
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 70
PRINCIPLES FOR THE SAFETY ASSESSMENT OF
FOOD ADDITIVES AND CONTAMINANTS IN FOOD
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 United Nations Environment Programme, the International
Labour Organisation, the World Health Organization, or the Food
Agriculture Organization of the United Nations
Published under the joint sponsorship of
the United Nations Environment Programme,
the International Labour Organisation,
and the World Health Organization in
collaboration with the Food and Agriculture
Organization of the United Nations
World Health Orgnization
Geneva, 1987
The International Programme on Chemical Safety (IPCS) is a
joint venture of the United Nations Environment Programme, the
International Labour Organisation, and the World Health
Organization. The main objective of the IPCS is to carry out and
disseminate evaluations of the effects of chemicals on human health
and the quality of the environment. Supporting activities include
the development of epidemiological, experimental laboratory, and
risk-assessment methods that could produce internationally
comparable results, and the development of manpower in the field of
toxicology. Other activities carried out by the IPCS include the
development of know-how for coping with chemical accidents,
coordination of laboratory testing and epidemiological studies, and
promotion of research on the mechanisms of the biological action of
chemicals.
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CONTENTS
PRINCIPLES FOR THE SAFETY ASSESSMENT OF FOOD ADDITIVES AND CONTAMINANTS
IN FOOD
FOREWORD
PREFACE
1. INTRODUCTION
2. HISTORICAL BACKGROUND
2.1. Introduction
2.2. Periodic review
2.2.1. Concept of periodic review
2.2.2. Mechanism of periodic review
3. CRITERIA FOR TESTING AND EVALUATION
3.1. Criteria for testing requirements
3.1.1. Estimating exposure
3.1.2. Predicting toxicity from chemical structure
3.1.3. Other factors to consider when developing criteria
3.2. Priorities for testing and evaluation
3.3. Quality of data
4. CHEMICAL COMPOSITION AND THE DEVELOPMENT OF SPECIFICATIONS
4.1. Identity and purity
4.2. Reactions and fate of food additives and contaminants in food
4.3. Specifications
5. TEST PROCEDURES AND EVALUATION
5.1. End-points in experimental toxicity studies
5.1.1. Effects with functional manifestations0
5.1.2. Non-neoplastic lesions with morphological manifestations
5.1.3. Neoplasms
5.1.4. Reproduction/developmental toxicity
5.1.5. In vitro studies
5.2. The use of metabolic and pharmacokinetic studies in safety
assessment
5.2.1. Identifying relevant animal species.
5.2.2. Determining the mechanisms of toxicity
5.2.3. Metabolism into normal body constituents
5.2.4. Influence of the gut microflora in safety assessment
5.2.4.1 Effects of the gut microflora on the chemical
5.2.4.2 Effects of the chemical on the gut microflora
5.3. Influence of age, nutritional status, and health status on the
design and interpretation of studies
5.3.1. Age
5.3.1.1 History
5.3.1.2 Usefulness of studies involving in utero exposure
5.3.1.3 Complications of aging
5.3.2. Nutritional status
5.3.3. Health status
5.3.4. Study design
5.4. Use of human studies in safety evaluation
5.4.1. Epidemiological studies
5.4.2. Food intolerance
5.5. Setting the ADI
5.5.1. Determination of the no-observed-effect level
5.5.2. Use of the safety factor
5.5.3. Toxicological versus physiological responses
5.5.4. Group ADIs
5.5.5. Special situations
5.5.6. Comparing the ADI with potential exposure
6. PRINCIPLES RELATED TO SPECIFIC GROUPS OF SUBSTANCES
6.1. Substances consumed in small amounts
6.1.1. Food contaminants
6.1.2. Food flavouring agents
6.2. Substances consumed in large amounts
6.2.1. Chemical composition, specifications, and impurities
6.2.2. Nutritional studies
6.2.3. Toxicity studies
6.2.4. Foods from novel sources
REFERENCES
ANNEX I. GLOSSARY
ANNEX II. STATISTICAL ASPECTS OF TOXICITY STUDIES
REFERENCES TO ANNEX II
ANNEX III. GUIDELINES FOR THE EVALUATION OF VARIOUS GROUPS OF FOOD
ADDITIVES AND CONTAMINANTS
REFERENCES TO ANNEX III
ANNEX IV. EXAMPLES OF THE USE OF METABOLIC STUDIES IN THE SAFETY
ASSESSMENT OF FOOD ADDITIVES
REFERENCES TO ANNEX IV
ANNEX V. APPROXIMATE RELATION OF PARTS PER MILLION IN DIET TO MG/KG PER
DAY
ANNEX VI. REPORTS AND OTHER DOCUMENTS RESULTING FROM MEETINGS OF
THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES
WHO TASK GROUP ON UPDATING THE PRINCIPLES FOR THE SAFETY ASSESSMENT OF
FOOD ADDITIVES AND CONTAMINANTS IN FOOD
1985 and/or 1986 JECFA Members
a,b,c,d,e Dr H. Blumenthal, Division of Toxicology, Center for
Food Safety and Applied Nutrition, US Food and
Drug Administration, Washington DC, USA
c,d Dr I. Chakravarty, Department of Biochemistry and
Nutrition, All India Institute of Hygiene and
Public Health, Calcutta, India
c Dr W.H.B. Denner, Food Composition and Information
Unit, Food Sciences Division, Ministry of Agri-
culture, Fisheries and Food, London, United
Kingdom
c Dr A.H. El-Sebae, Pesticides Division, Faculty of
Agriculture, Alexandria University, Alexandria,
Egypt
c Professor P.E. Fournier, Hpital Fernand-Widal,
Paris, France
a,c,d,f Dr S. Gunner, Food Directorate, Health Protection
Branch, Health and Welfare Canada, Ottawa,
Ontario, Canada
d Mr J. Howlett, Food Science Division, Ministry of
Agriculture, Fisheries and Food, London, United
Kingdom
c,d,f Professor K. Kojima, College of Environmental Health,
Azabu University, Sagamihara-Shi, Kanagawa-Ken,
Japan
c Dr W. Kroenert, Food Chemistry Division, Max von
Pettenkofer Institute, Federal Office of Public
Health, Berlin (West)
a,b,c,d,f Dr B. MacGibbon, Division of Toxicology, Environ-
mental Pollution and Prevention, Department of
Health and Social Security, London, United
Kingdom
c,d Dr R. Mathews, Food Chemicals Codex, National
Academy of Sciences, Washington DC, USA
d Mrs I. Meyland, National Food Institute, Ministry of
the Environment, Soborg, Denmark
c,d,e Dr J. Modderman, Food Additives Chemistry Evaluation
Branch, Center for Food Safety and Applied
Nutrition, US Food and Drug Administration,
Washington DC, USA
d Dr G. Nazario, Ministry of Health, National Health
Council, Rio de Janeiro, Brazil
c,d Professor K.A. Odusote, College of Medicine, Univ-
ersity of Lagos, Lagos, Nigeria
c,d Professor F. Pellerin, Facult de Pharmacie de
l'Universit Paris-Sud, Hpital Corentin Celton,
Issy-les-Moulineaux, France
c,f Dr P. Pothisiri, Food Control Division, Food and
Drug Administration, Ministry of Public Health,
Bangkok, Thailand
b,c,d Professor M.J. Rand, Department of Pharmacology,
University of Melbourne, Parkville, Victoria,
Australia
a,b,d,e,g Dr P. Shubik, Green College, Oxford, United Kingdom
d Dr A. Slorach, Food Research Department, The National
Food Administration, Uppsala, Sweden
d Dr V.A. Tutelyn, Institute of Nutrition, Academy of
Medical Sciences of the USSR, Moscow, USSR
Secretariat
d Dr Y.K. Al-Mutawa, Division of Public Health Labor-
atory, Ministry of Public Health, Safat, Kuwait
a Dr E.A. Bababunmi, University of Ibadan, Ibadan,
Nigeria
e Dr A. Br, Department of Vitamin and Nutrition
Research, F. Hoffmann-LaRoche and Company, Ltd.,
Basel, Switzerland
a,b,d,f Dr J. Cabral, Unit of Mechanisms of Carcinogenesis,
International Agency for Research on Cancer,
Lyons, France
e Dr J. Caldwell, St. Mary's Hospital Medical School,
London, United Kingdom
a,e Dr D.M. Conning, British Nutrition Council, London,
United Kingdom
f Dr J.L. Emerson, External Technical Affairs, The
Coca-Cola Company, Atlanta, Georgia, USA
d Mr A. Feberwee, Committee on Food Additives, Nutri-
tion and Quality Affairs, Ministry of Agriculture
and Fisheries, The Hague, The Netherlands
d Professor C.L. Galli, Toxicology Laboratory,
Institute of Pharmacology and Pharmacognosy,
University of Milan, Milan, Italy
e Dr M.J. Goldblatt, Consumer Nutrition Affairs,
General Foods Corporation, White Plains,New
York, USA
f Dr W. Grunow, Divison of Food Toxicology, Max von
Pettenkofer Institute, Federal Office of Public
Health, Berlin (West)
d Mr R. Haigh, Commission of the European Communities,
Brussels, Belgium
a,d,f Dr Y. Hayashi, Division of Pathology, Biological
Safety Research Center, National Institute of
Hygienic Sciences, Tokyo, Japan
b,d,e,g Dr J. Herrman, Division of Food and Color Additives,
Center for Food Safety and Applied Nutrition,
US Food and Drug Administration, Washington DC,
USA
f Dr D. Krewski, Environmental Health Directorate,
Health Protection Branch, Health and Welfare
Canada, Ottawa, Ontario, Canada
e Mr P.N. Lee, Consultant in Statistics and Adviser in
Epidemiology and Toxicology, Surrey, United
Kingdom
b Dr M. Mercier, International Programme on Chemical
Safety, World Health Organization, Geneva,
Switzerland
d Dr R.W. Moch, Center for Food Safety and Applied
Nutrition, US Food and Drug Administration,
Washington DC, USA
a Dr V.H. Morgenroth, Chemicals Division, Environment
Directorate, Organization for Economic Cooper-
ation and Development, Paris, France
a Dr I. Nir, Department of Pharmacology and Experi-
mental Therapeutics, The Hebrew University
Hadassah Medical School, Jerusalem, Israel
d Dr E. Poulsen, National Food Institute, Institute of
Toxicology, Soborg, Denmark
b,d,f Dr A.W. Randell, Food Policy and Nutrition Division,
Food and Agricultural Organization of the United
Nations, Rome, Italy
d Dr N. Rao Maturu, Joint FAO/WHO Food Standards
Programme, Food and Agricultural Organization of
the United Nations, Rome, Italy
e,f Dr A.G. Renwick, Clinical Pharmacology Group, Univ-
ersity of Southampton, Southampton, United
Kingdom
e,f Dr F.J.C. Roe, Consultant in Toxicology and Adviser
in Experimental Pathology and Cancer Research,
London, United Kingdom
d,e Dr S.I. Shibko, Division of Toxicology, Center for
Food Safety and Applied Nutrition, US Food and
Drug Administration, Washington DC, USA
a,b,e,f Dr V. Silano, Department of Comparative Toxicology,
High Institute of Health, Rome, Italy
d Professor A. Somogyi, Department of Drugs, Animal
Nutrition and Residue Research, Institute for
Vetinary Medicine, Berlin (West)
b,d Professor R. Truhaut, Facult des Sciences Pharma-
ceutiques et Biologiques de Paris Luxembourg,
Laboratoire de Toxicologie et Hygiene Indus-
rielle, Universit Rene Descartes, Paris, France
f Dr G.J. Van Esch, National Institute for Public
Health and Environmental Hygiene, Bilthoven,
The Netherlands
a,b,d Dr G. Vettorazzi, International Programme on Chemical
Safety, World Health Organization, Geneva,
Switzerland
e Dr M.J. Wade, Division of Toxicology, Center for
Food Safety and Applied Nutrition, Washington DC,
USA
a,b,d,e,g Dr R. Walker, Department of Biochemistry, University
of Surrey, Guildford, Surrey, United Kingdom
-----------------------------------------------------------------
a Present at strategy meeting, Oxford, United Kingdom, 19-23
September, 1983.
b Present at pre-consultation of contributors, Geneva,
Switzerland, 29-31 May, 1985.
c Member of JECFA-85, Geneva, Switzerland, 3-12 June, 1985.
d Participant in JECFA-86, Rome, Italy, 2-11 June, 1986.
e Consultant who contributed written material.
f Submitter of written comments.
g Member of Editorial Committee.
NOTE TO READERS OF THE CRITERIA DOCUMENTS
Every effort has been made to present information in the
criteria documents as accurately as possible without unduly
delaying their publication. In the interest of all users of the
environmental health criteria documents, readers are kindly
requested to communicate any errors that may have occurred to
the Manager of the International Programme on Chemical Safety,
World Health Organization, Geneva, Switzerland, in order that
they may be included in corrigenda, which will appear in
subsequent volumes.
* * *
FOREWORD
The WHO activities concerned with the safety assessment of
food chemicals were incorporated into the International
Programme on Chemical Safety (IPCS) in 1980. Since this time, a
keen interest has developed in all aspects pertaining to the
toxicological evaluation of food additives and contaminants,
including the methodological aspects. These activities are part
of the responsibilities of the Programme insofar that its
objectives include the formulation of "guiding principles for
exposure limits, such as acceptable daily intakes for food
additives and pesticide residues, and tolerances for toxic
substances in food, air, water, soil, and the working
environment".
The present publication on "Principles for the Safety
Assessment of Food Additives and Contaminants in Food" has been
developed in response to repeated recommendations by the Joint
FAO/WHO Expert Committee on Food Additives (JECFA). Its
inclusion in the methodology section of the Environmental Health
Criteria series will make it readily available to both Member
States and the food industry.
The IPCS gratefully acknowledges the financial support of
the United Kingdom Department of Health and Social Security
(DHSS), and the US Food and Drug Administration (FDA), which was
indispensable for the completion of the project.
Dr M. Mercier
Manager
International Programme on
Chemical Safety
PREFACE
For the last thirty years, the internationally sponsored
committee known as the Joint FAO/WHO Expert Committee on Food
Additives (JECFA) has played a major role in providing a unique
international mechanism for the identification and safety
assessment of food chemicals, including food additives, food
contaminants, and residues of veterinary drugs. With no
regulatory aspirations, this Committee has probably contributed
more to the elaboration of sound national food regulation in
this area than any other international body aimed at harmonizing
or normalizing often divergent national approaches to the
problem of food safety, food technology, and food control.
JECFA achieved this by providing recommendations based on
scientific evidence and by establishing a rational model of
safety assessment that is widely reputed and accepted.
Hundreds of highly skilled international specialists have
given, and continue to give, freely of their time and talents to
foster advances in toxicological methodologies and analytical
procedures, to consolidate accessible presentations of data, and
to keep abreast with scientific developments, which often
requires readjustment of previous conclusions. Through reports,
toxicological monographs, and profiles of chemical
specifications by JECFA, national food regulatory authorities
and the Codex Alimentarius Commission are provided with all the
necessary elements for making the best decisions on the rational
use of chemicals in food.
The present undertaking has several precedents in the
history of JECFA. For example, in 1957, the second report of
the Committee elaborated on procedures for the testing of
intentional food additives to establish their safety for use
and, in 1960, the fifth report contained a series of guidelines
for the evaluation of the carcinogenic hazards of food
additives. It should also be mentioned that, in 1966, the
Committee commissioned a special scientific group to develop
procedures for investigating intentional and unintentional food
additives. Finally, in 1981, after realizing that a significant
interval of time had elapsed since previous methodological
updatings, the Committee called for a state-of-the-art review of
methodology. A favourable answer was received from the newly
established International Programme on Chemical Safety (IPCS), a
cooperative programme sponsored by the International Labour
Organisation (ILO), the United Nations Environment Programme
(UNEP), and the World Health Organization (WHO). It should be
noted that the implementation of the recommendation by the IPCS
was significantly facilitated by the fact that the toxicological
component of the JECFA came within the scope of the programme.
The contents of this publication are the result of sustained
efforts of an IPCS Task Group during a number of meetings
including: a strategy meeting in 1983, a consultation of
contributors in 1985, and the JECFA Working Groups at their
annual meetings in 1985 and 1986. The members of the Task Group
contributed either written material or comments, or both. An
Editorial Board was responsible for preparing the final draft
for publication. The Task Group benefited widely from the large
number of recommendations and observations on the methodology of
testing and assessing chemicals in food, found in the previous
reports of JECFA and related scientific groups.
Thus, this publication reflects faithfully the
recommendations of the Committee regarding the safety assessment
of food additives and contaminants by reaffirming the validity
of recommendations that are still appropriate, while pointing
out the problems associated with those that, in the light of
modern advances in methodology, are no longer valid. New
recommendations are also made, as might be expected, with the
advancing state of the toxicological sciences. Particularly
enlightening are the section dealing with the principles related
to the safety assessment of substances consumed in large
amounts, and Annexes II and IV dealing with the statistical
aspects of toxicity studies and examples of the use of metabolic
studies in the safety assessment of food additives,
respectively. An Index has also been included at the end of the
book.
It is the most earnest wish of all concerned with the
production of this publication that it should make an important
contribution to the field of food toxicology and that it will be
found useful by the members of JECFA, national food regulatory
authorities, and industry, who are involved in the development
of safety data and in making the consumer aware of the problems
of the safe use of food additives.
Dr G. Vettorazzi
Dr A. Randell
1. INTRODUCTION
This publication is concerned with reviewing the basis for
decision-making by the Joint Expert Committee on Food Additives
(JECFA) of the Food and Agriculture Organization of the United
Nations (FAO) and the World Health Organization (WHO). Because
the toxicological and chemical characteristics of food additives
are the primary concern of the Committee, both aspects are dealt
with in this publication, which has been prepared by WHO- and
FAO-appointed consultants, assisted by the WHO and FAO
secretariats. The twenty-eighth JECFA report (1) includes a
summary of the areas that the Committee considered to be most in
need of evaluation.
The major concerns of this monograph are with the testing of
chemicals in food and with the evaluation of the test results.
In keeping with the approach developed during the past 30 years
by JECFA, the recommendations for test procedures and safety
assessment are discussed in broad terms taking into account the
latest scientific advances in the relevant fields. No effort
has been made to provide instructions for test procedures.
Differences in national approaches to toxicological evaluations
exist, and some variations in the data submitted must be
considered by JECFA in making their evaluations. However,
certain basic data requirements are necessary in order to enable
an expert committee to make sound judgements. The elements of
this base line are included in various sections of this report.
In essence, the problems under consideration fall into three
general categories: first, the determination of the chemical and
toxicological test requirements for individual chemicals that
are added to or occur in food; second, the assessment methods
that are to be applied; and third, the updating of the test
procedures and methods of assessment as the science progresses.
Methods for testing and assessment have changed considerably
during the life of JECFA. However, the Committee has, by no
means, been a static organization. Not only have various
approaches been continuously updated at the individual meetings
of the Committee, but intervening meetings of scientific groups
have been held to consider the impact of new scientific
developments on the procedures used (2, 3). Thus, every effort
has been made in this publication to record the views of JECFA
and to detail the changes that have come about with the course
of time.
It is recognized that, with advances in science, it is
possible to obtain more complete toxicological profiles for
individual chemicals. For example, it is becoming increasingly
easy to learn more about the disposition and metabolic fate of
xenobiotics. Until relatively recently, the toxicologist in
this field had to rely almost entirely on a set of routine
tests, the results of which were then assessed and used to
establish arbitrarily-determined safe levels.
JECFA has long recognized that a number of factors can be
used to determine test requirements; these include the structure
of the chemical, its natural occurrence in foodstuffs, its
metabolic characteristics, and knowledge of its effects in man.
However, systematic guidelines incorporating these factors have
not been developed by JECFA. In recent years, some of the
problems posed to JECFA have concerned the testing and
evaluation of additives and food ingredients that are consumed
in large amounts. Other key problems in the determination of
the appropriate level of testing involve the largest group of
food additives, the flavouring agents that are used, generally,
at very low levels and that are often "nature identical" or
derived from natural sources. Therefore, both "high
consumption" substances and flavouring agents are discussed in
detail in sections 6.1 and 6.2
There have been considerable changes in laboratory studies
used in other areas of toxicology, which are yet to have a major
impact on the evaluation of food additives. Of particular
interest are the series of mutagenicity/clastogenicity tests,
often, but not invariably, using sub-mammalian test organisms in
vitro. The number, diversity, and uses of these tests have
increased rapidly in the past decade. In general, such tests
are effective for measuring an intended genetic end-point. How
effectively these tests identify chemical carcinogens is much
less clear. In the absence of a clear correlation with
carcinogenicity, it is difficult to know how such tests should
be interpreted and used in safety evaluations. Even though
these in vitro tests may not be required for the evaluation of
the safety of food additives, it is becoming more and more
frequent for chemicals to be tested in this way for other
reasons, e.g., to detect potential environmental or occupational
hazards. Thus, JECFA may have to decide on the relevance of
such information (section 5.1.5).
During the past decade, there has been a major increase in
the number of chemicals tested routinely for chronic toxicity in
standardized in vivo tests. Although these tests may not be
designed to evaluate food additives, the results have to be
carefully considered at JECFA meetings. Among the major
problems that occur are those arising from results obtained when
chemicals are administered to animals by routes other than the
diet or drinking-water. For example, some years ago, JECFA
faced the problem of assessing the significance of the induction
of subcutaneous sarcomas at the site of injection of certain
food chemicals into rodents. It was found that many substances,
including inert plastics, could give rise to similar sarcomas on
implantation. As a result, the Committee concluded that such
findings could not be used in a definitive manner for assessing
the safety of food additives (4, pp. 16-17). However, these
findings cannot be totally ignored and may be an indicator of
the need for further carcinogenicity studies using the oral
route. Another problem with interpretation arises with many of
the more recent "routine" in vivo studies that record the
enhancement of a variety of common "spontaneous" tumours in
rodents, including lymphomas, hepatomas, and pheochromocytomas
(section 5.1.3).
The scope of long-term toxicity tests has been discussed
extensively by JECFA. For example, several food chemicals have
been tested in 2-generation studies rather than the commonly-
used single-generation study. While the use of this more
extensive test is advisable under certain conditions, it should
not necessarily be a routine procedure (section 5.3).
Many of the chemicals of concern to those responsible for
food safety evaluation are present in food at very low levels
and may be present as environmental contaminants or may result
from the migration of substances from food packaging or residues
from the use of solvents, pesticides, or veterinary drugs.
These situations often require very different approaches to test
requirements than those used for intentional food additives
(Annex III). One case, the use of anabolic agents in livestock,
has posed various problems for JECFA that cannot be answered
within the scope of current procedures (Annex III). JECFA will
soon be developing methodology for the testing and evaluation of
veterinary drug residues in support of a new committee on
residues of veterinary drugs in foods that has been established
by the Codex Alimentarius Commission.
In assessing the significance of data, a major issue to be
resolved concerns the distinctions that should be made among
different toxicological manifestations. The carcinogenic
potential of chemicals has been emphasized in the past few
decades to the exclusion of most other toxic end-points. There
was a general consensus that chemicals found to be carcinogenic
were not appropriate as food additives at any level whatsoever.
More recently, however, it has become widely accepted that the
term "carcinogen" has become harder and harder to define
(section 5.1). It is apparent that cancer can be induced by a
variety of chemicals acting by very different mechanisms and
that the mechanism should be an important consideration when
determining whether a safe level can be established. Other
questions concern whether high-dose animal data are relevant to
human exposure to low levels, and how teratogenicity data in the
absence of maternal toxicity are to be interpreted (section
5.1.4).
2. HISTORICAL BACKGROUND
2.1. Introduction
The Joint FAO/WHO Expert Committee on Food Additives (JECFA)
was established following recommendations made to the Directors-
General of FAO and WHO by the Joint FAO/WHO Expert Committee on
Nutrition at it fourth session (5), and the subsequent first
Joint FAO/WHO Conference on Food Additives was held in Septem-
ber, 1955 (6). The terms of reference of the earlier meetings
of JECFA related to the formulation of general principles gov-
erning the use of food additives and consideration of suitable
uniform methods for evaluating the safety of food additives.
For these purposes, food additives were defined by the Joint
Conference as "non-nutritive substances added intentionally to
food, generally in small quantities, to improve its appearance,
flavour, texture, or storage properties."a Following recom-
mendations of the third Joint FAO/WHO Conference on Food Addi-
tives (8), these terms of reference were broadened to include
substances unintentionally introduced into human food and JECFA
has subsequently considered and evaluated such materials,
including growth promoters, components of packaging materials,
solvents used in food processing, aerosol propellants, enzymes
used in food processing, and metals in foods. Novel foods and
ingredients that may be incorporated into foods at levels higher
than those previously envisaged for food additives have also
been referred to JECFA and pose special problems in safety
evaluation, which will be discussed later in this report
(section 6.2).
The first (9), second (10), and fifth Reports (4) of JECFA
established principles for the use of food additives and made
recommendations on methods for establishing the safety-in-use of
food additives and for the evaluation of the carcinogenic
hazards of food additives. From the outset, JECFA recognized
that:
"no single pattern of tests could cover adequately, but
not wastefully, the testing of substances so diverse in
structure and function as food additives" and that
"the establishment of a uniform set of experimental
procedures that would be standardized and obligatory is
therefore undesirable" (10).
----------------------------------
a From a practical standpoint, the "food additive" definition
has been expanded since the time it was drafted, as a
variety of compounds, including nutritive substances
consumed in high amounts, have been brought under the
umbrella of food additives. Indeed, the second Joint
FAO/WHO Conference on Food Additives (7) recommended that
the scope of the JECFA programme be expanded beyond the
substances included in the original definition.
Accordingly, this Committee concluded "that it was only possible
to formulate general recommendations with regard to testing
procedures." The Committee also recognized that advances in the
basic sciences might suggest new approaches to toxicological
investigations and that these might be used immediately by the
scientist but would take longer to become incorporated into any
officially recommended testing procedures. Subsequent meetings
of JECFA have consistently adopted this approach and have
avoided the adoption of fixed protocols for the testing and
evaluation of all classes of intentional and unintentional food
additives. This has had the advantage of allowing the Committee
to respond to new problems as they have arisen, with minimal
inertia, and to encompass non-routine and ad hoc studies in the
safety evaluation process. Within this framework, the Committee
has found it possible to formulate guidelines for the evaluation
of several groups of intentional and unintentional food
additives that posed their own peculiar problems; several of
these guidelines, which serve as specific examples to support
general principles, are contained in Annex III.
The requirement to keep abreast of scientific developments
in toxicology and related scientific disciplines implies the
need for a periodic review of testing methodology. Following
recommendations to this effect made by the eighth (11) and ninth
(12) meetings of JECFA, a WHO Scientific Group on Procedures for
Investigating Intentional and Unintentional Food Additives was
convened in 1966
"to review, in the light of new scientific knowledge,
the criteria used in establishing acceptable daily
intakes. . . ." and "to suggest further studies on
toxicological procedures used for the evaluation of
intentional and unintentional food additives in order
to establish their safety to the consumer" (2).
Subsequent meetings of JECFA have taken cognisance of the report
of this Scientific Group and of the report of a more recent WHO
Scientific Group on the Assessment of Carcinogenicity and Muta-
genicity of Chemicals (3). Some aspects of the reports of other
WHO Scientific Groups on the Principles for the Testing and
Evaluation of Drugs for Carcinogenicity (13), Mutagenicity (14),
and Teratogenicity (15) are also pertinent to the methodology
of testing food additives. However, significant developments in
the science of toxicology and related disciplines led the
seventeenth meeting of JECFA to recommend that "the methods and
procedures for the toxicity testing of food additives should be
comprehensively reviewed and brought into line with advances in
toxicology and cognate disciplines" (16). This recommendation
was reiterated in the reports of the eighteenth (17) and
nineteenth (18) meetings, the latter of which called for the
convening of an appropriate meeting for the purpose of the
review, and reaffirmed at the twentieth meeting (19).
Safety evaluation of food additives is a 2-stage process.
The first stage involves the collection of relevant data
including the results of studies on experimental animals and,
where possible, observations in man. The second stage involves
the assessment of data to determine the acceptability of the
substance as a food additive. While the recommendations
referred to in the preceding paragraph emphasize the impact of
scientific advances on the first testing stage, the impact of
such advances extends also to the assessment stage. This was
made explicit in the twenty-first report of JECFA (20, p. 31),
which stated that:
"in view of the rapid progress of the science of toxi-
cology and the increasing refinement of evaluation pro-
cedures, the Committee felt strongly that the tradi-
tional concepts of setting ADIs, the application of
safety factors, and the relationship of these safety
factors to the observed toxicological manifestations in
animal experiments should be reconsidered".
This recommendation was endorsed by the twenty-fourth meeting of
JECFA (21).
Many features of toxicity testing and evaluation of advent-
itious food additives and contaminants that fall within the
terms of reference of JECFA, are common to pesticides that are
within the scope of the Joint FAO/WHO Meeting on Pesticide
Residues. In recognition of this, the twenty-fifth meeting of
JECFA (22) recommended that a group of experts should be
convened, as soon as possible, to study the application of
advances in methodology to the toxicological evaluation of food
additives and contaminants, and also of pesticide residues. The
urgency of the need to implement this recommendation was
stressed by the twenty-sixth (23) and twenty-seventh (24) meet-
ings of JECFA.
In response to the Committee's repeated recommendations, a
meeting of a group of experts to study the application of
advances in methodology to the toxicological evaluation of food
additives and contaminants was convened in September 1983. The
objectives of the meeting were to formulate specific recommend-
ations in order to bring up to date:
(a) the principles set out in earlier reports of JECFA
concerning safety evaluation in relation to specific
toxicological problems or specific chemical entities or
groups;
(b) the test methods used in the toxicological evaluation
of chemicals in food; and
(c) the assessment procedures adopted by JECFA in
determining quantitative end-points.
The report of the Working Group (Updating Principles of
Methodology for Testing and Assessing Chemicals in Food: Report
of a Strategy Meeting) (unpublished WHO document ICS(Food)/83.3)
and working papers on specific issues were considered by the
twenty-eighth meeting of JECFA (1). Several questions were
identified as remaining to be considered, including special
problems associated with:
(a) bulking agents and novel foods;
(b) food contaminants;
(c) animal feed additives and veterinary drug residues;
(d) test methods and principles (including alternative
methods of testing);
(e) testing for allergenicity;
(f) lesions observed in bioassays that are difficult to
interpret (a number of examples are cited in the
report); and
(g) assessment procedures; extrapolation and quantitative
assessment.
The Committee recommended that a unified document on these
issues should be prepared for consideration by JECFA at a future
meeting. The present publication has been prepared in response
to that recommendation.
In carrying out the review of methodology for the testing
and evaluation of intentional and unintentional food additives,
the working group has taken notice of recommendations, guide-
lines, and procedures adopted by national regulatory authorities
and international/supra-national organizations including the
Organization for Economic Cooperation and Development (OECD),
the International Agency for Research on Cancer (IARC), and the
European Economic Community (EEC) Scientific Committee for
Food. It is recognized that, where possible, a unified approach
should be adopted. However, the purposes for which these other
bodies have formulated guidelines differ in detail from those of
JECFA, and it is inappropriate to adopt these without modifi-
cation to meet the needs of JECFA.
2.2. Periodic Review
2.2.1. Concept of periodic review
JECFA has indicated that, in discharging its duty to eval-
uate the safety-in-use of intentional and unintentional food
additives, it may be necessary to carry out a periodic re-
evaluation of substances previously assessed by the Committee.
The first JECFA meeting, in looking ahead, envisaged, in
addition to the continuing evaluation of food additives, that
there would be a re-evaluation process associated with the
programme on food additive safety assessment (9). It stated:
"Permitted additives should be subjected to continuing
observation for possible deleterious effects under
changing conditions of use. They should be reappraised
whenever indicated by advances in knowledge. Special
recognition in such reappraisals should be given to
improvements in toxicological methodology."
This principle was endorsed in the third (25), seventh (26),
eighth (11), and ninth reports (12) of JECFA.
The "need for review of past recommendations" was high-
lighted in the thirteenth JECFA report as follows (27, p. 22):
"There is a widespread but fallacious belief that
clearance of an additive for use in food constitutes an
irrevocable decision. Such a view renders a grave dis-
service to the cause of consumer protection for it
fails to recognize the need for regular review of all
safety evaluations."
Periodic review of past decisions on safety is made
necessary by one or more of the following developments (27):
(a) A new manufacturing process for the food additive.
(b) A new specification.
(c) New data on the biological properties of the compound.
(d) New data concerning the nature, or the biological
properties, or both, of the impurities present in a
food additive.
(e) Advances in scientific knowledge germane to the nature
or mode of action of food additives.
(f) Changes in consumption patterns or level of use of a
food additive.
(g) Improved standards of safety evaluation. This is made
possible by new scientific knowledge and the quality
and quantity of safety data considered necessary in the
case of new additives. Since JECFA began the eval-
uation of food additives in 1956, the paucity of
information available on many food additives has been
such that assessments have often been difficult to
make. Tests of too short duration, conducted with a
very small number of animals at inappropriate dose
levels, and without adequate clinical, haematological,
chemical, or histopathological examinations have
frequently been encountered among the data submitted
for evaluation. Tests of this sort cannot be regarded
as having permanent validity; with the passing of time,
they need to be supplemented by studies carried out in
full accordance with the recommendations set out in the
report of the WHO Scientific Group on Procedures for
Investigating Intentional and Unintentional Food
Additives (2).
It should also be noted that the second Joint FAO/WHO
Conference on Food Additives (7) recommended that it (JECFA)
"should revise, if needed, the toxicological evaluation of all
additives considered in previous meetings of the Expert
Committee."
The seventeenth report of JECFA (16) reads, in part:
"The objective in assessing the toxicological data on
food additives is to ensure their safety for the con-
sumer on the basis of all the evidence available to the
Committee at the time. Future results with present
methods or with techniques yet to be developed will
necessitate reassessments that may lead to changes in
earlier decisions."
Other meetings reaffirmed the need to take advantage of
recent developments in toxicological techniques for research and
safety assessment.
These Committee recommendations and observations, the
rationale set forth in the thirteenth report (27) and other
reports for the need to review past decisions, and the ensuing
years of progress in the science of toxicology and refinement of
research, evaluation procedures, and changing consumption
patterns all point towards the advisability of periodic review
of this large class of substances.
2.2.2. Mechanism of periodic review
That a considerable amount of re-evaluation of substances is
already carried out within the system is evident when the year-
to-year agenda of JECFA is examined. Food additives are reass-
essed when new biological and chemical data are made available
to FAO and WHO. In fact, new data are mandated on timetables
established by JECFA when temporary ADIs are established (sec-
tion 5.5.5). In addition, re-evaluations are made at the request
of Member States and by the Codex Alimentarius Commission.
However, for many additives, the assessment has not been
conducted using the more recently adopted procedures for
investigating intentional and unintentional food additives. A
review of past decisions also reveals that some additives have
only had a cursory examination. The evaluation of these addi-
tives may have been based on limited data.
A periodic review programme on substances previously
reviewed by JECFA should be constituted to reflect the changing
state-of-the-art and to provide the best possible assurance to
consumers of food additive safety. However, a mechanism has not
yet been developed for the continuous systematic updating of
safety information on food additives. Of course, even in the
absence of a periodic review programme, if new data on a food
additive raises suspicion of significant hazard, then immediate
re-evaluation is conducted.
The use of an international forum to devise and implement a
system for the periodic review of chemicals used in or on food
and contaminants of food could also be of great economic and
practical value to Member States. It would ensure a uniform
approach to a complex toxicological problem, duplication of
effort would be minimized, and emphasis on such a programme
would give added reassurance to consumers throughout the world
that the food supply continues to be safe. Perhaps such a
programme could be developed in cooperation with the Codex
Alimentarius Commission.
3. CRITERIA FOR TESTING AND EVALUATION
JECFA has always operated on the principle that testing
requirements for all food additives should not be the same.
Such factors as expected toxicity, exposure levels, natural
occurrence in food (section 6.1), occurrence as normal body
constituents (section 5.2), use in traditional foods, and
knowledge of effects in man (section 5.4) should be taken into
account. In relation to carcinogenic hazards, the Committee has
stated that "the scope of the test required should depend on a
number of factors, such as the nature of the substance, the
extent to which it might be present in food, and the population
consuming it" (4). More generally, the Committee has requested
data on, inter alia, method(s) of manufacture, impurities, fate
in food, levels of use of additives in food, and estimates of
actual daily intake, and concluded that such information "was
important and relevant both for the toxicological evaluation and
for the preparation of specifications" (22). However, difficul-
ties arise when an attempt is made to determine testing require-
ments because of problems in predicting toxicity, estimating
levels of food additive use and natural occurrence, and
obtaining human data. As discussed below, criteria for testing
requirements can also be used to allocate priorities for the
testing and evaluation of food chemicals.
3.1. Criteria for Testing Requirements
The establishment of principles for determining the
appropriate amount of data that will be required to adequately
evaluate the safety of additives at their estimated consumption
levels is urgently needed to ensure consistency in decision-
making by the Committee and to provide guidance to sponsors of
food chemicals. Both exposure data and potential toxicity
should be important considerations in the establishment of these
principles. Consideration of only one of these elements to the
exclusion of the other leaves serious deficiencies. If only
exposure data are used, then no consideration is given to the
wide range of toxicities observed among chemicals and no
advantage is taken of the vast amount of bioassay data already
in existence. If, on the other hand, only toxicity information,
predicted or known, is used, then chemicals with known toxic
properties or those related to chemicals of known high toxicity,
particularly carcinogenicity, would automatically require the
most data, with no consideration given to relatively low
exposure levels.
3.1.1. Estimating exposure
For the purpose of this publication, exposure is defined as
the total intake of a chemical substance by human beings. For
the majority of substances evaluated by JECFA, the primary mode
of exposure is through ingestion of the substance in the food
supply.
Estimates of exposure used by Committees in previous years
are of three general types: per capita estimates, estimates from
dietary food intake surveys, and analytical values from market-
basket/total-diet surveys. For a detailed discussion of the
advantages and the use of these different types of estimates see
reference no. 28.
The per capita approach is an estimated value that repre-
sents the exposure level if a food additive or contaminant were
equally distributed across a population. For example, a per
capita intake for a nation may be calculated by dividing the
total yearly production volume, corrected for imports and
exports, of a chemical used in food, within a nation, by the
national population. Another form of per capita intake may be
computed from a nation's per capita disappearance of a certain
food commodity multiplied by the usual level of an additive or
contaminant in the food commodity. These per capita intakes can
be converted to daily intake per kilogram body weight.
In some countries, dietary surveys are performed on food-
stuffs consumed by a representative group of individuals, within
a national population, over a short period of time, e.g., 1 - 14
days. The intake of an additive or contaminant, per food type,
can be calculated by multiplying the usual additive or contam-
inant level in each type of food by the dietary intake of the
food. The intakes per food type can then be summed to derive a
total additive or contaminant intake. An advantage of the
dietary survey approach is that additive or contaminant intakes
for selected subpopulations, such as different age groups or
high-frequency consumers of certain foodstuffs, may be computed,
depending on the specificity of the dietary survey.
When considering intakes computed by the dietary survey
approach, the tenth meeting of JECFA (29, pp. 23-24) reaffirmed
the validity of calculating the average daily intake of a food
additive based on: (a) levels arising from good technological
practice; (b) average consumption of foods containing the
additive; and (c) average body weight. This Committee also
noted at the time that, while data on average food consumption
were available from many countries, high consumption data were
available from only two countries. The Committee recognized a
special need for determining how much of a food additive is
likely to be consumed by groups that have a high level of
consumption and strongly recommended that every effort should be
made to obtain such information on food consumption.
The fourteenth meeting of JECFA (30) considered methodol-
ogies for computing additive intakes from dietary food surveys,
and recognized the importance of experimental design so that
collective data can be used for calculating reliable intakes on
an individual basis. The Committee noted difficulties in common
descriptors for food items, when information is gathered in
surveys performed by different organizations, and in obtaining
confidential information about food additive use from industry.
Market-basket surveys (also called total-diet surveys)
involve analyses of representative diets for the usual level of
additive or contaminant in the diet. The analyses may be per-
formed on food mixtures or on individual foodstuffs. The
selection of foods represents a normal diet for a certain popu-
lation, such as the typical daily diet for a certain nation's
average consumer. Market-basket surveys can be used for
estimating the actual level of additive or contaminant in a
selected total diet, which is of value for substances present in
food at levels that are less than the amounts added. However,
the difficulties of analyses usually restrict this approach to
estimations of average intakes of contaminants in samples repre-
sentative of the average dietary habits of a nation's general
population rather than estimations of intakes for selected
subpopulations. In this regard, data on certain contaminants in
food are available from the Global Environmental Monitoring
System (GEMS).
These procedures are useful for estimating exposure to food
additives in individual countries. However, accurate estimation
is much more difficult when attempted on a global scale.
Clearly, consumption of a food additive will not be the same in
two countries in which it is regulated with differing restric-
tions or with very different food consumption patterns. To use
exposure estimates on such a scale as a criterion for testing
requirements or for setting priorities for the testing of food
additives is an extremely ambitious exercise that would require
extensive resources.
3.1.2. Predicting toxicity from chemical structure
Chemical structure determines to a great extent the attitude
of the toxicologist towards a compound. As a result, there have
been many efforts to systematize the use of chemical structure
as a predictor of toxicity. The use of such relationships has
been suggested by JECFA with certain classes of flavouring
agents (section 6.1.2), and chemical structure is an important
consideration in the selection of compounds for carcinogenicity
testing. Structure/activity relationships also form the basis
for establishing group ADIs (section 5.5.4).
Structure/activity relationships appear to provide a
reasonably good basis for predicting toxicity for some
categories of compounds, primarily carcinogens, which are char-
acterized by specific functional groups (e.g., nitrosamines,
carbamates, epoxides, and aromatic amines) or by structural
features and specific atomic arrangements (e.g., polycyclic
aromatic hydrocarbons and aflatoxins). However, all these chem-
ical groups have some members that do not seem to be carcino-
genic or are only weakly so. Since structure/activity relation-
ships are better established for carcinogens than for other
toxic end-points, dependence on such predictions emphasizes
suspect carcinogens at the expense of other forms of potential
toxicity. However, as more chemicals are tested for toxicity
and other end-points are identified in the future, the data base
will become larger, which should permit more valid comparisons
between structure and toxicity among more classes of compounds.
In terms of carcinogenic substances, another system that has
sometimes been used for predictive purposes is a battery of
tests for genotoxicity (possible applications of such tests are
discussed in section 5.1.5).
3.1.3. Other factors to consider when developing criteria
The value of structure/activity relationships and exposure
data in determining the extent of testing required may be
considerably enhanced by collateral information on metabolism
and pharmacokinetics. It has been previously accepted that:
"if a series of chemical analogues can be shown to give
rise to the same metabolic product. . . it may be suf-
ficient to carry out toxicological studies on a suit-
able representative of the series" and "where adequate
biochemical and toxicological data on closely related
chemicals are available, the objective (of toxicity
tests) becomes the detection of any deviation from the
established pattern. This can usually be determined by
intensive studies of a few months duration when these
are adequately designed and evaluated" (2).
More recently (31), JECFA has concluded that:
"if the chemical structure of a compound under consid-
eration did not closely resemble that of any known
toxic or carcinogenic compound, and, if the toxicolo-
gical data on it, its metabolites, and its homologues
did not give any cause for concern, these less exten-
sive toxicological data might be used for the evalua-
tion of the compound. . . . In the evaluation of a
series of structurally-related compounds, complete
toxicological data should be available for at least one
member of the series. Other compounds in the series
should be evaluated on the basis of these data, plus
data on their natural occurrence and metabolism, and on
the toxicology of their homologous compounds."
These principles can form the basis for determining the lim-
ited amount of testing that may be required for compounds that
are closely related structurally. If the toxicological data
base is adequate for the homologous compounds and suggests a low
intrinsic toxicity, metabolic and pharmacokinetic data alone may
be sufficient to make an evaluation of a related compound.
The results of studies on absorption, distribution, and
metabolism may either increase or decrease the health concern
from the use of the additive. For example, a relatively non-
toxic additive may be transformed by liver enzymes into a sub-
stance with a much greater toxic potential, or vice versa.
Correlations between structure and activity will often auto-
matically include these considerations, because substances of a
particular class will often be absorbed, distributed, and meta-
bolized in similar ways. However, this will not always be the
case, and these parameters should be specifically considered
when making such correlations.
Other factors influence the extent and type of testing
required for safety assessment. For example, the need for
extensive testing may be mitigated when the substance occurs
naturally in food and has a history of human use or when it is
metabolized into normal body constituents (section 5.2.3). More
extensive testing in animals may be necessary when the additive
will be used in special populations at risk, such as pregnant
women and very young infants (section 5.3). Human testing may
be needed if problems of intolerance arise (section 5.4.2). The
types of end-points, as discussed in section 5.1, must be
considered in any criteria system that is established.
The development of criteria for determining the extent of
required testing is worthy of extensive future study. Its value
would be considerably enhanced by including it in the context of
a priority-setting scheme, as discussed below, because additives
should not be considered in isolation from one another.
3.2. Priorities for Testing and Evaluation
The primary basis for establishing the list of substances to
be considered by JECFA is the recommendations of the Codex
Committee on Food Additives (CCFA) and Member Governments.
However, Committees have recognized the need for the estab-
lishment of a "priority list as a means of selecting the most
relevant compounds for future evaluation. In order to establish
priorities for the toxicological testing and evaluation of
intentional and unintentional food additives", JECFA recommended
at the twenty-second (32) and twenty-third (31) meetings that:
"FAO and WHO convene an inter-disciplinary group of
experts to establish an inventory of compounds that
have not yet been fully evaluated and to classify them
in terms of their potential hazard to health on the
basis of toxicological knowledge and extent of use."
The Committee has recognized that the most obvious need for
allocating priorities is for the testing and safety evaluation
of food flavouring agents (19). Committees continue to stress
the need to establish priorities for testing and evaluating food
additives (24,33).
One basis for establishing priorities for testing food
additives is by using an index based on exposure levels and
predicted toxicity. For examples of approaches using these
parameters, see references 34-38.
As discussed in section 3.1.1, valid exposure data are
extremely difficult to develop. However, comparative levels of
consumption may be sufficient for the purpose of setting prior-
ities for the testing of food additives. Therefore, even though
accurate consumption estimates of wide geographical relevance
will probably never be achieved, the lesser requirement of com-
parative estimates may be achievable to the extent necessary for
JECFA's use, if the Committee decides to develop the informa-
tion.
Because of the semiquantitative nature of much of the
biological data available for predicting toxicity, rigorous
analytical or statistical interpretation is not always possible.
Therefore, expert interpretation and evaluation of the data, a
time-consuming and expensive procedure, must be integrated into
any automated decision-making mechanism that is developed. To
ensure maximum usefulness, the priority-setting system should
take account of all available toxicity and other biological
information, including metabolic and human data. A properly-
devised system will be capable of considering new data and can
be modified using modern data processing methods and equipment.
3.3. Quality of Data
In recent years, various national regulatory agencies and
international bodies have instituted codes of Good Laboratory
Practice (GLP), the aim of which is to help underwrite the
validity of studies by ensuring that they can be verified and
reproduced. GLP codes require the maintenance of certain
records regarding the performance of studies, including data
from chemical and toxicological tests, which help ensure full
documentation of the conduct and results of studies. However,
GLP codes are not a substitute for scientific quality; an
inappropriate study may be conducted according to GLP standards.
On the other hand, a study that does not meet GLP criteria may
still be scientifically sound.
JECFA has always judged studies on their merits, the main
criteria being that the study was: (a) carried out with
scientific rigor, and (b) reported in sufficient detail to
enable comprehensive evaluation of the validity of the results.
Usually, studies that are published in the scientific
literature are subjected to peer review prior to publication,
and after publication, the results are open to refutation or
confirmation. Unpublished reports, on the other hand, are not
necessarily subjected to this scrutiny. For this reason, JECFA
has repeatedly recommended that data brought before it be
published (10, p. 6; 12, p. 7; 39, p. 7; 40). However, in point
of fact, JECFA does review many high-quality studies that remain
unpublished for proprietary and other reasons. Also, the
Committee often requests unpublished raw data when published
reports do not include sufficiently detailed data for an
adequate safety review. Studies performed in compliance with
GLP codes provide added assurance that the quality of
unpublished data is acceptable. For these reasons, it is
appropriate that JECFA experts continue to consider all the data
brought before them, published, and unpublished, and make
decisions about the validity of these data on an ad hoc basis.
This means that the studies reviewed by the Committee should be
fully documented.
4. CHEMICAL COMPOSITION AND THE DEVELOPMENT OF SPECIFICATIONS
The proper safety evaluation and use of food additives
requires that they be chemically characterized. Therefore, the
Committees review data relating to the identity of additives,
impurities that may be present, and possible reaction products
that may arise during storage or processing. "JECFA
specifications" are then elaborated, taking these and other
factors into account.
4.1. Identity and Purity
To establish the chemical identities of food additives, it
is necessary to know the nature of the raw materials, methods of
manufacture, and impurities (22). This information is used to
assess the completeness of analytical data on the composition of
additives and to assess the similarity of materials used in
biological testing with those commercially produced. From
information on raw materials and methods of manufacture,
potential impurities in commercially manufactured chemical
materials due to carry-over of contaminants in raw materials and
by-products of the manufacturing process can be predicted.
To evaluate biological testing data from multiple studies,
JECFA must have information on the chemical composition of the
tested materials, which necessitates manufacturing information.
Analytical data on the chemical composition of materials used in
biological testing should be more detailed than a standard
presentation of chemical specifications. Furthermore, materials
used in biological testing should be representative of
substances manufactured by actual commercial processes so that
the materials administered to experimental animals will
represent those ingested by consumers.
A food additive may be a single chemical substance, a
manufactured complex chemical mixture, or a natural product.
The need for complete information on chemical composition,
including description, raw materials, methods of manufacture,
and analyses for impurities, is equally valid for each type of
additive. However, implementation of the requirement for
chemical composition data may vary depending on the type of
substance. For additives that are single chemical substances,
it is virtually impossible to remove all impurities in their
commercial production; therefore, analyses are generally
performed on the major components and predicted impurities, with
the highest significance placed on potentially toxic impurities.
For commercially manufactured complex mixtures, such as mono-
and diglycerides, information is needed on the range of
substances commercially produced, with emphasis on descriptions
of manufacturing processes, supported by analytical data on the
components of the different commercial products. Natural pro-
ducts present particularly difficult problems because of their
biological variability and because the chemical constituents are
too numerous for regular analytical determinations; thus, the
analyst is starting with an "unknown". For additives derived
from natural products, it is vital that the sources and methods
of manufacture are precisely defined. Chemical composition data
should include analyses for general chemical characteristics,
such as proximate analyses for protein, fat, moisture, carbo-
hydrate, and mineral content, and analyses for specific toxic
impurities carried over from raw materials or chemicals used in
the manufacture of the substance. Further information necessary
for the evaluation of "novel foods", which are usually sub-
stances derived from natural products, is provided in sections
6.2.1 and 6.2.4.
4.2. Reactions and Fate of Food Additives and Contaminants in Food
Biological testing of food chemicals must relate to their
presence in food as consumed. This is an important consider-
ation when added substances undergo chemical change in food.
Therefore, data are necessary on the reactions and fate of addi-
tives or contaminants in food and their effects on nutrients
(22).
Certain food additives perform their functional effect by
reaction with undesirable food constituents (e.g., antioxidants
react with oxygen in food and EDTA reacts with trace metals) or
by reactions that modify food constituents (e.g., potassium
bromate reacts with dough constituents). Food additives may
also degrade under certain conditions of food processing, though
such degradation is detrimental to their functional effect. For
example, the sweetener aspartame is transformed to a diketo-
piperazine derivative at rates varying with the acidity and the
temperature of the food. In previous evaluations of "reactive"
additives, Committees have evaluated analyses for additive
reaction products in food, as consumed, and biological testing
data on either specific reaction products or samples of food
containing the reaction products as consumers would ingest
them.
For all intentional food additives proposed for evaluation,
Committees request submission of four types of data related to
reactivity:
(a) the general chemical reactivity of the additive;
(b) its stability during storage and reactions in model
systems;
(c) reactions of the additive in actual food systems; and
(d) the additive's fate in living systems. These data are
important for relating biological test data to the
actual use of the additive in food.
Processing aids are substances that come into contact with
food during processing and may unintentionally become part of
food because of their incomplete removal. Committees have eval-
uated a number of processing aids, such as extraction solvents
and enzyme preparations, for their safety in use. When evalua-
ting a processing aid, information should be provided on its use
and either analytical data on the amount of the processing aid
carried over into food or a computed estimate of the amount to
be expected in food. In some cases, a component of the proces-
sing aid may have the greatest potential for biological effects,
such as ethylenimine leaching from polyethylenimine, an immobil-
izing agent used in the preparation of immobilized enzyme
preparations.
Contaminants in food evaluated by previous Committees
include environmental contaminants and substances migrating from
food packaging. Of environmental contaminants, metals have been
considered the most often. Committees request information on
the chemical forms of metals in the food supply (e.g., ionic
form and/or covalently bonded chemical form) and their concen-
tration distribution in the food supply, as determined by
analyses of food or experimental models for carry-over from
environmental sources. For contaminants derived from food
packaging, data are required on the identification of chemicals
migrating from the packaging material and concentrations in food
(analysed or estimated from migration modelling studies).
4.3. Specifications
Specifications are a necessary product of Committee eval-
uations, the purposes of which are 3-fold:
(a) to identify the substance that has been biologically
tested;
(b) to ensure that the substance is of the quality required
for safe use in food; and
(c) to reflect and encourage good manufacturing practice.
The first Joint FAO/WHO Conference on Food Additives (6)
established a programme for the collection and dissemination of
information on the chemical, physical, pharmacological, toxico-
logical, and other properties of individual food additives. The
first two meetings of the Joint Expert Committee, in preparing
reports on "General Principles Governing the Use of Food
Additives" (9) and "Procedures for the Testing of Intentional
Food Additives to Establish Their Safety for Use" (10),
recommended that specifications should be prepared, citing the
need for:
(a) limiting impurities in food;
(b) identifying materials used in toxicity testing; and
(c) ensuring that the additive tested is the additive used
in practice.
The third meeting of JECFA was devoted in its entirety to dev-
eloping principles governing the elaboration of specifications
and developing provisional specifications for the first group of
additives evaluated by the Committee (25).
JECFA specifications are minimum requirements for the compo-
sition and quality of food-grade additives, allowing for accep-
table variation in their production (18). These specifications
are meant to be used internationally and to the extent that data
are available, specifications are elaborated to cover suitable
products manufactured in various parts of the world. The third
meeting considered the value of specifications with regard to
protection for the consumer, advice to regulatory organizations,
standards for the food industry, and establishment of safety for
use (relative to identification of materials used in biological
testing in comparison with materials produced for commercial
use). The format for specifications established by this meeting
continues to be used in current JECFA specifications, that is,
the additive is identified by synonym, definition (chemical
name, formula, relative molecular mass, etc.), and description,
its functional uses are listed, tests of identity and impurities
are provided, and an assay for the major component(s) is pro-
vided. The third meeting of JECFA, recognizing that practical
specifications could not specify every impurity, limited the
scope of impurity tests to constituents of commercially produced
substances that: (a) were related to the safe use of the
additive; (b) might affect the usefulness of the additive; or
(c) would serve as an indicator of good manufacturing practice.
Finally, the meeting concluded that, for specifications to be
acceptable on a global basis, they must be subject to continuing
review and evaluation to take into account the presentation of
new information, particularly with respect to different
manufacturing processes and improved analytical methods.
In detailing the purposes of JECFA specifications, Commi-
ttees have, through the years, refined the scope of their spec-
ifications and provided advice on how they should be used.
Specifications developed by each Committee should be read in
conjunction with the report of that Committee. JECFA specifi-
cations apply to the material(s) that was toxicologically
reviewed and take into account the uses of the additive (17,
41). Periodic review of specifications is required, because of
changes in patterns of additive use, in raw materials, and in
methods of manufacture. Comments on JECFA specifications by
national and international organizations are valuable sources of
information for periodic review (18, 27, 29).
JECFA specifications in their entirety describe substances
of food-grade quality, and as such, they are directly related to
toxicological evaluations and to good manufacturing practice.
However, though specifications may include criteria that are
important for commercial users of additives, they do not include
requirements that are of interest only to commercial users
(42).
Differences may exist between specifications prepared by
national and international organizations; however, the Committee
is not aware of any information indicating that these differ-
ences incur health risks for consumers (23). JECFA specifica-
tions are meant to be minimum requirements for the safe use of
additives, and not every component of commercially manufactured
chemical substances is subject to an impurity test (11). Test
requirements in JECFA specifications are sufficient to ensure
the safe use of commercially manufactured food additives. Sub-
stances of higher chemical purity (e.g., analytical grade
reagents) are not excluded from use in food, even though such
substances may deviate somewhat from the identification tests in
the specifications, provided that they meet the stated require-
ments for specified purity tests and are otherwise suitable for
use as food additives (18).
Since 1956, the meetings of JECFA have designated specifi-
cations as either full or tentative. Specifications were given
the tentative designation from the third to twenty-second
meetings because either the chemistry data needed to prepare
specifications were not adequate or a temporary ADI was assigned
to the additive. At, and since, the twenty-third meeting of
JECFA, the tentative designation has been assigned only when the
data necessary for preparing specifications were insufficient.
JECFA policy has been to prepare specifications for sub-
stances added to food, whenever constituents of the substance
had the potential to be present in food. Initially, specifica-
tions were prepared only for intentional food additives that
were added directly, to accomplish a functional effect in food.
The fourteenth JECFA (30) evaluated extraction solvents, the
first group of "processing aids" that had been reviewed by
JECFA. This Committee concluded that, although extraction sol-
vents are substantially removed from food, evaluation of the
conditions of safe use of these solvents depends on the identity
and purity of the solvents. Therefore, JECFA specifications
were prepared. Since then, specifications have been reported
for other processing aids such as anti-foaming/defoaming agents,
clarifying agents, decolourizing agents, enzyme preparations,
filtering aids, packing gases, propellants, lubricants/release
agents, odour/taste-removing agents, and yeast "food" (yeast
nutrients). The twenty-seventh JECFA (24) decided that chemical
reagents used in the preparation of food additives or processing
aids (such as glutaraldehyde in the preparation of immobilized
enzyme preparations or acetic anhydride in the manufacture of
modified starches) do not usually need specifications. Carry-
over of these reagents or their contaminants into food may be
controlled in the specifications for purity of the additive or
processing aid.
Food additives may be marketed as formulated preparations,
such as a mixture of a main ingredient with a solvent vehicle
and emulsifier. Specifications refer only to each ingredient in
the formulated preparation as individual commercially-manufac-
tured food additive substances. Mixtures should not be formu-
lated in such a way that the absorption or metabolism of any
ingredient is altered so that the biological data are invali-
dated (12, 42). Added substances such as anticaking agents,
antioxidants, and stabilizers may also influence the results of
tests given in specifications. Therefore, in its nineteenth
report, JECFA recommended that manufacturers of food additives
should indicate the presence of such added substances (18).
In considering whether specifications apply to food additive
quality as manufactured or as received, JECFA has decided to
prepare specifications to cover the normal shelf-life of the
product. Limits are set for decomposition products that may
form during normal storage. Manufacturers and users of food
additives should ensure good packaging and storage conditions
and use good handling practices to minimize deleterious changes
in quality and purity (18). Information on changes in the
composition of food additives during storage should be submitted
for evaluation by the Committee.
In addition to periodic reviews to examine the consistency
of specifications within classes of similar additives, JECFA
periodically reviews specification test methods to update the
analytical methodology of specifications. Two summaries of
specification test methods have been published (43, 44), which
provide guidelines for the application and interpretation of
specification requirements and test methods. JECFA has made
considerable progress in adopting modern analytical methodology
for specification tests, whenever equipment and other supplies
needed to perform the tests are accessible on a world-wide
basis. However, because JECFA specifications are elaborated for
world-wide use, certain analytical methods involving recently-
developed techniques or equipment cannot be included until such
techniques are available on an international scale. Alternative
methods of analysis can be used to test products for conformity
with specifications, provided that the methods and procedures
used produce results of equivalent accuracy and specificity.
In order to foster international agreement on specifications
for food-grade substances, JECFA seeks comments from member
countries and international organizations. The Codex Aliment-
arius Commission systematically provides comments on JECFA
specifications through the Codex Committee on Food Additives
(CCFA) and endorses certain JECFA specifications as "Codex
Advisory Specifications". The systematic review of JECFA
specifications by the CCFA has provided JECFA with valuable data
on novel manufacturing processes, previously unknown impurities,
updated methodology, and advice on the format of JECFA speci-
fications.
Although JECFA specifications and those of the Codex
Alimentarius Commission are elaborated for many of the same
purposes, the interpretation of these purposes may result in
differences in specific requirements or test methods for the
same food-grade substance. In replying to suggested changes in
JECFA specifications from the CCFA or other interested parties,
it may be decided to amend existing specifications, providing
that the requested changes do not significantly lessen the
assurance of food-grade quality embodied within the JECFA
specifications and that the requested change conforms with the
principles for elaboration of specifications established at
previous meetings. A requested change in an existing full JECFA
specification must be supported by scientific data.
5. TEST PROCEDURES AND EVALUATION
5.1. End-Points in Experimental Toxicity Studies
There are virtually no findings in experimental toxicology
that can be simply extrapolated to man without careful thought.
During the past two decades, there has been an increasing emph-
asis on carcinogenesis as a manifestation of chemical toxicity.
Most of the other manifestations of chemical intoxication, for
example, immunosuppression, have, by comparison, received rela-
tively little attention. This has resulted in an unbalanced
approach by the toxicologist in which the emphasis on end-points
bears a less and less obvious relationship with disease patterns
in man. For example, a survey of the recommendations for the
evaluation of food additives has revealed that little or no
attention is paid to the detection of cardiovascular lesions,
even though these lesions are the most common cause of fatal-
ities in the human population in developed countries. In
addition, certain lesions commonly found in rodents that are the
primary targets of the toxicologist do not have any counterpart
in man. It seems reasonable that an effort should be made to
relate toxicological findings more carefully to the human situ-
ation, recognizing that this will be a long-term project. In
the meantime, when conducting experimental animal tests, special
attention should be paid to alterations that indicate a poten-
tial for the test compound to adversely affect the cardio-
vascular, immunological, reproductive, or central nervous
systems. If such alterations are detected, they should be
investigated further using special studies aimed at clarifying
their significance.
The end-points discussed in this section have been grouped
for convenience into effects with:
(a) functional manifestations only;
(b) non-neoplastic morphological characteristics;
(c) neoplastic manifestations; and
(d) reproduction/developmental manifestations.
In view of the large number of effects encountered, it is
possible to summarize only some of the specific observations.
However, situations that have become controversial are dealt
with in more detail.
Finally, this section concludes with a short discussion on
the role of short-term in vitro tests in the safety assessment
of food additives.
5.1.1. Effects with functional manifestations
Generalized weight loss, although having causes that are not
solely physiological (section 5.5.3), does not necessarily
involve any particular pathological lesions (section 5.5.3).
Reduced weight gain has played a major role as an end-point in
toxicological determinations in various ways. In a sense, it
has often been used for determining various empirical indices in
the absence of other manifestations. The procedures followed by
JECFA for determining an ADI demand that a no-observed-effect
level should be established. For this level to be established,
it is necessary to establish an effect level and, when all else
has failed, a generalized decrement in weight gain has been used
for this purpose, provided reduced food intake is not the
obvious cause. The other major use of a decrease in weight gain
has been in establishing a maximum tolerated dose (MTD) (for a
definition of the MTD, see Annex I).
Among the commonest effects observed in studies on food
additives is a laxative effect; the physiological reasons for
this are usually quite apparent and can be taken into account
when considering the appropriate levels of use of additives
causing this effect. In most instances, additives have been
permitted that cause laxation at high levels in man when they
have been otherwise non-toxic and can be used effectively at
levels at which laxation does not occur.
Although a great deal has been said about the need to
evaluate food additives and contaminants for the induction of
possible behavioural changes, JECFA has hitherto devoted little
time to evaluating such changes. Since it has been suggested
that certain food constituents can produce behavioural changes
in man, JECFA will, in the future, undoubtedly have to consider
such effects. Unfortunately, good animal models have not been
developed, and objective human data are difficult to obtain. It
is not possible to recommend a simple series of tests at this
time, primarily because there is no clear consensus on the kinds
of studies that should be performed nor on the interpretation of
the results.
Intolerance to food additives should always be considered a
possibility, even though tests for reactions to food additives
are not part of the normal data package that JECFA considers
when assessing new food additives. Even when evidence of
widespread intolerance to a food additive appears, it may still
be very difficult to determine a cause-and-effect relationship.
Problems include the often-anecdotal nature of much of the
evidence, psychological factors, and problems with developing an
adequate central data collection system. These points are
discussed in more detail in section 5.4.
5.1.2. Non-neoplastic lesions with morphological manifestations
A number of lesions are relatively frequently observed in in
vivo studies, particularly in rodents, that often give rise to
controversy.
Non-specific liver enlargement or hypertrophy has been
discussed by a WHO scientific group (2, pp. 18-19). In the
past, this occurrence has been considered to be a manifestation
of toxicity. More recently, it has been realized that this can
often be a physiological response involving the induction of
microsomal enzymes in the detoxification process that is
reversible on removal of the test compound.
The formation of calculi in the urinary bladder is a
frequent finding in rodent studies. Often, the formation of
calculi may be followed by the formation of bladder tumours. It
is not uncommon to find calculus formation in one rodent species
and not in another. Under these circumstances, the nature of
the calculi can sometimes be associated with specific metabolic
changes that have led to their formation. This, in turn, may
allow for a scientifically-based extrapolation to man, providing
that human clinical studies are possible.
Caecal enlargement, a common finding in rodent studies, is a
normal finding in rodents maintained on standard laboratory
diets under germ-free conditions. It is also a common response
of rodents, especially rats, to diets that include non-nutrient
substances (e.g., certain permitted food colours and saccharin)
or certain nutrients in excessive concentrations (e.g., modified
starches, plant gums, lactose, and various polyols).
Most of the enlargement is attributable to increased luminal
contents; in addition, the weight of the caecal wall after
washing out the luminal contents is usually marginally more than
normal. In haematoxylin- and eosin-stained sections, the caecal
wall shows no remarkable features, and there is no evidence that
caecal enlargement predisposes to any form of neoplasm of the
caecum. Caecal enlargement may be due to osmotic effects, but
its mechanism is not well understood. In some cases, caecal
enlargement is an incidental finding, with the primary effect
being nephrocalcinosis (23, pp. 11-12). Various forms of
mineral deposition occur in the kidneys of laboratory rodents,
more commonly in rats than in mice or hamsters. Unless
appropriate diagnostic staining or chemical analysis is carried
out, it is not strictly justifiable to refer to these changes as
renal "calcinosis", though most of the mineral deposits do, in
fact, contain calcium in one form or another. Mineral
deposition can take place in almost any part of the nephron and
deposition may predominate in one or more areas of the kidney.
The main forms of renal mineralization are basement membrane
mineralization, corticomedullary nephrocalcinosis, pelvic neph-
rocalcinosis, and nephrocalcinosis associated with acute tubular
nephrosis. All these forms of nephrocalcinosis may co-exist,
and one and the same agent may cause more than one form of neph-
rocalcinosis.
Magnesium deficiency in standard laboratory diets undoubt-
edly contributes to the high incidence of corticomedullary neph-
rocalcinosis in rats. Excessive dietary phosphate and possibly
excessive dietary calcium may predispose to pelvic nephrocal-
cinosis. Such observations lead to the conclusion that more
attention needs to be paid in the future to the formulation of
diets for rodents with respect to physiologically-relevant
levels of calcium, magnesium, and phosphorus.
Testicular atrophy, which is sometimes observed in rodents,
may occur as a result of reduced caloric intake, frequently as a
result of the addition of an unpalatable chemical to the
animal's food or drinking-water. This should be distinguished
from testicular atrophy resulting from the direct action of the
chemical on the testicular cells. This distinction can be
achieved by undertaking paired feeding studies. It is important
that the function of the testes be investigated in reproduction
studies when atrophy is detected.
Manifestations of vitamin deficiencies, notably of the fat-
soluble vitamins, are sometimes observed in studies on agents
that may be fat solvents and are only partly absorbed in the
gastrointestinal tract; an example of such a substance is
mineral oil. Another effect that is sometimes observed is dis-
colouration of mesenteric lymph nodes after feeding a coloured
substance. This is a normal physiological response and should
not be considered a toxic end-point, as long as it is not asso-
ciated with proliferative reactions.
Hormonally-associated effects occur with certain additives
and require special endocrinological evaluations. Recently,
JECFA has been faced with the task of evaluating the use of
certain anabolic agents used in the raising of meat-producing
animals (22, 23). These agents result in the presence of low-
level residues of hormonally-active compounds in meat. The
evaluation of the potential toxic effects of such compounds
requires knowledge of the levels of naturally-occurring com-
pounds with similar effects (Annex III).
5.1.3. Neoplasms
The most difficult decisions facing the toxicologist arise
from the varied end-points found in long-term in vivo carcino-
genesis studies. These problems have existed for a long time,
but they have been greatly exacerbated by the large number of
effects observed in the many rote tests now performed on
chemicals including many that may be present in the food supply.
These chemicals include certain direct food additives, some
processing and carrier solvents, components of packaging
materials, and a variety of contaminants.
Oncologists now generally recognize that different mech-
anisms of carcinogenesis exist with different chemicals acting
on different tissues in the body (45, 46). Many believe that it
may be possible to determine tolerance levels for some carcino-
gens, though this is still not possible with any degree of cer-
tainty with the majority of them. The view that a tolerance may
be set for carcinogens giving rise to tumours through either a
hormonal mechanism or by the formation of bladder calculi has
been expressed by a WHO scientific working group (3, p. 11).
The perception that a chemical for which there is evidence
of "carcinogenicity" in any system should not be permitted for
use as a food additive at any dose whatsoever has become wide-
spread. Although this philosophy has never been promulgated or
officially adopted by JECFA, it has, in practice, influenced the
Committee's approach to decision making. Probably more experi-
mental work has been undertaken in cancer research over the past
two decades, since this view was first established, than in all
the preceding years, and clearly there is a great need to
clarify the issue in terms of practical interpretation.
The assessment of the evidence for the carcinogenicity of
chemicals is a major issue to be resolved by JECFA. Not only
have many chemicals been tested by a variety of routes of
administration that may not be relevant to food additive use
(such as the repeated injection of food colours and other addi-
tives in rats and mice with consequent development of subcut-
aneous sarcomas at the site of injection (27)), but, in addi-
tion, new end-points are continually revealed and interpretation
becomes more confusing as studies become more and more detailed.
Positive results may be obtained due, for example, to a carcino-
genic impurity. The extrapolation of such data has become very
complex. One possibility, to make the term "carcinogen" more
generalized, clearly would not solve the problem of how best to
interpret these data.
Much of this issue centres around the meaning of the various
types of enhancement of the tumours that occur in the rodents
used for in vivo bioassays, since the rodents in common use, the
mouse and the rat, develop extremely high incidences of a
variety of tumours in the untreated state. Many reports indi-
cate that one or more of these tumours has an increased inci-
dence or has appeared earlier (or both) in treated, compared
with untreated animals. One problem in interpreting the signi-
ficance of these tumours is the difficulty in deciding whether
these naturally-occurring tumours are spontaneously induced or
whether the agent is able to induce them. The problem is fur-
ther confounded by the fact that the incidence of tumours in the
untreated control animals varies considerably with time. As a
result, there is now a debate as to the importance of historical
as well as concurrent control animals. It appears without doubt
that both such controls are of importance (especially when the
historical control data come from the same laboratory, using the
same standardized diet, and do not go back in history beyond 5
years of the study under consideration) and that, if the chem-
ical in question has only enhanced the incidence of a commonly-
seen tumour to a level seen in historical controls, then the
level of concern will be much less than would otherwise be the
case.
The evaluation of studies in which these commonly-occurring
tumours are a complicating factor need careful individual
assessment. The tumours that have given rise to the most
controversy in recent years are hepatomas (particularly in the
mouse), pheochromocytomas in the rat (see below), lymphomas and
lung adenomas in the mouse, pancreatic adenomas and gastric
papillomas in the rat, and certain endocrine-associated tumours,
including pituitary, mammary, and thyroid tumours, in both rats
and mice. Some of these tumours, such as hepatomas and lung
adenomas, may occur in the majority of untreated animals.
With the exception of lymphomas, some of which are virally
associated, the endocrine-associated tumours, and possibly
hepatomas in high-incidence strains of mice, which may involve
oncogenes (47), there is no clue as to the origin of tumours
that occur commonly in experimentally-used rodents. Indeed,
there is not even any cogent speculation as to the mechanisms by
which these tumour incidences are increased.
Adrenal medullary lesions in rats provide a good example of
the problems encountered in interpreting the significance of
high tumour incidences. An overview of the literature indicates
that untreated rats of various strains may exhibit widely dif-
fering incidences of lesions described as "pheochromocytomas"
(24, 48, 49). There are no clear criteria for distinguishing
between prominent foci of hyperplasia and benign neoplasms, and
pathologists differ in the criteria that they use for disting-
uishing between benign and malignant adrenal medullary tumours.
Rats fed ad libitum on highly nutritious diets tend to
develop a wide variety of neoplasms, particularly of the
endocrine glands, in much higher incidences than animals
provided with enough food to meet their nutritional needs but
not enough to render them obese. The adrenal medulla is just
one of the sites affected by overfeeding. Controlled feeding,
especially early in life, reduces the life-time expectation of
developing either hyperplasia or neoplasia of the adrenal
medulla in rats.
A complicating factor in assessing carcinogenicity studies
is the question of how to consider benign tumours. If benign
and malignant tumours are observed in an animal tissue and there
is evidence of progression from the benign to the malignant
state, then it is appropriate to combine the tumour types before
performing statistical analysis. Assessment of the relative
numbers of benign and malignant tumours at the various dose
levels in the study can help determine the dose response of the
animal to the compound under test. On the other hand, if only
benign tumours are observed and there is no indication that they
progress to malignancy, then, in most cases, it is not appro-
priate to consider the compound to be a frank carcinogen, under
the conditions of the test (this finding may suggest further
study). Often, how benign tumours should be considered is much
less clear. Some clarification can be achieved by classifying
and analysing tumours on the basis of their histogenic origin.
This is helpful, not only for determining the significance of
benign tumours, but also for preventing different malignant
tumours occurring in the same organ from being grouped together
for statistical analysis. For further discussion of these
points, see (50), pp. 226-230.
The results of statistical analyses are often misunderstood
and misused. An effect may be statistically significant but not
be of any biological significance, because the animal's well-
being is not affected by its occurrence. On the other hand, an
event that is of biological significance, such as the occurrence
of one or two tumours of a very rare type in treated animals,
may not be significant by the usual battery of statistical
tests. This difference between biological and statistical
significance underscores the need for critical analysis of
statistical results rather than the blind acceptance of the
numbers obtained. The statistical aspects of the design and
interpretation of toxicity studies are discussed in Annex II.
Generally, it is becoming increasingly difficult to identify
a substance as a carcinogen with confidence. In particular,
when animals with a high background incidence of tumours are
involved, it is extremely difficult to know when to draw the
line between a result that indicates that a compound that is
potentially hazardous for man has been discovered compared with
a compound that is merely an experimental curiosity. The
International Agency for Research on Cancer (IARC) reviews
evidence for the carcinogenicity of chemicals on a continuous
basis and drafts monographs on many groups of substances.
However, faced with the difficulty of separating different
levels of "carcinogenicity", IARC working groups on the
Evaluation of the Carcinogenic Risk of Chemicals to Humans
designate compounds as being possessed of either "limited" or
"sufficient" evidence for carcinogenicity. Given the natural
desire of the food additive toxicologist to be as cautious as
possible, this terminology is not very practical. After a
compound has been designated as possessing "limited carcino-
genicity", it is very difficult from a regulatory point of view
to approve it as a food additive, even if extensive further work
on the compound shows it to be safe at expected levels of con-
sumption with no other evidence of carcinogenicity.
The decisions that are being made on the basis of the
present state of knowledge of carcinogenesis, may, in the
future, prove to have been excessively conservative. However,
it is now possible to make certain reasoned decisions, provided
that each instance where carcinogenicity is the problem is
examined individually and all relevant factors are taken into
account.
5.1.4. Reproduction/developmental toxicity
Most food additives are consumed by men and women during the
reproductive stages of their lives and by pregnant and lactating
women. Some food additives are also consumed by infants. Thus,
a thorough safety evaluation requires that the effects of the
substance on reproductive performance and development from
fertilization through weaning be studied. JECFA recognizes,
however, that it is unrealistic to expect that such studies be
performed in all cases (sections 5.3.1 and 5.3.4).
Adverse effects on reproduction may be expressed through
reduced fertility or sterility in either the parents or off-
spring due to morphological, biochemical, or physiological dis-
turbances. Adverse effects on development may be expressed
through structural or functional abnormalities due to either
mutations or to biochemical or physiological disturbances.
Mutations may occur in either somatic or germ cells. Mutations
in male or female germ cells represent potentially the most
long-lasting and severe effects on the human population that a
chemical could cause.
Adverse effects on reproduction or development induced by
chemicals may be expressed immediately or they may be delayed,
sometimes for many years, as exemplified by transplacental
carcinogens (section 5.3.1.2).
Structural or functional abnormalities are most likely to
develop during embryogenesis, the period of development during
which cells differentiate into the various organ systems.
Typical teratogenicity studies investigate the effects of expo-
sure to test substances during this period. Effects due to
exposure during fetogenesis, the developmental period after the
organ systems have formed, generally involve growth retardation
and functional disorders, though the external genitalia and the
central nervous system are also susceptible to injury during
this period (51, 52). Such structural or functional abnorm-
alities often do not become obvious until some time after birth
and, in some cases, not until adulthood.
Neonatal development may be influenced by the consumption of
milk containing chemicals (or their metabolites) that were
consumed by the mother. Agents may also affect neonatal
development by influencing maternal behaviour, hormonal balance,
or nutrition. Direct neonatal exposure to xenobiotic compounds
also occurs, but is less common, since JECFA considers it pru-
dent that food intended for infants younger than 12 weeks of age
should not contain any additives (42).
Guidelines for reproductive toxicity investigations have
been developed by various legislative and international organi-
zations, including the US Food and Drug Administration (US FDA),
US Environmental Protection Agency (US EPA), the United Kingdom
Committee on Safety of Medicines (CSM), Committee on Toxicity
(COT), and Pesticides Safety Precautions Scheme (PSPS), the
Japan Ministry of Agriculture, Forestry and Fisheries (MAFF)
and Ministry of Health and Welfare (MHW), the World Health
Organization (WHO), the Organization for Economic Cooperation
and Development (OECD) (all listed in (51)) and the Inter-
national Programme on Chemical Safety (IPCS) (53). A review of
the methodology for assessing the effects of chemicals on repro-
ductive function has been published under the auspices of the
IPCS and the Scientific Committee on Problems of the Environment
(SCOPE) of the International Council of Scientific Unions (54).
The procedures described in these publications are designed to
assess the reproductive and developmental toxicity potential of
test compounds using lower mammals as model systems. These pro-
cedures generally involve combining various stages of the life
cycle in one test, as it is usually not practicable to examine
the effects of a chemical in each separate stage of the repro-
ductive cycle. An exception is the so-called "teratogenicity"
study, where exposure is limited to the period of organogenesis
(see below).
The goal of reproduction/developmental toxicity studies is
to assess whether the organism is more sensitive to the agent
under test during its reproductive and developmental stages than
during its adult phase. Therefore, the highest dose of food
chemical that is administered is generally the amount that would
be expected to cause slight maternal toxicity, and the lowest
dose is the amount that is not expected to cause an effect in
either the mother or the conceptus. If profound toxicity is
observed in the offspring at the high dose (the dose that causes
only slight maternal toxicity), then the conclusion would be
that the substance is more toxic for offspring than for adults.
This conclusion would be reinforced by the appearance of adverse
effects in the conceptus at the mid- and/or low-dose levels. On
the other hand, if the test substance injures reproduction or
development at levels comparable with levels that cause toxicity
in adults, then no special concern should be attached to the
results of the reproduction/developmental toxicity studies.
Single-generation and multi-generation reproduction studies
are useful for assessing potentially deleterious effects on
reproduction and development through parturition and lactation.
However, because of the long-term exposure inherent in these
studies, detoxifying enzymes may be induced in mothers before
embryogenesis takes place. Under these circumstances, the
observed toxicity would be understated. Studies in which
mothers are exposed to the test substance only during organo-
genesis, as in teratogenicity studies, reduce the possibility of
the mother adapting to the test compound.
The range of effects arising from maternal exposure to
chemicals during organogenesis includes:
(a) death and resorption of the embryo;
(b) teratogenic defects (malformations of a structural
nature);
(c) growth retardation or specific developmental delays;
and
(d) decreased postnatal functional capabilities (55).
Which of these effects will be expressed depends on the level
and gestational timing of the dosage of the food chemical, and
the duration of the period of treatment (56). Thus, a substance
given at one dose level may result in growth retardation, while,
at a higher level, it may result in death and resorption of the
embryo. Sometimes, the slope of the dose-response curve of, and
between, these effects is very steep, making the interpretation
of the studies very difficult. Because all of these outcomes
are unacceptable, the most important consideration when evalu-
ating these studies should not be which effect is observed, but
rather, at what dose level the adverse effect became evident.
This dosage information can then be used to set exposure limits.
Because teratogenic effects are only one part of the total
spectrum of the embryotoxic effects that should be investigated
in such studies, a better term for "teratogenic studies" might
be "embryotoxicity studies". In those rare situations when the
studies are performed during the period of fetal development,
the term "fetotoxicity studies" should be used.
The appropriate role of studies involving in utero and
neonatal exposure in the evaluation of food additives is
discussed in section 5.3.
5.1.5. In vitro studies
In recent years, a great deal of effort has gone into the
development of in vitro test systems. Generally, these systems
are segregated according to two kinds of functions: (a) to
reveal whether a particular kind of toxicity is produced by the
agent under study; or (b) to help elucidate the mechanism of
toxicity displayed by a chemical. The former tests are being
developed to serve both as predictors of toxicity (section
3.1.2) and as substitutes for complex, lengthy in vivo pro-
cedures. The latter are more directly focused than the former,
and their value has been clearly demonstrated as a means of
establishing the metabolic mechanisms at the organ, tissue, or
cellular level (section 5.2).
Much effort has been devoted to the development of in vitro
test systems based on isolated cells, tissues, and organs. Some
of these systems are reported to be related to specific toxic
end-points such as mutagenicity and carcinogenicity (e.g., DNA
damage and repair in mammalian cells, covalent binding to DNA,
cell transformation, mitotic recombination, and gene conversion
in yeast (57)) and to embryotoxicity (e.g., whole embryo
cultures, cultures of embryonic tissues, teratocarcinoma cells,
and embyronated eggs (58)).
The number, diversity, and use of these tests have increased
rapidly in the past decade and are likely to continue to
increase in the future. However, correlations among results of
various in vitro tests and reported correlations between the
results of batteries of short-term in vitro tests and in vivo
carcinogenesis bioassays (which have been the primary thrust of
these assays) are not high. Such short-term in vitro tests are
generally effective at measuring their intended genetic end-
point, i.e., mutagenicity in the particular system under study.
However, the relevance of mutagenic effects to food additive
toxicity has not been established, and the results of many
current in vitro test procedures do not relate to genetic
effects in mammalian reproductive tissues. Neither is it clear
how well these tests identify chemical carcinogens or how they
should be used in the absence of corroborative data on carcino-
genicity.
In a similar fashion, culture techniques designed to measure
prenatal toxicity are extremely useful for research purposes,
but, at their present stage of development, they are not very
suitable for screening (58). By excluding the maternal-
placental-fetal relationship, such dissected systems permit the
compound to reach the target directly (membrane systems that
provide biological barriers are missing) without permitting the
potential moderating or activating influences of the maternal
tissues.
Attention should be paid to scientific developments in in
vitro test systems. However, because of the many experimental
uncertainties and controversial issues surrounding the efficacy
of these tests as predictors of specific toxic end-points, it
would be inappropriate for JECFA to request that all food
additives brought before it should be subjected to such tests on
a systematic basis. On the other hand, data obtained with in
vitro systems sometimes help to clarify the mechanism of action
of chemicals observed in in vivo systems. Therefore, JECFA
should continue to determine the relevance of available in
vitro data on an ad hoc basis when assessing the safety of
specific compounds.
5.2. The Use of Metabolic and Pharmacokinetic Studies in Safety Assessment
Chemical toxicity results from reactions between the
ingested toxic chemical, or its metabolites, with constituents
of the body. Therefore, the complete safety evaluation of a
substance such as a food additive must consider its metabolism
and pharmacokinetics. Unfortunately, a great deal more has been
said and recommended in this area than has been done in
practice. The importance of metabolic and pharmacokinetic data
in the proper planning and interpretation of in vivo toxicity
testing of chemicals is obvious, but the fact is that such data
are either inadequate or unavailable to aid in the interpreta-
tion of the majority of long-term studies undertaken with chem-
icals, including food additives.
Detailed metabolic studies have gained added importance in
determining the extent of appropriate toxicological testing
since the advent of novel and modified foods. This is con-
sidered in detail in section 6.2. However, it is necessary to
repeat some general aspects of the subject here.
Biochemical studies play two separate roles in the safety
evaluation of chemicals. These are:
(a) to design animal studies by identifying the appropriate
species for, and helping to determine the appropriate
level of, testing; and
(b) to extrapolate experimental animal toxicity data to
human beings, by elucidating the mechanism of toxicity
of the chemical, thus facilitating the establishment of
a no-observed-effect level; a comparison of biochemical
data between experimental animals and man helps
determine the relevance of any toxicity observed in
animals.
The ingested chemical itself may exert a toxic effect, or a
metabolite(s) may be the toxic agent. Many polar, non-
lipophilic chemicals are rapidly metabolized and/or excreted,
while lipophilic compounds may be stored, excreted into the
bile, or metabolized into more polar, water-soluble compounds,
which are eliminated from the body, in the urine, more rapidly
than the ingested additive.
Absorbed substances, except those that enter the lymphatic
system, are transported directly to the liver via the portal
vein. Many substances that are metabolized in the liver are
transported via the hepatic vein to the kidneys to be excreted
in the urine. Through enterohepatic circulation, some sub-
stances that are conjugated in the liver are excreted with the
bile, reabsorbed, and then excreted once again in either the
bile or the urine.
Metabolism, primarily involving enzymatic reactions, may:
(a) convert the additive into a body constituent or a
source of energy;
(b) lead to the detoxification of the ingested chemical and
the excretion of its metabolites; or
(c) result in activation of the chemical into reactive
intermediates that then react most importantly with
glutathione, tissue proteins, RNA, or DNA.
Biotransformation reactions are catalysed by intra- and extra-
cellular enzymes and by enzymes of the microflora of the gastro-
intestinal tract. Knowledge of the rates of formation, reaction
with tissue components, and excretion of various metabolites is
essential for full understanding of the disposition and elimin-
ation of the chemical from the body and of the mechanism and
extent of its toxicity.
This section contains a general discussion of the role of
metabolism and pharmacokinetic data in the safety assessment of
food additives. Simple guidelines have not been generated, as
it is doubtful that such guidelines are feasible or desirable.
Several food additives that have been extensively studied bio-
chemically are discussed in Annex IV. These examples are
designed to provide an appreciation of the value and problems
involved with investigating the metabolic bases for the bio-
logical responses to food additives.
5.2.1. Identifying relevant animal species
The occurrence of interspecies differences in response to
foreign compounds complicates the extrapolation of animal
toxicity data to human beings. The resolution of this problem
depends on an understanding of such interspecies variations in
the disposition of ingested compounds. In this context,
disposition is meant to encompass metabolism and pharmaco-
kinetics.
The rates of absorption, rates and sites of distribution,
and rates and routes of excretion determine the concentration-
time profiles of the parent molecule and metabolites in the
various tissues and organs of the body. The overall biological
response is thus the product of the fluxes of the unchanged
molecule and its metabolites occurring in the animal under
examination. Definition of the pharmacokinetic properties of a
food additive may require various routes of administration. The
influence of any vehicle to be used in long-term studies should
be determined, because the vehicle may influence the absorption,
metabolism, or toxicity of the test compound.
In order to extrapolate reliably from animals to man, the
ideal situation would be one in which the tissues of the animals
and of man would be exposed to identical fluxes of the compound
and its metabolites. This requires that the qualitative,
quantitative, and kinetic aspects of the disposition of the
compound be the same in animals and man. This ideal situation
is probably never achieved because of species variations.
The goal should be to select a species for testing that is
the most closely related to man in terms of the metabolism of
the compound under study, using a route of administration
similar to the anticipated human exposure. However, the list of
species that may realistically be used in a toxicity test is
very limited because of problems of availability, lack of
background pathological and physiological knowledge, and
experimental convenience. Thus, it is unlikely that a suitable
metabolic model species will fulfill other important criteria
used to select test species. Given these facts, metabolic
studies used for species selection should be prospectively
performed only on species suitable for toxicity testing. The
species is then selected that is closest to the human being in
terms of the metabolism of the compound. This, of course,
requires knowledge about its metabolism in the human being. In
many cases, in vivo human studies are not possible.
In general, the required metabolic and kinetic information
can best be obtained from in vivo studies. Pharmacokinetics can
obviously only be examined in vivo, since these studies deal
with whole animal phenomena. In vitro studies, such as organ
perfusions and tissue cell incubations, provide useful inform-
ation in some cases, but they do not provide information on the
absorption, distribution, and excretion of chemicals. The use
of isolated cells of human tissues may prove acceptable in some
cases, because, even considering their inherent limitations,
these systems may be the only ones available for examining the
metabolism of compounds that cannot be administered to human
subjects.
In a long-term toxicity test, the attainment of a steady
state depends on the relationship between the kinetic variables
of the compound and the dose interval. When a compound is
administered continuously in the diet, an approximate "steady
state" will sometimes be established. Therefore, marked differ-
ences seen in animals in single-dose pharmacokinetic studies may
change, or even disappear with long-term administration. How-
ever, a steady state will never truly be achieved because of
diurnal patterns of dietary intake by common laboratory animals.
On the other hand, compounds that are rapidly absorbed and have
a short half-life will show wide temporal variations in plasma
concentrations, as will compounds that are administered by
gavage or capsule.
In many cases, the metabolic profile of a compound is
determined by the amount administered, as well as the species in
question. The use of very high doses in toxicity testing may
give metabolic patterns, and therefore biological responses,
that are patently unrepresentative of the situation to be
expected with actual levels of exposure. Thus, data on the
influence of dose level on metabolism in the test animal should
be generated to determine whether absorptive, metabolic, or
excretory processes may have a threshold. An animal model,
apparently suitable at one level of exposure, may be less
suitable at a different level.
Consideration should be given to the possibility that the
metabolism of the compound will differ between long-term tests
and short-term metabolic studies. This could be because of
adaptation by gut microflora, which is discussed in section
5.2.4, or because of the induction of enzyme systems that
metabolize the substance.
An aim of toxicity testing should be to examine the possible
activities in animals of all of the human metabolites of a
compound that may induce toxicity. In many cases, this is best
achieved by combining data from several animal species, to
include all the metabolites of interest. In interpreting such
data, the closest attention should be paid to the similarities
of the mechanisms of toxicity in the various animal species, and
also to the possibility that toxicity may involve interactions
between the parent compound and its metabolite(s), which may not
be the same in all species and may be irrelevant for man.
5.2.2. Determining the mechanisms of toxicity
A great deal of research has been performed in an attempt to
explain the mechanism(s) by which certain test chemicals have
given rise to particular lesions. In the majority of cases,
these studies have concerned the development of tumours.
Clearly, it is likely to be most difficult to find any simple
mechanistic answer to carcinogenesis. However, it is usually
possible to determine factors of importance for a safety
evaluation, which are not necessarily complete solutions to the
mechanism of action.
If an additive has been determined to be an animal
carcinogen, it is extremely difficult to show that it is safe
for human beings. It is not easy to establish its safety, with
retrospective metabolic and pharmacokinetic studies, that an
exclusively secondary mechanism is operative and that a
threshold exists below which the use of the additive is safe.
If a carcinogenic impurity is present in an additive, the
impurity should be either removed or limited to such an extent
that consumption of the additive does not pose a carcinogenic
risk for consumers. The level of the impurity in the food
additive should be controlled within specifications.
In some cases, toxicity may occur as a result of the test
compound or a metabolite displacing endogenous substrates from
carrier proteins or receptor sites. Where such mechanisms are
indicated, ad hoc studies of relative binding affinities can
form a useful adjunct to routine pharmacokinetic studies and
assist in establishing safe levels of exposure. In vitro
studies are useful for determining the mechanism of toxicity,
especially when covalent binding to cellular macromolecules is
involved. In such cases, comparative binding studies using
preparations of metabolizing enzymes from various animal species
are desirable.
5.2.3. Metabolism into normal body constituents
The metabolism of an ingested compound into normal body
constituents does not provide assurance that the substance is
safe. Not only are many metabolites toxic (e.g., most excretory
products), but there are limits to the body's ability to process
even relatively non-toxic metabolites. These limits should be
known, and, if a toxic threshold for a substance has been
identified, its acceptance as a food additive will depend on
controlling intake so that toxic levels are not ingested by
human beings.
Knowledge that a substance is a natural metabolite or is
metabolized into normal body constituents is of great help in
evaluating its safety. However, without concomitant information
on the kinetics of the production and disappearance of
metabolites, the extent to which such information can be used is
limited.
When the additive provides only a small increment in levels
of metabolites compared with the ordinary consumption of food,
then the questions about safety are greatly simplified. The
report of the WHO Scientific Group on Procedures for Invest-
igating Intentional and Unintentional Food Additives (2, p. 7)
considered this situation and concluded that:
"if the biochemical evidence shows that the additive
makes only a small contribution to existing metabolic
pools from food components or in the tissues, there may
be no need for detailed toxicological studies on it,
provided that it conforms to adequate specifications."
JECFA has also considered this situation in some detail (29,
pp.12-13) and has suggested that:
"any food additive that is completely broken down in
the food or in the gastrointestinal tract to substances
that are common dietary or body constituents might be
satisfactorily evaluated. . . on the basis of appro-
priate biochemical and metabolic studies alone. . .".
This report summarizes the evidence required in such cases as
follows:
(a) "evidence that the substance is readily broken down in
the food or in the gastrointestinal tract to common
food constituents under the conditions of use;
(b) evidence to indicate the main factors concerned in this
breakdown, e.g, pH and enzymes;
(c) evidence, preferably including studies on human sub-
jects, that the material, when given in moderate
amounts and under conditions similar to those that will
prevail if used as a food additive, is absorbed to the
same extent as the food materials to which it gives
rise, and does not interfere with the absorption of
other nutrients;
(d) evidence that unhydrolysed or partly hydrolysed mat-
erial does not occur in significant amounts in the
stools, and that it does not cumulate in body tissues;
and
(e) evidence that the most important food components in the
additive are metabolized and utilized as effectively
when administered in composite form as when given
separately, and that overloading does not occur."
As long as adequate evidence along the above lines is
presented, the Committee concluded that:
"the food additive is handled in the body in a way
that is not significantly different from that required
for the component food materials. If so, no toxico-
logical studies need be demanded, since the problem now
becomes one involving the toxicology of foods them-
selves rather than the toxicology of a food additive."
The Committee allocated ADIs to such substances, calculated on
the basis that the food additive would not increase the food
component into which it is converted by more than about 5% of
the quantity in an average diet (29, p. 13).
These general principles have been accepted by the eleventh
and seventeenth meetings of JECFA (41, pp. 8-9; 16, p. 31), the
second of which confirmed that, if biochemical evidence shows
that the sole effect of the additive is to make a small
contribution to existing metabolic loads from food components,
there is no need for detailed toxicological studies. Examples
cited in the various reports include sucrose esters of fatty
acids, lactic and fatty acid ester of glycerol, and some esters
used as food flavours.
These principles are still valid. However, in some JECFA
reports, the combined evidence of breakdown in food and in the
gastrointestinal tract was considered. In contrast, studies on
the stability/breakdown pathways of the additive in food, under
the proposed conditions of use, may be needed to ensure that
significant quantities of toxic products are not formed during
food processing or storage, either through transformation of the
additive or through its reaction with food constituents. All
procedures designed to measure metabolites must be accurate and
they must have a high level of sensitivity for the compounds
under consideration, to draw the conclusion that further
toxicity studies are not necessary.
5.2.4. Influence of the gut microflora in safety assessment
The gut microflora may influence the outcome of toxicity
tests in a number of ways, reflecting their importance in
relation to the nutritional status of the host animal, to the
metabolism of xenobiotics prior to absorption, and to the
hydrolysis of biliary conjugation products. JECFA has recog-
nized this, and has drawn attention to the usefulness of studies
on metabolism, involving the intestinal microflora, in toxico-
logical evaluation (30, p. 7; 18, p. 10).
Interactions that may occur between food additives and the
bacterial flora of the gastrointestinal tract should be con-
sidered both in terms of the effects of the gut microflora on
the chemical and the effects of the chemical on the gut micro-
flora. Because the gut microflora are important in the meta-
bolic fate and toxicological activity of some food additives,
the safety assessment of food additives should include the
possibility that gut microflora modify the host response to the
food additive and/or that the food additive is affecting the
host microflora.
5.2.4.1 Effects of the gut microflora on the chemical
The spectrum of metabolic activity shown by the gut flora
contrasts markedly with that of the host tissues. While hepatic
metabolism of foreign compounds is predominantly by oxidation
and conjugation reactions, the gut bacteria perform largely
reductive and hydrolytic reactions, some of which appear to be
unique to the gut flora. Typical reactions include:
(a) the hydrolysis of glycosides (including glucuronide
conjugates), amides, sulfates, and sulfamates;
(b) the reduction of double bonds and functional groups;
and
(c) the removal of functional groups such as phenol and
carboxylic acid moieties.
Thus, from a structural point of view, many food additives are
potential substrates for microbial metabolism.
The gut bacteria are situated principally in the terminal
parts of the intestinal tract, and thus highly lipid-soluble
compounds that are absorbed in the upper intestine will not
undergo bacterial metabolism. However, tissue metabolism may
give rise to conjugates that are excreted into the bile and thus
available for bacterial hydrolysis. Clearly, then, the design
of appropriate investigations with the gut microflora must be
linked closely to in vivo studies on absorption and metabolism.
In vitro incubation of the food additive and/or its metabolites
with the bacteria of the caecum or faeces is a useful but diffi-
cult technique, with considerable potential for the generation
of spurious data. Some of the pitfalls of prolonged incubations
are that:
(a) the use of a nutrient medium may allow the growth of a
non-representative bacterial population; while
(b) the use of a non-nutrient medium may act as a powerful
selective force for organisms able to use the additive
as a source of carbon and energy.
There are three primary in vivo methods for studying the
role of the gut microflora in the metabolism of a compound:
(a) parenteral administration of the compound, which should
result in decreased microbial metabolism of poorly
absorbed polar compounds, compared with oral dosing;
(b) studies on animals in which the bacterial flora are
reduced by the use of antibiotics or by surgical
removal of the caecum; and
(c) studies on germ-free animals and on (formerly) germ-
free animals contaminated with known strains of
bacteria.
A number of factors may influence the metabolic activation
of foreign chemicals by the host microflora (see reference 59
for an expansion of these points):
(a) Host species
Species differences exist in the number and type of bacteria
found in the gut and in their distribution along the gut. In
this respect, the rat is a poor model for man, since significant
numbers of bacteria occur in the upper intestinal tract of the
rat, whereas this region is almost sterile in man.
(b) Individual variations
There is a great deal of variability among individuals
within a species in the extent to which some compounds undergo
metabolism by the gut flora. Many of these variations probably
arise from differences in the enzymatic capacity of the gut
flora rather than in the delivery of the chemical to the lower
intestine. Thus, if, in animal studies, a food additive is
shown to be metabolized by the gut flora to an entity of toxico-
logical significance, it is essential that its metabolic fate be
characterized in the human being.
(c) Diet
The composition of the gut flora depends on the diet, which
may influence the extent of microbial metabolism of a food
additive.
(d) Medication
The widespread oral administration of medications, such as
antibiotics and antacids, in the human population, is a cause of
variations in metabolism by the gut microflora.
(e) Metabolic adaptation
The metabolic capacity of the gut flora is far more flexible
than that of the host. Thus, long-term adminstration of foreign
chemicals can lead to changes in both the pattern and extent of
microbial metabolism of the chemical. Because prior exposure to
the compound under test may significantly alter the metabolic
potential of the gut microflora, metabolic studies should be
performed not only on previously unexposed animals but also on
animals that have been exposed to the test compound for some
time. For the same reason, any in vitro studies should be per-
formed with caecal contents that have been collected both prior
to and during long-term animal feeding studies.
5.2.4.2 Effects of the chemical on the gut microflora
During high-dose animal feeding studies, the gut microflora
may be affected in two ways:
(a) Development of antibacterial activity
A weak antibacterial activity may become significant after
long-term intake of near-toxic doses of a food additive. This
may manifest itself either as an alteration in the number of
bacteria present, which can be measured directly, or as an
abnormal microbial metabolic pattern. The latter can be studied
by measurement of certain endogenous metabolites produced only
by the gut flora, such as phenol and p-cresol, which provide
indirect evidence of alterations in the gut flora. Such
information may also be of value in the interpretation of other
variables such as nitrogen balance.
(b) Increased substrate for gut microflora
The food additive may act directly as a substrate for bac-
terial growth. This can be readily illustrated by appropriate
high-dose pharmacokinetic studies, coupled with in vitro meta-
bolic studies on the gut flora. Alternatively, the food addi-
tive may inhibit digestion or absorption of other dietary compo-
nents so that these become available to the bacteria in the
lower intestine in increased amounts.
Increased amounts of substrates in the lower intestine pro-
vide an increased osmotic effect in the caecum, which may be
detectable as caecal enlargement (section 5.1.2). The reason
for caecal enlargement must be studied before the significance
of the lesion can be assessed since it may indicative of:
(i) abnormal osmotic balance with consequent changes in
permeability to minerals in the caecum, which could
lead to nephrocalcinosis;
(ii) microbial metabolism of nutrients, which could result
in the formation of potentially toxic metabolites and
abnormalities in the nitrogen balance; or
(iii) microbial metabolism of the food additive, which
could lead to the formation of toxic products.
5.3. Influence of Age, Nutritional Status, and Health Status in the Design
and Interpretation of Studies
Animal toxicity studies are generally performed with healthy
animal populations that are in a state of over-nutrition in a
protected environment. This basic procedure is altered only
when there is a specific reason for doing so, for example, when
nutritional factors are being studied.
In order to establish the safety of food additives, experi-
mental protocols have tended towards more universal designs
encompassing populations during all stages of the life cycle,
e.g., reproduction studies are often included in long-term
studies. Such protocols are intended to mimic the type of expo-
sure in the bulk of the human population. Margins of safety and
medical advice are used to protect subpopulations at special
risk for one reason or another.
5.3.1. Age
It is well known that the age of a test animal can influence
the toxic response to a substance being tested. For example, an
enzyme activity that is involved in the metabolism of a sub-
stance in an adult may be virtually absent in an immature animal
or vice versa. Thus, a compound that is metabolized to a less
toxic metabolite in an adult animal would be more toxic for
young animals lacking the appropriate enzyme activity; obvi-
ously, the reverse would be true for a substance metabolized to
a more toxic metabolite. Differences in sensitivity between
mature and young animals may also result from differences in
intestinal flora as observed, for example, in the growth of
distinctive flora in the upper intestine in human infants that
render them sensitive to nitrate. Greater sensitivity may also
arise in young animals, because of the incomplete formation of
intestinal, blood-brain, or other tissue barriers, which leads
to the passage of potentially harmful substances through the
barriers.
5.3.1.1 History
The WHO Scientific Group on Procedures for Investigating
Intentional and Unintentional Food Additives discussed the
effects of age on toxicity (2, pp. 10-12) and found that "in
general, the young animal is more sensitive to the toxic effects
of exposure to chemicals". Among the reasons cited for the
increased sensitivity in neonates were differences in the dis-
tinctive flora of the upper bowel and differences in the levels
of the "drug-metabolizing enzymes", which are frequently low in
the neonate. Attention was drawn to interspecies differences in
the neonatal levels and in age-related changes in the levels of
these enzymes. The Scientific Group stated that "pertinent
information derived from reproduction (multi-generation) studies
provides some assurance on the safety of compounds that might be
present in the diet of babies" but felt that "since babies con-
stitute a special population, close observation of epidemiology
in this group is an important practical aspect of the evaluation
of the effects of exposure." The Scientific Group also saw the
need for "further information on the development of enzyme sys-
tems in the human young, with particular emphasis on those
enzymes responsible for dealing with foreign compounds." The
report concluded (2, p. 23) that "useful information may be
obtained from studies in newborn or young animals, from repro-
duction studies, and from biochemical studies" and called for
further research on "the development of enzyme systems in the
human young, with particular emphasis on those enzymes respon-
sible for dealing with foreign chemicals" (2, p. 25). With
respect to the latter research, the Scientific Group concluded
that "this information is essential in assessing the safety of
additives in baby food."
Subsequently, the tenth Report of JECFA (29, p. 24) recom-
mended that a special subcommittee of JECFA should be estab-
lished to study the special problems arising from exposure of
infants and young children to food additives. In response to
this recommendation, an FAO/WHO meeting on Additives in Baby
Foods was convened in 1971, and the report of this meeting was
included as Annex 3 to the fifteenth JECFA Report (42, pp. 29-
37). A distinction was made, on developmental grounds, between
children up to 12 weeks of age and children over 12 weeks. The
Subcommittee considered it prudent that food intended for
infants under 12 weeks of age should not contain any additives
at all. However, if it were deemed necessary to use additives
in food intended for young infants, the Subcommittee concluded
that "particularly for infants under 12 weeks, toxicological
investigations should be more extensive and include evidence of
safety to young animals."
With respect to contaminants, the Subcommittee concluded
that "the establishment of acceptable residue levels of pesti-
cides or other contaminants likely to be present in milk and
cereals for infant foods should be based on toxicological eval-
uation in very young animals" (42, p. 31). The report also made
observations on particular classes of food additives (42,
pp. 32-33). The vulnerability of very young infants was recog-
nized, and guidelines on toxicological testing were formulated
(42, p. 34). These include the following:
(a) Before a food additive is regarded as safe for use in
food intended for infants up to 12 weeks of age, the
toxicological studies should be extended to include
animals in the corresponding period of life.
(b) It is difficult to recommend precise toxicological
testing procedures until more basic research has been
undertaken. There are also difficulties in selecting
appropriate species. In these circumstances, short-
term studies should be conducted in several species and
should include the oral administration of the additive
under test, at suitable dose levels, to newly born
animals up to and including the end of the weaning
period.
(d) When life-span studies and multi-generation studies are
carried out, they should be extended to include oral
adminstration of the food additive at suitable dose
levels to a proportion of animals from the day of birth
throughout the pre-weaning period.
The practical difficulties and cost of implementing these
recommendations on a routine basis would be immense, involving,
as it would, artificial feeding of litters of newborn laboratory
animals. However, in situations in which young infants are a
target population for an additive, it seems reasonable that
studies such as these should be performed.
When considering glutamate, the fourteenth report of JECFA
(30, p. 8) noted that:
"any attempt to interpret these data in terms of human
neonates and infants involves the problem of how far
developmental stages in animal species and in man can
be considered equivalent in relation to vulnerability
to possible effects of food additives. Relevant infor-
mation would be of considerable value."
The sixteenth (60) and twentieth meetings (19, p. 22) of
JECFA recommended that a review should be made of the special
problems arising from the exposure of infants and children to
contaminants in food. This review was conducted at the twenty-
first meeting (20, pp. 9-12), which also considered food
additives. The Committee stated that:
"scientific evidence indicates that newborn and very
young children are particularly sensitive to the harm-
ful effects of foreign chemicals" due to, inter alia,
"immaturity of enzymatic detoxifying mechanisms, incom-
plete function of excretory organs, low levels of
plasma proteins capable of binding toxic chemicals, and
incomplete development of physiological barriers such
as the blood-brain barrier. Moreover, there appears to
be a general vulnerability of rapidly growing tissues,
which is particularly important with regard to the
developing nervous system."
The Committee reiterated that food intended for infants
under 12 weeks of age should (with certain exceptions) not
contain any additives but that "in assessing food additive
safety, the question of potential special hazards for the
newborn and infants should be kept in mind" and "toxicological
and metabolic studies of food additives should always include
investigations that permit the evaluation of safety for the
newborn and the infant." The Committee stated further that "in
order to gain more information about the long-term effects of
exposure in utero and in the post-natal period, appropriate
methodology must be developed," and the Committee emphasized
that "short- and long-term effects of exposure in utero and
during lactation should be taken into account for food additives
and contaminants evaluated by the Committee" and "this evalua-
tion might include a request for appropriate animal studies."
Implicit in these statements is a call for metabolic studies
on neonates and for toxicological studies involving in utero
exposure followed by long-term studies. Indeed, the twenty-
second report of JECFA (32, p. 30) reaffirmed the need for
testing the effects of exposure to food additives and contam-
inants in utero and on neonates during suckling. However,
"in view of the complexity of the testing procedures,"
the Committee recommended that "WHO should convene a
meeting of experts to assess: (a) the degree of any in-
crease in the sensitivity of toxicological testing
afforded by exposure in utero through lactation; and
(b) the need to include such expo-sure in toxicological
tests as a means of increasing public health protec-
tion."
Criteria for determining whether in utero exposure is to be
included in such studies should include such information that
the chemical crosses the placental barrier and/or is secreted in
breast milk. The Committee recommended further that:
"the experts should also propose the most appropriate
guidelines for experimentation, taking into account:
(a) the dosages used and the relative exposure of
mother and fetus to the agent under study; (b) the pos-
sibility of combining this modified long-term test with
reproduction studies; (c) the length of the studies re-
quired; and (d) the most appropriate species to use."
A meeting of experts has not yet been convened to consider these
issues.
A document of potential benefit to JECFA is one that has
recently been developed by the IPCS and the Commission of the
European Communities concerning the principles for evaluating
health risks from chemicals during infancy and childhood. Among
the objectives of this activity were to:
(a) "investigate whether and when there is a need for
specific approaches when evaluating the health risk
associated with exposure to chemicals . . . during
infancy and childhood"; and (b) "identify further
developments in methodology that are necessary for the
assessment of health risks associated with exposure to
chemicals during the early period of life" (61).
5.3.1.2 Usefulness of studies involving in utero exposure
In order to evaluate the usefulness of in utero studies, it
is important to review the available toxicity data relating to
this issue. Most studies including in utero exposure have
involved the use of known carcinogens. The IARC monograph
"Transplacental Carcinogenesis" (62) provides information on
much of the earlier research on these substances. In general,
the reports indicate that, although transplacental carcino-
genesis could occur, there have been no instances in which
compounds that were carcinogenic for the offspring were not also
carcinogenic for the adult and vice versa. However,:
(a) the carcinogenic effects in the offspring can occur at
sites different from those observed in the parent
(e.g., in transplacental rat studies, ethylnitrosourea
shows striking neuro-oncoselectivity, not observed in
the parent, and the induction of unique tumours of the
vagina in young women has been observed with
diethylstilboestrol treatment of mothers given the drug
for pregnancy maintenance); and
(b) the fetus may be more susceptible to tumour development
than the adult rat (observed with ethylnitrosourea).
On the other hand, in some cases, adults are more sensitive
than young offspring, as with certain nitrosamines (62, 63),
suggesting that enzyme systems capable of converting these
compounds into their ultimately carcinogenic forms are not fully
developed in fetal tissue.
Although transplacental carcinogenesis has been the major
interest of in utero studies, evidence is accumulating that non-
carcinogenic substances may be the cause of a variety of
biochemical and other toxic effects in the developing fetus.
Some of this information has come from studies of the toxic
effects of environmental contaminants such as methylmercury and
PCBs. Poisoning episodes of methylmercury in Japan and Iraq
indicate that the developing fetus shows toxic symptoms at
levels at which the mother is asymptomatic. This appears to be
because of selective localization of methylmercury in the brains
of exposed fetuses rather than a higher sensitivity of the fetus
itself (64). In the case of PCBs, monkeys with body burdens of
PCBs derived from previous exposure produced offspring that
showed behavioural and learning deficiencies; it was estimated
that approximately 40% of the body burden of PCBs in the
offspring was derived from placental transfer (65). A finding of
equal or greater interest in this study was that 60% of the body
burden of PCBs was transferred postnatally in the milk. This
result is consistent with the finding that the only route of
excretion of certain chemicals from the human body, particularly
the halogenated hydrocarbons that accumulate in fat, may be via
breast milk. Breast-fed infants, therefore, may be exposed to
very high levels of these compounds, far exceeding the accep-
table daily intake (ADI) or the provisional tolerable weekly
intake (PTWI) (66), pointing to the critical need for obtaining
data during the neonatal phase for these types of compounds.
The results of transplacental rat studies have also shown
the possibility that the course of enzyme development in the
fetus may be markedly altered by exposure to foreign substances.
This so-called programming may alter the time of development of
specific enzymes or change the pattern of development of sex-
dependent enzymes, i.e., male offspring may develop enzyme pro-
files more characteristic of female offspring (67). Studies
designed so that the progeny of exposed parents are used in the
long-term phase serve as a fitness test to detect subtle effects
of this type.
Possible differences in the placenta structure in human
beings and experimental animals should be considered in the
interpretation of in utero studies. Structural differences may
result in significantly different rates of transfer of chemicals
across the placental membrane in experimental animals compared
with human beings. This should be considered when selecting
appropriate dose levels during the in utero phase of animal
studies.
5.3.1.3 Complications of aging
The Scientific Group on Procedures for Investigating
Intentional and Unintentional Food Additives (2) concluded that
it is "better to carry out toxicity studies before the
complications of senescence arise" but, nevertheless, called for
"more basic information . . . on toxicity in aged as well as in
young animals."
Older animals may be especially sensitive to certain
substances, because of reduced functioning of vital organs such
as the kidney or liver. The ability to metabolize certain
substances in the liver may decrease in aged animals (68-71),
resulting in an accumulation of toxic substances and consequent
effects that would not be seen in young animals. Normally-
occurring lesions of old age such as tumours or kidney lesions
may mask subtle compound-related pathology and may render aged
animals a poor model system for assessing some lesions.
Conversely, some lesions may require long exposure to develop or
may only be manifested in older animals. Such old animals are
used routinely, and more research and documentation are needed
in this area.
5.3.2. Nutritional status
JECFA has not directly addressed the issue of over-nutrition
in laboratory animals. Excessive food intake and, in parti-
cular, ad libitum feeding of animals can complicate the inter-
pretation of studies. Considerable research indicates that
altered caloric intake and qualitative changes in diet can have
a profound effect on various disease processes, particularly on
the occurrence of neoplasms (72, 73). Additional research is
needed leading to better nutritional designs of experimental
models for safety evaluation.
In considering the effects of nutritional status on toxi-
city, the WHO Scientific Group on Procedures for Investigating
Intentional and Unintentional Food Additives (2, pp. 12-13)
recognized that nutritional status can influence toxicity, posi-
tively or negatively, depending on the substance, but felt that
"it is wise to maintain all the animals on a diet that is nutri-
tionally adequate in every way, unless there is some specific
reason for doing otherwise." While noting that "further work is
needed on the effects of various states of malnutrition or
undernutrition on the toxicity manifested by chemical com-
pounds," the Scientific Group concluded that "an effort to
simulate conditions of malnutrition in man . . . is not
considered advisable in routine toxicological investigations
intended for the evaluation of safety" and "the evaluation of
safety is best carried out by using healthy animals on adequate,
balanced diets."
In the seventeenth JECFA report (16, p. 31), it is noted
that reactions of food additives with food constituents may
affect the nutritional value of the food and that this
possibility can be studied by chemical or biological assay
methods (section 4.2). However, it is further reported that "it
may be necessary to undertake a toxicological investigation of
treated food materials; here, a margin of safety may be
introduced by conducting the test with food that has been
deliberately over-treated to a measured extent." In addressing
this problem of interaction between additives and food
constituents in the twenty-fourth report of JECFA (21, pp. 10-
11), it is pointed out that such reactions may occur during food
manufacture, storage, and cooking, and it is re-emphasized
"that a better perspective of the safety of food addi-
tives would be gained if information on their manufac-
ture and technological use were more readily available.
Such information should cover . . . any available data
on the chemical fate of each additive in those foods
and on the effects of additives on nutrients . . . ."
It "may even be necessary sometimes to carry out a
study on the technological versus nutritional effects
of certain additives and to present this information to
the Committee."
Similar conclusions were reached by the twenty-fifth meeting of
JECFA (22, pp. 11-12).
In the twenty-fourth report of JECFA (21, p. 10), increasing
concern is also expressed with "the development of materials
designed as substitutes for normal components of food" and noted
that "questions of nutritional adequacy arise in such instances
and must not be overlooked." The Committee believed that "the
problems associated with the designing of tests to assess toxi-
city of these substances and with their interpretation and
extrapolation to man require special consideration".
In considering the particular case of acceptable daily
intakes (ADIs) with regard to nutrients such as ascorbic acid
used as food additives, it is noted in the eighteenth report of
JECFA that the lower limits corresponding to the requirements
for such nutrients are determined by expert committees "con-
cerned with adjusting the ADI if a food additive is shown to
interfere with nutritional requirements in one form or another"
(17, footnote p. 9).
Interference with nutritional requirements can occur by
antagonizing the normal physiological function of a vitamin,
trace metal, or other micronutrient, either through destruction
of the micronutrient before ingestion (such as thiamine
destruction by sulfur dioxide) or through antagonism or
inactivation in the body after ingestion. Testing regimes with
both nutritionally-supplemented and unsupplemented animals will
show whether the antagonism is reversible and will separate the
toxic potential of the agent under study from its effects on
nouriture. Such studies will aid in an ultimate assessment of
safety in that normal levels of the micronutrient in human
populations may be so in excess of absolute requirements as to
make this effect of the agent of little consequence. On the
other hand, if the nutrient is often at marginal levels in human
diets, then clearly its evaluation will have to take this effect
into account.
Other specific nutritional problems have been considered in
relation to phosphates (23, p. 13) and metals occurring in food
(23, p. 14). The problems of the former include alteration of
dietary calcium:phosphorus (Ca:P) ratios with consequent compli-
cations of, for example, nephrocalcinosis, and the Committee
recommended that "further studies should be carried out on the
consequences of high dietary intakes of phosphate, with parti-
cular reference to the Ca:P ratio." The association between
caecal enlargement and nephrocalcinosis also indicates that fur-
ther complications may arise in relation to calcium and phos-
phorus absorption due to other food additives.
With respect to the presence of metals in foods, the
Committee noted that:
"toxicological evaluation of metals in food calls for
carefully balanced consideration of. . . ( inter alia)
. . . nutritional requirements, including nutritional
interactions with other constituents of food in res-
pect of . . . absorption, storage in the body and
elimination" and in the case of essential elements,
tentative tolerable daily intakes "should not be con-
strued as an indication of any change in recommended
daily requirements, but as reflecting permissible
human exposure. . . ." (23, pp. 14-15).
5.3.3. Health status
Health status of test animals is of key importance in
assessing the results of any toxicity study. Health of animals
should be routinely monitored during testing. Animals in poor
health from a viral or bacterial infection may be especially
sensitive to the test substance. Early deaths from infectious
diseases may leave insufficient time for chronic toxicity of the
test compound such as carcinogenicity to manifest itself. Path-
ological lesions from infectious disease may also mask compound-
related pathology. For example, lung toxicity could be masked
by a respiratory infection. The reverse can also occur. Cer-
tain respiratory infections in the rat predispose the rat lung
to lymphoreticular neoplasms (74: acesulfame potassium, pp. 22-
23). Antibiotics and other drugs should not be used unless
absolutely necessary to control infections because their use
complicates the interpretation of the study.
5.3.4. Study design
When designing toxicity studies on food chemicals where
factors of age, nutritional status, and animal health are likely
to affect the results, the investigator should design the study
appropriately with foreknowledge of these factors, bearing in
mind the population likely to be exposed. For example, if the
substance is to be used in infant formulas or baby foods, an
appropriate animal model to mimic the human infant should be
used. The miniature pig may be a useful model in this regard,
because it can be bottle-fed and many aspects of its metabolism
are similar to those of man.
Most food additives are also consumed by pregnant women, so
the factors discussed above with respect to in utero exposure
should be considered when assessing their safety. Animal
studies designed to parallel human exposure should include the
important phases of exposure that occur during fetal development
and suckling of the infant. Exposure of the test animal can
then involve both the parent compound and maternal metabolites
that can either cross the placental barrier or enter the
mother's milk; it will also permit the assessment of metabolites
formed in the developing embryo, which may differ from those
formed in the maternal system.
The need for guidelines, as recommended by the twenty-second
JECFA (32, p. 30), continues (section 5.3.1.1). The exposure
level and its relationship to the no-observed-effect level in
animal studies, the type of additive (e.g., nutritive versus
non-nutritive), information about whether the additive crosses
the placental barrier or is secreted in milk, and other data
relating to the reproductive or developmental toxicity of
similar compounds should be considered in determining the need
for in utero studies.
The age of test animals is an important factor to consider
when designing carcinogenicity studies (75, pp. 57-107). If the
study is terminated too soon, the possibility of detecting
carcinogenicity that manifests itself late in the animal's life
span is lessened. Conversely, in studies with long exposure
times (more than 104 weeks in rats and mice), the background
incidence of naturally-occurring tumours may "swamp out"
compound-related tumours occurring at some sites. One way to
resolve this problem is to add additional groups of animals for
interim sacrifice. However, this increases the cost and
complexity of the study. Knowledge of the test strain with
regard to longevity and tumour incidence is necessary in the
design and interpretation of carcinogenicity studies. In any
case, final termination should take place while there are still
enough survivors among the exposed animals and concomitant
controls to make a statistical evaluation.
It generally is not feasible to test all food additives for
their effects on all age groups and disease states. In cases
where certain populations, such as phenylketonuric patients, are
known to be sensitive to a food additive, warning labels or edu-
cation through other means may be necessary. When substances
are included in special medical foods used in treating certain
diseases, it would be prudent to examine closely any reported
physiological or toxicological effects of the substances, to
determine whether they can be safety ingested by the intended
population. Specialized testing on animal models may be neces-
sary. If the additive is to be used in infant formulae or
"junior foods", this fact should be kept in mind at the time of
the safety assessment. This point is often overlooked. Large
amounts of such a food additive may be consumed, because the
formula may constitute the entire diet of the infant and because
infants take in much more food than an adult on a kg body weight
basis. If specific subpopulations are identified as being at
higher risk than the general population, these groups can be
protected by adjusting the ADI to take their special needs into
account.
In general, the safety of a food additive, as far as limited
special populations not readily identified are concerned, must
rely on the conservatisms built into the safety assessment
process, the analysis of the data, and the safety factors used
in setting an ADI.
5.4. Use of Human Studies in Safety Evaluation
Human studies are not normally included in the data packages
that JECFA reviews in its evaluation of new food additives.
However, the Committee recognizes the value of human data, has
sometimes requested such data, and has always used it in its
evaluations when available. Data from controlled human exposure
studies are useful in confirming the safety indicated by animal
studies after the establishment of ADIs. Such data are also
useful in subsequent periodic reviews, and might facilitate a
re-evaluation of the safety factors that are applied in
calculating ADIs.
Investigation in human subjects was addressed by the WHO
Scientific Group on Procedures for Investigating Intentional and
Unintentional Food Additives (2, pp. 9-10). The Group felt that
"prediction and prevention of possible toxic hazards to
the community that might arise from the introduction of
a chemical into the environment can be made more cer-
tain if information from meaningful studies in human
subjects is available." Three particular aspects of
toxicology were identified in this connection, "the
choice of the most appropriate animal species for. . .
the prediction of human responses; secondly, the inves-
tigation of a reversible specific effect observed in
the most sensitive animal species to determine whether
it represents a significant hazard to man; thirdly, the
study of effects specific to man."
The Group pointed to:
"the need, at a relatively early stage, to obtain in-
formation on the absorption, distribution, metab-
olism, and elimination of the chemical in human sub-
jects, since this makes it possible to compare this
information with that obtained in various animal spe-
cies and to choose the species that are most likely to
have a high predictive value for human responses."
This need has been reiterated by subsequent meetings of JECFA
(27, p. 23; 16, p. 31; 32, p. 13) and in WHO Environmental
Health Criteria 6 (76). However, the WHO Scientific Group
acknowledged that "it is necessary to have adequate short-term
toxicological information in several species before even low
doses of a new chemical are administered to human subjects" (2,
p. 9).
In relation to ascertaining whether the safety margin
predicted from animal data is valid, the WHO Scientific Group
decided that it might be helpful to administer a chemical to
human volunteers, but emphasized the conditions that should be
fulfilled with regard to such a study (2, p. 10). Inter alia,
these conditions include:
(a) The effect or effects studied should be reversible.
(b) The dose levels used should be based on full inform-
ation of the toxicological properties of the substance
in animals.
(c) The investigation should be terminated immediately the
effect has been unequivocally demonstrated.
With regard to effects specific to man, the WHO Scientific
Group (2, p. 10) considered it unacceptable to study such
effects by means of volunteers (in an analogous manner to
clinical trials with drugs) but thought that toxicological
studies could be made on those who are occupationally exposed to
the chemical or in patients suffering from accidental poisoning.
A need was identified for "more critical epidemiological and
toxicological investigations in such situations." Such studies
could be of particular value in relation to hypersensitivity or
other idiosyncratic reactions since no suitable animal model has
yet been developed. In relation to hypersensitivity, the
seventeenth and eighteenth meetings of JECFA (16; 17, p. 10)
stated that "no approval would be given for the use of a sub-
stance causing serious or widespread hypersensitivity reac-
tions". However, such information can be derived only from
studies on human beings.
The WHO Scientific Group has raised an apparent contra-
diction in its different recommendations with regard to con-
firming animal studies and investigating effects specific to
man. As stated above, the Group recommended that controlled
human studies be performed to confirm animal studies, but that
it is inappropriate to study effects specific to man by the use
of human volunteers. This is all the more perplexing, because
controlled human studies, despite their limitations, are the
only means available, at present, for studying effects in man
that are not observed in animals. JECFA may wish to reconsider
the question of using human volunteers to identify specific
responses, which would be done only after the usual battery of
toxicological investigations had been completed. The words of
Paget (77) are cogent in this regard:
"The question is not whether or not human subjects
should be used in toxicity experiments but rather
whether such chemicals, deemed from animal toxicity
studies to be relatively safe, should be released first
to controlled, carefully monitored groups of human sub-
jects, instead of being released indiscriminately to
large populations with no monitoring and with little or
no opportunity to observe adverse effects."
The ethical problems associated with toxicological studies
on human beings have been reviewed succinctly in WHO Environ-
mental Health Criteria No. 6 (76, pp. 41-42).
Information relating to human exposure to a food additive
during its pre-marketing stage can be obtained through the
health monitoring of employees coming into contact with it,
either in the laboratory or the manufacturing plant. Because
the route of exposure in such a situation is through either
contact with the skin or vapour in the lungs, immunological
sensitivity and anaphylactoid reactions (mediator-release ana-
phylaxis-like reactions), often involving histamine release, are
the adverse effects most likely to occur. Thus, any observa-
tions indicating the potential for these effects should be
recorded at the time they are observed.
5.4.1. Epidemiological studies
Most studies of the effects of food additives on human
populations are performed after the additive has been placed on
the market. In nearly all cases, the impetus for the perfor-
mance of human studies on a food additive is that the safety of
its use has been brought into question for one reason or ano-
ther. For example, retrospective investigations have revealed
effects such as "beer drinkers cardiomyopathy", resulting from
exposure to cobalt salts. Adverse findings in these studies may
be used for bringing an additive back to JECFA for re-evaluation
of its safety.
Epidemiological studies designed to assess the safety of
food additives have been performed in several instances, but,
generally, definitive results have not been obtained because of
the lack of sensitivity of such studies and problems in ident-
ifying control populations. For example, long-term low-level
nitrite exposure has been very difficult to study epidemiolog-
ically because of its ubiquitous nature and the consequent dif-
ficulty of finding subpopulations with little or no exposure to
nitrite. With saccharin, an extensive data base involving
retrospective epidemiological studies and case-control studies
has been developed. This data base has been generated using
different subpopulations located in different geographical
areas, and the results for human bladder cancer have usually
been negative (78).
In many cases, the purpose of an epidemiological study is to
confirm in human beings a positive finding observed in animals.
Thus, when food additives are used extensively and when exposed
or unexposed populations cannot be identified, negative results
are usually deemed not to be of much value for regulatory
purposes. This is because epidemiological studies, are, on the
whole, less sensitive than well-designed animal feeding studies.
However, when the number of individuals studied becomes very
large, this lack of sensitivity is somewhat ameliorated, and
safety decisions can be made on the basis of the human studies.
For example, in the case of saccharin, negative results in
epidemiological studies have been considered important by JECFA
for deciding that its continued use is acceptable (1).
Of course, much more can be said about a positive result
than a negative one, especially with epidemiological studies,
which are usually relatively insensitive. An undetectable
adverse effect in a study involving a few thousand individuals
could affect a very large number of people in a population of
hundreds of millions.
5.4.2. Food intolerance
For the purposes of this discussion, food intolerance is
defined as a reproducible, unpleasant reaction to a food or food
ingredient, including reactions due to immunological effects,
biochemical factors such as enzyme deficiencies, and anaphy-
lactoid reactions, which often include histamine release. Food
allergy, sometimes used synonymously with food sensitivity, is a
form of food intolerance in which there is evidence of abnormal
immunological reaction to the food. Immunological reactions may
be further characterized on the basis of the timing of the onset
of symptoms following ingestion of the offending food and on the
type of response involved. Reactions occurring within minutes
to hours of food ingestion are characterized as immediate aller-
gic reactions, which are mediated by Immunoglobulin E (IgE),
while reactions beginning several hours to days after food expo-
sure are characterized as delayed allergic, or cell-mediated,
reactions.
Various dietary factors may be responsible for food intol-
erance. These may be naturally-occurring dietary constituents
or, in some cases, food additives. Two notable examples of food
additives that have been implicated are tartrazine, which may
induce urticaria and bronchoconstriction in asthmatic patients,
and sodium metabisulfite, which has been associated with
bronchospasm, flushing, hypotension, and even death due to
anaphalaxis after ingestion by some asthmatic patients. Mono-
sodium glutamate (MSG) gives rise to "Chinese Restaurant
Syndrome", manifested largely by violent headache. Certain
subpopulations appear to be sensitive to MSG, but the mechanism
is unknown. Despite these examples, there is little to suggest
that food additives are likely to cause more problems of food
intolerance than are components naturally present in food.
Satisfactory animal models to predict food intolerance in
human beings have not been developed. At the same time, many
difficulties are associated with human studies, and interpret-
ation is difficult at least partly because of the anecdotal
nature of much of the evidence. Any interpretation of food
intolerance is complicated by psychological factors, making it
extremely important that blind trials be performed to assess the
nature of the problem.
The most unambiguous method of demonstrating food intol-
erance is to use challenge feeding in a double-blind study; the
diagnosis of food intolerance can only be established if the
symptoms disappear with an elimination diet and if a controlled
challenge then leads to either recurrence of symptoms or to some
other clearly identified change associated with the intolerance.
If delayed allergic reactions are being studied, such effects
may take several weeks to disappear and then redevelop after
challenge feeding. Challenge feeding is most reliable when the
ingestion of food is associated with development of symptoms
within one to two hours.
No oral food challenge, even if blind, can be perfect for a
number of reasons. Presentation of food in capsules may avoid
the possibility of reactions in the mouth, pharynx, and oeso-
phagus and may decrease early digestion of the food by salivary
enzymes. Small amounts of food may be regurgitated or eructated
and identified by taste and smell. Unknown relationships may
exist between suspected foods and periods of abstinence from
that food before challenge. The presence of other foods eaten
with a suspected food may have facilitated or inhibited diges-
tion and absorption (79).
The simplest and most commonly used test for demonstrating
IgE antibodies is the direct skin test. However, this test is
unreliable as used, because standard dosages of food extracts
have not been developed, and, with sufficiently concentrated
food extracts, it is possible to evoke positive skin test
results in any person tested. Therefore, skin testing results
should be verified by testing the extract on non-sensitive
individuals (80).
Other methods for testing IgE antibodies to food include the
in vitro radioimmunoassay and the leukocyte histamine release
assay (81). The former assay is limited by an inadequate stan-
dardized reporting procedure, making a comparison of results
between investigators very difficult (82). The latter has found
only limited application, because it requires fresh blood, and
only a limited number of allergens can be tested from a single
blood aliquot.
Published studies concerning the usefulness of either skin
testing or immunoassay to diagnose clinical adverse reactions to
food have shown a marked discrepancy in results. In most of
these studies, the investigators have relied on the clinical
history for determining the false-positive or false-negative
rates for skin tests or immunoassays. Such reports are unreli-
able. Negative results obtained in skin tests or immunoassays
should be treated with more confidence than positive results
(83, 84).
If evidence of widespread intolerance to a food additive
appears in a country that permits its use, procedures should be
established for the centralized reporting of such information,
if one is not already in place. Medical professionals should be
alerted, and appropriate medical tests performed on affected
individuals to determine the nature of intolerance. If the
problem arises with an additive previously considered and given
an ADI by JECFA, ideally the results will be relayed to the
Committee so that the safety of the additive can be recon-
sidered. Remedies may range from no action to a recommendation
that the additive be removed from the market. Factors, such as
its natural occurrence in food, should be taken into account in
such deliberations. Because food intolerance is not spread
throughout the general population, but is restricted to small
subpopulations or individuals, one of the usual remedies is to
label the food containing the additive prominently, so that
sensitive individuals can avoid it.
5.5. Setting the ADI
Almost any substance at a high enough test level will pro-
duce some adverse effect in animals. Evaluation of safety
requires that this potential adverse effect be identified and
that adequate toxicological data be available to determine the
level at which human exposure to the substance can be considered
safe.
At the time of its first meeting, JECFA recognized that the
amount of an additive used in food should be established with
due attention to "an adequate margin of safety to reduce to a
minimum any hazard to health in all groups of consumers" (9, pp.
14-15). The second JECFA, in outlining procedures for the
testing of intentional food additives to establish their safety
for use, concluded that the results of animal studies can be
extrapolated to man, and that
"some margin of safety is desirable to allow for any
species difference in susceptibility, the numerical
differences between the test animals and the human pop-
ulation exposed to the hazard, the greater variety of
complicating disease processes in the human population,
the difficulty of estimating the human intake, and the
possibility of synergistic action among food additives"
(10, p. 17).
This conclusion formed the basis for establishing the "accept-
able daily intake", or ADI, which is the end-point of JECFA
evaluations for intentional food additives. In the context in
which JECFA uses it, the ADI is defined as an estimate (by
JECFA) of the amount of a food additive, expressed on a body
weight basis, that can be ingested daily over a lifetime without
appreciable health risk.
The ADI is expressed in a range, from 0 to an upper limit,
which is considered to be the zone of acceptability of the
substance. JECFA expresses the ADI in this way to emphasize
that the acceptable level it establishes is an upper limit and
to encourage the lowest levels of use that are technologically
feasible.
Substances that accumulate in the body are not suitable for
use as food additives (39, p. 8). Therefore, ADIs are estab-
lished only for those compounds that are substantially cleared
from the body within 24 h. Data packages should include meta-
bolism and excretion studies designed to provide information on
the cumulative properties of food additives.
JECFA generally sets the ADI of a food additive on the basis
of the highest no-observed-effect level in animal studies. In
calculating the ADI, a "safety factor" is applied to the no-
observed-effect level to provide a conservative margin of safety
on account of the inherent uncertainties in extrapolating animal
toxicity data to potential effects in the human being and for
variation within the human species. When results from two or
more animal studies are available, the ADI is based on the most
sensitive animal species, i.e., the species that displayed the
toxic effect at the lowest dose, unless metabolic or pharmaco-
kinetic data are available establishing that the test in the
other species is more appropriate for man (section 5.5.1).
Generally, the ADI is established on the basis of toxico-
logical information and provides a useful assessment of safety
without the need for data on intended or actual use and con-
sumption. However, in setting ADIs, an attempt is made to take
account of special subpopulations that may be exposed. There-
fore, general information about exposure patterns should be
known at the time of the safety assessment (section 5.5.6). For
example, if a food additive is to be used in infant formulae,
the safety assessment is not complete without looking carefully
at safety studies involving exposure to very young animals.
JECFA uses the risk assessment process when setting the ADI,
i.e., the level of "no apparent risk" is set on the basis of
quantitative extrapolation from animal data to human beings.
Generally, JECFA does not undertake risk management, in that it
leaves it to national governments to use the quantitative
assessments in a manner appropriate to their own situations.
However, this has not always been the case, in that sometimes
JECFA has taken into consideration, in peripheral ways, benefits
(e.g., hydrogen peroxide as an alternative to pasteurization in
developing countries (12; 21)) and economic need (e.g, polymer
packaging materials that contain potentially hazardous migrants
should be limited to situations where no satisfactory alter-
natives exist (1)). In this context, risk assessment and risk
management are more broadly used than, e.g., they are often used
in the context of carcinogenesis.
5.5.1. Determination of the no-observed-effect level
A determination of a no-observed-effect level for a study
depends primarily on the proper selection of doses, such that
the highest dose produces an adverse effect that is not observed
at the lowest dose. Several dose levels are used to determine
the dose-effect relationship. Knowing the nature of the toxic
response to a compound at the high level, a more confident
assessment of a no-observed-effect level at the lower test
levels can be made by focusing more clearly on the target
tissues. Great care must be taken in dose selection, because
the no-observed-effect level must be one of the experimental
doses; it is not an inherent property of the animal system. For
a discussion of items to consider when selecting doses, see
reference 75, pp. 9-49.
The following discussion concerns the performance of long-
term studies, because these studies are the type most often
performed in support of intentional food additives, and they
give rise to much controversy. However, 90-day studies are
sometimes sufficient for establishing safety, as, for example,
with substances that are closely related to food additives of
known low toxicity (section 5.5.4). Many of the points
discussed below in relation to long-term studies are also
appropriate for shorter studies when such studies serve as the
basis for safety determinations.
When long-term studies are indicated, short-term range-
finding studies should first be performed to ensure the proper
selection of dosage regimen. Care must be taken in applying
this approach to dose level selection, because doses that
produce signs of toxicity in short-term testing may be rever-
sible on more long-term exposure. In such situations, the
highest dose selected from range-finding studies may not produce
an adverse effect with long-term exposure, precluding a deter-
mination of the no-observed-effect level in the longer study
(the significance of this transient effect should be taken into
account when evaluating the data). A situation of perhaps
greater frequency is one in which the dosages in the long-term
study are too high, so that, even the lowest dose results in
adverse effects, and a no-observed-effect level cannot be
established.
Ideally, in a long-term study, the high-dose level should be
sufficiently high to elicit signs of toxicity without causing
excessive mortality or some exaggerated pharmacological effect,
such as sedation. Although doses of non-nutritive additives as
high as 5% of the total diet do not always produce adverse
effects, higher doses should not be tested, because they may
produce a significant nutritional imbalance. Therefore, if no
adverse effects are observed at 5% of the diet, this dose should
be considered the no-observed-effect level. On the other hand,
nutritive additives may be fed at higher doses as long as the
nutritional balance is effectively preserved in both the test
animal and controls (section 6.2.3).
Ordinarily, the middle dose should be selected to be
sufficiently high to elicit minimal toxic effects or it should
be set midway between the high and low doses. However, if
significant differences exist in the pharmacokinetic or
metabolic profile of the test substance between the high and low
doses, then an additional dose should be included in the study
to provide more assessment points.
The lowest dose should not interfere with morphology,
development, normal growth, or longevity or produce adverse
functional alterations.
The determination of an adverse effect in a particular study
depends on the doses tested, the types of parameters measured,
and the ability to distinguish between real adverse effects and
false positives. If, for example, only a slight change in a
particular parameter is noted at the highest dose that is not
observed at the lower doses, then it is difficult to distinguish
between a real adverse effect and a spurious positive finding.
In addition, a reduction in body-weight gain coupled with
decreased food consumption is difficult to interpret as an
adverse effect, because palatability of the chow might be
affected by the presence of high levels of the test compound.
However, as noted in section 5.1.1, generalized decrement in
weight gain has sometimes been used for setting an effect level
in the absence of other toxic manifestations.
When two or more studies are performed on an additive in
different animal species, no-observed-effect levels are calcu-
lated from each study. The overall no-observed-effect level
used for calculating the ADI is the no-observed-effect level
from the animal study that displayed a toxic effect at the
lowest dose. The species on which this study was performed is
then considered to be the most sensitive species. This approach
is reasonable when the animal studies are of similar length (in
relation to the expected life span of the species) and quality,
and no other data relating to this issue are available. How-
ever, if the quality of one study is obviously superior to the
others and/or the studies differ with respect to length (long-
term versus short-term), extra weight should be given to the
longer better-quality studies when determining the overall no-
observed-effect level. If metabolic and pharmacokinetic data
are available, the species most similar to man with respect to
the toxic effect should be used in calculating the overall no-
observed-effect level, rather than the most sensitive species.
5.5.2. Use of the safety factor
The safety factor has been used by JECFA, since its
inception. It is intended to provide an adequate margin of
safety for the consumer by assuming that the human being is 10
times more sensitive than the test animal and that the differ-
ence of sensitivity within the human population is in a 10-fold
range. In determining an ADI, a safety factor is applied to the
no-observed-effect level determined in an appropriate animal
study.
JECFA traditionally uses a safety factor of 100 (10 x 10) in
setting ADIs based on long-term animal studies, i.e., the no-
observed-effect level is divided by 100 to calculate the ADI for
an additive. The no-observed-effect level is usually expressed
in terms of mg compound per kg body weight per day, and the ADI
is expressed in the same units. A food additive is considered
safe for its intended use if its human exposure is less than, or
is approximately, the same as the ADI. The ADI generally
includes both its natural occurrence and deliberate addition to
food (17, pp. 8-10), except when the substance occurs naturally
in a chemical form different from that employed as a food addi-
tive, or when its natural occurrence was not considered when
setting the ADI and the substance naturally present in the diet
contributes significantly to its total intake (as with nit-
rates). Because in most cases, data are extrapolated from life-
time animal studies, the ADI relates to life-time use and pro-
vides a margin of safety large enough for toxicologists not to
be particularly concerned about short-term use at exposure
levels exceeding the ADI, providing the average intake over
longer periods of time does not exceed it.
National governments are responsible for regulating food
additives in such a way that consumption from natural occurrence
and deliberate addition to food does not exceed the ADI for each
additive that is permitted. As stated by the WHO Scientific
Group on Procedures for Investigating Intentional and Uninten-
tional Food Additives (2, p. 6), "it is desirable that national
governments should maintain a check on the total intake of each
food additive, based on national dietary surveys, to determine
whether the total load in the diet approaches the acceptable
daily intake." Individual governments have the discretion of
determining whether they will base their regulatory decisions on
the "average" consumer or the "high" consumer of food
additives.
A safety factor of 100 should not be considered immutable.
When setting the ADI, various test data and judgemental factors
should be considered. These include:
(a) Inadequate data base
In this case, a larger safety factor may be appropriate
(section 5.5.5).
(b) Reversibility of the observed effect in
embryotoxicity studies
If irreversible developmental effects, such as skeletal
abnormalities (as opposed to retarded skeletal growth), are seen
in the fetuses of animals administered the substance in utero, a
study on a second species is indicated. If similar irreversible
effects are not confirmed in the second animal species, pharm-
acokinetic studies would be useful to determine relevance to
human beings. Judgement would then be needed to set an appro-
priate safety factor. If frank teratogenic effects are observed
in both studies, judgement would be needed to decide whether
either a larger safety factor should be considered or it should
be recognized that the use of the substance as a food additive
is not appropriate. If only reversible developmental effects
are seen, such as retarded skeletal and soft tissue development
or decreased fetal weight, the usual safety factor of 100 may be
applied.
(c) Age-related effects in reproduction studies
Such studies may demonstrate different toxic responses in
young animals compared with older ones. Metabolic studies may
demonstrate that the differences in sensitivity are due to such
factors as incomplete development of enzyme systems used for
metabolizing xenobiotic compounds or differences in intestinal
flora. Safety factors should be set on the basis of the target
population. If young children are likely to consume the addi-
tive, the ADI should be based on the no-observed-effect level
from the phase of the study in which young animals were exposed,
if the no-observed-effect level was lower than in the adult
phase. If, on the other hand, it is shown that children will
not be exposed to the additive, it may be appropriate to set the
ADI on the basis of the no-observed-effect level established in
the adult phase of the study.
(d) Finding of carcinogenicity
Carcinogens vary in the magnitude of risk they present for
man, because they act via different mechanisms. Even though no
basis exists for the exact extrapolation of risk from
experimental animals to man, the degrees of risk from different
carcinogens can often be inferred from the data. However, with
the present state of knowledge, it would be appropriate to
consider the use of a carcinogenic substance as an intentional
food additive only under very restricted circumstances. For
example, if cancer is shown to be a secondary effect, such as
bladder tumours occurring secondary to the induction of bladder
stones, and there is evidence of a threshold below which the
additive is safe, then it would be appropriate to use a safety
factor for determining the safe level of use of the additive.
Under extenuating circumstances, such as an unambiguous demon-
stration that the health benefits exceed the risk, it may also
be possible to use a carcinogenic additive.
(e) If reasons exist for setting a lower safety factor
If toxicity and dose-response effects in human beings are
known, such data should take precedence over extrapolation from
animal studies; a 10-fold safety factor would be appropriate if
there is no evidence that human sensitivity to the agent varies
more than 10-fold among individuals. A lower safety factor may
also be appropriate when the additive is similar to traditional
foods, is metabolized into normal body constituents, and/or
lacks overt toxicity. Also, a 100-fold safety factor often
would not provide a high enough level of nutrients required to
satisfy nutritional needs and to maintain health (toxicity for
some essential nutrients such as Vitamin A, Vitamin D, certain
essential amino acids, and iron may be reached at levels less
than 10 times higher than those recommended for optimal nutri-
tion). A substance that serves as a significant source of
energy in the human diet obviously cannot fit into the con-
straints of a 100-fold safety factor.
The use of standardized safety factors based on no-observed-
effect levels for establishing the acceptable level of use of
food additives is a crude procedure, given the known wide
variability in toxic responses. For example, the nature of the
dose response usually is not used. In part, this is a reflec-
tion of the fact that good dose-response data are not available
for many compounds. Attempts to use the dose-response behaviour
of compounds in establishing quantitative end-points must con-
tend with this limitation.
In the broadest sense, the procedures used by JECFA take
into account the nature of the biological effects observed in
animal bioassays only to the extent that a distinction is made
between carcinogens and non-carcinogens, i.e., ADIs are estab-
lished for non-carcinogens, while most carcinogens are con-
sidered to unacceptable for use as intentional food additives.
Otherwise, the nature of the observed effect is not an explicit
component of the quantitative assessment of food additives.
However, the nature of the effect and a determination of its
significance are often implicitly considered by scientists, when
reviewing the data.
JECFA should take these and other factors into account when
determining acceptable daily intakes of food additives.
However, in situations in which little is known beyond the
empirical finding of toxicity in animal studies, the traditional
approach for calculating ADIs would seem to be appropriate.
This may be an issue for future consideration by JECFA.
5.5.3. Toxicological versus physiological responses
When analysing a toxicological study and setting a no-
observed-effect level, a distinction must be drawn between
reversible changes that are due entirely to normal physiological
processes or homeostasis-maintaining mechanisms, and to toxic
responses themselves (section 5.1). Examples of the former
include: laxative effects from osmotic or faecal overload,
liver hypertrophy and microsomal enzyme induction from high
doses of substances metabolized by the liver, decreased body
weight gain or caecal enlargement from high levels of non-
nutritive substances, alteration in renal weight that is
directly related to the amount of water being processed by the
kidney, and decreased growth rate and food consumption related
to the dietary administration of an unpalatable substance.
However, care must be taken in interpreting these changes, and
they should not automatically be dismissed as being unimportant
from a toxicological point of view. For example, microsomal
enzyme induction in the liver may result in alterations in the
metabolism of compounds unrelated to the administered substance,
which could result in a toxic effect. A decrease in the rate of
body-weight gain coupled with a corresponding reduction of food
intake could be due to toxic anorexia, rather than a palat-
ability defect.
The dose at which the effect occurs should be compared with
the amount of the substance consumed by human beings. Thus, it
would ordinarily be acceptable to permit the use of a substance
that causes diarrhoea only at very high levels of consumption in
rats, but the use of such a substance should be severely
restricted or not permitted if it causes diarrhoea at normal
levels of consumption in human beings. Sometimes, physiological
adaptation may progress through overload to frank toxicity.
Further studies are indicated in situations in which it is
difficult to draw a clear distinction between a toxic and a
physiological response. Special studies such as paired feeding,
caloric balance comparisons between food consumption and body-
weight gain, or, in the case of reproduction studies, cross
fostering, can be performed to decide issues such as reduced
food intake and reduced body-weight gain related to unpalatable
test substances. Metabolic and pharmacokinetic studies may be
of use in providing information on the distribution of the test
compound and its metabolites or the dose at which a change in
metabolism occurs.
5.5.4. Group ADIs
If several compounds that display similar toxic effects are
to be considered for use as food additives, it may be appro-
priate in establishing an ADI to consider the group of compounds
in order to limit their cumulative intake. For this procedure
to be feasible, the additives must be in the same range of toxic
potency. Flexibility should be used in determining which no-
observed-effect level is to be used in calculating the ADI. In
some cases, the average no-observed-effect level for all the
compounds in the group may be used for calculating the group
ADI. A more conservative approach is to base the group ADI on
the compound with the lowest no-observed-effect level. The
relative quality and length of studies on the various compounds
should be considered when setting the group ADI. When the no-
observed-effect level for one of the compounds is out of line
with the others in the group, it should be treated separately.
When considering the use of a substance that is a member of
a series of compounds that are very closely related chemically
(e.g., fatty acids), but for which toxicological information is
limited, it may be possible to base its evaluation on the group
ADI established for the series of compounds. This procedure can
only be followed if a great deal of toxicological information is
available on at least one member of the series and if the known
toxic properties of the various compounds fall along a well-
defined continuum. Interpolation, but not extrapolation, can be
performed by this procedure. The use of this procedure by JECFA
represents one of the few situations in which the Committee has
used structure/activity relationships in its safety assess-
ments.
In some instances, group ADIs can be established primarily
on the basis of metabolic information. For example, the safety
of esters used as food flavours could be assessed on the basis
of toxicological information on their constituent acids and
alcohols, provided that it is shown that they are quantitatively
hydrolysed in the gut.
The calculation of a group ADI is also appropriate for
compounds that cause additive physiological or toxic effects,
even if they are not closely related chemically. For example,
it may be appropriate to establish a group ADI for additives
such as bulk sweeteners that are poorly absorbed and cause
laxation.
5.5.5. Special situations
There are occasions when JECFA considers the use of an ADI
in numerical terms not to be appropriate. This situation arises
when the estimated consumption of the additive is expected to be
well below any numerical value that would ordinarily be assigned
to it. Under such circumstances, JECFA uses the term "ADI not
specified". The Committee defines this term to mean that, on
the basis of available data (chemical, biochemical, toxico-
logical, and other), the total daily intake of the substance,
arising from its use at the levels necessary to achieve the
desired effect and from its acceptable background 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 in numerical
form is not deemed necessary (e.g., 1, Annex II). 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 inferior food quality
or adulteration, and it should not create a nutritional imbal-
ance (16, pp. 10-11). That the background occurrence of the
chemical must be taken into account in the evaluation of its
safety was articulated by the WHO Scientific Group on Procedures
for Investigating Intentional and Unintentional Food Additives
(2, p. 7).
JECFA has encountered several situations in which either the
body of data before it on a new additive was limited, or the
safety of a food additive for which the Committee previously
assigned an ADI was brought into question by the generation of
new data. When the Committee feels confident that the use of
the substance is safe over the relatively short period of time
required to generate and evaluate further safety data, but is
not confident that its use is safe over a lifetime, it often
establishes a "temporary" ADI, pending the submission of appro-
priate data to resolve the safety issue on a timetable estab-
lished by JECFA. When establishing a temporary ADI, the
Committee often uses a higher-than-usual safety factor, usually
increasing it by a factor of 2. The additional biochemical and
toxicological data required for the establishment of an ADI are
clearly stated, and a review of these new data is conducted
before the expiration of the provisional period.
This approach seems to have worked reasonably well in
practice in that it has encouraged necessary research without
creating any known safety problems. In many cases, long-term
studies are requested, but timetables are not met, which means
that JECFA has had to extend temporary ADIs for long periods of
time. JECFA has withdrawn ADIs, in instances where data were
not forthcoming, as a safety precaution.
5.5.6. Comparing the ADI with potential exposure
When establishing ADIs, collateral exposure information is
often useful for determining the relationship between the two
values. The agreement between exposure and acceptable daily in-
take helps to determine whether an "ADI not specified" should be
established. Exposure information is also indispensable when:
(a) performing risk assessments for food contaminants and
processing aids; and
(b) assessing the safety of added substances that may be
naturally present in food to determine their relative
contributions to the diet (17, pp. 8-10).
In order to accurately compare exposure and acceptable
intake, similar assumptions should be used for making each
estimate or, at least, the differences and similarities in the
estimates should be understood. For example, if an ADI is
computed from lifetime dosage, then the estimated human exposure
should represent lifetime exposure to the additive. Sometimes,
acceptable intakes are computed for specific age groups or for
certain dosage conditions when short-term exposure should be
limited, such as with certain food additives that cause laxative
effects at high dose levels. Under such circumstances, the
estimated human exposure should represent the same age group or
dosage conditions. In practice, however, estimates of exposure
do not represent exposure for individual consumers in the same
way that toxicological data represent dosages for individual
animals. Data bases on food and food additive intakes provide
composite data for subpopulations, such as the average dietary
habits of a particular nation's population.
For effective comparison of exposure estimates with accept-
able intakes, the assumptions used to compute exposure estimates
should always be stated. Data on the functional use(s) of
intentional food additives and information on approaches used to
compute intake estimates, such as analytical studies on food
constituents or migration (carry-over) models for certain con-
taminant situations, should be provided if possible.
Each estimate of exposure represents a facet of actual human
exposure and, thus, each estimate represents useful scientific
data. However, it is not possible to describe specific pro-
cedures for estimating exposure for all food additive and con-
taminant situations. However, JECFA is able to provide guidance
by describing the types of estimation procedures that have been
accepted by previous Committees, which are discussed in section
3.1.1
6. PRINCIPLES RELATED TO SPECIFIC GROUPS OF SUBSTANCES
6.1. Substances Consumed in Small Amounts
Many of the substances that come before JECFA for its eval-
uation are present in food in only trace amounts. Testing
requirements generally take these low exposures into account
(section 3.1). However, as discussed below, special safety
concerns are raised by the use of many of these substances,
despite the low exposures to both the parent compounds and their
residues.
In some cases, these substances have no technological func-
tion in the food itself. Some are used in food processing. For
example, residues from extraction solvents used inter alia in
extracting fats and oils, in defatting fish and other meals, and
in decaffeinating coffee and tea may be present in the final
food product because of incomplete removal. The same is true
for enzymes and immobilizing agents (and their residues) used
in immobilized enzyme preparations. Residues arising from the
use of xenobiotic anabolic agents and from the use of packaging
materials may also occur in food.
Residues belonging to all these classes of substances have
been evaluated by JECFA, and the Committee has developed guide-
lines concerning their safety evaluation. The guidelines, which
are reproduced in Annex III, are intended to serve as examples
of guidance by JECFA for evaluating these specific categories of
substances. Further discussion of such substances and others
follows in section 6.1.1.
Flavouring agents constitute a category of substances that
have a functional effect in food, but are generally added in
small amounts. The safety evaluation of flavours has presented
special problems for JECFA, and these are discussed in detail in
section 6.1.2.
6.1.1. Food contaminants
JECFA has considered the presence of food contaminants on
many occasions since 1972, when mercury, lead, and cadmium were
first assessed (60, pp. 11-24). These food contaminants have
included, in addition to heavy metals, environmental contam-
inants such as mycotoxins, impurities arising in food additives,
solvents used in food processing, packaging material migrants,
and residues arising from the use of animal feed additives
and/or veterinary drugs. Each of these classes of food contam-
inants possesses its own unique characteristics and evaluation
requirements. Thus, JECFA has recognized through the years that
evaluation principles should pertain to classes or groups of
contaminants rather than to food contaminants in toto. JECFA
has published guidelines, which are reproduced in Annex III, for
the evaluation of various classes of contaminants; these
guidelines are still valid.
At the time that JECFA considered mercury, cadmium, and
lead, in 1972, it established the concept of "provisional
tolerable weekly intake" (PTWI), which is a departure from the
traditional ADI concept (60, pp. 9-11). JECFA has continued to
use this concept, with some modifications, ever since.
ADIs are intended to be used in allocating the acceptable
amounts of an additive for necessary technological purposes.
Obviously, trace contaminants have no intended function, so the
term "tolerable" was seen as a more appropriate term than
"acceptable", which signifies permissibility rather than accep-
tability for the intake of contaminants unavoidably associated
with the consumption of otherwise wholesome and nutritious
foods.
In this convention, tolerable intakes are expressed on a
weekly basis, because the contaminants given this designation
may accumulate within the body over a period of time. On any
particular day, consumption of food containing above-average
levels of the contaminant may exceed the proportionate share of
its weekly tolerable intake. JECFA's assessment takes into
account such daily variations, its real concern being prolonged
exposure to the contaminant, because of its ability to accumu-
late within the body over a period of time.
The use of the term "provisional" expresses the tentative
nature of the evaluation, in view of the paucity of reliable
data on the consequences of human exposure at levels approaching
those with which JECFA is concerned.
A tolerable intake, as defined above, represents the maximum
acceptable level of a contaminant in the diet; the goal should
be to limit exposure to the maximum feasible extent, consistent
with the PTWI. However, potent carcinogens, such as certain
mycotoxins, cannot be made to fit within the confines of a PTWI
because, using the traditional approach, safe levels cannot be
set. JECFA addressed this issue in 1978 and introduced the
concept of an "irreducible level", which it defined as "that
concentration of a substance which cannot be eliminated from a
food without involving the discarding of that food altogether,
severely compromising the ultimate availability of major food
supplies" (32, pp. 14-15).
Another JECFA end-point, the "provisional maximum tolerable
daily intake" (PMTDI) has been established for food contam-
inants that are not known to accumulate in the body, such as tin
(23), arsenic (24), and styrene (1). The value assigned to the
PMTDI represents permissible human exposure as a result of the
natural occurrence of the substance in food and in drinking-
water.
In 1982, JECFA decided to change the methodology of
assessment for trace elements that are both essential nutrients
and unavoidable constituents of food, such as copper and zinc.
The Committee concluded that, in such situations, the use of one
number for expression of the tolerable intake was not suffi-
ciently informative, so expression in a range was instituted
(23, Annex II). The lower value represents the level of essen-
tiality and the upper value the PMTDI. Thus, the upper value
should not be construed as representing its normal daily
requirement.
With the use of increasingly sensitive analytical tech-
niques, it is becoming clear that many food additives also con-
tain trace levels of carcinogenic contaminants. JECFA addressed
this issue in 1984 when it considered the low-level migration of
carcinogenic contaminants from food packaging materials (1).
The Committee did not consider it appropriate to allocate ADIs
on the basis of the information available. For further eval-
uation, the twenty-eighth JECFA stated that it would need the
following information:
(a) the lowest levels of potential migrants from within the
polymeric system(s) that are technologically attainable
with improved manufacturing processes for food-contact
materials;
(b) the resulting levels of the migrants in foods;
(c) the intake of the foods; and
(d) the most appropriate statistical design that will
enable the implications for health to be interpreted
from adequate and relevant toxicological data.
In the meantime, the Committee recommended that "human exposure
to migrants from food-contact materials be restricted to the
lowest levels technologically attainable" (1, p. 23).
6.1.2. Food flavouring agents
The special problems associated with the safety evaluation
of food flavouring agents have been apparent to JECFA for a
considerable time and the tenth meeting of the Committee
recommended that "further meetings of JECFA should be convened
to draw up specifications for flavouring substances, . . . used
as food additives, and to evaluate the toxicological hazards
involved in their use" (29). The Committee referred to the
extent and magnitude of these problems in the report of the
eleventh meeting (41) but, despite the time that has elapsed,
the problems remain largely unresolved. Much of the difficulty
arises from the fact that a very large number of substances are
used as food flavouring agents, many of which occur in natural
products, but their level of use is generally low and self-
limiting. The compounds and other materials (extracts,
oleoresins, essential oils) used to impart flavour to foods can
be classified into four groups (19), viz:
(a) artificial substances unlikely to occur naturally in
food;
(b) natural materials not normally consumed as food, their
derived products, and the equivalent nature-identical
flavourings;
(c) herbs and spices, their derived products, and the
equivalent nature-identical flavourings; and
(d) natural flavouring substances obtained from vegetable
and animal products and normally consumed as food
whether processed or not, and their synthetic equi-
valents.
Most of these food flavouring materials have not been
subjected to detailed and comprehensive toxicity tests, though
with the flavouring agents in classes (c) and (d) above there
may be a long history of use and limited evidence of safety-in-
use. In some cases, there may also be evidence of adverse con-
sequences of human exposure such as hypersensitivity and idio-
syncratic intolerance, which have been observed with capsaicin,
zingiberin, and menthol. However, the natural origin of a food
flavouring agent is no guarantee of safety, nor does traditional
use of a material constitute unequivocal evidence of safety; the
flavour saffrole is of natural origin (oil of sassafras) and
had a long history of use before it was demonstrated to be hepa-
toxic and carcinogenic (22). Consequently, natural flavouring
compounds cannot be exempted from the toxicological evaluation
applicable to food flavouring agents in general. Conversely,
there is no basis for assuming that compounds that interact with
gustatory or olfactory receptors are more likely to interact
with other physiological receptor sites than non-flavoured com-
pounds. In principle, therefore, the safety evaluation of food
flavouring compounds is similar to that for other food addi-
tives, and this fact was recognized in the twentieth report of
JECFA (19). However, this Committee concluded that evaluation
should be flexible and "may require extensive toxicological
testing or be made simply from available data". In the evalua-
tion process, the work of other bodies, such as national govern-
ments and the Council of Europe, should be considered.
In view of the very large number of substances used as food
flavouring agents and the fact that they are generally applied
in low and self-timing concentrations in foods, it is considered
impractical and unreasonable to require that each food flavour-
ing material be subjected to the same and extensive toxico-
logical evaluation within a reasonable period (32). Further-
more, human data may be available that have a bearing on the
extent of toxicity testing required and the urgency with which
data from such tests are needed. Thus, there is a need to
develop a rational basis for allocating priorities for the
testing and safety evaluation of food flavouring agents and for
determining the extent of the testing required. Previous
Committees have drawn attention to this need on several occa-
sions (19, 32, 31, 24). Several of the factors that influence
the allocation of priorities are also relevant to the extent of
testing required and some of these are discussed in general
terms elsewhere in this report (sections 3, 5.2, and 5.4). The
following discussion is specifically concerned with food
flavouring compounds.
Factors that should be considered in the allocation of
priorities and the determination of the extent of testing
required include:
(a) nature and source of the material;
(b) data on usage and on the extent and frequency of
exposure of the average consumer and of subpopulations,
who may be highly exposed (including exposure from
natural sources such as herbs and spices but excluding
bizarre eating behaviour);
(c) structure/activity relationships and the similarity of
compounds of known toxicological and biochemical prop-
erties;
(d) similarity to compounds of known biological activity in
metabolism and pharmacokinetics;
(e) information from short-term tests for mutagenicity and
clastogenicity;
(f) prior experience of human use; and
(h) toxicological status/regulatory status previously det-
ermined by national regulatory agencies or supra-
national organizations such as the European Economic
Community (EEC) Scientific Committee for Foods and the
International Agency for Research on Cancer (IARC).
Information on the nature and source of the flavouring
material is clearly necessary in order to assess overall
exposure to its components, and such information may assist in
the allocation of priorities and level of testing required.
Data obtained on synthetic compounds may assist in the evalua-
tion of limited data on extracts or essential oils that contain
the compounds naturally. For example, knowledge of the compo-
sition of an extract or essential oil may indicate that no
further testing is necessary; conversely, the presence of a
known toxic compound as a major component in an extract or
essential oil (e.g., saffrole in oil of sassafras, thujone in
oil of wormwood, tansy, etc.) may alter the priority given to
that extract or essential oil and determine the nature of
specific toxicity tests required. It must be stressed that any
assessment of priorities will depend on the availability of
adequate specifications which, in the case of complex mixtures
such as spice extracts, may include maximum limits for known
toxic constituents.
In considering the extent of exposure, the eleventh meeting
of JECFA (41) suggested that flavouring compounds with an esti-
mated per capita consumption exceeding 3.65 mg per annum (sus-
tained intake exceeding 10 g per day), and/or use in food at a
level higher than 10 mg/kg food, should be given priority. How-
ever, exposure must be considered in the light of other avail-
able information and it is inappropriate to classify flavouring
compounds for priority evaluation solely on the basis of expo-
sure levels.
In the absence of other data or considerations, a combina-
tion of exposure levels and structure/activity relationships can
be used to establish both priorities and requirements for the
level of testing. A number of schemes that take these factors
into account have been proposed to accomplish this (34 - 38).
The general principles regarding structure/activity rela-
tionships are discussed in section 3.1.2. With particular ref-
erence to food flavouring compounds, these principles have been
applied already by JECFA in a limited number of cases, both in
relation to high levels of concern (flavouring compounds
structurally-related to saffrole) and acceptance of limited
toxicological data (simple structural analogues, homologues, or
derivatives such as esters). In the latter case, detailed
toxicological data on one member of a group of related compounds
together with metabolic and pharmacokinetic data can be used to
allocate an ADI or group ADI to structurally related compounds
without the need for further testing. On this basis, a sub-
stantial number of esters used as food flavouring agents would
warrant the allocation of a low priority for testing and
acceptance for an ADI on the basis of metabolic studies alone.
This would be true in cases in which flavouring compounds are
shown to be rapidly and quantitatively hydrolysed to toxico-
logically known alcohols and acids, the safety of which have
been established.
The use of short-term tests for mutagenicity/clastogenicity
cannot at present be considered a substitute for carcinogenicity
bioassays and hence negative results in such tests need to be
considered in the light of the total toxicological data base in
allocating priorities. In general, such negative results would
not justify waiving the requirement for long-term carcino-
genicity studies on food flavouring compounds, but they may be
useful supplementary information to that discussed above. Con-
versely, positive results in a suitable battery of short-term
mutagenicity/clastogenicity tests would indicate a higher
priority for more detailed testing.
Prior experience of human use will influence the allocation
of priorities, as indicated earlier, and even the limited
evidence of safety-in-use may support judgements based on the
other criteria discussed above, to allocate a low priority to
testing or to accept limited toxicological data. Conversely,
observations of human idiosyncratic intolerance and/or allergies
would indicate a need for adequate information relating to the
extent and severity of the problem.
Systematic treatment of the above data (structure/activity
relationships, exposure, and human data) and the application of
reasonable criteria for the adequacy of existing data, could
provide a useful approach in focusing effort on substances that
should receive priority from a scientific point of view. Other
criteria that influence the priority that JECFA allocates to
food flavours include requests from, or previous evaluation by,
national governments, the European Economic Community Scientific
Committee for Foods, the Codex Alimentarius Committee on Food
Additives, and the need to re-evaluate previously established
temporary ADIs. Earlier JECFAs have recommended the setting up
of a working group of experts specifically to consider the
allocation of priorities for the testing and evaluation of food
flavours, and this recommendation still appears to be appro-
priate. There is an implicit need for adequate resources to
perform this task and cooperation with other interested groups
to avoid wasteful duplication of effort.
6.2. Substances Consumed in Large Amounts
The safety assessment of substances that are consumed in
relatively large amounts presents a number of special problems.
Such materials include defined chemical substances such as
sorbitol and xylitol (23, 24), modified food ingredients such as
modified starches (23), and foods from novel sources.
The safety assessment of such substances should differ from
that of other food additives, such as colouring and flavouring
agents, and antioxidants, for the following reasons:
(a) Many will have a high daily intake and, thus, minor
constituents and processing impurities assume greater-
than-usual significance.
(b) Even though they are often structurally similar or even
identical to natural products used as food and thus may
appear to be of low toxicity, many may require
extensive toxicity testing, because of their high daily
intake.
(c) Some may be metabolized into normal body constituents.
(d) Some substances, particularly foods from novel sources,
may replace traditional foods of nutritional importance
in the diet.
(e) Many are complex mixtures rather than defined chemical
substances.
(f) The difference between the quantity that can be fed to
animals in feeding tests and the amount consumed by
human beings is often relatively small.
6.2.1. Chemical composition, specifications, and impurities
Thorough chemical analysis should be performed on high-
consumption substances to measure potential impurities and to
provide information on nutritional adequacy, especially when
such substances replace traditional food.
It is not possible to provide a checklist of necessary
chemical studies to cover all high-consumption compounds.
However, the substance should be subjected to a full proximate
analysis and particular attention should be paid to the points
discussed in the following paragraphs.
Because the intake of undesirable impurities concomitant
with the intake of high-consumption materials is potentially
high, special effort should be made to identify the impurities.
Information on the production process, including the materials
and procedures involved, will point to the types of contaminants
for which limits may need to be specified. The specifications
should be accompanied by details of product variability and of
the analytical methods used to check the specifications and
details of the sampling protocols. If the substance is so
complex that comprehensive product specifications on chemical
composition are impracticable (as it might be for a microbial
protein), the description of the substance in the specifications
may include relevant aspects of its manufacturing process. If
manufacturing data are based on production on a pilot scale, the
manufacturer should demonstrate that, when produced in a large-
scale plant, the substance will meet the specifications estab-
lished on the basis of pilot data.
The permissible limits for impurities may in some cases
correspond to the levels accepted for natural foods that have
similar structure or function, or that are intended to be
replaced by the new material. If the substance is prepared by a
biological process, special attention should be paid to the
possible occurrence of natural toxins (e.g., mycotoxins).
The substance should be analysed for the presence of toxic
metals. Depending on the intended use, analysis for metals of
nutritional significance may also be appropriate.
If the nature of the substance or manufacturing process
indicates the possible presence of naturally-occurring or adven-
titious antinutritional factors (phytate, trypsin inhibitors,
etc.), or toxins (haemagglutinins, mycotoxins, nicotine, etc.),
the product should be analysed for them specifically. Biolog-
ical tests, either as part of the nutritional evaluation in the
case of enzyme inhibitors or more specifically as part of a
mycotoxin screening programme, will provide useful back-up evi-
dence concerning the presence or absence of these contaminants.
Finally, if under the intended conditions of use the sub-
stance may be unstable or is likely to interact chemically with
other food components (e.g., degradation or rearrangement of the
substance during heat processing), data should be provided on
its stability and reactivity. The various tests should be
conducted under conditions relevant to the use of the substance
(e.g., at the acidity and temperature of the environment and in
the presence of other compounds that may react).
6.2.2. Nutritional studies
With some substances, particularly with novel foods, nutri-
tional studies may be necessary to forecast the likely impact of
their introduction on the nutritional status of consumers. In
addition to affecting the nutritional content of the diet, such
substances may influence the biological availability of
nutrients in the diet. The nutritional consequences of the
introduction of such a substance in the diet can only be judged
in the light of information about its intended use. As much
information as possible should therefore be obtained about
potential markets and uses, and the likely maximum consumption
by particular subpopulations should be estimated.
6.2.3. Toxicity studies
When testing high-consumption additives, animals should
generally be fed the highest levels possible consistent with
palatability and nutritional status. Therefore, before begin-
ning such studies, it is desirable to investigate the palat-
ability of the test diet in the test animals. If a palatability
problem is encountered, it may be necessary to increase the
amount of the test substance to the required level gradually.
Paired-feeding techniques should be used, if the problem cannot
be overcome. It should always be borne in mind that there are
practical limits to the amounts of certain foods that can be
added to animal diets without adversely affecting the animal's
nutrition and health.
To ensure that the nutritional status of the test animal is
not distorted, the test and control diets should have the same
nutritive value in terms of both macronutrients (e.g., protein,
fat, carbohydrate, and total calories) and micronutrients (e.g.,
vitamins and minerals). When feeding substances at high levels,
it is usually advisable to formulate diets from individual
ingredients (rather than adding the test material to a standard
laboratory diet) to provide the same nutrient levels in the
control and test diets. Comprehensive nutrient analyses of the
test and control diets should be performed to ensure that they
are comparable nutritionally. Sometimes nutritional studies are
advisable before toxicological studies are performed to ensure
that test diets are correctly balanced. Without due regard to
nutritional balance, excessive exposure may investigate the
toxicological end-points of long-term dietary imbalance rather
than the toxic effects of the substance.
The establishment of a precise no-observed-effect level will
not usually be feasible on account of the relative non-toxicity
of high-consumption additives and the impracticability of
achieving an adequate safety margin between the no-observed-
effect level in animals and the expected consumption of such
substances by human beings. Therefore, it is particularly
important that the variables for assessing the safety of the
substance, such as body weight, food and water consumption,
haematological parameters, ophthalmology, blood chemistry, urine
analysis, faecal analysis, mineral and vitamin excretory levels,
etc., are chosen carefully to include monitoring of all its
possible toxic effects.
Metabolic studies are useful and necessary for assessing the
safety of high-consumption additives. With complex mixtures,
studies on the metabolic fate of every constituent would be
impracticable. However, if contaminants or minor components are
suspected as the cause of toxicity, their metabolism should be
investigated. Consideration should also be given to the effects
that the new constituents may have on the ability of the host to
withstand other toxic agents, for example, effects on the
metabolism of xenobiotic compounds. If the material, or a major
component of it, consists of a new chemical compound that does
not normally occur in the diet (e.g., a novel carbohydrate),
studies of the metabolic fate of the new compound would be
appropriate.
If biochemical and metabolic studies show that the test
material is completely broken down in the food or in the gastro-
intestinal tract to substances that are common dietary or body
constituents, then other toxicity studies may not be necessary.
The results of metabolic studies can stand on their own if it is
shown: that breakdown into these common constituents occurs
under the conditions of normal consumption of the material, that
the material contributes only a small proportion of these common
constituents in the daily diet, and that side reactions giving
rise to toxic products do not occur.
Analysis of urine and faeces may provide important inform-
ation relating to changes in normal excretory functions caused
by the test substance. For example, the gut flora may be
altered, or preferential loss of a mineral or vitamin may occur,
resulting in detrimental effects on the health of the test
animals. If the substance is incompletely or not degraded by
the digestive enzymes of the stomach or the small intestine,
appreciable concentrations may be found in the faeces or in the
distal gut compartments. As a result, changes in the absorption
of dietary constituents or changes in the composition and meta-
bolic activity of the intestinal flora may be observed. Because
of species-dependent anatomical differences in the digestive
tract and because of considerable differences in the composition
of the basal diet, such effects may occur only in man but not in
rodents, or vice versa. Therefore, short-term biochemical
studies should be performed in animals and man (if possible) in
which variables likely to be affected by the test compound are
examined in detail. It is especially important to investigate
questions relating to whether the eventual effects are progres-
sive or transient and whether they occur in subjects exposed to
the compound for the first time and/or in subjects adapted to a
daily intake of the substance. Clearly, no standard design for
such studies can be devised. Only a thorough knowledge of the
nutritional and biochemical literature can serve as a guide-
line.
When establishing an ADI, the traditional concept of a 100-
fold safety factor cannot operate when the human consumption
level is high and feeding studies do not produce adverse effects
(except for effects arising from the physical properties of the
additive, such as its bulk and hydrophilicity), even when the
substance is added to the diet in the maximum possible propor-
tion, consistent with reasonable nutrition. In such cases, new
approaches are indicated, including setting the ADI on the basis
of a smaller safety factor, which may be permissible when fac-
tors such as similarity to traditional foods, metabolism into
normal body constituents, lack of overt toxicity, etc., are
considered. For a compound, such as a bulking agent, that may
influence the nutritional balance or the digestive physiology by
its mere bulk and which may be absorbed from the gut only incom-
pletely or not at all, it may be more appropriate to express the
dosage level in terms of the percentage inclusion in the diet.
In using this approach, a direct comparison between the propor-
tion in the human diet, with a small safety factor, can be made.
If several similar types of compounds are likely to be consumed,
a group ADI (limiting the cumulative intake) should be allo-
cated.
The results of human studies, which are discussed in rela-
tion to novel foods in section 6.2.4, may permit the use of a
lower safety factor than that obtained from animal studies.
Separate toxicological tests should be performed on toxico-
logically suspect impurities or minor components present in the
test material. If any observed toxicity can be attributed to
one of the impurities or minor components, its level should be
controlled by use of specifications and manufacturing controls.
6.2.4. Foods from novel sources
Over the past 25 years, a series of developments has made
possible the production of foods from unconventional sources
(e.g., fungal mycelia and yeast cells) and foods produced by
genetic techniques. These foods are intended for consumption,
either directly, or after simple physical modification to pro-
vide a more acceptable product. They may be consumed in large
amounts, even by infants and children, particularly if they are
permitted for use as protein supplements in otherwise protein-
deficient diets.
Complete chemical identification of such materials may not
be feasible, but specifications are necessary to ensure that
levels of potentially hazardous contaminants, such as mycotoxins
and heavy metals, and other substances of concern, such as
nucleic acids, are kept to a minimum. Toxicological evaluations
must be closely related to well-defined materials, and evalua-
tions may not be valid for all preparations from the same source
material, if different processing methods are used.
When a novel food is intended to replace a significant por-
tion of traditional food in the diet, its likely impact on the
nutritional status of consumers requires special consideration.
The influence of the introduction of the new substance on the
nutrient composition of the diet as a whole should be identi-
fied, particularly with respect to groups such as children, the
elderly, and "captive populations", e.g., hospital patients and
school children. In order not to adversely affect the nutri-
tional quality of the diet, it may be necessary to fortify the
substance with vitamins, minerals, or other nutrients.
The nutritional value of the novel food should be assessed
initially from its chemical composition with respect to both
macronutrients and micronutrients, taking into account the
effects of any further processing and storage. The possible
influence of components in the novel food, such as antinutri-
tional factors (e.g., inhibitors of enzyme activity or mineral
metabolism), on the nutritional value or keeping quality of the
remainder of the diet should also be established.
Depending on the nature and intended uses of the novel food,
studies in animals may be needed to supplement the chemical
studies. If the novel food is intended to be an alternative
significant supply of protein, tests on its protein quality will
be necessary. In vivo studies will also be needed when it is
appropriate to determine (a) the availability of vitamins and
minerals in the novel food in comparison with the food it would
replace; and (b) any interaction the novel food might have with
other items of the diet that would reduce the whole diet's
nutritional value. If the novel food is expected to play an
important role in the diet, it may be necessary to verify that
the results of animal studies can be extrapolated to human
beings by measuring the availability of nutrients to human
subjects.
In most cases, novel foods constitute a large percentage of
the daily diet in animal studies because they are of a non-toxic
nature. Therefore, the considerations discussed in section
6.2.3 apply to the toxicological testing and evaluation of foods
from novel sources. The sixteenth and seventeenth meetings of
the FAO/WHO/UNICEF Protein Advisory Group (PAG) and the United
Kingdom Department of Health and Social Security (DHSS) have
developed guidelines on the development and testing of foods
from novel sources, which should be consulted for a detailed
discussion (85 - 87).
With certain classes of substances, such as foods that are
modified by recombinant DNA or hybridization techniques to pro-
duce what effectively are new cultivars or modified traditional
foods, the use of the ADI is not appropriate. However, with
many foods produced from novel sources, the use of the ADI is
appropriate, because these foods bear little relationship to
foods that have been consumed traditionally. The allocation of
an ADI is useful to permit the establishment of specifications
to ensure microbiological purity and to control chemical contam-
inants.
After the appropriate animal tests have been performed and a
tentative ADI has been established, human volunteer studies to
test for specific human effects should be undertaken. The first
human study should involve the feeding of a single meal con-
taining the novel food at a known dose level to one volunteer at
a time. If no harmful effects are observed with several volun-
teers, studies involving the feeding of the novel food for a
short period (initially about four weeks with follow-up studies
of longer duration) should be performed. Different diets incor-
porating different levels of the novel food should be related to
the anticipated levels of human exposure. The closest attention
should be paid to matching groups with respect to age, height,
weight, sex, alcohol intake, and smoking habits. In addition to
having normal control groups, it may be useful to organize
studies in which the test groups are fed diets incorporating and
not incorporating the novel food in sequential periods, so that
each volunteer acts as his own control; blind crossover trials
are the most satisfactory. Once it has been determined that the
novel food is tolerated well by volunteers at fixed dietary
levels, it may be useful to feed it ad libitum, for a short
period of time, in order to assess its acceptability.
If the novel food is intended for use by a certain community
or section of the community (e.g., among a particular ethnic
group or by diabetic patients), at least one study should be
conducted in the group of people for whom the food is intended.
It may be necessary to conduct allergenicity studies on the
novel food because of its composition (e.g., if it is highly
proteinaceous) or because the results of animal or human feeding
studies suggest that the food might produce hypersensitivity in
some people. Important information can be gained by monitoring
the health of workers coming into contact with the novel food,
such as laboratory staff and employees in the manufacturing
plant. In order to detect possible allergenicity of the novel
food in the general population, it will generally be essential
to monitor a large number of people.
Large-scale acceptability and marketing trials should be
undertaken only after the novel food's safety has been demon-
strated by the studies indicated above. It may be most useful
to restrict the trial to a defined geographical area. The local
medical services responsible for the area in which the substance
is tested should be alerted so that they may take it into
account when evaluating any unusual disease patterns that may
appear during or after the test period. Because large numbers
of people will be involved in the trials, it may be possible to
obtain information about rare food intolerance (e.g., allergic
reactions) that may not have been observed in earlier human
studies. The extent to which health monitoring should be per-
formed will depend on the nature of the substance and the
results of previous toxicological investigations.
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ANNEX I. GLOSSARY
I.1 Abbreviations Used in this Document
ADI: Acceptable Daily Intake (see definition)
CCFA: Codex Committee on Food Additives (see definition of
Codex Alimentarius Commission)
COT: Committee on Toxicity (United Kingdom)
CSM: Committee on Safety of Medicines (United Kingdom)
EEC: European Economic Community
EPA: Environmental Protection Agency (USA)
FAO: Food and Agriculture Organization of the United Nations
FDA: Food and Drug Administration (USA)
FSC: Food Safety Council, Washington, DC, USA
GEMS: Global Environmental Monitoring System
GLP: Good Laboratory Practice
IARC: International Agency for Research on Cancer
IPCS International Programme on Chemical Safety
JECFA: Joint FAO/WHO Expert Committee on Food Additives (see
definition)
LD50 Lethal Dose, median
MAFF: Ministry of Agriculture, Forestry and Fisheries (Japan)
MHW: Ministry of Health and Welfare (Japan)
MTD: Maximum Tolerated Dose (see definition)
OECD: Organisation for Economic Cooperation and Development
PMTDI: Provisional Maximum Tolerable Daily Intake (see definition)
PSPS: Pesticides Safety Precautions Scheme (United Kingdom)
PTWI: Provisional Tolerable Weekly Intake (see definition)
SCOPE: Scientific Committee on Problems of the Environment of
the International Council of Scientific Unions
UNICEF: United Nations Childrens' Fund
WHO: World Health Organization
I.2 Definitions of Terms Used in this Document
Acceptable daily intake: An estimate by JECFA of the amount of
a food additive, expressed on a body weight basis, that can be
ingested daily over a lifetime without appreciable health risk
(standard man = 60 kg).
Acceptable daily intake not allocated: See no ADI allocated.
Acceptable daily intake not specified: A term applicable to a
food substance of very low toxicity which, on the basis of the
available data (chemical, biochemical, toxicological, and
other), the total dietary intake of the substance arising from
its use at the levels necessary to achieve the desired effect
and from its acceptable background in food does not, in the
opinion of JECFA, represent a hazard to health. For that
reason, and for reasons stated in individual evaluations, the
establishment of an acceptable daily intake 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 inferior food quality
or adulteration, and it should not create a nutritional
imbalance.
Codex Alimentarius Commission: The Commission was formed in
1962 to implement the Joint FAO/WHO Food Standards Programme.
The Commission is an intergovernmental body made up of more than
120 Member Nations, the delegates of whom represent their own
countries. The Commission's work of harmonizing food standards
is carried out through various committees, one of which is the
Codex Committee on Food Additives (CCFA). JECFA serves as the
advisory body to the Codex Alimentarius Commission on all
scientific matters concerning food additives.
Conceptus: All products of conception derived from and
including the fertilized ovum at any time during pregnancy,
including the embryo or fetus and embryonic membranes.
Developmental toxicity: Any adverse effects induced prior to
attainment of adult life, including effects induced or
manifested in the embryonic or fetal period and those induced or
manifested postnatally (before sexual maturity).
Effect: A biological change in an organism, organ, or tissue.
Elimination (in metabolism): The expelling of a substance or
other material from the body (or a defined part thereof),
usually by a process of extrusion or exclusion, but sometimes
through metabolic transformation.
Embryo/fetotoxicity: Any toxic effect on the conceptus resul-
ting from prenatal exposure, including structural or functional
abnormalities or postnatal manifestation of such effects.
Embryonic period: The period from conception to the end of
major organogenesis. Generally, the organ systems are identi-
fiable at the end of this period.
Enterohepatic circulation: Intestinal reabsorption of material
that has been excreted through the bile followed by transfer
back to the liver, making it available for biliary excretion
again.
Fetal period: The period from the end of embryogenesis to the
completion of pregnancy.
Food allergy: A form of food intolerance in which there is
evidence of an abnormal immunological reaction to the food.
"Immediate allergic reactions" are those which occur within
minutes to hours after ingestion of the offending food, while
reactions beginning several hours to days after food exposure
are characterized as "delayed allergic reactions".
Food intolerance: A reproducible, unpleasant reaction to a food
or food ingredient, including reactions due to immunological
effects, biochemical factors such as enzyme deficiencies, and
anaphylactoid reactions that often include histamine release.
Group acceptable daily intake: An acceptable daily intake
established for a group of compounds that display similar toxic
effects, thus limiting their cumulative intake.
Irreducible level (of a food contaminant): That concentration
of a substance which cannot be eliminated from a food without
involving the discarding of that food altogether, severely
compromising the ultimate availability of major food supplies.
JECFA: JECFA is a technical committee of specialists acting in
their individual capacities. Each JECFA is a separately-
constituted committee, and when either the term "JECFA" or "the
Committee" is used, it is meant to imply the common policy or
combined output of the separate meetings over the years.
Long-term toxicity study: A study in which animals are observed
during the whole life span (or the major part of the life span)
and in which exposure to the test material takes place over the
whole observation time or a substantial part thereof. The term
chronic toxicity study is used sometimes as a synonym for "long-
term toxicity study".
Maximum tolerated dose: A term in common use in carcinogenicity
testing meaning a dose that does not shorten life expectancy nor
produce signs of toxicity other than those due to cancer
(operationally, the MTD has been set as the maximum dose level
at which a substance induces a decrement in weight gain of no
greater than 10% in a subchronic toxicity test).
No ADI allocated: Terminology used by JECFA in situations where
an ADI is not established for a substance under consideration
because (a) insufficient safety information is available; (b) no
information is available on its food use; or (c) specifications
for identity and purity have not been developed.
No-observed-effect level: The greatest concentration or amount
of an agent, found by study or observation, that causes no det-
ectable, usually adverse, alteration of morphology, functional
capacity, growth, development, or lifespan of the target.
Novel food: A food or food ingredient produced from raw
materials not normally used for human consumption or food that
is severely modified by the introduction of new processes not
previously used in the production of food.
Processing aid: A substance added to food during processing,
but subsequently removed. Traces of a processing aid may remain
with the food.
Provisional maximum tolerable daily intake: The end-point used
by JECFA for contaminants with no cumulative properties. Its
value represents permissible human exposure as a result of the
natural occurrence of the substance in food and in drinking
water. In the case of trace elements that are both essential
nutrients and unavoidable constituents of food, a range is
expressed, the lower value representing the level of
essentiality and the upper value the PMTDI.
Provisional tolerable weekly intake: The end-point used by
JECFA for food contaminants such as heavy metals with cumulative
properties. Its value represents permissible human weekly
exposure to those contaminants unavoidably associated with the
consumption of otherwise wholesome and nutritious foods.
Reproductive effects: To test for the effects of exposure to
low levels of chemicals exceeding the life span of one gener-
ation, tests have been developed covering several reproductive
cycles. In the three-generation test, the animals are exposed
through three complete reproductive cycles (starting with the F0
generation at weaning). These tests, which include exposure in
utero and through the milk, have been used in particular for
assessing toxic effects related to reproduction.
Safety factor: A factor applied by JECFA to the no-observed-
effect level to derive an acceptable daily intake (the no-
observed-effect level is divided by the safety factor to
calculate the ADI). The value of the safety factor depends on
the nature of the toxic effect, the size and type of population
to be protected, and the quality of the toxicological
information available.
Short-term toxicity study: An animal study (sometimes called a
subacute or subchronic study) in which the effects produced by
the test material, when administered in repeated doses (or
continuously in food or drinking-water) over a period of about
90 days, are studied.
Temporary acceptable daily intake: Used by JECFA when data are
sufficient to conclude that use of the substance is safe over
the relatively short period of time required to generate and
evaluate further safety data, but are insufficient to conclude
that use of the substance is safe over a lifetime. A higher-
than-normal safety factor is used when establishing a temporary
ADI and an expiration date is established by which time
appropriate data to resolve the safety issue should be submitted
to JECFA.
Teratogen: An agent which, when administered prenatally,
induces permanent abnormalities in structure.
Teratogenicity: The property (or potential) to produce struc-
tural malformations or defects in an embryo or fetus.
Transplacental carcinogenesis: The appearance of neoplasia in
the progeny of females exposed to chemical agents during
pregnancy.
ANNEX II. STATISTICAL ASPECTS OF TOXICITY STUDIES
II.1 Summary
Statistical design and analysis should aim at eliminating
sources of potential bias and minimizing the role of chance.
The application of these principles in the experimental design
and conduct of toxicological studies is discussed under 10
headings: choice of species, dose levels, number of animals,
duration of the study, accuracy of determinations, stratifica-
tion, randomization, adequacy of control groups, animal place-
ment, and data recording. A number of general considerations to
be borne in mind when conducting statistical analyses are also
discussed: experimental and observational units, types of res-
ponse variable, types of between-group comparisons, stratifica-
tion, age adjustment, multiple observations per animal, hypo-
thesis testing and probability values, and multiple comparisons.
Finally, some recommended methods of statistical analysis are
summarized.
II.2 Introduction
These guidelines are intended primarily to provide the
experimental scientist without statistical qualifications with
some insight into statistical aspects of toxicological studies.
Considerations relating to the design and conduct of studies and
to the analysis and interpretation of results are discussed,
emphasizing the principles involved rather than the mathematical
details. While the experimentalist should have sufficient
information to deal with many standard situations, the need for
the advice of an expert statistician, when dealing with toxico-
logical data, cannot be overemphasized. Scientific journals
frequently contain papers describing studies in which the con-
clusions of the author(s) cannot be supported because of defi-
ciencies in statistical methodology, which could have been
avoided had the advice of a qualified statistician been avail-
able to the researcher.
II.3 Sources of Difference Between Treated and Control Groups
An objective of many toxicity studies is to determine
whether a treatment elicits a response. However, the observa-
tion of a difference in response between a treated and a control
group does not necessarily mean that the difference is a result
of the treatment. There are two other potential causes of dif-
ference, bias and chance.
Bias implies systematic differences other than treatment
between the groups, in other words, failure to compare like with
like. Properly conducted studies analysed appropriately can
eliminate bias.
Chance factors cannot be wholly excluded, because identi-
cally-treated animals will not all respond identically, however
carefully the study is conducted. While it is impossible to be
absolutely certain that even very extreme differences in res-
ponse are not due to chance, appropriate statistical analysis
will allow the experimentalist to assess the probability of a
"false positive", that is, the probability of the observed
difference having occurred had there been no effect of treatment
at all. The smaller the probability, the greater the confidence
of having found a real effect. To improve the likelihood of
detecting a true effect with confidence, it is necessary to try
to minimize the role of chance by seeking to ensure that the
"signal" can be recognized above the "noise".
II.4 Experimental Design and Conduct
Ten aspects of the experimental design and conduct of toxi-
city studies are considered below, the first six being involved
primarily with minimizing the role of chance and the last four
being particularly relevant to the avoidance of bias. For con-
venience, the principles are illustrated with reference to a
long-term carcinogenicity study.
II.4.1 Choice of species
While maximizing the "signal" means avoiding a species in
which the response of interest is very rare, the use of an over-
responsive species also has problems. Thus, to achieve the same
level of statistical significance in comparing a treated group
with a 5% response and a control group with a 0% response
requires only one tenth as many animals as when the responses
are 55 and 50%, respectively. Furthermore, it is not certain
that an increased incidence of a lesion that is a common spon-
taneous finding in the animal species used (such as pituitary
tumours in Wistar rats) provides biological evidence of an
effect that can be extrapolated to other species. Other con-
siderations related to the choice of species, whether they be
practical (short life span, small size, availability, existence
of detailed knowledge of the species) or more theoretical (bio-
chemical, physiological, or anatomical similarity to man), do
not really pose statistical problems.
II.4.2 Dose levels
Dose selection is an important and controversial element in
the development of a protocol for a toxicity bioassay. On bio-
logical grounds, it would be ideal to test only at dose levels
comparable with those to which human beings are exposed. On
statistical and economic grounds, this is not usually practic-
able because the effect will be too small to detect without very
large numbers of animals. To avoid the possibility of missing
an effect that would occur in a small proportion of millions of
exposed human beings in a study on hundreds or even thousands of
animals, it is normally appropriate to test animals at dose
levels many times higher than the maximum human exposure level.
Then, assuming any effect that exists is dose-related, the dem-
onstration of a non-significant increase in response at a high
dose level, though not providing evidence of absolute safety (an
impossible goal), can give reasonable grounds for believing that
any effects that might occur at a very much lower dose level
would be, at most, very slight.
A particular problem with this procedure is to decide how
high the dose level should be. In long-term carcinogenicity
studies, the dose should clearly be one that is not so great
that the animals die from toxic effects before they have a
chance to get cancer. On the basis of these principles, the
International Agency for Research on Cancer (1) has recommended
that the high dose should be one expected on the basis of an
adequate short-term study to produce some toxicity when admin-
istered for the duration of the study, but should not induce:
(a) overt toxicity, i.e., appreciable death of cells or organ
disfunction, as determined by appropriate methods; (b) toxic
manifestations that are predicted materially to reduce the life
span of the animals, except as a result of the development of
neoplasms; or (c) 10% or greater retardation of body-weight gain
compared with that in control animals.
If the substance seems completely non-toxic, the high dose
may represent about 5% of the diet, or even more for substances
such as some nutritive food ingredients.
It is important to have more than one dose level for a
number of reasons. One is to compensate for the possibility
that a misjudgment has occurred and that the highest dose may
prove to be toxic. A second is that the metabolic pathways may
differ at the various dose levels. A third reason is that the
whole point of the study may be to obtain dose-response inform-
ation. Finally, it may be necessary to ensure that an effect
does not occur at dose levels in the range to be used by man.
II.4.3 Number of animals
The number of animals to be used is clearly an important
determinant of the precision of the findings. The calculation
of the appropriate number depends on:
(a) The critical difference, i.e., the size of the effect
to be detected;
(b) the false positive rate, i.e., the probability of an
effect being detected when none exists (known as the
"type I error" or the " alpha level"); and
(c) the false negative rate, i.e., the probability of no
effect being detected when one of exactly the critical
size exists (known as the "type II error" or the "
level").
A reduction in any of these factors means an increase in the
number of animals required.
The method of calculation of the number depends on the exp-
erimental design and the type of statistical analysis envisaged.
Tables are available for a number of standard situations. To
give an idea how the numbers depend on the critical difference
and on the alpha and levels, Tables 1 and 2 give examples of two
common situations, both of which are related to a study in which
there is a control and a treated group. The first is related to
a continuous variable assumed to be normally distributed, with
the critical difference expressed in terms of the number of
standard deviations () by which the treated group differs from
the control group. Thus, given a control response known from
past experience to have a mean value of 50 units with a standard
deviation of 20 units, two groups of 36 animals each would be
needed to have a 90% chance ( = 0.10) of detecting a differ-
ence in response of 10 units (delta = 10/20 = 0.5) at the 95%
confidence level (alpha = 0.05).
In the second situation, two proportions are compared. Here
the numbers of animals depend not only on the ratio of propor-
tions, but also on the assumed proportion in the controls. Thus,
when the control response is expected to be 10%, the numbers of
animals required in each group to detect an increased response
by a factor of 1.5 (r) is 920, assuming again an alpha level of
0.05 and a level of 0.1, whereas if the control response is
expected to be 50%, the numbers required would be 79 per group.
For more complex situations, the advice of a professional
statistician should be sought, though a general rule is that to
increase precision (i.e., decrease the size of the critical
difference) by a factor n, the number of animals required will
have to be increased by a factor of approximately n squared.
Table 1. Number of animals required in each of a control and treated
group in order to have a probability (1 - ) of picking up a difference
of delta standard deviations as significant at the 100 (1 - alpha) percent
confidence level for a normally distributed variable
-----------------------------------------------------------------------------
Single-side test a alpha = 0.005 alpha = 0.025 alpha = 0.05
Double-sided test a alpha = 0.01 alpha = 0.05 alpha = 0.1
= 0.01 0.1 0.5 0.01 0.1 0.5 0.01 0.1 0.5
-----------------------------------------------------------------------------
delta = 0.5 100 63 30 76 44 18 65 36 13
0.75 47 30 16 35 21 9 30 17 7
1.0 28 19 10 21 13 6 18 11 5
1.5 15 11 7 11 7 9 6
2.0 10 8 5 7 5 6
-----------------------------------------------------------------------------
a See section II.5.7 for definitions of single-sided and double-sided
tests.
Table 2. Number of animals required in each of a control and treated
group in order to have a probability (1 - ) of picking up a
proportional increase by a factor r as significant at the 100 (1 - alpha)
percent confidence level for a binomially distributed variable
-----------------------------------------------------------------------------
Single-side test a alpha = 0.005 alpha = 0.025 alpha = 0.05
Double-sided test a alpha = 0.01 alpha = 0.05 alpha = 0.1
= 0.01 0.1 0.5 0.01 0.1 0.5 0.01 0.1 0.5
-----------------------------------------------------------------------------
Control level = 10%
r = 1.25 7679 4754 2120 5871 3358 1228 5039 2737 865
1.5 2103 1302 581 1608 920 337 1380 750 237
2.0 613 380 170 469 268 98 403 219 69
Control level = 20%
r = 1.25 3353 2076 926 2563 1466 536 2200 1195 378
1.5 902 558 249 689 395 145 592 322 102
2.0 253 157 70 193 111 41 166 90 29
Control level = 50%
r = 1.25 757 469 209 579 331 122 497 270 86
1.5 181 112 50 138 79 29 119 65 21
2.0 37 23 10 28 16 6 24 13 5
-----------------------------------------------------------------------------
a See section II.5.7 for definitions of single-sided and double-sided
tests.
When a number of treatments are to be tested in a study,
each to be compared with a single untreated control group, it is
advisable that more animals be included in the control group
than in each of the treated groups, because the precision of the
control group results is relatively more important. When all
groups are of equal interest, it is appropriate to have approx-
imately the square root of k times as many animals in the con-
trol group as in each of the k treated groups.
One point frequently misunderstood by the experimental
scientist is related to the number of animals required in
studies in which more than one treatment is investigated in a
crossed design. If, for example, compounds A and B are being
compared, and each is dissolved in two different solvents, in a
2 x 2 design with 4 groups, calculations of sample size to gain
an overall verdict on the difference between the two compounds
should generally be based on the overall numbers of animals
treated with each compound for both solvents combined, unless
there is reason to expect compound-solvent interaction, i.e.,
that the compound A/compound B difference depends on which
solvent is used. Conversely, if it has been decided that 2
groups of 100 animals each are sufficient for attaining a given
level of precision concerning the differences in effects of a
treatment, additional information and another factor (or
factors) of interest can be obtained without requiring any
additional animals.
II.4.4 Duration of the study
The duration of the study can also markedly affect the
sensitivity of tests. This is particularly so in long-term
carcinogenicity studies in which the great majority of cancers
are seen in the latter half of an animal's lifetime. Thus,
while studies should not be terminated too early, it is also
important that they do not go on too long. This is because the
last few weeks or months may produce relatively little data at a
disproportionate cost, and diseases of extreme old age may be of
little interest in themselves but may render it more difficult
to detect tumours and other conditions that are of interest.
Where the study is of the prevalence of an age-related non-
lethal condition observable only at death that ultimately occurs
in all or nearly all of the animals, early termination is
required. In this situation the greatest sensitivity is
obtained when the average prevalence is about 50%.
II.4.5 Accuracy of determinations
Accuracy of observations is clearly important in minimizing
error. The advent of good laboratory practice and quality
control units has done much to improve the quality of recording
observations, but the quality of the study still depends on
interested and diligent personnel.
II.4.6 Stratification
To detect a treatment difference with accuracy, the groups
being compared should be as homogeneous as possible with respect
to other known causes of the response of interest. Consider,
for example, a set of animals thought to be homogenous (but
which, in fact, consist of two genetically different substrains)
in which the following measurements of body weight were obtained
in groups of 10 treated and 10 control animals, the underlined
readings relating to the first of the two substrains:
Control: 181 192 217 290 321 292 307 347 276 256
Treated: 222 249 232 284 270 215 265 378 328 391.
If the substrain is ignored, the variability of the data
increases so that more controls are required to detect a
treatment effect and, if unequal numbers of each substrain are
present in each group, may bias the comparison. In the example
given the means are as follows:
Substrain 1 Substrain 2 Total
Control 196.7 298.4 267.9
Treated 248.1 365.7 283.4
Difference 51.5 67.2 15.5
Although in each substrain the treatment results in an increase
in body weight of over 50 units, the greater number of the
lower-weight strain in the treated groups means that the
difference observed is much less.
There are two ways to take account of the substrain differ-
ence and to achieve a more precise answer. One is to use sub-
strain as a "stratifying variable" at the analysis stage. This
involves carrying out separate analyses at each level of the
variable considered and combining the results for an overall
conclusion about the treatment effect. However, it does not
preclude the possibility that the proportions of each substrain
in each group are so different that the data provide substan-
tially less comparative information than might otherwise be
achieved. In the extreme case, if all control animals were of
substrain 1 and all treated animals were of substrain 2, the
study would be worthless to determine whether differences were
due to treatment or substrain. To obviate this possibility,
substrain can be used as a "blocking factor" in the design. In
this case, animals in each substrain are allocated equally to
control and treated groups. Although this removes bias, it is
still necessary to treat strain as a stratifying variable in
statistical analysis to increase precision.
When more than one known factor affects the response, all
can be taken into account simultaneously. Both at the design
stage, or retrospectively in the analysis, the results are
treated at each combination of levels of the factors. Thus, to
block for substrain, sex, and room where 3 experimental rooms
were needed to house the animals, 12 mini-studies, one for each
of the 2 (substrains) x 2 (sexes) x 3 (rooms) combinations
would be set up.
II.4.7 Randomization
Random allocation, or randomization, of animals in treatment
groups is an essential of good experimental design. If not
carried out, it is not strictly possible to tell whether a
difference between groups is a result of differences in the
treatment applied or is due to some other relevant factor. A
fundamental on which statistical methodology is based is that
the probability of a particular response occurring is equal for
each animal, regardless of group. The ability to randomize
easily is a major advantage that animal studies have over
epidemiological studies.
The process of randomization eliminates bias, so that
statistical analysis is concerned only with assessing the
probability of an observed difference happening by chance. The
smaller the probability, the more it suggests a true treatment
effect. The procedure used for randomization should genuinely
ensure that all possible assignments of animals to treatment
groups are equally probable. Such equal probabilities are best
achieved with pseudorandom numbers, as found in tables or
produced by computer, it being difficult to ensure that
apparently random devices such as dice or playing cards really
are random. Randomization should never be based on a system of
testing animals haphazardly, as they come, and assigning them to
successive treatment groups. Not only do human beings find it
virtually impossible to generate random sequences unaided, but
it is well known that the first animals selected may differ
markedly from the last, who are more active and avoid being
caught.
In many experimental situations it is adequate to randomly
allocate all the animals to treatment groups, but, in some, the
technique of stratified random sampling is preferred. In this
technique, the animals are first divided into subgroups
("strata"), according to factors known or believed to be
strongly related to the response, with random allocation to
treatment groups then being carried out within each stratum.
Sex is normally treated as a stratifying variable. In a large
study in which animals are delivered in batches, batch could
also be treated in this way, each batch forming a smaller study,
the results from which can be combined in the analysis.
The above discussion on randomization and stratification has
been concerned primarily with the allocation of animals to
treatment groups. The same principles apply to anything that
can affect the recorded response. Thus, in a two-group study,
measurements of some biochemical parameter should not be made
for the first group in the morning and for the second group in
the afternoon. While the major part of such potential bias can
be averted fairly easily by various simple procedures, such as
doing alternate measurements on treated and control animals,
randomization is preferable. Although many different procedures
throughout a study (feeding, weighing, observation, clinical
chemistry, and pathological examinations) require consideration
in this way, the same random number can usually be applied to
all the procedures. Thus, if the cage position of the animals
is randomly allocated and does not depend on treatment, the
animals can always be handled in the same cage sequence.
II.4.8 Adequacy of control groups
The principle of comparing like with like implies that con-
trol groups should be randomly allocated from the same control
source as the treatment groups. While historical control data
can be of value in the interpretation of rarer findings in
treated animals, there is so much evidence of quite large syste-
matic differences in response between apparently identical un-
treated control groups tested at different times that it is
often impossible to be sure whether a difference seen between a
treated group and a historic group is really due to treatment at
all.
It is also essential to be sure that the treated group
differs from the control group only with respect to the
treatment of interest. Thus, if a treatment is applied in a
solvent, an untreated control is not a proper basis for
comparison, as one cannot be sure whether observed differences
are a result of the treatment or the solvent. In this case, the
appropriate control group would be one in which animals are
given only the solvent.
II.4.9 Animal placement
The general underlying requirement to avoid systematic dif-
ferences between groups other than their treatment also demands
that attention be given to the question of animal placement. If
all treated animals are placed on the highest racks or are at
one end of the room, differences in heating, lighting, or venti-
lation may produce effects that are erroneously attributed to
treatment. Such systematic differences should be avoided, and,
in many cases, randomization of cage positions is desirable.
This may not be possible in some circumstances, such as with
studies involving volatile agents where cross-contamination can
occur.
II.4.10 Data recording
The application of the principle of comparing like with like
means the avoidance of systematic bias in data recording prac-
tices. Two distinctly different types of bias can occur. The
first is a systematic shift in the standard of measurement with
time, coupled with a tendency for the time of measurements to
vary from treatment to treatment. The second is that awareness
of the treatment may affect the values recorded by the measurer,
consciously or subconsciously. The second bias is circumvented
by the animals' treatment not being known to the measurer, i.e.,
the readings being carried out "blind". Although not always
practical (that an animal is treated may be obvious from its
appearance), laboratories should organize their data recording
practices so that, at least for subjective measurements, the
observations are made "blind".
The problem of avoidance of bias due to differences in time
of observation is a particularly important one in histopatho-
logical assessment, especially for the recording of lesions of a
graded severity, and in large studies in which the slides may
take the pathologist more than a year to read. When more than
one pathologist reads the slides, there should be discussion
between them as to standardization of terminology and data to be
recorded, and each should read a random or a stratified set of
the slides to avoid bias.
II.5 Statistical Analysis - General Considerations
In the simplest situation, animals are randomly assigned to
a treated, or a control, group and one observation is made on
each animal, the objective of the statistical analysis being to
determine whether the distribution of responses in the treated
group differs from that in the control group. Before summar-
izing some of the appropriate techniques for analysis, a number
of more general points underlying the choice of the correct
method and interpretation of the results will be discussed.
II.5.1 Experimental and observational units
In the simple example cited above, the animal is both the
"experimental unit" and the "observational unit". This is not
always so. In the case of feeding studies, the cage, rather
than the animal, is usually the experimental unit in that it is
the cage, rather than the animal, that is assigned to the treat-
ment. In the case of histopathology, observations are often
made from multiple sections per animal in which case the section
rather than the animal is the observational unit. For the pur-
pose of determining treatment effects by the methods described
below, it is important that each experimental unit provides only
one item of data for analysis, as the methods are all based on
the assumption that individual data items are statistically
independent. If multiple observations per experimental unit are
made, these observations should be combined in some suitable way
into an overall observation for that experimental unit before
analysis. Thus, in a study in which 20 animals were assigned to
two treatment groups of 10 animals each and in which measure-
ments of the weight of both kidneys were made individually, it
would be wrong to carry out an analysis in which the 20 weights
in group 1 were compared with the 20 weights in group 2, because
the individual kidney weights are not independent observations.
A valid method would be to carry out an analysis comparing the
10 average kidney weights in group 1 with the 10 average kidney
weights in group 2.
II.5.2 Types of response variable
Responses measured in toxicological studies can normally be
classified as being one of three types:
(a) Presence/absence: A response either occurs or it does
not.
(b) Ranked: A response may be present in various degrees.
Thus, severity may be classed as minimal, slight,
moderate, severe, or very severe.
(c) Continuous: A response may take any value, at least
within a given range.
Each type of response demands a different sort of statis-
tical technique. While analysis of presence/absence data, often
referred to as "contingency table analysis", can be applied to
ranked or continuous data by defining values above a given cut-
off point as "present", this is not generally recommended,
because it wastes information.
Continuous data are usually analysed by "parametric"
methods, which assume that the statistical distribution under-
lying the response variable (or some transformation of it, e.g.,
its logarithm) has a specific form, traditionally the well-known
bell-shaped Normal or Gaussian distribution. While such methods
are best when the distribution assumed is correct, they can give
misleading conclusions when the assumption is grossly incorrect.
For this reason, when there is doubt about the underlying dist-
ribution, it is often preferable to analyse continuous data by
methods appropriate for ranked data, since these "non-para-
metric" methods make no such underlying assumption and their
conclusions are generally more valid.
II.5.3 Types of between-group comparisons
While in the two-group study only one comparison is pos-
sible, this is not the case when more than two groups are being
compared. Two particularly important types of test made in the
k (> 2) group situation are the test for heterogeneity and the
test for dose-related trend.
The test for heterogeneity determines whether, taken as a
whole, there is significant evidence of departure from the
(null) hypothesis that the groups do not differ in their effect.
It is generally applicable but is not very informative, because
it does not specifically take into account the likely pattern of
response.
The test for dose-related trend is applicable only in
studies in which the groups receive different doses of the same
substance (or have some other natural ordering). It determines
whether there is a tendency for response to rise in relation to
the dose of the test substance. Graphically, the test for trend
can be seen as determining whether a sloped straight line
through the dose (x-axis)/response (y-axis) relationship fits
the data significantly better than a horizontal straight line.
That the trend statistic is significantly positive does not
necessarily imply that the treatment increases response at all
dose levels, though it is a particularly good test if the true
situation is a linear non-threshold model. A trend test often
detects a significant true effect when individual comparisons of
treated groups with the control group fail to give signifi-
cance.
Sometimes, there is significant departure from trend, e.g.,
when there is significant heterogeneity but no evidence of
trend. This may arise because a response increases with dose at
lower dose levels and then reduces at high dose levels, perhaps
due to competing risks. In this situation, it may be appro-
priate to test for trend using only the data from the control
and lower-dose groups.
II.5.4 Stratification
In the simplest situation all the animals in the study
differ systematically only with respect to the treatment
applied. However, often there are a number of sets of animals,
each of which differs systematically only in respect to treat-
ment, but where the characteristics of sets differ. The
commonest situation relates to male and female animals, but
there are many other possibilities, such as different conditions
under which the response variable is measured. While it is
often useful to look within each set of animals or "stratum", as
it is often called, to determine the effects of treatment in
each situation, it is also useful to determine whether, on the
basis of the data from all the strata, an effect of treatment
can be seen overall. In some situations, a relatively small
number of animals in each stratum can make it difficult to pick
up an effect of treatment as significant within individual
strata, and a clear result can be seen only when results are
combined over the strata. The essence of stratification lies in
making comparisons within strata and then accumulating treatment
differences over strata. Pooling data over strata and then
making a single comparison can lead to erroneous conclusions.
To illustrate this, consider a hypothetical study in which, in
one batch of animals, 5/10 controls and 12/30 test animals
responded, while in a second batch, 6/30 controls and 1/10 test
animals responded. If batch were ignored, it would be noted
that 11/40 controls compared with 13/40 test animals responded,
leading to the erroneous conclusion that treatment tended to
increase response. An appropriate analysis would consider batch
as a stratifying factor and note that within both batch 1 (50%
versus 40%) and batch 2 (20% versus 10%) the response in the
control group was higher than that in the test group so that,
combining these two differences, a conclusion would be reached
that treatment tended to decrease response.
II.5.5 Age adjustment
For many conditions, such as tumour incidence, frequency
increases markedly with age (and concomitantly with length of
exposure to the agent), and the overall frequency in a treatment
group can depend as much on the proportion of animals surviving
a long time as on the actual ability of the treatment to cause
the condition. To adjust for differential survival, age is
usually treated as a stratifying variable so that between-group
comparisons are made of animals at similar ages, results of the
comparisons being combined over the different age strata. Age
adjustment is normally applied to presence/absence conditions
and, as discussed at length by Peto et al. (2), the correct
method depends on the context of observation of the condition.
There are three different situations:
(a) Conditions visible in-life: Here comparisons are made
of the number of animals developing the condition in
the time period as a proportion of those without the
condition at the beginning of the period.
(b) Conditions visible only at death and assumed to cause
death (fatal): Here comparisons are made of the number
of animals dying from the condition in the time period
as a proportion of those alive at the beginning of the
period.
(c) Conditions visible only at death and assumed not to
cause death (incidental): Here comparisons are made of
the number of animals dying with the condition in the
time period as a proportion of all those dying in the
time period.
II.5.6 Multiple observations per animal
When multiple observations are made on one animal there are
a number of additional types of statistical analysis, depending
on the experimental situation and the objectives. It is
impossible to cover all the possibilities in this summary, but
the following situations are of reasonable frequency:
(a) Association between variables: Here two or more
different variables are recorded on an animal and the
objective is to determine whether the values are
independent or are correlated.
(b) Variation in association between variables by treat-
ment: For each treatment group an indicator of asso-
ciation between variables is calculated and the statis-
tical problem is to test whether this indicator varies
significantly by treatment group.
(c) Multiple observations of the same variable: For body
weight and clinical chemistry data it is common to take
measurements on the same animals at regular intervals
throughout a long-term study. While between-group
comparisons can be carried out on the basis of data at
any specific point in time, this limits the amount of
information in any one analysis. Statistical tech-
niques are also available to compare groups with res-
pect to change in response between two points in time
or, more generally, with respect to the general pattern
of response over a period of time.
(d) Within-animal comparisons: In most toxicological stud-
ies, different animals receive the different treatments
and comparisons are made on a between-animal basis. In
some studies, the same animal receives more than one
treatment. In such studies, it is important to use
appropriate statistical methods based on within-animal
comparisons.
II.5.7 Hypothesis testing and probability values
Reports of toxicity studies often include statements such as
"the relationship between treatment and blood glucose levels was
statistically significant (P = 0.02)". What does this actually
mean? Three points must be made.
First, there is a difference in meaning between biological
and statistical significance. It is quite possible to have a
relationship that is unlikely to have happened by chance and
therefore statistically significant but of no biological
consequence at all, the animals' well-being being unaffected.
On the other hand an observation may be biologically, but not
statistically, significant, such as when one or two tumours of
an extremely rare type are seen in treated animals. Overall
judgement of the evidence must take into account both biological
and statistical significance.
Second, "P = 0.02" does not mean that the probability that
there is no treatment effect is 0.02. The true meaning is that
given the treatment actually had no effect whatsoever (or to
phrase it more technically, under the null hypothesis) the
probability of observing a difference as great or greater than
that actually seen is 0.02.
Third, there are two types of probability (P)-value. A
"one-sided" (or one-tailed) P-value is the probability of
getting, by chance alone, a treatment effect in a specified
direction as great or greater than that observed. A "two-sided"
(two-tailed) P-value is the probability of obtaining by chance
alone, a treatment difference in either direction, positive or
negative, as great or greater than that observed. Whenever a P-
value is quoted, it should be made clear which is being used.
Normally, two-tailed P-values are appropriate. However, when
there is prior reason to expect a treatment effect in one
direction only, a one-tailed P-value is normally used. If a
one-tailed P-value is used, differences in the opposite
direction to that assumed should be ignored.
While a P-value of 0.001 or less can, on its own, provide
very convincing evidence of a true treatment effect, less
extreme P-values such as P = 0.05 should be viewed as providing
indicative evidence of a possible treatment effect, to be
reinforced or supported by other evidence. If the difference is
similar to one found in a previous study, or if the response,
based on biochemical considerations, is expected, a less extreme
P-value would suffice than if the response was unexpected or not
found at other dose levels.
Some laboratories, when presenting results of statistical
analyses, assign an almost magical relevance to the 95%
confidence level (P > 0.05), simply marking results significant
or not significant at this level. This is very poor practice,
because it gives insufficient information and does not enable
the distinction between an undeniable effect and one that
requires other confirmatory evidence. While it is not necessary
to give P-values exactly, it is essential to give some idea of
the degree of confidence. A useful method is to use plus signs
to indicate positive differences (and minus signs to indicate
negative differences), with ++++ meaning P < 0.001, +++ meaning
0.001 < P < 0.01, ++ meaning 0.01 < P < 0.05, and + meaning 0.05
< P < 0.1. This makes it easier to assimilate findings when
results for many variables are presented.
II.5.8 Multiple comparisons
Toxicological studies frequently involve making treatment/
control comparisons for large numbers of variables. If no true
treatment effect exists, it is possible that, purely by chance,
one or more variables will show differences significant at the
95% confidence level. For example, with 100 independent
variables, at least one variable would show significance 99.4%
of the time. Because of this, it has been suggested that the
critical value required to achieve significance should be made
more stringent with increasing numbers of variables studied, so
that, in testing at the 95% confidence level, 19 times out of
20, all the variables in the test show non-significance. This
approach is not recommended, because frequently in toxicological
studies a compound has only one or two real effects and has no
effect on a large number of other variables studied. Such
multiple comparison tests would make it much more difficult to
demonstrate statistical significance for the real effects. In
any case, there is something unsatisfactory about a situation in
which the relationship between a treatment and a particular
response arbitrarily depends on which other response happens to
be investigated at the same time. For this reason, no reference
is made below to any such procedures.
II.6 Statistical Analysis - Some Recommended Methods
A number of recommended methods of statistical analysis are
listed below. For mathematical details the reader is referred
to Peto et al. (2) and to Breslow & Day (3) for analysis of
presence/absence data, to Siegel (4) and to Conover (5) for non-
parametric analysis, and to Johnson & Leone (6) and Bennett &
Franklin (7) for analysis of continuous data. When details are
not found in these volumes, specific references are given.
II.6.1 Presence/absence data
II.6.1.1 Between-animal comparisons
Individual group Fisher exact test (unstratified data) 2 x 2
comparisons corrected chi-squared test (stratified or
unstratified data)
Heterogeneity 2 x k chi-squared test (stratified or
unstratified data)
Dose-related Armitage test (stratified or unstratified
trend data).
See reference 3 for details of stratified tests and for tests of
constancy of association over strata. See reference 2 for age-
adjusted tests.
II.6.1.2 Within-animal comparisons
Individual group McNemar test or sign test
comparisons
Heterogeneity Cochran test
Association Fisher exact test
between variables 2 x 2 corrected chi-squared test
II.6.2 Ranked data
II.6.2.1 Between-animal comparisons
Individual group Mann Whitney U test
comparisons
Heterogeneity Kruskal-Wallis one-way analysis of variance
Dose-related See reference 8
trend
II.6.2.2 Within-animal comparisons
Individual group Wilcoxon matched pairs signed-rank test
comparisons
Heterogeneity Friedman two-way analysis of variance
Dose-related Page test
trend
Association Spearman's rank correlation coefficients
between variables
II.6.3 Continuous data
Methods assume normality and homogeneity of variance between
groups.
Before using the methods:
Test for outliers See reference 9
Test for Bartlett test
homogeneity
of variance
Consider transformation of data by logarithms and/or square
roots if untransformed data show heterogeneity of variance.
If variance is still heterogeneous after transformation, use
methods of ranked data.
II.6.3.1 Between-animal comparisons
Individual group Students t-test
comparisons
Heterogeneity One-way analysis of variance
Dose-related Linear regression analysis
trend
II.6.3.2 Within-animal comparisons
Individual group Paired t-test
comparisons
Heterogeneity Two-way analysis of variance
Dose-related Linear regression analysis
trend
II.6.3.3 Association between variables
Variation in Pearson correlation coefficient
association Analysis of convariance
between variables
over groups
Change in Analysis of variance to assess difference at
variable second time-point after adjusting for first.
over time
REFERENCES TO ANNEX II
1. FERON, V.C., GRICE, H.C., GRIESEMER, R., & PETO, R. (1980)
Basic requirements for long-term assays for carcinogenicity.
In: Long-term and short-term screening assays for
carcinogens: a critical appraisal, Lyons, International
Agency for Research on Cancer, pp. 21-83 (IARC Monographs on
the Evaluation of the Carcinogenic Risk of Chemicals to
Humans, Suppl. 2).
2. PETO, R., PIKE, M.C., DAY, N.E., GRAY, R.C., LEE, P.N.,
PARISH, S., PETO, J., RICHARDS, S., & WAHRENDORF, J. (1980)
Guidelines for simple, sensitive significance tests for
carcinogenic effects in long-term animal experiments. In:
Long-term and short-term screening assays for carcinogens: a
critical appraisal, Lyons, International Agency for Research
on Cancer, pp. 311-426 (IARC Monographs on the Evaluation of
the Carcinogenic Risk of Chemicals to Humans, Suppl. 2).
3. BRESLOW, N.E. & DAY, N.E. (1980) Statistical methods in
cancer research. I. The analysis of case-control studies,
Lyons, International Agency for Research on Cancer (IARC
Scientific Publication No. 32).
4. SIEGEL, S. (1956) Non-parametric statistics for the
behavioural sciences, New York, McGraw-Hill Book Company.
5. CONOVER, W.J. (1980) Practical non-parametric statistics,
2nd ed., New York, John Wiley and Sons.
6. JOHNSON, N.L. & LEONE, F.C. (1964) Statistics and
experimental design in engineering and the physical
sciences, New York, John Wiley and Sons, Vol. 1.
7. BENNETT, C.A. & FRANKLIN, N.L. (1954) Statistical analysis
in chemistry and the chemical industry, New York, John Wiley
and Sons.
8. MARASCUILO, L.A. & MCSWEENEY, M. (1967) Non-parametric
post hoc comparisons for trend. Psychol. Bull., 67: 401-
412.
9. BARNETT, V. & LEWIS, T. (1978) Outliers in statistical
data, New York, John Wiley and Sons.
ANNEX III. GUIDELINES FOR THE EVALUATION OF VARIOUS GROUPS OF
FOOD ADDITIVES AND CONTAMINANTS
These guidelines have been established by JECFA and are
reproduced here for easy reference. They are valid within the
context in which they were generated, and are intended to serve
as examples of guidance by JECFA for evaluating specific
categories of substances.
1. Enzyme Preparations Used in Food Processing (adapted from:
1, p. 49; 2)
(a) Toxicological evaluation
For the purpose of toxicological evaluation, enzyme
preparations used in food processing can be grouped into 5 major
classes:
(i) Enzymes obtained from edible tissues of animals com-
monly used as foods. These are regarded as foods and,
consequently, considered acceptable, provided that
satisfactory chemical and microbiological specifica-
tions can be established.
(ii) Enzymes obtained from edible portions of plants.
These are also regarded as foods and, consequently,
considered acceptable, provided that satisfactory
chemical and microbiological specifications can be
established.
(iii) Enzymes derived from microorganisms that are tradi-
tionally accepted as constituents of foods or are nor-
mally used in the preparation of foods. These products
are regarded as foods and, consequently, considered
acceptable, provided that satisfactory chemical and
microbiological specifications can be established.
(iv) Enzymes derived from non-pathogenic microorganisms
commonly found as contaminants of foods. These
materials are not considered as foods. It is neces-
sary to establish chemical and microbiological speci-
fications and to conduct short-term toxicity studies
to ensure the absence of toxicity. Each preparation
must be evaluated individually and an ADI must be
established.
(v) Enzymes derived from microorganisms that are less well
known. These materials also require chemical and
microbiological specifications and more extensive
toxicological studies, including a long-term study in
a rodent species.
Safety assessments for enzymes belonging to classes i - iii
will be the same regardless of whether the enzyme is added
directly to food or is used in an immobilized form. Separate
situations should be considered with respect to the enzymes
described in classes iv and v:
(i) Enzyme preparations added directly to food but not
removed.
(ii) Enzyme preparations added to food but removed from the
final product according to good manufacturing prac-
tice.
(iii) Immobilized enzyme preparations that are in contact
with food only during processing.
For (i) above, an ADI should be established to ensure that
levels of the enzyme product present in food are safe. The
studies indicated in these guidelines are appropriate for
establishing ADIs (the guidelines were originally drafted for
this situation). For (ii), an ADI "not specified" may be
established, provided that a large margin of safety exists
between possible residues and their acceptable intake. For
(iii), it may not be necessary to set an ADI for residues that
could occur in food as a result of using the immobilized form of
the enzyme. It is acceptable to perform the toxicity studies
relating to the safety of the enzyme on the immobilized enzyme
preparation, provided that information is given on the enzyme
content in the preparation.
(b) Specifications for identity and purity
Prior to revising existing specifications and developing new
specifications for enzyme preparations for food processing, the
following data are necessary:
(i) A comprehensive description of the main enzymatic act-
ivity (or activities), including the Enzyme Commission
number(s) if any.
(ii) A list of the subsidiary enzymatic activities, whether
they perform a useful function or not.
(iii) A clear description of the source.
(iv) A list of non-enzymatic substances derived from the
source material(s), with limits where appropriate.
(v) A list of added co-factors, with limits where appro-
priate.
(vi) A list of carriers and diluents, with limits where
appropriate.
(vii) A list of preservatives present from manufacture or
deliberately added, with limits where appropriate.
(c) Immobilizing agents
A number of procedures involving different chemical sub-
stances are used for immobilizing enzymes. These processes
include microencapsulation (e.g., entrapment in gelatin to form
an immobilized complex), immobilization by direct addition of
glutaraldehyde, immobilization by entrapment in porous ceramic
carrier, and complexation with agents such as DEAE-cellulose or
polyethylenimine. Several agents may be used in the immobil-
izing process. Substances derived from the immobilizing mat-
erial may be in the final product due to either the physical
breakdown of the immobilized enzyme system or to impurities
contained in the system. The amount of data necessary to
establish the safety of the immobilizing agent depends on its
chemical nature. The levels of residues in the final product
are expected to be extremely low.
Some of the substances used in the preparation of immobil-
izing systems are extremely toxic. The levels of these sub-
stances or their contaminants permitted in the final product
should be at the lowest levels that are technologically feas-
ible, provided that these levels are below those of any toxico-
logical concern. An ADI will not be established.
2. Natural and Synthetic Food Colours (Adapted from: Reference
1, Annex 6, p. 50)
For toxicological evaluation, natural colours should be
considered as falling within three main groups:
(a) A colour isolated in a chemically unmodified form from
a recognized foodstuff and used in the foodstuff from
which it is extracted at levels normally found in that
food. This product could be accepted in the same
manner as the food itself with no requirement for
toxicological data.
(b) A colour isolated in a chemically unmodified form from
a recognized foodstuff but used at levels in excess of
those normally found in that food or used in foods
other than that from which it is extracted. This
product might require the toxicological data usually
demanded for assessing the toxicity of synthetic
colours.
(c) A colour isolated from a food source and chemically
modified during its production or a natural colour
isolated from a non-food source. These products would
also require a toxicological evaluation similar to that
carried out for a synthetic colour.
It is recognized that natural colours may be reproduced by
chemical synthesis but it is noted that "nature-identical"
colours produced by chemical synthesis may contain impurities
warranting toxicological evaluation similar to that required for
a synthetically produced food colour.
The toxicological evaluation of synthetic food colours would
require the following minimum data:
(a) Metabolic studies in several species, preferably inclu-
ding man. These should include studies on absorption,
distribution, biotransformation, and elimination, and
an attempt should be made to identify the metabolic
products in each of these steps.
(b) Short-term feeding studies in a non-rodent mammalian
species.
(c) Multi-generation reproduction/teratogenicity studies.
(d) Long-term carcinogenicity/toxicity studies in two
species.
3. Solvents Used in Food Processing (Adapted from: Reference 1,
Annex 6, pp. 50-51)
Extraction solvents are used inter alia in the extraction of
fats and oils, defatting fish and other meals, and in decaf-
feinating coffee and tea. They are chosen mainly for their
ability to dissolve the desired food constituents selectively
and for their volatility, which enables them to separate easily
from the extracted material with minimum damage. The points
raised by their use relate to:
(a) toxicity of their residues;
(b) toxicity of any impurities in them;
(c) toxicity of substances such as solvent stabilizers and
impurities that may be left behind after the solvent is
removed; and
(d) toxicity of any substances produced as a result of a
reaction between the solvent and food ingredients.
Before any extraction solvent can be evaluated, information
is required on:
(a) identity and amount of impurities in the solvent
(including those that are formed, acquired, or concen-
trated owing to continuous reuse of the solvent);
(b) identity and amount of stabilizers and other additives;
and
(c) toxicity of residues of solvents, additives, and
impurities.
Impurities are particularly important because there are wide
differences in the purities of food grade and industrial grade
solvents. The food use of extraction solvents is frequently
much less than the industrial use, and hence their food-grade
requirements may receive insufficient consideration, both in
food use and in toxicological testing. Furthermore, the impur-
ities or stabilizers may not have the same volatility as the
solvent itself, and as a result, these may be left behind in the
food after the solvent is removed. Finally, the possibility of
any solvent, impurity, stabilizer, or additive reacting with
food ingredients should be checked.
When biological and toxicological data raise doubts about a
substance's safety, two approaches are possible: (a) to set an
ADI for the substance or (b) to discourage its use altogether.
Even when data indicate a wide margin of safety for a substance,
or when there is a paucity of toxicological data on the sub-
stance, but no problems concerning the impurities, residues, and
any chemical reaction with food ingredients, it would be appro-
priate to limit the use of the substance to the minimum possible
level.
When the data on a substance indicate the presence of cer-
tain impurities in the tested material, considerable problems
arise in its evaluation. This is especially true if industrial-
grade rather than food-grade material has been used in the
toxicological study. For example, when evaluating the solvents
1,1,1-trichloroethane, trichloroethylene, and tetrachloro-
ethylene, it was noted that the toxicological data indicated the
presence of certain known toxic and carcinogenic substances.
The interpretation of these data became extremely difficult
because industrial-grade material had been used in the studies.
Only food-grade material should be used in toxicological studies
and the impurities in the material should be fully identified.
Carrier solvents raise somewhat different issues. They are
used for dissolving and dispersing nutrients, flavours, anti-
oxidants, emulsifiers, and a wide variety of other food ingred-
ients and additives. With the exception of carrier solvents for
flavours, they tend to occur at higher levels in food than
extraction solvents, mainly because frequently no attempt is
made to remove them, and because some of them are relatively
nonvolatile. Since carrier solvents are intentional additives
and are often not removed from the processed food, it is impor-
tant to evaluate their safety together with the safety of any
additives or stabilizers in them.
4. Residues Arising from Use of Xenobiotic Anabolic Agents in
Animal Feed (Adapted from: Reference 1, p. 13 and reference
3, p. 15)
Many studies have established the importance and efficacy of
anabolic agents for meat production. Two categories of com-
pounds are used - namely:
(a) hormones that are identical to those occurring
naturally in food-producing animals and human beings,
including the esters of these hormones; and
(b) xenobiotic compounds, such as derivatives of hormones,
synthetic compounds with hormonal activity, natural-
product hormonally active agents that are not identical
with human endogenous hormones, and derivatives of such
compounds.
The toxicological evaluation of residues of anabolic agents
that are present in human food obtained from animals treated
with these agents must take into account whether the residue is
identical to a human endocrine hormone. In the latter case, the
possible endocrinological effects and carcinogenic potential of
the residue must be closely examined.
Chemically modified hormones, hormonally active agents from
plants, and synthetic anabolic agents present the following
specific problems:
(a) extreme potency and consequently the need to ensure
minimal residues;
(b) potential tumorigenic activity; and
(c) the presence of their metabolites in animal products
that might be of endocrinological or toxicological
consequence.
The evaluation for acceptance of the use of xenobiotic
anabolic agents in animal food production resembles in many
respects the evaluation of pesticides, since the two essential
elements required are:
(a) adequate, relevant toxicological data, and
(b) comprehensive data about the kinds and levels of
residues when the substances are used in accordance
with good animal husbandry practice, which requires
evidence as to the efficacy of the anabolic agent, the
amounts used to produce the effect, the residue levels
based on field trials, and information about methods of
analysis of residue levels that could be used for
control or monitoring purposes.
5. Metals in Food (Adapted from: Reference 1, pp. 14-15)
Toxicological evaluation of metals in foods calls for
carefully balanced consideration of the following factors:
(a) nutritional requirements, including nutritional inter-
actions with other constituents of food (including
other metals when the interactions are nutritionally or
toxicologically relevant) in respect of, for instance,
absorption, storage in the body, and elimination;
(b) the results of epidemiological surveys and formal toxi-
cological studies, including interactions with other
constituents of food (including other metals when the
interactions are nutritionally or toxicologically rele-
vant), information about pharmaceutical and other medi-
cinal uses, and clinical observations on acute and
chronic toxicity in human experience and veterinary
practice;
(c) total intake on an appropriate time basis (e.g., daily,
weekly, yearly or lifetime) from all sources (food,
water, air) of metals as normal constituents of the
environment, as environmental contaminants, and as food
additives of an adventitious or deliberate nature.
The tentative tolerable daily intakes proposed for certain
metals by the Committee provide a guideline for maximum
tolerable exposure. In the case of essential elements, these
levels exceed the normal daily requirements, but this should not
be construed as an indication of any change in the recommended
daily requirements. In the case of both essential and non-
essential metals, the tentative tolerable intake reflects
permissible human exposures to these substances as a result of
natural occurrence in foods or various food processing
practices, as well as exposure from drinking-water.
It is important that the proposed tolerable intakes are not
used as guidelines for fortifying processed food, since the
currently accepted values for required daily intake are
sufficient to meet the known nutritional requirements.
6. Packaging Materials (Adapted from: Reference 4, pp. 22-23)
Many substances may become food contaminants as a result of
the use of food-contact materials. The Committee considered
that the general principles governing the use of food additives
established by the WHO Scientific Group on Procedures for
Investigating Intentional and Unintentional Food Additives, and
the WHO Scientific Group on the Assessment of the Carcino-
genicity and Mutagenicity of Chemicals, should be applied in the
overall evaluation of substances migrating into food from food-
contact materials.
Many such materials are made of polymer systems and the
polymers themselves are usually inert, non-toxic, and do not
migrate into food. However, monomers, which are inevitably
present in the polymeric materials, residual reactants, inter-
mediates, manufacturing aids, solvents, and plastic additives,
as well as the products of side reactions and chemical degra-
dation, may be present. These substances may migrate into food
and may be toxic. Migration from food-contact materials may
arise during processing, storage, and preparation operations
such as heating, microwave cooking, or treatment with ionizing
radiation. For evaluation purposes, information on the
following is required:
(a) the chemical identity and toxicological status of the
substances that enter food;
(b) the possible exposure, details of which can be derived
from migration studies using suitable extraction
procedures, and/or the analysis of food samples; and
(c) the nature and amount of food in contact with the
packaging materials, and the intake of such food.
It is necessary, in many instances, to recommend that human
exposure to migrants from food-contact materials be restricted
to the lowest levels technologically attainable. One way to
achieve this is to draw up strict specifications limiting the
quantities in the materials. It is also necessary to determine
whether food processing has an effect in generating the poten-
tially toxic substances in food-contact materials.
Generally, evaluation will require the following:
(a) the lowest levels of potential migrants from within the
polymeric system(s) that are technologically attainable
with improved manufacturing processes for food-contact
materials;
(b) the resulting levels of the migrants in food;
(c) the intake of the foods; and
(d) the most appropriate statistical design that will
enable the implications for health to be interpreted
from adequate and relevant toxicological data.
A monitoring programme should be established, with a view to
supplementing the existing data on human exposure and providing
a means of demonstrating a reduction in such exposure as
techniques improve. Priority in the programme should be given
to the substances with the greatest potential for adversely
affecting human health.
REFERENCES TO ANNEX III
1. FAO/WHO (1982) 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).
2. FAO/WHO (1986) 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).
3. FAO/WHO (1981) Evaluation of certain food additives.
Twenty-fifth report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 669).
4. FAO/WHO (1984) 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).
ANNEX IV. EXAMPLES OF THE USE OF METABOLIC STUDIES IN THE
SAFETY ASSESSMENT OF FOOD ADDITIVES
As indicated in section 5.2, it is not feasible or desirable
to develop simple guidelines for pharmacokinetic and metabolic
studies. In view of this, the examples given below of several
food additives and contaminants on which a great deal of
biochemical work has been done will serve to highlight the value
and many of the problems associated with the use of these
studies for determining mechanisms. Clearly, further research
will be needed to solve these problems.
1. Sodium Cyclamate
This compound represents a unique situation in toxicology in
that it has been generally agreed that levels of a metabolite
rather than the parent compound should be used for the usual
safety determinations. The twenty-sixth Committee allocated an
ADI of 0 - 11 mg/kg body weight to cyclamate, calcium and sodium
salts, expressed as cyclamic acid (1).
It has been shown that a metabolite of cyclamate,
cyclohexylamine (an active pressor amine), appears in the urine
in variable amounts after variable time intervals from the
administration of cyclamate in rats (2, 3). This metabolite has
been found to be produced by bacterial action in the intestine
(3, 4), but only after intestinal flora have undergone undefined
adaptive changes (2). Therefore, it normally appears only after
a latent period. However, in certain human subjects, some
immediate converters have been found to be present (2, 5). In
both animals and man, the ability of intestinal flora to convert
cyclamate to cyclohexylamine varies widely with time in the same
individual. The number of individuals able to convert cyclamate
to cyclohexylamine and the level at which this conversion occurs
have been factored into ADI determinations using averages from
some studies (6). However, it is difficult to obtain really
consistent figures, and those in use represent compromises. The
ADI is based on subsequent studies, which demonstrated that
cyclohexylamine, administered orally, induced testicular atrophy
in rats (7, 8).
The primary reason for the decision by JECFA and various
national regulatory bodies to agree to the use of the levels of
this metabolite rather than the parent compound for toxico-
logical evaluation purposes appears to have been the nature of
the metabolite, in this instance, a compound that is pharmaco-
logically-active relative to the parent compound and is capable
of inducing testicular atrophy in rats (9). However, it is
questionable that the readiness of all these bodies to accept
this unique approach in this situation is, in fact, appropriate.
The presence of the metabolite certainly cannot be ignored;
however, it would seem more logical to demand that the effects
in question (testicular atrophy) be demonstrated following
feeding the parent compound (cyclamate). This approach is
complicated by the inconsistent nature of the appearance of the
metabolite.
The individuality of the response has necessitated a most
conservative attitude that has raised the important general
question of how the toxicologist can best protect vulnerable
individuals. In addition, this example has pointed to the
importance of studying metabolism by gut flora in toxicological
evaluations. Unquestionably, variations in gut flora are one of
the more important determinants of species differences, and the
example of cyclamate has pointed the way to studies in which
this factor has been reduced to a minimum.
2. Sodium Saccharin
JECFA has reviewed the safety of saccharin many times. In
1984, the twenty-eighth Committee allocated a temporary group
ADI of 0 - 2.5 mg/kg body weight to saccharin, including its
calcium, potassium, and sodium salts (10). Subsequent to the
demonstration in some long-term toxicity studies that sodium
saccharin could induce tumours of the urinary bladder in male
rats at high dose levels, much work was undertaken in an effort
to explain this phenomenon. Only two of the many reported
findings will be discussed in this section, as illustrations of
certain general principles.
On the basis of the first series of studies, it would seem
unquestionable that sodium saccharin is not metabolized in the
rat; this seems to be generally applicable to human beings and
other species (11). There is no postulated theoretical mech-
anism of chemical carcinogenesis that could fit this picture.
The second series of studies have established the most inter-
esting finding that, although saccharin is not metabolized, it
can modify the metabolic pathway of certain normal constituents
in the diet. A dose-related increase in certain tryptophan
metabolites - notably indoxyl sulfate - was found in the urine
of saccharin-treated rats (12). In contrast, these effects
could not be demonstrated in human beings consuming saccharin
(13). In view of previous interest in the association of
tryptophan and its metabolites with bladder tumour induction and
its occurrence in man, this observation was of great interest.
Although the further steps in this series of investigations have
not succeeded in establishing a convincing relationship between
the carcinogenicity finding and the metabolism of tryptophan,
nevertheless, a most important general principle in toxicology
has been demonstrated that remains to be exploited. The fact
that a compound that is not metabolized could change the
metabolism of other compounds provides a basis for studies of
mechanism of action not considered in the past. It seemed
likely, initially, that the possible bacteriostatic action of
saccharin might be affecting the gut flora; although this seems
to be a possible practical explanation, further study is needed
to explain the whole picture.
3. o -Phenylphenol (OPP)
This compound is a fungicide widely used on oranges, the use
of which results in low residue levels as a food contaminant.
The Joint Meeting on Pesticide Residues (JMPR) has allocated a
temporary ADI to OPP (and its sodium salt) of 0 - 0.02 mg/kg
body weight (14). It has been chosen as an example of a situ-
ation in which extensive metabolic studies have been correlated
with toxicological findings.
OPP has been found to give rise to tumours of the urinary
bladder when fed to rats at relatively high levels in the diet
(15). Two metabolites, the glucuronide conjugate of OPP and the
sulfate ester conjugate of OPP, have been identified in the
urine of rats after administration of different levels of OPP.
A third metabolite, tentatively postulated to be a conjugated
dihydroxybiphenyl compound, has been reported after the adminis-
tration of a high level of OPP, the same level required to
induce bladder tumours, but not after the administration of
lower levels of OPP (16). From this observation, some invest-
igators have concluded that, at lower levels of administration,
carcinogenicity does not occur, because the "proximate carcin-
ogen" (i.e., the high-level metabolite) has not been formed.
This fascinating study is, unfortunately, incomplete. There are
disputes as to the absence of the "active" metabolite (the
conjugated dihydroxybiphenyl compound) at the lower dose
levels; the detection limits of the various metabolites, which
are not clearly delineated in the available literature, are a
matter of considerable concern. In addition, the "active"
metabolite has no special chemical characteristics to suggest
that it would conform to any of the current theories of action
of chemical carcinogens. These results show the difficulties of
proving the mechanism of a tumourigen so that a safe dose can be
established, even though the available evidence points in that
direction. It is clear that much more information is needed
before the postulated change in metabolism can be related to
carcinogenesis.
4. Methylene Chloride
This compound, which is used as a food extraction solvent in
some countries, has been the subject of intensive carcino-
genicity, mutagenicity, metabolism, pharmacokinetic, and epi-
demiological studies, but questions still exist about its safety
of use. Because of the inadequacies of the studies available at
that time, the twenty-sixth Committee withdrew the previously-
allocated ADI for methylene chloride (17).
The safety of methylene chloride was brought into question
by a long-term inhalation study that produced very rare salivary
gland sarcomas in male rats in an apparent dose-response rela-
tionship at 1500 and 3500 mg/kg air; an increased incidence of
tumours was not observed in female rats or in hamsters in a
parallel study (unpublished studies by the Dow Chemical Company,
Midland, Michigan, USA, 1980). Also, preliminary results from a
mouse inhalation study indicate an increased incidence of liver
and lung neoplasms at 1000 and 4000 mg methylene chloride/kg air
(18). However, arguments have been made on the basis of meta-
bolic studies that the maximum tolerated dose (MTD) was exceeded
in these long-term inhalation studies in rats and mice (19).
Bioassays in which methylene chloride was administered at lower
levels by inhalation or in drinking-water have not resulted in a
significant increase in malignant tumours (unpublished studies
by the Dow Chemical Company, Midland, Michigan, USA, 1982 and by
the National Coffee Association, USA, 1982).
Methylene chloride is metabolized via two pathways. The
principle site of metabolism is the liver in all species
studied, including man. One pathway involves glutathione,
giving rise to formaldehyde, which is oxidized to formic acid
and then carbon dioxide. The other pathway is mediated by cyto-
chrome P-450 and involves dehydrochlorination to carbon monoxide
and hydrogen chloride. One of the intermediates in the first
pathway, a glutathione conjugate, has been implicated as the
DNA-reactive species responsible for the apparent mutagenicity
of methylene chloride in some tests. However, there is no evi-
dence of alkylation in animals (20, 21, 22).
There is a linear discontinuity in metabolite formation
(carbon dioxide and carbon monoxide) as exposure to methylene
chloride increases. For example, it has been shown that, on
inhalation of 174 mg methylene chloride/m3 (50 ppm), 95% is
metabolized, while at 1750 mg/m3 air (500 ppm), only 69% is
metabolized and at 5200 mg methylene chloride/m3 air (1500 ppm),
only 45% is metabolized to carbon dioxide and carbon monoxide.
Both oral and inhalation studies show that saturation of meta-
bolism occurs in all species examined (rat, mouse, hamster, and
man) (19).
Greater amounts of methylene chloride are metabolized when
the compound is presented in drinking water than when the same
daily dose is gavaged in a single dose, either in corn oil or in
water. Administration of a large number of doses in small
amounts, such as when methylene chloride is administered in the
drinking-water, yields greater amounts of metabolites than when
the total amount is given at one time, such as by gavage. The
vehicle used in gavage studies also plays a role in the clear-
ance of methylene chloride from various tissues; for example,
the compound is dissipated from both blood and liver in less
than 2 h after administration by gavage in water compared with a
residence time of about 8 h in venous blood and over 25 h in the
liver after administration by gavage in corn oil (19).
Despite tremendous efforts to study this compound biochem-
ically, a clear picture of the mechanism of its biological
effects has not emerged. This shows the difficulties of devel-
oping sufficient biochemical data to set a safe dose level for a
substance that causes cancer at high dose levels. The satur-
ation effect and the occurrence of tumours at high dose levels
may be related. An encouraging point is that primates meta-
bolize chlorinated solvents to a lesser extent than rats or
mice; thus, less of the glutathione reactive intermediate, which
has been postulated as being responsible for the genotoxic
effects of methylene chloride, should be present in man than in
the animals exhibiting the deleterious effects. Finally, the
differences in rates of metabolism of methylene chloride,
depending on the route of administration, point to the need for
very careful assessment of the appropriate route of administra-
tion to mimic exposure in man.
5. Trichloroethylene
This chemical is an extraction solvent. It has been
reviewed by JECFA, but an ADI has not been allocated (17).
Trichloroethylene has been found to cause an increased incidence
of hepatocellular carcinomas in mice, but not in rats (23,24).
The earlier bioassays were performed with industrial-grade
trichloroethylene, which contained epoxide stabilizers, at least
one of which is a potent mutagen (section 3 in Annex III). How-
ever, the results of later studies using non-epoxide stabilized
material confirmed the results of the earlier studies, indi-
cating that the stabilizers were not responsible for the hepato-
carcinogenicity observed in mice.
Trichloroethylene has been subjected to a great deal of
metabolic and pharmacokinetic research on mice and rats, and an
interesting story is emerging, which could explain the differ-
ence in response between these species and the relevance to man
of the rodent bioassays.
Electron micrographs of liver tissue from mice that had been
dosed with a high level of trichloroethylene for 10 days showed
a proliferation of peroxisomes; significant proliferation was
not observed in rats after the administration of the same amount
of trichloroethylene (25). Biochemical studies have shown that
cyanide-insensitive acyl CoA oxidase, an enzyme present within
the peroxisome that ultimately produces hydrogen peroxide, is
enhanced 5 - 16-fold in the peroxisome-proliferated state
compared within the control. However, only a small increase in
catalase activity, which catalyses the conversion of hydrogen
peroxide to water, has been observed in the proliferated cell
(25, 26, 27). It has been postulated that the large increase in
acyl CoA oxidase activity coupled with the marginal increase in
catalase activity leads to an increased steady-state concentra-
tion of hydrogen peroxide within the liver cell, which results
in cytotoxicity and DNA damage and eventually cancer in mice
(28).
Trichloroethylene appears to be metabolized via the
cytochrome P-450 system, in both the rat and mouse (29, 30).
The major metabolite, which is ultimately converted to carbon
dioxide, is trichloroacetic acid. Apparently, the enzymes in
this pathway are not induced to the same extent in rats as in
mice, as shown by gavage studies with trichloroethylene; mice
show linear kinetics with respect to metabolite formation over a
wide dose range, while rats show saturation kinetics; saturation
is observed at low levels relative to amounts required to
observed peroxisome proliferation and the induction of hepato-
cellular carcinomas in mice. In contrast, when trichloroacetic
acid is administered to rats and mice, a similar dose-dependent
large increase in peroxisomes is observed in both species. This
suggests that trichloroacetic acid is the proximate peroxisome
proliferator, and the reason that proliferation is not observed
after the administration of trichloroethylene to rats is that
not enough of the acid is produced to cause the effect. If
peroxisome proliferation (and a consequent increased steady-
state concentration of hydrogen peroxide in the cell) is res-
ponsible for hepatocellular carcinoma induction, then trichloro-
acetic acid should be a hepatocellular carcinogen in both
species. This hypothesis is at presently being tested (28).
Where does man fit into the picture? Preliminary work with
hepatocytes isolated from mice, rats, and man show that the
human hepatocyte is much more like that of the rat than that of
the mouse in terms of its ability to convert trichloroethylene
to trichloroacetic acid; in fact, the human hepatocyte is even
less active in converting trichloroethylene to trichloroacetic
acid than that of the rat (28). In addition, when trichloro-
acetic acid is administered to cultured human hepatocytes, there
is no evidence of peroxisome proliferation, as measured by
cyanide-insensitive acyl CoA oxidase activity. In contrast,
studies on mouse and rat hepatocytes have registered large,
dose-related increases in this enzyme activity. These data
suggest that the mouse bioassay data showing an increase in
hepatocellular carcinomas after the administration of trichloro-
ethylene are not appropriate for human beings because:
(a) trichloroethylene is not converted into trichloroacetic
acid at a high enough rate in man to cause peroxisome
proliferation; and
(b) trichloroacetic acid does not appear to cause perox-
isome proliferation in man at the levels at which it
causes the effect in rats and mice.
Further data are needed to obtain a firm conclusion that could
withstand regulatory scrutiny.
6. Estragole
This chemical is a naturally-occurring anisole derivative
that is used as a flavouring agent. Estragole has been reviewed
by JECFA, but an ADI has not been allocated (31). It has been
found to be a mouse carcinogen at a dose level of approximately
500 mg/kg body weight per day (32, 33). In contrast, the
estimated human daily intake of estragole in the diet is
approximately 1 g/kg body weight (349).
One of the routes of metabolism of estragole is through a
hydroxylated intermediate, 1'-hydroxyestragole (35). This "act-
ivated" intermediate is likely to undergo esterification reac-
tions with cellular constituents to form electrophilic conju-
gates. It has been postulated that the carcinogenic effect seen
with estragole is due to the formation of the "proximate carcin-
ogen", 1'-hydroxyestragole, which reacts to form the "ultimate
carcinogen", the electrophilic conjugate (32, 33).
Studies have been performed in which the level of 1'-
hydroxyestragole has been measured in the urine of mice exposed
to various levels of estragole. At the low dose, 0.5 mg/kg body
weight, 1 - 2% of the dose was excreted as 1'-hydroxyestragole,
while, at the high dose, 1000 mg/kg body weight, 10 - 15% of the
ingested dose of estragole was excreted as the hydroxylated
compound. In studies on human volunteers, fed 1 g estragole,
0.3% of the dose was excreted as 1'-hydroxyestragole (34).
These and other data suggest that only at very high and
overwhelming levels of estragole are significant amounts of the
activated intermediate formed, and that there appear to be over
6 orders of magnitude difference between levels of the
intermediate in the high-dose mouse study and the level present
in man at normal levels of consumption. This hypothesis, if it
holds, leads to the conclusion that the human carcinogenic risk
from the ingestion of normal levels of estragole is negligible.
REFERENCES TO ANNEX IV
1. FAO/WHO (1982) 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).
2. RENWICK, A.G. & WILLIAMS, R.T. (1972) The fate of
cyclamate in man and other species. Biochem. J., 129: 869-
879.
3. BICKEL, M.H., BURKARD, B., MEIER-STRASSER, E., & VAN DEN
BROEK-BOOT, M. (1974) Entero-bacterial formation of
cyclohexylamine in rats ingesting cyclamate. Xenobiotica, 4:
425-439.
4. DRASSAR, B.S., RENWICK, A.G., & WILLIAMS, R.T. (1972) The
role of the gut flora in the metabolism of cyclamate.
Biochem. J., 129: 881-890.
5. ASAHINA, M., YAMAHA, T., WATANABE, K., & SARRAZIN, G.
(1971) Excretion of cyclohexylamine, a metabolite of
cyclamate, in human urine. Chem. Pharm. Bull. (Tokyo), 19:
628-632.
6. RENWICK, A.G. (1983) The fate of cyclamate in man and rat.
In: Transcripts of the European Toxicological Forum, 18-22
October 1983, Geneva, pp. 301-312.
7. GAUNT, I.F., SHARRATT, M., GRASSO, P., LANSDOWN, A.B.G., &
GANGOLLI, S.D. (1974) Short-term toxicity of cyclohexyl-
amine hydrochloride in the rat. Food Cosmet. Toxicol., 12:
609.
8. MASON, P.L. & THOMPSON, G.R. (1977) Testicular effects of
cyclohexylamine hydrochloride in the rat. Toxicology, 8:
143.
9. FAO/WHO (1978) Evaluation of certain food additives.
Twenty-first report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 617).
10. FAO/WHO (1984) 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).
11. RENWICK, A.G. (1985) The disposition of saccharin in
animals and man: a review. Food Chem. Toxicol., 23: 429-
435.
12. SIMS, J. & RENWICK, A.G. (1985) The microbial metabolism
of tryptophan in rats fed a diet containing 7.5% saccharin
in a two-generation protocol. Food Chem. Toxicol., 23: 437-
444.
13. ROBERTS, A. & RENWICK, A.G. (1985) The effect of saccharin
on the microbial metabolism of tryptophan in man. Food Chem.
Toxicol., 23: 451-455.
14. FAO/WHO (1985) Pesticide residues in food. Report of the
Joint Meeting of the FAO Panel of Experts on Pesticide
Residues in Food and the Environment and a WHO Expert Group
on Pesticide Residues (FAO Plant Production and Protection
Paper No. 68).
15. HIRAGA, K. & FUJII, T. (1984) Induction of tumours of the
urinary bladder in F-344 rats by dietary administration of
o-phenylphenol. Food Cosmet. Toxicol., 22: 865-870.
16. REITZ, R.H., FOX, T.R., QUAST, J.F., HERMANN, E.A., &
WATANABE, P.G. (1983) Molecular mechanisms involved in the
toxicity of orthophenylphenol and its sodium salt. Chem.-
Biol. Interact., 43: 99-119.
17. FAO/WHO (1983) 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 and corrigenda).
18. FOOD CHEMICAL NEWS (1985) Methylene chloride is
carcinogenic in inhalation tests conducted for NTP, March
25, p. 38.
19. KIRSCHMAN, J. (1984) Methylene chloride: safety testing
overview. In: Transcripts of the European Toxicology Forum,
18-21 September 1984, Geneva, pp. 197-210.
20. AHMED, A. & ANDERS, M. (1976) Metabolism of dihalomethanes
to formaldehyde and inorganic halide. I. In vitro studies.
Drug Metab. Dispos., 4: 357-361.
21. AHMED, A., ET AL. (1980) Halogenated methanes: metabolism
and toxicity. Fed. Proc., 39: 3150-3155.
22. GREEN, T. (1984) Genotoxicity of methylene chloride. In:
Transcripts of the European Toxicology Forum, 18-21
September 1984, Geneva, pp. 211-215.
23. NCI (1976) Carcinogenesis bioassay of trichloroethylene
(CAS Registry No. 79-01-6), National Cancer Institute (DHEW
Publication No. (NIH) 76-802).
24. NTP (1983) Draft report abstracts on nine chemical
carcinogenesis bioassays, National Toxicology Program, pp.
767-768 (Chemical Regulation Report No. 6).
25. ELCOMBE, C.R., ROSE, M.S., & PRATT, I.S. (1985)
Biochemical, histological, and ultrastructural changes in
rat and mouse liver following the administration of
trichloroethylene: possible relevance to species differences
in hepatocarcinogenicity. Toxicol. appl. Pharmacol., 79:
365-376.
26. LALWANI, N.D., REDDY, M.K., MANGKORNKANOL-MARK, M., & REDDY,
J.K. (1981) Induction, immunochemical identity and
immunofluorescence localization of an 80 000-molecular-
weight peroxisome-proliferation-associated polypeptide
(polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of
mouse liver and renal cortex. Biochem. J., 198: 177-186.
27. REDDY, J.K., LALWANI, N.D., QURESHI, S.A., REDDY, M.K., &
MOEHLE, C.M. (1984) Induction of hepatic peroxisome
proliferation in non-rodent species, including primates. Am.
J. Pathol., 114: 171-183.
28. ELCOMBE, C.R. (1984) In: Transcripts of the European
Toxicology Forum, 18-21 September 1984, Geneva, pp. 134-
144.
29. GREEN, T. & PROUT, M.S. (1985) Species differences in
response to trichloroethylene. II. Biotransformation in rats
and mice. Toxicol. appl. Pharmacol., 401-411.
30. PROUT, M.S., PROVAN, W.M., & GREEN, T. (1985) Species
differences in response to trichloroethylene. I.
Pharmacokinetics in rats and mice. Toxicol. appl.
Pharmacol., 79: 389-400.
31. FAO/WHO (1981) Evaluation of certain food additives.
Twenty-fifth report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 669).
32. DRINKWATER, N.R., MILLER, E.C., MILLER, J.A., & PITOT, H.C.
(1976) The hepatocarcinogenicity of estragole (1-allyl-r-
methoxybenzene) and 1'-hydroxyestragole in the mouse and the
mutagenicity of 1'-acetoxyestragole in bacteria. J. Natl
Cancer Inst., 57: 1323-1331.
33. MILLER, E.C., SWANSON, A.B., PHILLIPS, D.H., FLETCHER, T.L.,
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.
34. BERNARD, B. & CALDWELL, J. (1984) Significance of dose-
dependent metabolism of flavour chemicals for their safety
evaluation. In: Transcripts of the European Toxicology
Forum, 18-21 September 1984, Geneva, pp. 124-131.
35. SWANSON, A.B., MILLER, E.C., & MILLER, J.A. (1981) The
side-chain epoxidation and hydroxylation of the
hepatocarcinogens safrole and estragole and some related
compounds by rat and mouse liver microsomes. Biochim.
Biophys. Acta, 673: 505-516.
ANNEX V. APPROXIMATE RELATION OF PARTS PER MILLION IN THE DIET
TO MG/KG BODY WEIGHT PER DAY a
--------------------------------------------------------------------------------
Animal Weight Food con- Type of 1 ppm in 1 mg/kg body
(kg) sumed per diet food = weight per
day (g) (mg/kg body day = (ppm
(liquids body weight of diet)
omitted) per day)
--------------------------------------------------------------------------------
Mouse 0.02 3 0.150 7
Chick 0.40 50 0.125 8
Rat (young) 0.10 10 Dry 0.100 10
labor-
Rat (old) 0.40 20 atory 0.050 20
chow
Guinea-pig 0.75 30 diets 0.040 25
Rabbit 2.0 60 0.030 33
Dog 10.0 250 0.025 40
--------------------------------------------------------------------------------
Cat 2 100 0.050 20
Moist,
Monkey 5 250 semi- 0.050 20
solid
Dog 10 750 diets 0.075 13
Man 60 1500 0.025 40
--------------------------------------------------------------------------------
Pig or sheep 60 2400 0.040 25
Rela-
Cow 500 7500 tively 0.015 65
(maintenance) dry
grain
Cow 500 15 000 forage 0.030 33
(fattening) mix-
tures
Horse 500 10 000 0.020 50
--------------------------------------------------------------------------------
a Lehman, A.J. (1954) Association of Food and Drug Officials
Quarterly Bulletin, 18: 66. The values in this table are
average figures, derived from numerous sources.
Example: What is the value in ppm and mg/kg body weight per day of 0.5%
substance X mixed in the diet of a rat?
Solution: I. 0.5% corresponds to 5000 ppm.
II. From the table, 1 ppm in the diet of a rat is equivalent to
0.050 mg/kg body weight per day. Consequently, 5000 ppm is
equivalent to 250 mg/kg body weight per day (5000 x 0.050).
ANNEX VI. REPORTS AND OTHER DOCUMENTS RESULTING FROM MEETINGS
OF THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES
1. FAO/WHO (1957) 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; WHO Technical Report Series No. 129) (out of
print).
2. FAO/WHO (1958) 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; WHO
Technical Report Series No. 144) (out of print).
3. FAO/WHO (1962) Specifications for identity and purity of
food additives (microbial 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. I. Antimicrobial preservatives and
antioxidants, Rome, Food and Agricultural Organization of
the United Nations (out of print).
4. FAO/WHO (1963) 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.
II. Food colours, Rome, Food and Agricultural Organization
of the United Nations (out of print).
5. FAO/WHO (1961) 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; WHO Technical Report Series No. 220) (out of
print).
6. FAO/WHO (1962) 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; WHO Technical Report Series
No. 228) (out of print).
7. FAO/WHO (1964) 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 Report Series No. 35; WHO
Technical Report Series No. 281) (out of print).
8. FAO/WHO (1965) 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 Report Series No. 38; WHO
Technical Report Series No. 309) (out of print).
9. FAO/WHO (1965) Specifications for identity and purity and
toxicological evaluation of some antimicrobials and
antioxidants (FAO Nutrition Meetings Report Series No. 38A;
WHO/Food Add/24.65 (out of print).
10. FAO/WHO (1966) Specifications for identity and purity and
toxicological evaluation of food colours (FAO Nutrition
Meetings Report Series No. 38B; WHO/Food Add/66.25).
11. FAO/WHO (1966) 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 Report Series No. 40; WHO Technical
Report Series No. 339) (out of print).
12. FAO/WHO (1967) 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. FAO/WHO (1967) 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 Report Series No. 43; WHO
Technical Report Series No. 373).
14. FAO/WHO (1968) 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 Report Series No. 44;
WHO Technical Report Series No. 383).
15. FAO/WHO (1968) Toxicological evaluation of some flavouring
substances and non-nutritive sweetening agents (FAO
Nutrition Meetings Report Series No. 44A; WHO/Food
Add/68.33).
16. FAO/WHO (1969) Specifications and criteria for identity
and purity of some flavouring substances and non-nutritive
sweetening agents (FAO Nutrition Meetings Report Series No.
44B; WHO/Food Add/69.31).
17. FAO/WHO (1969) 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 Report
Series No. 45; WHO Technical Report Series No. 430).
18. FAO/WHO (1969) Specifications for the identity and purity
of some antibiotics (FAO Nutrition Meetings Report Series
No. 45A; WHO/Food Add/69.34).
19. FAO/WHO (1970) 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 Report Series No. 46; WHO Technical Report Series
No. 445).
20. FAO/WHO (1970) Toxicological evaluation of some food
colours, emulsifiers, stabilizers, anticaking agents, and
certain other substances (FAO Nutrition Meetings Report
Series No. 46A; WHO/Food Add/70.36).
21. FAO/WHO (1970) 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; WHO/Food Add/70.37).
22. FAO/WHO (1971) Evaluation of food additives: specifica-
tions 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 Report Series No. 48; WHO Technical
Report Series No. 462).
23. FAO/WHO (1971) Toxicological evaluation of some extraction
solvents and certain other substances (FAO Nutrition
Meetings Report Series No. 48A; WHO/Food Add/70.39).
24. FAO/WHO (1971) Specifications for the identity and purity
of some extraction solvents and certain other substances
(FAO Nutrition Meetings Report Series No. 48B; WHO/Food
Add/70.40).
25. FAO/WHO (1971) A review of the technological efficacy of
some microbial agents (FAO Nutrition Meetings Report Series
No. 48C; WHO/Food Add/70.41).
26. FAO/WHO (1972) 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 Report Series No. 50; WHO
Technical Report Series No. 488).
27. FAO/WHO (1972) Toxicological evaluation of some enzymes,
modified starches, and certain other substances (FAO
Nutrition Meetings Report Series No. 50A; WHO Food Additive
Series No. 1).
28. FAO/WHO (1972) Specifications for the identity and purity
of some enzymes and certain other substances (FAO Nutrition
Meetings Report Series No. 50B; WHO Food Additive Series No.
2).
29. FAO/WHO (1972) A review of the technological efficacy of
some antioxidants and synergists (FAO Nutrition Meetings
Report Series No. 50C; WHO Food Additive Series No. 3).
30. FAO/WHO (1972) 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 Report Series No. 51; WHO
Technical Report Series No. 505 and corrigendum).
31. FAO/WHO (1972) Evaluation of mercury, lead, cadmium, and
the food additives amaranth, diethylpyrocarbamate, and octyl
gallate (FAO Nutrition Meetings Report Series No. 51A; WHO
Food Additives Series No. 4).
32. FAO/WHO (1974) 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
Report Series No. 53; WHO Technical Report Series No. 539
and corrigendum) (out of print).
33. FAO/WHO (1974) Toxicological evaluation of certain food
additives including anticaking agents, antimicrobials,
antioxidants, emulsifiers, and thickening agents (FAO
Nutrition Meetings Report Series No. 53A; WHO Food Additives
Series No. 5).
34. FAO/WHO (1978) Specifications for the identity and purity
of thickening agents, anticaking agents, antimicrobials,
antioxidants, and emulsifiers (FAO Food and Nutrition Paper
No. 4).
35. FAO/WHO (1974) Evaluation of certain food additives.
Eighteenth report of the Joint FAO/WHO Expert Committee on
Food Additives (FAO Nutrition Meetings Report Series No. 54;
WHO Technical Report Series No. 557 and corrigendum).
36. FAO/WHO (1975) Toxicological evaluation of some food
colours, enzymes, flavour enhancers, thickening agents, and
certain other food additives (FAO Nutrition Meetings Report
Series No. 54A; WHO Food Additive Series No. 6).
37. FAO/WHO (1975) Specifications for the identity and purity
of some food colours, flavour enhancers, thickening agents,
and certain food additives (FAO Nutrition Meetings Report
Series No. 54B; WHO Food Additives Series No. 7).
38. FAO/WHO (1975) 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 Report Series No. 55; WHO Technical Report Series
No. 576).
39. FAO/WHO (1975) Toxicological evaluation of some food
colours, thickening agents, and certain other substances
(FAO Nutrition Meetings Report Series No. 55A; WHO Food
Additive Series No. 8).
40. FAO/WHO (1976) Specifications for the identity and purity
of certain food additives (FAO Nutrition Meetings Report
Series No. 55B; WHO Food Additive Series No. 9).
41. FAO/WHO (1976) Evaluation of certain food additives.
Twentieth report of the Joint FAO/WHO Expert Committee on
Food Additives (FAO Food and Nutrition Series No. 1; WHO
Technical Report Series No. 599).
42. FAO/WHO (1976) Toxicological evaluation of certain food
additives (WHO Food Additives Series No. 10).
43. FAO/WHO (1977) Specifications for the identity and purity
of some food additives (FAO Food and Nutrition Series No.
1B; WHO Food Additive Series No. 11).
44. FAO/WHO (1978) Evaluation of certain food additives.
Twenty-first report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 617).
45. FAO/WHO (1977) Summary of toxicological data of certain
food additives (WHO Food Additives Series No. 12).
46. FAO/WHO (1977) Specifications for the identity and purity
of some food additives, including antioxidants, food
colours, thickeners, and others (FAO Nutrition Meetings
Report Series No. 57).
47. FAO/WHO (1978) 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).
48. FAO/WHO (1978) Summary of toxicological data of certain
food additives and contaminants (WHO Food Additive Series
No. 13).
49. FAO/WHO (1978) Specifications for the identity and purity
of certain food additives (FAO Food and Nutrition Paper No.
7).
50. FAO/WHO (1980) Evaluation of certain food additives.
Twenty-third report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 648 and
corrigenda).
51. FAO/WHO (1980) Toxicological evaluation of certain food
additives (WHO Food Additive Series No. 14).
52. FAO/WHO (1979) Specifications for the identity and purity
of food colours, flavouring agents, and other food additives
(FAO Food and Nutrition Paper No. 12).
53. FAO/WHO (1980) Evaluation of certain food additives.
Twenty-fourth report of the Joint FAO/WHO Expert Committee
on Food Additives (WHO Technical Report Series No. 653).
54. FAO/WHO (1980) Toxicological evaluation of certain food
additives (WHO Food Additives Series No. 15).
55. FAO/WHO (1980) Specifications for the identity and purity
of food additives (sweetening agents, emulsifying agents,
and other food additives) (FAO Food and Nutrition Paper No.
17).
56. FAO/WHO (1981) Evaluation of certain food additives.
Twenty-fifth report of the Joint FAO/WHO Expert Committee on
Food Additives (WHO Technical Report Series No. 669).
57. FAO/WHO (1981) Toxicological evaluation of certain food
additives (WHO Food Additives Series No. 16).
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