Guideline for Assessment of the Effect of Food on
Human Health Regarding Food Additives

May 2010

Food Safety Commission of Japan (FSCJ)

Table of Contents
Page<History of Evaluation> .......................................................................................................... 3<Food Safety Commission Members> .................................................................................... 3<Food Safety Commission Food Additives Expert Committee Members> ........................ 4
Chapter I.
Article 1.
Article 2.
Article 3.

General Provisions ............................................................................................. 5
Background ........................................................................................................ 5
Definition ............................................................................................................ 5
Purpose ............................................................................................................... 7

Article 4. Policies for assessments of the effect of food on health regarding foodadditives .............................................................................................................. 7
Article 5. Policies on materials needed for assessments .................................................. 9
Article 6. Disposition tests and toxicological tests .......................................................... 11
Article 7. Risk characterization ....................................................................................... 11
1. Setting ADI ......................................................................................................... 11
2. Determination of NOAEL ................................................................................. 12
3. Group ADI ......................................................................................................... 13
Article 8. Reassessment .................................................................................................... 13
Chapter II. Detailed Expositions......................................................................................... 14
Article 1. Information on the food additives to be assessed.......................................... 14
Article 2. Findings regarding safety................................................................................ 14
1. Disposition studies ............................................................................................. 14
2. Toxicological studies ......................................................................................... 15(1) Subchronic toxicity studies and chronic toxicity studies ............................... 15(2) Carcinogenicity studies .................................................................................. 16(3) Toxicity/carcinogenicity combination studies with one-year repeateddose administration ........................................................................................ 18(4) Reproductive toxicity studies......................................................................... 18(5) Prenatal developmental toxicity studies ........................................................ 18(6) Genotoxicity studies ...................................................................................... 19(7) Allergenic potential studies............................................................................ 20(8) General pharmacological studies ................................................................... 20

1

(9) Other studies .................................................................................................. 20
Article 3.
Article 4.
Article 5.
Article 6.
Article 7.

Findings in humans ......................................................................................... 20
Estimation of daily intake ............................................................................... 21
Assessment procedure for internationally commonly used flavors............. 21
Assessment methods for enzymes ................................................................... 21
Assessment methods for nutritional elements ............................................... 22

Appendix 1. Materials required for assessments of food additives (excludinginternationally commonly used flavors) ........................................................ 24
Appendix 2. Materials regarding toxicity required for assessments of enzymes(when it is scientifically proven [by considering the items in Table 2 in
the guideline published by the Ministry of Health and Welfare in 1996]
that the enzyme is broken down in the digestive tract to become acommon component of food) .......................................................................... 25

2

<History of Evaluation>
November 11, 2008: 64th meeting of the Food Additives Expert Committee
December 15, 2008: 65th meeting of the Food Additives Expert Committee
January 13, 2009:
66th meeting of the Food Additives Expert Committee
February 2, 2009:
67th meeting of the Food Additives Expert Committee
March 23, 2009:
69th meeting of the Food Additives Expert Committee
April 20, 2009:70th meeting of the Food Additives Expert Committee
May 18, 2009:
71st meeting of the Food Additives Expert Committee
September 7, 2009: 77th meeting of the Food Additives Expert Committee
October 22, 2009: 306th meeting of FSCJ (report by the Pesticides Expert Committee meeting)
October 22 toNovember 20, 2009: Public comments
December 15, 2009: 81st meeting of the Food Additives Expert Committee
May 25, 2010:
Report to the Chairman of FSCJ by the Chairman of the Pesticides Expert
Committee
May 27, 2010:
333rd meeting of FSCJ (reporting of the discussion results from the Pesticides
Expert Committee meeting)
Adapted and published as the Guideline for Assessment of the Effect of Food on
Human Health Regarding Food Additives<Food Safety Commission Members>(Until June 30, 2009)
Dr. Takeshi Mikami (Chair)
Dr. Naoko Koizumi (Deputy Chair)
Dr. Taku Nagao
Dr. Kazumasa Nomura
Dr. Keiko Hatae
Dr. Masao Hirose
Dr. Seiichi Honma(From July 1, 2009)
Dr. Naoko Koizumo (Chair)
Dr. Takeshi Mikami (Deputy Chair*)Dr. Taku Nagao
Dr. Kazumasa Nomura
Dr. Keiko Hatae
Dr. Masao Hirose
Dr. Masatsune Murata* From July 9, 20093

<Food Safety Commission Food Additives Expert Committee Members>(Until September 30, 2009)
Dr. Shoji Fukushima (Chairman)
Dr. Yasushi Yamazoe (Deputy Chairman)
Dr. Mayumi Ishizuka
Dr. Kazuhide Inoue
Dr. Katsumi Imaida
Dr. Takashi Umemura
Dr. Makoto Ema
Dr. Kikue Kubota
Dr. Masahiro Tokin
Dr. Dai Nakae
Dr. Emi Nakajima
Dr. Makoto Hayashi
Dr. Kunitoshi Mitsumori
Dr. Nobuo Yoshiike(From October 1, 2009)
Dr. Katsumi Imaida (Chairman)
Dr. Yasushi Yamazoe (Deputy Chairman)
Dr. Mayumi Ishizuka
Dr. Kiyomi Ito
Dr. Kazuhide Inoue
Dr. Takashi Umemura
Dr. Makoto Ema
Dr. Kikue Kubota
Dr. Tetsuya Tsukamoto
Dr. Masahiro Tokin
Dr. Dai Nakae
Dr. Makoto Hayasi
Dr. Kunitoshi Mitsumori
Dr. Akemi Morita
Dr. Masami Yamada[Adviser]Dr. Kiyomi Ito
Dr. Junichi Sawada
Dr. Kentaro Tanemura
Dr. Tetsuya Tsukamoto
Dr. Masato Naya
Dr. Masamichi Honma
Dr. Akemi Morita
Dr. Masami Yamada

4Chapter I. General Provisions
Article 1. Background
The Food Safety Commission of Japan (FSCJ), based on the “Basic Matters” referred to in
Article 21-1 of the Food Safety Basic Law (approved by the Cabinet on January 16, 2004), is
obliged to establish guidelines for assessing the effect of foods on human health. The
following guidelines have been established in accordance with this obligation: “Standards for
the Safety Assessment of Genetically Modified Foods (Seed Plants)” (January 29, 2004),“Guideline for Assessment of the Effect of Food on Health Regarding Official Standards ofGeneral Fertilizers” (March 18, 2004), “Standards for the Safety Assessment of Food
Additives Produced Using Genetically Modified Microorganisms” (March 25, 2004),“Guideline for Safety Assessment of Genetically Modified Animal Feed and Animal Feed
Additives” (May 6, 2004), “Guideline for Assessment of the Effect of Food on Human Health
Regarding Antimicrobial-Resistant Bacteria Selected by Antimicrobial Use in Food Animals”(September 30, 2004), and “Standards for the Safety Assessment of Genetically Modified
Foods (Microorganisms)” (June 26, 2008).
Guidelines for the assessment of the effect of food on human health are essential for
ensuring the scientific validity and fairness of assessments as well as clearly defining theassessment data for applicants. They also serve to assure parties both within and outside Japanof the transparency of assessments.
FSCJ of Japan has created this “Guideline for Assessment of the Effect of Food on
Human Health Regarding Food Additives” based on the results of past assessments of theeffect of food on health as well as policies regarding safety assessments developed by theJapanese government and governments of other countries. Going forward, this should be
followed whenever assessments are conducted.
It should be noted that this Guideline for Assessment will be reviewed in accordance withregulations as necessary in consideration of trends in international standards for assessments
and new scientific findings both in and outside Japan. Based on such reviews, the Guideline
may be revised.
Article 2. Definition
1. Food additives
As defined in Article 4-2 of the Food Sanitation Act (Law No. 233 of 1947), substances
which are used by being added, mixed or infiltrated into food or by other methods in theprocess of producing food or for the purpose of processing or preserving food.

5

3.

4.

5.

6.

2. Acceptable daily intake (ADI)
Based on contemporaneous scientific knowledge, the amount of a substance that can beingested on a daily basis over a lifetime without appreciable health risk to the consumer.
Tolerable upper intake level (UL)
The maximum level of habitual nutrient intake that is likely to pose no risk of adverse
effects.
No-observed-adverse-effect level (NOAEL)The highest dose of a substance that has been reported to have no harmful effects intoxicological tests involving different dosage levels.
Lowest-observed-adverse-effect level (LOAEL)The lowest does of a substance that has been reported to have harmful effects intoxicological tests involving different dosage levels.
Benchmark dose (BMD)

The intake level at a certain toxicological response rate calculated by applying amathematical model to the correlation between the toxicological response rate and theintake.
7. Virtually safe dose (VSD)
The dose of a substance at which the risk of cancer is no greater than normal if the foodcontaining the substance were consumed at the maximum residue level over a lifetime(i.e., low probabilities, such as 1/100,000 or 1/1,000,000). This dose is used forassessment methods based on the assumption that there is no threshold forgenotoxicological substances.
8. Toxicological indicator (Endpoint)
The observable or measurable biological incidence or chemical concentration that is used
as an indicator of exposure effects of the assessed substance.
9. Safety factor
The factor used to translate an NOAEL when setting the ADI and other levels to ensure
further safety.
10. Mode of action (MOA)
The mechanism by which the chemical substance affects organisms.
11. Assessment based on the weight of evidence (WOE)
An assessment based on the weight of the evidence examined.
12. Good laboratory practice (GLP)Standards of quality of practice at the testing facilities and equipment of a testing
institution, as well as its organization, staff and operational procedures. Set to ensure thereliability of results of safety tests of various chemical substances.
13. Epidemiology

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A field of study in which the incidence and distribution—and factors that influence these,such as dietary, smoking and drinking habits—of various health problems occurring inhuman populations is examined in order to establish effective measures for issues
relating to human health.
14. Joint FAO/WHO Expert Committee on Food Additives (JECFA)
A committee jointly organized by FAO and WHO to conduct risk assessments for foodadditives, pollutants, veterinary products, and other items, and provide recommendations
grounded in scientific findings to member states and the Codex
Alimentarius Commission.
15. The 1996 Guideline by the Ministry of Health and Welfare“Concerning the Guidelines for Designation of Food Additives and for Revision ofStandards for Use of Food Additives” (Notification No. 29 of March 22, 1996)
16. Internationally commonly used food additives
Food additives so designated based on the agreement reached by the Food Sanitation
Subcommittee of the Pharmaceutical Affairs and Food Sanitation Council on July 2002
for which (1) an international safety assessment has been completed by the JECFA and(2) the use has been widely permitted in the U.S. and EU member states and there is a
global consensus on the necessity of use. The Ministry of Health, Labour and Welfare ofJapan has indicated a policy to start reviewing substances for this designation without
waiting for requests to be made by private corporations and other organizations.
Article 3. Purpose
The purpose of this Guideline is to define the scope of data required to assess effects and theguidelines for assessment to be used, when additives are sought to be designated as notharmful for human health based on Article 10 of the Food Safety Law and, to define thestandards for additives based on Article 11-1 of the same Law.
Assessments must be conducted following this Guideline when specific names ofadditives are removed from the existing list of additives based on the Supplementary
Provision 2-2-1 of the Act for Partial Revision of the Food Sanitation Act and the Nutrition
Improvement Act (Law No. 101 of 1995).
Article 4. Policies for assessments of the effect of food on health regarding food additives
1. Safety factors will be applied to the values determined as a result of assessments ofthe effect of food on health regarding food additives after FSCJ finalizes the treatment.
For the time being, the Expert Committee shall have the responsibility of deciding how totreat the safety factors.

7

2. For “Internationally commonly used food additives” which are broadly usedinternationally and proven safe (excluding internationally commonly used flavors),
which have passed the safety assessment conducted by the JECFA and are approved forlong-term use in the U.S. and Europe, assessments based on assessment reports generated
by the JECFA, the U.S. and European countries (“report-based assessments”) shall be
conducted in principle after the latest scientific findings are examined.
3. Although the existence of a threshold for genotoxic carcinogens1 has been a topic ofinternational discussion for some time, no consensus has yet been reached. Assessment,
therefore, shall be conducted in principle based on the assumption that no such thresholdexists. Any examination of a substance for classification as a genotoxic carcinogen must
be conducted carefully and conclusions must based on the MOA and WOE.
4. In principle, food additives that are assessed and determined to be genotoxic carcinogens
should not be approved at this point in accordance with the previous paragraph. If thesubstance is foreign matter (including naturally occurring matter; the same hereinafter)that is unavoidably added during the manufacturing process of the food additive, or if a
byproduct of the substance is a genotoxic carcinogen, assessment shall becomprehensively conducted based on the concept of VSD, while the content level should
be lowered to the minimum that is technologically possible.
5. Substances that are to be used as an alternative to ordinary food ingredients or for the
purpose of nutritional enhancement or as a “food with nutritional claims,” must be
examined regarding their quality as a nutritional component as well as relative to theintake levels of the same nutritional components that are available in other foods. Dietary
Reference Intakes and other information must also be examined in order to make anassessment.
6. Effects on pregnant women and fetuses, infants, children and elderly people should be
examined as necessary in cases where sufficient information is available to examinerisks.
7. It is preferable to examine as necessary in vitro studies and other studies conductedduring pharmaceutical development or in other areas that are recommended for foodadditive studies (for example, when a metabolite has had a harmful reaction in an animal
test, the results must be extrapolated to effects on humans by conducting an in vitro test
using a human metabolic enzyme).
8. When interaction between the assessed substance and a medical product is likely to occur,
the interaction shall be examined only when necessary and when sufficient knowledge is
1
A genotoxic carcinogen is a substance that affects DNA either directly or by its metabolites to show inducibility for genetic mutation or
chromosomal aberration, and whose genotoxic reaction is considered a part of the carcinogenic mechanism. Its genotoxicity must be
confirmed in vivo (in the carcinogenic target organ, if possible).

8

available to examine the risk. It should be noted that people subject to such reactions are,in principle, under the care of healthcare professionals.
9. The necessity for assessment should be examined also for decomposition products,
mixed foreign matter, and metabolites that characteristically effect humans. The safety offood additives and their safe inclusion in food should be also examined and, if found to
be unstable, the main types of decomposition products and the levels at which they are
generated should also be examined.
10. To examine the harmful effects of intake of more than one food additive, it is consideredthat the effects of simultaneous intake of different food additives are examined andpractical safety can be assured through complete assessment of each food additive basedon the reports contained in the “Collection of Information and Survey on Effects ofIntake of Multiple Food Additives” compiled in the 2006 General Survey to Ensure Food
Safety conducted by FSCJ. However, when findings are unavailable with respect to therisk of taking multiple food additives, assessment should be carried out as necessary.
11. Tests using genetically modified animals call for careful treatment. These tests are rarely
used by the JECFA and other organizations except in a very few cases where they areused in risk assessments by FSCJ.
12. The JECFA considers it possible that food additives manufactured with nano materials orother new technologies may have different toxicological features and therefore theexisting standards and the ADI cannot be applied to these substances in general. Whenassessment of these substances becomes necessary, each case will be examined
separately.
Article 5. Policies on materials needed for assessments
1. The scope and notes on the data needed for assessments are shown in Chapter II andAppendix 1 and 2, and additional information is given below. Specific test procedures inprinciple follow the internationally recognized test guidelines published by theOrganization for Economic Co-operation and Development (OECD) and otherorganizations.
(1) Part of the testing can be omitted when the test food additive is known to be acommon ingredient of food or when the test food additive is scientifically known tobecome a common component of food after the food is broken down in the digestive
tract following consumption. The scientific validity of such information shall be
determined after examination of the items in Table 2 of the 1996 Guideline by the
Ministry of Health and Welfare.
(2) Assessments of “Internationally commonly used food additives” should take thelong history and experience of human dietary habits into consideration (see Chapter I,

9Article 4-2) and assessments of “Internationally commonly used” flavors, enzymes
or nutritional components should take the characteristics of the substance into
consideration (see Chapter II, Articles 5, 6 and 7).
(3) When a test food additive differs from a food additive already designated only in thebase moiety, when it is an isomer of such additive, or when there is a scientificallyrational reason for doing so, part of the testing can be omitted provided the reason for
doing so is clearly described.
2. Revision of usage standards and compositional standards should be conducted inaccordance with the notes below.
(1) Revision of usage standards should be done in accordance with the following notes.
(a) When an assessment of the effect of food on health by FSCJ has been completed
for the food additive in question, data for the estimated daily intake regarding therequest (addition of food items for which the additive is to be used or change of

3.

4.
5.

6.

usage dose) should be submitted. If there is a new toxicological finding in such acase, the data on that finding should be also submitted.
(b) When an assessment of the effect of food on health by FSCJ has not been
conducted for the food additive in question, materials needed for the assessment
for the designation of additives should be submitted, in principle.
(2) For the revision of compositional standards, the validity and safety of the revised
compositional standards should be demonstrated.
The applicant is responsible for submitting the materials required for the assessment andmust ensure the reliability of the content of the materials. In principle, the materials
submitted by the applicant must be: (1) results from tests conducted at an experimental
facility whose operational management is recognized as appropriate (i.e., a GLP facility)and by a method whose reliability is ensured; or (2) scientifically reliable materials, suchas assessment reports complied by international organizations. If materials indicating that
there is a concern regarding the safety of the food additive, such materials must be
submitted for examination regardless of their reliability.
Autopsy and histopathological assessments should be conducted by a specialist with
ample experience.
Raw data and samples used for animal tests should be maintained until the end of theperiod regulated under GLP or until the assessment is completed so that they can besubmitted if needed.
In principle, assessments should be conducted based on the materials submitted by therequesting party. If the materials submitted are considered insufficient, the requesting
party may be asked to submit additional materials.

10Article 6. Disposition tests and toxicological tests
Disposition tests are conducted to estimate the behaviors of food substances within the humanbody in terms of absorption, distribution, metabolism, and excretion (ADME). Therefore not
only compiling the result of animal tests, ADME behaviors within the human body and thepossible occurrence of harmful effects should be examined.
When examining the test data, the observed toxicity and residual level in the body should
be confirmed from a scientific point of view to ensure that it is a property of the additive itself
and not an incidental effect of other factors such as the nutritional condition of the subject.
When deciding an endpoint, the disposition within the body and differences among tests andtest animal species should be considered, and the findings in terms of general conditions,body weight, food intake, hematological tests, blood biochemical tests, urine tests,
pathological tests and other tests should be examined for statistical significance and dose
relations in order to ensure scientifically rational assessment. In these cases, the toxicologicalmechanism should be determined as clearly as possible.
Article 7. Risk characterization
1. Setting ADI(1) When more than one NOAEL is indicated as a result of comprehensive assessment of
toxicological tests, the ADI should be set based on the lowest NOAEL value.
(2) In principle, results of toxicological tests should be examined by taking sexual
differences into consideration, and separate NOAELs should be set for each sex.
(3) Taking species differences and individual differences into consideration, a safety
factor of 100 should be used (10 for species differences, 10 for individual
differences). It should be noted, however, that the safety factor of 100 is not a fixed,
constant value but rather should be set individually in each case based on the
toxicological property and test data and in consideration of the following.
(a) When the data are taken from tests on human subjects, species differences do nothave to be taken into consideration. Based on individual differences, a safety
factor of 1 to 10 should be used, depending on the surveyed populations.
(b) When sufficient information is not available and if the food additive underassessment is associated with serious toxicity 2 , the safety factor should be
multiplied by an additional value of 1 to 10.
(c) When the ADI is set based on the LOAEL, the safety factor should be multipliedby an additional value of 1 to 10. A benchmark dose can be also used in these2
The Principles for the Safety Assessment of Food Additives and Contaminants (IPCS, EHC70) lists the following two items as
examples:a) Irreversible reaction seen in a prenatal developmental toxicity testb) Finding of carcinogenicity

11cases.
(4) The phrasing of the assessment result should follow the pattern set out below.
Phrasing of the results of assessment by the Food Additives Expert Committee(1) “ADI is established. ADI
is established as ….”
ADI can beestablished(2) “No ADI has to beestablished.”
Assessment available(sufficient data
available)

Note: The substance is of no safety concern whenappropriately used as a food additive, and
ADI therefore does not have to be specified.

(3) “No ADI can be established.

No ADI can beestablished

Example (Madder color): Madder color has been shown tobe genotoxic as well as carcinogenic to the kidney. [TheFood Additives Expert Committee of the Food Safety
Commission concluded that] no ADI (acceptable daily
intake) could be established for this substance.

2. Determination of NOAEL
In order to determine the NOAEL, the prior establishment of an appropriate dose should
be investigated. In a toxicological test, the maximum dose should be set at the level atwhich a toxicological effect is recognized, and the minimum dose should be set at thelevel at which no toxicological effect is recognized. Also, different dose levels should be
set so that the dose-reaction relationship can be observed. When the substance isadministered by feeding, care should be taken to prevent nutritional disturbance. In
general, the ratio of the substance to the feed does not have to exceed 5% (W/W). Whenthe substance is given by gavage administration, the general maximum dose required is
the technically possible maximum dose or 1,000 mg/kg bw. If no effect is observed atthat dose, the administration of a higher dose is not required.
When different tests are conducted for different animal species, different NOAELs
should be determined for each test. To calculate the ADI, the NOAEL taken from theanimal test with the lowest dose that shows a toxic effect should be used. However, when12

a certain test is obviously more appropriate in terms of its design or results than others
and the test periods were different among the tests, a test with a longer period and more
appropriate design should be given more weight in determining the NOAEL used to
calculate the ADI. When metabolic data or pharmacokinetic data are used, the NOAEL
used to calculate the ADI can be determined based on the animal species that mostresembles human beings.
3. Group ADIWhen several substances that have a structurerstructure activity correlation or do nothave a structural activity correlation but have a similar level of toxicology (e.g., thosethat can generate additive physiological/toxicological reactions), the ADI should be set
for the substances as a group in order to manage the accumulated intake. Whenestablishing a group ADI, the lowest NOAEL among the NOAELs of all the substancesin the group should be used, in principle. The relative quality of the test data and the test
period should be taken into consideration when establishing the NOAEL. If one NOAEL
is significantly higher or lower than the other NOAEL values of the substances in thegroup, that substance should be removed from the group.
Article 8. Reassessment
Even after a food additive is approved, it should be continuously observed for potential
harmful effects and if any harmful effect is identified as a result of progress in toxicologicalunderstanding or other factors the additive should be reassessed.
When an important set of data indicating a safety concern is newly obtained regarding afood additive that has previously been assessed, reassessment of that additive should be
carried out promptly.

13Chapter II. Detailed Expositions
The materials needed for assessments are listed in Appendix 1 and 2. For detail, the notes
below should be followed.
Article 1. Information on the food additives to be assessed
1. Name and usage
2. Origin or process of discovery
3. Usage in other countries
4. Assessments by international organizations and other organizations
5. Physiochemical properties
Chemical name, (generic names in Japanese and English, CAS number), molecular
structure, molecular weight, structural formula, manufacturing method, chemical nature,stability (including stability in food), suggestions for compositional standards, etc.
6. Suggestions for usage standards(1) When the establishment of usage standards to regulate usage, such as the food items
for which the additive can be used and the maximum amount of additive to be used,
is considered necessary based on a comprehensive examination of the safety andefficacy of the food additive, the reasons for setting such usage standards must be
clearly explained. When establishing the standards, such information as the estimateddaily intake (see Chapter II, Article 4) and the ADI obtained from toxicological tests
should be taken into consideration.
(2) When the establishment of usage standards is determined to be unnecessary, thereasons for such determination should be clearly indicated.
7. Other (Information useful for assessments of the effect of food on health)
Article 2. Findings regarding safety
1. Disposition studies
Studies to examine the disposition within the body should comply with the disposition
study guideline published by the Ministry of Health and Welfare in 1996. They also
should follow the notes below.
(1) The food additive or substance labeled by an isotope should be used as the test
substance. When an isotope-labeled substance is used, the species and location of theisotope should be clearly indicated.
(2) It is preferable to conduct tests on more than two species (more than one rodent
species [typically rats] and more than one non-rodent species [typically dogs]).
(3) In principle, the test substance should be administered orally. Absorption,
distribution, metabolism, and excretion should be estimated after single-dose

14

administration and repeated-dose administration. Additional tests with intravenous
administration and other tests may be carried out when necessary in order tocalculate accurate ratio of absorption or for other purposes.
(4) Each process of absorption, distribution, metabolism, and excretion must be
examined and values recorded, such as concentration of the active ingredient in the
blood; amount of the substance in urine, feces and other excretory matter; andsuccessive changes in the concentration in each organ; metabolites found inorganisms, as well as factors that are influential in each step.
(5) The results regarding absorption, distribution metabolism and excretion (e.g., highest
concentration in blood plasma, successive change in concentration in each organ, andelimination half-life) should be used to determine the organ(s) that can be a target of
toxicological tests. In such cases, the feasibility of extrapolating the results to obtain
the effects on the human body must be examined with regard to differences among
animal species and species specificity.
(6) For tests using a racemic body, it is preferable to examine the disposition of each
optical isomer within the body if it is necessary to understand the association with
toxicity.
(7) In principle, the existence of human-specific metabolites must be examined andtoxicological tests of such metabolites must be carried out as necessary.
2. Toxicological studies(1) Subchronic toxicity studies and chronic toxicity studies(a) Tests should be conducted on one rodent species (generally rats) and one
non-rodent species (generally dogs). In principle, the same number of male andfemale animals should be used.
(b) The administration period should be 28 days or 90 days for subchronic
toxicology tests and more than 12 months for chronic toxicology tests. The
28-day test can be omitted when a test with a 90-day administration period is
carried out.
(c) In principle, the test substance should be orally administered 7 days a week. The
substance should be administered in animal feed or water, but it can be also
administered by gavage.
(d) At least three groups receiving different levels of the administration dose shouldbe established in addition to the control group. The reasons for choosing eachdose level should be clearly indicated. Proper ratios should be chosen so that an
appropriate NOAEL can be obtained.
(e) Care should be taken to prevent nutritional disturbance among test animals when15

feeding them the substance. Usually, the amount of the substance as a proportionof the feed does not have to exceed 5% (W/W). When the substance is given bygavage administration, the general maximum dose needed is the technicallypossible maximum dose or 1,000 mg/kg bw. If no effect is observed at that dose,
the administration of a higher dose is not required.
(f) When the frequency or severity level of a naturally occurring pathologicalchange that is also observed within the control groups increases due to theadministration of the substance, even within the context of the background data
it should, in principle, be taken as an effect caused by the administration of thesubstance if biological some significance, such as a relationship between thedose and the frequency or severity level, is recognized.
(g) When neurotoxicity or immunotoxicity3 is suspected, the need for additional
tests as described in the OECD test guideline or ICH (International Conference
on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use) guideline should be examined.
(h) The procedure to extrapolate the findings of toxicological tests to humans should
be examined carefully by analyzing the endpoints separately and for different
factors, such as functional changes, non-oncological morphological changes,
oncological morphological changes, and changes to reproductive functions.
(i) When a combination test for chronic toxicity and carcinogenicity is carried out
using one rodent species, a chronic toxicity test and carcinogenicity test on
another rodent species can be omitted. .
(j) The need to add an in utero exposure phase should be examined where
necessary.
(2) Carcinogenicity studies(a) Tests should be conducted on more than two rodent species (rats, mice or
hamsters are used generally). In principle, the same number of male and female
animals should be used.
(b) In principle, administration should be carried out orally 7 days a week. For rats,
the period should be between 24 months or longer and 30 months or shorter. For
mice, the period should be between 18 months or longer and 24 months or
shorter. The test substance should be orally administered in animal feed or withwater, but it can be also administered by gavage if oral administration is difficult.
(c) At least three groups receiving different levels of the administration dose should3
In this guideline, “immunotoxicity” is defined as toxicity resulting from suppressed immune function caused by a substanceunintentionally ingested by a living organism in a non-antigen-specific way.

16

be established in addition to the control group. The reasons for choosing eachdose level should be clearly indicated. Proper ratios should be chosen so that an
appropriate NOAEL can be obtained.
(d) Care should be taken to prevent nutritional disturbance among test animals when
feeding them the substance. Usually, the amount of the substance as a proportionof the feed does not have to exceed 5% (W/W). When the substance is given bygavage administration, the general maximum dose needed is the technicallypossible maximum dose or 1,000 mg/kg bw. If no effect is observed at that dose,
the administration of a higher dose is not required.
(e) If the test for carcinogenicity is positive, the ADI cannot be established inprinciple if genotoxicity is positive and the substance is determined to be a
genotoxic carcinogen. If the test for carcinogenicity is negative, the ADI can beestablished if genotoxicity is negative and the substance is determined not to be a
genotoxic carcinogen. Even if the food additive being assessed unavoidably
generates/contains a byproduct/residue that is suspected of being genotoxic, theADI may be established in some cases after a required examination (see Chapter
I, Article 4-3, 4-4).
(f) If the incidence rate of lesions is relatively low, carcinogenicity may be
determined during the assessment by conducting a significance test using either:(1) the sum of benign tumor-like lesions and malignant tumor-like lesions; or (2)the sum of precancerous lesions, benign tumor-like lesions and malignant
tumor-like lesions. Assessment of carcinogenicity, including precancerous
lesions, is especially preferable where there is an increase in endocrine system
tumors, a type of lesion that frequently occurs with rodent species.
(g) If an increase in tumors in a region where tumor incidence is not normally highor when an increase in rare tumors is recognized it is preferable to include the
carcinogenic mechanism in the assessment.
(h) Factors that modify the development of cancer (suppression of weight increaseor decrease of survival rate) should be taken into consideration for theassessment.
(i) Special attention should be paid to species-specific toxicological findings (e.g.,
hypertrophy, hyperplasia and tumor of thyroid follicle epithelium [epithelium[specific to rodents] and renal disorder and tumor [specific to male rats]).
(j) When a combination test for chronic toxicity and carcinogenicity is carried out
using one rodent species, a chronic toxicity test and carcinogenicity test on
another rodent species can be omitted.
(k) The need to add an in utero exposure phase should be examined where17necessary.
(3) Toxicity/carcinogenicity combination studies with one-year repeated-dose
administration
Notes in (1) and (2) should be followed.
(4) Reproductive toxicity studies
Studies to examine reproductive toxicity should comply with the reproductive
toxicity study guideline published by the Ministry of Health and Welfare in 1996.
They also should follow the notes below.
(a) Tests should be conducted on more than one rodent species (rats are usedgenerally). In principle, the same number of male and female animals should be
used.
(b) In principle, administration should be carried out orally 7 days a week. The test
substance should be orally administered in animal feed or with water, but it canbe also administered by gavage if oral administration is difficult.
(c) At least three groups receiving different levels of the administration dose shouldbe established in addition to the control group. The reasons for choosing eachdose level should be clearly indicated. Proper ratios should be chosen so that an
appropriate NOAEL can be obtained.
(d) Care should be taken to prevent nutritional disturbance among test animals when
feeding them the substance. Usually, the amount of the substance as a proportionof the feed does not have to exceed 5% (W/W). When the substance is given bygavage administration, the general maximum dose needed is the technicallypossible maximum dose or 1,000 mg/kg bw. If no effect is observed at that dose,
the administration of a higher dose is not required.
(e) When neurotoxicity or immunotoxicity is suspected, the need for additional tests
as described in the OECD test guideline or ICH guideline should be examined.
(5) Prenatal developmental toxicity studies
Studies to examine prenatal developmental toxicity should comply with theteratogenetic study guideline published by the Ministry of Health and Welfare in
1996 and the notes below. The minimum period of administration should be from thedate of implantation to the estimated delivery date, and the substance should be
administered daily to the pregnant animals.
(a) Tests should be conducted on more than two species (more than one rodent
species [typically rats] and more than one non-rodent species [typically rabbits]).

18

(b) The test substance should be orally administered by gavage.
(c) At least three groups receiving different levels of the administration dose shouldbe established in addition to the control group. The reasons for choosing eachdose level should be clearly indicated. Proper ratios should be chosen so that an
appropriate NOAEL can be obtained.
(6) Genotoxicity studies
Studies to examine genotoxicity should comply with the mutagenicity test guideline
published by the Ministry of Health and Welfare in 1996. But the examination should
not be limited to the narrow definition of “mutagenicity” and the assessment should
be carried out based on the test results regarding genotoxicity in general. Among thetests included in the standard combination (i.e., combination of bacterial reverse
mutation tests, chromosome aberration tests using cultured cells of mammals, andmicronucleus tests on rodents), the chromosome aberration tests using mammaliancultured cells can be replaced with a mouse lymphoma TK assay (MLA) or in vitro
micronucleus test. In order to supplement the results from the standard test
combination, single cell gel electrophoresis (“Comet Assay”) and in vivo transgenic
animal mutation assay can be used, in addition to those described in the Ministry of
Health and Welfare guideline of 1996.
If one of the tests in the standard combination cannot be conducted due totechnical constraints, the reason should be explained backed up by scientificevidence. One of the internationally validated tests can be used as a replacement.
The test results should be judged in accordance with the following procedure.
(a) If the results of the bacterial reverse mutation tests are positive, a comprehensive
judgment should be made by fully considering the results of in vivo tests that use
genetic mutation or DNA damage (Comet Assay, in vivo transgenic animal
mutation assay) as an indicator.
(b) If the results of the chromosome aberration tests using mammalian cultured cells
are positive and the effect is also confirmed with rodent micronucleus tests, thesubstance can be determined as positive for genotoxicity.
(c) Even if the results of the chromosome aberration tests using mammalian cultured
cells are positive, if the results of the rodent micronucleus tests (preferably withevidence to show exposure of the target organ) are negative, the substance can be
determined as negative for genotoxicity.

19(7) Allergenic potential studies4
Studies to examine the allergenicity of food additives should follow the antigenicity
tests guideline published by the Ministry of Health and Welfare in 1996. There is nowell-established method for predicting the allergenicity of chemical substances whenorally ingested, particularly for predicting the immediate type of allergenicity.
Therefore, studies should be carried out with sensitization and induction methods
approved by specialists. For the time being, allergenicity studies using delayedallergy as an indicator should at least be carried out. Examples of tests for suchstudies include skin sensitization tests on guinea pigs (e.g., guinea pig maximization
test [GPMT] in the OECD test guideline 406) and lymph node reaction tests on mice(e.g., the local lymph node assay [LLNA] in the OECD test guideline 429).
Allergenicity assessment of food additives containing protein should follow the“Standards for the Safety Assessment of Genetically Modified Foods(Microorganisms)” (FSCJ decision, June 26, 2008).
(8) General pharmacological studies
Studies to examine general pharmacological properties of food additives should
follow the general pharmacological test guideline published by the Ministry of
Health and Welfare in 1996.
(9) Other studies
When neurotoxicity is suspected following a subchronic toxicity test and other tests,
additional tests should be conducted as necessary in compliance with the OECD test
guideline and other materials.
When immunotoxicity is suspected following a subchronic toxicity test andother tests, proper immunofunctional tests should be added as necessary inaccordance with the ICH guideline and other materials. Immunofunctional tests
should be also carried out as necessary when immunotoxicity in humans is suspectedbased on existing findings.
Article 3. Findings in humans
When available, appropriate clinical tests, epidemiological data and other information
regarding humans must be actively used. When allergenicity is suspected, findings in humans
should be especially valued because it is often infeasible to extrapolate the results of animal
tests to humans.
4

Also referred to as “allergenicity”

20Article 4. Estimation of daily intake
1. The daily intake should be determined based on the Japanese diet. Care should be taken
to avoid intake estimations that are too small. In principle, the estimated daily intake iscalculated by multiplying the daily intake of the food items for which the additive is to be
used by the amount of additive used. The daily intake of food should be properly
estimated based on the food group intakes given in the National Health and Nutrition
Survey or other materials. Estimations based on data gathered using other reliable
methods, such as market basket surveys and production analysis, can also be used. The
daily intake should be estimated for body weight of 50 kg.
2. The estimated daily intake should be compared with the ADI obtained from toxicologicaltests, and the results of such comparison should be examined. Where necessary, thesafety of food additives should also be examined in cases where more than one item of
the same kind of food additive, etc. is simultaneously consumed. This can be done bycomparing the sum of estimated daily intake to the group ADI, or by any other method.
3. Where considered necessary based on food consumption habits in Japan, theoverconsumption of nutritional elements and effects on electrolyte balance should also be
examined along with other relevant effects.
Article 5. Assessment procedure for “Internationally commonly used flavors
Assessments of internationally commonly used flavors should be conducted based on the
Safety Assessment Procedures regarding internationally commonly used Flavors (last report,
second revision) (November 4, 2003). Although this material indicates the need for in vivo
tests in cases where genotoxicity in living organisms is suspected following an in vivo
genotoxicity test using microorganisms and mammalian cells, additional in vivo chromosome
aberration tests are not needed if the results of an in vivo micronucleus test are alreadyavailable.
For the estimation of intake, the JECFA is planning to adopt the single portion exposuretechnique (SPET) method, a method for estimating the total intake by estimating the
proportional amount of additives contained in foods in each food group, in addition to theconventionally used per capita intake times ten (PCTT) method. The results of the SPET
method will be taken into account in future assessments. For Japan, where the estimation of
the proportional amount of food additives is not practical for new additives, the PCTT method
will continue to be used as the assessment method and the adaptation of the SPET method
will be discussed as an issue.
Article 6. Assessment methods for enzymes

21

Safety assessments of enzymes are, in principle, carried out based on the data in Appendix 1
and other information. When the safety of a production strain is not known for enzymes
obtained from microorganisms, appropriate tests must be conducted to assess the safety of theoriginal microorganism. Pathogenic or toxin-producing production bacteria should not in
principle be used for the production of enzymes.
When it is scientifically proven that the enzyme is broken down in the digestive tract tobecome a common component of food*, the materials regarding toxicity listed in Appendix 1
can be omitted. The materials regarding toxicity listed in Appendix 2 should be submitted.
* Such judgment should be made by considering the items in Table 2 in the guideline
published by the Ministry of Health and Welfare in 1996
Article 7. Assessment methods for nutritional elements
For the safety assessment of nutritional elements that are biologically essential or nutritional
elements that are proven to have positive effects on human health when consumed at a certain
level, materials listed in Appendix 1 are required, in principle. A Model for Establishing
Upper Levels of Intake for Nutrients and Related Substances, Report of a Joint FAO/WHOTechnical Workshop on Nutrient Risk Assessment (WHO Headquarters, Geneva, Switzerland,
2-6 May, 2005) should be also referred to for assessment.
Assessments of nutritional elements should be conducted by considering the following
notes.
1. Assessment should be conducted comprehensively based on the findings in humans fromclinical tests, epidemiological studies, and case reports. In assessments, backgroundfactors and variation in quality of studies should be taken into consideration and thefindings obtained from meta-analysis should be weighted higher.
2. The range of the amounts required and the amounts consumed in humans are frequently
relatively close to the LOAELs or NOAELs reported for humans. This fact, as well as thehomeostatic function specific to the nutritional element, should be taken into
consideration in the adoption of uncertainty factors 5 that differ depending on the
nutritional element.
3. When an excessive intake amount is likely to have serious effects on human health and inother cases where the amount of habitual intake from food is taken into consideration as
background, the distribution of habitual intake, in addition to the average value, should
be examined where necessary.

5

The safety factor used for establishing a tolerable upper limit for nutritional elements in food intake standards.

22

4. When a tolerable upper limit of the nutritional element is shown in the Dietary Reference
Intakes for Japanese established by the Ministry of Health, Labour and Welfare, thefigures shown and relevant background data should be examined.

23

Appendix 1. Materials required for assessments of food additives (excluding“Internationally commonly used flavors)

Items
Information on the additive subject to assessment
1. Name and usage
2. Origin or process of discovery
3. Usage in other countries
4. Assessments by international organizations andother organizations
5. Physiochemical properties
6. Suggestions for usage standards
7. Others (Information useful for assessments of theeffect of food on health)Findings regarding safety
1. Tests for disposition in organisms
2. Toxicity(1) Subchronic toxicity studies and chronic
toxicity studies(2) Carcinogenicity studies(3) Toxicity/carcinogenicity combination studies
with one-year repeated-dose administration(4) Reproductive toxicity studies(5) Prenatal developmental toxicity studies(6) Genotoxicity studies(7) Allergenic potential studies(8) General pharmacological studies(9) Other studies
3. Findings in humans
4. Estimation of daily intake, etc.

Designation

Revision ofstandard

Required
Required
Required
Required

Required*
Required*

Required
Required**
Required*

Required*

Required*

Required
Required**

Required
Required
Required
Required
Required*
Required
Required

*
*
*
*
*
*
*
Required

Note 1. When requesting a division of usage standards for a food additive for which assessment of theeffect of the food on health has already been carried out by FSCJ, the materials required for“Revision of standard” should be submitted. When requesting a division of usage standards
for a food additive for which assessment of the effect of the food on health has not been
carried out by FSCJ, documents required for designation should be submitted, in principle.
Note 2. Materials marked “Required” should be submitted whenever applicable. Materials marked
with an asterisk (*) should be submitted as necessary (when there is a new finding, forexample).
Note 3. When a combination test for chronic toxicity and carcinogenicity is carried out using one
rodent species, a chronic toxicity test and carcinogenicity test on another rodent species canbe omitted.

24Appendix 2. Materials regarding toxicity required for assessments of enzymes (when it
is scientifically proven [by considering the items in Table 2 in the guideline
published by the Ministry of Health and Welfare in 1996] that the enzyme
is broken down in the digestive tract to become a common component of
food)

Item

Designation(1) Toxicity tests on rats with 90-day repeated administration(2) Genotoxicity tests(3) Allergenicity tests

Required
Required
Required

Note:

Revisionofstandard***

Until further notice, the tests necessary for allergenicity assessment should be determined byreferring to the “Standards for the Safety Assessment of Genetically Modified Foods(Microorganisms)” (FSCJ decision, June 26, 2008).

25