Handbook of
Feed Additive Designation(Edition of Chemical Substances)

May 2016
Animal Products Safety DivisionTable of Contents
I Purpose of the Handbook････････････････････････････････････････････････

2

1 Introduction…………...…………………………………………………………………

2

2 About Feed Additives……………………………………………………………………

3

II Basic Ideas in the Preparation of the Required Documents…...……………………...

9

1 Flow of the Procedure for a Feed Additive Designation….……………………………

9

2 Outline of the Required Documents…….…………………………………………………

15

III Items to be Described in the Required Documents (Abstract)･･･････････････

16

1 Origin or Background of the Discovery, Status of Authorization and Use as a Feed
Additive in Foreign Countries, etc. ……..………………………………………….

17

2 Items Concerning Standards……….………………………………………………..

19(1) Name…………………………………………………………………………………...

19(2) Chemical structure…………………………………………………………………

19(3) Manufacturing process…………………………………………………………….

19(4) Biological and physicochemical properties………………………………………

20(5) Quantitation in feeds……………………..…………………………………………..

21(6) Changes with time……………………………………………………………..…….

22

3 Items Concerning Efficacy…………….……………………………………………...

26(1) Basic tests to prove efficacy………...……………………………………………..

26(2) Field application tests to prove efficacy………..…………………………………...

26

4 Items Concerning Residue…………………………………………………………..

30

5 Items Concerning Safety……………………………………………………………..

32(1) Toxicity tests………………………………………………………………………...

32(2) Feeding tests using targeted animals, etc.………………………………

49(3) Tests concerning the emergence of resistant bacteria………………………..

52(4) Others……..……………………………………………………………………………

52

IV Exemplary Abstract････････････････････････････････････････････････････

58

V Closing･･･････････････････････････････････････････････････････････････

75

VI Checklist for the Submission of the Required Documents･･････････････････････

76

Supplements[Reference 1] Glossary………………………………………………………………...………

4[Reference 2] Considerations for Descriptions of the Test Data…………………………...

16[Reference 3] Cases of Discussed/Authorized Substances in Foreign Countries as Additives…..........

17[Reference 4] Considerations for the Description of the Manufacturing Process………..

20[Reference 5] Considerations for the Description of the Test Results…………………….

27[Reference 6] Conditions to be Complied with in Conducting Animal Tests…….............

29[Reference 7] Tests for Analyzing In vivo Kinetics.………..………………………………...

49[Reference 8] Style Reference for the Description of Items Concerning Standards…….

53

1

I Purpose of the Handbook
1. Introduction
Animal feed must be safe to humans, in terms of the safety of the livestock products
made from the livestock that consumes the feed, not to mention the safety to thelivestock themselves that consume the feed. For the same reason, feed additives, whichmust serve purposes such as preventing the quality deterioration of feed as a matter ofcourse, must be safe to humans as well as to livestock.
The feed additive designation is granted by the Minister of Agriculture, Forestry and
Fisheries through consultations with the Agricultural Materials Council, under the policyof a substantial necessity, only to those substances that are in high-demand and havebeen proved to be apparently effective and safe. The designation is not a matter of an
approval issued in response to an application.
In other words, any substances that fall into the following categories cannot bedesignated as feed additives: substances having no apparent efficacy as a feed
additive; substances lacking safety proofs; and substances with poor levels of demand.
When the production or distribution, etc., of a non-designated feed additive is planned,
the documents required for the deliberation by the council will have to be prepared andsubmitted to the Animal Products Safety Division, Food Safety and Consumer Affairs
Bureau of the Ministry of Agriculture, Forestry and Fisheries (hereinafter referred to as“the authorities”). Those submissions which are judged worthy of a hearing by the
council are then sent to the council for deliberations.
The Agricultural Materials Council will conduct deliberations regarding the substance’s
efficacy as a feed additive and its impact on livestock and humans. When preparing the
documents, it should be borne in mind that the abstract that summarizes the test resultsof the efficacy of the substance as a feed additive and its safety, etc., is a particularly
important document for the purposes of the deliberation. There may be difficulties in
preparing the documents that meet the requirements of the deliberation and in offering
an explanation in front of the deliberation council because these documents containtechnical subjects, such as scientific test results and manufacturing process of thesubstances, and so on.
Based on the above consideration, this handbook explains the procedures and points ofcaution in preparing the required documents (in both abstract and specific terms) for the
deliberations on a feed additive by the Agricultural Materials Council in reference to theprocess for a feed additive designation. However, note that this handbook is written for a
supposed typical case of the feed additive designation of a chemical substance, andthat the required documents for seeking a designation of live microbial agents or
antibiotics, etc., are quite different from those explained herein. Please additionally notethat following the procedures described in this handbook for the preparation of therequired documents does not necessarily ensure success in the granting of adesignation.

22. About Feed Additives
Definition of a Feed Additive
A feed additive is a substance that is used with animal feeds in the form of an addition,
mixture, infiltration, etc. under the “Act Concerning the Safety Assurance and Quality
Improvement of Feed” (hereinafter referred to as the “Feed Safety Act”) for preventing
the feed quality deterioration and for other purposes specified in the ordinances of theAgriculture, Forestry and Fisheries Ministry, and as designated by the Minister for
Agriculture, Forestry and Fisheries through consultations with the Agricultural
Materials Council.
(Legal basis: Article 2, Paragraph 3 of the Feed Safety Act)
Intended Use of Feed Additives
The intended use of feed additives is limited to the following three uses outlined in the
Ordinance for Enforcement of the Feed Safety Act. Any substances intended for otheruses than these three are not designated as feed additives:
1) Preventing feed quality deterioration
2) Supplementing feeds with nutrients and other active ingredients
3) Facilitating the effective utilization of the nutrient ingredients of the feed
(Legal basis: Article 2, Paragraph 3 of the Feed Safety Act, Article 1 of the
Ministerial Ordinance for Enforcement of the Act Concerning the Safety
Assurance and Quality Improvement of Feed)
A feed additive designation is granted, within the limits of the minimum necessity, tothose substances that are in high-demand and have proved to be apparently effective
and safe, as has been conventionally exercised. Therefore, those who are planningthe manufacturing, importation, etc., of a non-designated feed additive as a feed
additive need to consult with the authorities in full, well before any actions are taken,
and receive instructions from them.
Relevant laws, ordinances and others
・ Law: the Act Concerning the Safety Assurance and Quality Improvement of Feed
(Law No. 35 issued on April 11, 1953)
・ Cabinet Order: the Order for Enforcement of the Act Concerning the Safety
Assurance and Quality Improvement of Feed (Cabinet Order No. 198 issued on
July 16, 1976)
・ Ordinance of the Ministry: the Ministerial Ordinance for Enforcement of the Act
Concerning Safety Assurance and Quality Improvement of Feed (Ordinance of the
Ministry of Agriculture, Forestry and Fisheries No. 36 issued on July 24, 1976),
and the Ministerial Ordinance on the Specifications and Standards of Feed and3Feed Additives (Ordinance of the Ministry of Agriculture, Forestry and Fisheries
No. 35 issued on July 24, 1976)
・ Public Announcement: the Ministerial Public Announcement Regarding the
Designation of Feed Additives (Ministry of Agriculture, Forestry and Fisheries
Public Announcement No. 750 issued on July 24, 1976)
・ Notification ： the Notification of the Establishment of the Standard for the
Evaluation of Feed Additives (Ministry of Agriculture, Forestry and Fisheries, Food
Safety and Consumer Affairs Bureau Director-General Notification No.
4-Chiku-A-201 issued on March 16, 1992), and the Notification Concerning theDocuments, etc., Required for the Feed Additive Designation (Ministry ofAgriculture, Forestry and Fisheries, Division Notification No. 54-Chiku-A-5002,
54-Suishin-3381 issued on February 4, 1980)
Note that substances that have been designated as a food additive cannot be used as
feed additives unless such a use has been designated by the Minister of Agriculture,
Forestry and Fisheries in accordance with Article 2, Paragraph 3 of the Feed Safety
Act and Article 1 of the Ordinance for Enforcement of the Feed Safety Act (refer to theabove listed laws, etc.).
[Reference 1] Glossary
Explanations of the technical terminologies are provided as follows.
Source: “Glossary concerning Food Safety” posted on the website of the Food Safety
Commission (http://www.fsc.go.jp)“Guideline for Total Diet Studies” posted on the website of the Ministry ofAgriculture, Forestry and Fisheries (http://www.maff.go.jp)
 Safety factor
A factor applied to the no-observed-adverse-effect-level (NOAEL), for thedetermination of the Acceptable Daily Intake (ADI), etc., of a substance from theperspective of further safety considerations. The ADI is given as a quotient of the
NOAEL/Safety factor. A safety factor is determined in consideration of the species
differences between animals and humans, and the differences in individual
humans, and is given as a product of the species difference and the individual
difference. Typically, a factor of 10 times for the species difference and the same
10 times for the individual difference is applied; thereby, the product of thosevalues (100) is used as the safety factor.
 Acceptable daily intake (ADI)

4

The estimated amount of a substance per day that a human can take every day
during her/his life span without incurring any supposed adverse health impacts
 General toxicity
Toxicity which can be assessed by general test methods (blood tests,
histopathological tests, etc.) in acute toxicity tests and chronic toxicity tests
 In vivo test
A test conducted in living organisms, typically meaning animal tests.
 In vitro test
A test conducted in test tubes
 Ames test
A test using salmonella to assess the incidence of gene mutations caused by a
substance Acute toxicity
Toxicity that emerges in a short period of time (from the same day, to within about
two weeks) after a single dose of a substance or multiple doses have been
administered in a short period
 Acute toxicity test
A test to assess the signs of acute toxicity by administering a substance to animals
 Limit of detection (LOD)
The minimum detectable concentration of a substance in a method used for the
analysis of the substance. There are several definitions of the LOD, including thefollowing:
i. AOAC (Association of Official Analytical Chemists) International: aconcentration equivalent to the sum of the mean blank value and 3σ (wherein σ
is a standard deviation of the distribution of a blank measurement)
ii. IUPAC (International Union of Pure and Applied Chemistry): a concentrationequivalent to the sum of the mean blank value and kσ (wherein k is a factor
determined based on the reliability requirement, and σ is a standard deviation of
the distribution of a blank measurement)
It must be clearly expressed in the test report which definition is being used forthe calculation. Note that the formula “LOD = LOQ” is scientifically false.
 Maximum residue limit (MRL)5

The maximum permissible concentration of agricultural chemicals, feed additives,
etc., remaining in food.
 Good Laboratory Practice (GLP)
A set of principles and rules to be complied with in conducting tests concerning thesafety and persistence of feed additives, formulated to achieve a higher reliabilityof the documents, and a more accurate and strict performance of the safetyassessment. (For details, please refer to the Reference 6 “Conditions to beComplied with in Conducting Animal Tests” on page 28).
 Micronucleus test
One form of mutagenicity tests, to assess the chromosome aberrations byexamining the emergence of a micronucleus (cellular fragment) In vivo kinetics test
A test to analyze the kinetics of the substance in the body of the animals (itsabsorption, distribution, metabolism, excretion, etc.) by administering thesubstance to animals
 Transgenerational reproductive test (Transgenerational reproductive toxicity
test)
A test to assess the reproductive toxicity of a substance by administering it toanimals Single dose toxicity test
A test that administers only a single dose of a substance to animals
 Limit of quantitation (LOQ)
The minimum concentration of a substance that can be quantitated by the method
used for the analysis of the substance with an appropriate accuracy and precision.
There are several definitions of the LOQ, including the following:
i. AOAC International: a concentration equivalent to the sum of the mean blankvalue and 10σ (wherein σ is a standard deviation of the distribution of a blankmeasurement)
ii. Codex (a definition in the criteria approach): a concentration equivalent to thesum of the mean blank value and 6σ, or the sum of the mean blank value and10σ (wherein σ is a standard deviation of the distribution of a blankmeasurement)
 Spike recovery test

6

A test to spike a predetermined amount of an objected substance to analyze andexamine whether the spiked amount can be accurately quantitated Special toxicity
Toxicity which is assessed by a special administration method (inhalation,transdermal delivery, etc.) and with special observation items (mutagenicity,
carcinogenicity, etc.)
 Carcinogenicity
Toxicity that causes cancers inside living organisms and can facilitate cancer
progression by the intake of a substance Developmental toxicity (Teratogenicity)
Toxicity that causes an impact on a fetus by way of the mother’s ingestion of thesubstance
 Developmental toxicity test (Teratogenicity test)
A test to assess the developmental toxicity (teratogenicity) of a substance byadministering the substance to animals. The doses are administered during theorganogenesis stage.
 Reproductive toxicity (Reproduction toxicity)
Toxicity that causes damage to the reproductive potential, embryo and fetus
 Lethal dose 50 (LD50)
An index of acute toxicity, where the amount of the substance is estimated to kill
statistically 50% of the animals exposed to the substance Repeated dose toxicity test
A test that administers repeated doses to animals
 Repeatability
Errors between the values of measurements, each of which is obtained from themeasurement of the samples which are identified as the same, conducted by thesame method and by the same experimenter, and under conditions that arecontrolled to produce independent results within a short period of time Mutagenicity (Genotoxicity)7

Toxicity that affects genes and chromosomes and causes mutations and damage
to genes and chromosomal aberrations. These aberrations can result in
carcinogenicity.
 Mutagenicity test (Genotoxicity test)
A test to assess the mutations and damage to genes and chromosomal
aberrations
 Positive list system
A system to ban the sale, etc., of a food in principle, in which the residue of feed
additives (and other agricultural chemicals, veterinary medicinal products, etc.) arefound to be in excess of the predetermined level (0.01 ppm). As to a substance forwhich the standard of a food ingredient is prescribed, the criteria specified in thatstandard will apply. Otherwise, the value of 0.01 ppm (a concentration causing nopotential harm to human health) is applied as the regulatory value for any
substances which are out of the scope of any specific standards.
 Chronic toxicity
Toxicity that is caused by the continuous or repeated administration of a substanceover an extended period of time (typically 6 months or more) Chronic toxicity test
Test to assess the signs of chronic toxicity by the administration of a substance toanimals
 No-observed-effect-level (NOEL)
The maximum dose of a substance at which no biological impact is found No-observed-adverse-effect-level (NOAEL)The maximum dose of a substance at which no adverse impact is found Pharmacological test
A test to clarify the actions of a substance that has been administered to livingorganisms
 Lot
A set of foods which are regarded as having been produced under the same
conditions based on the characteristics of the place of origin, place of production,variety, packer, packing form, label, consignee, etc., within a group of traded
goods in a single delivery

8

II Basic Ideas in the Preparation of the Required Documents
1. Flow of the Procedure for a Feed Additive Designation
The flow of the procedure for a new feed additive designation is illustrated below. The
designation is made through the deliberations by the Agricultural Materials Council(Ministry of Agriculture, Forestry and Fisheries), the Ministry of Health, Labour andWelfare and the Food Safety Commission.
Procedure for a feed additive designation⑦

Feed additive business operator (Submitter)
Sample provision
Submitting documents

Reporting analysis result

⑧
⑥

FAMIC

④

Deliberation at Feed
Safety Committee ofAgricultural Materials
Council(Feed additive, Efficacy and Safety)(Feed additive, Standard)

Article 59 ofFeed SafetyAct①
Notice ofDeliberation Council⑥⑥

Request for theassessment on
impact to human healththrough foodHearing on thefeed additive
designation, etc.

Ministry of Health,Labour and Welfare

Request for theassessment on
impact to human healththrough food

Article 24 of Food
Safety Basic Act

Food Safety Commission(Deliberation at Fertilizer, Feed
Additive, etc. Expert Panel)(Deliberation at Pharmaceutical
Affairs and Food Sanitation Council)

Notice ofDeliberation
Result

Notice ofDeliberation
ResultReply on the feed additive
designation, etc.

⑩

Re-deliberation
when needed

Article 2, 3 ofFeed Safety
Act

Notice of application invitation

Consult about theefficacy and safety
to livestock

Findings

Article 2, 3 ofFeed Safety
Act

JSFA

Ministry of Agriculture, Forestry and Fisheries

Request foranalysis

⑤
⑨

③

②

⑩

Ministry of Agriculture, Forestry and Fisheries

Deliberation at the Feed Subcommitteeof Agricultural Materials Council⑪⑫ Report⑬

Ministry of Agriculture,
Forestry and Fisheries⑭

Public Comment and Report to WTO/SPS

9

Feed Additive Designation(Announcement on
Government Gazette)

A submitter first needs to prepare the documents required for the deliberations by the
Agricultural Materials Council, etc. Refer to the Section “II-2 Outline of the Required
Documents” on page 15 and the following for details of the required documents. The
flow of the procedure from the preparation of the required documents to thedeliberation council is as follow:〈Preliminary consultation〉
It is essential for a business operator who is planning to start manufacturing orimporting any non-designated feed additives to consult with the authorities
thoroughly beforehand. To receive a consultation, a document that outlines theobjected substance, including descriptions of what livestock are involved andwhich of the three effects provided for in the Feed Safety Act is expected, shall be
prepared, at a minimum.
↓〈Preparation of the required documents〉
While the consultation with the authorities is available anytime, any substances
other than those whose applications have been received within the application
acceptance period for the deliberation council won’t be subjected to thediscussions concerning the application’s eligibility for a deliberation council.
↓〈Application acceptance period for the deliberation council〉 The application shallbe submitted to the authorities
The authorities will invite the application for a deliberate council through the Japan
Scientific Feed Association (JSFA). Please make sure that all required documents
have been properly prepared before submitting the application to the authorities.
Note that the deliberation will not be conducted for the following cases:
・The required documents have deficiencies
・An application contains many subjects pertaining to matters of re-deliberationfrom previous deliberations.
・The objected substance does not fulfill the requirements for a feed additive
designation, etc.
↓
When the authorities decide to deliberate in the deliberation council〈Period between the acceptance of the application and before the deliberation
council〉
The submitted documents shall be scrutinized. The copies of the documents forthe deliberation council (about 20 sets) will have to be prepared before thedeliberation.
↓〈Deliberation council at the Feed Safety Committee of the Agricultural Materials

10Commission (efficacy and safety)〉
Explanation (within 10 minutes), and questions and answers (about 10 minutes) inthe deliberation council
With regard to a new substance, (i) the efficacy and safety of the feed additive will
be deliberated by the Feed Safety Committee of the Agricultural Materials
Commission.*↓〈Deliberation Council of the Feed Safety Committee of the Agricultural Materials
Commission (standards)〉
After the efficacy and safety of the objected substance have been endorsed by
the Feed Safety Committee, (ii) the standards of the feed additive will bedeliberated in the deliberation council of the same committee at a later date.**The submitter will have to prepare responses to the findings of the council
members if questions are raised about the contents of the documents in the
deliberation council, and the subject matter will be included in the continuous
deliberation. When the responses are ready, the deliberation will be conductedagain.
After the endorsement of the substance by the Feed Safety Committee, discussions
on the residue limit in foods and human dietary risk assessments will be conducted by
the Ministry of Health, Labour and Welfare (Pharmaceutical Affairs and Food
Sanitation Council) and the Food Safety Commission (Fertilizer, Feed, etc., Expert
Panel). The Ministry of Health, Labour and Welfare and the Food Safety Commission
will also refer to the documents prepared by the submitter in their deliberations.
Although the submitted documents won’t be made public as they are, the submitter is
asked to black out any portions containing confidential information beforehand when
the assessment reports are published, because the assessment reports, etc. drawn up
by the deliberation council, etc., may contain information that is relevant to patents andthe submitter’s secrets.
Ministry of Agriculture, Forestry and Fisheries (Agricultural Materials Council)
When the Minister of Agriculture, Forestry and Fisheries makes a decision on the feed
additive designation and thereby makes a change in the specifications and standards,
etc., in accordance with the Feed Safety Act, the opinions of the Agricultural Materials
Council shall be heard. After the deliberations of the Feed Safety Committee of theAgricultural Materials Council (Feed Additives: Efficacy and Safety) and (Feed
Additives: Standards), the feed additive documents will be sent to the discussion at theFeed Subcommittee.

11

The deliberations of the council will be
conducted based on the submitted
documents prepared by the submitter.
The council members will discuss in ascientific manner whether the efficacy
and safety of the objected substance areassured in their practical use as a feed
additive, and to what extent such
efficacy and safety is assured, etc. With
regard to the standards, the adequacy ofthe quantitative methods, etc., that havebeen employed will be discussed.

Agricultural Materials Council
Feed Subcommittee
Feed Safety Committee:
Feed Additives (Efficacy and Safety)
Feed Additives (Standards)
Livestock feed
Pisciculture feed
Pet animal feed
Genetically modified feed committee
Feed nutrition committee
Livestock feed
Pisciculture feed

Based on the submitted documents, the authorities will scrutinize whether the objected
substance is eligible for deliberations in light of the evaluation standard of the feed
additive and whether the contents of the submitted documents have fulfilled therequirements.
Furthermore, if the objected substance is a substance (enzyme, etc.) made fromrecombinant microorganisms (bacteria, etc.), it must be subjected to further
deliberations regarding genetically modified feed additives (at a genetically modified
feed committee) in addition to the normal deliberations (on the efficacy and safety,
standards, etc.).
Feed Subcommittee
This subcommittee will consolidate the deliberation results of the Feed Safety
Committee (Feed Additives: Efficacy and Safety, as well as Standards), will hold ahearing in public and will issue a report.
Feed Safety Committee: Feed Additives (Efficacy and Safety)
This committee will conduct closed-door deliberations regarding the efficacy andsafety to livestock of the feed additive.
Feed Safety Committee: Feed Additives (Standards)
This committee will conduct closed-door deliberations about the adequate
specifications and standards for the feed additive based on the deliberation
results of the Feed Safety Committee (Feed Additives: Efficacy and Safety).

Ministry of Health, Labour and Welfare, Pharmaceutical Affairs and Food Sanitation
Council
The Ministry of Health, Labour and Welfare sets the residue limits and the standards
for foods, etc., and oversees these in accordance with the Food Sanitation Act.
The Ministry conducts deliberations about the maximum residue limit (MRL) of thefeed additive in foods as part of the Pharmaceutical Affairs and Food Sanitation
Council, which is an advisory body to the Ministry of Health, Labour and Welfare Food
Safety, based on the results of the dietary risk assessment (ADI) provided by the Food
Safety Commission. When necessary, the Ministry may set the MRL of the feed
additive in foods.
Food Safety Commission (Fertilizers, Feed, etc., Expert Panel)
The Food Safety Commission was established on July 1, 2003 in accordance with theFood Safety Basic Act. It conducts risk assessments regarding the impact ofhazardous elements, such as additives and agricultural chemicals, etc., which may becontained in foods, on human health based on the scientific knowledge and from an
objective, neutral and fair stand point (based on dietary risk assessment: settings of
the ADI, etc.).
Particularly, subjects such as what intake amount of a hazardous element can cause aserious adverse impact on human health and to what degree of probability this will
occur, are scientifically assessed. The dietary risk assessment is a requisite for thedesignation of new feed additives, as well as changes of standards for labeling andmanufacturing, etc., and changes in the standards of purity, etc.
Incorporated Administrative Agency, the Food and Agricultural Materials Inspection
Center (FAMIC)
The FAMIC assures the safety of consumers and foods, as well as fertilizers and feeds,
registers agricultural chemicals and oversees their safety, and oversees the labeling offood products. In terms of the feed additive designation, the FAMIC examines themethods of analysis based on the submitted documents. More specifically, it
determines whether the methods of analysis have been adequately described in theitems relating to the standards of the feed additive. In addition, it may conduct aninspection of the facility to determine whether it complies with the principles of Good
Laboratory Practice (GLP) on an as needed basis. (Refer to Reference 6 “Conditions
to be Complied with in Conducting Animal Tests” on page 29 for information about
GLP inspections.)
General Incorporated Association, the Japan Scientific Feed Association (JSFA)The JSFA is an entity which conducts experiments and research for the manufacturing

13

and supply of “safe and high quality feed” through the rational and economical
utilization of scientific feeds, and by doing so it aims to promote the development oftechnologies and the broader use of such technologies. It is organized by themembers of companies, etc., which manufacture feeds and feed additives.
The announcement of the invitation of an application by the deliberation council ismade by the authorities through the JSFA.

14

2. Outline of the Required Documents(1) The test items required for a feed additive designation are specified in the “Standard
for the Evaluation of Feed Additives.”*1 In addition, the proof of an appropriate test
performance is required from the facility used for the toxicity tests and residue
tests.*2(2) Once the test results are obtained, an abstract will be drawn up by filling out thedesignated forms with the summary of the test results.*3 The following documents
shall be submitted:
i. Abstract
ii. Original papers included in the abstractiii. Checklist*
Note that if any findings are pointed out by the council members in the deliberation, theresponses to the findings shall be presented in the following deliberation. The required
documents for the following deliberation are as follows:i. Summary of the response paper to the findings
ii. Original papers included in the response paper to the findingsiii. Revised abstract based on the findings
iv. Original papers included in the abstract
v. Checklist*
Note 1: Notification of the Establishment of the Standard for the Evaluation of Feed Additives(Ministry of Agriculture, Forestry and Fisheries, Fisheries Agency Director-General
Notice No. 4-Chiku-A-201 issued on March 16, 1992)
http://www.famic.go.jp/ffis/feed/tuti/4_201.html
Note 2: Notification of the Standard Concerning the Performance of Animal Tests for Feed
Additive Assessments (Ministry of Agriculture, Forestry and Fisheries, Fisheries
Agency Director-General Notification No. 63-Chiku-A-3039 issued on July 29, 1988)
http://www.famic.go.jp/ffis/feed/tuti/1_3039.html
Note 3: Notification Concerning the Documents, etc., Required for a Feed Additive Designation(Ministry of Agriculture, Forestry and Fisheries, Division Notification No.
54-Chiku-A-5002)
http://www.famic.go.jp/ffis/feed/tuti/54_a5002.html

In order to facilitate the smooth deliberation of the council, the authorities will check thesubmitted documents before holding the deliberation council, and will confirm whether
the items are described in conformity with the Standard for the Evaluation of Feed
Additives and are objectively described based on the test results. Insufficientdescriptions in the submitted documents will cause an extended time for the document
checks by the authorities. In addition, the submitters may be requested to revise thedocuments. Please be advised that such circumstances may result in a delay of thedeliberation by the council and of the feed additive designation.
*Although the submission of the checklist attached at the end of this handbook (VIChecklist for the Submission of the Required Documents) on page 76 and following is
not mandated by the notification or an equivalent, the submitters are kindly asked tosubmit this checklist in order to shorten the time for the document checks by theauthorities.
In the following chapter, the procedures for the preparation of the required documents15

(especially the abstract) are explained with reference to examples.

III Items to be Described in the Required Document (Abstract)
This chapter provides concrete explanations about the items to be described in the
abstract, with reference to the “Notification Concerning the “Documents, etc., Required
for the Feed Additive Designation” and the “Notification of the Establishment of theStandard for the Evaluation of Feed Additives”. In this handbook, a substance for which
the feed additive designation is being sought is referred to as an “objected substance”
for purposes of convenience. Before entering this chapter, please be advised that the“Requirements” only summarize the main items to be described, and the “Example”
merely represents one of many examples.
ii. In vivo test(2) Field application tests to prove
efficacy
4. Items Concerning Residue
Residue tests in targeted animals, etc.
5. Items Concerning Safety(1) Toxicity tests
i. General toxicity tests
a. Single dose toxicity test
b. Repeated dose toxicity test
(Short term)c. Repeated dose toxicity test
(Long term)ii. Special toxicity tests
a. Transgenerational reproductive
test
b. Developmental toxicity testc. Carcinogenicity test
d. Mutagenicity teste. Other tests
iii. Pharmacological tests
iv. In vivo kinetics tests(2) Feeding tests using targeted animals,etc.
(3) Tests concerning the emergence ofresistant bacteria(4) Other tests

1. Origin or Background of the Discovery,
status of Authorization and Use as a Feed
Additive in Foreign Countries, etc.
2. Items Concerning Standards(1) Names
i. General nameii. Chemical nameiii. Trade name(2) Chemical structure(3) Manufacturing process(4) Biological and physicochemical
properties
i. Physical and chemical properties
ii. Identification testiii. Purity test
iv. Content and quantitative method(5) Quantitation in feed(6) Changes with timei. Ambient temperature storage test
ii. Heat resistance testiii. Humidity resistance test
iv. Light resistance testv. Accelerated test
vi. In-feed stability test
3. Items Concerning Efficacy(1) Basic tests to prove efficacy
i. In vitro test

[Reference 2] Considerations for descriptions of the test data (omission oftests, use of new knowledge, etc.)
The notification of the “Establishment of the Standard for the Evaluation of Feed
Additives” states the following: items concerning the safety of a substance that isdesignated as a food additive, or is widely used in foods, can be omitted; also, someof the toxicity tests can be omitted if the conditions are met. However, the reasons
and adequacy of an omission must to be presented. Please bear in mind that reports
and literature to support the reasons and adequacy must be submitted to explain theadequacy of the omission
In addition, with regard to substances already used as food additives or foods, if new
knowledge about the substance (such as toxicity data for a newly found metabolite)

16

have been found at the time of seeking the feed additive designation, which was notknown at the time of the substance’s designation as a food additive, the data
concerning the safety must still be described. Therefore, please always try to collect
the latest available knowledge.

1. Origin or Background of the Discovery, Status of Authorization and
Use as a Feed Additive in Foreign Countries, etc.
[Requirements]○ Origin or background of the discovery: Describe the origin or background of the discoveryof the objected substance that has resulted in the idea to use it as a feed additive.
Describe what advantages it will bring about and how the current conditions will improvewhen the feed additive designation is granted to the objected substance. Also, write
about the following things: (1) Which of the three effects provided for in the Feed Safety
Act is achieved; (2) Which livestock animals are targeted and at which administration
stage (period of the dose); (3) What is the recommended dose (method of use).
○ Status of authorization and use as a feed additive in foreign countries, etc.：If the objected
substance is used as a feed additive in Europe and America, etc., describe the reasonsfor its authorization (targeted animals, doses, etc.). (see the [Example] below)○ Status of the manufacturing and distribution authorization, and its importation as aveterinary medicinal product：If the objected substance is used for any purpose, including
but not limited to its use as a veterinary medicinal product, describe the substance’s
intended use, method of use and dose, etc.
○ Comparison to related substances (generic substances having the same effect)：Write
about the substances that are already designated as a feed additive and have similar
attributes as the objected substance, such as having the same structure, same effect,
etc. (see the [Example] below)[Example]
The status of the substance as a feed additive in foreign countries is summarized in thetable below.
Table 1 Status of the substance’s designation in foreign countries, etc.
Country
United States of America
EU

Status of studies and authorization
Designated in xxxx
Pending (submitted in xxxx)(omitted)

Target livestock animals
Pigs and cattle
Pigs (planned)[Example]
Related substance:
Weigh out 0.01 g (0.005–0.014g) of the substance and dissolve it in 1.0 mL of methanol tomake a solution. Measure 10µL of the solution and spot it onto a thin layer of achromatography silica gel plate. Then, spread out the spots into a span of about 10 cm
using the mixture of ether and chloroform (50:50) as a spreading solvent, and dry the platein the air. … (omitted)…
[Reference 3] Case of a Discussed/Authorized Substance in Foreign Countries
as an Additive
The documents used in the assessment for the substance’s authorization as anadditive in foreign countries may substitute for the documents required for the17

deliberation of its designation in Japan, depending on the test items. When using
these documents, submitters are often requested to also provide information about
the status of the discussion/authorization (date of the designation, standards, etc.) in
foreign countries, as well as the status of the standard setting and assessment atinternational institutes, such as the Codex. Therefore, review such information
thoroughly and be prepared for such requests. Note that because the deliberation isconducted according to the Standard for the Evaluation of Feed Additives of Japan,
the documents used for the substance’s designation in foreign countries may not
suffice.

18

2. Items Concerning Standards
To ensure the safety of livestock and humans, any hazardous impurities which can becontained in feed additives and the purity of the product must be specified.
The specifications and standards specified in the Ministerial Ordinance on theSpecifications and Standards of Feeds and Feed Additives shall be described in line withthe writing style and definition of the terminology set out in the Appendix Table 2-1 “Generalrules for feed additives” attached to the above-mentioned ordinance. Therefore, pleasedraw up the required documents according to the rules set out in the general rules.
([Reference 8] Style Reference for the Description of Items Concerning Standards, on page
53 and following.)(1) Namei. General name
Describe the general name as it is used in the feed additive listing in the Ministry ordinance.
ii. Chemical name
The IUPAC name shall be described.
iii. Trade name
The name in foreign countries shall be described, if the substance is sold overseas, etc.
(2) Chemical structure
The chemical formula shall be described for an organic compound. (If a substance has achemical structure which cannot be expressed, such as an enzyme, it is allowable to omit
the description of the formula with a note.) In addition, the molecular formula shall be
described. For the calculation of the molecular weight, the Table of Standard Atomic
Weights 2007 shall be referred to.
(3) Manufacturing process
The manufacturing process of the ingredients used for the manufacturing and formulations
shall be described in enough detail including the following items, so that the detailed
manufacturing process can be replicated: test laboratory, place where the test was
conducted, manager responsible for the test, test method, lot number and manufactured
date. A detailed flowchart of the manufacturing process shall also be attached so that the
production of by-products as well as the purity of the ingredients for manufacturing, etc. canbe understood. The use of a recombinant technique or production bacteria in the
manufacturing process shall be noted with detailed descriptions.
[Example]
Oxidize Substance A (99.5%) with air using a platinum catalyst to produce Substance B.
Initiate hydrolysis by adding sodium hydroxide in the special grade ethanol to produce
unrefined xxxx. Refine this through a solvent extraction using hexane, then dehydrate toyield the objected substance xxxx.
Manufacturing
Process
Flowchart

Substance A (99.5％)↓ platinum catalyst
Substance B↓ sodium hydroxide↓(special grade ethanol)
19xxxx(unrefined), (by-product C)↓ solvent extraction by hexane
objected substance xxxx

[Reference 4] Considerations for the Description of the Manufacturing Process
Information about the by-products derived from the ingredient for manufacturing, or
produced by mixing with diluents, is essential because the ingestion of certain
by-products produced in the manufacturing process may affect the health of the
livestock.
(4) Biological and physicochemical properties
i. Physical and chemical properties
Information about the objected substance shall be described.
Describe the appearance and physical and chemical properties of the objected substance.
・ Appearance of the objected substance itself (color; state: powder, liquid, etc.)
・ Solubility in a solvent (degree of dissolution in an organic solvent or water)
・ Information about the denaturation of the objected substance (deliquesce, melting
point,
photo-degradation, etc.)[Example]
Physical and chemical properties
a. Appearance: colorless or white crystal, or white crystalline powder
b. Chemical properties: freely soluble in water, soluble in methanol, or practicallyinsoluble in hexane… (omitted) …
ii. Identification test
A test to identify the objected substance in the analyte by conducting tests based on its
specific properties.
For a new objected substance, the possibility of employing previously prescribed methods(methods described in the “List of the Specifications and Standards of Feed Additives”)
shall be discussed.
[Example]
Identification test
This substance shows an IR absorption peak at near 1,750 cm-1 of a wavenumber in theinfrared absorption spectrum by the potassium bromide tablet method of analysis. The
aqueous solution of this substance (1→10) shows a quantitative reaction with xx salt.
… (omitted) …
iii. Purity test
A test to detect impurities contained in the analyte other than the objected substanceFor the test to detect other items (lead, arsenic, etc.) for which standards have been
specified in relation to other substances, the possibility of employing previously prescribedmethods shall be discussed.
[Example]

Purity test
a. Clarity and color of a solution: An aqueous solution prepared with 1.0 g (0.95–1.04 g)of this substance dissolved in 20 mL of water shows a light tan color and is practically20

clear.
b. Chloride limit: When conducting a chloride limit test with 1.0 g (0.95–1.04 g) of thissubstance, the turbidity of the test solution should not exceed that of the control
solution prepared with 0.5 mL of 0.01 mol/L hydrochloric acid.
… (omitted) …
iv. Content and quantitative method
A method to quantitate the objected substance contained in the analyte[Example]

Content: This substance contains 99.5% or more of the objected substance (describe thechemical formula of the objected substance) in the quantitation after drying.
Quantitative method: Dry the substance, weigh out 0.5 g of the material, dry down to 0.001
g and record the reading. Dissolve the weighed substance in 50 mL of water, and add 5 mLof formalin and titrate with 0.5 mol/L sodium hydroxide solution (use three drops of thephenolphthalein indicator). Perform a blank test with the same method for correction.
1 mL of 0.5 mol/L sodium hydroxide solution = equivalent weight in mg, chemical formula ofthe objected substance… (omitted) …
(5) Quantitation in feeds
The information about the quantitation limit and the result of the spike recovery test shall be
provided so that the accuracy of the analysis can be demonstrated. The identification
method (method of analysis) is not prescribed for the quantitation in feed, as it is not amatter specified in the composition standard. However, from the standpoint of efficacy andsafety, the quantitation in feed is an important matter in order for feed manufacturers topractice quality control of the proper additive amount when the additive amount needs to becontrolled.
[Method to prove the adequacy of the quantitative method]
The following method is intended for the instrumental analysis method.
○Selectivity: Process a sample that does not contain the objected substance (thesubstance intended for analysis) as the blank sample, to confirm that there is no peakthat will disturb the quantitative method. A compound feed to which the objected
substance is planned to be added, the main basic ingredients and the feed additives
included in the same compound feed are studied as the blank sample. The test datasuch as a chromatograph, obtained in the studies, shall be attached to the test reportbecause it is important to determine the adequacy of the analysis method.
○Accuracy and repeatability: Perform the spike recovery test and calculate the mean
recovery rate and the relative standard deviation in order to obtain the accuracy andrepeatability. Note that the spike recovery test shall be performed in accordance withthe following conditions, in principle:･Sample: three kinds of compound feeds (choose those that correspond to the speciesof the targeted
livestock for the objected substance)･Additive concentration: arrange at minimum two levels of concentration based on the
additive concentration of the objected substance in the feed (the recommended
additive
concentration).
･Number of repeats: three times

21

○Limit of the quantitation: Perform the spike recovery test with the sample that has
one-tenth or less of the minimum of the objected substance concentration in the feed(recommended additive concentration), then calculate the mean recovery rate andrelative standard deviation to obtain the accuracy and repeatability.
(6) Changes with time
Tests are needed to assess the stability of the objected substance based on the conditionsof its actual use and handling. The changes in the properties of the objected substance(formulation) under severe conditions, as well as in the ambient temperature, shall betested. From the results of this test, the suitable storage conditions shall be discussed.
[Requirements]
The test results of the ingredient for its manufacturing and formulation shall be described
respectively. The information about the ingredients, etc., tests, and other things shall be
described, including at least the following:○Test conditions, place of storage (temperature, humidity, container, etc.): Describe which
testing laboratory in Japan or overseas was used, and what storage conditions were set
for the tests.
○Test period: Conduct the tests for the corresponding period and apply this to each test
item.
○Information about the test samples (purity, lot number, etc.): The lot number shall bealways recorded, as the purity may vary greatly depending on the lot. Tests shall be
performed with samples from different lots.
○Method of analysis for the quantitation of the content: The method of analysis for thequantitation of the content shall be described, because the substance may decompose
with time.
○Test results: The tests results shall be described in a properly arranged manner and using
tables, as there are many test items.
i. Storage test in an ambient temperature
The actual storage shall be conducted in an ambient temperature (1–30℃) to assess thestability of the substance under normal conditions.
Storage tests shall be conducted using samples from at least three different lots of theingredient, for manufacturing and for the formulation respectively. Put an appropriate
amount of the objected substance in the packaging containers that are regularly used andstore these in an indoor warehouse for each of the following periods, to assess the stability
over the corresponding storage period: 0, 3, 6, 9, 12, 18 and 24 months (these test periods
can be extended or shortened depending on the specifications of the shelf lives or the
expiration date). Examine any quality deteriorations of the substance as a feed additive.
Note that the size of the packaging container used for tests can be scaled-down as needed.
[Example] (A case where the objected substance is a white powder)
Table 2 Storage tests in an ambient temperature
Test conditions: air temperature 35℃; humidity 40% (for 24 months); in a 20 kg paper bag
Lot No.

Parameter
AppearanceA

Identification test
Purity test

At the start

3 mos.

6 mos.

9 mos.

12 mos.

24 mos.

white
powder
fit
fit

white
powder
fit
fit

white
powder
fit
fit

white
powder
fit
fit

white
powderfit
fit

white mud

22

1

fit
fit

Amount of active
ingredient (g)Loss on drying (g)Amount ofmoisture (g)B

Appearance

214.6

212.1

210.4

208.3

205.4

207.9

5.2
32.3

4.9
30.0

3.7
29.4

5.0
35.1

6.3
33.8

4.7
35.3

white
powderwhite
white
powderpowder(omitted)

white
powderwhite
powderwhite
powder

Note 1: “White mud” indicates a white paste condition.
Although the appearance of the substance turned to a white mud (paste condition) after 24months, it passed both the identification test and the purity test. In addition, no significant
changes in the amount of the active ingredient were found. No other problems were found;thus, it can be concluded that the substance remains stable for at least 24 months.
ii. Heat resistance test
The outdoor air temperature rises, especially in the summer season. The indoor air
temperature may also become high. Therefore, test the stability of the objected substancein such high temperatures, and ensure that no adverse changes are caused in its quality asa feed additive.
Storage tests shall be conducted with samples from at least three different lots of each of
the ingredients for the manufacturing and the formulation. Put an appropriate amount of theobjected substances in a sealed glass container or a tight container and keep them at 40℃
for each of the following periods to assess their stability over the corresponding period: 0, 1,
2, 3 and 6 months (these test periods can be extended or shortened depending on theconditions or the physical properties under which the objected substance will be used as afeed additive).
iii. Humidity resistance test
The reaction of the objected substance to humidity (moisture) shall be tested, in addition to
the reaction to temperature, to ensure its stability and thereby no adverse changes in its
quality as a feed additive.
Storage tests shall be conducted with samples from at least three different lots of each of
the ingredients for the manufacturing and the formulation. Put an appropriate amount of theobjected substances in separate Petri dishes and keep them at a constant temperature
anywhere between 25–30 ℃ , with two or more levels of relative humidity at thattemperature and without the lid on the Petri dishes, for each of the following periods toassess their stability over the corresponding period: 0, 1, 2, 3 and 6 months (these test
periods can be extended or shortened depending on the conditions or the physicalproperties under which the objected substance will be used as a feed additive). With regard
to the level of relative humidity, more than two levels of humidity are set within the range inwhich the substance shows no apparent quality deterioration, such as changes in its
appearance, growth of mold, degradation, deliquescence, consolidation, etc., in the
preliminary test, and one of those levels will be determined to be near the upper-limit of thesaid range.
iv. Light resistance test
The reaction of the objected substance to light shall be tested. In cases where excess
reactions against light are found, light shielded storage shall be arranged for quality
assurance.
Storage tests shall be conducted with samples from at least three different lots of each of
the ingredients for the manufacturing and the formulation. Put an appropriate amount of the23

objected substances in separate Petri dishes, cover the Petri dishes with lids and seal the
joint parts with tape or paraffin, then expose them to 500-lux fluorescent light at an ambient
temperature (1–30℃) for each of the following periods to assess their stability over thecorresponding period: 0, 1, 2, 3 and 6 months (these test periods can be extended or
shortened depending on the conditions or the physical properties under which the objected
substance will be used as a feed additive). After each period of time, check if any adversechanges have been caused in its quality as a feed additive.
[Example] (A case where the objected substance is a white powder)
Table 3 Light resistance test
Test conditions：air temperature 25℃; humidity 50% (for 6 months); kept under a 500-lux
fluorescent light
Lot No.
A

B

Parameter
Appearance
Identification test
Purity test
Amount of active
ingredient (g)Appearance

At the start
white powder
fit
fit
210.6

1 mo.
white powder
fit
fit
205.2

2 mos.
white powder
fit
fit
204.6

3 mos.
white powder
fit
fit
202.3

6 mos.
white powder
fit
fit
200.4

white powder

white powder

white powder

white powder

white powder(omitted)

The test results are summarized in the table above. The amount of the active ingredientdecreased after 6 months of the substance’s exposure to the light. According to the paperby xx et al. (xxxx), the amount of the active ingredient will decrease to below the limit ofquantitation after 12 months. Based on the above results and information, it can be said
that the amount of the active ingredient does not decrease following up to three months of
exposure to the light, and thereby the substance is stable. Nonetheless, we have specified
the use of light-shielded storage in the standards.
v. Accelerated test
The deterioration of a substance may be accelerated under conditions of high temperatureand high humidity (especially in the summer season). Therefore, the stability of theobjected substance under such severe conditions (in principle, 40℃ air temperature with a
75％ relative humidity) shall be tested. After the test period, check if any adverse changes
have been caused in its quality as a feed additive.
Storage tests shall be conducted on samples from at least three different lots of each of theingredients for the manufacturing and the formulation. Put an appropriate amount of theobjected substances in the packaging containers that are regularly used to store them at
40℃ with a 75％ relative humidity, in principle, as well as in an indoor warehouse (ambient
temperature) for each of the following periods to assess their stability over thecorresponding storage period: 0, 1, 3 and 6 months (these test periods can be extended or
shortened depending on the conditions or the physical properties under which the objected
substance will be used as a feed additive). Note that the size of the packaging container
used for the tests can be scaled-down as needed.
vi. In-feed stability test
Feed additives are added to feeds for their use. Therefore, the stability of the feed additives
in feeds is essential. Any possible interactions between the feed additive and the feed, orwith other feed additives, shall be tested.
The objected substance (formulation) is added to at least three kinds of typically24manufactured feeds at the regularly applied concentration. Put an appropriate amount of
the samples of the feeds with the objected substance in the regularly used packaging
container and store them in the indoor warehouse for each of the following periods toassess their stability over the corresponding storage period: 0, 0.5, 1, 2 and 3 months (and6 months when needed). The method used for the quantitation of the objected substance inthe feeds shall meet the following three conditions, in principle:
a. The mean recovery rate is 90% and higher. Repeatability (the value of the standard
deviation plus the error of repeated tests in a laboratory plus the error of interlaboratorybias) is 0.1 or less in the coefficient of variation. Note that the recovery tests are
conducted with the sample of feeds added with the object formulation at a regularly
applied concentration in at least three laboratories, three times for each laboratory andwith a parallel implementation in two laboratories on different days, to obtain the mean
recovery rate and repeatability.
b. The limit of the quantitation shall be precise enough to quantitate the content equivalent
to one-tenth or less of the regularly applied concentration in the feeds.
c. The amount of the active ingredient can be discriminated from degradation products
and other impurities.
[Example]
Table 4 In-feed stability test
Test conditions: Put Feed I, Feed II and Feed III in separate regularly used packaging
containers (packaging paper bags) and store in an indoor warehouse (air temperature 25℃,
humidity 50%) for 3 months. (Data are the ratio to the content at the start of the test
expressed in a 100-point scale.)
SampleFeed I
Feed II

Run
1
2
Average
1

At the start
100
100
100
100

0.5 mo.
99.8
99.2
99.5
98.8

1 mo.
98.3
98.3
98.3
97.5

2 mos.
97.9
96.9
97.4
97.6

3 mos.
97.1
96.2
96.6
95.1

(omitted)

For all the tested feeds, the residue rates remained at 95% and more. It can therefore beconcluded that the objected substance is highly stable, even in feeds, for up to 3 months.
Item of measurement
All items that are planned to be specified in the composition standard shall be measured atthree different time points or more, including at the start and the end of the test. At othertime points, the amount of the active ingredient shall be measured in addition to theobservation of any abnormalities in the appearance. Furthermore, for the in-feed stability
test, ambient temperature storage test and humidity resistance, other items of
measurement may be added for each test, such as measuring the loss on drying or
moisture, as needed.
Statistical analysis of the test results
A model that is thought to be the best for characterizing the relation between the test period
and the amount of active ingredient shall be used for the analysis. A regression analysis
shall be used to calculate the 90% confidence interval for the population mean.

25

3. Items Concerning Efficacy
The following items concerning the efficacy of the objected substance shall be assessed.
(1) Efficacy as a feed additive: which of the three effects specified in the Feed Safety Act
is attained.
(2) Targeted livestock animals and the administration stage (time of feeding)(3) Most effective dose (method for using)(1) Basic tests to prove the efficacy
i. In vitro test
A test to prove, or to extrapolate the efficacy of the objected substance in a test tube. In
addition, the test aims to determine the optimum amount of the addition. A comparison withpreviously designated feed additives shall be made on an as-needed basis.
ii. In vivo test
A test to assess whether the expected efficacy can be brought about, even in the body of
livestock, using laboratory animals or targeted livestock animals, etc.
(2) Field Application tests to prove efficacy
A test to assess whether the expected efficacy (effectiveness) can be brought about under
the conditions of the actual use of the objected substance. Any livestock animals for which
the efficacy is not confirmed cannot be designated as a target animal.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals: Specify the species, age, etc. of the livestock animals for which the objected
substance was planned to be applied. Also, describe the information about the test, suchas the number of animals in a group.
○Method of administration and dose ： Describe the additive amount of the objected
substance contained in the sample feeds (mg/kg-feed), the method and period of theadministration, as well as the daily intake amount of the objected substance per unit ofbody weight consumed by the test animals (mg/kg-BW/day; calculated from the bodyweight of the test animal and the daily consumption of feed). In principle, arrange threedifferent dose groups (except for control groups) including doses that are equivalent to
the maximum and minimum of the optimum additive amount range. If there was a
non-exposure, describe this accordingly. Also, arrange a period of administration that isequal to the planned application period of the objected substance.
○Test results and observations: Describe the body weight (live weight gain), the intake
amount of feeds, feed efficiency, survival rate, etc., for each of the different dose groups
separately. The efficacy of the administration of the objected substance shall beassessed using an analysis of variance for each testing laboratory, in principle.
Consolidate the results for concluding the assessment with a statement confirming thesignificant differences. Provide a summary of the test results for each test group in the
abstract, while describing the results in each individual test animal in the original paper.
Refer to the Appendix Form 1 for details of the observations. Note that the date shall beclearly stated if blood and enteruria, etc. were sampled, or if a slaughter was conducted.

26

As a summary of the tests concerning efficacy, estimate the optimum additive amountbased on the results from the tests and describe it.
[Example] A case of an application to newly weaned piglets
A field application test to prove the efficacy was conducted at the xx laboratory in xx
prefecture. Weaned piglets reared in an indoor pig house (Strain: xx, four pens each ofmales and females; 25–30 days old; average body weight: 10.3 kg) were fed feeds
continuously with the objected substance added at the following different concentrations:20, 40, 80, 100, 150 and 200 mg/kg (each concentration corresponding to the following
daily intake amounts of the objected substance per unit of body weight: 0.4, 0.8, 1.6, 2.0,
3.0 and 4.0 mg/kg-BW/day) for four weeks, which is an intended period in the actual use.
The consumption of water and feeds by the test animals was discretionary.
[Results]
The test results are shown in the form below. (Appendix Form 1) The live weight gainsignificantly increased at the concentrations of 40 mg/kg and higher (p<0.05). The live
weight gain at 100 mg/kg did not show any difference to that at 80 mg/kg. Thus, 80 mg/kg
can be thought to be the most effective concentration, even from an economical
perspective.
Succeeding tests were conducted at a concentration of 80 mg/kg as optimum additive
amount. With regard to the intake amount of feed, this peaked at 100 mg/kg and the
proportional increase to the concentration of the objected substance was not marked. Noneof test animals died during this test.

[Reference 5] Considerations for the Description of the Test Results
The council deliberates about target animals for which the efficacy is proved, theperiod of feeding, concentrations, etc., based on the submitted documents (testdata, etc.). Therefore, test results that demonstrate the conditions in which theobjected substance produces effects shall be described in a well-organized manner.
Any livestock animals for which the efficacy of the objected substance has not been
proved cannot be designated as a target animal.
Furthermore, a daily intake amount of the objected substance per unit body weight(mg/kg-BW/day), as well as the additive concentration to the feed (mg/kg-Feed) in
the feeding tests, shall be described for easy comparisons to the toxicity tests. In
cases where body weights or the intake amount of the feeds are not known, refer to
the Japan Feeding Standard and describe the cited data clearly.

27

Appendix Form 1 Tests concerning efficacy (Livestock)
Test animal
Document
No.

Testinglaboratory,
Testing place Species
and period

xx laboratory
Fr. xx xx, xxxx xx strain
To xx xx, xxxx

Test group assignment

Test results
Average live
weight gain*3(actual no.)(%)

Dose of test
No. ofTotal no.
*1
Feeding
substance
No. ofanimals in Test groupofperiod(mg/kg-Feed repeatsa groupanimals)

8

Control
20
40

0
20
40

5

35

4
weeks

(kg)
0.38a
0.49a
0.51b
*2

100
129
134

Average feed
Average intake*3
demand rate
amount of feed
(actual no.)(actual no.)
(%)
(kg)
0.60c
0.62c
0.69d

(omitted)(omitted)

Note 1: Describe the dose of the tested live microbial agent for the live microbial agent.
2: Test data denoted by a different superscript indicates that the differences in those data are significant.
3: Describe the ratio of the data of the samples to the data of the control group in a 100-point scale as well.
4: Describe the conditions of feeding and other noticeable observations, etc.

28

1.58e
1.27f
1.35f

100
124
117

*4
Survival
Pathological Notes
rate
test findings
(%)

100
100
100

-

[Reference 6] Conditions to be Complied with in Conducting Animal Tests
The Notification of the “Standards Concerning the Performance of Animal Tests for
Feed Additives Assessment” sets out the conditions to be complied with in conducting
tests concerning the safety and persistence of feed additives. This standard wasestablished to ensure a higher reliability of the required documents, and thereby
ensure the accurate and strict performance of safety assessments by the Agricultural
Materials Council in their deliberations for the designation of feed additives and theamendment of specifications and standards, etc. The standards are intended for thefollowing tests:
General toxicity tests (single dose toxicity test, repeated dose toxicity test),
Special toxicity tests (transgenerational reproductive toxicity test,developmental toxicity test, carcinogenicity test, mutagenicity test, other tests)
Feeding tests using targeted livestock animals, etc.
Residue tests using targeted livestock animals, etc.
To prepare the required documents, the submitter must collect the data in the animal
tests conducted by him/herself at the animal test laboratories, or from the tests
contracted out to a third party. The data must be collected only for the tests that are
conducted in conformity with the provisions of this standard, or the principles of the
GLP developed by the OECD (Organization for Economic Co-operation andDevelopment), which this standard conforms to. The laboratories used for the animal
tests must be GLP compliant facilities.
Furthermore, the Ministry of Agriculture, Forestry and Fisheries, or an Incorporated
Administrative Agency, the Food and Agricultural Materials inspection Center (FAMIC)
will conduct inspections of the test laboratories used for the tests, according to the
Notification of the Establishment of Inspection Proceeding Guidelines based on theStandard Concerning the Performance of Animal Tests for Feed Additive
Assessments*1 to confirm whether the tests concerning the safety and persistence ofthe feed additive have been conducted in conformity with the GLP.
The feed additive GLP compliant laboratories which have undergone inspections
during the past three years are posted on the website of FAMIC.*2
Note 1: Notification of the Establishment of Inspection Proceeding Guidelines based on the Standard
Concerning the Performance of Animal Tests for Feed Additive Assessments (Ministry of Agriculture,
Forestry and Fisheries, Fisheries Agency Director-General Notice, Gen-chiku A No. 4 3441 issued on
January 16, 1990)
http://www.famic.go.jp/ffis/feed/tuti/1_3441.html
Note 2: List of Feed Additive GLP Compliant Laboratories
http://www.famic.go.jp/ffis/feed/sub11_glp.html

29

4. Items Concerning Residue
Residue tests using targeted livestock animal, etc. (Subject to the Feed additive GLP)
When a livestock animal ingests a chemical substance, a certain amount of the chemical
substance may remain within the animal’s body, such as in its muscles, fat and liver, andnot be discharged in the excreta. In the residue tests, the objected substances shall beadministered to livestock animals and the amounts of the objected substance that remain ineach organ, etc. of the animal shall be assessed.
Although the feed additives are ingested by livestock animals, humans may also ingest
them through livestock products if they remain in the animal’s body. For this reason, theresidue test is one of the most important tests for proving the additive’s safety to humans.
Besides the residue test, a test to analyze the kinetics of the objected substance in the
animal’s body (III-5 (1) iv. In vivo kinetics tests on page 47) is required as well. By
conducting the kinetics test prior to the residue test, it can be roughly determined whether
the objected substance has remained in the body of the livestock animal. If it is confirmedthat the objected substance ingested by the animal has been completely discharged in theexcreta and does not remain in the animal’s body through the in vivo kinetics test, theresidue test may be omitted.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals: Specify the species, age, etc. of the livestock animals for which the objected
substance was planned to be applied. Also, describe the information about the test, suchas the number of animals in a group.
○Method of administration and dose ： Describe the additive amount of the objected
substance contained in the sample feeds (mg/kg-feed), the method and period ofadministration, as well as the daily intake amount of the objected substance per unit ofbody weight consumed by the test animal (mg/kg-BW/day; calculated from the bodyweight of the test animal and the daily consumption of feed). The minimum dose for thetest shall be set as equal to the maximum level of the dose in the actual use, and thedose shall be administered several dozens of times higher than the said minimum dosein the test (except for the control group).
○Method of analysis: Sampling locations will be arranged in the edible parts (muscles, fat,liver, eggs, milk, etc.) in principle, and the sampling will be conducted so that the
distribution of the objected substance is demonstrated. Although the analysis is intended
to focus on the objected substance, an analysis of the metabolite may be needed as wellin cases where the persistence of the metabolite needs to be examined. Describe theinformation about the metabolites (name, whether they have bioactive effects, etc.). In
order to discuss the persistence of the substance in livestock bodies, the method forquantitating both the substance remaining in the body and the substance excreted tooutside of the body is needed. Besides the sensitivity, high accuracy and repeatability are
required in the method of analysis. In particular, the following specifications are required:limit of quantitation ≤ 0.05 mg/L; recovery rate in the spike recovery test on 1–2 mg/L
sample ≥ 70%; and the coefficient of variation (standard deviation divided by the meanvalue) ≤ 0.1. These specifications need to be described. However, note that the limit ofdetection must be equal to, or lower than, the residue limit of the objected substance infoods if the said residue limit is set at below 0.05 mg/kg.
○Results of the analysis: If the results indicate that the amount of residue is smaller than30

the limit of quantitation, describe as “< limit of quantitation.”[Example] A case of an application to weaned cows
A field application test to assess the persistence of the objected substance was conductedusing cows at the xx laboratory in xx prefecture. The cows reared in the indoor cow houseof the laboratory (strain: xx; ten pens each of males and females; 2 years old; average bodyweight: 450 kg) were given feeds continuously with the objected substance added at thefollowing different concentrations: 100, 300 and 1,000 mg/kg (each concentration
corresponding to the following daily intake amounts of the objected substance per unit ofbody weight: 0.3, 0.9 and 3.0 mg/kg-BW/day) for four weeks. (The consumption of waterand the feeds by the test animals was discretionary.) After four weeks of feeding, the cows
were slaughtered for an analysis of the residue in each part of the anatomy. (There was nonon-exposure period.) The tissues of the muscles (skeletal muscles), liver, kidneys and fat(from the abdominal area) were sampled and analyzed by the liquid chromatograph mass
spectrometer (LC/MS) method. The limit of quantitation of this method (LC/MS) was 2.41
ng/g for the muscles, fat, liver, kidneys and milk. The average recovery rate was 85.7%,
and the coefficient of variation was 0.1.
The details of the LC/MS method are as follows:
Equipment: xx (time-of-flight type), ultraviolet detector (280 nm)
Column: octadecylsilyl silica gel (inside diameter: 5–6 mm; length: 200–300 mm; particle
diameter: 5 µm)
Column temperature: 25℃
Mobile phase: mixture of water and ethanol (90:10); gradient: 0 min. (90:10) → 30 min.
(60:40); flow: 0.5 mL/min.; sample: 10 µL; ionization method: ESI (+), 5.0 kV, 500℃
… (omitted) …
[Result]
The results of the analysis are summarized in the table below (Table 5). The residue in themilk and muscles (skeletal muscles) were below the limit of quantitation even in thesamples from the cows that had consumed the feed with 1,000 mg/kg of the additive
amount. The maximum residue, 8 mg/kg, was detected in the liver and kidneys from thecows that had consumed the feed with 1,000 mg/kg of the additive amount. In the fat of theabdominal area, residue was identified in the cows which had consumed the feed with 300
mg/kg and more of the additive amount. However, the amount of residue was equal to orless than the limit of quantitation in all parts of the body of the cows that had consumed thefeed with 100 mg/kg of the additive amount, which is the maximum additive amount toproduce the efficacy of the objected substance.
Table 5 Animal residue test of the objected substance in cows
Concentration of theobjected substance inthe feed (mg/kg)
100
300
1,000

Analyzed body partMuscle(skeletalmuscle)
<2
<2
<2

Liver

Kidneys

<2
<2
5–7

<2
<2
5–8

Fat
(abdominal
area)
<2
10–15
40–50

Limit of quantitation: 1µg/kg for milk and 2µg/kg for other parts

31

Milk
<1
<1
<1

5. Items Concerning Safety(1) Toxicity tests
Toxicity tests in laboratory animals such as mice and rats are very important, as they
provide a tool to assess the toxicity of the objected substance with a high degree of
accuracy because the tests are conducted under a genetic control (inbred line),
microbiological control and environmental control. The toxicity tests are conducted to obtainthe following information:･Information about the effect of the initial dose on livestock animals･Information about the safe administration period･Information about the toxic effects (symptoms, toxic dose, etc.)
i. General toxicity tests (subject to the feed additive GLP)
a. Single dose toxicity test
A test to assess the toxic effects from both a quality and a quantity aspect by administeringa single dose of the objected substance to the test animals and estimating the medianlethal dose LD50. Also the level of intoxication and the appearance period of the symptoms
shall be observed, and thereby the whole picture of the toxicity shall be clarified.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals: Use young and healthy rodents (such as rats). Specify the strain, age inweeks, sex, body weight at the beginning of test, etc., of the test animals. Also, describe
the information about the test, such as the number of animals in a group.
○Method of administration and dose: Administer a single dose by an oral gavage andspecify the dose. Conduct an exploratory test to get a whole picture of the toxicity andapproximate the lethal dose. Set the initial dose as equal to a dose by which an apparent
sign of toxicity is expected to be caused, within 2,000 mg/kg-BW of the maximum limit.
○Test results and observations: Feed the test animals for at least 14 days and estimate theapproximate lethal dose. Observe the level of intoxication and the appearance period of
the symptoms, transitions, reversibility, etc. Record any kinds of signs of toxicity thatwere observed by the gross observation and the appearance period of symptoms forevery single test animal, for all the involved animals. Refer to the Appendix Form 2 fordetails of the items in the observation.
[Example] A case of a single dose toxicity test in mice
A single dose toxicity tests in mice was conducted at the xx laboratory in xx prefecture. The
mice (strain: xx; eight mice each of males and females; eight weeks old; average bodyweight: 21.2 g) were administered a single dose of the objected substance by an oral
gavage. The doses were arranged in the following different concentrations: 100, 200, 400,
1,200 and 2,000 mg/kg-BW. The consumption of the water and feeds by the test animals
was discretionary. Careful observations were made of the test mice for a period of two
weeks to check whether general symptoms and abnormalities, such as death, were shown.
The interval of the observations was 30 minutes during the period immediately after thestart of test, then every 12 hours every day.
[Results]
On the seventh day of the test, a female mouse in the group of 200 mg/kg-BW was found32

dead. On the day before the death, symptoms of anorexia and hypopraxia were observedin the mouse. Also, three dead cases (one male and two females) in the group of 1,200mg/kg-BW, and six dead cases (three males and three females) in the 2,000 mg/kg-BW
group occurred. Based on these results, the LD50 is estimated to be between 1,200 and
2,000 mg/kg-BW. The details of the results are summarized in the form below (Appendix
Form 2).
Appendix Form 2 Single dose toxicity test
Document No.
Testing laboratory
and testing period

xx laboratory
Fr. xx xx, xxxx to xx xx, xxxx
Test method

Species of animal(name of strain, etc.)

Mice : xx strain

Method of administration

Oral gavage

Purity of the test substance

99.8%

Dose (mg/kg-BW)

100–2,000

Observation period

2 weeks

Approximate lethal dose
(mg/kg-BW)

1,200–2,000

General symptoms

On the 3rd day, one male showed poor feeding (nobody weight gain), and was confirmed dead on the
5th day(omitted)

Period of occurrence,prosperity and decline of thesigns of toxicity, andperiod of death

Dead cases
7th day: 1 fe. in 200 mg/kg-BW gr.
2nd day: 1 m. and 1 fe. in 1,200 mg/kg-BW gr.
4th day: 1 fe. in 1,200 mg/kg-BW gr.
2nd day: 2 fes. in 2,000 mg/kg-BW gr.
5th day: 1 m. in 2,000 mg/kg-BW gr.
7th day: 1 m. in 2,000 mg/kg-BW gr.
9th day: 2 fes. in 2,000 mg/kg-BW gr.
(omitted)(omitted)

Notes
Note: “-” indicates no abnormality was observed.

33

b. Repeated dose toxicity test (short term)
A test to estimate the dose that will evoke apparent toxic changes and to clarify the detailsof toxic changes by administering the objected substance to the test animals continuously
for 3 or more months
c. Repeated toxicity test (long term)
A test to assess the toxicity by administering the objected substance to the test animals
continuously for an extended period[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
Test periods are 90 days for short-term tests and 12 months for long-term tests.
○Test animals: Use young and healthy animals. Specify the strain, age in weeks, sex, bodyweight at the beginning of test, etc., of the test animals. A sooner start of the test isdesirable.
○Method of administration and dose: Add the objected substance to feed or water, inprinciple, and administer the feed by a continuous oral gavage. Arrange at least threedifferent dose groups, including a dose that causes no effect in the animals and anotherdose that will cause a certain sign of toxicity. If solvents are not used, a non-treatedgroup is the control. When a solvent is used, a group fed the solvent is the control. Notethat the concentration of the additive amount shall be 5 w/w% or lower.
○Test results and observations: Measure the items listed in the appendix to clarify thewhole picture of the toxicity. Refer to the Appendix Form 3 for the details of the items in
the observation.
[Example] A case of a long term repeated dose toxicity test in mice
A long term repeated dose toxicity test in mice was conducted at the xx laboratory in xx
prefecture. The mice (strain: xx; 16 mice each for males and females; 5 weeks old; average
body weight: 20.7 g) were fed for 12 months. The objected substance was added to thefeeds at the following variant concentrations: 100, 200, 500, 1,000 and 2,000 mg/kg. The
consumption of the water and feeds by the test animals was discretionary. After the test
period, clinical tests and pathological tests were conducted. For dead cases, a dissection
was performed.
[Results]
The details of the results are summarized in the form below (Appendix Form 3).
The following dead cases occurred in the groups of 1,000 mg/kg and higher concentrations:
one male in the 1,000 mg/kg group; one male and three females in the 2,000 mg/kg group.
A tumor was found in the female (in the lungs) in the 1,000 mg/kg group. A discussion was
conducted on whether the tumor was caused by the objected substance or not. Any
dose-response relationships were not confirmed. In one of the three dead females in the
2,000 mg/kg group, symptoms of anorexia and akinesis were observed two days before its
death. A dissection examination was performed, which provided no noticeable findings in
the gross observation. It was therefore thought to be a natural death. In the higher dosegroups (1,000 mg/kg and higher), the test animals showed a constant intake of the feeds
and did not show a significant difference in live weight gain among the higher dose groups.
Clinical tests were performed for each test animal, in addition to dissections for the deadanimals, which provided no noticeable differences (no significant differences). Based on34

these results, the NOAEL is estimated 500 mg/kg.

35

Appendix Form 3 Repeated dose toxicity test (short term or long term)
Document
No.

Testing laboratory
and testing period
xx laboratory
Fr. xx xx, xxxx to xx xx, xxxx

Species of animal(strain, etc.)
Mice : xx strain

Test group and dose
(mg/kg-feed; mg/kg-BW/day)General symptoms and death rate

Average live weight gain (g/day)Average intake of feed (g/day)
Feed
Feed efficiency
Total dose administered of the test substance(mg/animal)

Clinical test
findings

Pathologicaltest findings

No. of animals
per group
32 (16/16)

Method ofadministration

Purity of thetest substance

Mixed feeding

99.8%

0 (Control group)
General symptom
Male: Bleeding on the 57th day (bitewound), cured a few days later
Male: Loss of fur was found on the112th day(omitted)
0.6
0.8
0.67
0

Hematological test-

Blood biochemical test

(omitted)

Urine test-

Gross observation-

Weight of the organ-

Histological test-

NOAEL and toxic dose

-

Notes
Note: “-” indicates no abnormality was observed.

36

ii. Special toxicity tests (subject to the feed additive GLP)
a. Transgenerational reproductive toxicity test
A test to assess the effects on the reproductive potential, as well as the trans-generational
effects, by administering the objected substance to both male and female test animals
over multiple generations.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals and number of generations: Specify the strain, age in weeks, sex, bodyweight at the beginning of test, etc., of the test animals. Conduct the test over two
generations, in principle, and extend the test to three generations if necessary. Also,
describe the information about the test, such as the number of animals in a group.
○Method of administration and dosage: Add the objected substance to the feeds or waterand administer the feed/water by a continuous oral gavage. Arrange at least threedifferent dose groups to assess the dose-response relationship. The maximum doseshall be set as equal to the dose that invokes a sign of toxicity in the parent generation
but does not kill them. The minimum dose shall be set as the dose that causes no sign of
toxicity in both the parents and children. If solvents are not used, a non-treated group is
the control. When a solvent is used, the group fed the solvent is the control. Note that theconcentration of the additive amount shall be 5 w/w% or lower.
○Test results and observations: Measure the items listed in the appendix to clarify thewhole picture of the toxicity. Refer to the Appendix Form 4 for the details of the items in
the observation.
[Example] A case of a transgenerational reproductive toxicity test in mice
A transgenerational reproductive toxicity test in mice was conducted at the xx laboratory inxx prefecture. The mice (Generation: p; strain: xx; 30 females; 6 weeks old; average bodyweight: 20.4 g) were fed over two generations (F1 and F2 generations). The objected
substance was added to the feeds at the following variant concentrations: 100, 500, 1,000
and 2,000 mg/kg. Among the mice prepared for the test, 22 mice became pregnant. These
22 pregnant females were used for the test.
[Results]
The details of the results are summarized in the form below (Appendix Form 4). The
addition of the objected substance to the feed showed a contribution to a body weight gain
and the increase of the intake of feed in the P generation mice (p<0.1). In the F1 generation,a significant difference was indicated in both the initial body weight and the live weight gain
(p<0.01, p<0.02). The intake of feed also increased (p<0.02). Similar results were obtainedin the F2 generation (p<0.01). There was no significant difference between the F1 and F2
generations. With regard to the performance of reproduction, no significant differenceswere indicated except for an increase in the average body weight of the newborns (p<0.01).
A female of the second generation died at the age of one week old. This individual was
dissected and examined. No noticeable findings were obtained in the microscopic andclinical tests. Therefore, it can be thought that this was a natural death.
Based on the above, it was confirmed that the administration of the objected substancecauses a transgenerational effect, promotes live weight gain and affects the reproductive
potential.

37

Appendix Form 4 Transgenerational reproductive toxicity test
Testing laboratory
Method ofSpecies of animal No. of animals
and testing period
administration
Document(strain, etc.)
per groupxx laboratory
No.
Mice : xx strain
30 (female)
Fr. xx xx, xxxx to xx xx, xxxx
Mixed feeding

Purity of thetest substance

F1
Feeding period:days

F2
Feeding period:
days(omitted)(omitted)

Reproductive parameters

General feeding
parameters

Test group and dose
(mg/kg-feed; mg/kg-BW/day)General symptoms
Death rate
Average live weight gain (g)Average intake of feed (g/day)Average feed efficiency
Findings
Period of the estrous cycle
before copulation, normality(Female)No. of copulations
Rate of copulation
No. of pregnancies
Rate of pregnancy
No. of live births
Average body weight of thenewborns
No. of stillborns
Birth rate
Average litter size
Average live weight gain
(at 21 days of age; g)Surviving rate of the babies
at 21 days of age
Sex distribution (%)
Findings

0

100

500

0
1.2
2.6
0.46
-

0
1.8
2.7
0.67
-

0
2.3
3.1
0.74
-

25

25

25

14
88
10
63
82

16
100
13
81
90

15
94
11
73
88

0.5

0.7

0.8

0
100
6

0
100
10

0
100
10

10.2

11.2

10.8

100

100

100

64
-

64
-

73
-

Notes
Note: “-” indicates no abnormality was observed.

38(omitted)P
Feeding period: 300 days

Generation

99.8%

b. Developmental toxicity test
A test to assess the effects of the substance on the birth of fetuses, especially on theirteratogenicity, by administering the objected substance to pregnant animals in theorganogenetic period of the fetus.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals: Specify the strain, age in weeks, sex, body weight at the beginning of thetest, etc. of the test animals. Also, describe the information about the test, such as thenumber of animals in a group. Young and healthy nulliparous female animals shall be
used.
○Period of administration: Administer the objected substance in the organogenetic period of
the fetus.
○Method of administration and dosage: Administer the objected substance by an oral
gavage, in principle. Arrange at least three different dose groups to assess thedose-response relationship. The maximum dose shall be set within the extent of theconfinement that the physicochemical properties define, as equal to the dose thatinvokes a certain sign of toxicity in the mother animals, such as an inhibition of the bodyweight gain. The minimum dose shall be set as the dose that causes no damage in boththe mother and fetus. If solvents are not used, a non-treated group is the control. When asolvent is used, the group fed with the solvent is the control. The solvent shall not havean effect on the developmental toxicity and reproduction. Note that the dose should
desirably be determined through the preliminary test and be within 1,000 mg/kg-BW of
the maximum limit.
○Test results and observations: Measure the items listed in the appendix to clarify thewhole picture of the toxicity. Refer to the Appendix Form 5 for the details of the items in
the observation.
[Example] A case of a developmental toxicity test in mice
A developmental toxicity test in mice was conducted at the xx laboratory in xx prefecture.
The mice (strain: xx; 40 mice each for males and females; 6 weeks old; average bodyweight: 21.3 g) were administered the objected substance on the tenth day of pregnancy byan oral gavage. The objected substance was added at the following variant concentrations:50, 100, 400 and 1,000 mg/kg. The consumption of the water and feeds by the test animals
was discretionary. Among the 40 mice of each males and females (total 80 mice) prepared
for the test, 27 mice succeeded in an implantation and were used for the test.
[Results]
No noticeable changes were observed in either the mother animals or the fetuses. A deadcase of a fetus occurred in a female in the 400 mg/kg-BW dose group. A dissection
examination was conducted, the result of which suggested a crushing death, which was not
the result of the intake of the objected substance.
Based on the above, it can be concluded that the dose administered during the pregnancy
period caused no effects in either the mother or the fetuses. The details of the results are
summarized in the form below (Appendix Form 5).

39

Appendix Form 5 Developmental toxicity test
Testing laboratory
and testing period
Species of animal
Document
xx laboratory(strain, etc.)
No.
Fr. xx xx, xxxx to xx xx, xxxx
Mice : xx strain

0 (Control group)

No. of mother animals

25

General symptoms(omitted)

Average body weight (g)20.4

Average intake of feed (g/day)

3.3

Average feed efficiency

0.62

Death rate

0

No. of corpus luteum per dam

15.9

Implantation findings

Test group and dose
(mg/kg-feed; mg/kg-BW/day)

No. of implantation/dam
Average implantation no.

Rate of live fetuses (%)
Average no. of live fetuses

Fetal resorption
Dead fetuses
Macerated fetuses
Others

14.2
8.2
78
6.4
1
0
0
0

No. of animals
per group
80 (40/40)

Method ofadministration
Oral gavage(omitted)

Fetal sex distribution(percentage of males)

48 (male)

Fetal body weight
Average ± standard deviation (g)0.5±0.08

External abnormalities

Hydrocephalus (male)

Skeletal abnormalities-

Internal organ abnormalities-

Developmental abnormalities ofnewborns-

Notes
Note: “-” indicates no abnormality was observed.

40

Purity of
the test
substance
99.8%

c. Carcinogenicity test
Administer the objected substance across the life span of animals and assess the
oncogenicity in specific among other effects that may be caused.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
○Test animals: Specify the strain, age in weeks, sex, body weight at the beginning of thetest, etc., of the test animals. Also, describe the information about the test, such as thenumber of animals in a group.
○Period of administration: Typically, the objected substance is administered for a period of
24 months. Adequate reasons for a change shall be explained if the period is changed.
○Method of administration and dose: Add the objected substance to the feeds or water, inprinciple, and administer these continuously via an oral route. Arrange at least threedifferent dose groups to assess the dose-response relationship. The maximum doseshall be set as equal to the dose that is expected to cause an apparent effect on thefrequency of tumor occurrence, and thereby on the lifespan of the test animals. Notethat the concentration of the additive amount must be 5 w/w% or lower.
○Test results and observations: Measure the items listed in the appendix to clarify thewhole picture of the toxicity. Refer to the Appendix Form 6 for the details of the items in
the observation.
[Example] A case of a carcinogenicity test in mice
An oncogenicity test in mice was conducted at the xx laboratory in xx prefecture. The mice
(Strain: xx; 50 mice each of males and females; 5 weeks old; average body weight: 20.5 g)
were fed for 24 months. The objected substance was added in the following variant
concentrations: 100, 1,000 and 2,000 mg/kg.
[Results]
Two dead cases occurred (one each for males and females) during the test. A tumor was
found in the dead female (in the stomach). No tumor, etc., was found in the dead male,which suggests a natural death. In addition to these results, no change was indicated in thedeath rate. Based on the above, it can be concluded that the objected substance does nothave an oncogenicity effect. The details of the results are summarized in the form below(Appendix Form 6).

41

Appendix Form 6 Carcinogenicity test
Testing laboratory
Species of animal
Document
and testing period(strain, etc.)
No.
xx laboratory
Mice : xx strain
Fr. xx xx, xxxx to xx xx, xxxx
Test group and dose
(mg/kg-feed; mg/kg-BW/day)

0 (Control group)

Cumulative death rate0

Average live weight gain (g/day)

1.5

Average intake of feed (g/day)

2.3

General symptoms(omitted)

Weight of the organs (g)(Average value)

Body 22.1
Brain 0.42
Heart 0.12
Lungs 0.15
Kidney 0.27
(left) 0.13, (right) 0.14
Liver 0.95

Histopathological findings

Incidence of tumors(and incidence of a specific tumor)

Findings of other tests(omitted)

0

( 0 )

185th day: dead (1 m.)
241st day: dead (1 fe.)

Note

42

No. ofanimals
per group
100 (50/50)

Method ofadministration

Purity of thetest substance

Mixed feeding

99.8%(omitted)d. Mutagenicity test
A test to assess the mutagenicity of the objected substance by conducting in vitro reverse
mutation tests and in vitro chromosome aberration tests. A micronucleus test shall be
conducted if any abnormalities are found in the foregoing tests.
(d-1) Reverse mutation test
A test to assess the mutagenicity by way of examining the gene mutation inducibility (effecton the DNA base pairs) using Salmonella typhimurium and Escherichia coli bacteria.
[Requirements]○Test strains: Conduct the tests using five or more kinds of strains including the following
typical examples: Salmonella typhimurium—TA1535, TA1537, TA1538, TA98, TA100;
Escherichia coli bacteria—WP2uvrA.
○Doses used in the test: Arrange five or more different dose groups. The maximum dose is
5 mg/plate, in principle. Prepare both a negative control and a positive control. The
negative control is the group administered a solvent, and the positive controls are thegroups administered a known mutagenic agent. For the positive control groups, both thesubstance requiring the presence of the S9 mix and the substance not requiring the S9mix are prepared. Conduct the test using suitable metabolic activation methods (S9 mix)
and observe the results.
○Observations: Record the actual numbers of revertant colonies and their mean value.
[Example] A case of a reverse mutation test using Salmonella typhimurium and Escherichia
coli bacteria
Conduct the test for gene mutation inducibility using six Salmonella typhimurium strains(TA98, TA100, TA102, TA1535, TA1537 and TA1538) and Escherichia coli bacteria
WP2uvrA. Six dose levels of the objected substance were arranged with the 5,000 µg/plateas the maximum dose. The test strains were cultured at 37℃ for three days. Three plates
were tested for each of all possible combinations of the following contents and conditions:test strains each with the objected substance added at pre-arranged six dose levels; withand without the presence of the S9 mix metabolic activation. The negative control andpositive control groups (three plates each) were included as well. After the culture, therevertant colonies were counted for each individual plate.
[Results]
The results are summarized in the table below (Table 6).
Table 6 Number of revertant colonies of Salmonella typhimurium

(-)

(+)

Concentration(µg/plate)
0*1
1.6
8.0
40.0
200.0
1,000.0
5,000.0
Positive
control
0*1

Salmonella typhimurium
TA100
TA102

TA98
13
15
14
13
15
14
11
12

160
155
152
162
161
158
169
163

TA1535(omitted)(omitted)(omitted)

43(omitted)

S9mix

(*1) Negative control
TA98: 2-nitrofluorene (5.0 µg/plate)
TA100: sodium azide (2.0 µg/plate)TA1535: sodium azide (2.0 µg/plate)TA1537: 9-aminoacridine (50.0 µg/plate)
TA102: Mitomycin (0.2 µg/plate)
TA98: benzo[α]pyrene (10.0 µg/plate)
TA100: 2-aminoanthracene (5.0 µg/plate)TA1535: 2-aminoanthracene (5.0 µg/plate)TA1537: 2-aminoanthracene (5.0 µg/plate)
WP2uvrA: 2-aminoanthracene (20.0 µg/plate)

No increase in the number of revertant colonies was found in any of the dose groups,
including the maximum dose group, regardless of the presence of S9 mix for metabolic
activation.
Based on the above results, it can be concluded that the objected substance does not havemutagenicity.
(d-2) Chromosome aberration test
Chromosome aberrations caused by damage to the DNA and the proteins can be detectedby a microscopic observation in the metaphase of the cell division.
[Requirements]○Test cells: Primary or passage cultured cells of mammals, including humans○Doses used in test: Arrange at least three different dose groups with the maximum dose
equivalent to the concentration at which a proliferation or division of the cells is inhibitedby ≥ 50%. In a case where cell cytotoxicity is not identified, a 10 mm equivalent or 5
mg/mL of the concentration is the maximum limit. The negative control is the group
administered the solvent, in principle, and the positive controls are the groups
administered known chromosome aberration inducing agents. Conduct the test using
suitable metabolic activation methods (S9mix) and observe the results.
○Observations: Observe the incidence of chromosomally aberrant cells and the incidenceof polyploids, and record these values.
[Example] A case of a chromosome aberration test using CHL cells<Method>
A test was conducted for chromosome aberrations using the CHL cells of a Chinese
hamster. CHL cells were disseminated on 10 mm dishes at 5×104 cells/mL of theconcentration, and were cultured at 37℃. In the continuous treatment method, the objected
substance was added on the third day from the dissemination and was treated for 24 hours.
In the short time treatment method, the cells were treated with and without the S9 mix formetabolic activation for 12 hours on the third day from the dissemination, and were further
cultured.
Four dose levels of the objected substance (500, 1,000, 2,000 and 5,000 µg/plate) werearranged, with the 5,000 µg/plate as the maximum dose. The test cells were each given an
additional pre-arranged dose of the objected substance and were cultured at 37℃ for threedays, with and without the presence of the S9 mix for metabolic activation, along with thenegative and positive control groups. All test pieces were treated with both the continuous
treatment method (Test 1-1) and the short treatment method (Test 1-2). The structural
chromosome aberrations (chromatid gaps, chromosome gaps, etc.) were examined foreach dish.
(Test 2)
Prior to the determination of the four dose levels, a cytostatic test was conducted to assess44

the concentration at which the proliferation of cells is inhibited by ≥ 50 %. The CHL cells
were treated with the objected substance at concentrations of 500–2,000 µg/plate. The
numbers of cells were counted on the fifth day from the start of the treatment.
<Results>
Test 2
The results of the cytostatic test are summarized in the table below (Table 7). The ratio of
the cell proliferation rate to that of the negative control (survival rate [ratio in % against thenegative control]), which was calculated from the number of live cells, was used as theindex. From the results, the concentration at which the proliferation of cells was inhibited by≥ 50 % was 1,000 µg/mL ≤. Based on this result, 1,000 µg/mL of the concentration wasused for Test 1.
Table 7 Results of the cytostatic test
Observations
Live cell no.
(×104 cells)
Dead cell no.
(×104 cells)
Surviving rate (%)
Surviving rate
(ratio in % against thenegative control )0*172

Additive concentration of the objected substance (µg/mL)
500
1,000
56
40

74

28

55

98.6
100

66.7
67.6

42.1
42.7

(omitted)

Test 1-1
The results of the chromosome aberration test (short time treatment method) aresummarized in the table below (Table 8). Regardless of the presence of the S9 mix formetabolic activation, neither structural chromosome aberrations nor abnormal cells weredetected.
Table 8 Incidence of chromosome aberrations
S9 mixTreatment
period
(h)

(-)

12

(+)

12

No. ofcells
observed(cells)
200
200
200
200
200
200
200

Concentration(µg/dish)
0(*1)
500
1,000
2,000
5,000
Positive
control0(*1)

Incidence of structural chromosome
aberrations (%)

Judgment
(*8)

gap

ctb

cte

csb

cse
(*6)

(*7)

Total

0
1
0
0
0
0

0
0
0
0
0
1

0
0
0
1
0
0

0
0
0
0
2
0

0
0
0
1
0
0

0
0
0
1
0
1

0
1
0
3
2
2

-

0

0

0
0
(omitted)

0

0

0

-

(*2)

(*3)

(*4)

(*5)

f

(omitted)S9 mix(-)

Treatment
period(h)12No. ofcells
observed(cells)
200
200
200
200

Concentr
ation(µg/dish)
0(*1)
500
1,000
2,000

0
1
0
0

Incidence of chromosomallyaberrant cells (%)
Chromatid gap
(-)
(+)
0
0
0
1

45

Polypoid
0.0
0.0
0.0
0.0

Judgment
(*8)

-

200
200
(+)

12

200

5,000
Positive
control
0(*1)

0
0

0
1

0.0
0.0

-

0

0
(omitted)

0.0-(omitted)(*1) negative control, (*2) gap: chromatid gap, (*3) ctb: chromatid break, (*4) cte: chromatid exchange,(*5) csb: chromosome break, (*6) cse: chromosome exchange, (*7) f: fragmentation,(*8) Judgment (-): negative… (omitted) … Results of Test 1-2, Continuous treatment method, are omitted as well.

(d-3) Micronucleus test
Test to assess the inducibility of chromosome aberrations by examining the incidence of
micronucleated immature erythrocytes in mammalian bone marrow smears as an index of
the toxicity.
[Requirements]○Test animals: Mice or rats
○Method of administration and dosage: Use an intraperitoneal administration, or an oral
administration (oral gavage, in principle). Administer a single dose, as well as a repeateddose 4–5 times. Prescribe an appropriate dose for the repeated dose. Arrange at least
three different dose groups. Set the maximum dose as equal to the dose that causes any
signs of toxicity, such as an inhibition of the body weight gain. Use 2,000 mg/kg as the
maximum dose if no toxic signs occur. In addition, prepare negative and positive controls.
The negative control is the group administered the solvent, in principle, and the positive
controls are the groups administered the known micronucleus-inducing agents.
○Observations: After the administration, kill all the test animals and collect the bone marrowfrom each of them to prepare smears. In principle, count the micronuclei in a minimum
2,000 of immature erythrocytes per test animal. At the same time, examine the incidenceof polychromatophilic erythrocytes to all the erythrocytes.
[Example]
The mice (strain: xx; 5 mice each of males and females; 8 weeks old; average body weight:
23.1 g) were administered a single dose of the objected substance (by oral gavage). Threedifferent doses (500, 1,000 and 2,000 mg/kg) were arranged, with 2,000 mg/kg as the
maximum dose. Bone marrow was collected at 24 hours and at 48 hours from the time of
the administration, and 2,000 immature erythrocyte cells were examined per test mouse todetect the micronuclei.
[Results]
The results are summarized in the table below (Table 9).
The incidence of micronucleated cells was calculated (by the number ofmicronuclei/number of examined cells x 100).
No significant difference was identified in the incidence of polychromatophilic erythrocytes
between the treated groups and the negative control group. Also, there were no significant
differences between the treated groups and the positive control group.
Based on the above, it can be concluded that the objected substance does not haveinducibility of micronuclei.
Table 9 Incidence of micronucleated erythrocytes

46

Dose
(mg/kg-BW)
0 (controls)24

No. ofcells
observed(cells)2,000

No. ofpolychromatophilic
erythrocytes(cells)1,898

No. oforthochromaticerythrocytes(cells)102

1.0Incidenceof
micronuclei
(%)
0.10

48

2,000

1,914

86

1.6

0.08

Treatment
period(h)No. ofmicronuclei(cells)(omitted)e. Other tests(e-1) Tests to assess the inducibility of gene mutation as an index･Gene mutation test using cultured mammalian cells･Test in drosophila･Spot test in mice･Specific-locus test in mice(e-2) Test to assess the inducibility of chromosome aberrations as an index･Chromosome aberration test using rodent germ cells･Dominant lethal test in rodents･Test of interphase chromosome locus displacement in mice(e-3) Test to assess the damage to DNA as an index･Bacteriophage test using bacteria･DNA repair test using bacteria･Test of unscheduled DNA synthesis using bacteria･Test of sister chromatid exchanges using mammalian cells(e-4) Other tests･Somatic recombination and gene exchange test using fermentum･Sperm morphology aberration test in miceiii. Pharmacological test
Test conducted to assess the pharmacological effect of the objected substance when it isanticipated. Unless it is anticipated, this test can be omitted by indicating so. Any
antimicrobials which were proved to be effective in other things (disease treatments, forexample) than the inhibition of the productivity in livestock, caused by the particularpathogenicity organisms in their juvenile period, cannot be designated as feed additives.

iv. Test concerning in vivo kinetics
Test to clarify the in vivo kinetics by tracing the objected substance in its absorption,
distribution, metabolism, excretion, etc., when the animals ingest the objected substance.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
The test period shall be equal to the period of applying the objected substance as a feed
additive.
○Test animals: Use the targeted animals of the objected substance, and add rats andrabbits, etc., when needed.
○Method of administration and dose: Administer a single dose via an oral route, in principle.
If possible, consider a continuous administration as well. Select a dose that is suited to47

the method of analysis so that the test substance, etc., can be quantitated in the bodytissues and/or in the excreta.
○Method of analysis: Employ an adequate method capable for the analysis of the in vivo
kinetics.
○Results: The items to be observed are described in detail later. ([Reference 7] Tests forAnalyzing In vivo Kinetics, on page 49.)
[Example] A case of an in vivo kinetics test in pigs
A field application test was conducted to prove the efficacy of the substance at the xx
laboratory in xx prefecture. Weaned piglets reared in an indoor pig house (strain: xx; 12pens each of males and females; 14 days old; average body weight: 11.7 kg) wereadministered a single dose of the objected substance at a 100 mg/kg concentration via an
oral route (gavage). 12C of the objected substance was replaced by 14C, and the dynamic
states of the radio isotopes were traced inside the body for a three-day period from the timeof the administration.
[Results]
The results of the quantitative analysis of the discharged 14C in the excreta are summarizedin Table 10 below. The data shows that approximately 78% of 14C was excreted within 24
hours. In addition, 83% was discharged to the outside of the body as the three day total.
The test results indicate that the persistence of the objected substance inside the body islow, and that the objected substance is excreted mainly in the feces.
Table 10 Results of the quantitative analysis of 14C in the feces in the single dose
administration test of the 14C labeled objected substance via an oral route (ratio in % to thetotal administered 14C)
24
0.48
77.29
77.77

Urine
Feces
Daily total

Elapsed time (hrs.)
48
0.66
2.34
3.00

72
0.32
1.91
2.23

3 day total
1.46
81.54
83.00

A quantitative analysis was also performed on the objected substance contained in theexcreta. (Table 11)
Table 11 Ratio of the 14C labeled subject substance in the feces. (Ratio in % to the total
administered 14C)
Objected substance

After 24 hours
5

Metabolite A (N-hydroxide)

13

Metabolite B (Demethylated substance)

56

14

The ratio of C in the feces was 56% at 24 hours from the time of the administration.
Besides the objected substance, mainly Substance A and Substance B were detected. It isconsidered that these substances were produced by the metabolization process in the liver
after the administration. These substances didn’t have a physiological activation effect.
Based on these results, it is clarified that the objected substance is metabolized within 24
hours after it is administered and loses its physiological activation effect.

48

[Reference 7] Tests for Analyzing In vivo Kinetics
To analyze the kinetics inside the body of livestock animals, such as whether
metabolization takes place and what amount of the substance is excreted, theobjected substance is labeled by a radioactive material such as 14C: that is, theisotope-labeled compound produced by replacing a part of 12C of the objected
substance with 14C, is administered to animals and the radioactive materials aretraced in the organs and the excreta. Even if the structural formula has changed,
the kinetics can be analyzed by tracing the 14C.
○ Absorption and excretion test, the analysis of the distribution inside the livestock
animal’s body
Measurements are conducted of the blood concentration, residual amount in thedigestive tracts and the amount excreted in the urine and feces of the objected
substance and its main metabolites, and the change of those values with time, andan analysis is performed of the absorption rate in the digestive tracts, route of
excretion and the rate of excretion based on the data. In addition, an analysis isperformed of the distribution of those substances in the muscles, fat, liver, kidneys,
and the other organs and anatomies, and its time-dependent changes, and thebiological half-life is calculated on an as-needed basis. (For example, the combined
use of autoradiography, etc., after the administration of the radio isotope-labeled
compound is also effective.) Furthermore, the chemical type of the isotope-labeled
compound recovered in urine, feces, each organ etc. can be identified. (There may
be a possibility of the compound having been metabolized, if the identified chemical
type differs from that of the original.)○Identification of metabolites
Identify the main metabolites and analyze the production rate of those metabolites
when the metabolization of the objected substance in the animal’s body is
confirmed. (Typically the in vitro test using the cells of the organs and tissues thatare involved in the metabolization is conducted for the analysis.) When a differencein the production rate of the main metabolites is found between animal species, it isdesirable to conduct further similar tests in other species of animals.

(2) Feeding tests using targeted animals, etc. (Subject to the feed additive GLP)
Based on the target animal and the effective dose, both of which are supposed to act on theefficacy of the objected substance as a feed additive, conduct a test to administer theobjected substance to the targeted animal practically and continuously, and assess theeffects on the animal.
[Requirements]○Testing laboratory and period, testing place and conditions: Describe which testinglaboratory in Japan or overseas was used, and what conditions the animals were fed in.
Set the test period as equal to the applicable period of the objected substance whenused as a feed additive.
○Test animals: Use livestock animals for which the objected substance is planned to be
applied. Specify the strain, age, etc., of the test animals. Also, describe the information
about the test, such as the number of animals in a group.
○Method of administration and dosage: Describe the additive amount of the objected49

substance contained in the sample feeds (mg/kg-feed), the method and period of theadministration, as well as the daily intake amount of the objected substance per unit ofbody weight ingested by the test animals (mg/kg-BW/day; calculated from the bodyweight of the test animal and its daily consumption of feed). Arrange at least two dosegroups including a dose equivalent to the maximum of the optimum dose range, andanother dose of approximately 10 times said dose (except for the control groups).
Method of analysis:○Test results and observations: Describe the body weight (live weight gain), the intake
amount of the feeds, the feed efficiency, surviving rate, etc. for each dose groupseparately. When any abnormalities are found in these parameters, perform ahematological test, biochemical test, pathological test, etc., on an as-needed basis. Referto the Appendix Form 7 for the details of the items in the observation.
[Example] A case of an application to weaned piglets
A field application test was conducted to prove the efficacy at the xx laboratory in xx
prefecture. Pigs reared in an indoor pig house (strain: xx; 15 pens each of males andfemales; 30 days old; average body weight: 8.2 kg) were given feeds continuously for fourweeks, which is the intended period in actual use. The objected substance was added to
the feeds at the following different concentrations: 50 mg/kg; 80 mg/kg as the optimum
dose, which is equivalent to 0.4 mg/kg-BW/day of a daily intake amount of the objected
substance per unit of body weight; 160 mg/kg as two times the optimum dose, which isequivalent to 0.8 mg/kg-BW/day; 400 mg/kg as five times the optimum dose, which isequivalent to 2.0 mg/kg-BW/day; and 800 mg/kg as ten times the optimum dose, which isequivalent to 4.0 mg/kg-BW/day. The consumption of the water and feeds by the test
animals was discretionary.
[Results]
The results are summarized in the table below (Appendix Form 7). The live weight gain of
the 80 mg/kg group was 15.4 kg larger than that of the control group (p<0.01). Although theincrease in both the live weight gain and the intake of feeds was shown in the 800 mg/kg(10 times the optimum dose) group, a significant difference was not indicated between thisgroup and the group without the additive. Two female pigs became anorexic, while no
noticeable changes were observed as general symptoms. One of these two pigs showed amild gastric erosion, which was cured in a week or so. After the cure, the live weight gainincreased. The other pig did not show any noticeable symptoms.

50Appendix Form 7 Feeding test on targeted animals, etc. (livestock)
Test animal

Document
No.

Testinglaboratory,
Testing place
and period

xx laboratory
Fr. xx xx, xxxx
To xx xx, xxxx

Speciesxx breed

No. ofanimals ina group30(15/15)

Test group assignment

Test
group

Dose of the test
substance*1(mg/kg-feed)

0
50
80
160

0
50
80
160

No. ofrepeats2

Total no. ofanimals

60

Test results

Average live
weight gain*3(actual no.)(%)

Feeding
period

(kg)
0.41a
0.43a
0.55b
0.62c

4
weeks

100
105
134
151

Average intake
amount of feed
(actual no.)

(kg)
0.71d
0.68de0.61de0.60e

Average feeddemand rate*3(actual no.)(%)

Surviving
Hematologicalratetest findings
(%)

1.73f
1.58g
1.11h
0.97i

100
100
100
100

(omitted)(omitted)

Note: Test data noted with a different superscript indicate that the differences between those data are significant.

51

100
91
64
56

Blood
biochemical
findings(omitted)

Pathologicaltest findings

Notes*4

(3) Tests concerning the emergence of resistant bacteria
Perform quality and quantity assessments on the items concerning the emergence ofdrug-resistant strains among the effects caused by the use of antimicrobial substances.

(4) Other tests
Perform assessments on the effects of the use of the objected substance on the naturalenvironment by way of the excreta, etc., of livestock animals.

52

[Reference 8] Style Reference for the Description of Items Concerning Standards
Once the objected substance has been designated as a feed additive, the items set
forth in “III-2 Items Concerning Standards”, on page 19 and following, are listed in the
Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives(Ordinance of the Ministry of Agriculture, Forestry and Fisheries No. 35 issued on July24, 1976) as composition standards. The instructions for the use of the symbols, etc.,
set forth in the Ministerial Ordinance on the Specifications and Standards of Feed andFeed Additives shall be referred to for the description of these items. The descriptions
for previously designated feed additives, which can be found in the “List of theSpecifications and Standards of Feed Additives,” may be referred to as well.
- e-gov: Electronic information provision services concerning laws (Ministerial
Ordinance on the Specifications and Standards of Feed and Feed Additives)
http://law.e-gov.go.jp/htmldata/S51/S51F00601000035.html- List of the Specifications and Standards of Feed Additives (13th Edition)

Source: Appendix 2-1 to the “Ministerial Ordinance on the Specifications and Standards ofFeed and Feed Additives”(1) Inspections of feed additives are made in accordance with the composition
specifications and the standards of the manufacturing process, etc., of each feed
additive (hereinafter referred to as “each article”), and in line with the test methods
prescribed in the general rules on feed additives and the test method for the feed
additives (hereinafter referred to as the “general test method”). Note that thephysicochemical properties, such as the odor, taste, crystalline form, solubility, acidity
or alkalinity of a solution, stability, light absorbance, freezing point, refractive index,
optical rotation, viscosity, specific gravity and the melting point described in thepertinent section are for reference purposes only and do not constitute the elementsof the inspection criteria. Note also that stabilizing agents, lubricating agents, binding
agents, moistening agents, emulsifying agents, covering agents, dispersing agents,
disintegrating agents, preserving agents, or solubilizing agents can be used for theformula prescribed in each article to increase the efficacy or the stability of each feed
additive.
(2)

The substance name followed by the molecular formulas in parentheses ( ) indicates achemically pure substance.

(3)

The following signs are used for the main measurement units.
metermcentimetercm
millimetermm
micrometerµm
nanometernm
square centimeter
cm2
liter
L
milliliter
mL
microliterµLton
T (1,000 kg)
kilogramkg
gramg
milligrammg
microgramµg
kilopascal
kPa
mole
mol
micromoleµmol mole per liter
mol/L
degrees Celsius℃(4)

The mass percentage is denoted using a percentage sign %. In addition, the following
signs are used for each measurement: % w/v for the content of the substance (g) in53

100 mL of solution; % v/v for the content of the substance (mL) in 100 mL ofsolution; % v/w for the content of the substance (mL) in 100 g of solution.
(5)

With regard to the expression of the amount of antibiotics and enzymes, the potencyof the discussed antibiotics and the enzyme per liter unit of the discussed enzyme areused, respectively.

(6)

The term “standard temperature” indicates a temperature of 20℃. Similarly, “ordinary
temperature” is a temperature of 15–25℃, “ambient temperature” is a temperature of
1–30℃, and “lukewarm” is a temperature of 30–40℃. The term “cold place” indicatesa place of ≤15℃, unless otherwise specified. Likewise, “cold water” indicates water of≤10℃; “lukewarm water” indicates water of 30–40℃; “warm water” indicates 60–
70℃; and “hot water” indicates water of approximately 100℃. The term “heat on or ina water bath” indicates heating in a boiling water bath or a steam bath atapproximately 100℃, unless otherwise specified.

(7)

Purified water shall be used for the tests of the feed additives, unless otherwise
specified.

(8)

A dropping device which delivers 20 drops of purified water weighing 0.90–1.10g at
20℃ shall be used for measuring the number of drops.

(9)

The value of the "n+ 1" figure shall be rounded off for obtaining a value of the "n"figures.

(10) The table of the “Standard Atomic Weights 2007” shall be referred to for the atomic
masses. The molecular weight shall be calculated using the values from this table andshall be rounded off to two decimal points.
(11) The term “reduced pressure” indicates a pressure not exceeding 2.0 kPa, unlessotherwise specified.
(12) The acidity or alkalinity of a solution is determined by the use of blue or red litmus
papers, unless otherwise specified. The pH value shall be used for a precise
expression.
(13) Solutions expressed with the word “solution” following the name of the solute, and notstating the name of the solute, indicates a water solution.
(14) Solutions denoted using the style of (1→3), (1→10) or (1→100) indicate that eachsolution has a concentration corresponding to 3 ml, 10 ml and 100 mL of solution, allof which contain 1 g of the dissolved solid solute or 1 mL of the dissolved liquid solute,respectively. Mixtures denoted by (1:10), (5:3:1), etc., are mixtures containing two
kinds of liquids at a 1:10 ratio, and three kinds of liquids at a 5:3:1 ratio, respectively.
(15) The tests of the feed additives shall be conducted at an ordinary temperature, and theobservations shall be made immediately after the operation, unless otherwise
specified. When examining temperature-sensitive matters, the conditions at thestandard temperature shall be examined.

54

(16) The term “white” used in the item of physicochemical properties indicates a white orpractically white color. Similarly, the term “colorless” indicates a colorless or practicallycolorless item. Unless otherwise specified, the test of the color tone is performed byplacing 1 g of the test feed additive on a sheet of white paper, or in a watch glass
placed on white paper for solid feed additives. A liquid feed additive is put into acolorless test tube measuring 15 mm as the inside diameter, and is observed in frontof a white background through a 30 mm liquid layer. For the clarity test of liquid feed
additives, the above-mentioned procedure is used with either a black or a whitebackground. Only a black background shall be used for testing the fluorescence of aliquid feed additive.
(17) The term “odorless” used in the item of physicochemical properties indicates anodorless or practically odorless item. Unless otherwise specified, the test of odor shallbe performed by placing 1 g of the solid or liquid feed additive in a 100 ml beaker.
(18) The terms used to indicate the solubility in the item of physicochemical properties aredefined in the table below. Unless otherwise specified, solubility means the degree of
dissolution of a feed additive, previously powdered in the case of a solid feed additive,
within 30 minutes of its immersion in a solvent at 20 ± 5℃, with 30 seconds ofvigorous shaking repeated at 5-minute intervals.
TermVery soluble
Freely soluble
SolubleSparingly soluble
Slightly soluble
Very slightly soluble
Practically insoluble, or
insoluble

Volume of solvent required for
dissolving 1 g or 1 mL of the solute< 1 mL
1 mL ≤< 10 mL
10 mL ≤
<30mL
30 mL ≤
<100mL
100 mL ≤<1,000mL
1,000 mL ≤<10,000mL
10,000 mL ≤(19) In the test of feed additives, the term “dissolve in a solvent” or “mix with a solvent”
indicates that the feed additive is dissolved in, or mixed with the solvent to form aclear solution or mixture. A fraction of fibers or dust should be considered as within theallowance.
(20) The identification test is the test to identify feed additives or the main ingredients offeed additives.
(21) The purity test aims to detect impurities in the feed additives. The test is intended to
specify the purity of feed additives, together with the other test items specified in eacharticle. It is typically performed to control the type and amount of impurities. The
impurities subject to the impurity test shall include those anticipated to become mixed
in during the manufacturing process or the storage of the feed additives, as well as
hazardous impurities such as heavy metals and arsenic. If foreign substances areused or are predicted to be added, this test shall be performed on those substances.
(22) The terms “clear”, “practically clear”, “very slightly turbid”, “slightly turbid” and “turbid”
are standardized by the following procedures respectively.
Turbidity standard stock solution: Add water to 14.1 mL of 0.1 mol/L of hydrochloric

55acid to make 50 mL of solution. One ml of this solution contains 1 mg of chlorine (Cl).
Turbidity standard solution: Measure 10 ml of the turbidity standard stock solution,
and add water to make 1,000 mL of solution. One ml of this solution contains 0.01 mg
of Cl.
i. Clear: Measure 0.2 ml of the turbidity standard solution, and add water to make 20
ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.2 ml of 2% w/v dextrin
solution, and 1 ml of 2% w/v silver nitrate solution. Allow the solution to stand for 15
minutes to prepare the reference solution. To be described as “clear” the test
solution shall have the same or lower turbidity than that of the reference solution.
Note that impurities such as floating substances should not be observed.
ii. Practically clear: Measure 0.5 ml of the turbidity standard solution, and add water tomake 20 ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.2 ml of 2% w/v
dextrin solution, and 1 ml of 2% w/v silver nitrate solution. Allow the solution to standfor 15 minutes to prepare the reference solution. To be described as “practically
clear” the test solution shall have the same turbidity as that of the reference solution.
Note that impurities such as floating substances should not be observed.
iii. Very slightly turbid: Measure 1.2 ml of the turbidity standard solution, and add
water to make 20 ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.2 ml of 2%
w/v dextrin solution, and 1 ml of 2% w/v silver nitrate solution. Allow the solution to
stand for 15 minutes to prepare the reference solution. To be described as “veryslightly turbid” the test solution shall have the same turbidity as that of the reference
solution.
iv. Slightly turbid: Measure 6 ml of the turbidity standard stock solution, and add water
to make 20 ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.2 ml of 2% w/v
dextrin solution, and 1 ml of 2% w/v silver nitrate solution. Allow the solution to standfor 15 minutes to prepare the reference solution. To be described as “slightly turbid”
the test solution shall have the same turbidity as that of the reference solution.
v. Turbid: Measure 0.3 ml of the turbidity standard solution, and add water to make 20
ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.2 ml of 2% w/v dextrin
solution, and 1 ml of 2% w/v silver nitrate solution. Allow the solution to stand for 15
minutes to prepare the reference solution. To be described as “turbid” the test
solution shall have the same turbidity as that of the reference solution.
(23) In drying or ignition, the term “constant mass”, unless otherwise specified, means thatthe difference of the masses measured before and after an additional 1 hour of dryingor ignition is not more than 0.10% of the preceding mass of the dried substance orignited residue. In the cases of differences of masses ≤ 0.5 mg in a chemical balance,≤ 0.05 mg in a semi-microbalance, and 0.005 mg in a microbalance, those masses
are regarded as the constant mass.
(24) The quantitative method is the method of testing to determine the composition of feed
additives, the content or concentrations, etc., of the ingredients by the use of physical,
chemical or biological procedures.

56

(25) The quantity of test samples or standard substances preceded by the word
“approximate” indicates a quantity that is within ±10% of the specified mass. The word“dry” indicates drying under the same conditions as are specified in each article, or in
the section of the drying loss of the standard substances when it is simply used inconjunction with the test samples or standard substances. Similarly, the word “ignite”
indicates igniting under the same conditions as are specified in the section of theigniting loss of each article.
(26) For the content of an ingredient determined by the quantitative method specified ineach article, when it is expressed simply as "equal to or more than a certain
percentage" without indicating its upper limit, 101.0% is understood as the upper limit.
For example, content specified with the expression “contains a pure substance of acontent equal to 90–110% of the labeled content” indicates that the substance isprepared so as to contain chemically pure substances or their equivalent at a 100%concentration, and the quantitation provides the percentage point within said range.
Content expressed as “contains a potency of 85–125% of the labeled potency”
indicates that the substance is prepared so as to maintain the labeled potency during
its shelf life, and the quantitation provides the percentage point within said range.
(27) Any test methods may be employed to substitute for the general test methods andmethods specified in each article when the same level of or a higher accuracy andprecision are achieved by those methods; however, it is provided that the test using
the specified methods shall be performed for the final determination if any questions
have arisen as to the results.
(28) The term “container” indicates the container of the feed additives and includes allparts that constitute the container such as the cover and the lid.
(29) A “sealed container” indicates a container that is capable of protecting the contained
feed additives from extraneous solids and from the loss of the feed additives under
the ordinary or customary conditions of handling and storage. Where a sealedcontainer is specified, it may be replaced by a tightly sealed container or by ahermetically sealed container.
(30) A “tightly sealed container” indicates a container that is capable of protecting thecontained feed additives from extraneous solids, liquids or moisture, from the loss ofthe contents, and from efflorescence, deliquescence, or evaporation under theordinary or customary conditions of handling and storage. Where a tightly sealedcontainer is specified, it may be replaced by a hermetically sealed container.
(31) A “hermetically sealed container” indicates a container that is impervious to any gases
and microbes under the ordinary or customary conditions of handling and storage.
(32) A “light-shielded container” indicates a container that is made to shield light or having
a light shielding encasement.

57

IV Exemplary Abstract
Referring to the chapter “III Items to be Described in the Required Documents (Abstract”,
on page 16 and following, an abstract shall be drawn up following the style exhibited on
page 58 and following.
The exemplary abstract, exhibited on page 58 and following, contains blank spacesthat are marked with “…(omitted) …” where descriptions of the required items areomitted to simplify the explanation. In an actual abstract prepared for submission, all of
the required items shall be described. If any items are omitted the reasons andgrounds shall be presented.
Insufficient descriptions in the submitted documents, such as in the abstract, will cause an
extended time for the document checks by the authorities, which may result in a delay ofthe deliberation in the council and the designation of the feed additive. Even if thedeliberation is conducted in the council, the submitters may be requested to submit
additional tests or documents, which will also defer the process. The abstract submitted as
the very first action of the application is very important to avoid the above-mentioned
situations.
In the preparation of the abstract, the cited test reports, academic papers, etc., shall benumbered by material numbers and submitted as attachments to the abstract.
Additionally, please refer to the chapter “VI Checklist for the Preparation of the Required
Documents”, on page 76 and following, which summarizes the notes of caution for thewording and descriptions of the required items. Keep this checklist handy and make use of
it during the preparation of the abstract. The checklist shall also be submitted together withthe abstract after each of the items has been checked.

58

Cover

Abstract of the Test Results, etc., of
the Objected Substance (xxxx)

mmmm, dd, yyyy
Feed Additive Business Operator

59

General name…(omitted)…
Intended use and dosage…(omitted)…

Chemical name…(omitted)…

Trade name…(omitted)…

Chemical structure…(omitted)…

List of Items to be Described in the Abstract
1. Origin or Background of the Discovery, Status of Authorization and Use as a
Feed Additive in Foreign Countries, etc.
(1) Origin or background of the discovery………………………………………
(2) Status of authorization and use in foreign countries………………………
(3) Status of authorization and use as veterinary drugs and importation……
(4) Comparison to related substances……………………………………………
2. Items Concerning Standards(1) Name………………………………………………………………………………
i. General name…………………………………………………………………
ii. Chemical name………………………………………………………………
iii. Trade name……………………………………………………………………
(2) Chemical structure………………………………………………………………
(3) Manufacturing process…………………………………………………………
(4) Biological and physicochemical properties……………………………………
i. Physical and chemical properties……………………………………………
ii. Identification tests……………………………………………………………
iii. Purity tests……………………………………………………………………
iv. Content and quantitative method……………………………………………
(5) Quantitation in feeds…………..…………………………………………………
(6) Changes with time………………………………………………………………
i. Storage test in an ambient temperature……………………………………
ii. Heat resistance test……………………………………………………………
iii. Humidity resistance test………………………………………………………
iv. Light resistance test…………………………………………………………
v. Accelerated test………………………………………………………………
vi. In-feed stability test…………………………………………………………
3. Items Concerning Efficacy(1) Basic tests to prove effectiveness………………………………………………
i. In vitro test………………………………………………………………………
ii. In vivo test………………………………………………………………………
(2) Field application test to prove effectiveness…………………………………
4. Items Concerning Residue
Residue test in targeted animals, etc.…………………………………………
5. Items Concerning Safety(1) Toxicity tests………………………………………………………………………
i. General toxicity test……………………………………………………………
a. Single dose toxicity test……………………………………………………
b. Repeated dose toxicity test (short term)…………………………………
c. Repeated dose toxicity test (song term)…………………………………
ii. Special toxicity tests…………………………………………………………
a. Transgenerational reproduction toxicity test………………………………
b. Developmental toxicity test…………………………………………………
c. Carcinogenicity test…………………………………………………………

60(page)

61
61
61
61

61
61
61
61
62
62
62
62
62
63
63
63
63
64
64
65
65
65
65
65
65
65
65
66
67
67
67
68
68
68
68
68
68

d. Mutagenicity test……………………………………………………………
e. Other tests……………………………………………………………………
iii. Pharmacological test…………………………………………………………
iv. Test concerning the in vivo kinetics and metabolism………………………
(2) Feeding test using targeted animals, etc.……………………………………
(3) Test concerning the emergence of resistant bacteria…………………………
(4) Others...…………………………………………………………………………

61

68
69
69
70
71
71
71

Document
No.

Item
1. Origin or
Backgroundof theDiscovery,
Status of
Authorizationand Use as a
Feed
Additive in
Foreign
Countries,
etc.

Specific item
(1) Origin or
background of the
discovery(development)(1) Origin or background of the discovery (development)… (omitted) …

(2) Status of its
authorization anduse as a feed
additive in foreigncountries, etc.

(2) Status of the substance’s authorization and use as a feed additive in foreign countries,etc.
… (omitted) …

(3) Status of its
manufacturing anddistribution
authorization, andits importation as aveterinary
medicinal product(4) Comparison torelated substances(generic
substances having
the same effect)

2．Items
ConcerningStandards

Examined item and summary of results(1) Namei. General nameii. Chemical
nameiii. Trade name(3) Status of the substance’s manufacturing and distribution authorization, and its importation
as a veterinary medicinal product… (omitted) …

(4) Comparison to related substances (generic substances having the same effect)… (omitted) …

(1) Namei. General nameii. Chemical name… (omitted) …
… (omitted) …

iii. Trade name… (omitted) …
62

(2)Chemical
structure(2) Chemical structure
Definition … (omitted) …
Potency… (omitted) …

Structural formula, molecular formula, molecular weight, … (omitted) …
(3) Manufacturing
process(3) Manufacturing process
Oxidize Substance A (99.5％) with air using a platinum catalyst to produce Substance B.
Initiate hydrolysis by adding sodium hydroxide in the special grade ethanol to produce theunrefined xxxx. Refine it through a solvent extraction using hexane, then dehydrate it to yield
the objected substance xxxx
Substance A (99.5%)↓ platinum catalyst
Substance B↓ sodium hydroxide (in special grade ethanol)xxxx (unrefined) ,(by-product C)↓ solvent extraction by hexane
objected substance xxxx

(4) Biological andphysicochemical
properties
i. Physical andchemical properties(4) Biological and physicochemical properties

ii. Identification test

ii. Identification test
This substance shows an IR absorption peak at near 1,750 cm-1 of the wavenumber in thei. Physical and chemical properties
Physical and chemical properties
a. Appearance: colorless or white crystal, or a white crystalline powder
b. Chemical properties: freely soluble in water, soluble in methanol and practicallyinsoluble in benzene.
… (omitted) …

63

infrared absorption spectrum by the potassium bromide tablet method. The aqueous solutionof this substance (1→10) shows a quantitative reaction with xx salt. … (omitted) …
iii. Purity testiii. Purity test
a. Clarity and color of the solution: an aqueous solution prepared by dissolving 1.0 g(0.95–1.04 g) of this substance in 20 mL of water shows a light tan color and ispractically clear.
b. Chloride limit: When conducting a chloride limit test with 1.0 g (0.95–1.04 g) of thissubstance, the turbidity of the test solution does not exceed that of the control solution
prepared with 0.5 mL of 0.01 mol/L hydrochloric acid.
… (omitted) …

iv. Content andquantitative method iv. Content and quantitative method
Content: This material contains 99.5% or more of the objected substance (describe thechemical formula of the objected substance) in the quantitation after drying.
Quantitative method: Dry the material, weigh out 0.5 g of the material, dry down to 0.001 g
and record the reading. Dissolve the weighed material in 50 mL of water, and add 5 mL offormalin and titrate with 0.5 mol/L sodium hydroxide solution (use three drops of thephenolphthalein indicator). Perform a blank test using the same method for correction.
1 mL of 0.5 mol/L sodium hydroxide solution = equivalent weight in mg, chemical formulaof the objected substance… (omitted) …
(5) Quantitation in(5) Quantitation in feed
feed
Micronize the feed with the added substance, then weigh out 10 g (9.5–10.4 g) of the
micronized feed. Dissolve the weighed material in 100 mL of a mixture of chloroform andether (1:9). Extract the solution three times with 50 mL of a mixture of ethanol and methanol(6:4). Add 500 mL of purified water, then measure 100 mL of the obtained solution to yield thetest solution. Add 2 mL of formalin to 10 mL of the test solution and titrate with 0.5 mol/L
sodium hydroxide solution (use three drops of the phenolphthalein indicator). Perform a
blank test using the same method for correction. … (omitted) …
(6) Changes with
(6) Changes with time
time

64

i. Storage test in an
ambient
temperaturei. Storage test in an ambient temperature
Pack the substance in a typical package (such as a plastic bag), then store the package in anindoor warehouse for 24 months at 25℃, 50% humidity to assess the stability. The observedchanges with time are shown below.
[Results]
Table 1 Storage test in an ambient temperature
Test conditions: air temperature 25℃; humidity 50% (for period of 24 months); in a 20 kg
paper bag
Lot
No.

Parameter
AppearanceA

B

ii. Heat resistance
test

Identification
test
Purity test
Amount ofactiveingredient (g)Drying loss(g)Amount ofmoisture (g)Appearance

At thestart

3 mos.

6 mos.

9 mos.

12 mos.

24 mos.

white
powderfit

white
powderfit

white
powderfit

white
powderfit

white
powder
fit

white
mud1
fit

fit
214.6

fit
212.1

fit
210.4

fit
208.3

fit
205.4

fit
207.9

5.2
32.3

4.9
30.0

3.7
29.4

5.0
35.1

6.3
33.8

4.7
35.3

white
powderwhite
white
powderpowder(omitted)

white
powderwhite
powderwhite
powder

Note 1: “White mud” indicates a white paste condition.
Although the appearance of the substance turned to a white mud (paste condition) after aperiod of 24 months, it passed both the identification test and the purity test. In addition, no
significant changes in the amount of the active ingredient were found. No other problems
were found; thus, it can be concluded that the substance remains stable for at least 24
months.
… (omitted) …
ii. Heat resistance test65

… (omitted) …
iii. Humidity
resistance test
iv. Light resistancetest
v. Accelerated
test
vi. In-feed stabilitytest

3. Items
Concerning
Efficacyiii. Humidity resistance test
… (omitted) …
iv. Light resistance test
… (omitted) …
v. Accelerated test
… (omitted) …
vi. In-feed stability test
… (omitted) …

(1) Basic tests toprove the efficacy
i. In vitro test

(1) Basic tests to prove the efficacy

ii. In vivo test

ii. In vivo test
… (omitted) …

(2) Field application
tests to prove
efficacy(2) Field application tests to prove efficacy
Testing laboratory and place: xx laboratory in xx prefecture (indoor pig house)
Test animals: Weaned piglets (Strain: xx; 25–30 days old; average body weight: 10.3 kg),four pens each of males and females in a group
Dose and method of administration: The test animals were given feeds continuously with theobjected substance added at the following different concentrations: 20, 40, 80, 100, 150 and
200 mg/kg, for four weeks which is an intended period in actual use. There was nonon-exposure period. After four weeks, all of the test animals were slaughtered andsubjected to a dissection examination.
The test animals were given feeds continuously with the objected substance added at thei. In vitro test
… (omitted) …

66

following different concentrations: 20, 40, 80, 100, 150 and 200 mg/kg (each concentration
corresponding to the following daily intake amounts of the objected substance per unit ofbody weight: 0.4, 0.8, 1.6, 2.0, 3.0 and 4.0 mg/kg-BW/day) for four weeks, which is anintended period in actual use.

4．Items
Concerning
Residue[Results]
The test results are shown in the form below. (Appendix Form 2) The live weight gainsignificantly increased at the concentration of 40 mg/kg and higher (p<0.05). The live weightgain at 100 mg/kg did not show any difference to that at 80 mg/kg. Thus, 80 mg/kg can bethought to be the most effective concentration, even from an economical perspective.
Succeeding tests were conducted at a concentration of 80 mg/kg as optimum additive
amount. With regard to the intake amount of feed, this peaked at 100 mg/kg and the
proportional increase to the concentration of the objected substance was not marked. …
(omitted) …
4．Items Concerning Residue
Testing laboratory and place: xx laboratory in xx prefecture (indoor cow house)
Test animals: Cows (Strain: xx; 2 years old; average body weight: 450 kg), 10 pens each ofmales and females in a group
Dose and method of administration: The objected substance was mixed in the feed and fed
continuously to the animals for 4 weeks. Feeds were given two times a day with the objected
substance added at the following different concentrations: 100, 500, 1,000 and 5,000 g/kg.
There was no non-exposure period. After four weeks, all of the test animals were slaughteredand subjected to a dissection examination.
Method of analysis: The analysis was performed using the liquid chromatograph mass
spectrometer (LC/MS) method. The limit of quantitation of this method (LC/MS) was 2.41
ng/g for the muscles, fat, liver, kidneys and milk. The average recovery rate was 85.7%, andthe coefficient of variation was 0.1. The details of the method used are as follows.
… (omitted) …
[Result]

The results of the analysis are summarized in the table below (Table 3). The residue in themilk and muscles (skeletal muscles) were below the limit of quantitation even in the samples
from the cow that had consumed the feed with 1,000 mg/kg of the additive amount. The
maximum residue, 8 mg/kg, was detected in the liver and kidneys from the cows that had

67consumed the feed with 1,000 mg/kg of the additive amount. In the fat of the abdominal
area, residue was identified in the cows which had consumed the feed with 300 mg/kg andmore of the additive amount. However, the amount of residue was equal to or less than thelimit of quantitation in all parts of the body of the cows that had consumed the feed with 100
mg/kg of the additive amount, which is the maximum additive amount to produce the efficacyof the objected substance.
Table 3 Animal residue test of the objected substance in cows
Analyzed body partConcentration of
the objected
MusclesFat
substance in the(skeletal
Liver
Kidneys(abdominal
feed (mg/kg)
muscles)
area)
100
<2
<2
<2
<2
10–15
300
<2
<2
<2
5–7
5–8
40–50
1,000
<2
Limit of quantitation: 1µg/kg for milk and 2µg/kg for other parts

Milk
<1
<1
<1

… (omitted) …
5. Items
Concerning
Safety(1) Toxicity tests
i. General toxicity
tests
a. Single dose
toxicity test

(1) Toxicity tests
i. General toxicity tests
a. Single dose toxicity test
Testing place and environment: xx laboratory in xx prefecture (indoor rearing facility)
Test animals: Mice (Strain: xx; 3 weeks old; average body weight: 7.9 g), 12 mice each ofmales and females in a group
Method of administration and dosage: Consumption of the water and feeds by the test
animals was discretionary. The objected substance was administered at 100–2,000
mg/kg-BW by an oral gavage. General symptoms and any abnormalities, such as death,
were observed for 2 weeks after the administration.
[Results]
On the seventh day of the test, a female mouse in the group of 200 mg/kg-BW was founddead. On the day before the death, symptoms of anorexia and hypopraxia were observed in
the mouse. .… (omitted) …
Based on these results, the LD50 is estimated to be between 1,200 and 2,000 mg/kg-BW.

68

The details of the results are summarized in the form below (Appendix Form 4)… (omitted) …

ii. Repeated dose
toxicity test
(short term)iii. Repeatedtoxicity test
(long term)ii. Special toxicity
tests

ii. Repeated dose toxicity test (short term)… (omitted) …
iii. Repeated toxicity test (long term)… (omitted) …
ii. Special toxicity tests

a. Transgenerational reproductive toxicity test (three generations)a.
… (omitted) …
Transgenerational
reproductive toxicity
test
b. Developmental toxicity test
b. Developmental… (omitted) …
toxicity testc. Carcinogenicity test
… (omitted) …
c. Carcinogenicity
test
d. Mutagenicity test
d. Mutagenicity test Conduct the test for gene mutation inducibility using six Salmonella typhimurium strains(TA98, TA100, TA102, TA1535, TA1537 and TA1538) and Escherichia coli bacteria
WP2uvrA. Six dose levels of the objected substance were arranged with the 5,000 µg/plateas the maximum dose. Three plates for each test strains were cultured with the respective
dose levels of the objected substance, and each of those plates were conditioned with andwithout the presence of the S9 mix for metabolic activation, along with negative control andpositive control groups, at 37℃ for 3 days. After the culture, the revertant colonies were69

counted for each individual plate.

[Results]
The results are summarized in the table below (Table 5
Table 5 Number of revertant colonies of Salmonella typ
S9mix

(-)

(+)

Concentration(µg/plate)
0*1
1.6
8.0
40.0
200.0
1,000.0
5,000.0
Positive
control
0*1

Salmonella ty
TA100

TA98
13
15
14
13
15
14
11
12

160
155
152
162
161
158
169
163

(omi
(omitted)e. Other tests(*1) Negative control
No increase in the number of revertant colonies wa
including the maximum dose group, regardless of theactivation.
Based on the above results, it can be concluded that t
mutagenicity.
… (omitted) …
e. Other tests
Other tests were not conducted.

iii. Pharmacological
test

70

iii. Pharmacological test
The pharmacological test was omitted because the objected substance does not have any
iv. Test concerning pharmacological effects.
the in vivo kinetics
iv. Test concerning the in vivo kinetics and metabolism
and metabolism
A single dose of the objected substance 12C was replaced by 14C, and was administered via
an oral route to a mouse, cow, dog and a human. All of the urine and feces excreted within 24
hours from the time of the administration were collected for the analysis of the dynamic stateof 14C inside the bodies. Information about the tested individual mouse, cow dog and humanis as follows. … (omitted) …
[Results]
Mouse: The 14C excretion in 24 hrs was 0.48% in the urine and 89.2% in the feces; almost all
the 14C was excreted into the excreta in 72 hrs.; and 97.5% of the total excreted radioactive
substance in the excreta was in the form of metabolites with 1.5% of the original substanceat the 72 hr time point from the administration. The metabolite had experienced an
O-demethylation reaction and had bioactivity. The level of the metabolite bioactivity wasmeasured at 0.01, compared to that of the objected substance being 1.0. Almost all of theobjected substance (approximately 98%) was metabolized in the liver and excreted as
metabolites. Similar data was obtained in the cow: 98% of the objected substance wasmetabolized in the liver and was excreted as metabolites.
Table 6 Results of the quantitative analysis of 14C in the feces in the single dose
administration test of the 14C labeled objected substance via an oral route (ratio in a
100-scale against the total administered 14C)
Animalspecies
Mouse
Cow

Test sample
Urine
Feces
Urine
Feces

24
0.48
89.29
0.61
89.44

Elapsed time (hrs.)
48
72
0.66
0.32
4.34
3.91
0.52
0.10
5.78
2.98

3 days total
1.46
97.54
1.23
98.20

(omitted)

Scintigraphy for the analysis of the dynamic state of the radioactive substance inside thebody, as well as an infrared spectroscopy and nuclear magnetic resonance spectroscopy for71

(2) Feeding test
using targetedanimals(3) Test concerning
the emergence ofresistant bacteria(4) Other tests

the identification of the chemical structure were used. The details of the method are as
follows. … (omitted) …

(2) Feeding test using targeted animals
Testing place and environment: xx laboratory in xx prefecture (indoor pig house)
Test animals: Pigs (Strain: xx; 30 days old; average body weight: 8.2 kg), 15 pens each ofmales and females in a group
Dose and method of administration: Continuous administration was given by mixing theobjected substance in the feeds for 3 months. Consumption of the water and feeds by thetest animals was discretionary. The dose was 50–800 mg/kg (with 80 mg/kg as the optimumdose).
… (omitted) …
[Results]
The results are summarized in the table below (Appendix Form 7). The live weight gain of the
80 mg/kg group was 15.4 kg larger than that of the control group (p<0.01). Although anincrease in both the live weight gain and the intake of feeds was shown in the group given a
10 times larger dose than the optimum dose, a significant difference was not indicated whencompared to the group without the additive. Two female pigs became anorexic, while no
noticeable changes were observed as general symptoms. One of these two pigs showed amild gastric erosion, which was cured in a week or so. After the cure, the live weight gainincreased.
… (omitted) …
(3) Test concerning the emergence of resistant bacteria
This test was not conducted because the objected substance is not an antimicrobial agent,
nor a live microbial agent.

(4) Other tests
This test was not conducted because no effects are anticipated by the use of the objected
substance as a feed additive.

72

List of the original papers used for the preparation of the Abstract
No. oforiginal
paper

Title of original paper

1(omitted)

2(omitted)

3(omitted)

4(omitted)

5(omitted)(omitted)73

１

Title of Original Paper 1*The original papers (attachments), etc., cited in the abstract are exhibited on the next page
and the following pages.

74

V Closing
This handbook has been drawn up for those who are preparing the documents
required for the designation of a mainly chemical substance as a feed additive
for the first time.
For a feed additive designation to be granted, substantial documents are
required to prove the substance’s efficacy, safety, etc. Additional documents
may be requested during the professional deliberations conducted by the
council members. Therefore, it should be borne in mind that the submission of
the documents that are prepared in full conformity to the requirements of thishandbook does not necessarily guarantee the substance’s successful
designation, even if the intended substance is a chemical substance. If theintended substance is an antibiotic or a live microbial agent, significantly
different documents are required from those needed for chemical substances.
Please contact the authorities or the Japan Scientific Feed Association withany questions.
In closing, we hope that this handbook will come in useful for those who arepreparing such documents.
We would like to thank the Japan Scientific Feed Association and many othergroups and individuals for their support and advice during the preparation ofthis handbook.

Contact
Feeds Safety Standard Group,
Animal Products Safety Division,
Food Safety and Consumer Affairs Bureau,
Ministry of Agriculture, Forestry and Fisheries
Postal Code: 100-8950
1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo
Phone: 03-3502-8111, Ext.4546
General Incorporated Association
Japan Scientific Feed Association
Postal Code: 104-0033
2-6-16 Shinkawa, Chuo-ku, Tokyo
Bajichikusan Kaikan Bldg.
Phone: 03-3297-5631

75

VI Checklist for the Submission of the Required Documents
Review and make sure that following items are properly completed, then enter a check mark in each check box (bold-outlined box) andsubmit it with the abstract.
Leave check boxes blank (no check mark) for items that are not relevant or are not confirmed.

Checked by:
Check
box

Items to be checked[In writing the abstract]
Ensure unity of the character style (Japanese characters, alphanumeric characters), with no mixing of one-byte and two-bytecharacters.
Use a 12 point or larger font size, and do not use characters that are extremely difficult to read.
Ensure there are no typographical errors, omissions, or transcription errors.
Use proper punctuation (spaces, paragraphs, line feeds, etc.) for better readability. Split long sentences into two sentences.
Use the unit signs commonly used in Japan (if not, convert these).
[Example] pound→kilogram; ° → ℃; ppm→mg/kg or µg/mL
Use general names, not trade names.
Ensure uniformity of the writing style.
[Example] Use general names; xx sodium salt (hereafter referred to as “xx”); xx salt of hydrochloric acid or hydrochloric acidxx salt
Use tables and figures effectively for easy-to-understand explanations.
[Example] Number tables and figures (Table 1, Figure 1, etc.).
Clearly distinguish facts from presumptions. Where facts are stated, supporting documents have to be attached.
The abstract must have a Table of Contents with a page index.
Bind the documents with a front cover and back cover, both having the title of the document and the company name.
The abstract is laid out in line with the “Notification on the Documents, etc., Required for the Submission for the Designationof Feed Additives” (issued on February 4, 1980).
If there are findings from a previous deliberation, those findings and the response papers have to be included.
Any data and the descriptions of items which are the same as previously submitted documents must be noted accordingly,
76and the relevant documents will still have to be attached and included.
[Attachments – Original papers, reports, etc.]
Make sure that there are no potential copyright infringements in the use of original papers.
Number original papers (attachments) with serial numbers, create a list of these, and bind them using indexes, fileseparators, etc., for easy-to-read documents.
Make sure that the document numbers correspond accurately to the original papers (attachments). Make sure that there are
no mistakes nor gaps in the numbering.
Make sure that cited texts from original papers (attachments) are marked using underlines, highlights, etc., for easy
searching.
(This is required only for the documents submitted to the authorities. The documents prepared for committee members thatare otherwise instructed, or the documents for the deliberation by the council do not require this marking.)
Make sure that all cited original papers (attachments) are included.
Make sure that all technical terms written in English are translated into the correct Japanese terms.
Make sure that the information and contents of the original papers (attachments), etc. are not misconstrued.
[Standards]
Check whether the tone of the description of items concerning the standards for a new substance accord with those for
known substances.
[Reference] List of the Specifications and Standards of Feed Additives (General Incorporated Association, Japan ScientificFeed Association)
Are the identification tests simple enough to be repeated in an actual operating environment?
Has the possibility of performing the identification test by a known identification test method been discussed?
Are the manufacturing processes of the ingredients for the manufacturing and the formulation described in detail?
Has the provision of samples to the FAMIC been arranged?
[Tests]Have the tests that are subject to the “feed additive GLP” been performed in accordance with these principles?
Are the facts and discussions clearly separated in the description of the test results?
Is the test program described in enough detail to be duplicated?
Are the values of the concentration in the feeds converted into the daily intake amount per unit of body weight?
[Example] ppm, mg/kg → mg/kg-BW/day[Reference] Cite the data of the daily feed intake and the body weight of the livestock animals from the literature, etc., (or77from the Japan Feed Standard if no relevant literature can be found) and then calculate the converted value on an as-neededbasis: concentration in feeds (mg/kg-feed) x daily feed intake/body weight.
Example of the feeds added at a concentration of 100mg/kg (daily feed intake: 2.89 kg/day; animal body weight: 93 kg-BW),
100 mg/kg×2.89 kg/day/93 kg-BW = 3.11 mg/kg-BW/day (round off the value on an as-needed basis)
Have the test results been statistically analyzed? When a significant difference is shown, the details of the analysis must be
described.
[Example]…(omitted)… a significant difference was shown (t-test: p<0.05)
Are appropriate significant digits used for the data description?
Difference between “1” and “1.0”: “1” indicates a value in the range of 0.5–1.4; “1.0” indicates a value in the range of 1.05–
1.14.
Check for any missing items to be described in each test: number of test animals, period of administration, tests omitted even
if an omission not permissible, conformity to the evaluation standards, etc.
Items Concerning Standards, Changes with Items Concerning Efficacy
Items
Concerning
Time
Residue
Each storage test
Stability tests
Basic tests
Field application tests Residue test
Items Concerning Safety
Single dose toxicity Repeated dose toxicity Repeated dose toxicity Pharmacological test
test
test (short term)
test (long term)
Transgenerational
reproductive
toxicity test

Developmental
test

toxicity Carcinogenicity test

Mutagenicity test

In vivo kinetics test

Feeding test

In cases where tests, etc., have been omitted, has the adequacy of the omission been presented, or is the submitter ready topresent this when requested?

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