WHO Technical Report Series

934

This report represents the conclusions of a Joint FAO/WHO Expert

EVALUATION OF CERTAIN

EVALUATION OF CERTAIN FOOD ADDITIVES

Committee convened to evaluate the safety of various food additives,
with a view to recommending acceptable daily intakes (ADIs) and
FOOD ADDITIVES
to prepare specifications for the identity and purity of food additives.
The first part of the report contains a general discussion of the principles
governing the toxicological evaluation of food additives (including
flavouring agents), assessments of intake, and the establishment and
revision of specifications for food additives.
A summary follows of the Committee’s evaluations of toxicological and
intake data on various specific food additives (Beeswax, Candelilla
wax, Calcium L-5-Methyltetrahydrofolic acid (L-5-MTHF), Phospholipase
A1 from Fusarium venenatum expressed in Aspergillus oryzae, Pullulan,
Quillaia extract Type 1, Quillaia extract Type 2) and seven groups Sixty-fifth report of the joint
of flavouring agents. Annexed to the report are tables summarizing
the Committee’s recommendations for ADIs of the food additives,
FAO/WHO Expert Committee on food additives
recommendations on the flavouring agents considered, changes in the
status of specifications, and further information requested or desired.

WHO Technical Report Series – 934

ISBN 92 4 120934 8
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This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the
stated policy of the World Health Organization or of the Food and Agriculture Organization of the United Nations

WHO Technical Report Series

934

EVALUATION OF CERTAIN
FOOD ADDITIVES

Sixty-fifth report of the

Joint FAO / WHO Expert Committee on
Food Additives

World Health Organization

Geneva 2006

iii
 WHO Library Cataloguing-in-Publication Data

Joint FAO/WHO Expert Committee on Food Additives. Meeting (65th : 2005 : Geneva,
Switzerland)
Evaluation of certain food additives : sixty-fifth report of the Joint FAO/WHO
Expert Committee on Food Additives.

(WHO technical report series; 934)

1. Food additives — toxicity 2. Food additives — analysis 3. Flavouring

agents — toxicity. 4. Flavouring agents — analysis. 5. Risk assessment.
I. Title : Sixty-fifth report of the Joint FAO/WHO Expert Committee on Food
Additives. II. Series. III. World Health Organization

ISBN 92 4 120934 8 (NLM classification: WA 712

ISSN 0512-3054

© World Health Organization 2006

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Printed in Singapore

ii
Contents
1. Introduction ...................................................................................... 1

2. General considerations ....................................................................... 1

2.1 Modification of the agenda ...................................................... 1
2.2 Principles governing the toxicological evaluation of
compounds on the agenda ...................................................... 2
2.2.1 Safety evaluation of flavouring agents .......................... 2
2.2.2 Safety evaluation of enzymes produced by
genetically modified microorganisms ............................ 6
2.3 Food additive specifications .................................................... 7
2.3.1 Compendium of food additive specifications ................. 7
2.3.2 Residual solvents .......................................................... 8
2.3.3 Standard curves in analytical methods ......................... 8
2.3.4 Use of the terms 'anhydrous' and 'dried basis' in
specifications ................................................................. 9
2.3.5 Withdrawal of certain food additive specifications ......... 9
2.3.6 Revision of specifications for ethyl maltol
and maltol ...................................................................... 10
2.4 Project to update the principles and methods for the
risk assessment of chemicals in food ...................................... 10
2.5 FAO/WHO workshop on nutrient risk assessment .................. 10
2.6 Application of approaches to thresholds of
toxicological concern for risk characterization ......................... 11

3. Specific food additives and ingredients (other than flavouring agents) 12

3.1 Safety evaluations ................................................................... 12
3.1.1 Beeswax ........................................................................ 12
3.1.2 Candelilla wax ............................................................... 15
3.1.3 Calcium L-5-methyltetrahydrofolate .............................. 17
3.1.4 Phospholipase A1 from Fusarium venenatum
expressed in Aspergillus oryzae .................................... 22
3.1.5 Pullulan ......................................................................... 25
3.1.6 Quillaia extract type 2 .................................................... 28
3.1.7 Quillaia extract type 1: Assessment of
dietary exposure ............................................................ 31
3.2 Revision of specifications ........................................................ 34
3.2.1 Aspartame–acesulfame salt .......................................... 34
3.2.2 Hexanes ........................................................................ 34
3.2.3 Laccase from Myceliophthora thermophila
expressed in Aspergillus oryzae .................................... 34
3.2.4 Monomagnesium phosphate and trisodium
diphosphate ................................................................... 35
3.2.5 Sucrose esters of fatty acids ........................................ 35

4. Flavouring agents ............................................................................... 35

4.1 Flavouring agents evaluated by the Procedure for
the Safety Evaluation of Flavouring Agents ........................... 36
4.1.1 Maltol and related substances ...................................... 36

iii
 4.1.2 Furan-substituted aliphatic alcohols, aldehydes,
ketones, carboxylic acids and related esters,
sulfides, disulfides and ethers ....................................... 42
4.1.3 Eugenol and related hydroxyallylbenzene
derivatives ..................................................................... 49
4.1.4 Anthranilate derivatives ................................................ 54
4.1.5 Miscellaneous nitrogen-containing substances ........... 64
4.1.6 Epoxides ....................................................................... 82
4.1.7 Aliphatic and aromatic amines and amides ................. 89
4.2 Specifications of identity and purity for flavouring
agents ..................................................................................... 108
4.2.1 Specifications for flavouring agents evaluated
for the first time ............................................................. 108
4.2.2 Revision of existing specifications for
flavouring agents .......................................................... 109

5. Recommendations ............................................................................. 110

Acknowledgement ......................................................................................... 111

References .................................................................................................... 112

Annex 1
Reports and other documents resulting from previous meetings
of the Joint FAO/WHO Expert Committee on Food Additives ....................... 115

Annex 2
Acceptable daily intakes, other toxicological information and
information on specifications ......................................................................... 125

Annex 3
Further information required .......................................................................... 135

Annex 4
Summary of safety evaluations of secondary components of
flavouring agents with minimum assay values of less than 95% .................. 137

Annex 5
Flavouring agents for which use level or reported poundage data
are required ................................................................................................... 141

Annex 6
Divergent opinion on safety assessment of flavouring agents ..................... 145

iv
Joint FAO/WHO Expert Committee on Food Additives
Geneva, 7–16 June 2005

Members

Professor J.R. Bend, Faculty of Medicine and Dentistry, University of Western

Ontario, London, Ontario, Canada
Professor Y. El-Samragy, Food Science Department, Ain Shams University, Cairo,
Egypt
Dr Y. Kawamura, National Institute of Health Sciences, Tokyo, Japan
Dr A. Knaap, National Institute of Public Health and the Environment, Bilthoven,
The Netherlands
Dr P.M. Kuznesof, US Food and Drug Administration, College Park, Maryland, United
States
Dr J.C. Larsen, Danish Institute of Food and Veterinary Research, Søborg, Denmark
(Joint Vice-Chairman)
Dr A. Mattia, US Food and Drug Administration, College Park, Maryland, United
States
Mrs I. Meyland, Danish Institute of Food and Veterinary Research, Søborg, Denmark
(Chairman)
Dr G. Pascal, Institut National de la Recherche Agronomique, Paris, France
Dr M. Veerabhadra Rao, Central Laboratories Unit, United Arab Emirates University,
Al Ain, United Arab Emirates
Dr J. Schlatter, Swiss Federal Office of Public Health, Zürich, Switzerland
Dr P. Verger, Institut National de la Recherche Agronomique, Paris, France
Mrs H. Wallin, National Food Agency, Helsinki, Finland
Dr D.B Whitehouse, Bowdon, Cheshire, United Kingdom

1
Secretariat

Dr P.J. Abbott, Food Standards Australia New Zealand, Canberra, Australia (WHO
Temporary Adviser)
Dr A. Bruno, Joint FAO/WHO Food Standards Programme, Secretariat of the Codex
Alimentarius Commission, Food and Agriculture Organization of the United
Nations, Rome, Italy
Dr R.C. Cantrill, American Oil Chemists' Society, Champaign, Illinois, United States
(FAO Consultant)
Dr R. Charrondiere, Food and Nutrition Division, Food and Agriculture Organization
of the United Nations, Rome, Italy (FAO Staff Member)
Dr M.deL. Costarrica, Food and Nutrition Division, Food and Agriculture Organization
of the United Nations, Rome, Italy (FAO Staff, Acting FAO Joint Secretary)
Dr M. DiNovi, US Food and Drug Administration, College Park, Maryland, United
States (WHO Temporary Adviser)
Dr C.E. Fisher, Hatfield, Hertfordshire, United Kingdom (FAO Consultant, Acting
FAO Joint Secretary)
Dr C.A. Lawrie, Food Standards Agency, London, United Kingdom ( FAO Consultant)
Dr C. Leclercq, National Research Institute for Food and Nutrition, Rome, Italy
(FAO Consultant)

1
Unable to attend: Dr Z. Olempska-Beer, Center for Food Safety and Applied Nutrition, US
Food and Drug Administration, College Park, Maryland, USA; Dr Monica Olsen, Food and
Nutrition Division, Food and Agriculture Organization of the United Nations, Rome, Italy

v
 Dr G. Moy, Food Safety Department, World Health Organization, Geneva,
Switzerland (WHO Staff Member )
Dr I.C. Munro, CanTox Health Sciences International, Mississauga, Ontario, Canada
(WHO Temporary Adviser)
Dr A. Nishikawa, Division of Pathology, National Institute of Health Sciences, Tokyo,
Japan (WHO Temporary Adviser)
Dr S. Page, International Programme on Chemical Safety, World Health
Organization, Geneva, Switzerland (WHO Staff Member)
Mrs Ir M.E.J. Pronk, Center for Substances and Integrated Risk Assessment,
National Institute for Public Health and the Environment, Bilthoven, The
Netherlands (WHO Temporary Adviser)
Professor A.G. Renwick, Clinical Pharmacology Group, University of Southampton,
Southampton, United Kingdom (WHO Temporary Adviser)
Professor I.G. Sipes, Department of Pharmacology, College of Medicine, University
of Arizona, Tucson, Arizona, United States (WHO Temporary Adviser)
Professor L.M. Valenta Soares, Food Science Department, State University of
Campinas, Campinas, Brazil (FAO Consultant)
Professor I. Stankovic, Institute of Bromatology, Faculty of Pharmacy, Belgrade,
Serbia and Montenegro (FAO Consultant)
Dr A. Tritscher, International Programme on Chemical Safety, World Health
Organization, Geneva, Switzerland (WHO Joint Secretary)
Dr L.G. Valerio, Jr, Center for Food Safety and Applied Nutrition, US Food and
Drug Administration, College Park, Maryland, United States ( WHO Temporary
Adviser)
Professor G.M. Williams, Environmental Pathology and Toxicology, New York
Medical College, Valhalla, New York, United States ( WHO Temporary Adviser)

vi
Monographs containing summaries of relevant data and toxicological
evaluations are available from WHO under the title:
Safety evaluation of certain food additives. WHO Food Additive Series, No.
56, in preparation.

Specifications are issued separately by FAO under the title:

Compendium of food additive specifications, Addendum 13. FAO Food and
Nutrition Paper, No. 52, Add. 13.

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

The preparatory work for toxicological evaluations of food

additives and contaminants by the Joint FAO/WHO Expert
Committee on Food Additives (JECFA) is actively supported by
certain of the Member States that contribute to the work of the
International Programme on Chemical Safety (IPCS).
The IPCS is a joint venture of the United Nations Environment
Programme, the International Labour Organization and the World
Health Organization. One of the main objectives of the IPCS is to
carry out and disseminate evaluations of the effects of chemicals
on human health and the quality of the environment.

vii
1. Introduction
The Joint FAO/WHO Expert Committee on Food Additives (JECFA) met
in Geneva from 6 to 17 June 2005. The meeting was opened by Dr Kerstin
Leitner, Assistant Director-General for Sustainable Development and
Healthy Environment, on behalf of the Directors-General of the Food and
Agriculture Organization of the United Nations and the World Health
Organization. Dr Leitner noted that the work of the Committee plays an
important role in improving global food safety, by providing a scientific
foundati(on for international food standards. Equally important is its work
on formulating principles for assessing the safety of food chemicals. In
order that the Committee continue to respond to the increased need for
international, independent scientific advice, despite limited financial
resources, priorities must be set. Strengthening the system for the provision
of scientific advice is a central part of FAO’s and WHO’s normative work,
and there is a clear commitment to continue support for this activity.

2. General considerations
As a result of the recommendations of the first Joint FAO/WHO Conference
on Food Additives, held in September 1955 (1), there have been sixty-four
previous meetings of the Expert Committee (Annex 1). The present meeting
was convened on the basis of the recommendation made at the sixtieth
meeting (Annex 1, reference 163).
The tasks before the Committee were:

— to elaborate further principles for evaluating the safety of food

additives and contaminants (section 2);

— to undertake toxicological evaluations of certain food additives and

flavouring agents (sections 3 and 4 and Annex 2); and
— to review and prepare specifications for selected food additives and
flavouring agents (sections 3 and 4 and Annex 2).

2.1 Modification of the agenda

Three substances, Arpink red, sucralose and the flavouring agent guanilic
acid, were withdrawn from the agenda as no data had been received. Arpink
red and guanilic acid had been referred to the Committee by the Codex
Committee on Food Additives and Contaminants for safety evaluation and
sucralose for revision of specifications. Evaluation of Arpink red had been
scheduled as a priority for the second time, but no data were submitted on

1
 either occasion. The JECFA Secretariat re-emphasized that, when the Codex
Committee requests evaluations by JECFA, it must apply the established
criteria for priorities and ensure that data are available.

2.2 Principles governing the toxicological evaluation of compounds

on the agenda

In making recommendations on the safety of food additives and

contaminants, the Committee took into consideration the principles
established and contained in WHO Environmental Health Criteria, No. 70,
Principles for the safety assessment of food additives and contaminants in
food (Annex 1, reference 76), as well as the principles elaborated at
subsequent meetings of the Committee (Annex 1, references 77, 83, 88, 94,
101, 107, 116, 122, 131, 137, 143, 149, 152, 154, 160, 166), including the
present one. WHO Environmental Health Criteria, No. 70, contains the
most important observations, comments and recommendations made, up to
the time of its publication, by the Committee and associated bodies in their
reports on the safety assessment of food additives and contaminants.

2.2.1 Safety evaluation of flavouring agents

Estimating dietary exposure to flavouring agents
At its forty-sixth meeting (Annex 1, reference 122), the Committee adopted
a procedure for the safety evaluation of flavouring agents. In formulating
this procedure, the Committee recognized the need for an approach that
could be used efficiently for a large class of substances. In view of the
availability from industry of data on production volumes for several thousand
flavouring ingredients, the Committee decided that a method for calculating
per capita exposure, the maximum survey-derived daily intake (MSDI),
could be readily used for assessing exposure as part of the procedure for
safety evaluation. The Committee re-endorsed the MSDI approach at its
forty-ninth (Annex 1, reference 131), fifty-fifth (Annex 1, reference 149),
sixty-first (Annex 1, reference 166) and sixty-third (Annex 1, reference
173) meetings.
The Committee has also discussed limitations to use of the MSDI for
estimating dietary exposure. At its fifty-fifth meeting (Annex 1, reference
149), the Committee noted that use of the MSDI could result in
underestimates of the dietary exposure of persons with high levels of
consumption of certain foods. At its sixty-first meeting (Annex 1, reference
166), the Committee decided that it would not evaluate flavouring agents
for which poundage data had not been reported; at its sixty-third meeting

2
(Annex 1, reference 173), the Committee recognized that the estimates of
current dietary exposure are difficult to reconcile with reported maximum
use levels of some flavouring agents in foods.

At its current meeting, the Committee considered how better to identify and
deal with flavouring agents for which the MSDI estimates, as used in the
procedure, are substantially lower than the dietary exposures estimated from
the levels of use. The Committee anticipated that, in most cases, the existing
data would provide assurance about safety at levels of exposure higher than
the MSDI (particularly for flavouring agents that are not used in a wide
range of food products). Nevertheless, this assumption would need to be
confirmed case by case.
Implications for toxicological data requirements of using model diets for
estimating exposure to flavouring agents

Estimates of exposure based on levels of use (model diets) are higher than
MSDI estimates. Thus, with this method, the exposure thresholds of more
flavouring agents would be exceeded at steps A3 and B3 of the decision
tree, which are central to the procedure for evaluating flavouring agents.
The Committee at its present meeting explored alternative approaches for
estimating dietary exposure , on the basis of use levels1 for about 90% of
the flavouring agents submitted for evaluation. These data made it possible
to prepare conservative estimates of dietary exposure by several methods,
including a model diet. Dietary exposure to most of the flavouring agents
was above the threshold for the respective structural class when estimated
by methods based on use levels, while this was true for only a few compounds
when exposure was estimated as the MSDI. A preliminary comparison of
the dietary exposure estimates with the no-observed effect level (NOEL)
values for selected agents indicated, however, that additional, more
conservative estimates of dietary exposure would suggest a safety concern
in only a few cases. Comprehensive risk characterizations based on dietary
exposure estimates from use levels could not be performed for all agents at
the present meeting because appropriate toxicological data were not required
for assessment of safety with the procedure and had therefore not been
submitted.
The Committee will have to consider carefully the most appropriate approach
for evaluating the safety of flavouring agents on the basis of conservative
methods for estimating dietary exposure .

1
Use levels were taken from the ‘GRAS [generally recognized as safe] lists’ published by
the Expert Panel of the Flavor and Extract Manufacturers Association. of the United States

3
Recommendation to the JECFA Secretariat to form a working group
To address concerns raised by the Committee at its fifty-fifth meeting (Annex
1, reference 149), at a recent FAO/WHO workshop on dietary exposure
assessment (see section 2.4) and in several recent publications, the
Committee recommended that the Secretariat form a small working group,
shortly after the conclusion of the present meeting, to consider further all
relevant aspects of use of an additional screening method based on use
levels, to complement the MSDI, the method used by JECFA for estimating
dietary exposure to flavouring agents. The Committee also recommended
that experts on exposure should work with the temporary advisers during
the preparation of monographs.
The terms of reference for this working group will be determined by the
JECFA Secretariat but might include:

(i) a detailed analysis of the effects of various methods for estimating

dietary exposure on the safety assessment of flavouring agents
according to the procedure;
(ii) formulation of an approach based on dietary exposure estimates
derived from the MSDI and use levels to be used before a meeting to
identify flavouring agents that require special consideration at the
meeting;

(iii) revision of the dietary exposure sections of the safety evaluations of

flavouring agents, for discussion by JECFA at its next meeting; and

(iv) consideration of an approach for estimating combined dietary

exposure to a group of substances using use level-based and model
diets.

Interactions with industry and requests for data

The Committee emphasized that flavouring agents should be evaluated on
the basis of complete, up-to-date information. It therefore welcomed a
proposal from industry to update and extend the existing surveys of flavour
usage.

The Committee recommended that data on poundage be collected regularly

for all flavouring agents, so that rolling averages of poundage can be
calculated. This information should be collected with attention to adequate
quality control.
The apparent discrepancy in dietary exposure to some flavouring agents
between that estimated from reported poundage and that estimated from

4
published use levels requires further investigation to ensure that the safety
evaluations are based on exposure estimates that reflect current (and future)
practice in the food and flavouring industries. The Committee recommended
that studies be undertaken in this area, giving priority to substances of
potential toxicological concern, for which there is only a low margin of
safety between the potential exposure level and the NOEL observed in
studies in experimental animals with the same compound or a structural
analogue.

In a recent analysis made available to JECFA, the margins of safety for 808
flavouring agents previously evaluated by JECFA were compared by use
of estimates derived from either the MSDI or a model diet. The margin of
safety was less than 100 for 16 of 808 substances evaluated by the model
diet method and for one (methyl salicylate) identified by MSDI analysis.
On the basis of estimates of dietary exposure and data on toxicity, a subset
of these flavouring substances were identified as priorities for further
investigation: para-ethylphenol, 2,5-xylenol, 2,6-xylenol, 3,4-xylenol, para-
vinylphenol, 2,3,6-trimethylphenol, 4-phenyl-2-butyl acetate, heptanal
dimethyl acetal, thiamine hydrochloride, 4-[(2-furanmethyl)thio]-2-
pentanone, 4,8-dimethyl-3,7-nonadien-2-one, 2-(methylthio)ethanol, 2,3,5-
trithiahexane, 3-L -menthoxypropane-1,2-diol and 3-(l-menthoxy)-2-
methylpropane-1,2-diol.
The Committee recommended that the JECFA Secretariat ensure that data
on use levels are included in submissions from sponsors for safety evaluation
of flavouring agents, as requested in the call for data. The Committee noted
that such data were not submitted by the sponsors at the current meeting.
Subsequent submissions that do not contain this information will not be
evaluated by the Committee.

Anticipated poundage data

The Committee noted that no poundage was recorded in either the European
Union or the United States of America (USA) for an increasing number of
flavouring agents submitted for evaluation, and MSDI values could be
calculated only on the basis of the annual poundage anticipated by the
manufacturer. This was the situation for 60 of 135 flavouring agents on the
agenda of the present meeting and for a number of those evaluated at the
fifty-ninth, sixty-first and sixty-third meetings. As these MSDI estimates
contain additional uncertainty, the Committee decided that dietary exposure
to such substances, in the future, should either be assessed by an alternative
approach or the assessment should be deferred until actual poundage data
become available.

5
 The Committee requested actual use levels or poundage data for the
flavouring agents that it had previously assessed on the basis of an MSDI
calculated from anticipated poundage. These include substances for which
the MSDI based on anticipated poundage for one region (the European
Union or the USA) was higher than that based on recorded poundage in the
other region. A list of these flavouring agents will be published by the
Secretariat, and the existing assessments will be revoked if the necessary
data are not forthcoming by December 2007.

The Committee decided that the procedure would be applied to evaluate

the safety of the flavouring agents for which data were submitted to the
present meeting, including those for which anticipated poundage data for
the European Union or the USA were submitted. For this group, the
evaluations were made conditional if based on MSDI values derived from
anticipated poundage estimates. The results of the conditional assessments
will be revoked if use levels or poundage data are not provided before
December 2007. This decision was not unanimous, and two members
registered a minority opinion (see Annex 6).
2.2.2 Safety evaluation of enzymes produced by genetically modified
microorganisms

In 1987, the Committee outlined criteria for evaluating the safety of enzymes
(Annex 1, reference 76) and proposed to categorize enzyme preparations
into five main groups on the basis of their origin: animal tissues, portions
of edible plants, microorganisms traditionally accepted as constituents of
food or normally used in the preparation of foods, non-pathogenic
microorganisms commonly found as contaminants of foods or micro-
organisms that are less well known. At the same time, the Committee
envisaged three cases for assessing the safety of enzymes—those added
directly to food and not removed, those added to food but removed and
immobilized enzyme preparations—and indicated guidelines appropriate
for evaluations of safety in each case.

In 1987, enzymes produced by genetically modified microorganisms were

not considered. Subsequently, the Committee evaluated several enzymes
in this category, including laccase from Myceliophthora thermophila
expressed in Aspergillus oryzae and xylanase from Thermomyces
lanuginosus expressed in Fusarium venenatum. The Committee evaluated
the safety of these enzyme preparations on the basis of toxicological data
files, both of which included a 90-day study in rats, a test for reverse mutation
in vitro and a test for chromosomal aberrations. The Committee allocated
an ADI ‘not specified’ to these enzyme preparations.

6
 The present Committee evaluated an enzyme preparation of phospholipase
A1 produced by the same host strain of A. oryzae that has been modified to
produce other enzymes. It could not, however, assess the safety of this
preparation by comparison with the information available on one of the
other enzymes. Therefore, the Committee concluded that guidelines should
be drawn up for the safety assessment of enzymes produced by genetically
modified microorganisms. These guidelines should include the essential
information for various situations and details of molecular characterization
of the producing microbial strain necessary to allow adequate assessment
of the safety of the preparation. These guidelines should be used by the
Committee to adopt a coherent approach to evaluations and should be useful
for sponsors in preparing their applications. The Committee reiterated that
the existing General specifications and considerations for enzyme
preparations used in food processing (Annex 1, reference 156) should be
revised at the same time as guidelines for the safety evaluation of enzyme
preparations.
The report from the Joint FAO/WHO Expert Consultation on Safety
Assessment of Foods Derived from Genetically Modified Microorganisms
(2) should constitute a starting point for this task.

2.3 Food additive specifications

2.3.1 Compendium of Food Additive Specifications

The current Compendium of food additive specifications was published in

1992 (Annex 1, reference 96), consolidating all the specifications for food
additives that had been elaborated by the Committee up to that time.
Specifications that were updated and developed at subsequent meetings
have been published in a series of addenda (Annex 1, references 103, 109,
118, 124, 133, 139, 145, 151, 156, 162, 168, 172). FAO now plans to
consolidate all the specifications in a second edition of the Compendium.

At its current meeting, the Committee considered a report on a number of

issues that had arisen during drafting of an introduction to the second edition.
The new introduction is intended to update the current one and also to serve
as the basis for revising those sections of the Principles for the safety
assessment of food additives and contaminants in food (Annex 1, reference
76) dealing with specifications. The Committee noted that the new
introduction emphasizes the importance of setting specifications as an
inherent part of the risk assessment of food additives, and that the safety
evaluation of an additive should therefore always be read in conjunction
with the specifications of identity and purity for that additive.

7
 The Committee also discussed the conditions under which a ‘tentative’
designation is given to additive specifications and the possible link with the
‘temporary ADI’ designation. It agreed that, although a clear link between
the specifications and the safety assessment is essential, the situations in
which the ‘tentative’ specifications and the ‘temporary ADI’ designations
are used should continue to be judged case by case. The Committee also
reaffirmed that these designations should be time-limited.
2.3.2 Residual solvents

At its sixty-first meeting (Annex 1, reference 166), the Committee recognized

that the general method for determining residual solvents in food additives,
published in FAO Food and Nutrition Paper, No. 5 (3), should be revised. A
tentative general method based on head-space capillary gas chromatography
with flame ionization detection for determination of residual solvents was
published in FAO Food and Nutrition Paper, No. 52, Add. 11 (Annex 1,
reference 168).

At its present meeting, the Committee recognized that several issues had to
be addressed before a single method could be decided upon. For example,
the method should be widely applicable, and its sensitivity should meet the
requirements of the specifications. In addition, preference would be given
to methods studied collaboratively; in the absence of such studies, methods
with other validation would be considered.
In reviewing the responses to the call for data and comments on the tentative
general method, the Committee noted that the critical aspects of the
determination are liberation of solvent residues from the food additive and
their capture by head-space sampling before the gas chromatographic step.
Therefore, the Committee decided that the critical steps should be included
in future specifications rather than in the general method. The Committee
also considered that the tentative general method for determination of
residual solvents by gas chromatography should be revised to include more
solvents.

The Committee further recommended that methods for the analysis of many
common solvents used in the preparation of food additives should be
reviewed during further revision of Guide to specifications—general notices,
general analytical techniques, identification tests, test solutions and other
reference materials(3).

2.3.3 Standard curves in analytical methods

The Committee noted that many specifications mention analytical methods

in which standard or calibration curves are used. These curves are typically

8
 constructed by plotting instrument readings against known concentrations
of a series of standard solutions. It was noted that metrologists reserve the
term ‘calibration’ for the metrological relationship between a reading and a
traceable standard. An example of such a relationship is calibration of an
analytical balance with weights that have been calibrated during an unbroken
chain of comparisons with a national or international measurement standard
for mass. The Committee agreed that the term ‘standard curve’ should be
used in the analytical methods in preference to ‘calibration curve’.
2.3.4 Use of the terms ‘anhydrous’ and ‘dried basis’ in specifications

The Committee agreed that use of the terms ‘anhydrous’, ‘dried basis’ and
‘dry basis’ in food additive specifications has been a source of misunder-
standing. To clarify its position, the Committee agreed to discontinue use of
the term ‘dry basis’ and recommended that provisions in future food additive
specifications should refer to either ‘anhydrous’ or ‘dried basis’. It was agreed
to interpret these terms as described below.

‘Anhydrous’ refers to:

– the calculated amount of substance, adjusted for the known
stoichiometric number of molecules of water of hydration

– the amount of substance adjusted for the measured amount of water,

as determined by a method such as the Karl Fischer method described
in Guide to specifications—general notices, general analytical
techniques, identification tests, test solutions and other reference
materials(3).
‘Dried basis’ refers to:
– the amount of substance remaining after a sample has been subjected
to the stated conditions for loss on drying (e.g. duration, temperature,
pressure, presence of a desiccant).

2.3.5 Withdrawal of certain food additive specifications

Eugenol, methyl anthranilate, methyl N-methylanthranilate, ethyl 3-

phenylglycidate and ethyl methylphenylglycidate were on the agenda for
evaluation as flavouring agents and were found to have specifications in
the standard food additive format. As these substances are used solely as
flavouring agents, the Committee prepared specifications in the format for
flavouring agents and withdrew the specifications in the standard food
additive format.

9
2.3.6 Revision of specifications for ethyl maltol and maltol

Ethyl maltol and maltol were on the agenda for evaluation as flavouring
agents and were found to have specifications in the standard food additive
format. Specifications in the format for flavouring agents were prepared.
As the substances have uses other than as flavouring agents, the Committee
decided to revise the specifications in the standard food additive format but
to designate them as ‘tentative’, pending information on functional uses
and methods of assay.

2.4 Project to update the principles and methods for the risk
assessment of chemicals in food
The Committee was informed of progress on the Joint FAO/WHO project
to update the principles and methods for the risk assessment of chemicals
in foods, including a recent workshop convened to draft guidance on how
to perform and interpret dietary exposure assessments at international,
regional, national and local levels. The workshop participants reviewed the
report of the Joint FAO/WHO Consultation on Food Consumption and
Exposure Assessment of Chemicals (4), and, on this basis, drafted additional
guidance for determining the concentrations of chemicals in food, food
consumption and dietary exposure. The final draft of the report of the
workshop is in preparation. Once finalized, it will be incorporated into the
draft General principles and methods for the risk assessment of chemicals
in food. The Committee took note of this important activity, the outcome of
which will affect the work of JECFA.
The Committee recommended that all the chapters of the General principles
and methods for the risk assessment of chemicals in food and the comments
received during public review undergo external peer review before they
are considered and applied by JECFA.

2.5 FAO/WHO workshop on nutrient risk assessment

The Committee was informed of a joint FAO/WHO workshop on nutrient
risk assessment, held in Geneva on 2–6 May 2005, which was convened to
formulate a model for establishing upper levels of exposure to nutrients
and related substances. The goal was to specify a general approach for
scientific risk assessment that could be used internationally. It is anticipated
that the report of the workshop will be available in autumn 2005.

The Committee took note of this important activity, the outcome of which
might affect the work of JECFA.

10
2.6 Application of approaches to thresholds of toxicological
concern for risk characterization
A threshold of toxicological concern is the level of human exposure below
which no significant risks to health are anticipated, even in the absence of
toxicological data. Risk assessments based on this approach include a variety
of scientific data, such as information on the structure of the substance;
data on absorption, distribution, metabolism and excretion and on the toxicity
of compounds in the same structural class; and, most importantly, data on
exposure. Such pragmatic risk assessments can be used when more
comprehensive evaluations are not possible, to provide timely advice for
risk managers. They are also useful in making priorities for risk management.
The approach should not be used if there are sufficient, chemical-specific
toxicological data for hazard characterization. Furthermore, it should be
used only for defined chemical entities of low relative molecular mass.
The approach of using thresholds of toxicological concern is based on
analyses of the relationships between chemical structures and long-term
toxicity, including carcinogenicity (5–10). Similar approaches have been
proposed for other toxicological end-points (11–13).

JECFA has adopted a decision-tree approach which is based on a series of

considerations of threshold of toxicological concern for the evaluation of
flavouring agents (Annex 1, references 107, 116, 131).
The Committee noted that the following considerations should be taken
into account for further application of ‘threshold of toxicological concern’
approaches:
– The approaches should be used in conjunction with conservative
estimates of dietary exposure.
– Additional data on the toxicity of structurally related substances might
be required.
The Committee reaffirmed use of this approach for flavouring agents. It
recommended that guidance be drawn up on application of the approach
with regard to substances present in the diet in small amounts, such as
certain residues of processing aids, packaging materials and contaminants,
to provide advice on the risk assessment of substances for which full
toxicological datasets are not available or are unnecessary. The Committee
recommended that such guidance be developed by a special task group
appointed by the Joint FAO/WHO secretaries and incorporated into the
Principles and methods for the risk assessment of chemicals in food.

11
3. Specific food additives and ingredients (other than
flavouring agents)
At its present meeting, the Committee evaluated three food additives for
the first time, i.e. calcium L -5-methyltetrahydrofolate, phospholipase A1
from Fusarium venenatum expressed in Aspergillus oryzae and pullulan.
Four others that had been considered at prevous meetings, i.e. beeswax,
candelilla wax, quillaia extract type 1 and quillaia extract type 2, were re-
evaluated. In additon, five substances that had been evaluated at previous
meetings, i.e. aspartame-acesulfame salt, hexanes, laccase from
Myceliophthora thermophila expressed in Aspergillus oryzae, mono-
magnesium phosphate, trisodium diphosphate and sucrose esters of fatty
acids, were considered for review of specifications only. Details are given
in sections 3.1 and 3.2, and the conclusions are summarized in Annex 2.
3.1 Safety evaluations
3.1.1 Beeswax

Explanation

Beeswax was first evaluated by the Committee at its thirty-ninth meeting

(Annex 1, reference 101). At that time, the only toxicological data available
were an LD50 in rats of > 5 g/kg body weight (bw) per day and results
showing lack of mutagenic potential in microbial assays in vitro. The
Committee concluded at that time that “although an evaluation in the
traditional manner could not be carried out, the long history of use of natural
yellow beeswax without apparent adverse effects provided a degree of
assurance that its present functional uses (release and glazing agent in bakery
products, glazing agent on fresh and frozen fruit, glazing agent on candy,
carrier for flavours and component of chewing-gum bases) did not raise
any toxicological concerns.” The processing necessary to obtain bleached
white beeswax was considered not to alter this conclusion. The Committee
also noted that “beeswax might have allergenic potential and that the
consumer should be made aware of its presence in foods” and that “attention
should be paid to the possibility that toxic substances present in honey in
some parts of the world might also occur in beeswax”.
Beeswax was evaluated at the present meeting at the request of the Codex
Committee on Food Additives and Contaminants at its Thirty-sixth Session,
in order to consider the acceptability of its use as a carrier for flavours in
‘water-based flavoured drinks, including “sport”, “energy” or “electrolyte”
drinks and “particulated” drinks, based on adopted provisions in the GSFA
[Codex General Standard for Food Additives]’ (14).

12
Chemical and technical considerations
Beeswax (INS no. 901) is marketed in two forms: yellow beeswax (CAS
no. 8006-40-4) and white beeswax (CAS no. 8012-89-3). Yellow beeswax
is a yellow or light-brown solid that is somewhat brittle when cold and has
a characteristic odour of honey. White beeswax is a white or yellowish-
white solid (thin layers are translucent) with a characteristic, but faint, odour
of honey.
Beeswax is obtained from the honeycombs of honeybees (Apis mellifera L,
family Apidae) after removal of the honey. The combs are melted with hot
water, steam or solar heat. After removal of insoluble impurities, the liquid
wax is cast into cakes for further purification to obtain food-grade yellow
beeswax. Yellow beeswax can be bleached with e.g. hydrogen peroxide,
sulfuric acid or sunlight to produce white beeswax.

The composition of beeswax depends to some extent on the subspecies of

bee, the age of the wax and the climatic circumstances of its production.
The variations in composition are, however, mainly in the quantitative ratios
of the components and not in their chemical identity. Beeswax consists
primarily of five main groups of components: (1) free fatty acids (typically
12–14%), most of which (about 85%) are saturated and have a chain length
of C24–C32; (2) free primary fatty alcohols (about 1%) with a chain length
of C28–C35; (3) linear wax monoesters and hydroxymonoesters (35–45%)
with chain lengths generally of C40–C48, which are derived almost
exclusively from palmitic acid, 15-hydroxypalmitic acid and oleic acid; (4)
complex wax esters (15–27%) containing 15-hydroxypalmitic acid or diols,
which, through their hydroxyl group, are linked to another fatty acid
molecule; and (5) odd-numbered, straight-chain hydrocarbons (12–16%)
with a predominant chain length of C27–C33.

The food applications of beeswax include its use as a component in dietary

food supplements (soft gelatine capsules and tablets), as a glazing and coating
agent, as a texturizer for chewing-gum base and as a carrier for food additives
(including flavours and colours). When used as a flavour carrier in water-
based flavoured drinks, beeswax imparts a cloudy appearance.

At its sixty-third meeting (Annex 1, reference 173), the Committee revised

the limit for lead established for beeswax at its thirty-ninth meeting. At the
present meeting, the Committee incorporated this change to the limit into
the full specification monograph and added a purity test for the presence of
carnauba wax; it also modified the section on functional uses to include the
technical effect of clouding.

13
Toxicological data
The Committee evaluated additional biochemical and toxicological studies
on the main components of beeswax (linear monoesters, complex esters,
hydrocarbons, free fatty acids and free fatty alcohols) and considered the
use of beeswax in water-based, flavoured drinks. The toxicological studies
conducted on the various components of beeswax included short-term studies
with oral administration, long-term studies of toxicity and carcinogenicity
and studies of reproductive toxicity. The components, which are common
in other foods, were not toxic. A search of the literature did not reveal the
presence of naturally occurring toxic substances in commercial beeswax. It
was noted that beeswax administered topically or by intracutaneous injection
did not induce an allergic response in humans.
Assessment of dietary exposure

Information was submitted on the food uses and resulting exposures to

beeswax. Dietary exposure would be about 350 mg per person per day for a
person with 90th percentile exposure to foods containing beeswax, in
addition to consumption as a component of food supplement tablets or
capsules. The Committee received information on the poundage of beeswax
sold to the food market in the European Union in 2003. If it is assumed that
1–10% of the population consumes products containing beeswax, the average
dietary exposure to beeswax per consumer would be 10–100 mg per person
per day.

The Committee estimated the dietary exposure to beeswax from reports of

its current use and maximum levels of use in foods, including as a flavour
carrier in water-based drinks. On the basis of the conservative assumption
that a person would consume all foods (and food supplement tablets or
capsules) containing beeswax at the 95th percentile in each food category
and that all those foods would contain beeswax, exposure to beeswax would
be < 650 mg per person per day. Addition of use as a carrier for flavours in
water-based drinks would result in an increase in the estimated dietary
exposure of 200 mg per person per day, about 50% higher than the estimated
exposure from current uses (450–650 mg per person per day).

Evaluation
The Committee concluded that current uses of beeswax, including that as a
carrier for flavours and as a clouding agent in water-based drinks, would
not result in dietary exposure that raised concern about safety, especially in
view of the long history of use of beeswax and the absence of toxicity of the
main components. As the available information was very limited, the

14
 Committee was unable to reach a conclusion about the potential allergenicity
of beeswax noted by the Committee at its thirty-ninth meeting.
A revised toxicological monograph and a chemical and technical assessment
were prepared. The existing specifications were revised.

3.1.2 Candelilla wax

Explanation
Candelilla wax was first evaluated by the Committee at its thirty-ninth
meeting (Annex 1, reference 101). At that time, the Committee evaluated a
number of studies in mice, rats and dogs. No compound-related toxicity
was observed in a 6-month study in dogs or in a 2-year study in rats treated
orally, even at the highest dietary levels, equivalent to 600 and 750 mg/kg
bw per day, respectively. These studies were considered to be of fundamental
importance in the safety assessment. Furthermore, candelilla wax was not
mutagenic in microbial assays in vitro with or without metabolic activation.
Deficiencies in the studies in experimental animals, in the light of current
criteria, were considered by the Committee at that time to be counterbalanced
to some extent by the consistent absence of adverse effects.

Candelilla wax was evaluated at the present meeting at the request of the
Codex Committee on Food Additives and Contaminants at its Thirty-sixth
Session, in order to consider the acceptability of its use as a carrier for
flavours in ‘water-based flavoured drinks, including “sport”, “energy” or
“electrolyte” drinks and “particulated” drinks, based on adopted provisions
in the GSFA [Codex General Standard for Food Additives]’ (14).
Chemical and technical considerations
Candelilla wax (INS no. 902; CAS no. 8006-44-8) is a yellowish-brown,
hard, brittle, lustrous solid with an aromatic odour when heated. It consists
primarily of odd-numbered saturated straight-chain hydrocarbons (C29–
C33) and esters of acids and alcohols with even-numbered carbon chains
(C28–C34). The most abundant n-alkane, C31, comprises more than 80%
of total n-alkanes. Free acids, free alcohols, sterols, neutral resins and mineral
matter (< 1%) are also present.
Candelilla wax is obtained from the slender, leafless, wax-coated, cylindrical
stalks of several species of Euphorbiacea; the primary source is E.
antisyphilitica. The crude wax is obtained by boiling the dried stalks in
water acidified with sulfuric acid to release the wax. The molten wax, known
as ‘cerote’, is then skimmed off and allowed to solidify. It is refined by
treatment with sulfuric acid and subsequent passage through filter presses.

15
The principal food applications of candelilla wax previously considered by
the Committee were as a glazing and surface-finishing agent, as a texturizer
for chewing-gum and as a carrier for food additives (including flavours and
colours). When used as a flavour carrier for water-based flavoured drinks,
candelilla wax imparts a cloudy appearance.

At its sixty-third meeting (Annex 1, reference 173), the Committee revised

the limit for lead established for candelilla wax at its thirty-ninth meeting.
At the present meeting, the Committee incorporated this change into the
full specification monograph, expanded the definition to give more
information on the production method, and modified the section on
‘functional uses’ to include the technical effect of clouding agent.
Toxicological data
No new information was available to the Committee on the safety of
candelilla wax.
Assessment of dietary exposure

The only information submitted to the Committee was poundage data in

the European Union for 2004 and in the USA for 1995. The per capita
dietary exposure in the European Union (379 million persons) on the basis
of this poundage would be 0.02 mg per person per day, while that for the
USA (260 million persons) would be 0.01 mg per person per day. These
poundages do not include use of candelilla wax in tablets or capsules for
dietary supplements.

Although the poundage data suggest little current use, candelilla wax is
currently listed in the Codex General Standard for Food Additives for use
within good manufacturing practice in the same food categories as beeswax
(also reviewed at the present meeting). As no other information was available,
the Committee estimated dietary exposure to candelilla wax on the basis of
the data submitted for beeswax, including use as a carrier for flavours in
water-based drinks. Using the conservative assumption that an individual
would consume all the foods (and tablets or capsules) containing candelilla
wax at the highest percentile in each food category and that all those foods
contained candelilla wax, the Committee calculated that the dietary exposure
would be < 650 mg per person per day. Addition of use as a carrier for
flavours in water-based drinks would result in an increase in the estimated
dietary exposure to 200 mg per person per day, approximately 50% higher
than the estimated exposure from current uses (450–650 mg per person per
day).

16
 Evaluation
The Committee concluded that the uses of candelilla wax, including as a
carrier for flavours and as a clouding agent in water-based drinks, would
not result in dietary exposure that raises concern about safety.
No toxicological monograph was prepared. The existing specifications were
revised, and a chemical and technical assessment was prepared.

3.1.3 Calcium L-5-methyltetrahydrofolate

Explanation

Calcium L-5-methyltetrahydrofolate is the calcium salt of L -5-methyltetra-

hydrofolic acid, which is the predominant naturally occurring form of folate.
It contains a reduced and methylated pteridine ring system. This compound
has not been evaluated previously by the Committee.
Calcium L -5-methyltetrahydrofolate is structurally analogous to the reduced
form of folic acid (pteroyl-L-glutamic acid), which is the nutritionally active
form. The form of naturally occurring reduced folate found predominantly
in food is a polyglutamyl folic acid. L-5-Methyltetrahydrofolic acid is a co-
factor for key enzymatic reactions in the transfer and processing of the one-
carbon units needed for re-methylation of homocysteine to methionine, to
serve as the methyl donor for numerous methyltransferases, which methylate
a range of biological substrates (lipids, proteins, myelin, dopamine). It also
serves as a carbon donor in the pathway leading to nucleotide synthesis,
supporting the biosynthesis of DNA.

The safety of folic acid was evaluated by the European Commission

Scientific Committee for Food (15), which established a tolerable upper
intake level of folate at 1 mg per adult per day in order to avoid masking
vitamin B12 deficiency. The same tolerable upper intake level for folate was
established by the Institute of Medicine in the USA (16) and by the FAO/
WHO consultation on human vitamin and mineral requirements (17). The
Scientific Panel on Food Additives, Flavourings, Processing Aids and
Materials in Contact with Food of the European Food Safety Authority
concluded that use of calcium L-5-methyltetrahydrofolate as a source of
folate in foods for specific nutritional uses, food supplements and foods
intended for the general population, with a tolerable upper intake level of
1 mg per adult per day, was not a concern with regard to safety (15).
For calcium, the tolerable upper intake level established by the European
Commission Scientific Committee for Food and the Institute of Medicine

17
was 2.5 g, and that established by the FAO/WHO consultation on human
vitamin and mineral requirements was 3 g.
At the request of a WHO Member State, the Committee was asked to evaluate
the safety of calcium L-5-methyltetrahydrofolic acid as an alternative to
folic acid in food fortification and supplementation.

Chemical and technical considerations

Calcium L-5-methyltetrahydrofolate is a white to light-yellowish, almost
odourless crystalline powder. Its full chemical name is N-{4-[[((6S)-2-amino-
3,4,5,6,7,8-hexahydro-5-methyl-4-oxo-6-pteridinyl)methyl]amino]-
benzoyl}-L-glutamic acid, calcium salt. It is also referred to as:
— L-methylfolate, calcium;
— L-5-methyltetrahydrofolic acid, calcium salt;
— (6S)-5-methyltetrahydrofolic acid, calcium salt;
— (6S)-5-methyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid, calcium
salt; and
— L-5-methyltetrahydrofolic acid, the cation being unspecified.

Calcium L-5-methyltetrahydrofolate is proposed for use as an alternative to

folic acid in dry crystalline or microencapsulated form in dietary
supplements, foods for special dietary uses and other foods, in food
supplements (providing 400 µg of folate per day), meal replacements
(providing 200 µg of folate per meal), starch-based fortified foods
(containing folate at 400 µg/kg of prepared food) and milk-type products
(containing folate at 300 µg/l).
Calcium L-5-methyltetrahydrofolate is synthesized by reduction of folic
acid to tetrahydrofolic acid, followed by methylation and diastereoselective
crystallization (in water) of L-5-methyltetrahydrofolic acid as its calcium
salt. The product contains variable amounts of water of crystallization.

The purity of the substance evaluated at the present meeting was stated to
be 95% or greater. Specifications for the identity and purity of the material
intended for addition to food include limits for water content, calcium, lead
and organic folate impurities and related substances. The organic impurities,
which are determined by liquid chromatography, can be categorized as minor
amounts of other folates, breakdown and oxidation products and other
products, including the D stereoisomer of 5-methyltetrahydrofolic acid and
the dimethylated form of tetrahydrofolic acid. The specifications set by the
Committee include a combined limit of 2.5% for the first two classes and a
limit of 1.0% for the D diastereoisomer of 5-methyltetrahydrofolic acid.

18
Calcium L-5-methyltetrahydrofolate in crystalline micronized form is stable
during food processing and long-term storage under conditions of high
temperature and humidity. It is stable in multivitamin tablets and in micro-
encapsulation and food matrix systems. It is less stable in aqueous solution
at elevated temperature, forming products with and without folate vitamin
activity as a result of oxidative degradation.
Toxicological data
Studies in humans indicate that L-5-methyltetrahydrofolic acid is the only
form of folate normally taken up by cells and appearing in plasma, and that
cellular uptake is mediated by a reduced folate carrier and folate receptors,
which are integral plasma membrane proteins. At exposure to folate of
> 200–300 µg/day per person, the metabolic capacity of the human intestinal
mucosa for folic acid begins to be exceeded, resulting in small amounts of
unaltered folic acid in circulating blood.
In humans given 3H- and 14C-folic acid orally, the bioavailability of synthetic
folic acid was estimated to be 90–95%. A study of the absorption of calcium
L-5-methyltetrahydrofolate indicated that it dissociates in aqueous media
into Ca2+ and L -5-methyltetrahydrofolic acid. After absorption, the latter
enters the circulation directly, becoming indistinguishable from other
absorbed and metabolized natural folates or from L-5-methyltetrahydrofolate
formed from synthetic folic acid. The bioavailability of calcium L-5-
methyltetrahydrofolate and synthetic folic acid (400 µg/day per person as
folate) was compared in a randomized, double-blind, cross-over study of
21 healthy women. The bioavailability of the two compounds was found to
be similar. In a 24-week placebo-controlled study in women, the appearance
of folate derived from equimolar concentrations of calcium L -5-methyl-
tetrahydrofolate and folic acid was compared in plasma and erythrocytes
by a microbiological assay: similar values were found for the two
supplements.
A comparison of the bioavailability of naturally occurring folate from food
and synthetic folic acid in humans showed significant differences, with a
lower level of bioavailability from natural folate than from synthetic folic
acid. Another study found that synthetic folic acid appeared in human plasma
more slowly than natural folate. The Committee noted that the differences
in bioavailability might reflect rate-limiting kinetics for the metabolic
conversion of folic acid to L-5-methyltetrahydrofolic acid, as low doses of
radiolabelled compounds showed similar short-term distribution, metabolism
and kinetics in vivo. Moderately high doses (several hundred micrograms)
of folic acid are likely to result in significant hepatic uptake, enterohepatic
circulation, tissue distribution and urinary reabsorption.

19
Calcium L-5-methyltetrahydrofolate was not acutely toxic to rats after a
single oral dose (LD50 > 2000 mg/kg bw): no gross changes in organs were
observed at necropsy, and all animals gained weight and survived until end
of the 15-day observation period. In a short-term study of toxicity in male
and female Wistar rats given calcium L-5-methyltetrahydrofolate orally for
13 weeks, no adverse effects were seen. The NOEL was 400 mg/kg bw per
day, the highest dose tested. A study of embryotoxicity and teratogenicity
in Wistar rats given the compound showed no effects up to the highest dose
tested (1000 mg/kg bw). The results of a battery of assays for genotoxicity
in vitro and in vivo did not indicate any genotoxic potential.

No long-term studies of toxicity or carcinogenicity were submitted; however,

the Committee noted that, given the well-characterized metabolism and
nutritional function and the known fate of naturally occurring reduced L -5-
methyltetrahydrofolic acid as an essential vitamin in humans, such studies
were not required.
The Committee took note of a case report in which L-5-methyltetrahydrofolic
acid did not mask the clinical features of vitamin B12 deficiency. Vitamin
B12 is essential for the activity of methionine synthase, which converts
homocysteine to methionine, with L-5-methyltetrahydrofolic acid as a co-
factor. Recycling of homocysteine back to methionine is part of the
methylation cycle necessary for methyltransferases, which methylate a wide
range of substrates, such as hormones, lipids and proteins, including neural
myelin basic protein. In vitamin B 12 deficiency, the recycling of
homocysteine back to methionine diminishes with the level of methionine
synthase activity, resulting in neuropathy. Owing to the diminished activity
of the enzyme, administration of L-5-methyltetrahydrofolic acid has no effect
on the methylation cycle. Likewise, administration of synthetic folic acid
has no effect on the methylation cycle because it is not a substrate of the
enzyme. The pernicious anaemia arising from vitamin B12 deficiency is
corrected by synthetic folic acid because it replenishes the supply of
tetrahydrofolate and thereby restores the metabolic pathway leading to DNA
biosynthesis and red blood cell formation. Folic acid does not restore
methylation reactions via methionine synthase, so that neuropathy can
progress in the absence of pernicious anaemia (masking). L -5-Methyltetra-
hydrofolic acid is not expected to correct the pernicious anaemia caused by
vitamin B12 deficiency because diminished methionine synthase activity
leads to failure to convert L-5-methyltetrahydrofolic acid to tetrahydrofolate,
the pathway leading to DNA biosynthesis and red blood cell formation. No
data were available to determine whether long-term administration of L -5-
methyltetrahydrofolic acid would not mask vitamin B12 deficiency.

20
Epidemiological studies provided evidence that high plasma homocysteine
levels are a risk factor for cardiovascular disease. A meta-analysis of clinical
trials showed that 0.5–5 mg of folic acid could reduce blood homocysteine
concentrations by 25–33%. In three intervention studies, lasting up to
24 weeks, groups of healthy persons were given folic acid, vitamin B12 or
calcium L-5-methyltetrahydrofolate at a dose of ≤ 950 µg per person per
day. Significantly elevated plasma folate levels were found in response to
folate treatment, accompanied by significantly reduced (by 9–19%) levels
of plasma homocysteine. Calcium L-5-methyltetrahydrofolate and synthetic
folic acid had similar effects.

Methylenetetrahydrofolate reductase is a key enzyme in folate metabolism,

converting methylenetetrahydrofolate to L-5-methyltetrahydrofolic acid.
Persons homozygous for a mutation in the gene encoding 5-methyltetra-
hydrofolic acid reductase have decreased specific enzymatic activity (~ 34%
of normal), lower plasma folate levels and higher plasma homocysteine
concentrations than persons who express the wild-type gene. The prevalence
of this mutant genotype is related to ethnic group, but elevated homocysteine
levels are not a specific indicator of inadequate intake of folate.

In a series of studies with healthy persons who had not been not genotyped
for 5-methyltetrahydrofolic acid reductase activity, L-5-methyltetrahydro-
folic acid was found to be as effective as folic acid in lowering plasma
homocysteine, at doses as low as 400 µg/day as folate.
In a case–control study of the risk for colorectal adenoma associated with
two polymorphisms in thymidylate synthase, a key enzyme in folate
metabolism downstream of L-5-methyltetrahydrofolic acid, an intake of
folic acid > 440 µg/day was associated with a 1.5-fold increase in risk for
colorectal adenomas in polymorphic individuals with a double-repeat in
the enhancer region of the thymidylate synthase gene and an estimated
threefold decreased risk in individuals with a more common triple repeat.
The Committee noted the existence of several common inherited
polymorphisms in folate-metabolizing enzymes; however, the influence and
human health significance of such gene–nutrient interactions on overall
folate status is unclear.
No controlled studies on human tolerance to calcium L-5-methyltetrahydro-
folate were submitted to the Committee. Circumstantial evidence for high
tolerance to the compound and to calcium DL-5-methyltetrahydrofolate was
provided by studies in which oral doses of 15–17 mg/day for up to 6 months
were given to haemodialysis or psychiatric patients. Although no toxic
effects were reported, the scope and design of the studies were inadequate
to contribute to a safety evaluation.

21
 Assessment of dietary exposure
Both in Europe and the USA, the average folate intake from food sources is
about 300 µg/day for men and 250 µg/day for women. Assessments of
exposure to folate were available from three countries with a history of
supplementation and food fortification with folic acid. It was assumed that
calcium L-5-methyltetrahydrofolate would be substituted for synthetic folic
acid in the same products and at the same levels. Supplementation leads to
increases in the average intake of folate in the adult population of
15–90 µg/day (Ireland and the United Kingdom), and mandatory fortification
of foods could increase the average intake of folate by about 200 µg/day
(USA). Overall intake from natural foods, fortified foods and supplements
could reach 1 mg or more per day for some segments of the adult population.
The calcium provided by 1 mg of calcium L -5-methyltetrahydrofolate
amounts to 0.08 mg per adult per day which is insignificant in comparison
with the tolerable upper intake levels for calcium.
Evaluation
The Committee concluded that, in humans, the bioavailability of calcium
L -5-methyltetrahydrofolate is similar to that of folic acid and that synthetic
calcium L-5-methyltetrahydrofolate has the same metabolic fate as other
absorbed natural folates. The Committee evaluated the intended use of
calcium L-5-methyltetrahydrofolate as a substitute for folic acid but did not
evaluate the safety of folate fortification and supplementation. The
Committee had no concern about the safety of the proposed use of calcium
L -5-methyltetrahydrofolate in dry crystalline or microencapsulated form as
an alternative to folic acid in dietary supplements, foods for special dietary
uses and other foods.
In view of a number of common inherited polymorphisms in folate
metabolism, the Committee recommended that the health effects of folates
be evaluated further when there is better understanding of the role of relevant
genetic polymorphisms in the population.

A toxicological monograph and a chemical and technical assessment were

prepared. New specifications were drawn up.

3.1.4 Phospholipase A1 from Fusarium venenatum expressed in Aspergillus

oryzae

Explanation

At the request of the Codex Committee on Food Additives and Contaminants

at its Thirty-sixth Session (14), the Committee evaluated the enzyme

22
phospholipase A1 (phosphatidylcholine 1-acylhydrolase, E.C. 3.1.1.32),
which it had not evaluated previously. Phospholipase A1 is an enzyme that
acts specifically on the fatty acid in position 1 in phospholipid substrates,
resulting in the formation of lysophospholipids and free fatty acids. It is
intended to be used in the dairy industry as a processing aid in the manufacture
of cheese.
Genetic modification
The phospholipase A1 enzyme preparation under evaluation is produced by
submerged fermentation of Aspergillus oryzae carrying a gene encoding
phospholipase A1 from Fusarium venenatum. The host organism A. oryzae
is not pathogenic and has a long history of safe use in food. The specific
host strain used, A. oryzae BECh2, and the production strain derived
therefrom, A. oryzae PFJo142, were claimed to constitute a safe strain lineage,
given the genetic modifications to remove genes encoding amylases and
proteases and to remove or strongly reduce the potential for producing
secondary metabolites (aflatoxins, cyclopiazonic acid, 3-β-nitropropionic
acid and kojic acid). Furthermore, the expression plasmid was fully
characterized, as known DNA sequences were used in the construction and
the DNA derived from F. venenatum was limited to the phospholipase A1
coding sequence. The plasmid does not contain antibiotic resistance genes,
nor does it contain any unidentified DNA or DNA sequences that would
result in the production of toxic substances. Phospholipase A1 expressed by
the production strain has no significant amino acid sequence homology with
known allergens or toxins. Two test batches of the phospholipase A1 enzyme
preparation were shown not to contain secondary metabolites.

Chemical and technical considerations

Phospholipase A1 is produced by submerged fed-batch pure culture
fermentation of the A. oryzae PFJo142 production strain. It is secreted into
the fermentation medium, from which it is recovered and concentrated. It is
subsequently stabilized, formulated and standardized with glycerol, sucrose,
water, sodium benzoate and potassium sorbate. The phospholipase A1
enzyme preparation conforms to the General specifications and
considerations for enzyme preparations used in food processing prepared
by the Committee at its fifty-seventh meeting (Annex 1, reference 156).
The enzyme preparation is free from the production organism and
recombinant DNA.
The phospholipase A1 preparation is added to milk before the coagulation
step in the manufacture of cheese, in order to modify phospholipids in milk.
The modified phospholipids have improved emulsification properties and
help to retain more solids in the cheese. After coagulation, most of the enzyme

23
is drained off with the whey stream, which is pasteurized, causing
inactivation of phospholipase A1. Any enzyme remaining in the cheese can
no longer function, either because there is no substrate left or because the
substrate is occluded by the solid cheese matrix and therefore unavailable
to the enzyme. Cheese can contain the reaction products, lysophospholipids
and free fatty acids, which are considered normal constituents of the diet.
The recommended dosage is up to 10 lecitase units/g milk fat, corresponding
to 350 lecitase units (or 3.5 mg of total organic solids) per litre of milk if the
milk contains 3.5% milk fat.
Toxicological data

Only two toxicological studies were performed in vitro with the

phospholipase A1 enzyme under evaluation, because the A. oryzae BECh2
host strain and the production strain A. oryzae PFJo142 derived therefrom
were considered to constitute a safe strain lineage. Additionally, summaries
were provided of toxicological studies performed with four enzymes derived
from the same host strain, A. oryzae BECh2: a modified lipase, glucose
oxidase and two xylanases. The DNA introduced into the production strains
of these enzymes is essentially the same as that introduced into the
phospholipase A1 production strain, except for the sequence encoding the
specific enzyme. At the request of the Committee, full toxicological data
were also provided on one of the four enzymes, a xylanase on which studies
had recently been conducted. The studies were a 13-week study of oral
toxicity in rats, an assay for mutagenicity in bacteria in vitro and two assays
of cytogeneticity in human lymphocytes in vitro.
In the two toxicological studies with the phospholipase A1 enzyme, a test
batch of the liquid enzyme concentrate was used, without stabilization,
formulation or standardization. The liquid enzyme concentrate was not
cytotoxic in an assay in mammalian cells in vitro. Considerable cytotoxicity
was, however, observed in bacteria in vitro, making it impossible to interpret
the result. The experiment was not repeated. Although no explanation was
given for the observed cytotoxicity, the Committee considered that it might
have been the result of enzymatic activity on the cells. In contrast to the
finding for phospholipase A1, no cytotoxicity was observed when the enzyme
xylanase, which is also derived from the host strain A. oryzae BECh2, was
tested in the same assay for mutagenicity in bacteria in vitro.

The Committee noted that the materials added to the phospholipase A1

liquid enzyme concentrate for stabilization, formulation and standardization
have either been evaluated previously by the Committee or are common
food constituents and do not raise safety concerns.

24
 Assessment of dietary exposure
When phospholipase A1 is used as a processing aid in the production of
cheese, most of the enzyme is drained off with the whey, and only a small
amount remains in cheese. Although whey derivatives are known to be used
as ingredients in processed foods, it is difficult to assess potential dietary
exposure because of the wide variety of uses. On the basis of a conservative
estimate of daily consumption of 375 g of cheese by a 60-kg adult and on
the assumption that the enzyme is used at the recommended dosage and all
total organic solids originating from enzyme preparation remain in the
cheese, the dietary exposure would be to 0.22 mg of total organic solids per
kg bw per day.
Evaluation
The Committee concluded that the information provided on the enzyme
phospholipase A1 was too limited to allow an assessment of its safety. Only
two test batches of the enzyme preparation were analysed for secondary
metabolites, and, of the two toxicological studies provided, one, the assay
for mutagenicity in bacteria in vitro, could not be interpreted owing to
considerable cytotoxicity. Given that cytotoxicity was not observed when
xylanase, an enzyme derived from the same host strain A. oryzae BECh2,
was tested in the same assay, the Committee decided not to use the
toxicological data provided on xylanase to assess the safety of phospholipase
A1. The Committee concluded that, in order to make a proper safety
assessment, the results of two adequate studies of genotoxicity (including a
test for chromosomal aberration in mammalian cells in vitro) and a study of
toxicity in vivo would be needed. Alternatives to toxicity testing in vivo
would be the demonstration that no unintended compounds are present in
the enzyme preparation or better molecular characterization of the genetically
modified microorganism.
A toxicological monograph and a chemical and technical assessment were
prepared, and new specifications were established.

3.1.5 Pullulan

Explanation
Pullulan is a naturally occurring, fungal polysaccharide produced by
fermentation of liquefied corn starch by Aureobasidium pullulans, a
ubiquitous yeast-like fungus. It has a linear structure, consisting
predominantly of repeating maltotriose units, which are made up of three
α-1,4-linked glucose molecules, linked by α-1,6-glycosidic bonds. The

25
maltotriose units are interspersed with some maltotetraose units (about 6%)
consisting of four α-1,4-linked glucose molecules; occasionally, there are
branch points at which poly-maltotriosyl side-chains are attached to the
main chain by a 1,3-glycosidic bond.
Pullulan is used as a glazing agent, as a film-forming agent, as a thickener
or as a carrier for additives in the production of capsules for dietary
supplements (substitute for gelatin), coatings for coated tablets (dietary
supplements), edible flavoured films (breath fresheners), jams and jellies,
confectionery and some meat and fruit products. It is also used as a texturizer
in chewing-gum and as a foaming agent in milk-based desserts.
The Codex Committee on Food Additives and Contaminants at its Thirty-
sixth Session (14) requested that pullulan be reviewed by the Committee,
which has not evaluated the substance previously.

Chemical and technical considerations

Pullulan is produced by fermentation from a food-grade hydrolysed starch
with a non-toxin-producing strain of A. pullulans. After completion of the
fermentation, the fungal biomass is removed by microfiltration, the filtrate
is heat-sterilized, and pigments and other impurities are removed by
adsorption and ion-exchange chromatography. The product contains not
less than 90% glucan on a dried basis. The main impurities are mono-, di-
and oligosaccharides from the starting material. The average relative
molecular mass of pullulan can vary considerably, depending on culture
conditions; a commercially available product has an average relative
molecular mass of 200 000 Da.
Pullulan is stable in aqueous solution over a wide pH range (pH 3–8). Upon
dry heating, pullulan decomposes and carbonizes at 250–280 °C. It dissolves
readily in water but is insoluble in organic solvents. Aqueous solutions of
pullulan are viscous but do not form gels. Upon drying, pullulan forms
transparent, water-soluble, fat-resistant, odourless, anti-static, flavourless
films.

Toxicological data
Pullulan is largely resistant to digestion in the gastrointestinal tract as a
result of the occasional presence of 1,3-glycosidic linkages and the high
percentage of α-1,6-glycosidic linkages, which are resistant to hydrolysis
by salivary and pancreatic amylases. The degree of digestion appears to
depend on the relative molecular mass. A commercially available pullulan
(relative molecular mass, 200 000 Da) releases only a small amount of
reducing sugar after salivary amylase treatment but is converted to a

26
substance with a lower relative molecular mass (about 70 000 Da) after
treatment with an intestinal enzyme preparation.
Pullulan is fermented in the colon in vitro and in vivo by intestinal microflora,
to produce short-chain fatty acids, although the degree of fermentation
depends on the degree of polymerization of the pullulan. In humans, pullulan
(relative molecular mass, 50 000 Da) could not be detected in faeces after
daily consumption of 10 g for 14 days, suggesting that it was completely
fermented. In contrast to maltodextrin, pullulan reduced the glycaemic
response in healthy non-diabetic persons.
Although no studies were conducted to examine the effect of pullulan on
the bioavailability of vitamins and minerals, there is no evidence from the
published literature that similar polysaccharides of high relative molecular
mass have adverse effects on vitamin or mineral bioavailability. When fed
to rats at 20% in the diet, pullulan reduced intestinal calcium absorption but
did not affect serum calcium levels.

The oral LD50 of A. pullulans was reported to be > 24 g/kg bw. In rats, a
single oral dose of A. pullulans lysate at 10 or 20 g/kg bw caused no signs
of toxicity. Other studies indicate that A. pullulans does not produce toxins
and is not toxic when fed to rats.
The oral LD50 of pullulan was reported to be > 14 g/kg bw in mice. Short-
term studies in rats showed that pullulan has little toxicity. In a 13-week
study in rats given diets containing up to 10% pullulan (relative molecular
mass, 200 000 Da), no evidence of treatment-related toxicity was found.
The study showed a dose-dependent increase in caecum weight (full and
empty) as a result of an increased level of poorly digested polysaccharide in
the diet. This effect is considered to be a physiological response common to
indigestible polysaccharides and of no toxicological significance. The NOEL
was 10% in the diet, equal to 7900 mg/kg bw per day, on the basis of the
highest dose used in this study. The results of other short-term studies in
rats (9 and 62 weeks) support these conclusions. No long-term studies of
toxicity or of reproductive toxicity were available on pullulan. Assays for
genotoxicity with pullulan in vitro and in vivo gave negative results.
In a 14-day study in humans, daily consumption of 10 g of pullulan (relative
molecular mass, 50 000 Da) had no adverse effects. The faecal Bifidobacteria
population and short-chain fatty acid concentration increased, but no other
clinical changes were observed. Abdominal fullness was the only clinical
symptom reported. After a single dose of 50 g pullulan (relative molecular
mass, 100 000 Da), the frequency of flatulence was increased for 24 h.

27
 Assessment of dietary exposure
Pullulan is used as a substitute for gelatin in the production of capsule shells,
as an ingredient of coated tablets and in edible, flavoured films (breath
fresheners). The amount of pullulan ingested from one unit of each of these
products is 135 mg per capsule, 30 mg per tablet and 29 mg per film.

Data on consumption of food supplements were available from France and

the United Kingdom. For consumers at the 97.5th percentile, intake of seven
capsules per day was reported to correspond to a dietary exposure to pullulan
of 950 mg/day. As dietary supplements for children are usually formulated
as tablets, the consumption of pullulan by children was estimated to be
lower than that of adults and typically not to exceed 90 mg/day on the basis
of intake of three tablets per day, as reported in the United Kingdom. If
regular maximum daily consumption of seven capsules (950 mg/day of
pullulan) and of one standard packet of breath-freshening films (700 mg/day
of pullulan) is assumed, the maximum daily exposure would be 1.65 g.

Pullulan is used in Japan in various foodstuffs, at levels ranging from 2 g/

kg in ham and sausages to 30 g/kg in various processed products; use of
50 g/kg was reported in hard sweets. A conservative estimate of dietary
exposure from various foods by the budget method, assuming the presence
of pullulan at the maximum reported level in a limited fraction of the diet
(30 g/kg in 1/16 of the diet, corresponding to 187 g/day), resulted in a value
of about 6 g/day. Consumption of sweets by children was considered
separately, with consumption figures for France and the USA, resulting in
an estimate of about 2.5 g/day.
The Committee recognized that the conservative estimates should not be
summed.
Evaluation

The Committee concluded that the current uses of pullulan as a food additive
and the studies on its safety provided sufficient information to allocate an
ADI ‘not specified’.

A toxicological monograph, new specifications and a chemical and technical

assessment were prepared.

3.1.6 Quillaia extract type 2

Explanation
Quillaia extracts (synonyms: quillaja extracts, bois de Panama, Panama bark
extracts, quillaia extracts, Quillay bark extracts, soapbark extracts; CAS

28
No. 68990-67-0, INS No. 999) are obtained by aqueous extraction of the
milled inner bark or whole wood of Quillaja saponaria Molina (family
Rosaceae), which is a large evergreen tree with shiny, leathery leaves and a
thick bark, native to China and several South American countries, particularly
Bolivia, Chile and Peru. Quillaia extracts (types 1 and 2) are used in foods,
primarily for their foaming and emulsifying properties, which are attributed
to their saponin content.
Quillaia extracts were considered previously by the Committee, at its twenty-
sixth, twenty-ninth, fifty-seventh and sixty-first meetings (Annex 1,
references 59, 70, 154 and 166) At its fifty-seventh meeting, the Committee
assessed all relevant information on toxicity and dietary exposure, as
requested by the Codex Committee on Food Additives and Contaminants at
its Thirty-second Session (14). At that time, the Committee gave temporary
status to the ADI of 0–5 mg/kg bw allocated previously to the unpurified
extract, pending further clarification of the specifications. The existing
specifications were revised and designated ‘tentative’.
At its sixty-first meeting, the Committee reviewed new information relating
to the chemical characterization of quillaia extracts and further information
for specifications. The Committee agreed that separate specifications were
needed for the two forms of quillaia extract, type 1 (‘unpurified’) and type
2 (‘semi-purified’). Specifications for the total saponin content of type-1
extract were set at 20–26% (dried weight basis), and the Committee agreed
that, while there was some variation in saponin content, the material tested
toxicologically was representative of the material specified as type-1 extract.
The ‘temporary’ assignment to the ADI of 0–5 mg/kg bw was therefore
removed. Specifications for the total saponin content of type-2 extract after
a concentration step of ultra-filtration or chromatography were set at 75–
90% (dried weight basis). The saponin profile of the type-2 extract when
prepared by membrane ultrafiltration was similar to that of the extract assayed
according to the specifications. No information was available on the saponin
profile of the extract prepared by chromatography. Limited information was
available on the composition of the non-saponin fraction. Although the
Committee established separate specifications for type-1 and type-2 quillaia
extracts, it was unable to establish an ADI for the type-2 extract owing to
the limited information on the qualitative and quantitative composition of
this extract. A decision about which additional toxicological studies were
needed on the type-2 extract remained suspended in the absence of further
data on composition.
Further evaluation of quillaia extract type 2 was requested by the Codex
Committee on Food Additives and Contaminants at its Thirty-sixth Session
(14). At its present meeting, the Committee considered additional information

29
on the production and composition of type-2 extract prepared by membrane
ultrafiltration in Chile and by chromatography in Japan. The Committee
also considered data on the acute toxicity in rats of preparations of quillaia
type-1 and type-2 extracts.
Chemical and technical considerations

Quillaia extracts (types 1 and 2) can contain over 100 tri-terpenoid saponins,
the glycosides of the aglycone quillaic acid. Other constituents are
polyphenols, tannins, oxalate salts, simple sugars and trace amounts of fats
and nitrogen compounds. Quillaia extract type 2 is derived from type 1
extract subjected to chromatography or ultrafiltration to reduce the amount
of non-saponin soluble solids, such as polyphenols and tannins. Type 2
extract is used in food, primarily for its foaming and emulsifying properties,
which are attributed to the saponin content. The chemical compositon and
manufacture of type-1 extract were discussed by the Committee at its sixty-
first meeting.

Chromatographic analysis indicated that the saponin profile of type-2 extract

prepared by membrane ultrafiltration or chromatography is similar to that
of type-1 extract. The new information indicated that the range of saponin
contents is broader than established in the specifications at the sixty-first
meeting; therefore, the specification for total saponin content of type-2
extract was revised to 65–90% on a dried basis at the present meeting.
Toxicological data

The Committee considered data on the acute oral toxicity in rats of two
preparations of quillaia extract from Chile: a type-2 extract prepared by
ultrafiltration and a type-1 extract. Groups of five Sprague-Dawley rats of
each sex were given single oral doses ranging from 3000 to 20 000 mg/kg
bw and were observed for clinical signs of toxicity for 14 days. The LD50
for the type-2 extract was 6600 mg/kg bw; when expressed in relation to
the 14% saponin content of the standardized test material (72% on dry matter
basis), the LD50 was 900 mg/kg bw. The LD50 for the type-1 extract was 11
400 mg/kg bw; when expressed in relation to the 8.8% saponin content of
the standardized test material (20% w/w on dry matter basis), the LD50 was
1000 mg/kg bw. On the basis of the saponin content, the LD50s for the two
extracts were the same: about 900 mg/kg bw. No other new studies of toxicity
were available.

Assessment of dietary exposure

Quillaia extracts are used as foaming agents in soft drinks and cocktail
mixes and as emulsifiers in foods such as baked goods, sweets, frozen dairy
products, gelatine and puddings. Their major food use is in soft drinks. If a

30
 similar technological function based on saponin content and the
interchangeability of type-1 and type-2 extracts are assumed, the estimated
dietary exposure to saponins from type-1 and type-2 extracts would be the
same.
Evaluation

The previous requirement of the Committee for information on the

qualitative and quantitative composition of the type-2 extract was considered
to have been met, and the existing specifications for type-2 extract were
revised. On the basis of the new information on composition, the Committee
assessed the need for additional toxicological studies on this extract, as it
had decided at its previous meeting, and concluded that no additional studies
were necessary.
The Committee noted that there was no difference between type-1 and type-
2 extracts with respect to acute toxicity when expressed in relation to the
quillaia saponin content of the extracts.

On the basis of the conclusion of the Committee at its previous meeting

that the material tested toxicologically was representative of the material
specified as type-1 extract, and assuming that the toxicity is due to the
saponin content, the Committee agreed that the ADI for quillaia type-1
extract could be changed to an ADI based on the saponin content and
incorporated into a group ADI for type-1 and type-2 extracts. On the basis
of the lower end of the range of the saponin content (20%), the ADI for
type-1 extract would be 0–1 mg/kg bw expressed as quillaia saponins.
The Committee established a group ADI of 0–1 mg/kg bw for type-1 and
type-2 extract expressed as quillaia saponins. The previously established
ADI of 0–5 mg/kg bw for type-1 extract was withdrawn.
No toxicological monograph was prepared. The existing specifications and
the chemical and technical assessment were revised.

3.1.7 Quillaia extract type 1: Assessment of dietary exposure

Explanation
Quillaia extracts were considered previously by the Committee, at its twenty-
sixth, twenty-ninth, fifty-seventh and sixty-first meetings (Annex 1,
references 59, 70, 154 and 166). At its fifty-seventh meeting, the Committee
assessed all relevant information on toxicity and dietary exposure and
allocated a temporary ADI of 0–5 mg/kg bw to the unpurified extract,
pending clarification of the specifications. At its sixty-first meeting, the

31
Committee reviewed new information relating to the chemical
characterization of quillaia extracts and further information for
specifications. The Committee agreed that separate specifications were
needed for the two forms of quillaia extract, type 1 (‘unpurified’) and type
2 (‘semi-purified’) and also concluded that the data submitted for
toxicological and dietary exposure assessment were specific to the material
described as type-1 extract. The ‘temporary’ assignment to the ADI of
0–5 mg/kg bw for quillaia extract type 1 was therefore removed.

At its present meeting, the Committee decided to express the ADI on the
basis of the saponin content. Taking the lower end of the specified range, a
group ADI of 0–1 mg/kg bw, expressed as quillaia saponins, was established
(see section 3.1.6).
Quillaia extracts can be used as foaming agents in soft drinks and cocktail
mixes and as emulsifiers in foods such as baked goods, sweets, frozen dairy
products, gelatine and puddings. Their main food use is in soft drinks. The
Codex Alimentarius Commission adopted a provision for the use of quillaia
extract as a foaming agent at a level of 100 mg/kg in food category 14.1.4,
‘water-based flavoured drinks’, of the Codex General Standard for Food
Additives.
At its fifty-seventh meeting, the Committee estimated dietary exposure to
quillaia extracts by a stepwise procedure, assuming a quillaia concentration
of 500 mg/kg in all water-based flavoured drinks. On the basis of this use
level, the Committee concluded that consumption at the 95th percentile of
the distribution of consumption of soft drinks, particularly by children, could
exceed the ADI. Estimates of exposure based on consumption of soft drinks
in the USA that are likely to contain quillaia at a level of 100 mg/kg were
below the ADI.

The previous exposure estimates considered by JECFA did not explicitly

include use of quillaia extract in semi-frozen carbonated and non-carbonated
beverages. The Codex Committee on Food Additives and Contaminants at
its Thirty-sixth Session (14) therefore requested further information on
dietary exposure to quillaia extract type 1 at levels up to 500 mg/kg in these
products.
The composition of semi-frozen carbonated and non-carbonated beverages
is similar to that of the corresponding unfrozen beverages. They differ in
the content of foaming agents and carbonation or addition of air to expand
their volume up to 180% of the original. Therefore, a use level of 500 mg/kg
in unexpanded semi-frozen carbonated and non-carbonated beverages
corresponds to 295 mg/l as consumed, expressed as quillaia extract.

32
Assessment of dietary exposure based on model diets
Dietary exposure to quillaia extracts was estimated after assuming the
presence of the additive at 295 mg/l in all water-based flavoured drinks.
High-percentile consumption of soft drinks is 446–1600 ml/day, resulting
in a level of exposure to quillaia extracts of 130–470 mg per person per day
or 44–160% of the group ADI of 0–1 mg/kg bw expressed as quillaia
saponins (see section 3.1.6).
The Committee noted that this assessment assumes that semi-frozen
carbonated and non-carbonated beverages are the only source of quillaia
extracts. This assumption is based on the fact that no data were submitted
about levels of quillaia extracts in solid foods and that it is unlikely that a
person who consumes semi-frozen carbonated and non-carbonated beverages
would also consume other beverages potentially containing the same
additive.
Assessment of dietary exposure based on a probabilistic approach

A probabilistic exposure assessment was submitted, combining the frequency

of consumption of semi-frozen carbonated and non-carbonated beverages
with the amounts consumed per eating occasion. The amounts were estimated
from the size of the containers available and on the consumption of ‘frozen
novelties’ in the USA, used as a surrogate for semi-frozen carbonated and
non-carbonated beverages. Assuming that the frequency and the amount
per eating occasion are independent variables, dietary exposure to quillaia
extracts is below the group ADI of 0–1 mg/kg bw, expressed as quillaia
saponins, at the 90th percentile.
The hypothesis of independence between the amount consumed and the
frequency of consumption could not be verified from the available
information. Therefore, the possibility that a consumer of large amounts of
semi-frozen carbonated and non-carbonated beverages is also a frequent
consumer cannot be excluded. Assuming 100% dependence between the
frequency and the amount of consumption, it is possible to estimate the
number of consumers who potentially exceed the group ADI of 0–1 mg/kg
bw, as follows: semi-frozen carbonated and non-carbonated beverages are
consumed in the USA by 1–7% of the total population, which corresponds
to 10 000–70 000 consumers per million. Of those consumers, 15% consume
semi-frozen carbonated and non-carbonated beverages at least once a day,
corresponding to 1500–10 500 individuals per million, and 1% drink
> 1 l/day. Thus, the consumption of 15–100 individuals per million in the
whole population could exceed the group ADI under these conditions.
An addendum to the monograph was prepared.

33
3.2 Revision of specifications
3.2.1 Aspartame-acesulfame salt

Aspartame-acesulfame salt was placed on the agenda for revision of

specifications at the request of the Codex Committee on Food Additives
and Contaminants (14). The Committee agreed to delete the text ‘after
adjusting the pH to alkalinity’ in the definition section of the monograph, to
change the text under solubility to ‘sparingly soluble in water, slightly soluble
in ethanol’ and to correct the value for specific rotation.

3.2.2 Hexanes

The Committee was asked to review the specifications for hexanes in view
of a submission that indicated that the present provision for the refractive
index was not consistent with the major components present in the current
article of commerce. As used in the food industry, hexanes, obtained by
fractionation of petroleum, are a mixture of components ranging in chain
length from C5 to C7, with a preponderance of C6 components. Recent
changes in environmental regulations have led to a reduction of the n-hexane
content of hexanes since the original specifications were prepared. In
addition, the composition of hexanes depends on the region of production,
the source of the raw material and the site of production. Therefore, the
Committee concluded that the present articles of commerce differ from those
previously evaluated and that the composition of the residues and their levels
in foods might not be the same as those evaluated originally.

The Committee decided there was insufficient information available to

change the current specifications and recommended a re-evaluation of
hexanes at a future meeting.

3.2.3 Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae

Laccase was reviewed by the Committee at its sixty-first meeting and was
placed on the agenda of the present meeting at the request of the Codex
Committee on Food Additives and Contaminants at its Thirty-sixth Session
(14) for further revision of the specifications. In response to the call for
data, the Committee received a request to revise the text in the ‘sources’
section by removing reference to the ancestral strain number. The Committee
agreed to this request and also removed the phrase ‘non-pathogenic and
non-toxigenic’, as these requirements are described in the General
specifications and considerations for enzyme preparations used in food
processing (3). Furthermore, the description of the production and isolation
of the enzyme was amplified.

34
3.2.4 Monomagnesium phosphate and trisodium diphosphate

At its sixty-first meeting (Annex 1, reference 166), the Committee

recommended that the tentative specifications for these two substances
should be withdrawn if details of the methods and levels for ‘loss on drying’
and ‘loss on ignition’ were not received by the end of 2004. As the Committee
received no additional information, it withdrew the specifications.

3.2.5 Sucrose esters of fatty acids

In response to a request from the Codex Committee on Food Additives and

Contaminants (14), the Committee replaced the test methods in the
specifications for the determination of free sucrose and propylene glycol
by new methods. The Committee noted, however, that the method for
determining free sucrose is still unsatisfactory and decided that it should be
replaced by either a capillary gas chromatography or a high-performance
liquid chromatography method. Furthermore, as the methods for free sucrose
and propylene glycol currently require the use of pyridine, the Committee
decided to request information on substitution of pyridine by a less toxic
solvent.
The Committee also noted that the method for residual dimethyl sulfoxide
called for use of a packed gas chromatography column and decided to request
an updated method that does not have this requirement. The Committee
assigned a tentative designation to the specifications and agreed to withdraw
them if the requested information was not received by the end of 2006.

4 Flavouring agents
The Committee was asked to evaluate seven groups of flavouring agents at
its present meeting. It agreed to evaluate six by the Procedure for the Safety
Evaluation of Flavouring Agents (Annex 1, reference 131) but decided that
it could not evaluate a seventh group with the Procedure because of concerns
about the data on safety for some members of the group. The Committee
also reviewed the specifications for 135 substances submitted for evaluation
and reviewed and revised the existing ‘tentative’ specifications for four
flavouring agents evaluated at previous meetings. Details are given in
sections 4.1 and 4.2, and the conclusions and details of further information
required are summarized in Annexes 2–6.

35
4.1 Flavouring agents evaluated by the Procedure for the Safety
Evaluation of Flavouring Agents
4.1.1 Maltol and related substances

The Committee evaluated a group of seven flavouring agents (see Table 1)

comprising maltol and related substances. The evaluations were conducted
according to the Procedure for the Safety Evaluation of Flavouring Agents
(Annex 1, reference 131). The Committee has evaluated two members of
the group previously. Maltol (No. 1480) was evaluated at the eleventh
meeting (Annex 1, reference 15), when a temporary ADI of 0–1 mg/kg bw
was established because no results of long-term studies were available. At
the eighteenth meeting (Annex 1, reference 35), the Committee withdrew
the temporary ADI because the results of the long-term studies of toxicity
that had been requested at the previous meeting had not been made available.
At the twenty-second meeting (Annex 1, reference 47), the Committee
evaluated new data on toxicity and established a temporary ADI of 0–0.5 mg/
kg bw. At its twenty-fifth meeting (Annex 1, reference 56), the Committee
evaluated additional data and assigned an ADI of 0–1 mg/kg bw. Ethyl
maltol (No. 1481) was evaluated at the fourteenth meeting (Annex 1,
reference 22), when the Committee established an ADI of 0–2 mg/kg bw.
At its eighteenth meeting (Annex 1, reference 35), the Committee re-
evaluated ethyl maltol and confirmed the previous ADI of 0–2 mg/kg bw.
One of the seven substances, maltol (No. 1480), has been reported to occur
naturally in a wide variety of foods, including wheaten and rye bread, milk,
butter, uncured pork, beer, cocoa, coffee, peanuts, soya proteins, beans,
and clams. Under conditions of baking (e.g. bread, beans) and roasting
(cocoa, coffee, peanuts), simple sugars are partly converted to maltol.
Estimated daily per capita exposure

Annual volumes of production have been reported for six of the seven
flavouring agents in the group (Nos 1480, 1481, 1482, 1484, 1485 and
1486). With respect to the remaining substance (No. 1483), anticipated
annual volumes of production have been given for its proposed use as a
flavouring agent. The total reported and anticipated annual volumes of
production of the seven flavouring agents in this group is about 38 000 kg
in Europe and 73 000 kg in the USA. More than 99% of the total reported
and anticipated annual volumes of production in Europe and the USA is
accounted for by maltol and ethyl maltol. The per capita exposure to maltol
in Europe and the USA is approximately 3600 and 2900 µg/day, respectively,
and that of ethyl maltol in Europe and the USA is approximately 1900 and
6700 µg/day, respectively. The per capita exposure to the remainder of the

36
 Table 1. Summary of results of safety evaluations of maltol and related substances used or proposed for use as flavouring agents

Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate NOEL for on current intake
exceed the agent or are its substance or
threshold for metabolites related substance?
human intake?b endogenous?

Structural class II

Maltol 1480 118-71-8 Yes No Yes. The NOEL of Note 1 At its 25th meeting,
Europe: 3585 100 mg/kg bw per day JECFA established
USA: 2898 (Annex 1, reference 56) an ADI of 0–1 mg/
is > 1600 times the kg bw (Annex 1,
estimated daily intake reference 56).
of maltol when used
as a flavouring agent.

Ethyl maltol 1481 4940-11-8 Yes No Yes. The NOEL of Note 1 At its 18th meeting,
Europe: 1851 200 mg/kg bw per day for JECFA established
USA: 6692 ethyl maltol in rats (Annex an ADI of 0–2 mg/
1, reference 35) is kg bw (Annex 1,
> 1800 times the reference 35).
estimated daily intake
of ethyl maltol when used
as a flavouring agent.

37
 Table 1 (contd)

38
Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate NOEL for on current intake
exceed the agent or are its substance or
threshold for metabolites related substance?
human intake?b endogenous?

Maltyl isobutyrate 1482 65416-14-0 No NR NR Note 2 No safety concern

Europe: 23
USA: 38

2-Methyl-3-(1-oxo- 1483 68555-63-5 No NR NR Note 2 No safety concern

propoxy)-4H-pyran- Europe: ND (conditional)
4-one USA: 26b

Structural class III

2-Amyl-5 or 6-keto- 1485 65504-96-3 No NR NR Note 3 No safety concern
1,4-dioxane Europe: ND
USA: 0.2
 Table 1 (contd)
Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate NOEL for on current intake
exceed the agent or are its substance or
threshold for metabolites related substance?
human intake?b endogenous?

Structural class III

2-Butyl-5- or -6-keto- 1484 65504-95-2 No NR NR Note 3 No safety concern
1,4-dioxane Europe: ND

USA: 0.5

2-Hexyl-5 or 6-keto- 1486 65504-97-4 No NR NR Note 3 No safety concern

1,4-dioxane Europe: ND

USA: 0.5

CAS: Chemical Abstracts Service; ND: no intake data reported; N/R: not required for evaluation because intake of the substance was determined to be of no
safety concern at Step A3 of the procedure.
Step 2: All the agents in this group can be predicted to be metabolized to innocuous products. The evaluation of these flavouring agents therefore proceeded
via the A-side of the Procedure.
a
The thresholds for human intake for structural classes II and III are 540 µg/day and 90 µg/day, respectively. All intake values are expressed in µg/day. The
combined per capita intakes of flavouring agents in structural class II are 5459 µg/day in Europe and 9655 µg/day in the USA. The combined per capita
intake of flavouring agents in structural class III is 1.2 µg/day in the USA.
b
Intake estimate based on anticipated annual volume of production
Notes:
1. Conjugation with glucuronic acid or sulfate followed by excretion in urine
2. Hydrolysis to maltol and the corresponding carboxylic acid, followed by conjugation with glucuronic acid or sulfate and excretion in urine
3. Hydrolysis to a hydroxycarboxylic acid, followed by excretion as the glucuronic acid

39
flavouring agents in the group is 0–23 µg/day in Europe and 0.2–38 µg/day
in the USA, most of the values being at the low end of these ranges. Per
capita exposure to each agent is reported in Table 1.

Absorption, distribution, metabolism and elimination

Chemically, maltol is classified as a γ-pyrone. It is a hydroxyl-substituted
4H-pyran-4-one and is expected to be metabolized similarly to phenol,
primarily undergoing phase II conjugation of the free hydroxy substituent.
Maltol (2-methyl-3-hydroxy-4H-pyran-4-one) and ethyl maltol (2-ethyl-3-
hydroxy-4H-pyran-4-one) are predominantly metabolized to sulfate and
glucuronic acid conjugates, which are then eliminated in the urine. Maltol
esters (Nos 1482 and 1483) are predicted to be hydrolysed to ethyl maltol
and the corresponding simple aliphatic carboxylic acid (propionic acid or
isobutyric acid) and to undergo further metabolism similar to that of maltol
and ethyl maltol.
The remaining three substances (Nos 1484, 1485, and 1486) in the group
are α-pyrone derivatives and contain a saturated 3H-pyranone nucleus. These
three substances are lactones and are readily hydrolysed to yield the
corresponding ring-opened hydroxy acid derivatives. In nature, lactones
are formed by acid-catalysed intramolecular cyclization of four- or five-
carbon hydroxycarboxylic acids to yield five- (γ-) or six- (δ-) membered
lactone rings, respectively. The stability of the lactone ring in an aqueous
environment is pH-dependent. In basic media such as blood, lactones
hydrolyse rapidly to the open-chain hydroxycarboxylic acid. Studies of
structurally related lactones indicate that the aliphatic lactones would be
hydrolysed to yield the corresponding hydroxycarboxylic acid. These acids
can undergo further oxidation to yield polar, excretable metabolites or enter
the fatty acid pathway and undergo β-oxidative cleavage to yield polar
metabolites of lower relative molecular mass, which are also excreted either
unchanged or conjugated in the urine.
Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported

and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated
from it exceeded the exposure estimated from the anticipated volume of
production and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure

40
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.
Step 1. In applying the Procedure to this group of flavouring agents, the
Committee assigned four of the seven agents (Nos 1480, 1481, 1482 and
1483) to structural class II and the remaining three agents (Nos 1484, 1485
and 1486) to structural class III.
Step 2. All the flavouring agents in this group are expected to be
metabolized to innocuous products. The evaluation of all agents in this group
therefore proceeded via the A-side of the Procedure.
Step A3. The estimated daily per capita exposure to two of the four agents
in structural class II (Nos 1482 and 1483) and of all three agents in structural
class III is below the threshold of concern for their respective class (i.e.
class II, 540 µg/day; class III, 90 µg/day). Four of these five substances
(Nos 1482, 1484, 1485 and 1486) are reported to be used as flavouring
agents. According to the Procedure, use of these four agents would not raise
concern about safety at estimated daily exposure. The other substance (No.
1483) is proposed for use as a flavour. Although the Procedure indicates no
safety concern with use of this flavouring agent at the estimated daily
exposure derived from the anticipated annual volume of production, more
reliable exposure estimates are needed. Estimated daily exposure to the
remaining two agents in structural class II, maltol (No. 1480) and ethyl
maltol (No. 1481), exceed the threshold of concern for structural class II.
Exposure to maltol is 3600 µg per person per day in Europe and 2900 µg
per person per day in the USA. Exposure to ethyl maltol is 1900 µg per
person per day in Europe and 6700 µg per person per day in the USA.
Accordingly, the evaluation of these two agents proceeded to step A4.
Step A4. Maltol (No. 1480) and ethyl maltol (No. 1481) are not endogenous.
Therefore, their evaluation proceeded to step A5.
Step A5. At its twenty-fifth meeting, the Committee established an ADI of
0–1 mg/kg bw for maltol (No. 1480) on the basis of a NOEL of 100 mg/kg
bw per day in a 2-year dietary study in rats (Annex 1, reference 56). This
NOEL is more than 1800 times the estimated daily exposure to this agent
from its use as a flavouring agent in Europe or the USA. At its eighteenth
meeting, the Committee established an ADI of 0–2 mg/kg bw for ethyl
maltol (No. 1481) on the basis of a NOEL of 200 mg/kg bw per day in a 2-
year dietary study in rats (Annex 1, reference 35). This NOEL is more than
1800 times the estimated daily exposure to this substance from its use as a
flavouring agent in Europe or the USA. The Committee therefore concluded
that exposure to flavouring agents in this group would not raise concern
about safety.

41
 The exposure considerations and other information used to evaluate maltol
and six related derivatives according to the Procedure are summarized in
Table 1.

Consideration of combined exposure from use as flavouring agents

In the unlikely event that all four agents in structural class II were to be
consumed concurrently on a daily basis, the estimated combined exposure
would exceed the human exposure threshold for class II (540 µg per person
per day). All four agents in this group are, however, expected to be efficiently
metabolized and would not saturate metabolic pathways. Their safety is
also indicated by the results of studies on the toxicity of maltol and ethyl
maltol. An evaluation of all the data indicates that combined exposure would
not raise concern about safety.
In the unlikely event that all three agents in structural class III were to be
consumed concurrently on a daily basis, the estimated combined exposure
would not exceed the human exposure threshold for class III (90 µg per
person per day). Their safety is also indicated by the results of studies of
toxicity. An evaluation of all the data indicates that combined exposure
would not raise concern about safety.

Conclusions
The Committee maintained the previously established ADIs of 0–1 mg/kg
bw for maltol and 0–2 mg/kg bw for ethyl maltol. The Committee concluded
that use of the flavouring agents in this group of maltol and related substances
would not present a safety concern at estimated daily exposure. For one
agent (No. 1483), the evaluation was conditional, because the estimated
daily exposure was based on the anticipated annual volume of production.
The conclusion about the safety of this substance will be revoked if use
levels or poundage data are not provided before December 2007. The
Committee noted that the available data on the toxicity and metabolism of
the maltol derivatives were consistent with the results of the safety evaluation
made with the Procedure.

A monograph summarizing the safety data on this group of flavouring agents

was prepared.

4.1.2 Furan-substituted aliphatic hydrocarbons, alcohols, aldehydes,

ketones, carboxylic acids and related esters, sulfides, disulfides and
ethers

A group of 40 furan-substituted substances was considered by the

Committee (see Table 2). The Committee took note of the extensive evidence

42
Table 2. Furan-substituted aliphatic hydrocarbons, alcohols, aldehydes,
ketones, carboxylic acids and related esters, sulfides, disulfides and ethers

Flavouring agent No. CAS No. and

structure

Structural class II
2-Methylfuran 1487

534-22-5

2,5-Dimethylfuran 1488

625-86-5

2-Ethylfuran 1489

3208-16-0

2-Butylfuran 1490

4466-24-4

2-Pentylfuran 1491

3777-69-3

2-Heptylfuran 1492

3777-71-7

2-Decylfuran 1493

83469-85-6

43
Table 2 (contd)

Flavouring agent No. CAS No. and

structure

3-Methyl-2-(3-methylbut-2-enyl) furan 1494

15186-51-3

3-(2-Furyl)acrolein 1497

623-30-3

3-(5-Methyl-2-furyl)prop-2-enal 1499

5555-90-8

2-Furyl methyl ketone 1503

1192-62-7

2-Acetyl-5-methylfuran 1504

1193-79-9

2-Acetyl-3,5-dimethylfuran 1505

22940-86-9

44
Table 2 (contd)

Flavouring agent No. CAS No. and

structure

2-Butyrylfuran 1507

4208-57-5

(2-Furyl)-2-propanone 1508

6975-60-6

2-Pentanoylfuran 1509

3194-17-0

1-(2-Furyl)butan-3-one 1510

699-17-2

4-(2-Furyl)-3-buten-2-one 1511

623-15-4

Ethyl 3-(2-furyl)propanoate 1513

10031-90-0

Isobutyl 3-(2-furan)propionate 1514

105-01-1

45
Table 2 (contd)

Flavouring agent No. CAS No. and

structure

Isoamyl 3-(2-furan)propionate 1515

7779-67-1

Isoamyl 4-(2-furan)butyrate 1516

7779-66-0

Phenethyl 2-furoate 1517

7149-32-8

Furfuryl methyl ether 1520

13679-46-4

Ethyl furfuryl ether 1521

6270-56-0

Difurfuryl ether 1522

4437-22-3

2,5-Dimethyl-3-furanthiol acetate 1523

55764-22-2

46
Table 2 (contd)

Flavouring agent No. CAS No. and

structure

Furfuryl 2-methyl-3-furyl disulfide 1524

109537-55-5

3-[(2-Methyl-3-furyl)thio]-2-butanone 1525

61295-44-1

O-Ethyl S-(2-furylmethyl)thiocarbonate 1526

376595-42-5

Structural class III

2,3-Dimethylbenzofuran 1495

3782-00-1

2,4-Difurfurylfuran 1496

64280-32-6

2-Methyl-3-(2-furyl)acrolein 1498

874-66-8

3-(5-Methyl-2-furyl)-butanal 1500

31704-80-0

47
Table 2 (contd)

Flavouring agent No. CAS No. and

structure

2-Furfurylidenebutyraldehyde 1501

770-27-4

2-Phenyl-3-(2-furyl)prop-2-enal 1502

65545-81-5

3-Acetyl-2,5-dimethylfuran 1506

10599-70-9

Pentyl 2-furyl ketone 1512

14360-50-0

Propyl 2-furanacrylate 1518

623-22-3

2,5-Dimethyl-3-oxo-(2H)-fur-4-yl butyrate 1519

114099-96-6

CAS, Chemical Abstracts Service

48
 for the genotoxicity of several members of this group of flavouring agents
related to furan, including the clastogenicity of 2-furyl methyl ketone (No.
1503) in mouse bone marrow. Furan, which is carcinogenic, is known to
undergo epoxidation and ring opening to form a reactive 2-ene-1,4-
dicarbonyl intermediate. Accordingly, there is concern that the observed
genotoxicity might be due to formation of a reactive metabolite. No data
were available on the potential of members of this group to form reactive
metabolites, and no role of metabolism has been identified in the observed
genotoxicity. Moreover, there were few data on genotoxicity in vivo, and
specific in-vivo assays to address potential carcinogenicity were lacking.

The Committee concluded that the Procedure could not be applied to this
group, because of the above concerns. Studies that would assist in resolving
the concerns include studies of metabolism and assays for DNA reactivity,
mutagenicity and carcinogenic potential in vivo of members of this group
with representative structures.

4.1.3 Eugenol and related hydroxyallylbenzene derivatives

The Committee evaluated a group of seven hydroxyallylbenzene flavouring

agents (Table 3), including eugenol (No. 1529). The evaluations were
conducted according to the Procedure for the Safety Evaluation of
Flavouring Agents (Annex 1, reference 131). The Committee has evaluated
one member of the group previously: eugenol (No. 1529) was evaluated at
the twenty-sixth meeting (Annex 1, reference 59), and an ADI of
0–2.5 mg/kg bw was assigned.
Three of the seven flavouring agents (Nos 1527, 1529 and 1531) have been
reported to occur naturally in various foods. They have been detected in
wheaten bread, clove buds, leaves and stems, oregano, tarragon, dill, basil,
rosemary, pimento leaf and berry, cinnamon bark and leaf, laurel, apples,
cherries, whisky and red and white wine.
Estimated daily per capita exposure
Annual volumes of production have been reported for four of the seven
flavouring agents in this group (Nos 1529–1531 and 1533). For the
remaining three substances (Nos 1527, 1528 and 1532), anticipated annual
volumes of production have been given for their proposed use as flavouring
agents. The total reported and anticipated annual volumes of production of
eugenol and the six related hydroxyallylbenzenes are about 7900 kg in
Europe and 26 000 kg in the USA. Approximately 98% of the total reported
and anticipated annual volume of production in Europe and approximately
97% of that in the USA is accounted for by eugenol (No. 1529). Estimated

49
 Table 3. Summary of results of safety evaluations of eugenol and related hydroxyallylbenzene derivatives

50
Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the substance Adequate NOEL on current intake
exceed the or its metabolites for substance or
threshold for endogenous? related substance?
human intake?

Structural class I

4-Allylphenol 1527 501-92-8 No NR NR See notes 1, 2 No safety concern

Europe: 0.09a and 3 (conditional)
USA: 0.2a

2-Methoxy-6-(2-pro- 1528 579-60-2 No NR NR See notes 1, 2 No safety concern

penyl)phenol Europe: 0.1a and 3 (conditional)
USA: 0.2a

Eugenol 1529 97-53-0 Yes No Yes. The NOEL of 300 mg/kg bw See notes 1, 2 An ADI of 2.5 mg/kg
Europe: 1107 per day (National Toxicology and 3 bw was established
USA: 3364 Program, 1983) is > 16 000 and for eugenol (Annex 1,
5000 times the estimated daily reference 59), which
intakes of 18 µg/kg bw in Europe was maintained at
and 56 µg/kg bw in the USA,
respectively, when eugenol is used
as a flavouring agent.

Eugenyl formate 1530 10031-96-6 No NR NR See notes 1, 2, No safety concern

Europe: 0.01 3 and 4
USA: 0.05
 Table 3 (contd)

Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the substance Adequate NOEL on current intake
exceed the or its metabolites for substance or
threshold for endogenous? related substance?
human intake?

Eugenyl acetate 1531 93-28-7 No NR NR See notes 1, 2, No safety concern

Europe: 23 3 and 4
USA: 90

Eugenyl isovalerate 1532 61114-24-7 No NR NR See notes 1, 2, No safety concern

Europe: 0.4a 3 and 4 (conditional)
USA: 0.5a

Eugenyl benzoate 1533 531-26-0 No NR NR See notes 1, 2, No safety concern

Europe: 0.003 3 and 4
USA: 0.9

CAS, Chemical Abstracts Service; NR, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step A3
of the Procedure.
Step 1: All the agents in this group are in structural class I (Cramer et al., 1978).
Step 2: All the agents in this group can be predicted to be metabolized to innocuous products.
a
The threshold for human intake for structural class I is 1800 µg/day. All intake values are expressed in µg/day. The combined per capita intake of the flavouring

51
agents in structural class I is 1130 µg per day in Europe and 3456 µg per day in the USA.
b
Intake estimate based on anticipated annual volume of production
Table 3 (contd)

Notes:
1.The phenolic hydroxyl group forms a conjugate with glucuronic acid and is readily
excreted in the urine.
2.Minor amounts of epoxide are formed on the allyl moiety, which undergoes hydrolysis,
followed by conjugation and subsequent excretion.
3.Formation of quinone methide may occur, followed by conjugation with glutathione.
4.The ester group is hydrolysed by carboxyl esterases.

per capita exposure to eugenol is approximately 1100 µg/day in Europe and

3400 µg/day in the USA. Estimated per capita exposure to all the other
flavouring agents in the group (Nos 1527, 1528, 1530–1533), on the basis
of reported or anticipated annual volumes of production, are 0.003–23 µg/day
in Europe, and 0.05–90 µg/day in the USA, most of the values being at the
lower end of the ranges. The estimated per capita exposure to each flavouring
agent is reported in Table 3.
Absorption, distribution, metabolism and elimination

In humans and rodents, orally administered eugenol and related

allylhydroxyphenol derivatives are rapidly absorbed from the gastrointestinal
tract and efficiently extracted by the liver, where they mainly undergo phase
II conjugation. The resulting glucuronide and sulfate conjugates are
subsequently excreted in the urine. To a lesser extent, eugenol is metabolized
to polar products, some of which are more reactive than the parent molecule.
These products are also conjugated and eliminated, primarily in the urine.
Minute amounts (< 1%) of eugenol are excreted unchanged. The primary
urinary metabolites of the other agents containing an unsubstituted phenolic
group also form glucuronic acid and sulfate conjugates. Eugenyl esters are
hydrolysed to eugenol and the corresponding carboxylic acid. These
metabolites are readily excreted, primarily in the urine.

Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported
and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated
from it exceeded the exposure estimated from the anticipated volume of
production, and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.

52
Step 1. In applying the Procedure, the Committee assigned all seven
agents (Nos 1527–1533) to structural class I.
Step 2. All the flavouring agents in this group are predicted to be
metabolized to innocuous products. The evaluation of all the agents in this
group therefore proceeded via the A-side of the Procedure.

Step A3. Estimated daily per capita exposure to six of the seven flavouring
agents in structural class I are below the threshold of concern (i.e.
1800 µg/day for class I). Three of these six substances (Nos 1530, 1531
and 1533) are reported to be used as flavouring agents; according to the
Procedure, use of these three agents raises no safety concern at estimated
daily exposure. The other three substances (Nos 1527, 1528 and 1532) are
proposed for use as flavouring agents. Although, according to the Procedure,
use of these three flavouring agents raises no safety concern at the exposure
levels estimated on the basis of the anticipated annual volumes of production,
less uncertain exposure estimates are needed. Estimated daily exposure to
the remaining agent in this group, eugenol (No. 1529), which is 1107 µg/day
in Europe and 3364 µg/day in the USA, exceeds the threshold of concern
for class I. Accordingly, the evaluation of eugenol proceeded to step A4.

Step A4. Eugenol and its metabolites are not endogenous. Accordingly, the
evaluation of this agent proceeded to step A5.

Step A5. At its twenty-sixth meeting, the Committee established an ADI of

0–2.5 mg/kg bw per day for eugenol on the basis of the results of a 19-week
study in rats (Annex 1, reference 59). At its current meeting, the Committee
considered the results of a bioassay in rodents, in which the NOEL was
300 mg/kg bw per day. This NOEL for eugenol, which is consistent with
the previous evaluation leading to the ADI, is more than 16 000 and 5000
times the estimated daily exposure to eugenol from its use as a flavouring
agent in Europe (18 µg/kg bw) and the USA (56 µg/kg bw), respectively.
The Committee therefore concluded that eugenol would not present a safety
concern at the estimated daily exposure.

Considerations on exposure and other information used to evaluate eugenol

and the six related hydroxyallylbenzene derivatives according to the
Procedure are summarized in Table 3.
Consideration of secondary components
One member of this group of flavouring agents, eugenyl formate (No. 1530),
has an assay value of < 95%. The secondary component in No. 1530, eugenyl
formate, is eugenol (No. 1529), which was evaluated at the present meeting
and considered not to present a safety concern at estimated current levels
of exposure. The Committee also concluded that the flavouring agent as

53
 specified would not present a safety concern at the estimated levels of
exposure.
Consideration of combined exposure from use as flavouring agents

In the unlikely event that all seven agents in structural class I were to be
consumed concurrently on a daily basis, the estimated combined exposure
would exceed the human exposure threshold for class I (i.e. 1800 µg per
person per day). All seven agents in this group are, however, expected to be
efficiently metabolized and would not saturate metabolic pathways.
Moreover, the combined exposure to all seven agents would be well below
the ADI of 0–2.5 mg/kg bw for eugenol. Overall evaluation of the data
indicates that combined exposure would not raise concerns about safety.
Conclusions
The Committee maintained the previously established ADI of 0–2.5 mg/kg
bw for eugenol. It concluded that use of the flavouring agents in the group
of eugenol and related hydroxyallylbenzene derivatives would not present
a safety concern at the estimated exposure levels. For three flavouring agents
(Nos 1527, 1528 and 1532), the evaluation was conditional because the
estimated exposure was based on anticipated annual volumes of production.
The conclusion of the safety evaluation of these three agents will be revoked
if use levels or poundage data are not provided by December 2007. The
Committee noted that the available data on the toxicity and metabolism of
the hydroxyallylbenzenes are consistent with the results of the safety
evaluation with the Procedure.
A monograph summarizing the safety data on this group of flavouring agents
was prepared.

4.1.4 Anthranilate derivatives

The Committee evaluated a group of 19 anthranilate derivatives (Table 4)

by the Procedure for the Safety Evaluation of Flavouring Agents (see
Figure 1, Annex 1, references 116, 122 and 131). The Committee had
previously evaluated two members of this group. Methyl anthranilate (No.
1534) was evaluated at the eleventh meeting (Annex 1, reference 14) and
was assigned a conditional ADI1 of 0–1.5 mg/kg bw. At its twenty-third
meeting (Annex 1, reference 50), the Committee re-evaluated the conditional
ADI of methyl anthranilate and recommended that it be converted to an

‘Conditional ADI’, which signifies an ADI with special considerations, is a term no longer used by
JECFA.

54
 Table 4. Summary of results of safety evaluations of anthranilate derivatives used or proposed to be used as flavouring agents

Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

Structural class I

Methyl anthranilate 1534 134-20-3 Yes No Yes. The NOEL of 150 See note 1 An ADI of 0–1.5 mg/kg
Europe: 804 mg/kg bw per day bw was established for
USA: 3764 (Annex 1, reference 50) methyl anthranilate by
is > 11 000 and > 2300 the Committee at its
times the estimated daily twenty-third meeting
intakes of 13 and 63 (Annex 1, reference
µg/kg bw in Europe and 50), which was
the USA, respectively, maintained at the
when used as a flavouring present meeting.
agent.

Ethyl anthranilate 1535 87-25-2 No NR NR See note 1 No safety concern

Europe: 14
USA: 39

Butyl anthranilate 1536 7756-96-9 No NR NR See note 1 No safety concern

Europe: 0.003
USA: 14

55
 Table 4 (contd)

56
Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

Isobutyl anthranilate 1537 7779-77-3 No NR NR See note 1 No safety concern

Europe: 1
USA: 0.4

cis-3-Hexenyl 1538 65405-76-7 No NR NR See note 1 No safety concern

anthranilate Europe: ND (conditional)
USA: 53b

Citronellyl anthranilate 1539 68555-57-7 No NR NR See note 1 No safety concern

Europe: 7b (conditional)
USA: 9b

Linalyl anthranilate 1540 7149-26-0 No NR NR See note 1 No safety concern

Europe: 0.04
USA: 0.07
 Table 4 (contd)

Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

Cyclohexyl 1541 7779-16-0 No NR NR See note 1 No safety concern

anthranilate Europe: ND
USA: 0.007

β-Terpinyl 1542 14481-52-8 No NR NR See note 1 No safety concern

anthranilate Europe: 0.004
USA: 1

Phenylethyl 1543 133-18-6 No NR NR See note 1 No safety concern

anthranilate Europe: 2
USA: 7

β-Naphthyl 1544 63449-68-3 No NR NR See note 1 No safety concern

anthranilate Europe: ND
USA: 2

57
58
Table 4 (contd)

Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

Methyl N-methyl- 1545 85-91-6 No NR NR See note 2 An ADI of 0–0.2 mg/kg

anthranilate Europe: 60 bw was established for
USA: 120 methyl N-methyl-
anthranilate by the
Committee at its
twenty-third meeting
(Annex 1, reference
50), which was main-
tained at the present
meeting.

Ethyl N-methyl- 1546 35472-56-1 No NR NR See note 2 No safety concern

anthranilate Europe: 0.03b (conditional)
USA: 0.04b

Ethyl N-ethyl- 1547 38446-21-8 No NR NR See note 3 No safety concern

anthranilate Europe: 0.07b (conditional)
USA: 0.09b
 Table 4 (contd)

Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

Isobutyl N-methyl- 1548 65505-24-0 No NR NR See note 2 No safety concern

anthranilate Europe: 0.07b (conditional)
USA: 0.09b

Methyl N-formyl- 1549 41270-80-8 No NR NR See note 4 No safety concern

anthranilate Europe: 0.1b (conditional)
USA: 0.2b

Methyl N-acetyl- 1550 2719-08-6 No NR NR See note 5 No safety concern

anthranilate Europe: 0.05b (conditional)
USA: 0.06b

Methyl N, N-dimethyl- 1551 10072-05-6 No NR NR See note 6 No safety concern

anthranilate Europe: 15b (conditional)
USA: 18b

59
 Table 4 (contd)

60
Flavouring agent No. CAS No. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the flavouring Adequate margin on estimated daily
exceed the agent or are its of safety for the intake
threshold for metabolites flavouring agent
human intake? endogenous? or related
substance?

N-Benzoylanthranilic 1552 579-93-1 No NR NR See note 7 No safety concern

acid Europe: 1b (conditional)
USA: 2b

CAS: Chemical Abstracts Service; ND: no intake data reported; N/R: not required for evaluation because intake of the substance was determined to
be of no safety concern at Step A3 of the procedure.
Step 2: All the agents in this group can be predicted to be metabolized to innocuous products. The evaluation of these flavouring agents therefore
proceeded via the A-side of the Procedure.
a
The threshold for human intake for structural class I is 1800 µg/day. The combined per capita intake of flavouring agents in this group are 904 µg/
day in Europe and 4030 µg/day in the USA.
b
Intake estimate based on anticipated annual volume of production

Notes:
1. Hydrolysed to anthranilic acid, followed by rapid excretion in the urine in conjugated form with glycine (as ortho-aminohippuric acid) or glucuronic
acid. The alcohols formed on hydrolysis would be oxidized or conjugated with glucuronic acid or sulfate, followed by excretion in the urine.
2. Hydrolysed to N-methylanthranilic acid, followed by excretion in the urine
3. Hydrolysed to N-ethylanthranilic acid, followed by excretion in the urine
4. Hydrolysed to N-formylanthranilic acid, followed by excretion in the urine
5. Hydrolysed to N-acetylanthranilic acid, followed by excretion in the urine
6. Hydrolyzed to N,N-dimethylanthranilic acid, followed by excretion in the urine
7. Conjugated at the carboxylic acid group to glycine and acyl-glucuronic acid conjugates, followed by excretion in the urine
(unconditional) ADI of 0–1.5 mg/kg bw. Methyl N-methylanthranilate (No.
1545) was evaluated at the twenty-third meeting (Annex 1, reference 50)
and was assigned an ADI of 0–0.2 mg/kg bw.

Four of the 19 flavouring agents (Nos 1534, 1535, 1545 and 1546) have
been reported to occur naturally in foods. They have been detected in, for
example, starfruit, orange juice, grapefruit juice, strawberries and orange,
mandarin and tangerine peel oils. The substance that occurs naturally most
frequently is methyl anthranilate (No. 1534).
Estimated daily per capita exposure
Annual volumes of production have been reported for 10 of the 19 flavouring
agents in this group (Nos 1534–1537 and 1540–1545). For the remaining
nine substances (Nos 1538, 1539, 1546–1552), anticipated annual volumes
of production were given for their proposed use as flavouring agents. The
total reported and anticipated annual volume of production of the 19
flavouring agents in this group is about 6300 kg in Europe and 30 000 kg in
the USA. Methyl anthranilate (No. 1534) accounts for approximately 89%
of the total reported and anticipated annual volume of production in Europe
and 94% in the USA. Estimated daily exposure to methyl anthranilate in
Europe and the USA is approximately 800 and 3800 µg per person,
respectively. Ethyl anthranilate (No. 1535) and methyl N-methylanthranilate
(No. 1545) account for most of the remaining total reported and anticipated
annual volumes of production (approximately 8% in Europe and 4% in the
USA). Estimated daily per capita exposure to ethyl anthranilate is 14 µg in
Europe and 39 µg in the USA; that to methyl N-methylanthranilate is 60 µg
in Europe and 120 µg in the USA; and that to the remaining flavouring
agents in this group is 0.003–15 µg in Europe and 0.007–53 µg in the USA.
The estimated daily per capita exposure to each agent is reported in Table 4.

Absorption, distribution, metabolism and elimination

The 11 anthranilic acid esters (Nos 1534–1544) and the five N-alkyl
anthranilic acid esters (Nos 1545–1548 and 1551) in this group are expected
to be readily absorbed, either unchanged or in hydrolysed form. Once
absorbed, the unchanged esters are hydrolysed in the liver to their
corresponding alcohols and carboxylic acids (anthranilic acid, N-methyl-
anthranilic acid, N-ethylanthranilic acid or N,N-dimethylanthranilic acid).
These anthranilic acid derivatives are then rapidly excreted in the urine.

Given the relative resistance of the amide bond to hydrolysis, the two
combined amides–esters in this group (Nos 1549 and 1550) are expected to
be hydrolysed at the methyl ester bond, with rapid excretion of the

61
corresponding carboxylic acids (N-formylanthranilic acid or N-acetylanthra-
nilic acid) in the urine, either unchanged or in conjugated form. Rather than
undergoing hydrolysis at the amide bond, N-benzoylanthranilic acid (No.
1552) will be conjugated with glycine or glucuronic acid at the free
carboxylic acid group, before excretion in the urine.

Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported
and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated
from it exceeded the exposure estimated from the anticipated volume of
production and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.

Step 1. In applying the Procedure, the Committee assigned all 19

flavouring agents in this group to structural class I.

Step 2. All the flavouring agents in this group are expected to be

metabolized to innocuous products. The evaluation of all the agents in this
group therefore proceeded via the A-side of the Procedure.
Step A3. The estimated daily exposure to 18 of the 19 flavouring agents
(Nos 1535–1552) is below the threshold of concern (i.e. 1800 µg per person
per day for class I). Nine of these 18 substances (Nos 1535–1537 and 1540–
1545) are reported to be used as flavouring agents; according to the
Procedure, use of these nine flavouring agents raises no safety concern at
estimated current exposure. The other nine substances (Nos 1538, 1539 and
1546–1552) are proposed for use as flavouring agents. Although, according
to the Procedure, use of these nine flavouring agents raises no safety concern
at estimated exposure on the basis of anticipated annual volumes of
production, less uncertain estimates are needed. Estimated daily exposure
to the remaining agent in this group, methyl anthranilate (No. 1534), which
is 804 µg per person in Europe and 3764 µg per person in the USA, exceeds
the threshold of concern for class I. Accordingly, the evaluation of methyl
anthranilate proceeded to step A4.

Step A4. Methyl anthranilate is not endogenous in humans. Therefore, its

evaluation proceeded to step A5.

62
Step A5. At its twenty-third meeting, the Committee established an ADI of
0–1.5 mg/kg bw for methyl anthranilate on the basis of a NOEL of 150 mg/kg
bw per day in a short-term study in rats (Annex 1, reference 50). This NOEL
is > 11 000 and > 2300 times greater than the estimated daily exposure to
methyl anthranilate from its use as a flavouring agent in Europe (13 µg/kg
bw) and the USA (63 µg/kg bw), respectively. The Committee therefore
concluded that methyl anthranilate would not present a safety concern at
the estimated daily exposure.

The exposure considerations and other information used to evaluate the 19

anthranilate derivatives in this group according to the Procedure are
summarized in Table 4.
Consideration of secondary components
All 19 flavouring agents in this group have minimum assay values of ≥ 95%.
Hence, it is not necessary to consider secondary components.
Consideration of combined exposure from use as flavouring agents

In the unlikely event that all 19 agents in this group were to be consumed
concurrently on a daily basis, the estimated combined exposure would exceed
the human exposure threshold of 1800 µg per person per day for class I. All
these agents are, however, expected to be efficiently metabolized and would
not saturate metabolic pathways. Overall evaluation of the data indicated
that combined exposure to these agents would not raise a safety concern.
Conclusions

The Committee maintained the previously established ADIs of 0–1.5 mg/kg

bw for methyl anthranilate and 0–0.2 mg/kg bw for methyl N-
methylanthranilate (Annex 1, reference 50). The Committee concluded that
use of the flavouring agents in this group of anthranilate derivatives would
not present a safety concern at the estimated exposure levels. For nine
flavouring agents (Nos 1538, 1539 and 1546–1552), the evaluation was
conditional because the estimated exposure was based on anticipated annual
volumes of production. The conclusions of the safety evaluations of these
agents will be revoked if use levels or poundage data are not provided before
December 2007. The Committee noted that the available data on the toxicity
and metabolism of the anthranilate derivatives were consistent with the
results of the safety evaluation conducted with the Procedure.
A monograph summarizing the safety data on this group of flavouring agents
was prepared.

63
4.1.5 Miscellaneous nitrogen-containing substances

The Committee evaluated a group of 16 flavouring agents (Table 5) by the

Procedure for the Safety Evaluation of flavouring Agents (Annex 1, reference
131). This group comprised five structurally related isothiocyanates (Nos
1560–1564) that included allyl isothiocyanate (No. 1560); six alkylated
oxazole analogues (Nos 1553–1557 and 1569); two methylated oxazoline
analogues (Nos 1558–1559); two pyrimidines (Nos 1565–1566); and one
pyrazole (No. 1568).
None of these agents has previously been evaluated by the Committee.
Fourteen of the 16 substances (Nos 1553–1565 and 1569) have been reported
to occur naturally in foods and have been detected in a variety of vegetables,
cooked meats, cocoa, coffee, pineapple and papaya.
Estimated daily per capita exposure

Annual volumes of production have been reported for six of the 16 flavouring
agents in this group (Nos 1559, 1560, 1564–1566 and 1568). For the
remaining 10 substances (Nos 1553–1558, 1561–1563 and 1569), anticipated
annual volumes of production have been given for their proposed use as
flavouring agents. The total reported and anticipated annual volume of
production of the 16 flavouring agents in this group is about 10 600 kg in
Europe and 1400 kg in the USA. More than 98% of the total reported and
anticipated annual volume of production in Europe and more than 70% in
the USA is accounted for by one substance in this group, allyl isothiocyanate
(No. 1560). Estimated per capita exposure to this substance is approximately
1500 µg/day in Europe and 130 µg/day in the USA. Per capita exposure to
all the other flavouring agents in the group is 0.03–13 µg/day in Europe and
0.01–52 µg/day in the USA, most of the values being at the lower end of
the range (Table 5).

Absorption, distribution, metabolism and elimination

Data on structurally related substances indicate that oxazoles, oxazolines,
pyrimidines and pyrazoles will be rapidly absorbed, metabolized and
excreted in the urine. The metabolism of oxazoles involves two pathways:
oxazole ring cleavage and, if the ring is properly substituted, ring
hydroxylation. The presence of a substituent at the 2-position tends to
stabilize the oxazole ring.
Isothiocyanates are readily absorbed and distributed to all the main tissues
in rodents, peak concentrations in the tissues being achieved 2–8 h after
dosing. At comparable doses, there are clear sex- and species-specific
differences in the distribution, metabolism and excretion of substituted
isothiocyanates.

64
 Table 5. Summary of results of safety evaluations of miscellaneous nitrogen-containing substances
Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
Structural class II
Trimethyloxazole 1553 20662-84-4 No Yes. The NOEL of 2.3 NR See note 3 No safety concern
Europe: 1b mg/kg bw per day for the (conditional)
USA: 1b related substance 2-
ethyl-4,5-dimethyl-
oxazole (Griffiths et al.,
1979) is 115 000 times
the estimated daily intake
of trimethyloxazole of
0.02 µg/kg bw in both
Europe and the USA,
when used as a flavouring
agent.
2,5-Dimethyl-4- 1554 30408-61-8 No Yes. The NOEL of 2.3 NR See note 3 No safety concern
ethyloxazole Europe: 0.2b mg/kg bw per day for the (conditional)
USA: 0.2b related substance 2-ethyl-
4,5-dimethyloxazole
(Griffiths et al., 1979) is
575 000 times the esti-
mated daily intake 2,5-
dimethyl-4-ethyloxazole
of 0.004 µg/kg bw in
both Europe and the
USA, when used as a

65
flavouring agent.
 Table 5 (contd)

66
Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
2-Ethyl-4,5-di- 1555 53833-30-0 No Yes. The NOEL of 2.3 NR See note 3 No safety concern
methyloxazole Europe: 0.03 mg/kg bw per day (Griffiths (conditional)
USA: 0.7b et al., 1979) is 4 600 000
and 230 000 times the
estimated daily intake of
2-ethyl-4,5-dimethyloxazole
of 0.0005 µg/kg bw in
Europe and 0.01 µg/kg
bw in the USA, when used
as a flavouring agent.
2-Isobutyl-4,5-di- 1556 26131-91-9 No Yes. The NOEL of 2.3 NR See note 3 No safety concern
methyloxazole Europe: 0.2b mg/kg bw per day for the (conditional)
USA: 0.2b related substance 2-ethyl-
4,5-dimethyloxazole
(Griffiths et al., 1979) is
575 000 times the esti-
mated daily intake of
2-isobutyl-4,5-dimethyl-
oxazole of 0.004 µg/kg
bw in both Europe and
the USA, when used as
a flavouring agent.
 Table 5 (contd)

Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
2-Methyl-4,5- 1557 95-21-6 No Yes. The NOEL of 2.3 NR See note 1,2 No safety concern
benzo-oxazole Europe: 0.1b mg/kg bw per day for the (conditional)
USA: ND related substance 2-ethyl-
4,5-dimethyloxazole
(Griffiths et al., 1979) is
1 150 000 times the esti-
mated daily intake of
2-methyl-4,5-benzo-oxa-
zole of 0.002 µg/kg bw in
both Europe and the USA,
when used as a flavouring
agent.
2,4-Dimethyl-3- 1558 77311-02-5 No Yes. The NOEL of 41 NR See note 3 No safety concern
oxazoline Europe: 0.07b mg/kg bw per day for the (conditional)
USA: ND related substance 2,4,5-
trimethyl-∆-3-oxazoline
(Morgareidge,1972) is
41 000 000 times the esti-
mated daily intake of 2,4-
dimethyl-3-oxazoline of
0.001 µg/kg bw in both
Europe and the USA, when
used as a flavouring agent.

67
 Table 5 (contd)

68
Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
2,4,5-Trimethyl-∆- 1559 22694-96-8 No Yes. The NOEL of 41 NR See note 3 No safety concern
3-oxazoline Europe: 0.04 mg/kg bw per day
USA: 0.01 (Morgareidge, 1972) is
approximately 60 000 000
and 205 000 times the esti-
mated daily intake of 2,4,5-tri-
methyl-∆-3-oxazoline of
0.0007 µg/kg bw in Europe
and 0.0002 µg/kg bw in the
USA, when used as a fla-
vouring agent.
Allyl isothiocyanate 1560 57-06-7 Yes Yes. The NOEL of 12 See notes 4 No safety concern
Europe: 1502 mg/kg bw per day (National and 5
USA: 133 Toxicology Program, 1982)
is > 400 and > 5000 times
the estimated daily intake of
allyl isothiocyanate of
25 µg/kg bw in Europe and
2.2 µg/kg bw in the USA,
when used as a flavouring
agent.
 Table 5 (contd)

Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
Butyl isothio- 1561 592-82-5 No Yes. The NOEL of 12 NR See notes 4 No safety concern
cyanate Europe: 2b mg/kg bw per day for the and 5 (conditional)
USA: ND related substance allyl
isothiocyanate (National
Toxicology Program, 1982)
is 400 000 times the esti-
mated daily intake of butyl
isothiocyanate of 0.03 µg/
kg bw in Europe, when
used as a flavouring agent.
Benzyl isothio- 1562 622-78-6 No Yes. The NOEL of 5 NR See note 4 No safety concern
cyanate Europe: 1b mg/kg bw per day for the (conditional)
USA: 0.4b related substance phenethyl
isothiocyanate (Ogawa et al.,
2001) is 250 000 and about
700 000 times the estimated
daily intake of benzyl isothio-
cyanate of 0.02 µg/kg bw in
Europe and 0.007 µg/kg bw
in the USA, when used as a
flavouring agent.

69
 Table 5 (contd)

70
Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
Phenethyl isothio- 1563 2257-09-2 No Yes. The NOEL of 5 NR See note 4 No safety concern
cyanate Europe: 0.4b mg/kg bw per day (Ogawa (conditional)
USA: 0.5b et al., 2001) is 700 000 and
about 600 000 times the
estimated daily intake of
phenethyl isothiocyanate
of 0.007 µg/kg bw in
Europe and 0.008 µg/kg
bw in the USA, when used
as a flavouring agent.
3-Methylthiopropyl 1564 505-79-3 No Yes. The NOEL of 30 NR See note 4 No safety concern
isothiocyanate Europe: 13 mg/kg bw per day (Harper
USA: 52 et al., 1961) is 150 000 and
30 000 times the estimated
daily intake of 3-methylthio-
propyl isothiocyanate of
0.2 µg/kg bw in Europe and
0.9 µg/kg bw in the USA,
when used as a flavouring
agent.
 Table 5 (contd)

Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?
4-Acetyl-2-methyl- 1565 67860-38-2 No Yes. The NOEL of 1 mg/kg NR See note 4 No safety concern
pyrimidine Europe: ND bw per day (Peano, 1981)
USA: 0.01 is 5 000 000 times the esti-
mated daily intake of 4-
acetyl-2-methylpyrimidine
of 0.0002 µg/kg bw in the
USA, when used as a
flavouring agent.
4,5-Dimethyl-2- 1569 53833-32-2 No Yes. The NOEL of 2.3 NR See note 3 No safety concern
propyloxazole Europe: 0.1b mg/kg bw per day for the (conditional)
USA: 0.1b related substance 2-ethyl-
4,5-dimethyloxazole
(Griffiths et al., 1979) is
1 150 000 times the esti-
mated daily intake of 4,5-
dimethyl-2-propyloxazole
of 0.002 µg/kg bw in both
Europe and the USA,
when used as a flavouring
agent.

71
72
Table 5 (contd)

Flavouring agent No. CAS no. and Step B3a Step B4 Are additional data Comments Conclusion based
structure Does intake Adequate margin of available to perform a on estimated daily
exceed the safety for the flavouring safety evaluation for intake
threshold for agent or related substances with an
human intake? substance? intake exceeding the
threshold of concern?

Structural class III

5,7-Dihydro-2- 1566 36267-71-7 No Yes. The NOEL of 6.6 NR See note 2 No safety concern
methylthieno(3,4-d)- Europe: ND mg/kg bw per day
pyrimidine USA: 0.4 (Shellenberger, 1970) is
1 100 000 times the esti-
mated daily intake of 5,7-
dihydro-2-methylthieno-
(3,4-d)pyrimidine of
0.006 µg/kg bw in the
USA, when used as a
flavouring agent.
1-Phenyl-3- or -5- 1568 65504-93-0 No Yes. The NOEL of 25 NR See note 2 No safety concern
propylpyrazole Europe: ND mg/kg bw per day
USA: 0.2 (Posternak et al., 1969)
is about 6 000 000 times
the estimated daily intake
of 1-phenyl-3- or -5-propyl-
pyrazole of 0.004 µg/kg bw
in the USA, when used as
a flavouring agent.
 Table 5 (contd)

CAS, Chemical Abstracts Service; ND, no intake data reported; NR, not required for evaluation because consumption of the substance was determined not
to exceed the threshold of concern at Step B3 of the Procedure
Step 1: Fourteen flavouring agents are in structural class II (Nos 1553–1565 and 1569), and two are in structural class III (Nos 1566 and 1568).
Step 2: None of the miscellaneous nitrogen derivatives (Nos 1553–1566, 1568 and 1569) can be predicted to be metabolized to innocuous products.
a
The thresholds for concern for structural classes II and III are 540 and 90 µg/day, respectively. All intake values are expressed in µg per day. The
combined per capita intake of the flavouring agents in structural class II is 1520 µg per day in Europe and 188 µg per day in the USA. The combined per
capita intake of the flavouring agents in structural class III is 0.6 µg per day in the USA (no intake data reported for Europe).
b
Intake estimate based on anticipated annual volume of production
Notes:
1. Predicted to be absorbed rapidly, followed by ring cleavage and excretion in the urine
2. Predicted to be readily absorbed, followed by ring hydroxylation and excretion in the urine
3. Predicted to be readily absorbed, followed by side-chain oxidation and excretion in the urine
4. Readily absorbed, principally conjugated with glutathione, followed by formation of mercapturic acid conjugate and excretion in the urine
5. Hydrolysis followed by excretion in the urine

73
Metabolic studies in humans, mice and rats indicate that isothiocyanates
react readily with reduced glutathione (GSH) to form a conjugate as the
principal metabolite and that the reaction is catalysed enzymatically by
glutathione S-transferase enzymes and non-enzymatically (at a slower rate),
both reactions occurring in a pH-dependent equilibrium. The isothiocyanate–
GSH conjugates formed are subsequently excreted into bile, and corres-
ponding N-acetylcysteine adducts appear as the major metabolite in urine.
A key element of isothiocyanate metabolism is the highly electrophilic,
reactive central carbon in the group, as it drives Michael addition reactions
with N-, O- or S-based nucleophiles (e.g. GSH), giving rise to the relatively
stable but reversible conjugates implicated in its toxicity. In humans and
rats, aromatic isothiocyanates are metabolized mainly to the corresponding
mercapturic acid conjugates, which subsequently hydrolyse to the
corresponding cysteine conjugates as the major urinary metabolites. The
lability of glutathione conjugates under the conditions in the rodent bladder
can lead to formation of unconjugated, ‘free’ isothiocyanate and GSH. The
presence of free isothiocyanates can increase irritation of the rat bladder
epithelium. In rabbits, mice and guinea-pigs, however, the cysteine conjugate
is hydrolysed and then undergoes transamination and cyclization to form a
substituted thiazolidine-2-thione as the main urinary metabolite.

Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported
and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated
from it exceeded the exposure estimated from the anticipated volume of
production and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.
Step 1. In applying the Procedure, the Committee assigned 14 agents in
this group (Nos 1553–1565 and 1569) to structural class II and the remaining
two agents (Nos 1566 and 1568) to structural class III.
Step 2. None of the flavouring agents in this group can be predicted to be
metabolized to innocuous products. The evaluation of all flavouring agents
in this group therefore proceeded via the B-side of the Procedure.

74
Step B3. Estimated daily per capita exposure in Europe and the USA of 13
of the flavouring agents in structural class II (Nos 1553–1559, 1561–1565
and 1569) and both of the flavouring agents in structural class III (Nos
1566 and 1568) is below the threshold of concern for their respective class
(i.e. class II, 540 µg/day; class III, 90 µg/day). Accordingly, the evaluation
of these 15 agents proceeded to Step B4. The estimated per capita exposure
to one of the agents in structural class II, allyl isothiocyanate (No. 1560),
which is 1502 µg/day in Europe and 133 µg/day in the USA, exceeds the
threshold of concern (540 µg per day) for its class. In accordance with the
Procedure, more extensive data are needed to evaluate the safety of
flavouring agents the exposure to which exceeds the threshold of concern
for their structural class at Step B3. Additional data on allyl isothiocyanate
were therefore considered.

Step B4. For 2-ethyl-4,5-dimethyloxazole (No. 1555), the NOEL of 2.3 mg/
kg bw per day in a 91-day study in rats treated by gavage is 4 600 000 times
the estimated exposure from its use as flavouring agent in Europe (0.0005
µg/kg bw per day) and 230 000 times the estimated exposure from its
proposed use in the USA (0.01 µg/kg bw per day).

The NOEL for 2-ethyl-4,5-dimethyloxazole is also appropriate for the

structurally related agents trimethyloxazole (No. 1553), 2,5-dimethyl-4-
ethyloxazole (No. 1554), 2-isobutyl-4,5-dimethyloxazole (No. 1556), 2-
methyl-4,5-benzo-oxazole (No. 1557) and 4,5-dimethyl-2-propyloxazole
(No. 1569), all of which are oxazole analogues and as such are expected to
be metabolized via similar metabolic pathways. The NOEL of 2.3 mg/kg
bw per day is 115 000 times the estimated exposure to trimethyloxazole
from its proposed use as a flavouring agent in both Europe and the USA
(0.02 µg/kg bw per day), 575 000 times the estimated exposure to 2,5-
dimethyl-4-ethyloxazole and 2-isobutyl-4,5-dimethyloxazole from their
proposed use as flavouring agents in both Europe and the USA (0.004 µg/
kg bw per day) and 1 500 000 times the estimated exposure to 2-methyl-
4,5-benzo-oxazole and 4,5-dimethyl-2-propyloxazole from their proposed
use as flavouring agents in both Europe and the USA (0.002 µg/kg bw per
day).
The NOEL of 41 mg/kg bw per day for 2,4,5-trimethyl-∆-3-oxazoline (No.
1559) in a 90-day feeding study in rats is approximately 60 000 000 times
the estimated exposure to this substance from its use as flavouring agent in
Europe (0.0007 µg/kg bw per day) and 205 000 000 times that in the USA
(0.0002 µg/kg bw per day).
The NOEL for 2,4,5-trimethyl-∆-3-oxazoline is also appropriate for the
structurally related agent 2,4-dimethyl-3-oxazoline (No. 1558), as this is an

75
oxazoline analogue and is therefore expected to be metabolized via similar
metabolic pathways. The NOEL of 41 mg/kg bw per day is 41 000 000
times the estimated exposure to 2,4-dimethyl-3-oxazoline from its proposed
use as a flavouring agent in Europe (0.001 µg/kg bw per day).
Although no NOEL is available for butyl isothiocyanate (No. 1561), the
NOEL of 12 mg/kg bw per day for the structurally related agent allyl
isothiocyanate (No. 1560; see below) is also appropriate for butyl
isothiocyanate, as they are both isothiocyanates, which will be metabolized
via similar metabolic pathways. This NOEL is 400 000 times the estimated
exposure to butyl isothiocyanate from its proposed use as a flavouring agent
in Europe (0.03 µg/kg bw per day).
Although no NOEL is available for benzyl isothiocyanate (No. 1562), the
NOEL of 5 mg/kg bw per day for the structurally related agent phenethyl
isothiocyanate is also appropriate for benzyl isothiocyanate, as both are
isothiocyanates, which will be metabolized via similar metabolic pathways.
This NOEL is 250 000 times the estimated exposure to this substance from
its proposed use as a flavouring agent in Europe (0.02 µg/kg bw per day)
and about 700 000 times that in the USA (0.007 µg/kg bw per day).
For phenethyl isothiocyanate (No. 1563), the NOEL of 5 mg/kg bw per day
in a 91-day feeding study in rats is approximately 700 000 times the
estimated exposure to this substance from its proposed use as a flavouring
agent in Europe (0.007 µg/kg bw per day) and about 600 000 times that in
the USA (0.008 µg/kg bw per day).
For 3-methylthiopropyl isothiocyanate (No. 1564), the NOEL of 30 mg/kg
bw per day in an 84-day feeding study in rats is 150 000 times the estimated
exposure to this substance from its use as a flavouring agent in Europe
(0.2 µg/kg bw per day) and about 30 000 times that in the USA (0.9 µg/kg
bw per day).
For 4-acetyl-2-methylpyrimidine (No. 1565), the NOEL of 1 mg/kg bw per
day in a 91-day study in rats treated by gavage is 5 000 000 times the
estimated exposure to this substance from its use as flavouring agent in the
USA (0.0002 µg/kg bw per day).

For 5,7-dihydro-2-methylthieno(3,4-d)pyrimidine (No. 1566), the NOEL

of 6.6 mg/kg bw per day in a 90-day feeding study in rats is 1 100 000 times
the estimated exposure to this substance from its use as a flavouring agent
in the USA (0.006 µg/kg bw per day).
For 1-phenyl-3- or -5-propylpyrazole (No. 1568), the NOEL of 25 mg/kg
bw per day in a 90-day feeding study in rats is about 6 000 000 times the

76
estimated exposure to this substance from its use as a flavouring agent in
the USA (0.004 µg/kg bw per day).
The Committee concluded that the margins between the estimated daily
exposure to the five substances reported to be used as flavouring agents
(Nos 1559, 1564–1566 and 1568) and the NOELs for these agents were
adequate, and that their use would not present a safety concern. The
Committee also concluded that the margins between the exposure estimated
from anticipated annual volumes of production for the other 10 substances
proposed for use as flavouring agents (Nos 1553–1558, 1561–1563 and
1569) and the NOELs for these agents were adequate. Although their use
would raise no safety concern at estimated exposure levels, more accurate
estimates of exposure are required.
The exposure considerations and other information used to evaluate these
miscellaneous nitrogen-containing flavouring agents are summarized in
Table 5.

Consideration of flavouring agents with high exposure evaluated on the B-

side of the Procedure
As stipulated in the Procedure, more extensive data on metabolism and
toxicity were considered to complete the safety evaluation of allyl
isothiocyanate (No. 1560), as the exposure level estimated from use of this
compound as a flavouring agent in Europe exceeded the threshold of concern
for structural class II (540 µg per person per day).

Short-term and long-term studies

Several short-term of studies of toxicity were conducted in rats and mice
given allyl isothiocyanate orally. Furthermore, allyl isothiocyanate was tested
in a series of short-term studies of toxicity and long-term studies of
carcinogenicity in both laboratory species.

In a 20-day study, groups of five weanling Osborne-Mendel rats of each

sex were given allyl isothiocyanate in corn oil at a dose of 0 (vehicle control),
20 or 50 mg/kg bw per day. Macroscopically, the non-glandular part of the
stomach was thickened, with occasional roughening of the lining, at both
doses. Minor inflammatory foci were reported in the livers of rats at the
higher dose.
Groups of male outbred Shoe:WIST rats (number of animals per group not
specified) were given allyl isothiocyanate at a dose of 0 (paraffin oil vehicle
control), 10, 20 or 40 mg/kg bw per day, 5 days per week for up to 6 weeks
by gavage. Hepatohistopathology showed diffuse ballooning of centrilobular
hepatocytes in some rats at the highest dose. The kidneys of rats at this dose

77
and at the middle dose showed dilatation of distal tubules and increased
desquamation.
In a 9-week study conducted to examine the possible effects of allyl
isothiocyanate on growth, groups of four weanling rats (strain not specified)
were fed basal diet (control) or basal diet with 0.1% allyl isothiocyanate for
5 weeks. This dietary level was calculated to provide an average daily
exposure to 100 mg/kg bw. During weeks 6–9, the rats were given allyl
isothiocyanate by gavage daily at the dose in food (about 100 mg/kg bw).
Only animals treated by gavage had lower body-weight gains than controls
(statistics not reported).
A dose-range-finding study was conducted in mice. Groups of five B6C3F1
mice of each sex were given allyl isothiocyanate in corn oil by gavage at a
dose of 3, 6, 12, 25 or 50 mg/kg bw per day for 14 days. One male at
50 mg/kg bw per day died. No dose-dependent change in body-weight gain
was found. Four of the five males and all females at the highest dose showed
thickened areas of mucosa in the non-glandular region of the stomach, and
four males and one female had a thickened urinary bladder wall.
In a 13-week study, groups of 10 B6C3F1 mice of each sex were given allyl
isothiocyanate by gavage at a dose of 0 (vehicle control), 1.5, 3, 6, 12 or
25 mg/kg bw per day on 5 days per week. The mean body weights of treated
and control animals were comparable, and no gross or histological changes
were reported at any dose.

In a study of carcinogenicity, groups of 50 male and 50 female B6C3F1

mice received allyl isothiocyanate by gavage at a dose of 0 (vehicle control),
12 or 25 mg/kg bw per day, 5 days per week for 103 weeks. Many of the
female mice that died before week 104 had suppurative inflammation of
the peritoneum, uterus or multiple organs, suggesting generalized infection.
The final mean body weights of treated and control animals were comparable
(statistics not reported). The incidence of primary tumours was not increased
in treated mice. Male mice showed a statistically significant, dose-related
increase in cytoplasmic vacuolization in the liver (control, 2/49; low dose,
8/49; high dose, 13/50). The severity of this lesion was similar in the three
groups. Most of the vacuoles were centrilobular and all contained fat. The
authors concluded that allyl isothiocyanate was not carcinogenic under the
conditions of this study.

A dose-range finding study was conducted in groups of five Fischer 344/N

rats of each sex given allyl isothiocyanate in corn oil by gavage at a dose of
25, 50, 100, 200 or 400 mg/kg bw per day for 14 days. Clinical signs,
including inactivity and ruffled fur, were observed at all doses, the severity
increasing with dose. All animals at the two highest doses died before the

78
end of the study. Reduced body weights were observed at 100 mg/kg bw
per day. Gross necropsy revealed a thickened mucosal surface of the stomach
and adhesion of the stomach to the peritoneum in treated animals.

Groups of 10 Fischer 344/N rats of each sex were given allyl isothiocyanate
by gavage at a dose of 0 (vehicle control), 1.5, 3, 6, 12 or 25 mg/kg bw per
day, 5 days per week for 13 weeks. All animals survived to scheduled
termination with no clinical signs of toxicity. No significant changes in
body weight and no gross or histological changes were reported at any
dose.
In a carcinogenesis bioassay, groups of 50 male and 50 female Fischer
344/N rats received allyl isothiocyanate by gavage at a dose of 0 (vehicle
control), 12 or 25 mg/kg bw per day, 5 days per week for 103 weeks. The
mean body weights of male rats at the higher dose were lower than those of
controls throughout the study. No clinical signs of toxicity were reported.
Survival (58–74%) was comparable in all groups, including controls. The
incidence of subcutaneous fibrosarcomas was increased in females at the
higher dose, with a significant positive trend. The incidence of
undifferentiated leukaemia was increased over that in controls in treated
male rats at both doses: control, 2/50; low dose, 6/50; high dose, 8/50.
Males at the higher dose also had a significantly increased incidence of
transitional-cell papillomas of the urinary bladder: control, 0/49; high dose,
4/49. Epithelial hyperplasia of the urinary bladder also occurred in males,
with a significant overall trend at the higher dose. Hyperplasia was not
observed in animals with papillomas. The authors concluded that, under
the conditions of this bioassay, allyl isothiocyanate was carcinogenic in
male rats, causing transitional-cell papillomas of the urinary bladder, but
that the evidence of an association with subcutaneous fibrosarcomas in
female rats was equivocal.
Genotoxicity
Numerous tests for genotoxicity were reported. The studies indicated mixed
results in vitro, but mainly negative results (8 of 10 studies) in vivo. The
two studies with positive results showed only weak activity.
Mode of action
In rats given allyl isothiocyanate at an LD50 dose of 112 mg/kg bw by
stomach tube, marked irritation of the lungs and gastrointestinal tract was
reported. In mice, the sensitizing effects of allyl isothiocyanates on the skin
correlated with a perturbation in the ratio of skin GSH to glutathione
disulfide, suggesting that the substance might induce oxidative stress in
mouse skin epithelia.

79
An underlying concept in the hypothesis for the mode of toxic action of
allyl isothiocyanate is the chemical reactivity of the highly electrophilic
central carbon in the isothiocyanate group (–N=C=S). It can efficiently
undergo Michael addition reactions with N-, O- or S-based nucleophiles
such as the thiol of GSH. The reaction gives rise to relatively stable but
reversible GSH adducts, which have been implicated in its cytotoxicity in
hepatocytes in vitro. Free and conjugated forms of allyl isothiocyanate are
in equilibrium, allowing for the presence of as much as 15–20% free allyl
isothiocyanate. The position of the equilibrium can be shifted in favour of
the free form under conditions of low GSH concentration and alkaline pH,
which may exist in the urinary bladder. Therefore, increased absorption of
free allyl isothiocyanate might occur in the bladder epithelium.
Data on the metabolism and disposition of allyl isothiocyanate show clear
sex- and species-specific differences. Mice excrete essentially all orally
administered allyl 14C-isothiocyanate within 96 h, while rats retain up to
20% in the carcass over the same interval. Blood concentrations of
radioactivity returned to background levels within 96 h in mice but persisted
up to 240 h in rats. In a separate experiment, mice excreted more than 80%
of an administered dose of allyl 14C-isothiocyanate in the urine within 3 days,
while rats excreted only 55%, indicating that rats selectively retain
thiocyanate ion. Studies in rats showed that females produce twice as much
urine as males, so that the male rat bladder is exposed to higher concentrations
of allyl isothiocyanate and its metabolites than the female rat bladder.
The histopathological evidence of male bladder epithelial hyperplasia
reported in the long-term study in rats correlates with the biochemical
evidence that the male rat bladder is subjected to longer exposure to high
concentrations of possible irritants, such as allyl isothiocyanate or its
metabolites.
Both the qualitative and the quantitative aspects of the molecular disposition
of allyl isothiocyanate and its associated toxicological sequelae have been
relatively well defined in studies in mammals and are similar to those
reported for other irritating substances in the urinary bladder. Epithelial-
cell papillomas are benign lesions on luminal surfaces.
On the basis of the observations that allyl isothiocyanate has strong irritant
and cytotoxic properties, is considered not to be genotoxic in vivo and
induces sex- and species-specific benign transitional-cell papillomas only
in male rats, it is highly probable that allyl isothiocyanate operates through
a secondary non-genotoxic mechanism, like other irritating bladder
carcinogens, such as sodium saccharin.

80
The two factors that distinguish human exposure to allyl isothiocyanate
from that of rats are daily exposure and bladder function. Rats in the 2-year
study were given 25 000 µg/kg bw daily, while human exposure from use
of allyl isothiocyanate as a flavouring agent is approximately 1000 times
less (25 µg/kg bw per day). Furthermore, humans excrete about 1500 ml of
urine per day. Therefore, the potential concentration of allyl isothiocyanate
or its metabolites in the human bladder is orders of magnitude lower than
that required to induce hyperplasia and papillomas in rats. As no toxicity
was observed in the bladder at a dose of 12 000 µg/kg bw per day in the
study in rats, it is highly unlikely that this mode of action of carcinogenicity
would operate in humans.
The NOEL for allyl isothiocyanate in the 2-year study in rats was 12 mg/kg
bw per day. This NOEL is more than 400 times the estimated daily exposure
to allyl isothiocyanate when used as a flavouring agent in Europe (25 µg/kg
bw per day) and more than 5000 times that in the USA (2.2 µg/kg bw per
day). Therefore, on the basis of the additional data on toxicity, the Committee
concluded that allyl isothiocyanate (No. 1560) would not be expected to
present a safety concern at estimated current exposure (Table 5).

Consideration of secondary components

One member of this group of flavouring agents, 2,4,5-trimethyl-∆-3-
oxazoline (No. 1559), has an assay value of < 95%. The secondary
component in this substance, trimethyloxazole (No. 1553), was evaluated
at the present meeting, where present levels of exposure were considered
to present no safety concern. The Committee also concluded that the
flavouring agent as specified would not present a safety concern at the
estimated levels of exposure.
Consideration of combined exposure from use as flavouring agents

In the unlikely event that all 14 agents in structural class II were to be

consumed concurrently on a daily basis, the estimated combined exposure
would exceed the human exposure threshold for class II (540 µg per person
per day). More than 98% of the total combined estimated exposure in Europe
(1520 µg/kg bw per day) is accounted for by allyl isothiocyanate (No. 1560),
for which toxicity data are available that adequately support the safety of
this substance at the exposure level estimated from its use as a flavouring
agent. In the unlikely event that both agents in structural class III were to
be consumed concurrently on a daily basis, the estimated combined exposure
would not exceed the human exposure threshold for class III (90 µg per
person per day). Overall evaluation of the data indicates that combined
exposure would not raise concerns about safety.

81
 Conclusions
The Committee concluded that use of the miscellaneous nitrogen-containing
substances would not present a safety concern at the estimated daily
exposure. For 10 flavouring agents (Nos 1553–1558, 1561–1563 and 1569),
the evaluation was conditional because the estimated exposure was based
on anticipated annual volumes of production. The conclusions of the safety
evaluations of these agents will be revoked if use levels or poundage data
are not provided before December 2007. The Committee noted that the
available data on the toxicity and metabolism of these miscellaneous
nitrogen-containing substances were consistent with the results of the safety
evaluation.
A toxicological monograph summarizing the safety data on this group of
flavouring agents was prepared.

4.1.6 Epoxides

The Committee evaluated a group of nine epoxide flavouring agents,

including ethyl methylphenylglycidate (No. 1577) (Table 6). The evaluations
were conducted according to the Procedure for the Safety Evaluation of
Flavouring Agents (Annex 1, reference 131). The Committee previously
evaluated two members of the group. Ethyl 3-phenylglycidate (No. 1576)
was evaluated at the twenty-fifth meeting (Annex 1, reference 56), when no
ADI was assigned; ethyl methylphenylglycidate (No. 1577) was evaluated
at the twenty-eighth meeting, when an ADI of 0–0.5 mg/kg bw was assigned
(Annex 1, reference 66).

Five of the nine flavouring agents (Nos 1570–1572, 1574 and 1575) have
been reported to occur naturally in various foods and have been detected in
fruits (e.g. citrus fruit, currants, mango and guava), beverages (beer) and a
wide variety of spices and essential oils (e.g. scotch spearmint oil, celery
seed, cinnamon bark and leaf oil, clove stem oil, ginger, peppermint oil,
cornmint oil, pepper, thyme, hop oil, calamus, basil, rosemary, lemon balm,
sage, pimento leaf, winter savoury, angelica seed oil, German camomile
oil, and mastic gum oil).

Estimated daily per capita exposure

Annual volumes of production have been reported for six of the nine
flavouring agents in this group (Nos 1572 and 1574–1578). For the remaining
three substances (1570, 1571 and 1573), anticipated annual volumes of
production were given for their proposed use as flavouring agents. The total
reported and anticipated annual volume of production of the nine epoxides

82
 Table 6. Summary of results of safety evaluations of epoxides used or proposed for use as flavouring agents

Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the agent or are Adequate NOEL on estimated daily
exceed the its metabolites for substance or intake
threshold for endogenous? related substance?
human intake?

Structural class III

4,5-Epoxy-(E)-2-decenal 1570 188590-62-7 No NR NR See note 1 No safety concern

Europe: 0.1b (conditional)
USA: 0.2b

β-Ionone epoxide 1571 23267-57-4 No NR NR See note 1 No safety concern

Europe: 0.09b (conditional)
USA: 0.1b

trans-Carvone-5,6-oxide 1572 18383-49-8 No NR NR See note 1 No safety concern

Europe: 0.01
USA: 0.2

Epoxyoxophorone 1573 38284-11-6 No NR NR See note 1 No safety concern

Europe: 0.1b (conditional)
USA: 0.2b

83
 Table 6 (contd)

84
Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the agent or are Adequate NOEL on estimated daily
exceed the its metabolites for substance or intake
threshold for endogenous? related substance?
human intake?

Piperitenone oxide 1574 35178-55-3 No NR NR See note 1 No safety concern

Europe: 0.01
USA: 0.2

β-Caryophyllene oxide 1575 1139-30-6 No NR NR See note 1 No safety concern

Europe: 0.01
USA: 0.1

Ethyl 3-phenylglycidate 1576 121-39-1 Yes No Yes. The NOEL for the See note 2 No safety concern
Europe: 114 related compound, ethyl
USA: 96 methylphenylglycidate, is
35 mg/kg bw per day
(Dunnington et al., 1981),
which is > 17 000 times the
estimated daily intake of
ethyl 3-phenylglycidate of
2 µg/kg bw in Europe and
the USA when used as a
flavouring agent.
 Table 6 (contd)

Flavouring agent No. CAS no. and Step A3a Step A4 Step A5 Comments Conclusion based
structure Does intake Is the agent or are Adequate NOEL on estimated daily
exceed the its metabolites for substance or intake
threshold for endogenous? related substance?
human intake?

Ethyl methylphenyl- 1577 77-83-8 Yes No Yes. The NOEL is 35 mg/kg See note 3 An ADI of 0–0.5
glycidate Europe: 240 bw per day (Dunnington et al., mg/kg bw was
USA: 1840 1981), which is > 8000 and established for
> 1000 times the estimated ethyl methylphe-
daily intakes of 4 µg/kg bw in nylglycidate by the
Europe and 31 µg/kg bw in Committee at its
the USA from use as a 28th meeting
flavouring agent. (Annex 1, refe-
rence 66), which
was maintained at
the present meeting.

Ethyl methyl-para - 1578 74367-97-8 No NR NR See note 4 No safety concern

tolylglycidate Europe: 23
USA: 0.009

CAS, Chemical Abstracts Service; ND, no intake data reported; NR, not required for evaluation because consumption of the substance was determined to be of no
safety concern at Step A3 of the Procedure.
Step 1: All the agents in this group are in structural class III (Cramer et al., 1978).
Step 2: All the agents in this group are expected to be metabolized to innocuous products.

85
Table 6 (contd)
a
The threshold for human intake for structural class III is 90 µg/day. All intake values are
expressed in µg/day. The combined per capita intakes of flavouring agents in structural
class III are 377 µg per day in Europe and 1937 µg per day in the USA.
b
Intake estimate based on anticipated annual volume of production

Notes:
1. Epoxide hydrolysed via epoxide hydrolase to form vicinal diol, which forms glucuronic
acid conjugate and is eliminated in the urine,or the epoxide is directly conjugated with
glutathione by glutathione transferase and is eliminated in the urine.
2. The ester group is hydrolysed by carboxyl esterases followed by loss of carbon dioxide
and rearrangement to phenacetaldehyde.
3. The ester group is hydrolysed by carboxyl esterases followed by loss of carbon dioxide
and rearrangement to 2-phenylpropanal.
4. The ester group is hydrolysed by carboxyl esterases followed by loss of carbon dioxide
and rearrangement to para-methyl-2-phenylpropanal.

is about 2600 kg in Europe and 14 800 kg in the USA. About 95% of the
total annual reported and anticipated volume in Europe and about 99% of
that in the USA are accounted for by ethyl methylphenylglycidate (No. 1577)
and ethyl 3-phenylglycidate (No. 1576). Estimated per capita exposure to
ethyl methylphenylglycidate are 240 µg/day in Europe and 1800 µg/day in
the USA, and those of ethyl 3-phenylglycidate are 114 and 96 µg/day,
respectively. Estimated exposure to all the other flavouring agents in the
group is 0.01–23 µg/day in Europe and 0.009–0.2 µg/day in the USA. The
estimated daily per capita exposure to each agent is reported in Table 6.
Absorption, distribution, metabolism and elimination

Epoxides are characterized by an oxygen-containing three-membered ring.

The inherent strain and polarity of the C–O bond in the epoxide ring are
factors that promote its cleavage in the presence of suitable nucleophiles.
They undergo chemical hydrolysis in gastrointestinal fluids. In vivo, epoxide
hydrolase, which has been identified in the cytosol, endoplasmic reticulum
(microsomes), mitochondria and nuclei of liver and to some extent kidney
cells, catalyses epoxide ring cleavage by water to yield vicinal trans-diols.
The diols are then excreted primarily in the urine unchanged or as glucuronic
acid or sulfate conjugates. Alternatively, epoxides can be conjugated with
glutathione, mediated by glutathione S-transferase, to yield the corresponding
mercapturic acid conjugates, which also are excreted in the urine.
Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported

and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated

86
from it exceeded the exposure estimated from the anticipated volume of
production and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.
Step 1. In applying the Procedure, the Committee assigned all nine
flavouring agents (Nos 1570–1578) to structural class III.
Step 2. All the flavouring agents in this group can be predicted to be
metabolized to innocuous products. The evaluation of all the agents in this
group therefore proceeded via the A-side of the Procedure.
Step A3. Estimated daily exposure to seven of the nine flavouring agents
(Nos 1570–1575 and 1578) are below the threshold of concern for structural
class III (90 mg per day). One substance (No. 1578) is reported to be used
as a flavouring agent in Europe and the USA, and three others (Nos 1572,
1574 and 1575) are reported to be used in one region only. The remaining
three substances (Nos 1570, 1571 and 1573) are only proposed for use as
flavouring agents. According to the Procedure, the use of these seven
flavouring agents and the estimated exposureraise no safety concern;
however, less uncertain exposure estimates are needed for those flavouring
agents for which only anticipated volume data were available (Nos 1570,
1571 and 1573).

Estimated daily per capita exposure to the remaining two substances, ethyl
3-phenylglycidate (No. 1576) and ethyl methylphenylglycidate (No. 1577),
for which annual volumes of production were reported, exceed the threshold
of concern for structural class III (90 µg per day). The per capita exposure
to ethyl 3-phenylglycidate (No. 1576) is 114 µg/day in Europe and 96 µg/day
in the USA, and that of ethyl methylphenylglycidate (No. 1577) is
240 µg/day in Europe and 1840 µg/day in the USA. Accordingly, the
evaluation of these agents proceeded to step A4 of the Procedure.
Step A4. These two agents and their metabolites are not endogenous.
Accordingly, the evaluation of this agent proceeded to step A5.

Step A5. At its twenty-eighth meeting, the Committee established an ADI

of 0–0.5 mg/kg bw for ethyl methylphenylglycidate on the basis of the
results of a long-term study, in which the NOEL was 35 mg/kg bw per day.
This NOEL is more than 8000 times the estimated daily exposure of 4 µg/kg

87
bw in Europe and more than 1000 times that of 31 µg/kg bw in the USA.
This NOEL is more than 17 000 times the estimated exposure to the related
substance, ethyl 3-phenylglycidate, from its use as a flavouring agent in
Europe and in the USA (both 2 mg/kg bw per day). The Committee therefore
concluded that these flavouring agents would not present a safety concern
at estimated daily exposure levels.
The exposure considerations and other information used to evaluate the
nine epoxides according to the Procedure are summarized in Table 6.

Consideration of secondary components

One member of this group of flavouring agents, 4,5-epoxy-(E)-2-decenal
(No. 1570), has an assay value of < 95%. The secondary component, 4-5-
epoxy-(Z)-2-decenal, is expected to have the same metabolic fate as the E
isomer. It was therefore considered not to present a safety concern at the
estimated levels of exposure.
Consideration of combined exposure from use as flavouring agents

In the unlikely event that all nine flavouring agents in this group were to be
consumed concurrently on a daily basis, the estimated combined exposure
would exceed the human exposure threshold for class III (90 µg per person
per day); however, all nine agents are expected to be efficiently metabolized
at the exposure levels estimated from their use as flavouring agents.
Specifically, epoxides primarily undergo epoxide hydrolase-catalysed ring
cleavage, resulting in the production of vicinal trans-diols, which are
subsequently excreted predominantly in the urine unchanged or as glucuronic
acid or sulfate conjugates. In an alternative pathway of metabolism, epoxides
can undergo conjugation with GSH to yield the corresponding mercapturic
acid conjugates, which are also excreted in urine. Theoretically, therefore,
simultaneous consumption of the epoxides (especially trans-epoxides) at
sufficiently high concentrations could result in depletion of GSH; however,
under normal conditions, intracellular GSH concentrations (1–10 mmol/l)
can be replenished and are sufficient to detoxify the concentrations of
epoxides resulting from their use as flavouring agents. Moreover, additional
cytoprotection is provided by the hydrolytic activity of epoxide hydrolase.
Therefore, at the exposure levels resulting from use of the nine epoxides
evaluated in this group as flavouring agents and due to the constant
replenishment of GSH by biosynthesis, the combined exposure to these
flavouring agents would not present a safety concern.
Conclusions

The Committee maintained the previously established ADI of 0–0.5 mg/kg

bw for ethyl methylphenylglycidate (No. 1577). It concluded that use of the

88
 flavouring agents in this group of epoxides would not present a safety concern
at estimated exposure. For three flavouring agents (Nos 1570, 1571 and
1573), the evaluation was made conditional because estimated daily exposure
was based on anticipated annual volumes of production. The conclusions
of the safety evaluations of these three agents will be revoked if use levels
or poundage data are not provided by December 2007. The Committee noted
that the available data on the toxicity and metabolism of these epoxides are
consistent with the safety evaluation made with the Procedure.

A monograph summarizing the safety data on this group of flavouring agents

was prepared.

4.1.7 Aliphatic and aromatic amines and amides

The Committee evaluated the group of 37 aliphatic and aromatic amine and
amide flavouring agents shown in Table 7. The group comprised 13 primary
aliphatic and aromatic amines (Nos 1579–1591), five tertiary aliphatic and
aromatic amines (Nos 1610–1614), four alicyclic amines (Nos 1607–1609
and 1615), four aliphatic and alicyclic imines (Nos 1603–1606) and 11
amides (Nos 1592–1602). The evaluations were conducted according to the
Procedure for the Safety Evaluation of Flavouring Agents (Annex 1,
reference 131). None of these flavouring agents has been evaluated
previously by the Committee.
The Committee noted that the available data on one of the compounds in
the group, acetamide (No. 1592), indicated that it was clearly carcinogenic
in both mice and rats; although the mechanism of tumour formation is
unknown, the possibility of a genotoxic mechanism cannot be discounted.
The Committee considered it inappropriate for such a compound to be used
as a flavouring agent or for any other food additive purpose, and agreed that
acetamide would not be evaluated according to the Procedure.
Twenty-eight of the 36 remaining flavouring agents (Nos 1579–1591, 1593,
1598, 1600, 1603, 1604 and 1607–1615) have been reported to occur
naturally in various foods. They have been detected in apple, banana,
cabbage, carrot, lettuce, rutabaga, tomato, radish, sweet corn, potato, kale,
celery, cauliflower, beetroot, rhubarb, sauerkraut, jackfruit, truffle, pepper,
laurel, garlic, blue cheeses, Cheddar, Swiss, Camembert, Limburger,
Manchengo, provolone, Russian and Tilset cheeses, caviar, fatty fish (raw,
smoked, tinned or salted), lean fish (raw, processed or cooked), clam, squid,
shrimp, oyster, crab, scallop, beef, pork, chicken, mutton, beer, red and white
wine, sherry, sake, cider, cocoa, coffee, black and green tea, barley, oats,
popcorn, rice, and wheat and rye breads.

89
 Table 7. Summary of results of safety evaluations of aliphatic and aromatic amines and amides used or proposed for use as flavouring

90
agents

Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

Structural class I

Ethylamine 1579 75-04-7 No NR NR See note 1 No safety concern

Europe: 0.1b (conditional)
USA: 0.2 b

Propylamine 1580 107-10-8 No NR NR See note 1 No safety concern

Europe: 0.01b (conditional)
USA: 0.02b

Isopropylamine 1581 75-31-0 No NR NR See note 1 No safety concern

Europe: 0.0b (conditional)
USA: 0.02b

Butylamine 1582 109-73-9 No NR NR See note 1 No safety concern

Europe: 104
USA: 0.01

Isobutylamine 1583 78-81-9 No NR NR See note 1 No safety concern

Europe: 0.07b (conditional)
USA: 0.09b
 Table 7 (contd)

Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

sec-Butylamine 1584 13952-84-6 No NR NR See note 1 No safety concern

Europe: 2 b (conditional)
USA: 2b

Pentylamine 1585 110-58-7 No NR NR See note 1 No safety concern

Europe: 0.1b (conditional)
USA: 0.2b

2-Methylbutylamine 1586 96-15-1 No NR NR See note 1 No safety concern

Europe: 0.01b (conditional)
USA: 0.02b

Isopentylamine 1587 107-85-7 No NR NR See note 1 No safety concern

Europe: 28
USA: 0.07

Hexylamine 1588 111-26-2 No NR NR See note 1 No safety concern

Europe: 0.006 b (conditional)
USA: 0.007b

91
 Table 7 (contd)

92
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

1-Amino-2-propanol 1591 78-96-6 No NR NR See note 1 No safety concern

Europe: ND (conditional)
USA: 16b

(+/–)-N,N-Dimethyl- 1602 544714-08-1 No NR NR See notes 2 No safety concern

menthyl succinamide Europe: 71 b and 3 (conditional)
USA: 88b

Trimethylamine 1610 75-50-3 No NR NR See note 4 No safety concern

Europe: 153
USA: 70

Triethylamine 1611 121-44-8 No NR NR See note 4 No safety concern

Europe: 0.7b (conditional)
USA: 0.9b

Tripropylamine 1612 102-69-2 No NR NR See note 4 No safety concern

Europe: 0.01b (conditional)
USA: 0.02b
 Table 7 (contd)

Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

Trimethylamine oxide 1614 1184-78-7 No NR NR See note 5 No safety concern

Europe: 0.07b (conditional)
USA: 0.09b

Structural class II

Phenethylamine 1589 64-04-0 No NR NR See note 6 No safety concern

Europe: ND
USA: 0.05

2-(4-Hydroxyphenyl)- 1590 51-67-2 No NR NR See note 7 No safety concern

ethylamine Europe: 0.01b (conditional)
USA: 0.02b

Butyramide 1593 541-35-5 No NR NR See notes 2 No safety concern

Europe: 0.001b and 8 (conditional)
USA: 0.002b

1-Pyrroline 1603 5724-81-2 No NR NR See note 9 No safety concern

Europe: ND (conditional)
USA: 0.4b

93
 Table 7 (contd)

94
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

2-Acetyl-1-pyrroline 1604 99583-29-6 No NR NR See note 10 No safety concern

Europe: 0.09b (conditional)
USA: 0.1b

2-Propionylpyrroline 1605 133447-37-7 No NR NR See note 10 No safety concern

Europe: 0.1b (conditional)
USA: 0.2b

Piperidine 1607 110-89-4 No NR NR See note 11 No safety concern

Europe: 103
USA: 96

2-Methylpiperidine 1608 109-05-7 No NR NR See note 11 No safety concern

Europe: 0.001b (conditional)
USA: 0.002b

Pyrrolidine 1609 123-75-1 No NR NR See note 11 No safety concern

Europe: 0.2
USA: 2
 Table 7 (contd)

Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

Piperazine 1615 110-85-0 No NR NR See note 11 No safety concern

Europe: 0.001 b (conditional)
USA: 0.002b

Structural class III

1,6-Hexalactam 1594 105-60-2 No Yes. The NOEL of See notes 2 No safety concern
Europe: 0.001 b 750 mg/kg bw per day and 8 (conditional)
USA: 0.002b (National Toxicology
Program, 1982) is at
least 2.5 x 1010 times
the estimated daily
intake of 0.00002
µg/kg bw in Europe
and 0.00003 µg/kg bw
in the USA) from its
proposed use as a
flavouring agent.

95
 Table 7 (contd)

96
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?
2-Isopropyl-N,2,3- 1595 51115-67-4 Yes Yes. There is a 14-day See notes 2 No safety concern
trimethylbutyramide Europe: ND study in rats (Nixon & and 8 (conditional)
USA: 1054b Alden, 1978) and two
14-week studies in rats
(Pence, 1980a; Cheng,
1982), as well as a study
of reproduction and tera-
togenicity in rats (Pence,
1980b). The NOEL of 5
mg/kg bw per day in
these studies is 280
times the estimated daily
intake of 18 µg/kg bw from
its proposed use as a
flavouring agent in the USA.
N-Ethyl (E)-2,(Z)-6- 1596 608514-56-3 No Yes. The NOEL of 572 See notes 2 No safety concern
nonadienamide Europe: ND mg/kg bw per day for the and 8 (conditional)
USA: 88b structurally related sub-
stance N-isobutyl-2,6,8-
decatrienamide is 600 000
times the estimated daily
intake of N-ethyl(E)-2,(Z)-
6-nonadienamide of 1 µg/
kg bw from its proposed
use as a flavouring agent
in the USA.
 Table 7 (contd)
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?
N-Cyclopropyl (E)-2, 1597 608514-55-2 No Yes. The NOEL of 572 See notes 2 No safety concern
(Z)-6-nonadienamide Europe: ND mg/kg bw per day for the and 8 (conditional)
USA: 40b structurally related sub-
stance N-isobutyl-2,6,8-
decatrienamide is
> 800 000 times the esti-
mated daily intake of N-
cyclopropyl-(E)-2,(Z)-6-
nonadienamide of 0.7
µg/kg bw from its propo-
sed use as a flavouring
agent in the USA.

N-Isobutyl (E,E)-2,4- 1598 18836-52-7 No Yes. The NOEL of 572 See notes 2 No safety concern
decadienamide Europe: 67 b mg/kg bw per day for the and 8 (conditional)
USA: 83b structurally related sub-
stance N-isobutyl-2,6,8-
decatrienamide is at least
600 000 times the esti-
mated daily intake of N-
isobutyl-(E,E)-2,4-deca-
dienamide of 1 µg/kg bw
from its proposed use as
a flavouring agent in both
Europe and the USA.

97
 Table 7 (contd)

98
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

Nonanoyl 4-hydroxy- 1599 2444-46-4 No Yes. The NOEL of 8.4 See note 12 No safety concern
3-methoxybenzylamide Europe: 7 mg/kg bw per day
USA: 0.07b (Posternak et al., 1969)
is at least 70 000 times
the estimated daily intake
from its reported use as a
flavouring agent in Europe
(0.12 µg/kg bw) and
8 400 000 times that in
the USA (0.001 µg/kg bw).

Piperine 1600 94-62-2 No Yes. The NOEL of 20 See note 13 No safety concern
Europe: 23 mg/kg bw per day (Bhat
USA: 0.07 & Chandrasekhara, 1986b)
is 50 000 times the estima-
ted daily intake from its
reported use as a flavouring
agent in Europe (0.4 µg/kg
bw) and 20 000 000 times
that in the USA (0.001
µg/kg bw).
 Table 7 (contd)

Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?

N-Ethyl-2-isopropyl- 1601 39711-79-0 Yes Yes. There is a 28-day See notes No safety concern
5-methylcyclohexane- Europe: 0.5 (Miyata, 1995) and a 22- 2 and 8
carboxamide USA: 127 week study in rats (Hunter
et al., 1975) and a 28-day
and a 52-week study in
dogs (James, 1974). The
NOEL of 8 mg/kg bw per
day in the studies in rats
(Miyata, 1995) is 1 000 000
times the estimated daily
intake of N-ethyl 2-isopropyl-
5-methylcyclohexanecar-
boxamide from its reported
use as a flavouring agent in
Europe (0.008 µg/kg bw)
and 4000 times that in the
USA (2 µg/kg bw).

99
 Table 7 (contd)

100
Flavouring agent No. CAS No. and Step A3/B3a Step B4 Are additional data Comments Conclusion based
structure Does the estimated Adequate margin of available to perform on estimated daily
intake exceed the safety for the a safety evaluation intake
threshold for flavouring agent for substances with
human intake? or related substance? an estimated intake
exceeding the
threshold of concern?
Isopentylidene iso- 1606 35448-31-8 No Yes. The NOEL of 115 See note 14 No safety concern
pentylamine Europe: 0.009 b mg/kg bw per day for the (conditional)
USA: 0.01b related substance sec-butyl-
amine (No. 1584) (Gage,
1970) is at least 5.75 x 108
times the estimated daily
intake of isopentylidene
isopentylamine from its
proposed use as a flavouring
agent in Europe (0.0001 µg/
kg bw) and in the USA
(0.0002 µg/kg bw).

N, N-Dimethylphen- 1613 19342-01-9 No Yes. The NOEL of 247 ppm See note 4 No safety concern
ethylamine Europe: 0.0b by inhalation (equivalent to (conditional)
USA: 0.09b 157 mg/kg bw per day) for
the related substance phen-
ethyl alcohol (No. 987)
(Lynch et al., 1990) is at
least 1 x 108 times the
estimated daily intake of N,N-
dimethylphenethylamine from
its proposed use as a
flavouring agent in Europe
and the USA (0.001 µg/kg bw).
 Table 7 (contd)
CAS, Chemical Abstracts Service; ND, no intake data reported; NR, not required for evaluation because consumption of the substance was deter-
mined to be of no safety concern at Step A3 of the Procedure.
Step 1: Sixteen flavouring agents in this group are in structural class I, 11 are in structural class II and 10 are in structural class III (Cramer et al.,
1978).
Step 2: Twenty-seven of the agents in this group (Nos 1579–1593, 1602–1605, 1607–1612, 1614 and 1615) are expected to be metabolized to
innocuous products. The remaining 10 agents (Nos 1594-1601, 1606 and 1613) are not expected to be metabolized to innocuous agents.
a
The thresholds for human intake of structural classes I, II and III are 1800, 540 and 90 µg/person per day, respectively. All intake values are
expressed in µg/person per day. The combined per capita intakes of the flavouring agents in structural class I is 359 µg/person per day in Europe
and 178 µg/person per day in the USA, that of the flavouring agents in structural class II is 103 µg/person per day in Europe and 99 µg/person per
day in the USA, and that of the flavouring agents in structural class III is 98 µg/person per day in Europe and 1392 µg per day in the USA.
b
Intake estimate based on anticipated annual volume of production
Notes:
1. Aliphatic primary amines readily undergo oxidative deamination, and the resulting aldehydes and ketones enter existing pathways of metabolism
and excretion.
2. Amides undergo limited hydrolysis with the corresponding ammonium ion or amines and enter known pathways of metabolism and excretion.
3. Anticipated to undergo hydrolysis at the ester moiety, followed by conjugate formation and subsequent elimination in the urine.
4. Tertiary amines primarily undergo N-oxidation to form the corresponding N-oxide, which is readily excreted in the urine.
5. Trimethylamine oxide is expected to be readily excreted in the urine.
6. Phenethylamine undergoes oxidative deamination and further oxidation to form phenylacetic acid, which is readily excreted in the urine in
conjugate form.
7. Tyramine undergoes rapid deamination by monoamine oxidase and is excreted as acidic metabolites.
8. Amides are expected to undergo oxidation and enter known pathways of metabolism.
9. Pyrroline, an imine, is anticipated to undergo hydrolysis to the corresponding iminoketone, which will be reduced to the corresponding alcohol.
10. The ketone moiety can be anticipated to be reduced to the corresponding alcohol, which will form glucuronic acid conjugates, which are excreted
in the urine.
11. Alicyclic amines undergo both N- and C-oxidation, followed by excretion of the polar metabolites in the urine.
12. This phenolic substance is anticipated readily to form glucuronic acid conjugates, which are excreted in the urine.
13. Hydrolysis of the amide group of piperine and subsequent oxidation of metabolites to form conjugates of piperonylic acid and vanillic acid are
expected.
14. This imine is expected to undergo hydrolysis to form isoamylamine and isoamyl aldehyde, which will enter known pathways of metabolism and
excretion.

101
Estimated daily per capita exposure
Annual volumes of production were reported for nine of the 36 flavouring
agents in this group (Nos 1582, 1587, 1589, 1599–1601, 1607, 1609 and
1610). For the remaining 27 substances, anticipated annual volumes were
given for their proposed use as flavouring agents. The total reported and
anticipated annual volume of the 36 aliphatic and aromatic amines and
amides is about 3900 kg in Europe and 9900 kg in the USA. About 64% of
the total reported and anticipated annual volume in Europe is accounted for
by butylamine (No. 1582), piperidine (No. 1607) and trimethylamine (No.
1610), and about 78% in the USA is accounted for by 2-isopropyl-N-2,3-
trimethylbutyramide (No. 1595), N-ethyl-2-isopropyl-5-methylcyclohexane
carboxamide (No. 1601) and piperidine (No. 1607). Estimated per capita
exposure in Europe to butylamine, piperidine and trimethylamine are 104,
103 and 153 µg/day, respectively. Estimated per capita exposure in the
USA to 2-isopropyl-N,2,3-trimethylbutyramide, N-ethyl-2-isopropyl-5-
methyl-cyclohexane carboxamide and piperidine is 1054, 127 and 96 µg
per person per day, respectively. Estimated per capita exposure to all the
other flavouring agents in the group is 0.001–71 µg/day in Europe and
0.002–88 µg/day in the USA, most of the values being at the lower end of
the ranges. The estimated per capita exposure to each agent is reported in
Table 7.
Absorption, distribution, metabolism and elimination

A number of the amines in this group are endogenous and have been
identified as normal constituents of urine from healthy individuals, as a
result of the catabolism of sarcosine, creatine and choline. These include
trimethylamine (No. 1610), ethylamine (No. 1579), isopentylamine (No.
1587), piperidine (No. 1607), pyrrolidine (No. 1609), phenethylamine (No.
1589) and trimethylamine oxide (No. 1614).
Aliphatic amines are metabolized primarily by flavin-containing
monooxygenases, monoamine oxidases or amine oxidases by a process
known as oxidative deamination. The initial step is hydroxylation of the
carbon adjacent to the nitrogen (C-oxidation), followed by formation of an
imine, with concomitant reduction of molecular oxygen to hydrogen
peroxide. The resulting imine is rapidly hydrolysed to the corresponding
aldehyde, which is oxidized to the corresponding carboxylic acid.
Representative primary aliphatic and aromatic amines in this group are
readily absorbed and are rapidly metabolized to carboxylic acids, which
are excreted in the urine.
Alicyclic secondary amines (Nos 1607–1609 and 1615) also undergo C-
oxidation at the α-carbon, but oxidation can also occur at other carbons on

102
the ring. The alicyclic imines in this group (Nos 1603–1606) are readily
absorbed and rapidly hydrolysed in aqueous solution to yield the
corresponding aminoaldehyde or iminoketone, both of which are further
metabolized.
Primary, secondary and tertiary amines can also undergo N-oxidation by
cytochrome P450 enzymes. Primary aliphatic amines with an accessible α-
substituted carbon atom can be N-oxidized to nitroso groups and
subsequently to oximes, which are labile and readily hydrolysed. Secondary
amines can be N-oxidized to reactive hydroxylamines, which are further
oxidized to form nitrones, which are readily hydrolysed. For tertiary amines,
N-oxidation by flavin-dependent monooxygenases is the primary route of
metabolism, resulting in the formation of stable N-oxides. Tertiary aliphatic
amines can also be metabolized by C-oxidation, leading to dealkylation
and formation of the corresponding primary and secondary amines and an
aliphatic aldehyde or ketone.

The aliphatic amides in this group are reported to undergo limited hydrolysis,
the extent of which depends partly on the chain length. They are well
absorbed and metabolized to polar metabolites, although there are limited
data on the actual metabolic routes of the amides in this group; a variety of
polar metabolites are detected in the urine of animals after an oral dose.

The available data on the aliphatic and aromatic amines in this group indicate
that they are likely to be rapidly absorbed in the gastrointestinal tract and
transformed by well-understood metabolic pathways to polar metabolites,
which are rapidly eliminated in the urine. The information on the amides in
this group is more limited.

Application of the Procedure for the Safety Evaluation of Flavouring Agents

In applying the Procedure to flavouring agents for which both a reported
and an anticipated volume of production were given, the Committee based
its evaluation on the reported volume of production if the exposure estimated
from it exceeded the exposure estimated from the anticipated volume of
production and applied no conditions to its decision on safety. If the exposure
estimated from the anticipated volume of production exceeded the exposure
estimated from the reported volume of production, the Committee based its
evaluation on the anticipated volume of production but considered its
decision on safety to be ‘conditional’, pending receipt of information on
use levels or poundage data by December 2007. In applying the Procedure
to flavouring agents for which only anticipated volumes of production were
given, the decision was likewise made conditional.
Step 1. In applying the Procedure for the Safety Evaluation of Flavouring
Agents to these flavouring agents, the Committee assigned 16 agents (Nos

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1579–1588, 1591, 1602, 1610–1612 and 1614) to structural class I, 10
flavouring agents (Nos 1589, 1590, 1593, 1603–1605, 1607–1609 and 1615)
to structural class II and the remaining 10 flavouring agents (Nos 1594–
1601, 1606 and 1613) to structural class III.
Step 2. Twenty-six flavouring agents in this group, namely all those in
structural classes I and II (Nos 1579–1591, 1593, 1602–1605, 1607–1612,
1614 and 1615), are predicted to be metabolized to innocuous products.
The evaluation of these agents therefore proceeded via the A-side of the
Procedure. For the 10 flavouring agents in structural class III, namely the
medium chain saturated and unsaturated aliphatic and alicyclic amides (Nos
1594–1601 and 1606) and N,N-dimethylphenethylamine (No. 1613), limited
metabolic data were available, and evaluation of these agents therefore
proceeded via the B-side of the Procedure.
Step A3. Estimated daily per capita exposure to all 16 flavouring agents in
structural class I are below the threshold of concern (1800 µg/day for class
I). Three of these 16 substances (Nos 1582, 1587 and 1610) are reported to
be used as flavouring agents, and, according to the Procedure, use of these
three agents and estimated current exposure raise no safety concern. The
other 13 substances (Nos 1579–1581, 1583–1586, 1588–1602 and 1611–
1614) are proposed for use as flavouring agents. Although, according to the
Procedure, use of these 13 flavouring agents raises no safety concern at the
exposure levels estimated from anticipated volumes of production, less
uncertain estimates are needed. Estimated daily per capita exposure to all
10 flavouring agents in structural class II are below the threshold of concern
(540 µg/day). Three of these 10 substances (Nos 1589, 1607 and 1609) are
reported to be used as flavouring agents, and, according to the Procedure,
their use raises no safety concern at estimated current exposure. The other
seven substances (Nos 1590, 1593, 1603–1605, 1608 and 1615) are proposed
for use as flavouring agents. Although, according to the Procedure, use of
these seven agents raises no safety concern at the exposure levels estimated
from anticipated volumes of production, less uncertain exposure estimates
are needed.

Step B3. Estimated per capita exposure to eight of the flavouring agents in
structural class III (Nos 1594, 1596–1600, 1606 and 1613) are below the
threshold of concern (90 µg/day). One of these substances (No. 1600) is
reported to be used as a flavouring agent in Europe and the USA, one (No.
1599) is reported to be used in Europe and to be proposed for use in the
USA, and six (Nos 1594, 1596–1598, 1606 and 1613) are proposed for use
in both regions. For those seven substances proposed for use in flavours in
one or more region (Nos 1594, 1596–1599, 1606 and 1613), less uncertain
exposure estimates are needed. In accordance with the Procedure, evaluation

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of these eight flavouring agents proceeded to Step B4. Per capita exposure
in the USA of the two remaining flavouring agents in structural class III, 2-
isopropyl-N-2,3-trimethylbutyramide (No. 1595; exposure, 1054 µg/day)
and N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide (No. 1601;
exposure, 127 µg/day), exceed the threshold of concern for their class. In
accordance with the Procedure, data must be available on these substances
or closely related substances for an evaluation of safety. For No. 1595, which
is proposed for use as a flavouring agent, a less uncertain exposure estimate
is needed.
Step B4. The NOEL of 750 mg/kg bw per day for 1,6-hexalactam (No.
1594) is at least 2.5 x 1010 times higher than the estimated exposure from its
proposed use as a flavouring agent in Europe (0.00002 µg/kg bw per day)
and in the USA (0.00003 µg/kg bw per day).

The NOEL of 572 mg/kg bw per day for the structurally related substance,
N-isobutyl-2,6,8-decatrienamide, is applicable to N-ethyl(E)-2,(Z)-6-
nonadienamide (No. 1596), to N-cyclopropyl(E)-2,(Z)-6-nonadienamide
(No. 1597) and to N-isobutyl(E,E)-2,4-decadienamide (No. 1598), as they
follow similar pathways of metabolism. This NOEL is 600 000 times the
estimated exposure to N-ethyl(E)-2,(Z)-6-nonadienamide (No. 1596) from
its proposed use as a flavouring agent in the USA (1 µg/kg bw per day) and
is more than 800 000 times the estimated exposure to N-cyclopropyl(E)-
2,(Z)-6-nonadienamide (No. 1597) from its proposed use as flavouring agent
in the USA (0.7 µg/kg bw per day) and at least 600 000 times the estimated
exposure to N-isobutyl(E,E)-2,4-decadienamide (No. 1598) from its
proposed use as flavouring agent in Europe and in the USA (both 1 µg/kg
bw per day).
The NOEL of 8.4 mg/kg bw per day for nonanoyl 4-hydroxy-3-methoxy-
benzylamide (No. 1599) is 70 000 times the estimated exposure from its
proposed use as a flavouring agent in Europe (0.12 µg/kg bw per day) and
8 400 000 times that in the USA (0.001 µg/kg bw per day).

The NOEL of 20 mg/kg bw per day for piperine (No. 1600) is 50 000 times
the estimated exposure to piperine from its reported use as a flavouring
agent in Europe (0.4 µg/kg bw per day) and 20 000 000 times that in the
USA (0.001 µg/kg bw per day).
The NOEL of 115 mg/kg bw per day for the structurally related substance
sec-butylamine (No. 1584) is applicable to isopentylidene isopentylamine
(No. 1606) and is at least 5.75 × 108 times the estimated exposure to
isopentylidene isopentylamine from its proposed use as flavouring agent in
Europe (0.0001 µg/kg bw per day) and in the USA (0.0002 µg/kg bw per
day).

105
The NOEL of 247 ppm in a study in rats treated by inhalation (equivalent to
an oral dose of 157 mg/kg bw per day) for the structurally related substance
triethylamine (No. 1611) is applicable to N,N-dimethylphenethylamine (No.
1613) and is at least 1 x 10 8 times the estimated exposure to N,N-
dimethylphenethylamine from its proposed use as flavouring agent in Europe
(0.001 µg/kg bw per day) and in the USA (0.001 µg/kg bw per day).
The Committee concluded that the margin between the estimated current
exposure to piperine (No. 1600), which is reported to be used as a flavouring
agent, and the NOEL for this agent was adequate, and its use would not
present a safety concern. The Committee also concluded that the margins
between estimated exposure to the other seven substances proposed for use
as flavouring agents in one or more regions (Nos 1594, 1596–1599, 1606
and 1613) based on the anticipated annual volumes of production, and the
NOELs for these agents were adequate. Although their use would raise no
safety concern at estimated exposure levels, less uncertain exposure estimates
are needed.
Consideration of flavouring agents with high exposure evaluated on the B-
side of the Procedure

In accordance with the Procedure, more data on toxicity were considered to

evaluate the safety of 2-isopropyl-N-2,3-trimethylbutyramide (No. 1595)
and N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide (No. 1601), as
the estimated exposure levels from proposed use (No. 1595) and reported
use (No. 1601) as flavouring agents were determined to exceed the threshold
of concern for structural class III (90 µg per person per day).
The results of three studies in Sprague-Dawley (CD®) rats treated by gavage
were available on 2-isopropyl-N-2,3-trimethylbutyramide: a 14-day study
in groups of six rats of each sex at a dose of 0, 5, 25 or 50 mg/kg bw in corn
oil twice daily; a 14-week study in groups of 30 rats of each sex at a dose of
0, 10, 50 or 100 mg/kg bw in corn oil once daily; and a 14-week study in
groups of 30 rats of each sex at a dose of 0, 1, 2, 5, 10 or 50 mg/kg bw in
corn oil once daily. The studies showed treatment-related hepatic and renal
toxicity at doses of 10 mg/kg bw and higher. The NOEL was 5 mg/kg bw
per day, on the basis of histopathological lesions in the kidneys of male rats
in the 14-week study. A study of reproductive and teratogenic toxicity in
rats at a dose of 0, 10, 50 or 100 mg/kg bw showed no reproductive effects
or fetal abnormalities. The NOEL of 5 mg/kg bw per day is 280 times the
estimated daily exposure to 2-isopropyl-N-2,3-trimethylbutyramide when
used as a flavouring agent in the USA (18 µg/kg bw per day).
Two studies were conducted on N-ethyl-2-isopropyl-5-methylcyclohexane-
carboxamide in rats treated by gavage: a 28-day study in groups of six

106
Crj:CD(SD) rats of each sex at a dose of 0, 8, 40, 200 or 1000 mg/kg bw per
day and a 22-week study in groups of 15 Sprague-Dawley (CFY) rats of
each sex at a dose of 0, 100, 300 or 725 mg/kg bw per day. Mild toxicity in
the liver and kidneys was observed at doses of 40 mg/kg bw and above.
Two further studies were conducted in beagle dogs given gelatine capsules:
a 28-day study in groups of one male and one female given a dose of 0, 600,
1000 or 1500 mg/kg bw per day and a 52-week study in groups of three
animals of each sex given a dose of 0, 100, 300 or 1000 mg/kg bw per day.
These studies showed mild toxic effects in the liver at all doses. The NOEL
of 8 mg/kg bw per day in these studies is 1 000 000 times the estimated
daily exposure to N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide
when used as a flavouring agent in Europe (0.008 µg/kg bw per day) and
4000 times that in the USA (2 µg/kg bw per day).

The additional toxicity data indicate that 2-isopropyl-N-2,3-trimethylbutyra-

mide (No. 1595) and N-ethyl-2-isopropyl-5-methylcyclohexanecarboxamide
(No. 1601) would not be expected to raise safety concerns at their estimated
levels of exposure when used as flavouring agents. For one of these agents
(No. 1595), however, less uncertain exposure estimates are needed, as the
existing estimate was based on anticipated poundage.
The stepwise evaluation of the 36 aliphatic and aromatic amines and amides
evaluated according to the Procedure is summarized in Table 7.
Consideration of secondary components

One member of this group of flavouring agents, isopentylidene isopentyl-

amine (No. 1606), has an assay value of < 95%. One of its secondary
components, 3-methylbutyraldehyde (No. 258), was evaluated by the
Committee at its forty-ninth meeting (Annex 1, reference 131) and
considered to be of no concern at estimated levels of exposure. The other
secondary component, diisopentylamine, has not been evaluated by the
Committee; however, it is structurally related to the primary and secondary
amines that were evaluated in this group of flavouring agents and is expected
to have the same metabolic fate. These amines are primarily oxidized to
imines by flavin-containing monooxygenases, monoamine oxidases or amine
oxidases, and the resulting imine can be further oxidized to produce the
corresponding aldehyde and ammonia. Moreover, the NOELs for the
structurally related compounds piperidine (No. 1607) and trimethylamine
(No. 1610) are 80 and 160 mg/kg bw per day, respectively. On this basis,
diisopentylamine was considered not to present a safety concern at estimated
levels of exposure. The Committee also concluded that the flavouring agent
as specified would not present a safety concern at the estimated levels of
exposure.

107
 Consideration of combined exposure from use as flavouring agents
In the unlikely event that all 16 agents in structural class I were to be
consumed concurrently on a daily basis, the estimated combined exposure
would not exceed the human exposure threshold for class I (1800 µg per
person per day). Likewise, in the unlikely event that all 10 agents in structural
class II were to be consumed concurrently on a daily basis, the estimated
combined exposure would not exceed the human exposure threshold for
class II (540 µg per person per day). In the unlikely event that all 10 agents
in structural class III were to be consumed concurrently on a daily basis, the
estimated combined exposure would exceed the human exposure threshold
for class III (90 µg per person per day); however, the toxicity data available
for these substances adequately supports their safety at the exposure levels
estimated from their use as flavouring agents. Overall evaluation of the
data indicates that combined exposure would not raise safety concerns.
Conclusions

On the basis of the available data on the toxicity of acetamide (No. 1592),
the Committee concluded that its use as a flavouring agent or for any other
food additive purpose would be inappropriate, and it was therefore not
evaluated by the Procedure.
The Committee concluded that use of the remaining 36 flavouring agents in
this group of aliphatic and aromatic amines and amides would not present a
safety concern at the estimated exposure levels. For 27 flavouring agents
(Nos 1579–1581, 1583–1586, 1588, 1590–1591, 1593–1598, 1602–1606,
1608 and 1611–1615), the evaluation was conditional because the exposure
was estimated on the basis of an anticipated annual volume of production.
The conclusions of the safety evaluations of these 27 flavouring agents will
be revoked if use levels or poundage data are not provided before December
2007. The Committee noted that the available data on the toxicity and
metabolism of these aliphatic and aromatic amines and amides were
consistent with the results of the safety evaluations.

A monograph summarizing the safety data on this group of flavouring agents

was prepared.

4.2 Specifications of identity and purity for flavouring agents

4.2.1 Specifications for flavouring agents evaluated for the first time

The Committee reviewed the specifications of 135 substances submitted

for evaluation (see Annex 2).

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 The Committee agreed that acetamide (No. 1592) should not be added to
food and decided not to include specifications for this substance. The
Committee thus prepared specifications for 134 substances.

The safety evaluations of 53 flavouring agents were made ‘conditional’,

and this decision is noted in the table of specifications for these agents.

Forty flavouring agents in the furan group (Nos 1487–1526) were not fully
evaluated, pending further toxicological information. Nevertheless, the
Committee decided to retain the specifications as a basis for future safety
evaluations of these substances.
Specifications for three flavouring agents, maltyl isobutyrate (No. 1482),
3-acetyl-2,5-dimethylfuran (No. 1506) and 2,4,5-trimethyl-∆-3-oxazoline
(No. 1559), were designated ‘tentative’, pending receipt of additional data.
New specifications were prepared for 131 flavouring agents (see Annex 2).
The Committee noted that seven agents had been evaluated previously as
food additives and had food additive specifications. The Committee had
already agreed that substances used as flavouring agents should comply
with existing food additive specifications. Two of the substances, maltol
(No. 1480) and ethyl maltol (No. 1481), were believed to have uses in
addition to flavouring agents. Therefore, new specifications were prepared
in the format for flavouring agents, and the existing food additive
specifications were revised (see section 2.3.6). Five of the substances,
eugenol (No. 1529), methyl anthranilate (No. 1534), methyl N-
methylanthranilate (No. 1545), ethyl 3-phenylglycidate (No. 1576) and ethyl
methylphenylglycidate (No. 1577), have no functional uses other than as
flavouring agents; therefore, the Committee decided that the specifications
presented in flavouring agent format should replace existing food additive
specifications (see section 2.3.5).

4.2.2 Revision of existing specifications for flavouring agents

The existing ‘tentative’ specifications for four flavouring agents, sodium 3-

methyl-2-oxobutanoate (No. 631.2), sodium 3-methyl-2-oxopentanoate (No.
632.2), sodium 4-methyl-2-oxopentanoate (No. 633.2) and sodium 2-oxo-
3-phenylpropionate (No. 1479), were reviewed and revised to include new
information on methods of assay. The ‘tentative’ designations of the
specifications were nevertheless maintained, pending more detailed
information on these methods.

109
5. Recommendations
1. 2006 will mark the fiftieth anniversary of the first meeting of the
Committee. In recognition of this event, the Committee recommended
that FAO and WHO take special note and consider plans for
acknowledging this milestone during the 2006 meeting of the
Committee.
2. The Committee recommended that all the chapters of the General
principles and methods for the risk assessment of chemicals in food
and the comments received during public review undergo external peer
review before the principles and methods are considered and applied
by JECFA.
3. The Committee reaffirmed use of the ‘threshold of toxicological
concern’ approach for flavouring agents. It recommended that guidance
be drawn up on application of the approach with regard to substances
present in the diet in small amounts, such as certain residues of
processing aids, packaging materials and contaminants, to provide
advice on the risk assessment of substances for which full toxicological
datasets are not available or are unnecessary. The Committee
recommended that such guidance be developed by a special task group
appointed by the Joint FAO and WHO secretaries and incorporated
into the General principles and methods for the risk assessment of
chemicals in food.

4. The General specifications and considerations for enzyme preparations

used in food processing were revised by the Committee at its fifty-
seventh meeting (Annex 1, reference 154) and published in FAO Food
and Nutrition Paper 52, Addendum 9 (Annex 1, reference 156). Over
the past few years, sections of these general specifications have become
out of date. The Committee recommended that the document be revised
at the next meeting.

5. The Committee noted that the current specifications and safety

evaluation for hexanes are not appropriate for the article of commerce
and recommended that they be re-evaluated at a future meeting.

6. The Committee recommended that the tentative general method for

determining residual solvents by gas chromatography be revised to
include more solvents, as part of a general review of the methods of
analysis of solvents used in the preparation of food additives, during
further revision of Guide to specifications—general notices, general
analytical techniques, identification tests, test solutions and other
reference materials(3).

110
 7. To address concerns raised at the fifty-fifth meeting (Annex 1, reference
149), at a recent FAO/WHO workshop on dietary exposure assessm-
ent (see section 2.4) and in several recent publications, the Committee
recommended that the Secretariat form a small group to consider all
relevant aspects of the use of an additional screening method based on
use levels, to complement the MSDI, the method used by JECFA for
estimating dietary exposure to flavouring agents. The Committee also
recommended that experts on intake work with the temporary advisers
during preparation of monographs.
8. The Committee recommended that data on poundage be collected
regularly for all flavouring agents, so that rolling averages of poundage
can be calculated. This information should be collected with attention
to adequate quality control.

9. The apparent discrepancy in dietary exposure to some flavouring agents

between that estimated from reported poundage and that estimated from
published use levels requires further investigation to ensure that the
safety evaluations are based on exposure estimates that reflect current
(and future) practice in the food and flavouring industries. The
Committee recommended that studies be undertaken in this area, giving
priority to substances of potential toxicological concern, for which there
is only a low margin of safety between the potential exposure level
and the NOEL in studies in experimental animals with the same
compound or a structural analogue.
10. The Committee recommended that the JECFA Secretariat ensure that
data on use levels are included in submissions from sponsors for safety
evaluation of flavouring agents, as requested in the call for data. The
Committee noted that such data were not submitted by the sponsors at
the current meeting. Subsequent submissions that do not contain this
information will not be evaluated by the Committee.
11. In view of a number of common inherited polymorphisms in folate
metabolism, the Committee recommended that the health effects of
folates be evaluated further when there is better understanding of the
role of relevant genetic polymorphisms in the population.

Acknowledgement
The Committee wishes to thank Mrs E. Heseltine, Lajarthe, Saint Léon-sur-
Vézère, France, for her assistance in the preparation of the report.

111
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9. Munro IC, Ford RA, Kennepohl E, Sprenger JG. Correlation of structural

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Food Additives and Contaminants, Rotterdam, Netherlands,
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Panel on Food Additives, Flavourings, Processing Aids and
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 Annex 1
Reports and other documents resulting from
previous meetings of the Joint FAO/WHO Expert
Committee on Food Additives

1. General principles governing the use of food additives (First report of the Joint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings
Report Series, No. 15, 1957; WHO Technical Report Series, No. 129, 1957 (out
of print).
2. Procedures for the testing of intentional food additives to establish their safety
for use (Second report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Report Series, No. 17, 1958; WHO
Technical Report Series, No. 144, 1958 (out of print).
3. Specifications for identity and purity of food additives (antimicrobial
preservatives and antioxidants) (Third report of the Joint FAO/WHO Expert
Committee on Food Additives). These specifications were subsequently revised
and published as Specifications for identity and purity of food additives, Vol. I.
Antimicrobial preservatives and antioxidants, Rome, Food and Agriculture
Organization of the United Nations, 1962 (out of print).
4. Specifications for identity and purity of food additives (food colours) (Fourth
report of the Joint FAO/WHO Expert Committee on Food Additives). These
specifications were subsequently revised and published as Specifications for
identity and purity of food additives, Vol. II. Food colours, Rome, Food and
Agriculture Organization of the United Nations, 1963 (out of print).
5. Evaluation of the carcinogenic hazards of food additives (Fifth report of the
Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
Meetings Report Series, No. 29, 1961; WHO Technical Report Series, No. 220,
1961 (out of print).
6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Report Series, No. 31, 1962; WHO Technical Report Series,
No. 228, 1962 (out of print).
7. Specifications for the identity and purity of food additives and their toxicological
evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Series, No. 35, 1964; WHO Technical Report Series, No.
281, 1964 (out of print).
8. Specifications for the identity and purity of food additives and their toxicological
evaluation: food colours and some antimicrobials and antioxidants (Eighth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Series, No. 38, 1965; WHO Technical Report Series, No.
309, 1965 (out of print).
9. Specifications for identity and purity and toxicological evaluation of some
antimicrobials and antioxidants. FAO Nutrition Meetings Report Series, No.
38A, 1965; WHO/Food Add/24.65 (out of print).
10. Specifications for identity and purity and toxicological evaluation of food
colours. FAO Nutrition Meetings Report Series, No. 38B, 1966; WHO/Food
Add/66.25.

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 11. Specifications for the identity and purity of food additives and their toxicological
evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flour
treatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Series, No. 40, 1966;
WHO Technical Report Series, No. 339, 1966 (out of print).
12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers,
stabilizers, flour treatment agents, acids, and bases. FAO Nutrition Meetings
Report Series, No. 40A, B, C; WHO/Food Add/67.29.
13. Specifications for the identity and purity of food additives and their toxicological
evaluation: some emulsifiers and stabilizers and certain other substances (Tenth
report of the Joint FAO/WHO Expert Committee on Food Additives). FAO
Nutrition Meetings Series, No. 43, 1967; WHO Technical Report Series, No.
373, 1967.
14. Specifications for the identity and purity of food additives and their toxicological
evaluation: some flavouring substances and non nutritive sweetening agents
(Eleventh report of the Joint FAO/WHO Expert Committee on Food Additives).
FAO Nutrition Meetings Series, No. 44, 1968; WHO Technical Report Series,
No. 383, 1968.
15. Toxicological evaluation of some flavouring substances and non nutritive
sweetening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968;
WHO/Food Add/68.33.
16. Specifications and criteria for identity and purity of some flavouring substances
and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series,
No. 44B, 1969; WHO/Food Add/69.31.
17. Specifications for the identity and purity of food additives and their toxicological
evaluation: some antibiotics (Twelfth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Series, No. 45, 1969;
WHO Technical Report Series, No. 430, 1969.
18. Specifications for the identity and purity of some antibiotics. FAO Nutrition
Meetings Series, No. 45A, 1969; WHO/Food Add/69.34.
19. Specifications for the identity and purity of food additives and their toxicological
evaluation: some food colours, emulsifiers, stabilizers, anticaking agents, and
certain other substances (Thirteenth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970;
WHO Technical Report Series, No. 445, 1970.
20. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking
agents, and certain other substances. FAO Nutrition Meetings Report Series,
No. 46A, 1970; WHO/Food Add/70.36.
21. Specifications for the identity and purity of some food colours, emulsifiers,
stabilizers, anticaking agents, and certain other food additives. FAO Nutrition
Meetings Report Series, No. 46B, 1970; WHO/Food Add/70.37.
22. Evaluation of food additives: specifications for the identity and purity of food
additives and their toxicological evaluation: some extraction solvents and certain
other substances; and a review of the technological efficacy of some
antimicrobial agents. (Fourteenth report of the Joint FAO/WHO Expert
Committee on Food Additives). FAO Nutrition Meetings Series, No. 48, 1971;
WHO Technical Report Series, No. 462, 1971.
23. Toxicological evaluation of some extraction solvents and certain other
substances. FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food
Add/70.39.
24. Specifications for the identity and purity of some extraction solvents and certain
other substances. FAO Nutrition Meetings Report Series, No. 48B, 1971; WHO/
Food Add/70.40.

116
25. A review of the technological efficacy of some antimicrobial agents. FAO
Nutrition Meetings Report Series, No. 48C, 1971; WHO/Food Add/70.41.
26. Evaluation of food additives: some enzymes, modified starches, and certain
other substances: Toxicological evaluations and specifications and a review of
the technological efficacy of some antioxidants (Fifteenth report of the Joint
FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings
Series, No. 50, 1972; WHO Technical Report Series, No. 488, 1972.
27. Toxicological evaluation of some enzymes, modified starches, and certain other
substances. FAO Nutrition Meetings Report Series, No. 50A, 1972; WHO Food
Additives Series, No. 1, 1972.
28. Specifications for the identity and purity of some enzymes and certain other
substances. FAO Nutrition Meetings Report Series, No. 50B, 1972; WHO Food
Additives Series, No. 2, 1972.
29. A review of the technological efficacy of some antioxidants and synergists. FAO
Nutrition Meetings Report Series, No. 50C, 1972; WHO Food Additives Series,
No. 3, 1972.
30. Evaluation of certain food additives and the contaminants mercury, lead, and
cadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on Food
Additives). FAO Nutrition Meetings Series, No. 51, 1972; WHO Technical
Report Series, No. 505, 1972, and corrigendum.
31. Evaluation of mercury, lead, cadmium and the food additives amaranth,
diethylpyrocarbamate, and octyl gallate. FAO Nutrition Meetings Report Series,
No. 51A, 1972; WHO Food Additives Series, No. 4, 1972.
32. Toxicological evaluation of certain food additives with a review of general
principles and of specifications (Seventeenth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Nutrition Meetings Series, No.
53, 1974; WHO Technical Report Series, No. 539, 1974, and corrigendum (out
of print).
33. Toxicological evaluation of some food additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers, and thickening agents. FAO Nutrition
Meetings Report Series, No. 53A, 1974; WHO Food Additives Series, No. 5,
1974.
34. Specifications for identity and purity of thickening agents, anticaking agents,
antimicrobials, antioxidants and emulsifiers. FAO Food and Nutrition Paper,
No. 4, 1978.
35. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Nutrition Meetings Series, No.
54, 1974; WHO Technical Report Series, No. 557, 1974, and corrigendum.
36. Toxicological evaluation of some food colours, enzymes, flavour enhancers,
thickening agents, and certain other food additives. FAO Nutrition Meetings
Report Series, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975.
37. Specifications for the identity and purity of some food colours, enhancers,
thickening agents, and certain food additives. FAO Nutrition Meetings Report
Series, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975.
38. Evaluation of certain food additives: some food colours, thickening agents,
smoke condensates, and certain other substances. (Nineteenth report of the
Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition
Meetings Series, No. 55, 1975; WHO Technical Report Series, No. 576, 1975.
39. Toxicological evaluation of some food colours, thickening agents, and certain
other substances. FAO Nutrition Meetings Report Series, No. 55A, 1975; WHO
Food Additives Series, No. 8, 1975.

117
 40. Specifications for the identity and purity of certain food additives. FAO Nutrition
Meetings Report Series, No. 55B, 1976; WHO Food Additives Series, No. 9,
1976.
41. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO
Expert Committee on Food Additives). FAO Food and Nutrition Meetings Series,
No. 1, 1976; WHO Technical Report Series, No. 599, 1976.
42. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 10, 1976.
43. Specifications for the identity and purity of some food additives. FAO Food and
Nutrition Series, No. 1B, 1977; WHO Food Additives Series, No. 11, 1977.
44. Evaluation of certain food additives (Twenty-first report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 617,
1978.
45. Summary of toxicological data of certain food additives. WHO Food Additives
Series, No. 12, 1977.
46. Specifications for identity and purity of some food additives, including
antioxidant, food colours, thickeners, and others. FAO Nutrition Meetings Report
Series, No. 57, 1977.
47. Evaluation of certain food additives and contaminants (Twenty-second report
of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 631, 1978.
48. Summary of toxicological data of certain food additives and contaminants. WHO
Food Additives Series, No. 13, 1978.
49. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 7, 1978.
50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/
WHO Expert Committee on Food Additives). WHO Technical Report Series,
No. 648, 1980, and corrigenda.
51. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 14, 1980.
52. Specifications for identity and purity of food colours, flavouring agents, and
other food additives. FAO Food and Nutrition Paper, No. 12, 1979.
53. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/
WHO Expert Committee on Food Additives). WHO Technical Report Series,
No. 653, 1980.
54. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 15, 1980.
55. Specifications for identity and purity of food additives (sweetening agents,
emulsifying agents, and other food additives). FAO Food and Nutrition Paper,
No. 17, 1980.
56. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 669,
1981.
57. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 16, 1981.
58. Specifications for identity and purity of food additives (carrier solvents,
emulsifiers and stabilizers, enzyme preparations, flavouring agents, food colours,
sweetening agents, and other food additives). FAO Food and Nutrition Paper,
No. 19, 1981.
59. Evaluation of certain food additives and contaminants (Twenty-sixth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 683, 1982.

118
60. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 17, 1982.
61. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 25, 1982.
62. Evaluation of certain food additives and contaminants (Twenty-seventh report
of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 696, 1983, and corrigenda.
63. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 18, 1983.
64. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 28, 1983.
65. Guide to specifications General notices, general methods, identification tests,
test solutions, and other reference materials. FAO Food and Nutrition Paper,
No. 5, Rev. 1, 1983.
66. Evaluation of certain food additives and contaminants (Twenty-eighth report
of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 710, 1984, and corrigendum.
67. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 19, 1984.
68. Specifications for the identity and purity of food colours. FAO Food and Nutrition
Paper, No. 31/1, 1984.
69. Specifications for the identity and purity of food additives. FAO Food and
Nutrition Paper, No. 31/2, 1984.
70. Evaluation of certain food additives and contaminants (Twenty-ninth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 733, 1986, and corrigendum.
71. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 34, 1986.
72. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 20. Cambridge University Press, 1987.
73. Evaluation of certain food additives and contaminants (Thirtieth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 751, 1987.
74. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 21. Cambridge University Press, 1987.
75. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 37, 1986.
76. Principles for the safety assessment of food additives and contaminants in food.
WHO Environmental Health Criteria, No. 70. Geneva, World Health
Organization, 1987 (out of print). The full text is available electronically at
www.who.int/pcs.
77. Evaluation of certain food additives and contaminants (Thirty-first report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 759, 1987 and corrigendum.
78. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 22. Cambridge University Press, 1988.
79. Specifications for the identity and purity of certain food additives. FAO Food
and Nutrition Paper, No. 38, 1988.
80. Evaluation of certain veterinary drug residues in food (Thirty-second report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 763, 1988.

119
 81. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 23. Cambridge University Press, 1988.
82. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41, 1988.
83. Evaluation of certain food additives and contaminants (Thirty-third report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 776, 1989.
84. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 24. Cambridge University Press, 1989.
85. Evaluation of certain veterinary drug residues in food (Thirty-fourth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 788, 1989.
86. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 25, 1990.
87. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/2, 1990.
88. Evaluation of certain food additives and contaminants (Thirty-fifth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 789, 1990, and corrigenda.
89. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 26, 1990.
90. Specifications for identity and purity of certain food additives. FAO Food and
Nutrition Paper, No. 49, 1990.
91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 799, 1990.
92. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 27, 1991.
93. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/3, 1991.
94. Evaluation of certain food additives and contaminants (Thirty-seventh report
of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 806, 1991, and corrigenda.
95. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 28, 1991.
96. Compendium of food additive specifications (Joint FAO/WHO Expert Committee
on Food Additives (JECFA)). Combined specifications from 1st through the
37th meetings, 1956–1990. Rome, Food and Agricultural Organization of the
United Nations, 1992 (2 volumes).
97. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 815, 1991.
98. Toxicological evaluation of certain veterinary residues in food. WHO Food
Additives Series, No. 29, 1991.
99. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/4, 1991.
100. Guide to specifications—General notices, general analytical techniques,
identification tests, test solutions, and other reference materials. FAO Food
and Nutrition Paper, No. 5, Ref. 2, 1991.
101. Evaluation of certain food additives and naturally occurring toxicants (Thirty-
ninth report of the Joint FAO/WHO Expert Committee on Food Additives).
WHO Technical Report Series No. 828, 1992.

120
102. Toxicological evaluation of certain food additives and naturally occurring
toxicants. WHO Food Additive Series, No. 30, 1993.
103. Compendium of food additive specifications: addendum 1. FAO Food and
Nutrition Paper, No. 52, 1992.
104. Evaluation of certain veterinary drug residues in food (Fortieth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 832, 1993.
105. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 31, 1993.
106. Residues of some veterinary drugs in animals and food. FAO Food and Nutrition
Paper, No. 41/5, 1993.
107. Evaluation of certain food additives and contaminants (Forty-first report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 837, 1993.
108. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 32, 1993.
109. Compendium of food additive specifications: addendum 2. FAO Food and
Nutrition Paper, No. 52, Add. 2, 1993.
110. Evaluation of certain veterinary drug residues in food (Forty-second report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 851, 1995.
111. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 33, 1994.
112. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/6, 1994.
113. Evaluation of certain veterinary drug residues in food (Forty-third report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 855, 1995, and corrigendum.
114. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 34, 1995.
115. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/7, 1995.
116. Evaluation of certain food additives and contaminants (Forty-fourth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 859, 1995.
117. Toxicological evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 35, 1996.
118. Compendium of food additive specifications: addendum 3. FAO Food and
Nutrition Paper, No. 52, Add. 3, 1995.
119. Evaluation of certain veterinary drug residues in food (Forty-fifth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 864, 1996.
120. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 36, 1996.
121. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/8, 1996.
122. Evaluation of certain food additives and contaminants (Forty-sixth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 868, 1997.
123. Toxicological evaluation of certain food additives. WHO Food Additives Series,
No. 37, 1996.

121
 124. Compendium of food additive specifications, addendum 4. FAO Food and
Nutrition Paper, No. 52, Add. 4, 1996.
125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 876, 1998.
126. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 38, 1996.
127. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/9, 1997.
128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 879, 1998.
129. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 39, 1997.
130. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/10, 1998.
131. Evaluation of certain food additives and contaminants (Forty-ninth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 884, 1999.
132. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 40, 1998.
133. Compendium of food additive specifications: addendum 5. FAO Food and
Nutrition Paper, No. 52, Add. 5, 1997.
134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, No. 888, 1999.
135. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 41, 1998.
136. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/11, 1999.
137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 891,
2000.
138. Safety evaluation of certain food additives. WHO Food Additives Series, No.
42, 1999.
139. Compendium of food additive specifications, addendum 6. FAO Food and
Nutrition Paper, No. 52, Add. 6, 1998.
140. Evaluation of certain veterinary drug residues in food (Fifty-second report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 893, in press.
141. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 43, 2000
142. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/12, 2000.
143. Evaluation of certain food additives and contaminants (Fifty-third report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series, in press.
144. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 44, 2000.
145. Compendium of food additive specifications, addendum 7. FAO Food and
Nutrition Paper, No. 52, Add. 7, 1999.

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146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, in press.
147. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 45, 2000.
148. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition
Paper, No. 41/13, 2000.
149. Evaluation of certain food additives and contaminants (Fifty-fifth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report
Series No. 901, 2001.
150. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 46, 2001.
151. Compendium of food additive specifications: addendum 8. FAO Food and
Nutrition Paper, No. 52, Add. 8, 2000.
152. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/
WHO Expert Committee on Food Additives). WHO Technical Report Series
No. 906, 2002.
153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series,
No. 47/FAO Food and Nutrition Paper 74, 2001.
154. Evaluation of certain food additives and contaminants (Fifty-seventh report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 909, in press.
155. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 48, 2002.
156. Compendium of food additive specifications: addendum 9. FAO Food and
Nutrition Paper, No. 52, Add. 9, 2001.
157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series No. 911, 2002.
158. Toxicological evaluation of certain veterinary drug residues in food WHO Food
Additives Series, No. 49, 2002.
159. Residues of some veterinary drugs in animals and foods. FAO Food and
Nutrition Paper, No. 41/14, 2002.
160. Evaluation of certain food additives (Fifty-ninth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series No. 913,
2002.
161. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 50, 2003.
162. Compendium of food additive specifications: addendum 10. FAO Food and
Nutrition Paper No. 52, Add. 10, 2002.
163. Evaluation of certain veterinary drug residues in food (Sixtieth report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 918, 2003.
164. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 51, 2003.
165. Residues of some veterinary drugs in animals and foods. FAO Food and
Nutrition Paper, No. 41/15, 2003.
166. Evaluation of certain food additives and contaminants (Sixty-first report of the
Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 922, 2004.
167. Safety evaluation of certain food additives and contaminants. WHO Food
Additives Series, No. 52, 2004.
168. Compendium of food additive specifications: addendum 11. FAO Food and
Nutrition Paper, No. 52, Add. 11, 2003.

123
 169. Evaluation of certain veterinary drug residues in food (Sixty-second report of
the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical
Report Series, No. 925, 2004.
170. Residues of some veterinary drugs in animals and foods. FAO Food and
Nutrition Paper, No. 41/16, 2004.
171. Toxicological evaluation of certain veterinary drug residues in food. WHO Food
Additives Series, No. 53, 2005.
172. Compendium of food additive specifications: addendum 12. FAO Food and
Nutrition Paper, No. 52, Add. 12, 2004.
173. Evaluation of certain food additives (Sixty-third report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 928,
2005.
174. Safety evaluation of certain food additives WHO Food Additives Series, No. 54,
2005.
175. Evaluation of certain food contaminants (Sixty-fourth report of the Joint FAO/
WHO Expert Committee on Food Additives). WHO Technical Report Series,
No. 930, 2005.
176. Safety evaluation of certain contaminants in food. WHO Food Additives Series,
No. 55/FAO Food and Nutrition Paper, No. 82, 2006.
177. Evaluation of certain food additives (Sixty-fifth report of the Joint FAO/WHO
Expert Committee on Food Additives). WHO Technical Report Series, No. 934
2006.
178. Compendium of food additive specifications: addendum 13. FAO Food and
Nutrition Paper, No. 52, Add.13 (in prepration).

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 Annex 2
Acceptable daily intakes, other toxicological
information and information on specifications

1. Food additives and ingredients evaluated

toxicologically or assessed for dietary exposure

Food additive Specifi- Acceptable daily intake (ADI) and other

cationsa toxicological recommendations

Beeswax R No safety concern at predicted dietary intake

(< 650 mg per person per day), based on long
history of use and lack of toxicity observed
with major components

Candelilla wax R No safety concern at predicted dietary intake

(< 650 mg per person per day)

Calcium L-5-methyltetrahydrofolate N No safety concern for proposed use in dry

crystalline or microencapsulated form as
alternative to folic acid used in dietary
supplements, foods for special dietary uses
and other foods.
Safety of folate fortification and
supplementation as such not evaluated.

Phospholipase A1 from Fusarium N Information provided too limited to assess

venenatum expressed in Aspergillus safety
oryzae

Pullulan N ADI ‘not specified’b

Quillaia extract type 1 S Previous ADI converted to an ADI based on

saponin content from the lower end of speci-
fied saponin range and established as group
ADI for quillaia extract type 1 and quillaia
extract type 2. Assessment of dietary
exposure included additional use of quillaia
extract type 1 in semi-frozen carbonated and
non-carbonated beverages (≤ 500 mg/kg
product). In a model diet approach, high-per-
centile consumption estimated to lead to
intake of 44–157% of ADI, assuming pre-
sence of quillaia extract type 1 at 295 mg/l in
all water-based flavoured drinks. In a proba-
bilistic exposure assessment and assuming
that the frequency and amount per eating
occasion are independent variables, the estima-
ted dietary exposure was below the ADI at the
90th percentile. Assuming 100% dependence
between frequency and amount consumed,
estimated that 100–700 individuals per million
in the entire population could exceed the ADI .

125
Food additive Specifi- Acceptable daily intake (ADI) and other
cationsa toxicological recommendations

Quillaia extract type 2 R Previous ADI established for quillaia extract

type 1 converted to an ADI based on saponin
content from the lower end of the specified
saponin range and established as a group ADI
for quillaia extract type 1 and type 2.
a
N: new specifications prepared; R: existing specifications revised; S: existing specifications
maintained.
b
ADI ‘not specified’ is used to refer to a food substance of very low toxicity which, on the basis of
the available data (chemical, biochemical, toxicological and other) and the total dietary intake of
the substance arising from its use at the levels necessary to achieve the desired effects and from its
acceptable background levels in food, does not, in the opinion of the Committee, represent a hazard
to health. For that reason, and for the reasons stated in the individual evaluations, the establishment
of an ADI expressed in numerical form is not deemed necessary. An additive meeting this criterion
must be used within the bounds of good manufacturing practice, i.e. it should be technologically
efficacious and should be used at the lowest level necessary to achieve this effect, it should not
conceal food of inferior quality or adulterated food, and it should not create a nutritional imbalance.

2. Food additives considered for specifications only

Food additive Specifications a

Aspartame acesulfame salt R

Hexanes See below
Laccase from Myceliophthora thermophila expressed in Aspergillus oryzae R
Monomagnesium phosphate and trisodium diphosphate Wb
Sucrose esters of fatty acids R, T

a
R: existing specifications revised; T: tentative specifications; W: existing specifications withdrawn.
b
As no information was received on these substances, the existing tentative specifications were
withdrawn.

Hexanes

As used in the food industry, ‘hexane’ is a mixture of hydrocarbons. Recent

changes in environmental regulations have led to a change in composition
of hexanes since the original specifications were established. In addition,
the composition of hexanes depends on the region of production, the source
of the raw material and the site of production. Therefore, the Committee
concluded that the present articles of commerce differ from those previously
evaluated by the Committee and that the composition of the residues and
their levels in foods may not be the same as those evaluated in the original
safety assessment. The Committee also concluded that there was insufficient
information available to change the current specifications, and therefore
recommended a re-evaluation of hexanes.

126
3. Flavouring agents evaluated with the Procedure for the
Safety Evaluation of Flavouring Agents

See also the discussion on the safety evaluation of flavouring agents in

Annexes 3 and 4.

A. Maltol and related substances

Flavouring agent No. Specifications a Conclusions based
on current intake

Maltol 1480 Nb See footnote c

Ethyl maltol 1481 Nb See footnote d
Maltyl isobutyrate 1482 N, T No safety concern
2-Methyl-3-(1-oxopropoxy)-4H-pyran-4-one 1483 N No safety concern
(conditional)e
2-Butyl-5- or 6-keto-1,4-dioxane 1484 N No safety concern
2-Amyl-5 or 6-keto-1,4-dioxane 1485 N No safety concern
2-Hexyl-5 or 6-keto-1,4-dioxane 1486 N No safety concern

a
N: new specifications prepared; T: tentative specifications.
b
Revised specifications for these substances in the standard additive format were also prepared.
c
The ADI of 0–1 mg/kg bw established at the twenty-fifth meeting was maintained.
d
The ADI of 0–2 mg/kg bw established at the eighteenth meeting was maintained.
e
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.

B. Furan-substituted aliphatic hydrocarbons, alcohols, aldehydes,

ketones, carboxylic acids and related esters, sulfides, disulfides
and ethers
The Committee took note of the extensive positive genotoxicity data for
several members of this group of flavouring agents related to furan. Furan,
which is carcinogenic, is known to undergo epoxidation and ring opening
to form a reactive 2-ene-1,4-dicarbonyl intermediate. Accordingly, concern
arises that the observed genotoxicity may be due to formation of a reactive
metabolite. Data on the potential of members of this group to form a reactive
metabolite were not available and the role of metabolism in the observed
genotoxicity has not been identified. Moreover, there was a paucity of in
vivo genotoxicity data to allay concern. Also, specific in vivo assays to
address potential carcinogenicity were lacking. Because of these concerns,
the Committee concluded that the Procedure could not be applied to this
group.

127
Flavouring agent No. Specificationsa

2-Methylfuran 1487 N
2,5-Dimethylfuran 1488 N
2-Ethylfuran 1489 N
2-Butylfuran 1490 N
2-Pentylfuran 1491 N
2-Heptylfuran 1492 N
2-Decylfuran 1493 N
3-Methyl-2-(3-methylbut-2-enyl)-furan 1494 N
2,3-Dimethylbenzofuran 1495 N
2,4-Difurfurylfuran 1496 N
3-(2-Furyl)acrolein 1497 N
2-Methyl-3(2-furyl)acrolein 1498 N
3-(5-Methyl-2-furyl)prop-2-enal 1499 N
3-(5-Methyl-2-furyl)-butanal 1500 N
2-Furfurylidenebutyraldehyde 1501 N
2-Phenyl-3-(2-furyl)prop-2-enal 1502 N
2-Furyl methyl ketone 1503 N
2-Acetyl-5-methylfuran 1504 N
2-Acetyl-3,5-dimethylfuran 1505 N
3-Acetyl-2,5-dimethylfuran 1506 N,T
2-Butyrylfuran 1507 N
(2-Furyl)-2-propanone 1508 N
2-Pentanoylfuran 1509 N
1-(2-Furyl)butan-3-one 1510 N
4-(2-Furyl)-3-buten-2-one 1511 N
Pentyl 2-furyl ketone 1512 N
Ethyl 3-(2-furyl)propanoate 1513 N
Isobutyl 3-(2-furan)propionate 1514 N
Isoamyl 3-(2-furan)propionate 1515 N
Isoamyl 4-(2-furan)butyrate 1516 N
Phenethyl 2-furoate 1517 N
Propyl 2-furanacrylate 1518 N
2,5-Dimethyl-3-oxo-(2H)-fur-4-yl butyrate 1519 N
Furfuryl methyl ether 1520 N
Ethyl furfuryl ether 1521 N
Difurfuryl ether 1522 N
2,5-Dimethyl-3-furanthiol acetate 1523 N
Furfuryl 2-methyl-3-furyl disulfide 1524 N
3-[(2-Methyl-3-furyl)thio]-2-butanone 1525 N
O-Ethyl S-(2-furylmethyl)thiocarbonate 1526 N

a
N: new specifications prepared; T: tentative specifications

128
C. Eugenol and related hydroxyallylbenzene derivatives

Flavouring agent No. Specificationsa Conclusions based

on current intake

4-Allylphenol 1527 N No safety concern

(conditional)b
2-Methoxy-6-(2-propenyl)phenol 1528 N No safety concern
(conditional)b
Eugenol 1529 Rc See footnote d
Eugenyl formate 1530 N No safety concern
Eugenyl acetate 1531 N No safety concern
Eugenyl isovalerate 1532 N No safety concern
(conditional)b
Eugenyl benzoate 1533 N No safety concern

a
N: new specifications prepared; R: existing specifications revised.
b
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.
c
As this substance is used only as a flavouring agent, the Committee considered that the existing
specifications in the standard food additive format should be deleted.
d
The ADI of 0-2.5 mg/kg bw established at the twenty-sixth meeting was maintained.

D. Anthranilate derivatives

Flavouring agent No. Specificationsa Conclusions based

on current intake

Methyl anthranilate 1534 Rb See footnote c

Ethyl anthranilate 1535 N No safety concern
Butyl anthranilate 1536 N No safety concern
Isobutyl anthranilate 1537 N No safety concern
cis-3-Hexenyl anthranilate 1538 N No safety concern
(conditional)d
Citronellyl anthranilate 1539 N No safety concern
(conditional)d
Linalyl anthranilate 1540 N No safety concern
Cyclohexyl anthranilate 1541 N No safety concern
β-Terpinyl anthranilate 1542 N No safety concern
Phenylethyl anthranilate 1543 N No safety concern
β-Naphthyl anthranilate 1544 N No safety concern
Methyl N-methylanthranilate 1545 Rb See footnote e
Ethyl N-methylanthranilate 1546 N No safety concern
(conditional)d
Ethyl N-ethylanthranilate 1547 N No safety concern
(conditional)d
Isobutyl N-methylanthranilate 1548 N No safety concern
(conditional)d
Methyl N-formylanthranilate 1549 N No safety concern
(conditional)d
Methyl N-acetylanthranilate 1550 N No safety concern
(conditional)d

129
Flavouring agent No. Specificationsa Conclusions based
on current intake

Methyl N,N-dimethylanthranilate 1551 N No safety concern

(conditional)d
N-Benzoylanthranilic acid 1552 N No safety concern
(conditional)d

a
N: new specifications prepared; R: existing specifications revised.
b
As this substance is used only as a flavouring agent, the Committee decided that the existing
specifications in the standard food additive format should be deleted.
c
The ADI of 0–1.5 mg/kg bw established at the twenty-third meeting was maintained.
d
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.
e
The ADI of 0–0.2 mg/kg bw established at the twenty-third meeting was maintained.

E. Miscellaneous nitrogen-containing substances

Flavouring agent No. Specificationsa Conclusions based
on current intake

Trimethyloxazole 1553 N No safety concern

(conditional)b
2,5-Dimethyl-4-ethyloxazole 1554 N No safety concern
(conditional)b
2-Ethyl-4,5-dimethyloxazole 1555 N No safety concern
(conditional)b
2-Isobutyl-4,5-dimethyloxazole 1556 N No safety concern
(conditional)b
2-Methyl-4,5-benzo-oxazole 1557 N No safety concern
(conditional)b
2,4-Dimethyl-3-oxazoline 1558 N No safety concern
(conditional)b
2,4,5-Trimethyl-δ-3-oxazoline 1559 N,T No safety concern
Allyl isothiocyanate 1560 N No safety concern
Butyl isothiocyanate 1561 N No safety concern
(conditional)b
Benzyl isothiocyanate 1562 N No safety concern
(conditional)b
Phenethyl isothiocyanate 1563 N No safety concern
(conditional)b
3-Methylthiopropyl isothiocyanate 1564 N No safety concern
4-Acetyl-2-methylpyrimidine 1565 N No safety concern
5,7-Dihydro-2-methylthieno(3,4-d)pyrimidine 1566 N No safety concern
1-Phenyl-3- or -5-propylpyrazole 1568 N No safety concern
4,5-Dimethyl-2-propyloxazole 1569 N No safety concern
(conditional)b
a
N: new specifications prepared; T: tentative specifications.
b
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.

130
F. Epoxides
Flavouring agent No. Specifications a Conclusions based
on current intake

4,5-Epoxy-(E)-2-decenal 1570 N No safety concern

(conditional)b
β-Ionone epoxide 1571 N No safety concern
(conditional)b
trans-Carvone-5,6-oxide 1572 N No safety concern
Epoxyoxophorone 1573 N No safety concern
(conditional)b
Piperitenone oxide 1574 N No safety concern
β-Caryophyllene oxide 1575 N No safety concern
Ethyl 3-phenylglycidate 1576 Rc No safety concern
Ethyl methylphenylglycidate 1577 Rc See footnote d
Ethyl methyl-para-tolylglycidate 1578 N No safety concern
a
N: new specifications prepared; R: existing specifications revised.
b
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.
c
As this substance is used only as a flavouring agent, the Committee decided that the existing
specifications in the standard food additive format should be deleted.
d
The ADI of 0–0.5 mg/kg bw established at the twenty-eighth meeting. was maintained.

G. Aliphatic and aromatic amines and amides

Acetamide (No. 1592)
The Committee noted that the available data on the toxicity of this substance
indicate that it is clearly carcinogenic in both mice and rats, and, although
the mechanism of tumour formation is unknown, the possibility of a
genotoxic mechanism cannot be discounted. The Committee considered it
inappropriate for such a compound to be used as a flavouring agent or for
any other food additive purpose and agreed that acetamide would not be
evaluated according to the Procedure. No specifications were prepared.

Other substances in this group

Flavouring agent No. Specifications a Conclusions based

on current intake

Ethylamine 1579 N No safety concern

(conditional)b
Propylamine 1580 N No safety concern
(conditional)b
Isopropylamine 1581 N No safety concern
(conditional)b
Butylamine 1582 N No safety concern
Isobutylamine 1583 N No safety concern
(conditional)b

131
Flavouring agent No. Specificationsa Conclusions based
on current intake

sec-Butylamine 1584 N No safety concern

(conditional)b
Pentylamine 1585 N No safety concern
(conditional)b
2-Methylbutylamine 1586 N No safety concern
(conditional)b
Isopentylamine 1587 N No safety concern
Hexylamine 1588 N No safety concern
(conditional)b
Phenethylamine 1589 N No safety concern
2-(4-Hydroxyphenyl)ethylamine 1590 N No safety concern
(conditional)b
1-Amino-2-propanol 1591 N No safety concern
(conditional)b
Butyramide 1593 N No safety concern
(conditional)b
1,6-Hexalactam 1594 N No safety concern
(conditional)b
2-Isopropyl-N,2,3-trimethylbutyramide 1595 N No safety concern
(conditional)b
N-Ethyl (E)-2,(Z)-6-nonadienamide 1596 N No safety concern
(conditional)b
N-Cyclopropyl (E)-2,(Z)-6-nonadienamide 1597 N No safety concern
(conditional)b
N-Isobutyl (E,E)-2,4-decadienamide 1598 N No safety concern
(conditional)b
Nonanoyl 4-hydroxy-3-methoxybenzylamide 1599 N No safety concern
Piperine 1600 N No safety concern
N-Ethyl-2-isopropyl-5-methylcyclohexane 1601 N No safety concern
carboxamide
(+/–)-N,N-Dimethyl menthyl succinamide 1602 N No safety concern
(conditional)b
1-Pyrroline 1603 N No safety concern
(conditional)b
2-Acetyl-1-pyrroline 1604 N No safety concern
(conditional)b
2-Propionylpyrroline 1605 N No safety concern
(conditional)b
Isopentylidene isopentylamine 1606 N No safety concern
(conditional)b
Piperidine 1607 N No safety concern
2-Methylpiperidine 1608 N No safety concern
(conditional)b
Pyrrolidine 1609 N No safety concern
Trimethylamine 1610 N No safety concern
Triethylamine 1611 N No safety concern
(conditional)b
Tripropylamine 1612 N No safety concern
(conditional)b
N,N-Dimethylphenethylamine 1613 N No safety concern
(conditional)b

132
Flavouring agent No. Specifications a Conclusions based
on current intake

Trimethylamine oxide 1614 N No safety concern

(conditional)b
Piperazine 1615 N No safety concern
(conditional)b

a
N: new specifications prepared.
b
Evaluation conditional because the estimated daily intake is based on the anticipated annual volume
of production. The conclusion of the safety evaluation of this substance will be revoked if use
levels or poundage data are not provided before the end of 2007.

4. Flavouring agents considered for specifications only

Flavouring agent No. Specifications a

Sodium salt of 3-methyl-2-oxobutanoic acid 631.2 R,T

Sodium salt of 3-methyl-2-oxopentanoic acid 632.2 R,T
Sodium salt of 4-methyl-2-oxopentanoic acid 633.2 R,T
Sodium 2-oxo-3-phenylpropionate 1479 R,T

a
R: existing specifications revised; T: tentative specifications

133
134
 Annex 3
Further information required

1. Need for use levels and reported poundage data for

flavouring agents
The evaluations of a number of flavouring agents were made conditional
because the estimated daily intake was based on the anticipated annual
volume of production. The safety evaluation of these substances will be
revoked if use levels or poundage data are not provided before the end of
2007. The Committee also requested use levels or poundage data to be
provided for the flavouring agents it had assessed previously on the basis of
an MSDI that was calculated from anticipated poundage. These would
include any substances for which the MSDI based on anticipated poundage
for one region (European Union or USA) was higher than the MSDI based
on recorded poundage in the other region.

The existing assessments will be revoked if such data are not forthcoming
by the end of 2007.

The Committee emphasized that data on level of use are required for all
flavouring agents listed in ‘calls for data’. Subsequent submissions that do
not contain this information will not be evaluated by the Committee.
Details of all the flavouring agents for which further data are required are
given in Annex 4.

2. Information for specifications

2.1 Flavouring agents
2.1.1 Sodium 3-methyl-2-oxobutanoate (No. 631.2), sodium 3-methyl-2-
oxopentanoate (No. 632.2), sodium 4-methyl-2-oxopentanoate (No.
633.2) and sodium 2-oxo-3-phenylpropionate (No. 1479)

The existing tentative specifications for these four flavouring agents were
revised to include new information on methods of assay. Nevertheless, the
tentative designations of the specifications were maintained, pending more
detailed information on these methods. For the first three substances,
information on an assay by high-performance liquid chromatography with
an ion-exchange column are required; for flavouring agent No. 1479,
information on an assay by high-performance liquid chromatography is
required.

135
2.1.2 Maltol (No.1480) and ethyl maltol (No. 1481)

New specifications were prepared for these substances in the flavouring

agent format. Both substances are, however, believed to have uses other
than as flavouring agents, and the existing specifications in the standard
food additive format were revised and made tentative. In both cases,
information is required on functional uses other than for flavouring and on
the method of assay.
2.1.3 Maltyl isobutyrate (No. 1482), 3-acetyl-2,5-dimethylfuran (No. 1506)
and 2,4,5-trimethyl-δ-3-oxazoline (No. 1559)
New tentative specifications were prepared for these substances. In each
case, information is required about why the quoted ranges for specific gravity
are wider than would be expected, given the level of purity of the substances.
In addition, further information is required on why the range of refractive
indexes for flavouring agent No. 1559 is wider than would be expected,
given the level of purity of the substance.
2.2 Sucrose esters of fatty acids

The specifications for sucrose esters of fatty acids were revised but
maintained as tentative. Information is required on:

— a method of analysis for the determination of free sucrose by

capillary gas chromatography or high-performance liquid
chromatography;
— an alternative and less toxic solvent than pyridine for preparing
the standard and sample solutions for determinations of free
sucrose and propylene glycol; and
— a method of analysis for the determination of dimethyl sulfoxide
that does not require a packed column.
The tentative specifications mentioned above will be withdrawn unless the
requested information is received before the end of 2006.

136
 Annex 4
Summary of safety evaluations of secondary components of flavouring agents with
minimum assay values of less than 95%
No. Name Minimum assay Other Comments on secondary components
value (%) requirements

Eugenol and related hydroxyallylbenzene derivatives

1530 Eugenyl formate 94 2–3% eugenol Eugenol (No. 1529) was evaluated at the current meeting (see
monograph). It has been examined for toxicity in studies lasting
from 30 days to 2 years. The NOELs in most of these studies were
> 400 mg/kg bw per day (Trubek, 1958; Hagan et al., 1965; Bar &
Griepentrog, 1967; Hagan et al., 1967; Miller et al., 1983; National
Toxicology Program, 1983; Hirose et al., 1987). In a 2-year study,
the NOEL was > 450 mg/kg per day in mice and 300 mg/kg per day
in rats (National Toxicology Program, 1983).

Miscellaneous nitrogen-containing substances

1559 2,4,5-Trimethyl-∆-3-oxazoline 94 2–3% trimethyloxazole Trimethyloxazole (No. 1553) was evaluated at the current JECFA
meeting. It is expected to have a similar metabolic fate and similar
toxicity as the primary material, 2,4,5-trimethyl-∆-3-oxazoline, and
the other oxazoles and oxazolines in this group. In a 90-day study
with the primary material, the NOEL was > 41 mg/kg per day
(Morgareidge, 1972).

137
 Annex 4 (contd)

138
No. Name Minimum assay Other Comments on secondary components
value (%) requirements

Epoxides
1570 4,5-Epoxy-(E)-2-decenal 87 8–10% Z isomer 4,5-Epoxy-(Z)-2-decenal is expected to have the same metabolic
fate as the E isomer and the other epoxides in this group.
Epoxide hydrolase present in the cytosol (Gill et al., 1974), endo
plasmic reticulum (Oesch et al., 1970) and nucleus (Bresnick et al.,
1977) catalyses epoxide ring cleavage by water to yield vicinal
trans-diols. Alternatively, glutathione transferase present in the
cytosol catalyses ring cleavage by glutathione to yield trans-
thioalcohol conjugates (Jakoby, 1978). In a 28-day study, the
NOEL for the structurally related compound cyclohexane oxide was
100 mg/kg bw per day (Sauer et al., 1997).

Aliphatic and aromatic amines and amides

1606 Isopentylidene isopentylamine 93 2–3% diisopentylamine; Diisopentylamine is expected to have the same metabolic fate as
1-2% 3-methylbutyr- the other primary, secondary and tertiary amines in this group.
aldehyde They are mainly oxidized to imines by flavin-containing mono-
oxygenases, monoamine oxidases or amine oxidases. The
resulting imine can be further oxidized to the corresponding
aldehyde and ammonia (Kearney et al., 1971). In 90-day studies
with structurally related materials, the NOELs were 80 mg/kg bw
per day for piperidine and 160 mg/kg bw per day for trimethylamine
(Amoore et al., 1978).
3-Methylbutyraldehyde (No. 258) was evaluated by the Committee
at its forty-sixth meeting and found to be of no safety concern at
current levels of intake.
 Annex 4 (contd)

References
Amoore, J.E., Gumbmann, M.R., Booth, A.N. & Gould, D.H. (1978) Synthetic flavors: Efficiency and safety factors for sweaty and fishy odorants. Chem. Senses
Flavour , 3, 307.
Bär, V.F. & Griepentrog, F. (1967) [Where we stand concerning the evaluation of flavouring substances from the viewpoint of health]. Med. Ernahr., 8, 244–251.
Bresnick, E., Stoming, T.A., Vaught, J.B., Thakker, D.K. & Jerina, D.M. (1977) Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8-
dihydrobenzo[a]pyrene—Comparative chromatographic analysis of alkylated DNA. Arch. Biochem. Biophys., 183, 31–37.
Gill, S.S., Hammock, B.D. & Casida, J.E. (1974) Mammalian metabolism and environmental degradation of juvenoid 1-(4´-ethylphenoxy)-3,7-dimethyl-6,7-epoxy-
trans-2-octene and related compounds. J. Agric. Food Chem., 22, 386–395.
Hagan, E.C., Jenner, P.M., Jones, W.I., Fitzhugh, O.G., Long, E.L., Brouwer, J.G. & Webb, W.K. (1965) Toxic properties of compounds related to safrole. Toxicol.
Appl. Pharmacol ., 7, 18–24.
Hagan, E.C., Hansen, W.H., Fitzhugh, O.G., Jenner, P.M., Jones, W.I., Taylor, J.M., Long, E.L., Nelson, A.A. & Brouwer, J.B. (1967) Food flavourings and
compounds of related structure. II. Subacute and chronic toxicity. Food Cosmet. Toxicol., 5, 141–157.
Hirose, M., Masuda, A., Imaida, K., Kagawa, M., Tsuda, H. & Ito, N. (1987) Induction of forestomach lesions in rats by oral administrations of naturally occurring
antioxidants for 4 weeks. Jpn. J. Cancer Res. (Gann), 78, 317–321.
Kearney, E.B., Salach, J.I., Walker, W.H., Seng, R. & Singer, T.P. (1971) Structure of the covalently bound flavin of monoamine oxidase. Biochem. Biophys. Res
Commun., 42, 490–496.
Miller, E.C., Swanson, A.B., Phillips, D., Fletcher, L.T., Liem, A. & Miller, J.A. (1983) Structure–activity studies of the carcinogenicities in the mouse and rat of
some naturally occurring and synthetic alkenylbenzene derivatives related to safrole and estragole. Cancer Res., 43, 1124–1134.
Morgareidge, K. (1972) 90-day feeding studies in rats with 2,4,5-trimethyl-∆3-oxazoline (31202). Food and Drug Research Laboratories. Unpublished report
submitted to WHO by the Food and Extract Manufacturers Association of the United States, Washington DC, USA.
National Toxicology Program (1983) Carcinogenesis Studies of Eugenol (CAS No. 97-53-0) in F344/N Rats and B6C3F1 Mice (Feed Studies) (NTP TR 223. NTP
84-1779), Research Triangle Park, North Carolina, USA, National Toxicology Program.
Oesch, F., Jerina, D.M. & Daly, J. (1970) A radiometric assay for hepatic epoxide hydrase activity with 7-3H-styrene oxide. Biochim. Biophys. Acta, 227, 685–691.
Sauer, J.-M., Bao, J., Smith, R.L., McClure, T.D., Mayersohn, M., Pillai, U., Cunningham, M.L. & Sipes, I.G. (1997) Absorption, disposition kinetics, and metabolic
pathways of cyclohexane oxide in the male Fischer 244 rat and female B6C3F1 mouse. Drug Metab. Disposition, 25, 371–378.
Trubek Laboratories, Inc. (1958) Toxicolgical screening of eugenol, p-methoxybenzaldehyde and piperonal in rats. Class IX. Aromatic aldehydes. Unpublished
report submitted to WHO by the Flavor and Extract Manufacturers Association of the United States, Washington DC, USA.

139
140
 Annex 5
Flavouring agents for which use level or
reported poundage data are required

The safety assessments of these flavouring agents will be revoked if data on

levels of use or reported poundage data are not provided before the end of
2007 (see Annex 3).

1. Flavouring agents evaluated at the present meeting that were assessed

as of ‘no safety concern’ on a conditional basis

No. Flavouring agent

1483 2-Methyl-3-(1-oxopropoxy)-4 H-pyran-4-one

1527 4-Allylphenol
1528 2-Methoxy-6-(2-propenyl)phenol
1532 Eugenyl isovalerate
1538 cis-3-Hexenyl anthranilate
1539 Citronellyl anthranilate
1546 Ethyl N-methylanthranilate
1547 Ethyl N-ethylanthranilate
1548 Isobutyl N -methylanthranilate
1549 Methyl N-formylanthranilate
1550 Methyl N-acetylanthranilate
1551 Methyl N,N-dimethylanthranilate
1552 N-Benzoylanthranilic acid
1553 Trimethyloxazole
1554 2,5-Dimethyl-4-ethyloxazole
1555 2-Ethyl-4,5-dimethyloxazole
1556 2-Isobutyl-4,5-dimethyloxazole
1557 2-Methyl-4,5-benzo-oxazole
1558 2,4-Dimethyl-3-oxazoline
1561 Butyl isothiocyanate
1562 Benzyl isothiocyanate
1563 Phenethyl isothiocyanate
1569 4,5-Dimethyl-2-propyloxazole
1570 4,5-Epoxy-(E)-2-decenal
1571 β-Ionone epoxide
1573 Epoxyoxophorone
1579 Ethylamine
1580 Propylamine
1581 Isopropylamine
1583 Isobutylamine
1584 sec -Butylamine
1585 Pentylamine
1586 2-Methylbutylamine
1588 Hexylamine
1590 2-(4-Hydroxyphenyl)ethylamine
1591 1-Amino-2-propanol
1593 Butyramide
1594 1,6-Hexalactam

141
No. Flavouring agent

1595 2-Isopropyl-N ,2,3-trimethylbutyramide

1596 N-Ethyl (E)-2(Z)-6-nonadienamide
1597 N-Cyclopropyl (E)-2(Z)-6-nonadienamide
1598 N-Isobutyl (E,E)-2,4-decadienamide
1602 (±)-N,N-Dimethyl menthyl succinamide
1603 1-Pyrroline
1604 2-Acetyl-1-pyrroline
1605 2-Propionylpyrroline
1606 Isopentylidene isopentylamine
1608 2-Methylpiperidine
1611 Triethylamine
1612 Tripropylamine
1613 N,N-Dimethylphenethylamine
1614 Trimethylamine oxide
1615 Piperazine

2. Flavouring agents evaluated at the 59th (2002), 61st (2003) and 63rd
(2004) meetings of JECFA, for which only anticipated poundage data
were available or for which the MSDI derived from anticipated poundage
data from one region (European Union or USA) was greater than the
MSDI derived from recorded poundage data for the other region

No. Flavouring agent Year Note

963 Ethyl cyclohexanecarboxylate 2002 a

986 10-Hydroxymethylene-2-pinene 2002 a
1063 2,5-Dimethyl-3-furanthiol 2002 b
1065 Propyl 2-methyl-3-furyl disulfide 2002 a
1066 Bis(2-methyl-3-furyl) disulfide 2002 b
1067 Bis(2,5-dimethyl-3-furyl) disulfide 2002 b
1068 Bis(2-methyl-3-furyl) tetrasulfide 2002 a
1070 2,5-Dimethyl-3-furan thioisovalerate 2002 a
1077 Furfuryl isopropyl sulfide 2002 b
1082 2-Methyl-3,5- or -6-(furfurylthio)pyrazine 2002 b
1085 3-[(2-Methyl-3-furyl)thio]-4-heptanone 2002 a
1086 2,6-Dimethyl-3-[(2-methyl-3-furyl)thio]-4-heptanone 2002 a
1087 4-[(2-Methyl-3-furyl)thio]-5-nonanone 2002 a
1089 2-Methyl-3-thioacetoxy-4,5-dihydrofuran 2002 a
1157 4-Hydroxy-4-methyl-5-hexenoic acid γ-lactone 2003 a
1158 (±) 3-Methyl-γ-decalactone 2003 a
1159 4-Hydroxy-4-methyl-7-cis-decenoic acid γ-lactone 2003 a
1160 Tuberose lactone 2003 a
1161 Dihydromintlactone 2003 a
1162 Mintlactone 2003 b
1163 Dehydromenthofurolactone 2003 b
1164 (±)-(2,6,6-Trimethyl-2-hydroxycyclohexylidene)acetic acid 2003 a
γ-lactone
1167 2-(4-Methyl-2-hydroxyphenyl)propionic acid γ-lactone 2003 a
1174 2,4-Hexadien-1-ol 2003 a
1176 (E,E)-2,4-Hexadienoic acid 2003 a

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No. Flavouring agent Year Note

1180 (E,E)-2,4-Octadien-1-ol 2003 a

1183 2,4-Nonadien-1-ol 2003 a
1188 (E,Z)-2,6-Nonadien-1-ol acetate 2003 a
1189 (E,E)-2,4-Decadien-1-ol 2003 a
1191 Methyl (E)-2-(Z)-4-decadienoate 2003 a
1193 Ethyl 2,4,7-decatrienoate 2003 a
1199 (±)-2-Methyl-1-butanol 2003 a
1217 2-Methyl-2-octenal 2003 a
1218 4-Ethyloctanoic acid 2003 a
1226 8-Ocimenyl acetate 2003 a
1228 3,7,11-Trimethyl-2,6,10-dodecatrienal 2003 a
1229 12-Methyltridecanal 2003 a
1232 1-Ethoxy-3-methyl-2-butene 2003 b
1236 2,2,6-Trimethyl-6-vinyltetrahydropyran 2003 b
1239 Cycloionone 2003 a
1245 2,4-Dimethylanisole 2003 a
1248 1,2-Dimethoxybenzene 2003 a
1265 4-Propenyl-2,6-dimethoxyphenol 2003 a
1289 Erythro- and threo-3-mercapto-2-methylbutan-1-ol 2003 b
1290 (±)-2-Mercaptomethylpentan-1-ol 2003 b
1292 3-Mercapto-2-methylpentanal 2003 b
1293 4-Mercapto-4-methyl-2-pentanone 2003 b
1296 spiro[2,4-Dithia-1-methyl-8-oxabicyclo(3.3.0)octane-3,3´- 2003 a
(1´-oxa-2´-methyl)-cyclopentane]
1299 2,3,5-Trithiahexane 2003 b
1300 Diisopropyl trisulfide 2003 b
1311 2-(2-Methylpropyl)pyridine 2004 a
1319 2-Propionylpyrrole 2004 b
1322 2-Propylpyridine 2004 a
1334 4-Methylbiphenyl 2004 b
1342 δ-3-Carene 2004 a
1343 α-Farnesene 2004 a
1344 1-Methyl-1,3-cyclohexadiene 2004 a
1367 trans-2-Octen-1-yl acetate 2004 b
1368 trans-2-Octen-1-yl butanoate 2004 b
1369 cis-2-Nonen-1-ol 2004 b
1370 (E)-2-Octen-1-ol 2004 a
1371 (E)-2-Butenoic acid 2004 a
1372 (E)-2-Decenoic acid 2004 a
1373 (E)-2-Heptenoic acid 2004 a
1374 (Z)-2-Hexen-1-ol 2004 a
1375 trans-2-Hexenyl butyrate 2004 a
1376 (E)-2-Hexenyl formate 2004 a
1377 trans-2-Hexenyl isovalerate 2004 a
1378 trans-2-Hexenyl propionate 2004 a
1379 trans-2-Hexenyl pentanoate 2004 a
1380 (E)-2-Nonenoic acid 2004 a
1381 (E)-2-Hexenyl hexanoate 2004 a
1382 (Z)-3- and (E)-2-Hexenyl propionate 2004 a
1384 2-Undecen-1-ol 2004 a
1407 Dihydronootkatone 2004 b
1409 β-Ionyl acetate 2004 a

143
No. Flavouring agent Year Note

1410 α-Isomethylionyl acetate 2004 a

1411 3-(l-Menthoxy)-2-methylpropane-1,2-diol 2004 a
1412 Bornyl butyrate 2004 a
1413 DL-Menthol(±)propylene glycol carbonate 2004 a
1414 L-Monomenthyl glutarate 2004 a
1415 L-Menthyl methyl ether 2004 a
1416 para-Menthane-3,8-diol 2004 a
1435 Taurine 2004 a
1438 L-Arginine 2004 a
1439 L-Lysine 2004 a
1447 Tetrahydrofurfuryl cinnamate 2004 a
1457 (±)-2-(5-Methyl-5-vinyltetrahydrofuran-2-yl)propionaldehyde 2004 a
1475 Ethyl 2-ethyl-3-phenylpropanoate 2004 a
1478 2-Oxo-3-phenylpropionic acid 2004 a

a
Flavourings for which only anticipated poundage data were available
b
Flavourings for which the MSDI derived from anticipated poundage data from the USA was greater
than the MSDI derived from recorded poundage data from the European Union

144
 Annex 6
Divergent opinion on safety assessment of
flavouring substances
Gérard Pascal and Philippe Verger
Institut National de la Recherche Agronomique, Paris, France

JECFA has adopted part of the concept of ‘threshold of toxicological concern’

for evaluating flavouring agents. The concept is based on the assumption
that, if the level of exposure is low, risk assessment can be based on data for
structurally related compounds. The data include those on absorption,
distribution, metabolism, excretion and toxicity for compounds of the same
structural class. The threshold of toxicological concern is defined as the
level of human exposure below which it can be anticipated there are no
significant risk for health even in the absence of data on the compound
itself.
The quality of the estimate of dietary exposure is therefore crucial for
reaching a conclusion about the safety of flavouring agents evaluated by
this Procedure.

The estimated dietary intake used by the Committee is based on the amount
of the flavouring agent produced per year by industry (also called poundage
data) divided by the number of consumers, assumed to be 10% of the
population.
During the sixty-fifth meeting, 135 flavouring substances were submitted
for safety assessment. Production figures were not available for 60 of them,
and industry provided the Committee with ‘anticipated production data’,
corresponding to volumes that might be produced in the future. The
Committee agreed that these data were not adequate for use in its procedure
for evaluating the safety of flavouring substances. Nevertheless, the
Committee came to conclusions about the safety of these substances by
applying the normal procedure, although making the conclusions conditional.

The minority opinion is that the safety of the 60 flavouring substances

without reported poundage data should not be evaluated by the normal
procedure, even on a conditional basis.

145