessed online at https://doi.
org/10.1080/10408444.2023.2270574.
ORCID
Mirlinda Tahiri http://orcid.org/0009-0006-7295-7882
Celine Johnsrud http://orcid.org/0009-0008-4855-0489
Inger-Lise Steffensen http://orcid.org/0000-0002-1859-0083
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Appendices
Appendix A. PRISMA-ScR checklist filled in for this
scoping review
Preferred Reporting Items for Systematic reviews and
Meta-Analyses extension for Scoping Reviews (PRISMA-
ScR) Checklist
562 M. TAHIRI ET AL.

SECTION ITEM PRISMA-ScR CHECKLIST ITEM REPORTED ON PAGE #

TITLE
Title 1 Identify the report as a scoping review. 1
ABSTRACT
Structured summary 2 Provide a structured summary that includes (as applicable): background, objectives, 1
eligibility criteria, sources of evidence, charting methods, results, and conclusions
that relate to the review questions and objectives.
INTRODUCTION
Rationale 3 Describe the rationale for the review in the context of what is already known. 2
Explain why the review questions/objectives lend themselves to a scoping
review approach.
Objectives 4 Provide an explicit statement of the questions and objectives being addressed with 4
reference to their key elements (e.g., population or participants, concepts, and
context) or other relevant key elements used to conceptualize the review
questions and/or objectives.
METHODS
Protocol and registration 5 Indicate whether a review protocol exists; state if and where it can be accessed 4
(e.g., a Web address); and if available, provide registration information, including
the registration number.
Eligibility criteria 6 Specify characteristics of the sources of evidence used as eligibility criteria (e.g., 4–5
years considered, language, and publication status), and provide a rationale.
Information sources� 7 Describe all information sources in the search (e.g., databases with dates of 4–5
coverage and contact with authors to identify additional sources), as well as the
date the most recent search was executed.
Search 8 Present the full electronic search strategy for at least 1 database, including any Appendices B and C
limits used, such that it could be repeated.
Selection of sources of evidence† 9 State the process for selecting sources of evidence (i.e., screening and eligibility) 5
included in the scoping review.
Data charting process‡ 10 Describe the methods of charting data from the included sources of evidence (e.g., 5–6
calibrated forms or forms that have been tested by the team before their use,
and whether data charting was done independently or in duplicate) and any
processes for obtaining and confirming data from investigators.
Data items 11 List and define all variables for which data were sought and any assumptions and 5–6
simplifications made.
Critical appraisal of individual sources 12 If done, provide a rationale for conducting a critical appraisal of included sources Not done
of evidence§ of evidence; describe the methods used and how this information was used in
any data synthesis (if appropriate).
Synthesis of results 13 Describe the methods of handling and summarizing the data that were charted. 6
RESULTS
Selection of sources of evidence 14 Give numbers of sources of evidence screened, assessed for eligibility, and included Section 3. Results, Figure 3
in the review, with reasons for exclusions at each stage, ideally using a flow
diagram.
Characteristics of sources of evidence 15 For each source of evidence, present characteristics for which data were charted Section 3. Results
and provide the citations.
Critical appraisal within sources of 16 If done, present data on critical appraisal of included sources of evidence (see Not done
evidence item 12).
Results of individual sources of 17 For each included source of evidence, present the relevant data that were charted Section 3. Results
evidence that relate to the review questions and objectives.
Synthesis of results 18 Summarize and/or present the charting results as they relate to the review Section 3. Results
questions and objectives.
DISCUSSION
Summary of evidence 19 Summarize the main results (including an overview of concepts, themes, and types Sections 4.2., 4.3., 4.4., pages
of evidence available), link to the review questions and objectives, and consider 28–31
the relevance to key groups.
Limitations 20 Discuss the limitations of the scoping review process. Section 4.5., page 32
Conclusions 21 Provide a general interpretation of the results with respect to the review questions Section 5, page 33
and objectives, as well as potential implications and/or next steps.
FUNDING
Funding 22 Describe sources of funding for the included sources of evidence, as well as Disclosure of interest
sources of funding for the scoping review. Describe the role of the funders of
the scoping review.
JBI ¼ Joanna Briggs Institute; PRISMA-ScR ¼ Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews.
� Where sources of evidence (see second footnote) are compiled from, such as bibliographic databases, social media platforms, and Web sites.
† A more inclusive/heterogeneous term used to account for the different types of evidence or data sources (e.g., quantitative and/or qualitative research, expert
opinion, and policy documents) that may be eligible in a scoping review as opposed to only studies. This is not to be confused with information sources (see
first footnote).
‡ The frameworks by Arksey and O’Malley (6) and Levac and colleagues (7) and the JBI guidance (4, 5) refer to the process of data extraction in a scoping review
as data charting.
§ The process of systematically examining research evidence to assess its validity, results, and relevance before using it to inform a decision. This term is used
for items 12 and 19 instead of "risk of bias" (which is more applicable to systematic reviews of interventions) to include and acknowledge the various sources
of evidence that may be used in a scoping review (e.g., quantitative and/or qualitative research, expert opinion, and policy document).

From: Tricco AC, Lillie E, Zarin W, O’Brien KK, Colquhoun H, Levac D, et al. PRISMA Extension for Scoping Reviews (PRISMAScR): Checklist and
Explanation. Ann Intern Med. 2018;169:467–473. doi: 10.7326/M18-0850.

Appendix B. First literature search
Scoping review on food additives and adverse effects on
the intestines
Contact person: Inger-Lise Steffensen
Performed the search: Ragnhild Agathe Tornes
Performed peer review: Marita Heintz
Duplicate check in EndNote: Before: 2318
After: 1153
Database: Ovid MEDLINE(R) and Epub Ahead of Print, In-
Process, In-Data-Review & Other Non-Indexed
Citations, Daily and Versions(R) <1946 to January
28, 2022>
Date: 31.01.22
Number of hits: 377
# Searches
1 Carrageenan/ or (carr#g?e?n� or genugel or killeen or
satiagum or seakem or viscarin or burtonite or
((organic sulfate? or organosulfate? or sulfate ester?
or Sulfuric Acid Ester?) adj5 (polygalactose? or
galactan?)) or processed Eucheuma seaweed?).tw,kf.
2 Carboxymethylcellulose Sodium/ or (((carboxylmethyl
or carboxy methyl or carboxymethyl) adj cellulose)
or carboxymethylcellulose or aquacel or aquaplast
or carmellose or croscarmellose sodium or cellolax
or cethylose or polycell or polycel or ruspol or
cellulose gum or CMC or almelose or apergel or
blandlax or bu lax or carbethox or carbose d or
carmellose or carmethose or cel o brandt or
cellofas or eskalose or gelaxin or glycocellon or
moventon or natulose or regucellulose or sodium
cellulose glycolate or thylose or (tylose adj (mga or
sodium)) or xylo mucine or xylomucin).tw,kf.
3 1 or 2
4 Intestines/ or (intestin� or bowel� or gut or colon or
rectum or anal canal?).tw,kf.
5 Homeostasis/ or Tight Junctions/ or exp Inflammatory
Bowel Diseases/ or Irritable Bowel Syndrome/ or
(homeostasis or (homeostatic adj (equilibrium or
mechanism)) or homoeostasis or homoeostasis or
autoregulation or ((tight or occluding) adj
junction?) or nexus or zonula occluden? or barrier
dysfunction� or permeable or permeability� or
integrity or inflammation? or innate inflammatory
response? or ((inflammatory bowel or chron� or
functional colonic) adj disease?) or ((chron� or
granulomatous) adj enteritis) or ((ulcerative or
granulomatous or gravis or spastic) adj1 colitis) or
ileocolitis or idiopathic proctocolitis or intestinal
transporter? or (regional adj (ileitis or iletides or
enteritis)) or terminal ileitis or irritable bowel
syndrom� or ((irritable or spastic or unstable) adj
colon) or colon spasm or colonospasm or ((mocus
or mucomembraneous or mucomembraneous) adj
colitides)).tw,kf.
6 Autoimmunity/ or autoimmun�.tw,kf.
7 Microbiota/ or (microbiota? or microbiome? or
(microbial adj (communit� or flora)) or
microflora�).tw,kf.
8 5 or 6 or 7
9 3 and 4 and 8
10 limit 9 to (danish or english or interlingua or
multilingual or norwegian or swedish)
CRITICAL REVIEWS IN TOXICOLOGY 563
Database: Embase <1974 to 2022 January 28>
Date: 31.01.22
Number of hits: 527
# Searches Results
1 carrageenan/ or (carr#g?e?n� or genugel or killeen 18364
or satiagum or seakem or viscarin or burtonite
or ((organic sulfate? or organosulfate? or sulfate
ester? or Sulfuric Acid Ester?) adj5
(polygalactose? or galactan?)) or processed
Eucheuma seaweed?).tw,kf.
2 Carboxymethylcellulose/ or (((carboxylmethyl or 24769
carboxy methyl or carboxymethyl) adj cellulose)
or carboxymethylcellulose or aquacel or
aquaplast or carmellose or croscarmellose
sodium or cellolax or cethylose or polycell or
polycel or ruspol or cellulose gum or CMC or
almelose or apergel or blandlax or bu lax or
carbethox or carbose d or carmellose or
Results carmethose or cel o brandt or cellofas or
eskalose or gelaxin or glycocellon or moventon
13897 or natulose or regucellulose or sodium cellulose
glycolate or thylose or (tylose adj (mga or
sodium)) or xylo mucine or xylomucin).tw,kf.
3 1 or 2 42789
4 Intestine/ or (intestin� or bowel� or gut or colon 983754
16462 or rectum or anal canal?).tw,kf.
5 homeostasis/ or tight junction/ or inflammatory 1637954
bowel disease/ or irritable colon/ or
(homeostasis or (homeostatic adj (equilibrium or
mechanism)) or homoeostasis or homoeostasis
or autoregulation or ((tight or occluding) adj
junction?) or nexus or zonula occluden? or
barrier dysfunction� or permeable or
permeability� or integrity or inflammation? or
innate inflammatory response? or ((inflammatory
bowel or chron� or functional colonic) adj
disease?) or ((chron� or granulomatous) adj
30200 enteritis) or ((ulcerative or granulomatous or
765275 gravis or spastic) adj1 colitis) or ileocolitis or
idiopathic proctocolitis or intestinal transporter?
1215034 or (regional adj (ileitis or iletides or enteritis)) or
terminal ileitis or irritable bowel syndrom� or
((irritable or spastic or unstable) adj colon) or
colon spasm or colonospasm or ((mocus or
mucomembraneous or mucomembraneous) adj
colitides)).tw,kf.
6 autoimmunity/ or autoimmun�.tw,kf. 305523
7 microflora/ or (microbiota? or microbiome? or 161273
(microbial adj (communit� or flora)) or
microflora�).tw,kf.
8 5 or 6 or 7 2008608
9 3 and 4 and 8 602
10 limit 9 to (danish or english or norwegian or 576
polyglot or swedish)
11 limit 10 to (conference abstracts or embase) 527
Database: Scopus
Date: 31.01.22
194987
137214 Number of hits: 817
12 #3 and #4 and #8 AND (EXCLUDE (LANGUAGE , 817
1484929 "japanese") OR EXCLUDE (LANGUAGE , "chinese")
394 OR EXCLUDE (LANGUAGE , "german") OR
377 EXCLUDE (LANGUAGE , "italian") OR EXCLUDE
(LANGUAGE , "portuguese") OR EXCLUDE
(LANGUAGE , "spanish") OR EXCLUDE
(LANGUAGE , "dutch") OR EXCLUDE (LANGUAGE
, "french") OR EXCLUDE (LANGUAGE , "greek")
OR EXCLUDE (LANGUAGE , "russian") OR
EXCLUDE (LANGUAGE , "turkish"))
11 #3 and #4 and #8 869
8 #5 or #6 or #7 2,872,804
(continued)
564 M. TAHIRI ET AL.
7 TITLE-ABS-KEY ((microbiota� OR microbiome� OR
(microbial PRE/0 (communit� OR flora)) OR
microflora�))
6 TITLE-ABS-KEY (autoimmun�)
5 TITLE-ABS-KEY ((homeostasis OR (homeostatic PRE/
0 (equilibrium OR mechanism)) OR homoeostasis
OR homoeostasis OR autoregulation OR ((tight
OR occluding) PRE/0 junction�) OR nexus OR
"zonula occluden�" OR "barrier dysfunction�" OR
permeable OR permeability� OR integrity OR
inflammation� OR "innate inflammatory
response�" OR (("inflammatory bowel" OR
chron� OR "functional colonic") PRE/0 disease�)
OR ((chron� OR granulomatous) PRE/0 enteritis)
OR ((ulcerative OR granulomatous OR gravis OR
spastic) W/0 colitis) OR ileocolitis OR "idiopathic
proctocolitis" OR "intestinal transporter�" OR
(regional PRE/0 (ileitis OR iletides OR enteritis))
OR "terminal ileitis" OR "irritable bowel
syndrom�" OR ((irritable OR spastic OR unstable)
PRE/0 colon) OR "colon spasm" OR colonospasm
OR ((mocus OR mucomembraneous OR
mucomembraneous) PRE/0 colitides)))
4 TITLE-ABS-KEY ((intestin� OR bowel� OR gut OR
colon OR rectum OR "anal canal�"))
3 #1 or #2
2 TITLE-ABS-KEY (((carboxylmethyl OR "carboxy
methyl" OR carboxymethyl) PRE/0 cellulose) OR
carboxymethylcellulose OR aquacel OR aquaplast
OR carmellose OR "croscarmellose sodium" OR
cellolax OR cethylose OR polycell OR polycel OR
ruspol OR "cellulose gum" OR cmc OR almelose
OR apergel OR blandlax OR "bu lax" OR
carbethox OR "carbose d" OR carmellose OR
carmethose OR "cel o brandt" OR cellofas OR
eskalose OR gelaxin OR glycocellon OR
moventon OR natulose OR regucellulose OR
"sodium cellulose glycolate" OR thylose OR
(tylose PRE/0 (mga OR sodium)) OR "xylo
mucine" OR xylomucin)
1 TITLE-ABS-KEY ((carrageen� OR carragen� OR
carrhagen� OR carragheen� OR carrogeen� OR
genugel OR killeen OR satiagum OR seakem OR
viscarin OR burtonite OR (("organic sulfate�" OR
organosulfate� OR "sulfate ester�" OR "sulfuric
acid ester�") W/4 (polygalactose� OR galactan�))
OR "processed eucheuma seaweed�"))
Database: Web of Science Core Collection (1987-present):
Science Citation Index Expanded
EXPANDED)–1987-present
Social Sciences Citation Index (SSCI)–1987-present
Arts & Humanities Citation Index (AHCI)–1987-
present
Emerging Sources Citation Index (ESCI)–2017-
present
Date: 31.01.22
Number of hits: 509
10 #9 and German or Japanese or Turkish (Exclude –
Languages)
9 #3 AND #4 AND #8
8 #5 OR #6 OR #7
7 TS¼("microbiota$" or "microbiome$" or ("microbial"
NEAR/0 ("communit�" or "flora")) or
"microflora�")
6 TS¼("autoimmun�")
5 TS¼(("homeostasis" or ("homeostatic" NEAR/0
("equilibrium" or "mechanism")) or
"homoeostasis" or "homoeostasis" or
"autoregulation" or (("tight" or "occluding")
NEAR/0 "junction$") or "nexus" or "zonula
occluden$" or "barrier dysfunction�" or
230,562 "permeable" or "permeability�" or "integrity" or
"inflammation$" or "innate inflammatory
response$" or (("inflammatory bowel" or
290,812 "chron�" or "functional colonic") NEAR/0
2,455,119 "disease$") or (("chron�" or "granulomatous")
NEAR/0 "enteritis") or (("ulcerative" or
"granulomatous" or "gravis" or "spastic") NEAR/0
"colitis") or "ileocolitis " or "idiopathic
proctocolitis" or "intestinal transporter$" or
("regional" NEAR/0 ("ileitis" or "iletides" or
"enteritis")) or "terminal ileitis" or "irritable
bowel syndrom�" or (("irritable" or "spastic" or
"unstable") NEAR/0 "colon") or "colon spasm" or
"colonospasm" or (("mocus" or
"mucomembraneous" or "mucomembraneous")
NEAR/0 "colitides")))
4 TS¼(("intestin�" or "bowel�" or "gut" or "colon" or 788,359
"rectum" or "anal canal$"))
3 #1 OR #2 50,184
2 TS¼(("carboxylmethyl" or "carboxy methyl" or 33,676
"carboxymethyl" NEAR/0 "cellulose") or
"carboxymethylcellulose" or "aquacel" or
"aquaplast" or "carmellose" or "croscarmellose
1,308,755 sodium" or "cellolax" or "cethylose" or "polycell"
or "polycel" or "ruspol" or "cellulose gum" or
78,931 "CMC" or "almelose" or "apergel" or "blandlax"
54,341 or "bu lax" or "carbethox" or "carbose d" or
"carmellose" or "carmethose" or "cel o brandt"
or "cellofas" or "eskalose" or "gelaxin" or
"glycocellon" or "moventon" or "natulose" or
"regucellulose" or "sodium cellulose glycolate" or
"thylose" or ("tylose" NEAR/0 ("mga" or
"sodium")) or "xylo mucine" or "xylomucin")
1 TS¼("carrageen�" or "carragen�" or "carrhagen�" or 16,869
"carragheen�" or "carrogeen�" or "genugel" or
"killeen" or "satiagum" or "seakem" or "viscarin"
or "burtonite" or (("organic sulfate$" or
"organosulfate$" or "sulfate ester$" or "Sulfuric
Acid Ester$") NEAR/4 ("polygalactose$" or
"galactan$")) or "processed Eucheuma
25,196 seaweed�")
Database: Cochrane Database of Systematic Reviews Issue 1
of 12, January 2022
Cochrane Central Register of Controlled Trials
Issue 12 of 12, December 2021
Date: 28.01.22
Number of hits: 22 (1 Cochrane review, 21 Trials)
(SCI-
ID Search Hits
#1 [mh ^"Carrageenan"] 36
#2 (carrageen� or carragen� or carrhagen� or 95
carragheen� or carrogeen� or genugel or killeen
or satiagum or seakem or viscarin or burtonite
or (("organic sulfate" or "organic sulfates" or
organosulfate? or "sulfate ester" or "sulfate
esters" or "Sulfuric Acid Ester" or "Sulfuric Acid
Esters") NEAR/5 (polygalactose? or galactan?)) or
("processed Eucheuma" NEXT seaweed?)):ti,ab
509 #3 [mh ^"Carboxymethylcellulose Sodium"] 274
#4 (((carboxylmethyl or "carboxy methyl" or 867
512 carboxymethyl) NEXT cellulose) or
1,915,701 carboxymethylcellulose or aquacel or aquaplast
200,879 or carmellose or "croscarmellose sodium" or
cellolax or cethylose or polycell or polycel or
ruspol or "cellulose gum" or CMC or almelose or
213,492 apergel or blandlax or "bu lax" or carbethox or
1,584,790 "carbose d" or carmellose or carmethose or "cel
o brandt" or cellofas or eskalose or gelaxin or
glycocellon or moventon or natulose or
regucellulose or "sodium cellulose glycolate" or
thylose or (tylose NEXT (mga or sodium)) or
"xylo mucine" or xylomucin):ti,ab
(continued) (continued)
 CRITICAL REVIEWS IN TOXICOLOGY 565

Continued. Database: Ovid MEDLINE(R) and Epub Ahead of Print, In-

ID Search
#5 {or #1-#4}
#6 [mh ^"Intestines"]
#7 (intestin� or bowel� or gut or colon or rectum or
(anal NEXT canal?)):ti,ab
#8 #6 or #7
#9 [mh ^"Homeostasis"]
#10 [mh ^"Tight Junctions"]
#11 [mh ^"Inflammatory Bowel Diseases"]
#12 [mh ^"Irritable Bowel Syndrome"]
#13 (homeostasis or (homeostatic NEXT (equilibrium or
mechanism)) or homoeostasis or homoeostasis
or autoregulation or ((tight or occluding) NEXT
junction?) or nexus or (zonula NEXT occluden?)
or (barrier NEXT dysfunction�) or permeable or
permeability� or integrity or inflammation? or
("innate inflammatory" NEXT response?) or
(("inflammatory bowel" or chron� or "functional
colonic") NEXT disease?) or ((chron� or
granulomatous) NEXT enteritis) or ((ulcerative or
granulomatous or gravis or spastic) NEAR/1
colitis) or ileocolitis or "idiopathic proctocolitis"
or (intestinal NEXT transporter?) or (regional
NEXT (ileitis or iletides or enteritis)) or "terminal
ileitis" or ("irritable bowel" NEXT syndrom�) or
((irritable or spastic or unstable) NEXT colon) or
"colon spasm" or colonospasm or ((mocus or
mucomembraneous or mucomembraneous)
NEXT colitides)):ti,ab
#14 [mh ^"Autoimmunity"]
#15 (autoimmun�):ti,ab
#16 [mh ^"Microbiota"]
#17 (microbiota? or microbiome? or (microbial NEXT
(communit� or flora)) or microflora�):ti,ab
#18 {or #9-#17}
#19 #5 and #8 and #18
Database: Epistemonikos
Comment: Simplified search strategy due to the limited search
functionality
Date: 31.01.22
Number of hits: 66
(carrageen� or carragen� or carrhagen� or carragheen� or carrogeen� or
genugel or killeen or satiagum or seakem or viscarin or burtonite or
"processed Eucheuma seaweed" or "processed Eucheuma seaweeds" or
"carboxylmethyl cellulose" or "carboxy methyl cellulose" or "carboxy­
methyl cellulose" or carboxymethylcellulose or aquacel or aquaplast or
carmellose or "croscarmellose sodium" or cellolax or cethylose or polycell
or polycel or ruspol or "cellulose gum" or CMC or almelose or apergel or
blandlax or "bu lax" or carbethox or "carbose d" or carmellose or carme­
those or "cel o brandt" or cellofas or eskalose or gelaxin or glycocellon
or moventon or natulose or regucellulose or "sodium cellulose glycolate"
or thylose or "tylose mga" or "tylose sodium" or "xylo mucine" or xylo­
mucin) AND (intestin� or bowel� or gut or colon or rectum or "anal
canal" or "anal canals")
Appendix C. Repeated literature search
Scoping review on food additives and adverse effects on
the intestines – repeated search
Hits Process, In-Data-Review & Other Non-Indexed
1088 Citations, Daily and Versions <1946 to December
877 06, 2022>
48546 Date: 07.12.22
Number of hits: 41
48644
973
15 1 Carrageenan/ or (carr#g?e?n� or genugel or killeen 14390
627 or satiagum or seakem or viscarin or burtonite
1312 or ((organic sulfate? or organosulfate? or sulfate
67949 ester? or Sulfuric Acid Ester?) adj5
(polygalactose? or galactan?)) or processed
Eucheuma seaweed?).tw,kf.
2 Carboxymethylcellulose Sodium/ or 17538
(((carboxylmethyl or carboxy methyl or
carboxymethyl) adj cellulose) or
carboxymethylcellulose or aquacel or aquaplast
or carmellose or croscarmellose sodium or
cellolax or cethylose or polycell or polycel or
ruspol or cellulose gum or CMC or almelose or
apergel or blandlax or bu lax or carbethox or
carbose d or carmellose or carmethose or cel o
brandt or cellofas or eskalose or gelaxin or
glycocellon or moventon or natulose or
regucellulose or sodium cellulose glycolate or
thylose or (tylose adj (mga or sodium)) or xylo
mucine or xylomucin).tw,kf.
3 1 or 2 31764
4 Intestines/ or (intestin� or bowel� or gut or colon 804107
86 or rectum or anal canal?).tw,kf.
5088 5 Homeostasis/ or Tight Junctions/ or exp 1299242
267 Inflammatory Bowel Diseases/ or Irritable Bowel
8508 Syndrome/ or (homeostasis or (homeostatic adj
(equilibrium or mechanism)) or homoeostasis or
78950 homoeostasis or autoregulation or ((tight or
22 occluding) adj junction?) or nexus or zonula
occluden? or barrier dysfunction� or permeable
or permeability� or integrity or inflammation? or
innate inflammatory response? or ((inflammatory
bowel or chron� or functional colonic) adj
disease?) or ((chron� or granulomatous) adj
enteritis) or ((ulcerative or granulomatous or
gravis or spastic) adj1 colitis) or ileocolitis or
idiopathic proctocolitis or intestinal transporter?
or (regional adj (ileitis or iletides or enteritis)) or
terminal ileitis or irritable bowel syndrom� or
((irritable or spastic or unstable) adj colon) or
colon spasm or colonospasm or ((mocus or
mucomembraneous or mucomembraneous) adj
colitides)).tw,kf.
6 Autoimmunity/ or autoimmun�.tw,kf. 206622
7 Microbiota/ or (microbiota? or microbiome? or 158802
(microbial adj (communit� or flora)) or
microflora�).tw,kf.
8 5 or 6 or 7 1594271
9 3 and 4 and 8 425
10 limit 9 to (danish or english or interlingua or 408
multilingual or norwegian or swedish)
11 (202202� or 202203� or 202204� or 202205� or 1705432
202206� or 202207� or 202208� or 202209� or
202210� or 202211� or 202212�).ep,ed,dt.
12 10 and 11 41

Contact person: Inger-Lise Steffensen

Performed the search: Ragnhild Agathe Tornes
Comment: Update of search performed 28. and 31.01.22
Duplicate check in EndNote: Before: 254
After: 127
566 M. TAHIRI ET AL.
Database: Embase <1974 to 2022 December 06>
Date: 07.12.22
Number of hits: 73
1 carrageenan/ or (carr#g?e?n� or genugel or killeen
or satiagum or seakem or viscarin or burtonite
or ((organic sulfate? or organosulfate? or sulfate
ester? or Sulfuric Acid Ester?) adj5
(polygalactose? or galactan?)) or processed
Eucheuma seaweed?).tw,kf.
2 Carboxymethylcellulose/ or (((carboxylmethyl or
carboxy methyl or carboxymethyl) adj cellulose)
or carboxymethylcellulose or aquacel or
aquaplast or carmellose or croscarmellose
sodium or cellolax or cethylose or polycell or
polycel or ruspol or cellulose gum or CMC or
almelose or apergel or blandlax or bu lax or
carbethox or carbose d or carmellose or
carmethose or cel o brandt or cellofas or
eskalose or gelaxin or glycocellon or moventon
or natulose or regucellulose or sodium cellulose
glycolate or thylose or (tylose adj (mga or
sodium)) or xylo mucine or xylomucin).tw,kf.
3 1 or 2
4 Intestine/ or (intestin� or bowel� or gut or colon
or rectum or anal canal?).tw,kf.
5 homeostasis/ or tight junction/ or inflammatory
bowel disease/ or irritable colon/ or
(homeostasis or (homeostatic adj (equilibrium or
mechanism)) or homoeostasis or homoeostasis
or autoregulation or ((tight or occluding) adj
junction?) or nexus or zonula occluden? or
barrier dysfunction� or permeable or
permeability� or integrity or inflammation? or
innate inflammatory response? or ((inflammatory
bowel or chron� or functional colonic) adj
disease?) or ((chron� or granulomatous) adj
enteritis) or ((ulcerative or granulomatous or
gravis or spastic) adj1 colitis) or ileocolitis or
idiopathic proctocolitis or intestinal transporter?
or (regional adj (ileitis or iletides or enteritis)) or
terminal ileitis or irritable bowel syndrom� or
((irritable or spastic or unstable) adj colon) or
colon spasm or colonospasm or ((mocus or
mucomembraneous or mucomembraneous) adj
colitides)).tw,kf.
6 autoimmunity/ or autoimmun�.tw,kf.
7 microflora/ or (microbiota? or microbiome? or
(microbial adj (communit� or flora)) or
microflora�).tw,kf.
8 5 or 6 or 7
9 3 and 4 and 8
10 limit 9 to (danish or english or norwegian or
polyglot or swedish)
11 limit 10 to (conference abstracts or embase)
12 (202202� or 202203� or 202204� or 202205� or
202206� or 202207� or 202208� or 202209� or
202210� or 202211� or 202212�).dd,dc.
13 11 and 12
Database: Scopus
Date: 07.12.22
Number of hits: 71
15 (#10 AND (LIMIT-TO (PUBYEAR , 2022)) 71
19015
10 (#9 (AND (LIMIT-TO (LANGUAGE , "english") OR 891
LIMIT-TO (LANGUAGE , "norwegian"))
9 #3 and #4 and #8 941
8 #5 or #6 or #7 3,068,111
7 TITLE-ABS-KEY ((microbiota� OR microbiome� OR 263,647
(microbial PRE/0 (communit� OR flora)) OR
26297
microflora�))
6 TITLE-ABS-KEY (autoimmun�) 307,615
5 TITLE-ABS-KEY ((homeostasis OR (homeostatic PRE/ 2,612,937
0 (equilibrium OR mechanism)) OR homoeostasis
OR homoeostasis OR autoregulation OR ((tight
OR occluding) PRE/0 junction�) OR nexus OR
"zonula occluden�" OR "barrier dysfunction�" OR
permeable OR permeability� OR integrity OR
inflammation� OR "innate inflammatory
response�" OR (("inflammatory bowel" OR
chron� OR "functional colonic") PRE/0 disease�)
OR ((chron� OR granulomatous) PRE/0 enteritis)
OR ((ulcerative OR granulomatous OR gravis OR
44943
spastic) W/0 colitis) OR ileocolitis OR "idiopathic
1038395
proctocolitis" OR "intestinal transporter�" OR
(regional PRE/0 (ileitis OR iletides OR enteritis))
1750819
OR "terminal ileitis" OR "irritable bowel
syndrom�" OR ((irritable OR spastic OR unstable)
PRE/0 colon) OR "colon spasm" OR colonospasm
OR ((mocus OR mucomembraneous OR
mucomembraneous) PRE/0 colitides)))
4 TITLE-ABS-KEY ((intestin� OR bowel� OR gut OR 1,371,737
colon OR rectum OR "anal canal�"))
3 #1 or #2 82,937
2 TITLE-ABS-KEY (((carboxylmethyl OR "carboxy 57,196
methyl" OR carboxymethyl) PRE/0 cellulose) OR
carboxymethylcellulose OR aquacel OR aquaplast
OR carmellose OR "croscarmellose sodium" OR
cellolax OR cethylose OR polycell OR polycel OR
ruspol OR "cellulose gum" OR cmc OR almelose
OR apergel OR blandlax OR "bu lax" OR
carbethox OR "carbose d" OR carmellose OR
carmethose OR "cel o brandt" OR cellofas OR
eskalose OR gelaxin OR glycocellon OR
moventon OR natulose OR regucellulose OR
"sodium cellulose glycolate" OR thylose OR
324053
(tylose PRE/0 (mga OR sodium)) OR "xylo
185571
mucine" OR xylomucin)
1 TITLE-ABS-KEY ((carrageen� OR carragen� OR 26,381
carrhagen� OR carragheen� OR carrogeen� OR
2153424
genugel OR killeen OR satiagum OR seakem OR
672
viscarin OR burtonite OR (("organic sulfate�" OR
646
organosulfate� OR "sulfate ester�" OR "sulfuric
acid ester�") W/4 (polygalactose� OR galactan�))
589
OR "processed eucheuma seaweed�"))
2107333
73
 CRITICAL REVIEWS IN TOXICOLOGY 567

Database: Web of Science Core Collection (1987-present): Database: Cochrane Central Register of Controlled Trials
Science Citation Index Expanded (SCI- Issue 11 of 12, November 2022
EXPANDED)–1987-present Cochrane Database of Systematic Reviews
Social Sciences Citation Index (SSCI)–1987-present Issue 12 of 12, December 2022
Arts & Humanities Citation Index (AHCI)–1987- Date: 08.12.22
present Number of hits: 2 (0 Cochrane review, 2 Trials)
Emerging Sources Citation Index (ESCI)–2017-
present #1 [mh ^"Carrageenan"] 39
Date: 08.12.22 #2 (carrageen� or carragen� or carrhagen� or 101
Number of hits: 52 carragheen� or carrogeen� or genugel or killeen or
satiagum or seakem or viscarin or burtonite or
(("organic sulfate" or "organic sulfates" or
organosulfate? or "sulfate ester" or "sulfate esters"
1 TS¼("carrageen�" or "carragen�" or "carrhagen�" or 17869 or "Sulfuric Acid Ester" or "Sulfuric Acid Esters")
"carragheen�" or "carrogeen�" or "genugel" or NEAR/5 (polygalactose? or galactan?)) or
"killeen" or "satiagum" or "seakem" or "viscarin" ("processed Eucheuma" NEXT seaweed?)):ti,ab
or "burtonite" or (("organic sulfate$" or #3 [mh ^"Carboxymethylcellulose Sodium"] 281
"organosulfate$" or "sulfate ester$" or "Sulfuric #4 (((carboxylmethyl or "carboxy methyl" or 933
Acid Ester$") NEAR/4 ("polygalactose$" or carboxymethyl) NEXT cellulose) or
"galactan$")) or "processed Eucheuma carboxymethylcellulose or aquacel or aquaplast or
seaweed�") carmellose or "croscarmellose sodium" or cellolax or
2 TS¼(("carboxylmethyl" or "carboxy methyl" or 36086 cethylose or polycell or polycel or ruspol or
"carboxymethyl" NEAR/0 "cellulose") or "cellulose gum" or CMC or almelose or apergel or
"carboxymethylcellulose" or "aquacel" or blandlax or "bu lax" or carbethox or "carbose d" or
"aquaplast" or "carmellose" or "croscarmellose carmellose or carmethose or "cel o brandt" or
sodium" or "cellolax" or "cethylose" or "polycell" cellofas or eskalose or gelaxin or glycocellon or
or "polycel" or "ruspol" or "cellulose gum" or moventon or natulose or regucellulose or "sodium
"CMC" or "almelose" or "apergel" or "blandlax" cellulose glycolate" or thylose or (tylose NEXT (mga
or "bu lax" or "carbethox" or "carbose d" or or sodium)) or "xylo mucine" or xylomucin):ti,ab
"carmellose" or "carmethose" or "cel o brandt" #5 {or #1-#4} 1161
or "cellofas" or "eskalose" or "gelaxin" or #6 [mh ^"Intestines"] 891
"glycocellon" or "moventon" or "natulose" or #7 (intestin� or bowel� or gut or colon or rectum or 52614
"regucellulose" or "sodium cellulose glycolate" or (anal NEXT canal?)):ti,ab
"thylose" or ("tylose" NEAR/0 ("mga" or #8 #6 or #7 52713
"sodium")) or "xylo mucine" or "xylomucin") #9 [mh ^"Homeostasis"] 995
3 #1 OR #2 53572 #10 [mh ^"Tight Junctions"] 15
4 TS¼(("intestin�" or "bowel�" or "gut" or "colon" or 839169 #11 [mh ^"Inflammatory Bowel Diseases"] 691
"rectum" or "anal canal$")) #12 [mh ^"Irritable Bowel Syndrome"] 1417
5 TS¼(("homeostasis" or ("homeostatic" NEAR/0 1713383 #13 (homeostasis or (homeostatic NEXT (equilibrium or 73278
("equilibrium" or "mechanism")) or mechanism)) or homoeostasis or homoeostasis or
"homoeostasis" or "homoeostasis" or autoregulation or ((tight or occluding) NEXT
"autoregulation" or (("tight" or "occluding") junction?) or nexus or (zonula NEXT occluden?) or
NEAR/0 "junction$") or "nexus" or "zonula (barrier NEXT dysfunction�) or permeable or
occluden$" or "barrier dysfunction�" or permeability� or integrity or inflammation? or
"permeable" or "permeability�" or "integrity" or ("innate inflammatory" NEXT response?) or
"inflammation$" or "innate inflammatory (("inflammatory bowel" or chron� or "functional
response$" or (("inflammatory bowel" or colonic") NEXT disease?) or ((chron� or
"chron�" or "functional colonic") NEAR/0 granulomatous) NEXT enteritis) or ((ulcerative or
"disease$") or (("chron�" or "granulomatous") granulomatous or gravis or spastic) NEAR/1 colitis)
NEAR/0 "enteritis") or (("ulcerative" or or ileocolitis or "idiopathic proctocolitis" or
"granulomatous" or "gravis" or "spastic") NEAR/0 (intestinal NEXT transporter?) or (regional NEXT
"colitis") or "ileocolitis " or "idiopathic (ileitis or iletides or enteritis)) or "terminal ileitis" or
proctocolitis" or "intestinal transporter$" or ("irritable bowel" NEXT syndrom�) or ((irritable or
("regional" NEAR/0 ("ileitis" or "iletides" or spastic or unstable) NEXT colon) or "colon spasm"
"enteritis")) or "terminal ileitis" or "irritable or colonospasm or ((mocus or mucomembraneous
bowel syndrom�" or (("irritable" or "spastic" or or mucomembraneous) NEXT colitides)):ti,ab
"unstable") NEAR/0 "colon") or "colon spasm" or #14 [mh ^"Autoimmunity"] 92
"colonospasm" or (("mocus" or #15 (autoimmun�):ti,ab 5579
"mucomembraneous" or "mucomembraneous") #16 [mh ^"Microbiota"] 327
NEAR/0 "colitides"))) #17 (microbiota? or microbiome? or (microbial NEXT 10058
6 TS¼("autoimmun�") 226912 (communit� or flora)) or microflora�):ti,ab
7 TS¼("microbiota$" or "microbiome$" or ("microbial" 231986 #18 {or #9-#17} 85816
NEAR/0 ("communit�" or "flora")) or #19 #5 and #8 and #18 24
"microflora�") #20 #19 with Cochrane Library publication date Between 0
8 #5 OR #6 OR #7 2077473 Feb 2022 and Dec 2022, in Cochrane Reviews
9 #3 AND #4 AND #8 564 #21 #19 with Cochrane Library publication date Between 1
10 #9 564 Feb 2022 and Dec 2022, in Trials
11 #9 and German or Japanese or Turkish (Exclude – 561 #22 #19 with Publication Year from 2022 to 2022, in Trials 2
Languages) #23 #20 or #21 or #22 2
12 #9 52
Timespan: 2022-02-01 to 2022-12-08
568 M. TAHIRI ET AL.

Database: Epistemonikos
Comment: Simplified search strategy due to the limited search
functionality
Date: 08.12.22
Number of hits: 15
(carrageen� or carragen� or carrhagen� or carragheen� or carrogeen� or genugel or killeen or satiagum or seakem or viscarin or burtonite or "proc­
essed Eucheuma seaweed" or "processed Eucheuma seaweeds" or "carboxylmethyl cellulose" or "carboxy methyl cellulose" or "carboxymethyl cellu­
lose" or carboxymethylcellulose or aquacel or aquaplast or carmellose or "croscarmellose sodium" or cellolax or cethylose or polycell or polycel or
ruspol or "cellulose gum" or CMC or almelose or apergel or blandlax or "bu lax" or carbethox or "carbose d" or carmellose or carmethose or "cel o
brandt" or cellofas or eskalose or gelaxin or glycocellon or moventon or natulose or regucellulose or "sodium cellulose glycolate" or thylose or "tylose
mga" or "tylose sodium" or "xylo mucine" or xylomucin) AND (intestin� or bowel� or gut or colon or rectum or "anal canal" or "anal canals")
Custom date range:
From: 01-02-22 To: 08-12-22

Appendix D. Publications excluded at full-text level with reasons

Publication Screen on Title and Abstract Screen on Full Text

Aiba (2002) (ID:68321635) -INCLUDE on title & abstract -EXCLUDE on Full Text
CMC given by enema (through anus), not orally. Mice
were treated with virus and their virus-infected lymph
nodes were transferred to other mice, where group 4
were given CMC enema. No dose of CMC is given.
Bagger (1985) (ID:68321721) -INCLUDE on title & abstract -EXCLUDE on Full Text
It seems that in the tablet emepronium carrageenate
(EC) (Cetiprin Novum), emepronium is the active
substance, not carrageenate, and the effects decribed
are related to emepronium, not to cargeenate.
Behall (1987) (ID:78874667) -INCLUDE on title & abstract -EXCLUDE on Full Text
Studied basic physiology and mineral balance.
Benjamin (1997) (ID:68321904) -INCLUDE on title & abstract -EXCLUDE on Full Text
Carrageenan is given by intraperitoneal injection to rats
to induce migration of neutrophils into the peritoneal
cavity, an administration route which is not relevant for
oral exposure to food additives.
Bertoli (1980) (ID:68322373) -INCLUDE on title & abstract -EXCLUDE on Full Text
Injected carrageenan in the paw or in the pleura to
induce inflammation experimentally, no oral exposure.
Bhattacharyya (2012) (ID: 68321775 -INCLUDE on title & abstract -EXCLUDE on Full Text
Effects were not on the intestines.
Bokov (2021) (ID:68322074) -INCLUDE on title & abstract -EXCLUDE on Full Text
Not on adverse effects, more about use etc.
Brown (2014) (ID:68322279) -INCLUDE on title & abstract -EXCLUDE on Full Text
About positive effects, not adverse effects.
Case (2011) (ID:68321753) -INCLUDE on title & abstract -EXCLUDE on Full Text
It seems that CMC is used to induce inflammation, at
the same time they say that it has protective effects.
Unclear message (abstract).
Chedid (2017) (ID:68322398) -INCLUDE on title & abstract -EXCLUDE on Full Text
Is injection in rat paw, thus, not affecting the intestines.
Not oral exposure.
Chin (2019) (ID:68322150) -INCLUDE on title & abstract -EXCLUDE on Full Text
Describe positive effects, decreased obesity, not adverse
effects.
Corino (2021) (ID:68322174) -INCLUDE on title & abstract -EXCLUDE on Full Text
Only information on positive effects of carrageenan,
not adverse effects.
Costa (2018) (ID:68322140) -INCLUDE on title & abstract -EXCLUDE on Full Text
Carrageenan was given to the mice by intraperitoneal
injections to induce peritonitis.
Ferreira (2020) (ID:78874628) -INCLUDE on title & abstract -EXCLUDE on Full Text
Effects on neurodevelopment and behaviour, not on the
intestines.
Furuhashi (2016) (ID:68321589) -INCLUDE on title & abstract -EXCLUDE on Full Text
CMC was given together with indomethacin.
Goto (2001) (ID:68321328) -INCLUDE on title & abstract -EXCLUDE on Full Text
No negative control group to compare with only CMC.
Gudiel-Urbano (2002) (ID:68321649) -INCLUDE on title & abstract -EXCLUDE on Full Text
The pulverised whole alga was given, of which Nori
contains variable acidic polysaccharides (carrageenans),
thus, very unspecific carrageenan content.
Hales (2022) (ID:78874605) -INCLUDE on title & abstract -EXCLUDE on Full Text
Results not given on CMC alone and no control group
to CMC.
(continued)

Continued.
Publication
Huang (2022) (ID:68321380)
Hurley and Willoughby (1973) (ID:68321270)
Kitano (1978) (ID:68322320)
Kitano (1981) (ID:68322314)
Kladnicka (2022) (ID:78874674)
Kusmardi (2021) (ID:78874614)
Kusmardi (2022) (ID:78874619)
Laffin (2016) (ID:68321918)
Lahaye (1997) (ID:68322280)
Miclotte (2021) (ID:68322271)
Mohamed (2021) (ID:68322270)
Nct (2015) (ID:68321868)
Nct (2020) (ID:68322024)
Neag (2022) (ID:78874697)
Ocek (2019) (ID:68321612)
Oestreicher (1991) (ID:68321908)
Onderdonk (1983) (ID:68321731)
Onderdonk (1987) (ID:68321514)
O’Sullivan (2010) (ID:68322175)
Patrascu (2017) (ID:68321790)
Pfeffer (1977) (ID:78874611)
Pick (1971) (ID:68322151)
Sabino (2017a) (ID:68321792)
Sabino (2017b) (ID:68322358)
Screen on Title and Abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
CRITICAL REVIEWS IN TOXICOLOGY 569
Screen on Full Text
-EXCLUDE on Full Text
Only on beneficial effects of carrageenans, not adverse
effects.
-EXCLUDE on Full Text
Carrageenan is injected in the cremaster muscle or in
the foot pad of rats. No oral exposure.
-EXCLUDE on Full Text
In Japanese language, which we do not understand.
-EXCLUDE on Full Text
In Japanese language most of it, not possible for us to
understand properly.
-EXCLUDE on Full Text
On obesity and glucose regulation, not effects on the
intestines.
-EXCLUDE on Full Text
The mice were infected with PbA, were given
deworming medicine, and have been given treatment
from previous published research.
-EXCLUDE on Full Text
All mice were infected with P. berghei.
-EXCLUDE on Full Text
Apparently no negative control group without CMC
included.
-EXCLUDE on Full Text
No clear information on adverse effects.
-EXCLUDE on Full Text
This abstract is on studies of other emulsifiers than CMC
(and not on carrageenan).
-EXCLUDE on Full Text
No groups were given nothing, since CMC was used as
control group and was vehicle for the other substance
studied (rupatadine). It is therefore not possible to
evaluate the effects of CMC compared with a group
without CMC.
-EXCLUDE on Full Text
The project will study only positive effects of
carrageenan.
-EXCLUDE on Full Text
Diet free of several food additives, not only
carageenan.
-EXCLUDE on Full Text
No negative control group to compare with CMC alone.
-EXCLUDE on Full Text
They mention carrageenan, but it is about use for
persons that have difficulties in swallowing liquids by
making it thicker (not on the intestines), positive effects.
-EXCLUDE on Full Text
The animals were injected with bacteria before given
carrageenan.
-EXCLUDE on Full Text
Both hamsters and mice were given bacteria before
carrageenan.
-EXCLUDE on Full Text
The control was a non-immunized guinea pig given 5%
degraded carrageenan ad libitum in drinking water. All
B. vulgatus whole-cell preparations were tested in
controls.
-EXCLUDE on Full Text
Only about beneficial effects, not about adverse effects
of carrageenan.
-EXCLUDE on Full Text
Not on adverse effects, more basic physiology.
-EXCLUDE on Full Text
Not on effects of CMC, only used to grow cells in.
-EXCLUDE on Full Text
The granulomas were induced with k-carrageenin
injected s.c. into the flank of rats, thus, not oral
exposure.
-EXCLUDE on Full Text
The healthy persons in the study were given a diet
without carrageenan. Only positive effects were
recorded.
-EXCLUDE on Full Text
The healthy persons in the study were given a diet
without carrageenan. Only positive effects were
recorded.
(continued)
570 M. TAHIRI ET AL.
Continued.
Publication
Singh (2020) (ID:68321992)
Solimabi (1980) (ID:68321358)
Tkachenko (2021) (ID: 68321798)
Tsuji (2003) (ID:68322329)
Upadhyay (2017) (ID:68322076)
Wang (2022) (ID:78874616)
Wine (2020) (ID:68321634)
Xie (2021) (ID:68322234)
Yang (2022a) (ID:78874587)
Yang (2022b) (ID:78874690)
Yates (1992) (ID:68322236)
Yermak (2020) (ID: 68321922)
Yilmaz-Ersan (2018) (ID:68321654)
Yue (2022) (ID:78874646)
Zangara (2022) (ID:78874663)
Zhang (2022) (ID:78874668)
Zheng (2023) (ID:78874687)
Screen on Title and Abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
-INCLUDE on title & abstract
Screen on Full Text
-EXCLUDE on Full Text
Nothing on adverse effects.
-EXCLUDE on Full Text
Reported positive effects (anti-spasmodic effect).
-EXCLUDE on Full Text
Only data on erythrocytes, not on the intestines.
-EXCLUDE on Full Text
Studied only positive changes in the immune system,
not negative effects.
-EXCLUDE on Full Text
Not on adverse effects.
-EXCLUDE on Full Text
Control group were given CMC. No group without CMC
to compare CMC with.
-EXCLUDE on Full Text
CMC and carrageenan are only mentioned once in this
commentary and it is more on the disease IBD.
-EXCLUDE on Full Text
Only on beneficial effects, therefore excluded.
-EXCLUDE on Full Text
Only on positive effects, except for content of heavy
metals and chemicals in the raw materials. No report of
adverse effects of carrageenan or CMC.
-EXCLUDE on Full Text
CMC was negative control group to puerarin. There was
no group without CMC to compare CMC with.
-EXCLUDE on Full Text
CMC here means mononuclear cells extracted from the
colon. Carboxymethylcellulose or carrageenan are not
mentioned in this paper.
-EXCLUDE on Full Text
Only positive effects reported, no groups with only CGN
included.
-EXCLUDE on Full Text
Examines how carrageenan and other gums can affect
growth of Bifidobacterium longum as a prebiotic. Thus,
no information on adverse effects.
-EXCLUDE on Full Text
CMC as negative control for other substances, but no
group without CMC to compare CMC with.
-EXCLUDE on Full Text
IL10KO mice were conditioned with fecal material from
NOD2KO mice to introduce a pro-inflammatory
microbiota and normalize disease onset between cages.
-EXCLUDE on Full Text
The rats were given infusion of toxic amyloid-beta
(Abeta), effects on memory function, glucose regulation
and microbiota studied. Control rats were on high-fat
diet.
-EXCLUDE on Full Text
Only on beneficial effects, not on adverse effects.
 CRITICAL REVIEWS IN TOXICOLOGY 571

Appendix E. Eppi-reviewer Code book

Eppi-Reviewer Code book for scoping review on adverse effects on the intestines of CGN (E 407)/processed
Eucheuma seaweed (E 407a) and CMC (E 466)

Code Child codes

Food additive
Carrageenan (CGN) (E 407)/Processed Eucheuma seaweed (PES) (E 407a) j-CGN, ί-CGN, k-CGN, Unspecified CGN, Degraded CGN (poligeenan (PGN),
C16, Ebimar), Modified CGN
Carboxymethylcellulose (CMC) (E 466) CMC non-modified, CMC modified
Publication type
Human studies Systematic review�, Randomised controlled trial (RCT), Non-randomised
controlled study, Prospective/retrospective cohort study, Case-control study,
Cross-sectional study, Case report, Incidence/prevalence study
Animal studies Rat, Mouse, Guinea pig, Hamster, Rabbit, Gerbil, Ferret, Pig, Dog, Monkey
In vitro studies Human cells or microbiota, Animal cells and microbiota, Other in vitro studies
Other types of studies Toxicokinetic study, Non-systematic review��, Commentary/Editorial/Letter,
Exposure study, Risk assessment opinions
Conference abstracts Conference abstracts
Human populations
Healthy general population Children, Adolescents, Adults
Patients Patients with inflammatory bowel disease (IBD), including ulcerative colitis
(UC) and Crohn’s disease (CD), Patients with irritable bowel syndrome (IBS),
Patients with other diseases
Publication year
1960-1965 1960, 1961, 1962, 1963, 1964, 1965
1966-1970 1966, 1967, 1968, 1969, 1970
1971-1975 1971, 1972, 1973, 1974, 1975
1976-1980 1976, 1977, 1978, 1979, 1980
1981-1985 1981, 1982, 1983, 1984, 1985
1986-1990 1986, 1987, 1988, 1989, 1990
1991-1995 1991, 1992, 1993, 1994, 1995
1996-2000 1996, 1997, 1998, 1999, 2000
2001-2005 2001, 2002, 2003, 2004, 2005
2006-2010 2006, 2007, 2008, 2009, 2010
2011-2015 2011, 2012, 2013, 2014, 2015
2016-2020 2016, 2017, 2018, 2019, 2020
2021-2023 2021, 2022, 2023
Hypotheses on adverse effects on the intestines Effects on barrier dysfunction or permeability of the intestines including
inflammation, Autoimmune effects, Effects on the intestinal microbiome,
Other hypotheses
�With literature search and quality assessment of included studies.
��With literature search but no quality assessment of included studies.