Safety Evaluation Of Certain Food Additives potx

Safety evaluation of certain
food additives
Prepared by the
Seventy-first meeting of the Joint FAO/WHO
Expert Committee on Food Additives (JECFA)
WHO FOOD
ADDITIVES
SERIES: 62
World Health Organization, Geneva, 2010
WHO Library Cataloguing-in-Publication Data
Safety evaluation of certain food additives / prepared by the Seventy-first meeting of
the Joint FAO/WHO Expert Committee on Food Additives (JECFA).
(WHO food additives series ; 62)
1.Food additives - toxicity. 2.Food contamination. 3.Flavoring agents - analysis.
4.Flavoring agents - toxicity. 5.Risk assessment. I.Joint FAO/WHO Expert Committee
on Food Additives. Meeting (71st : 2009 : Geneva, Switzerland). II.International
Programme on Chemical Safety. III.Series.
ISBN 978 92 4 166062 4 (NLM Classification: WA 712)
ISSN 0300-0923
© World Health Organization 2010
All rights reserved. Publications of the World Health Organization can be obtained from
WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: ). Requests
for permission to reproduce or translate WHO publications – whether for sale or for
noncommercial distribution – should be addressed to WHO Press, at the above address
(fax: +41 22 791 4806; e-mail: ).
The designations employed and the presentation of the material in this publication do
not imply the expression of any opinion whatsoever on the part of the World Health
Organization concerning the legal status of any country, territory, city or area or of its
authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on

maps represent approximate border lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply
that they are endorsed or recommended by the World Health Organization in preference
to others of a similar nature that are not mentioned. Errors and omissions excepted, the
names of proprietary products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify
the information contained in this publication. However, the published material is being
distributed without warranty of any kind, either expressed or implied. The responsibility
for the interpretation and use of the material lies with the reader. In no event shall the
World Health Organization be liable for damages arising from its use.
This publication contains the collective views of an international group of experts and
does not necessarily represent the decisions or the policies of the World Health
Organization.
Typeset in India
Printed in India
CONTENTS
Preface
Specific food additives
Branching glycosyltransferase from Rhodothermus obamensis expressed in
Bacillus subtilis
Cassia gum
Cyclamic acid and its salts: dietary exposure assessment
Ferrous ammonium phosphate
Glycerol ester of gum rosin
Glycerol ester of tall oil rosin
Lycopene from all sources
Octenyl succinic acid modified gum arabic
Sodium hydrogen sulfate
Sucrose oligoesters type I and type II
Annexes

Annex 1 Reports and other documents resulting from previous meetings of
the Joint FAO/WHO Expert Committee on Food Additives
Annex 2 Abbreviations used in the monographs
Annex 3 Participants in the seventy-first meeting of the Joint FAO/WHO
Expert Committee on Food Additives
Annex 4 Acceptable daily intakes and other toxicological information and
information on specifications
v
3
11
29
57
119
133
149
223
237
249
265
277
279
281

PREFACE
The monographs contained in this volume were prepared at the seventy-first
meeting of the Joint Food and Agriculture Organization of the United Nations (FAO)/
World Health Organization (WHO) Expert Committee on Food Additives (JECFA),
which met at WHO headquarters in Geneva, Switzerland, on 16–24 June 2009.
These monographs summarize the data on selected food additives reviewed by the
Committee.

The seventy-first report of JECFA has been published by the World Health
Organization as WHO Technical Report No. 950. Reports and other documents
resulting from previous meetings of JECFA are listed in Annex 1. The participants
in the meeting are listed in Annex 3 of the present publication.
JECFA serves as a scientific advisory body to FAO, WHO, their Member States
and the Codex Alimentarius Commission, primarily through the Codex Committee
on Food Additives, the Codex Committee on Contaminants in Food and the Codex
Committee on Residues of Veterinary Drugs in Foods, regarding the safety of food
additives, residues of veterinary drugs, naturally occurring toxicants and
contaminants in food. Committees accomplish this task by preparing reports of their
meetings and publishing specifications or residue monographs and toxicological
monographs, such as those contained in this volume, on substances that they
have considered.
The monographs contained in this volume are based on working papers that
were prepared by temporary advisers. A special acknowledgement is given at the
beginning of each monograph to those who prepared these working papers. The
monographs were edited by M. Sheffer, Ottawa, Canada.
Many unpublished proprietary reports are unreferenced. These were voluntarily
submitted to the Committee by various producers of the food additives under review
and in many cases represent the only data available on those substances. The
temporary advisers based the working papers they wrote on all the data that were
submitted, and all these reports were available to the Committee when it made its
evaluations.
The designations employed and the presentation of the material in this
publication do not imply the expression of any opinion whatsoever on the part of the
organizations participating in WHO concerning the legal status of any country,
territory, city or area or its authorities, or concerning the delimitation of its frontiers
or boundaries. The mention of specific companies or of certain manufacturers’
products does not imply that they are endorsed or recommended by the
organizations in preference to others of a similar nature that are not mentioned.

Any comments or new information on the biological or toxicological properties
of the compounds evaluated in this publication should be addressed to: Joint WHO
Secretary of the Joint FAO/WHO Expert Committee on Food Additives, Department
of Food Safety and Zoonoses, World Health Organization, 20 Avenue Appia, 1211
Geneva 27, Switzerland.
- v -

SPECIFIC FOOD ADDITIVES

BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS
OBAMENSIS EXPRESSED IN BACILLUS SUBTILIS
First draft prepared by
Dr U. Mueller,
1
Dr P. Verger,
2
Dr Z. Olempska-Beer,
3
Mrs I. Meyland
4
and
Professor R. Walker
5
1
Food Standards Australia New Zealand, Canberra, Australian Capital
Territory, Australia
2
French National Institute for Agricultural Research (INRA) – AgroParisTech,
Paris, France
3

Center for Food Safety and Applied Nutrition, Food and Drug Administration,
College Park, Maryland, United States of America (USA)
4
National Food Institute, Technical University of Denmark, Søborg, Denmark
5
Emeritus Professor of Food Science, School of Biomedical and Health
Sciences, University of Surrey, Guildford, England
1. Explanation
1.1 Genetic modification
1.2 Chemical and technical considerations
2. Biological data
2.1 Biochemical aspects
2.2 Toxicological studies
2.2.1 Acute toxicity
2.2.2 Short-term studies of toxicity
2.2.3 Long-term studies of toxicity and
carcinogenicity
2.2.4 Genotoxicity
2.2.5 Reproductive toxicity
2.3 Observations in humans
3. Dietary exposure
4. Comments
4.1 Assessment of potential allergenicity
4.2 Toxicological data
4.3 Assessment of dietary exposure
5. Evaluation
6. References
1. EXPLANATION
At the request of the Codex Committee on Food Additives at its fortieth
session (FAO/WHO, 2008), the Committee evaluated the enzyme branching

glycosyltransferase (1,4-Į-glucan branching enzyme; Enzyme Commission number
2.4.1.18), which it had not evaluated previously. Branching glycosyltransferase
catalyses the transfer of a segment of a 1,4-Į-D-glucan chain to a primary hydroxy
group in a similar glucan chain to create 1,6-linkages. The enzyme is intended for
3
4
4
4
4
5
5
5
6
6
7
7
7
7
7
8
8
8
9
- 3 -
use in starch processing to obtain modified starch with an increased number of
branch points and improved functional properties.
1.1 Genetic modification
Branching glycosyltransferase is manufactured by pure culture fermentation
of a genetically modified strain of Bacillus subtilis containing a synthetic gene coding
for branching glycosyltransferase from Rhodothermus obamensis. Bacillus

subtilis is a Gram-positive bacterium that is widely distributed in nature and is
considered to be non-pathogenic and non-toxigenic. It has a long history of use in
the production of enzymes used in food processing, including enzymes from
genetically engineered strains. It has also been granted a Qualified Presumption of
Safety status by the European Food Safety Authority (2008).
The gene encoding branching glycosyltransferase was originally cloned
from R. obamensis, a thermophilic bacterium that was isolated from a marine
hydrothermal vent. Based on the amino acid sequence of branching
glycosyltransferase translated from the R. obamensis gene, a synthetic gene was
designed that encodes branching glycosyltransferase with the same amino acid
sequence as that of the native R. obamensis enzyme. The gene was subsequently
placed under deoxyribonucleic acid (DNA) regulatory sequences derived from
several Bacillus species and introduced into the B. subtilis host strain JA1343 by
transformation. The chloramphenicol resistance gene (cat) was used in
transformation as a selectable marker, but it was subsequently deleted to make the
production strain marker free.
1.2 Chemical and technical considerations
Branching glycosyltransferase is secreted during fermentation into the
fermentation broth and is subsequently purified and concentrated. The final product
is formulated with sorbitol, glycerol and water and standardized to a desired activity.
The total organic solids (TOS) content of the branching glycosyltransferase
preparation is approximately 4%. The branching glycosyltransferase enzyme
preparation complies with the General Specifications and Considerations for
Enzyme Preparations Used in Food Processing (FAO/WHO, 2006).
The branching glycosyltransferase preparation is intended for use in the
production of modified starch with improved functional properties, such as higher
solubility, lower viscosity and reduced retrogradation (undesirable structural
changes). The recommended use levels range from 0.4 to 40 kg of the enzyme
preparation per tonne of starch dry substance. The branching glycosyltransferase
is likely to be inactivated and/or removed during starch processing steps. The

enzyme is not added directly to food, and any carryover to food products formulated
with modified starch is expected to be very low.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
Branching glycosyltransferase has been evaluated for potential allergenicity
using bioinformatics criteria recommended in the report of the Joint Food and
Agriculture Organization of the United Nations (FAO)/World Health Organization
4 BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS
(WHO) Expert Consultation on Allergenicity of Foods Derived from Biotechnology
(FAO/WHO, 2001). An amino acid sequence homology search between
branching glycosyltransferase and known allergens listed in the allergen database
at was conducted. No homology was found
for sequence fragments of six contiguous amino acids. However, when using a
sliding window of 80 amino acids, a 35% match was found to sequences of Asp o
21 allergen, which is the Į-amylase from Aspergillus oryzae (TAKA amylase A).
However, the sequence alignment of the two enzymes showed that there are
large differences in the loop regions, and the overall identity is only about 32%.
As the two enzymes belong to the same family of glycosylhydrolases (Family 13;
some homology is not surprising.
Although Į-amylase from A. oryzae is an occupational allergen (Skamstrup
Hansen et al., 1999), allergy symptoms after ingestion of the enzyme were reported
only for four individuals. Three of these individuals consumed bread baked with the
enzyme (Baur & Czuppon, 1995; Kanny & Moneret-Vautrin, 1995; Moreno-Ancillo
et al., 2004), and one had a positive response to the oral challenge with Į-amylase
(Losada et al., 1992). In other studies conducted with patients with documented
occupational or other allergies, no cases of food allergy to Į-amylase from A.
oryzae or other commercial enzymes used in food were identified (Skamstrup
Hansen et al., 1999; Bindslev-Jensen et al., 2006). Thus, food allergy to Į-amylase
from A. oryzae is extremely rare. Moreover, branching glycosyltransferase is a
bacterial protein, whereas nearly all known allergens listed in allergen databases

are eukaryotic proteins. Therefore, despite certain homology to Į-amylase from A.
oryzae, branching glycosyltransferase does not seem to have the characteristics of
a potential food allergen.
2.2 Toxicological studies
Bacillus subtilis is a non-pathogenic and non-toxigenic bacterium that has
been utilized as a source of enzymes used in food for many years.
Toxicological studies were performed with branching glycosyltransferase
using a representative batch (PPY 27209), which was produced according to the
procedure used for commercial production. The liquid enzyme preparation used in
the toxicological studies was a mixture of three preparations from fermentation sub-
batches. The final preparation (specific gravity 1.065 g/ml) had an activity of 89 200
branching enzyme units (BEU) per gram and a TOS value of 7.3%.
2.2.1 Acute toxicity
No information was available.
2.2.2 Short-term studies of toxicity
In a study conducted in accordance with Organisation for Economic Co-
operation and Development (OECD) Test Guideline 408 (Repeated Dose 90-Day
Oral Toxicity Study in Rodents) and Good Laboratory Practice (GLP) requirements,
a 10-ml aqueous suspension of branching glycosyltransferase (batch PPY 27209,
BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS 5
activity 89 200 BEU/g) was administered daily at 0, 77, 256 or 769 mg TOS/kg body
weight (bw) by gavage to groups of 20 Wistar WU [Crl:WI(WU), outbred] rats (10
per sex) for 13 weeks. Stability testing of the prepared preparations at weeks 1, 6
and 13 indicated that the enzyme activity was similar to that predicted. The
experimental parameters determined were clinical signs, body weight, food and
water consumption, neurobehavioural testing (WHO/International Programme on
Chemical Safety functional observational battery), ophthalmic end-points,
haematological parameters, clinical chemical end-points, gross and microscopic
appearance, and organ weights. Blood for haematology and clinical chemistry was
collected during necropsy from male rats on day 91 of treatment and from female

rats on day 92. Ophthalmoscopy was performed before treatment in all rats and
then only in the control and high-dose groups during the last week of treatment. All
other measurements were performed on day 91/92 only.
No treatment-related effects were observed for mortality, clinical signs, body
weight gain, food and water consumption, clinical chemistry, neurobehavioural
effects or ophthalmic end-points. A small, but statistically significant, reduction in
the mean corpuscular haemoglobin concentration, which was observed only in high-
dose males, was considered to have no toxicological significance, because it was
not corroborated by other related haematological parameters, such as packed cell
volume and haemoglobin concentration. A reduction in absolute and relative
weights of the epididymides in low-dose and mid-dose males was considered to be
unrelated to treatment because of the absence of any effects at a 3-fold higher
dose. The slightly increased relative liver weight (5%) and reduced absolute brain
weight (4%) in high-dose males together with the absence of corresponding
histopathological lesions identified in these organs were not considered to be
toxicologically relevant. In both sexes, macroscopic pathology and histopathology
were unaffected by treatment.
Overall, it can be concluded that no toxicologically relevant effects were
seen in this 13-week study of general toxicity in rats when branching glycosyl-
transferase was administered daily by gavage at doses up to 769 mg TOS/kg bw
per day. This dose, the highest dose tested, was therefore taken to be the no-
observed-adverse-effect level (NOAEL) (Appel & Van den Hoven, 2008).
2.2.3 Long-term studies of toxicity and carcinogenicity
No information was available.
2.2.4 Genotoxicity
The results of two studies of genotoxicity with branching glycosyltransferase
(batch PPY 27209) are summarized in Table 1. The first study was conducted in
accordance with OECD Test Guideline 471 (Bacterial Reverse Mutation Test),
whereas the second complied with OECD Test Guideline 487 (In Vitro Mammalian
Cell Micronucleus Test; draft). Both studies were certified for compliance with GLP

and quality assurance.
6 BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS
2.2.5 Reproductive toxicity
(a) Multigeneration studies
No multigeneration studies were available.
(b) Developmental toxicity
No developmental toxicity studies were available.
2.3 Observations in humans
No information was available.
3. DIETARY EXPOSURE
Branching glycosyltransferase can be used in a wide range of foodstuffs, but
it is not expected to be present in the final product. The following estimation is based
on the worst-case assumption that the enzyme is used in all processed food and
beverages and remains in the products consumed. The maximum amount of TOS
added to food is 48 mg/kg. Assuming a daily consumption of 750 g of food (50%)
and 1500 g of beverages (25%), according to the budget method, the amount of
TOS ingested would be about 2 mg/kg bw per day for an adult weighing 60 kg.
4. COMMENTS
4.1 Assessment of potential allergenicity
Branching glycosyltransferase was assessed for potential allergenicity by
comparing its amino acid sequence with the sequences of known allergens
according to the bioinformatics criteria recommended in the report of the Joint
Table 1. Genotoxicity of branching glycosyltransferase in vitro
End-point Test system Concentration Result Reference
Reverse mutation Salmonella
typhimurium TA98,
TA100, TA1535 and
TA1537 and
Escherichia coli
WP2uvrApKM101

156–5000 μg/ml
(liquid culture
method), ±S9
Negative Pedersen
(2008)
Clastogenicity/
aneuploidy
Human lymphocytes 1st and 2nd
experiments: 2813,
3750 or 5000 μg/
ml, ±S9
Negative Whitwell (2008)
S9, 9000 × g supernatant from rat liver.
BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS 7
FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotech-
nology. A 35% homology within a sliding window of 80 amino acids to Į-amylase
from Aspergillus oryzae was identified. Aspergillus oryzae is recognized as the
occupational allergen Asp o 21 and was also reported to cause allergy symptoms
in a few individuals after ingestion. However, no homology between branching
glycosyltransferase and Į-amylase from A. oryzae was found at the level of six
contiguous amino acid sequences. In addition, branching glycosyltransferase is a
bacterial protein, whereas nearly all known allergens are of eukaryotic origin. Thus,
branching glycosyltransferase does not seem to have the characteristics of a
potential food allergen.
4.2 Toxicological data
Toxicological studies were performed with branching glycosyltransferase
using a representative batch (PPY 27209), which was produced according to the
procedure used for commercial production. The liquid enzyme preparation used in
the toxicological studies was a mixture of three preparations from fermentation sub-
batches. The final preparation (specific gravity 1.065 g/ml) had an activity of 89 200

BEU/g and a TOS value of 7.3%.
In a 13-week study of general toxicity in rats, no toxicologically relevant
effects were seen when branching glycosyltransferase was administered daily by
gavage at doses up to 769 mg TOS/kg bw per day. This dose, the highest dose
tested, was therefore taken to be the NOAEL.
Branching glycosyltransferase was not mutagenic in an assay for muta-
genicity in bacteria in vitro and was not clastogenic in an assay for chromosomal
aberrations in human lymphocytes in vitro.
4.3 Assessment of dietary exposure
Branching glycosyltransferase can be used in a wide range of foodstuffs, but
it is not expected to be present in the final product. However, the following estimation
is based on the worst-case assumption that the enzyme is used in all processed
food and beverages and remains in the products consumed. The maximum amount
of TOS added to food is 48 mg/kg. Assuming a daily consumption of 750 g of food
(50%) and 1500 g of beverages (25%), according to the budget method, the amount
of TOS ingested would be about 2 mg/kg bw per day for an adult weighing 60 kg.
5. EVALUATION
The Committee allocated an acceptable daily intake (ADI) “not specified” for
branching glycosyltransferase from this recombinant strain of B. subtilis (JA1343)
used in the specified applications and in accordance with Good Manufacturing
Practice.
8 BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS
6. REFERENCES
Appel, M.J. & Van den Hoven, M.J. (2008) Repeated-dose (13-week) oral (gavage) toxicity
study with branching enzyme, PPY 27209 in rats. Unpublished report No. 7510/02 from
TNO, Zeist, the Netherlands. Submitted to WHO by Novozymes, Bagsvaerd, Denmark.
Baur, X. & Czuppon, A.B. (1995) Allergic reaction after eating alpha-amylase (Asp o 2)-
containing bread. A case report. Allergy, 50, 85–87.
Bindslev-Jensen, C., Skov, P.S., Roggen, E.L., Hvass, P. & Brinch, D.S. (2006) Investigation
on possible allergenicity of 19 different commercial enzymes used in the food industry.

Food Chem. Toxicol., 44, 1909–1915.
European Food Safety Authority (2008) The maintenance of the list of QPS microorganisms
intentionally added to food and feed. Scientific opinion of the Panel on Biological Hazards
adopted on 10 December 2008. EFSA J., 923, 1–48.
FAO/WHO (2001) Evaluation of allergenicity of genetically modified foods. Report of a Joint
FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology,
22–25 January 2001. Rome, Italy, Food and Agriculture Organization of the United Nations
( />FAO/WHO (2006) General specifications and considerations for enzyme preparations used
in food processing. Prepared at the sixty-seventh meeting of the Joint FAO/WHO
Expert Committee on Food Additives, Rome, 20–29 June 2006. Rome, Italy, Food and
Agriculture Organization of the United Nations (FAO JECFA Monographs, No. 3; http://
www.fao.org/ag/agn/jecfa-additives/docs/enzymes_en.htm).
FAO/WHO (2008) Report of the fortieth session of the Codex Committee on Food Additives,
Beijing, China, 21–25 April. Rome, Italy, Food and Agriculture Organization of the United
Nations, Codex Alimentarius Commission (ALINORM 08/31/12 Rev.; http://
www.codexalimentarius.net/web/archives.jsp?year=08).
Kanny, G. & Moneret-Vautrin, D A. (1995) Į-Amylase contained in bread can induce food
allergy. J. Allergy Clin. Immunol., 95, 132–133.
Losada, E., Hinojosa, M., Quirce, S., Sànchez-Cano, M. & Moneo, I. (1992) Occupational
asthma caused by alpha-amylase inhalation: clinical and immunologic findings and
bronchial response patterns. J. Allergy Clin. Immunol., 89, 118–125.
Moreno-Ancillo, Á., Dominguez-Noche, C., Gil-Adrados, A.C. & Cosmes, P.M. (2004) Bread
eating induced oral angioedema due to Į-amylase allergy. J. Investig. Allergol. Clin.
Immunol., 14, 346–347.
Pariza, M.W. & Johnson, E.A. (2001) Evaluating the safety of microbial enzyme preparations
used in food processing: update for a new century. Regul. Toxicol. Pharmacol., 33,
173–186.
Pedersen, P.B. (2008) Branching enzyme, PPY 27209: test for mutagenic activity with strains
of Salmonella typhimurium and Escherichia coli. Unpublished study no. 20078073 from
Novozymes, Bagsvaerd, Denmark. Submitted to WHO by Novozymes, Bagsvaerd,

Denmark.
Skamstrup Hansen, K., Vestergaard, H., Petersen, L.N., Bindslev-Jensen, C. & Poulsen, L.K.
(1999) Food allergy to fungal alpha-amylase in occupationally sensitized individuals.
Allergy, 54(Suppl. 52), 64–65.
Whitwell, J. (2008) Induction of micronuclei in cultured human peripheral lymphocytes
.
Unpublished report No. 1974/71 from Covance Laboratories, Harrogate, England.
Submitted to WHO by Novozymes, Bagsvaerd, Denmark.
BRANCHING GLYCOSYLTRANSFERASE FROM RHODOTHERMUS OBAMENSIS 9

CASSIA GUM
First draft prepared by
Dr S.M.F. Jeurissen,
1
Dr M. DiNovi
2
and Dr A. Mattia
2
1
Centre for Substances and Integrated Risk Assessment, National Institute
for Public Health and the Environment, Bilthoven, the Netherlands
2
Center for Food Safety and Applied Nutrition, Food and Drug Administration,
College Park, Maryland, United States of America (USA)
1. Explanation
1.1 Chemical and technical considerations
2. Biological data
2.1 Biochemical aspects
2.2 Toxicological studies
2.2.1 Acute toxicity

2.2.2 Short-term studies of toxicity
2.2.3 Long-term studies of toxicity and carcinogenicity
2.2.4 Genotoxicity
2.2.5 Reproductive toxicity
2.3 Observations in humans
3. Dietary exposure
3.1 Use in foods
3.2 Dietary exposure estimates
4. Comments
4.1 Toxicological data
4.2 Assessment of dietary exposure
5. Evaluation
6. References
1. EXPLANATION
At the request of the Codex Committee on Food Additives at its fortieth
session (FAO/WHO, 2008), the Committee evaluated cassia gum, which it had not
evaluated previously. Cassia gum is related to guar gum, locust (carob) bean gum
and tara gum in terms of structure and chemical properties. The galactomannans
of guar gum, locust (carob) bean gum and tara gum have mannose to galactose
ratios of 2:1, 4:1 and approximately 3:1, respectively. Each of these three gums was
previously allocated an acceptable daily intake (ADI) “not specified” (Annex 1,
references 39, 57 and 74).
1.1 Chemical and technical considerations
Cassia gum is the purified flour from the endosperm of the seeds of Cassia
tora and Cassia obtusifolia, which belong to the Leguminosae family. Cassia
gum is composed of at least 75% high relative molecular mass (approximately
11
11
12
12

12
12
13
15
15
18
20
20
20
21
23
23
24
25
25
- 11 -
200 000–300 000) polysaccharide, consisting primarily of a linear chain of 1,4-ȕ-D-
mannopyranose units with 1,6-linked Į-D-galactopyranose units. The saccharides
are composed of mannose (77.2–78.9%), galactose (15.7–14.7%) and glucose
(7.1–6.3%). The ratio of mannose to galactose is 5:1.
The manufacture of cassia gum includes cleaning of the source material, by
which the content of Cassia occidentalis (which is a naturally occurring contaminant)
is reduced to less than 0.05%, de-husking and de-germing by thermal mechanical
treatment, followed by milling and screening of the endosperm. The ground
endosperm is further purified by extraction with isopropanol. The concentration of
anthraquinones in cassia gum is below the 0.5 mg/kg detection limit. The food
additive under evaluation is cassia gum that is refined and complies with the
specifications established at the current meeting.
Cassia gum is used as a thickener, emulsifier, foam stabilizer, moisture
retention agent and/or texturizing agent in processed cheese, frozen dairy desserts

and mixes, meat products and poultry products.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
No specific absorption, distribution, metabolism or excretion data were
available on the galactomannans from cassia gum. However, from studies on guar
gum, locust (carob) bean gum and tara gum reviewed by the Committee at its
nineteenth, twenty-fifth and thirtieth meetings, respectively (Annex 1, references
39, 57 and 74), it appears that other galactomannans in related gums undergo no
or only minimal hydrolysis by digestive juices or enzymes, independent of the
specific mannose to galactose ratio. They can be partially fermented by large gut
microflora, but are largely excreted unchanged in faeces. The Committee concluded
that cassia gum will be largely excreted unchanged as well, although fermentation
by gut microflora may occur to some extent. If hydrolysis of cassia gum occurs, the
resulting oligosaccharides or monosaccharides would be expected to be absorbed
and metabolized in normal biochemical pathways.
2.2 Toxicological studies
Most available toxicological studies were performed with semi-refined cassia
gum. Semi-refined cassia gum is produced similarly to the cassia gum currently
under evaluation, with the exception of an additional isopropanol extraction step to
significantly reduce the level of anthraquinones in the latter. Anthraquinones are
impurities that occur naturally in the seeds from which cassia gum is produced,
some of which may display muscle-toxic, genotoxic or carcinogenic properties.
Semi-refined cassia gum contains approximately 70 mg total anthraquinones/kg.
2.2.1 Acute toxicity
Two studies of acute oral toxicity were available. In a limit test, five male
Wister-Han-Schering rats were given in total 5000 mg semi-refined cassia gum/kg
12 CASSIA GUM
body weight (bw) by oral gavage in two doses at a 2-h interval. The oral median
lethal dose (LD
50

value) in this study was >5000 mg/kg bw. The study was certified
for compliance with Good Laboratory Practice (GLP) and quality assurance (QA)
(Schöbel, 1986). In another limit test, 10 male and 10 female KM mice were given
in total 10 000 mg cassia gum/kg bw by oral gavage in four doses over 24 h. The
oral LD
50
value in this study was >10 000 mg/kg bw. Statements regarding
compliance with GLP and QA were lacking (Weidu, 2006).
2.2.2 Short-term studies of toxicity
In a 28-day study of toxicity (Zühlke, 1990), groups of five male and five
female Crl:CD (SD)BR Sprague-Dawley rats (aged 5–6 weeks) were administered
semi-refined cassia gum at dietary concentrations of 0, 2500, 10 000, 25 000 or
50 000 mg/kg feed (equal to doses of 0, 250, 1030, 2590 and 4960 mg/kg bw per
day for males and 0, 230, 1110, 2360 and 4590 mg/kg bw per day for females). A
sixth group received semi-refined cassia gum by gavage (in distilled water) 2 times
a day, at a total dose of 1000 mg/kg bw per day. The study was certified for
compliance with GLP and QA and was essentially performed as described in
Organisation for Economic Co-operation and Development (OECD) Test Guideline
407 (Repeated Dose 28-day Oral Toxicity Study in Rodents), although weekly
detailed clinical investigations and measurements of sensory reactivity were
omitted. Observations included mortality, clinical signs, behaviour, body weight,
food consumption, haematology, clinical chemistry, organ weights (adrenals, brain,
heart, kidneys, liver, ovaries and testes), macroscopic examination and
histopathology (on major organs of the animals in the control group, the 50 000 mg/
kg feed group and the group treated by gavage).
Five animals died during the experiment, but these deaths were incidental
or due to an intubation error or blood sampling procedure and were not
accompanied by signs of systemic target organ toxicity. No clinical changes that
could be attributed to the treatment were observed. Body weight gain was
statistically significantly reduced (–20%) in males of the 50 000 mg/kg feed group,

possibly related to a small (–11%) decrease in food intake in these animals. In
females, body weight gain was statistically significantly reduced (–17%) in the
10 000 and 25 000 mg/kg feed group and in the 1000 mg/kg bw per day group.
These changes are considered to be related to the viscous nature of cassia gum
and not considered to be of toxicological relevance.
Haematology and clinical chemistry findings included several statistically
significant changes that for the most part were small, were not dose related or
occurred in one sex only. They were also claimed to be within the normal range for
the species tested, but historical control data were not provided. The only changes
that were outside the historical control range and could have been related to
treatment were increased mean concentrations of glucose and triglyceride in both
sexes of the 10 000 mg/kg feed group (males 41% and 149% and females 56% and
46%, respectively) and 25 000 mg/kg feed group (males 53% and 168% and
females 74% and 67%, respectively). These findings were not dose related,
however, as they were not observed in the 50 000 mg/kg feed group or in the group
treated by gavage.
CASSIA GUM 13
No treatment-related effects were observed at necropsy or during
histopathological examination. In males, group mean absolute kidney weights were
statistically significantly reduced in the 10 000 mg/kg feed (–8%), 50 000 mg/kg feed
(–15%) and 1000 mg/kg bw per day (–7%) groups, but group mean relative kidney
weights were not affected. In females, in contrast, no changes were observed in
group mean absolute kidney weights, whereas the group mean relative kidney
weight was statistically significantly increased (+11%) in the 50 000 mg/kg feed
group. These inconsistent changes were not considered to be treatment related,
given also the absence of histopathological changes in the kidneys.
Overall, it can be concluded that, in the absence of dose relationships and
histopathological findings, the effects observed were of no toxicological relevance.
The no-observed-adverse-effect level (NOAEL) was 50 000 mg/kg feed, equal to
4590 mg/kg bw per day, the highest dose tested (Zühlke, 1990).

In a limitedly reported 30-day study of toxicity, groups of 10 male and 10
female SD rats were administered cassia gum via the diet at levels corre-
sponding to intakes of 0, 250, 500 and 1000 mg/kg bw per day. Statements
regarding compliance with GLP and QA were lacking. No treatment-related
effects on mortality, body weight gain, food consumption or food utilization were
observed. The investigated haematological (red and white blood cell counts,
haemoglobin) and biochemical parameters (albumin, cholesterol, creatinine,
alanine aminotransferase, aspartate aminotransferase, glucose, total protein,
triglyceride and urea nitrogen) were not affected. No gross findings were observed,
and investigated organ weights (liver, kidney, spleen, ovaries and testes) were not
affected. Histopathological examination of liver, kidney, spleen, stomach and
intestines, ovaries and testes also did not show treatment-related effects. It seems,
therefore, that no adverse effects were observed at doses up to and including 1000
mg/kg bw per day, the highest dose tested (Weidu, 2006).
Groups of four male and four female Beagle dogs were given semi-refined
cassia gum mixed into canned dog food at a dietary concentration of 7500 or
25 000 mg/kg for 90 days (equal to doses of 980 and 3290 mg/kg bw per day for
males and 1130 and 3890 mg/kg bw per day for females). A control group of the
same size was administered the canned dog food with 2300 mg/kg locust (carob)
bean gum. The study was essentially performed as described in OECD Test
Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents) and
was certified for compliance with GLP and QA. The only treatment-related effect
observed was a dose-dependent increase in water consumption. However, as this
was most likely associated with water retention in the gastrointestinal tract by
colloidally dissolved semi-refined cassia gum, it was not considered to be of
toxicological relevance. All other effects observed (for several haematological,
blood coagulation and biochemical parameters and some organ weights) were not
considered treatment related because they lacked a dose or time relationship,
occurred in one sex only and/or remained within the historical reference range.
Overall, it can be concluded that the NOAEL was 25 000 mg/kg feed, equal to

3290 mg/kg bw per day, the highest dose tested (Schuh, 1990).
14 CASSIA GUM
In a 13-week study of toxicity (certified for compliance with GLP and QA),
groups of five male and five female cats were given semi-refined cassia gum as part
of a canned food diet at a concentration of 0, 5000 or 25 000 mg/kg (equal to doses
of 0, 520 and 2410 mg/kg bw per day for males and 0, 530 and 2740 mg/kg bw per
day for females). The study was essentially performed according to OECD Test
Guideline 409, with some slight deviations. No adverse or treatment-related effects
on mortality, behaviour, clinical signs, body weight gain, food and water
consumption, haematology, clinical biochemistry, organ weights, macroscopy or
microscopy were observed. The no-observed-effect level (NOEL) was 25 000 mg/kg
feed, equal to 2410 mg/kg bw per day, the highest dose tested in this study
(Virat, 1984).
2.2.3 Long-term studies of toxicity and carcinogenicity
No information was available for cassia gum.
In a limited long-term study of toxicity with guar gum reviewed by the
Committee at its nineteenth meeting (Annex 1, reference 39), no adverse effects
were observed in rats administered guar gum at a dietary concentration of 5% for
24 months. In carcinogenicity studies reviewed by the Committee at its twenty-fifth
and thirtieth meetings (Annex 1, references 57 and 74), no significant adverse
effects were observed in rats and mice administered locust (carob) bean gum or
tara gum at dietary concentrations up to 5% for 103 weeks.
2.2.4 Genotoxicity
The results of five studies of genotoxicity in vitro with cassia gum and/or
semi-refined cassia gum (three bacterial reverse mutation assays, one
chromosomal aberration assay and one gene mutation assay) are summarized in
Table 1. The first bacterial reverse mutation study (Verspeek-Rip, 1998a) was
conducted with semi-refined cassia gum, the second with purified semi-refined
cassia gum (8.6 mg total anthraquinones/kg; Meerts, 2003) and the third with cassia
gum (Weidu, 2006). The first two studies followed OECD Test Guideline 471

(Bacterial Reverse Mutation Test) and were certified for compliance with GLP and
QA. For the third, limitedly reported study, no statements regarding compliance with
GLP and QA were available. The positive results obtained for semi-refined cassia
gum in strain TA100 at precipitating concentrations in the first reverse mutation
study (Verspeek-Rip, 1998a) were not observed in the second reverse mutation
study. The chromosomal aberration assay (Bertens, 1998) was performed
according to OECD Test Guideline 473 (In Vitro Mammalian Chromosome
Aberration Test), and the gene mutation assay (Verspeek-Rip, 1998b) was
conducted according to OECD Test Guideline 476 (In Vitro Mammalian Cell Gene
Mutation Test). Both studies were certified for compliance with GLP and QA and
used semi-refined cassia gum.
CASSIA GUM 15
Table 1. Results of studies of genotoxicity in vitro with cassia gum
End-point Test system Concentration Result Reference
Reverse mutation Salmonella typhimurium strain TA100,
Escherichia coli WP2uvrA
Range-finding study: 3–5000 μg/plate, ±S9 Positive/
negative
a
Verspeek-Rip (1998a)
S. typhimurium strains TA98, TA1535 and
TA1537
1st experiment: 4–1000 μg/plate, ±S9
S. typhimurium strains TA98, TA100,
TA1535 and TA1537, E. coli WP2uvrA
2nd experiment: 1.6–1000 μg/plate, ±S9
Reverse mutation S. typhimurium strains TA98, TA100,
TA1535 and TA1537, E. coli WP2uvrA
1st experiment: 0.3–100 μg/plate, ±S9
2nd experiment: 0.3–100 μg/plate, ±S9

Negative
b
Meerts (2003)
S. typhimurium strain TA100, E. coli
WP2uvrA
3rd experiment: 100–5000 μg/plate, ±S9
Reverse mutation S. typhimurium strains TA97, TA98, TA100
and TA102
1st experiment: 0.05–5 mg/plate, ±S9
2nd experiment: 0.05–5 mg/plate, ±S9
Negative
c
Weidu (2006)
Gene mutation Mouse lymphoma L5178Y TK
+/–
cells 1st experiment: 0.003–10 μg/ml, ±S9
2nd experiment: 0.003–10 μg/ml, ±S9
Negative
d
Verspeek-Rip (1998b)
Chromosomal
aberration
Human lymphocytes 1st experiment: 1–10 μg/ml, ±S9
2nd experiment: 1–10 μg/ml, ±S9
Negative
e
Bertens (1998)
S9, 9000 × g supernatant from rat liver.
a
With semi-refined cassia gum, with and without metabolic activation (S9), by the plate incorporation method, using dimethyl sulfoxide (DMSO) as a vehicle.

Slight precipitation occurred from 100 μg/plate upwards in the dose range–finding study and from 62.5 and 200 μg/plate upwards in the first and second
experiments, respectively. In none of the experiments was toxicity o
bserved. In two independent experiments, S. typhimurium strain TA100 showed a
dose-related increase in the number of revertants at precipitating concentrations in the presence and absence of metabolic acti
vation,
whereas E. coli WP2uvrA showed negative responses. Salmonella typhimurium strains TA98, TA1535 and TA1537 showed negative responses in the
first experiment, but in the second experiment they showed dose-related increases in the number of revertants at precipitating concentrations in
16 CASSIA GUM
Table 1 (contd)
the presence (TA1537 only) and absence of metabolic activation. However, the increases observed in strains TA98, TA1535 and TA1537 were within
the historical control range.
b
With purified semi-refined cassia gum (8.6 mg total anthraquinones/kg), with and without metabolic activation (S9), by the plate incorporation method.
In the first and second experiments, ultrapure water was used as a vehicle; slight precipitation occurred at 33 and 100 μg/plate, but no toxicity was
observed. In the third experiment, DMSO was used as a vehicle; slight precipitation occurred at all concentrations, but no toxicity was observed.
c
With cassia gum, with and without metabolic activation (S9), by the plate incorporation method, using sterilized distilled water as a vehicle. The study
was reported in a very limited manner, and no information was provided on the occurrence of precipitation or toxicity.
d
With semi-refined cassia gum, with and without metabolic activation (
S9), with DMSO as vehicle. In both experiments, precipitation occurred at 10
μg/ml. In experiment 1, cells were exposed for 3 h and harvested 3 days following exposure. No toxicity was observed. In experiment 2, in the absence
of metabolic activation, cells were exposed for 24 h and harvested 2 days later, whereas in the presence of metabolic activation, cells were exposed
for 3 h and harvested 3 days later. Without metabolic activation, the cell count and cloning efficiency were reduced by 42% and 81% at the highest
concentration tested, respectively, but with metabolic activation, no toxicity was observed.
e
With semi-refined cassia gum, with and without metabolic activation (
S9), with DMSO as vehicle. In both experiments, precipitation occurred at 10
μg/ml. In the first experiment, the cells were exposed for 3 h and harvested 21 h later. The highest tested concentration induced mitotic inhibition
(22%) in the presence, but not in the absence, of metabolic activation. In the second experiment, cells were exposed for 24 or 48 h without S9 and

harvested immediately after exposure. With S9, the cells were treated for 3 h and harvested another 45 h later. The highest tested concentration
induced mitotic inhibition (33%) in the presence, but not in the absence, of metabolic activation.
CASSIA GUM 17
The results of two limitedly reported studies of genotoxicity in vivo (a sperm
abnormality test and a micronucleus test in mice) are summarized in Table 2. These
studies were performed with cassia gum. No statements regarding compliance with
GLP and QA were available (Weidu, 2006).
Overall, the Committee concluded that cassia gum is not genotoxic.
2.2.5 Reproductive toxicity
In a two-generation study of reproductive toxicity, groups of 25 male and
25 female Ico:OFA.SD Sprague-Dawley rats were given diets containing 0, 5000,
20 000 or 50 000 mg semi-refined cassia gum/kg. These dietary concentrations
were equal to doses of 0, 510, 2060 and 5280 mg/kg bw per day for males and 0,
510, 2090 and 6120 mg/kg bw per day for females (calculated using the mean food
intake and mean body weights in weeks 1–10). An additional group received a diet
containing 50 000 mg of purified semi-refined cassia gum (resulting from an
additional isopropanol extraction step) per kilogram (equal to a dose of 5430 mg/kg
bw per day for males and 6230 mg/kg bw per day for females). All parental animals
(P) were treated for approximately 10 weeks before mating and during mating,
gestation and lactation. Pregnant females were allowed to rear their offspring (F
1a
)
to weaning. Rats in both 50 000 mg/kg diet groups exhibited low pregnancy rates,
and the non-pregnant rats were mated again with the same males. They were
allowed to litter, and the subsequent offspring (F
1b
) were terminated on days 5–7
postpartum. Selected F
1a
offspring were treated for a 10-week period of maturation

and during mating, gestation and lactation. Pregnant F
1a
females were allowed to
rear their offspring (F
2
) to weaning. The study was performed according to OECD
Test Guideline 416 (Two-Generation Reproduction Study) and was certified for
compliance with GLP and QA. The only significant findings were a slightly reduced
pregnancy rate in the 50 000 mg semi-refined cassia/kg diet group (pregnancy rate
Table 2. Results of studies of genotoxicity in vivo with cassia gum
End-point Test system Concentration Result Reference
Micronucleus test Bone marrow
of KM mice
(males and
females)
625–2500 mg/kg
bw, by oral gavage
(divided over 2
doses in 30 h)
Negative
a
Weidu (2006)
Sperm abnormality
test
Male KM mice 625–2500 mg/kg bw
per day, by oral
gavage for 5 days
Negative
b
Weidu (2006)

a
Study was performed with cassia gum and was reported in a very limited manner. Bone
marrow was collected 6 h after second gavage, and micronuclei of 1000 polychromatic
erythrocytes (PCE) per animal were counted, followed by determination of the ratio of PCE
to normal chromatic erythrocytes (NCE).
b
Study was performed with cassia gum and was reported in a very limited manner. Sperm
was collected 30 days after last administration, and aberrations were counted in 1000 sperm
cells per animal.
18 CASSIA GUM