Handbook of Feed Additive Designations (Chemical Substances Edition)

Handbook of Feed Additive Designations (Chemical Substances Edition) Edition No. 1.10 April 2021 Animal Products Safety Division 1 Contents Ⅰ Purpose of the Handbook (p. 3) 1. Introduction (p. 3) 2. About Feed Additives (p. 4) Ⅱ Basic Ideas in the Preparation of the Required Documents (p. 10) 1 Flow of the Procedure for a Feed Additive Designation (p. 10) 2 Outline of the Required Documents (p. 16) Ⅲ Items to be Described in the Required Document (Abstract) (p. 18) 1 Origin or Background of the Discovery, Status of Authorization and Use as a Feed Additive in Foreign Countries, etc. (p. 20) 2 Items Concerning Standards (p. 22) (1) Name (p. 22) (2) Chemical structure (p. 22) (3) Manufacturing process (p. 22) (4) Biological and physicochemical properties (p. 26) (5) Quantitation in feeds (p. 28) (6) Changes with time (p. 28) 3 Items Concerning Efficacy (p. 34) (1) Basic tests to prove efficacy (p. 34) (2) Field Application tests to prove efficacy (p. 34) 4 Items Concerning Residue (p. 43) 5 Items Concerning Safety (p. 46) (1) Toxicity tests (p. 46) (2) Feeding tests using targeted livestock, etc. (p. 69) (3) Tests concerning the emergence of resistant bacteria (p. 72) (4) Other tests (p. 72) Ⅳ Exemplary Abstract (p. 77) Ⅴ Closing (p. 94) Ⅵ Checklist (p. 95) 2 [Reference 1] Glossary (p. 5) [Reference 2] Points to note when describing the test data (omission of tests, use of new knowledge, etc.) (p. 19) [Reference 3] Case of a discussed/authorized substance in foreign countries as an additive (p. 21) [Reference 4] Points to note when describing the manufacturing process (p. 24) [Reference 5] Diluents and dilution materials (p. 24) [Reference 6] Stabilizers, etc., used to enhance the usability or stability of feed additives (p. 25) [Reference 7] Points to note when considering composition standards (p. 27) [Reference 8] Points to note when describing the test results (p. 39) [Reference 9] Phytase Efficacy Test (p. 40) [Reference 10] Conditions to be complied with in conducting animal tests (p. 42) [Reference 11] Points to note when recording safety test results (p. 47) [Reference 12] Tests for analyzing in vivo kinetics (p. 68) [Reference 13] Style reference for the description of items concerning standards (p. 72) 3 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 the livestock themselves that consume the feed. For the same reason, feed additives, which must serve purposes such as preventing the quality deterioration of feed as a matter of course, 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 policy of a substantial necessity, only to those substances that are in high-demand and have been 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 be designated 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 must be prepared and submitted to the Animal Products Safety Division of the Food Safety and Consumer Affairs Bureau of the Ministry of Agriculture, Forestry and Fisheries (hereinafter referred to as “the secretariat”). Those submissions which are judged worthy of a hearing by the council will then be sent to the council for deliberation. 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 results of 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 contain technical subjects, such as scientific test results and manufacturing process of the substances, and so on. Based on the above consideration, this handbook explains the procedures and points of caution in preparing the required documents (especially the abstract) for the deliberations on a feed additive by the Agricultural Materials Council in reference to the process 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, and that the required documents for seeking a designation of live microbial agents or antibiotics, etc., are quite different from those explained herein. Please additionally note that following the procedures described in this handbook for the preparation of the required documents does not necessarily ensure success in the granting of a designation. 4 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 the Agriculture, 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 other uses than these three are not designated as feed additives: a. Preventing feed quality deterioration b. Supplementing feeds with nutrients and other active ingredients c. 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 Ordinance for Enforcement of the Feed Safety Act) A feed additive designation shall be granted, to the minimum extent required, to those substances that are highly necessary and that have clearly proved to be effective and safe, as has been done in the past. Therefore, those who are planning to newly manufacture, import, etc., a non-designated feed additive as a feed additive must consult with the secretariat in full, well before any actions are taken, and receive instructions from it. 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 and Feed 5 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 the Documents, etc., Required for the Feed Additive Designation (Ministry of Agriculture, 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 the above listed laws, etc.).  Safety factor A factor applied to the no-observed-adverse-effect-level (NOAEL), for the determination of the Acceptable Daily Intake (ADI), etc., of a substance from the perspective 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 those values (100) is used as the safety factor.  Acceptable daily intake (ADI) 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. [Reference 1] Glossary Explanations of the technical terminologies are provided as follows. Source: “Glossary Concerning Food Safety” by the Food Safety Commission “Guideline for Total Diet Studies” by the Ministry of Agriculture, Forestry and Fisheries 6  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 the following: i. AOAC (Association of Official Analytical Chemists) International: a concentration 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 concentration equivalent 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 for the calculation. Note that the formula “LOD = LOQ*” is scientifically false. * See the “Limit of quantitation (LOQ)” below.  Maximum residue limit (MRL) The maximum permissible concentration of agricultural chemicals, feed additives, etc., remaining in food. 7  Good Laboratory Practice (GLP) A set of principles and rules to be complied with in conducting tests concerning the safety and persistence of feed additives, formulated to achieve a higher reliability of the documents, and a more accurate and strict performance of the safety assessment. (For details, please refer to the Reference 10 "Conditions to be complied with in conducting animal tests”).  Micronucleus test One form of mutagenicity tests, to assess the chromosome aberrations by examining 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 (its absorption, distribution, metabolism, excretion, etc.) by administering the substance to animals.  Transgenerational reproductive test (Transgenerational reproductive toxicity test) A test to assess the reproductive toxicity of a substance by administering it to animals.  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 blank value and 10σ (wherein σ is a standard deviation of the distribution of a blank measurement) ii. Codex (a definition in the criteria approach): a concentration equivalent to the sum of the mean blank value and 6σ, or the sum of the mean blank value and 10σ (wherein σ is a standard deviation of the distribution of a blank measurement)  Spike recovery test A test to spike a predetermined amount of an objected substance to analyze and examine whether the spiked amount can be accurately quantitated. 8  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 the substance.  Developmental toxicity test (Teratogenicity test) A test to assess the developmental toxicity (teratogenicity) of a substance by administering the substance to animals. The doses are administered during the organogenesis 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 the measurement of the samples which are identified as the same, conducted by the same method and by the same experimenter, and under conditions that are controlled to produce independent results within a short period of time.  Mutagenicity (Genotoxicity) 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. 9  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.) are found to be in excess of the predetermined level (0.01 ppm). As to a substance for which the standard of a food ingredient is prescribed, the criteria specified in that standard will apply. Otherwise, the value of 0.01 ppm (a concentration causing no potential 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 substance over 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 to animals.  No-observed-effect-level (NOEL) The maximum dose of a substance at which no biological impact was found.  No-observed-adverse-effect-level (NOAEL) The maximum dose of a substance at which no adverse impact was found.  Pharmacological test A test to clarify the actions of a substance that has been administered to living organisms.  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. 10 Ⅱ Basic Ideas in the Preparation of the Required Documents 1 Flow of the Procedure for a Feed Additive Designation The procedure up until a new feed additive designation is granted is, in principle, as illustrated below. The designation is made through deliberation by the Agricultural Materials Council (Ministry of Agriculture, Forestry and Fisheries), the Ministry of Health, Labour and Welfare, and the Food Safety Commission. Procedure for Safety Assessment of a Feed Additive Submission of data on efficacy, safety, etc. Request for cooperation in setting standards Cooperation Agricultural Materials Council Feed Subcommittee (deliberation on efficacy and safety) Subcommittee (deliberation on standards) Subcommittee Deliberation at a subcommittee meeting for agrichemicals and veterinary medicines Deliberation at the Food Sanitation Subcommittee Request for assessment Deliberation results Deliberation at the Food Safety Commission Report Entrustment Answer Report Genetically modified (GM) feed additives are also checked for safety. Deliberation results Ministry of Agriculture, Forestry and Fisheries Notice of Deliberation Council Notice of application invitation JSFA Submitting documents Consult about the efficacy and safety to livestock Hearing on the feed additive designation, etc. Request for the assessment on impact to human health through food Food Safety Commission (Deliberation at Expert Panel on Fertilizer, Feed, etc.) Ministry of Health, Labour and Welfare Pharmaceutical Affairs and Food Sanitation Council Ministry of Agriculture, Forestry and Fisheries Feed Additive Designation (Announcement on Government Gazette) Public Comment and Report to WTO/SPS Feed Additive Efficacy and Safety Feed Additive Standards Deliberation at the Feed Subcommittee (consisting of experts and consumer committee members) Feed Safety Committee 11 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” for details of the required documents. The flow of the procedure from the preparation of the required documents to the deliberation council is as follow: 〈Preliminary consultation〉 It is essential for a business operator who is planning to manufacture or import any non-designated feed additive to consult with the authorities beforehand. In order to do so, please prepare the attached document outlining the objected substance beforehand. ↓ 〈Preparation of the required documents〉 While the secretariat is available for consultation at any time, substances other than those whose applications have been received within the deliberation council’s application acceptance period won’t be subject to the discussions concerning the application’s eligibility as a case for the deliberation council. ↓ 〈Deliberation council application acceptance period〉 Application to the secretariat The secretariat takes applications through the Japan Scientific Feed Association (JSFA). Please make sure that all required documents have been properly prepared before submitting the application to the secretariat (*1). Note that deliberation will not be conducted in the following cases: ・When the required documents are inadequate. ・When an application contains too many subjects pertaining to matters of re deliberation from previous deliberations. ・When the objected substance does not fulfill the requirements for a feed additive designation, etc. ↓ When the secretariat decides 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 for the deliberation council (about 20 sets) will have to be prepared before the deliberation. ↓ 〈Deliberation Council of the Feed Additive Efficacy and Safety Subcommittee, Feed Safety Committee, Agricultural Materials Council〉 Explanation (within 10 minutes), and questions and answers before the deliberation council With regard to new substances, (i) the efficacy and safety of the feed additive will be deliberated by the Feed Additive Efficacy and Safety Subcommittee, Feed Safety Committee, Agricultural Materials Council. *2 12 ↓ 〈Deliberation Council of the Feed Additive Standards Subcommittee, Feed Safety Committee, Agricultural Materials Council〉 After the efficacy and safety of the objected substance have been endorsed by the Feed Additive Efficacy and Safety Subcommittee of the Feed Safety Committee, (ii) the standards of the feed additive will be deliberated at the Standard Subcommittee of the same Committee at a later date. *2 (*1) In principle, all of the required documents must be procured prior to application. However, in the case of ambient temperature storage tests for which a 24-month test period is required, if 12 months have passed since the test started, an application may be made for an efficacy and safety examination even when the test has not been completed. (*2) 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 conducted again. 13 After the acceptance of the substance by the Feed Additive Efficacy and Safety Subcommittee, 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 (Expert Panel on Fertilizer, Feed, etc.). 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, as the assessment reports, etc. drawn up by the deliberation council and others may contain information that is relevant to patents, classified information of the submitter, etc. 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 by the Feed Additive Efficacy and Safety Subcommittee and the Feed Additive Standards Subcommittee of the Feed Safety Committee under the Agricultural Materials Council, the feed additive will be discussed at the Feed Subcommittee. Agricultural Materials Council Feed Subcommittee Feed Safety Committee: Feed Additive Efficacy and Safety Subcommittee Feed Additive Standards Subcommittee Livestock feed Pisciculture feed Pet animal feed Genetically modified feed committee Feed nutrition committee Livestock feed Pisciculture feed The deliberations of the council will be conducted based on the submitted documents prepared by the submitter. The council members will discuss in a scientific manner whether the efficacy and safety of the objected substance are assured 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 of the quantitative methods, etc., that have been employed will be discussed. Based on the submitted documents, the secretariat will scrutinize whether the objected substance is eligible for deliberations in light of the Standard for the Evaluation of Feed Additives and whether the contents of the submitted documents have fulfilled their requirements. 14 Furthermore, if the objected substance is a substance (enzyme, etc.) made from genetically modified microorganisms (bacteria, etc.), it must be subjected to further deliberation as a genetically modified feed additive (at a genetically modified feed committee) in addition to the deliberation at the Feed Safety Committee. Deliberations by the Feed Safety Committee and the Genetically Modified Feed Committee can progress in parallel. When using genome-edited microorganisms such as bacteria, etc., please consult with the secretariat beforehand. 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 a hearing in public and will issue a report. Feed Safety Committee: Feed Additive Efficacy and Safety Subcommittee This subcommittee will conduct closed-door deliberations regarding the efficacy and safety to livestock of the feed additive. Feed Safety Committee: Feed Additive Standards Subcommittee This subcommittee will conduct closed-door deliberations regarding the appropriateness of the specifications and standards for the feed additive based on the deliberation results of the Feed Safety Committee’s Feed Additive Efficacy and Safety Subcommittee. 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 the feed 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 (Expert Panel on Fertilizer, Feed, etc.) The Food Safety Commission was established on July 1, 2003 in accordance with the Food Safety Basic Act. It conducts risk assessments regarding the impact of hazardous elements, such as additives and agricultural chemicals, etc., which may be contained 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 a serious adverse impact on human health and to what degree of probability this will 15 occur, are scientifically assessed. The dietary risk assessment is a requisite for the designation of new feed additives, as well as changes of standards for labeling and manufacturing, 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 of food products. In terms of the feed additive designation, the FAMIC examines the methods of analysis based on the submitted documents. More specifically, it determines whether the methods of analysis have been adequately described in the items relating to the standards of the feed additive. In addition, it may conduct an inspection of the facility to determine whether it complies with the principles of Good Laboratory Practice (GLP) on an as needed basis. (Refer to Reference 10: “Conditions to be complied with in conducting animal tests” 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 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 of technologies and the broader use of such technologies. It is organized by the members of companies, etc., which manufacture feeds and feed additives. Notifications about application invitations from the deliberation council will be made by the secretariat through JSFA. WTO/SPS Notifications The Agreement on the Application of Sanitary and Phytosanitary Measures (SPS Agreement) is an Agreement of the World Trade Organization (WTO). Under this agreement, when a member state modifies its sanitary and phytosanitary measures, the other member states must be notified of this (SPS notification). Similarly, in addition to public comments made domestically, notifications shall also be sent to member states in regards to new designations for feed additives, etc. 16 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 the designated forms with the summary of the test results.*3 (3) The documents to be submitted to each deliberation council are listed below. When preparing an abstract, refer to “IV. Exemplary Abstract.” Before submitting a proposal to the secretariat, check the checklist shown in the last part of this handbook closely. Feed Safety Committee, Feed Additive Efficacy and Safety Subcommittee a. Abstract (including appendix form) b. Original papers cited when preparing the abstract Food Safety Committee, Feed Additive Standards Subcommittee a. Abstract (items from “3. Items Concerning Efficacy” and below can be omitted in principle.) b. Original papers cited when preparing the abstract c. Comparative Tables 1 and 2 for deliberating standards and codes (forms are shown on the page after “IV. Exemplary Abstract” - Appendix Form 7.) d. Method of testing items specified as composition standards (Japanese) If the proposed test method is one specified by a ministerial order or an official method adopted overseas, please state this. Submit flow charts, if any (English versions are also acceptable.) e. Analysis results that prove conformance to standards as a result of the test method(s) In the case of a quantitative method: 3 lots x 3 points or more In the case of a qualitative method: 3 lots x 1 point or more Please also attach measurement conditions, raw data, chromatography, photos, etc. (Descriptive examples are shown next to the forms for Comparative Tables 1 and 2.) Note: You may be requested to submit spike recovery test results (e.g., concerning lead or arsenic) and third-party organization analysis results if a prior consultation with the secretariat (FAMIC) or a deliberation by the Feed Safety Committee (the Feed Additive Efficacy and Safety Subcommittee and the Feed Additive Standards Subcommittee) has led to the conclusion that they are necessary. If you have spike recovery test results, etc. as mentioned above, please submit them beforehand. Note that if any findings are pointed out by the council members in the deliberation, the responses to the findings shall be presented in the following deliberation. In such a case, examples of the required documents are as follows: a. Response paper regarding specified items (describe the test design, and fill in an appendix, etc., according to the guidebook.) b. Original papers cited when preparing the response paper regarding specified items 17 c. Revised abstract based on the specified items (when necessary) d. Original papers cited when preparing the abstract (when necessary) 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) 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) Note 3: Notification Concerning the Documents, etc., Required for the Feed Additive Designation (Ministry of Agriculture, Forestry and Fisheries Agency Director-General Notification No. 54-Chiku-A-5002 issued on February 4, 1980) In order to facilitate the smooth deliberation of the council, the secretariat will check the submitted documents before holding a meeting of the deliberation council and will confirm whether the items are described in conformity with the Standard for the Evaluation of Feed Additives and whether they are objectively described based on test results. Insufficient descriptions in the submitted documents will require that more time be spent on the document checks by the secretary. In addition, the submitters may be requested to revise the documents. Please be advised that such circumstances may result in a delay of the deliberation by the council and of the feed additive designation. In the following chapter, the procedures for the preparation of the required documents (especially the abstract) are explained with reference to examples. 18 Ⅲ Items to be Described in the Required Document (Abstract) This chapter provides concrete explanations about the items to be described in the abstract (including appendices), with reference to the “Notification Concerning the Documents, etc., Required for the Feed Additive Designation” and the “Establishment of the Standard 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. 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 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 test iii. Purity test iv. Content and quantitative method (5) Quantitation in feed (6) Changes with time i. Ambient temperature storage test 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 efficacy i. In vitro test ii. In vivo test (2) Field application tests to prove efficacy 4. Items Concerning Residue Residue tests using targeted livestock, 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 test c. Carcinogenicity test d. Mutagenicity test e. Other tests iii. Pharmacological tests iv. In vivo kinetics tests (2) Feeding tests using targeted livestock, etc. (3) Tests concerning the emergence of resistant bacteria (4) Other tests 19 [Reference 2] Points to note when describing the test data (omission of tests, 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 is designated as a food additive, or is widely used in foods, can be omitted; also, some of 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 the adequacy 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) have been found at the time of seeking the feed additive designation, which was not known 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. 20 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 discovery of the objected substance that has resulted in the idea to use it as a feed additive. Moreover, describe the advantages it will bring about and how the current conditions will improve when the feed additive designation is granted to the objected substance. Also, please list the following items as the basic information regarding the objected substance: (1) Which of the three uses specified in the Feed Safety Act the objected substance is to be used for; (2) What kinds of livestock are to be targeted and at which stage (period of the dose); (3) The recommended dose and method of use (describe the types of feedstuff the feed additive is added to). ○Status of authorization and use as a feed additive in foreign countries, etc.： If the substance is used as a feed additive in Europe and America, etc., describe the reasons for its authorization (targeted livestock, uses, additive amount, etc.). (See the [Example] below) ○Status of the manufacturing and distribution authorization, and its importation as a veterinary 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］ Table 1 summarizes targeted livestock and others. Table 1: Targeted livestock, etc. Targeted livestock Stage Recommended additive amount Target feedstuff Purpose Broiler About 3 weeks after incubation 12 mg/kg Feedstuffs deficient in inorganic phosphorus Somatic growth of livestock animals Pig Suckling period 8 mg/kg Feedstuffs deficient in inorganic phosphorus Somatic growth of livestock animals … Omitted … The status of the substance as a feed additive in foreign countries is summarized in table 2. Table 2: Designation status in foreign countries, etc. Country Status of studies and authorization Targeted livestock Additive amount Purpose United States of America Designated in xxxx Pigs and cattle 12 mg/kg (upper limit of additive amount) Somatic growth, etc. EU Pending (submitted in xxxx) Pigs (planned) 18 mg/kg (upper limit of additive amount) Somatic growth, etc. Related substance: This agent is an in-vivo metabolite of xxxx and is designated as a feed additive. This agent is intended to be added to feedstuffs as an alternative to xxxx, and because its bioavailability is higher than that of xxxx, it is possible to reduce the additive amount. 21 [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 an additive in foreign countries may be used as a substitute for the documents required for the deliberation of its designation in Japan, depending on the test items. When using these documents, submitters are often requested by the secretariat or the deliberation council to 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 settings and assessments at international institutes, such as the Codex. Therefore, please review such information thoroughly and be prepared for such requests. (In the case of a substance that has been already assessed by the EU, please attach the relevant EC regulatory rules and EFSA journal to the application document.) Note that because the deliberation is to be conducted according to the Japanese Standard for the Evaluation of Feed Additives, the documents used for deliberation on the substance’s designation in foreign countries may not suffice. 22 2 Items Concerning Standards To ensure the safety of livestock and humans, any hazardous impurities which can be contained in feed additives and the purity of the product must be specified. The specifications and standards specified in the “Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives” shall be described in line with the writing style and definition of the terminology set out in the Appendix Table 2-1 “General rules for feed additives” attached to the above-mentioned ordinance. Therefore, please draw up the required documents according to the rules set out in the general rules. ([Reference 13] Style reference for the description of items concerning standards) (1) Name i. 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 a chemical 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 List manufacturing methods and related flow diagrams for ingredients used in manufacturing, as well as any formulations. Please also provide the names of process aids, pH adjusters, and by-products. If a raw material manufactured by a recombinant technique or the like has been used, please clearly state this. Moreover, components contained in the formulations should be summarized in a table. ［Example］ Ingredients for manufacturing: Oxidize Substance A (99.5%) with air using a platinum catalyst to produce Substance B. Initiate hydrolysis by adding sodium hydroxide to special grade ethanol to produce unrefined xxxx. Refine this through solvent extraction using hexane, then dehydrate to yield the objected substance xxxx. Formulation 1: The ingredients for manufacturing are mixed with cornstarch, substance D, …. Moreover, substance E or F is added and mixed as necessary. Powder or particles obtained by powdering or pelletization are the formulation 1. Formulation 2: A hydrosoluble liquid substance obtained by mixing the ingredients for 23 manufacturing with substance G and purified water. Substances contained in formulation 1 Substance name Ratio (%) Diluent, etc. of formulation (Note 1) Stabilizer, etc. (Note 2) Other Ingredient for manufacturing Cornstarch ○ Substance D (designated feed additive) ○ Substance E (Ordinance for Enforcement of the Food Sanitation Act, Appendix No.1) ○ Substance F (Note 3) ○ (Note 1) Appendix 2 of the “Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives” (Note 2) Stabilizers, etc., specified in the “Application of the Act Concerning the Safety Assurance and Quality Improvement of Feed” (Note 3) Used for the purpose of …. Substance F is included as “xx agent” in the Pharmaceutical Excipient Standard of Japan. In xx (country), the use of substance F as “xx agent” in food has been allowed. Substances contained in formulation 2 Substance name Ratio (%) Diluent, etc. of formulation (note 1) Stabilizer, etc. (note 2) Other Ingredient for manufacturing Substance G (note 3) ○ Purified water ○ (Note 1)・・・ Substance A (99.5%) ↓platinum catalyst Substance B ↓sodium hydroxide (special grade ethanol) xxxx(unrefined), (by-product C) ↓solvent extraction by hexane Objected substance (ingredient for manufacturing) xxxx Manufacturing process flowchart Formulation 1: Ingredients for manufacturing ↓Mixed with cornstarch, etc. ↓Powdering or pelletization Formulation 1 Formulation 2: Ingredients for manufacturing ↓Mixed with substance G and purified water Formulation 1 24 [Reference 4] Points to note when describing the manufacturing process Some raw materials may be omitted in the final standards, but in the submitted documents, the names of any raw materials must be provided. Listings for standards provided in ministerial orders shall be arranged at the secretariat in consultation with business operators. In such an arrangement, the fundamental concepts are as follows: ・A substance that corresponds to a diluent shall be listed as a “diluent” and not named separately. ・There is no need to state the names of stabilizers (because they are usable as long as they conform to the conditions specified in the “Application of the Act Concerning the Safety Assurance and Quality Improvement of Feed”). [Reference 5] Diluents and dilution materials Appendix 2-3 (6) of the “Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives” shows substances corresponding to diluents, etc. The use of substances specified as “diluents” in the composition standards for formulations are allowed. However, liquid feed additives not specified in the articles cannot be used. This is because their stability as formulations may not be ensured if they are combined with diluents and dilution materials. (An example of composition standards for powder formulations) This substance is powder made by mixing an ingredient for manufacturing sodium alginate with a diluent. (An example of composition standards for liquid formulations) This product is an oily liquid or a water-soluble liquid substance made by mixing an ingredient for manufacturing ethoxyquin with glycerin, hardened oil, saturated higher fatty acid, fatty acid, vegetable oil, or animal fat. 25 [Reference 6] Stabilizers, etc., used to enhance the usability or stability of feed additives The “Application of the Act Concerning the Safety Assurance and Quality Improvement of Feed” (Ministry of Agriculture, Forestry and Fisheries, Fisheries Agency Director General Notification No. 12-Seichiku-1826 issued on March 30, 2001) specifies the following: Even if not specified in the Articles, stabilizers, etc., that correspond to the following can be used to the minimum extent required if they are intended to enhance the usability or stability of a feed additive. This also applies to liquid feed additives. * For concepts regarding substances used as diluents and dilution materials, refer to reference 5. An excerpt from the “Application of the Act Concerning the Safety Assurance and Quality Improvement of Feed” (Ministry of Agriculture, Forestry and Fisheries, Fisheries Agency Director-General Notification No.12-Seichiku-1826 issued on March 30, 2001) i. General rules on feed additives (Appendix No. 2 of the ministerial order concerning composition standards) The general rules state that stabilizers, lubricants, binders, moistening agents, emulsifiers, coating agents, dispersants, disintegrants, preservatives, or solubilizing agents can be used as formulations in order to enhance the usability or stability of feed additives. However, such substances must fall under category a, b or c below and satisfy the requirements of Appendix No. 2 3 (5). The amounts used must be restricted to the minimum necessary to manufacture the formulation. The names of those which were used must be indicated on feed additive packages according to the provisions of Appendix No. 2-5 (2), vi. They must be indicated using generic names. a. Natural products b. Feed additives (excluding antibacterial substances other than propionic acid, calcium propionate and sodium propionate) and diluents specified in the i i f A di N 2 8) 26 (4) Biological and physicochemical properties i. Physical and chemical properties Describe the physical and chemical properties of the objected substance. ・Appearance of the objected substance itself (color; state: powder, liquid, etc.) ・Information on solubility in a solvent and denaturation (deliquescency, melting point, photodegradability, etc.) ・There is no need to describe odor or taste. ・In the case of enzymes, regarding the pH at which the activity of a certain enzyme is maximized, list the reason(s) why the pH has been proposed. ［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 practically insoluble 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 the infrared 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 substance In principle, show standards for “lead” and “arsenic.” Even if the substance is described as a “heavy metal” in the reference feed additive, describe it as “lead” in principle when newly setting a standard. ［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 practically clear. b. Chloride limit: When conducting a chloride limit test with 1.0 g (0.95–1.04 g) of this substance, 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. c. Lead: When using 1.0 g (0.95 to 1.04 g) of this substance to perform the limit test for lead (Atomic Absorption Spectrophotometry, Method 1), it must not exceed 10 μg/g. 27 d. Arsenic: When using 0.40 g (0.395 to 0.404 g) of this substance …. … (omitted) … iv. Content and quantitative method A method to quantitate the objected substance contained in the analyte ［Example 1］ Content: This substance contains a fixed quantity of 99.5% or more of the objected substance (chemical formula of the objected substance) after drying at x ºC for x hours. Quantitative method: Dry the substance, weigh out 0.5 g of the material to the order of 0.001 g digits and record the reading. Dissolve the weighed substance in 50 mL of water and add 5 mL of acetone and titrate with 0.5 mol/L sodium hydroxide solution (use three drops of the phenolphthalein 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 of the objected substance … (omitted) … [Example 2] Content: When determining the quantity of this substance, a dried matter having been converted … (When performing corrections based on weight loss on drying, as in the case of peptide zinc) [Example 3] Content: When determining the quantity of this substance, a dehydrate having been converted … (When performing corrections based on a moisture value by means of Karl Fischer’s method) [Reference 7] Points to note when considering composition standards 1. Refer to the standards for the objected substance or for substances similar to it. Examples of reference materials are as follows: ・Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives (Appendix 2) ・Japanese Standards of Food Additives ・Japanese Pharmacopoeia ・European Pharmacopoeia ・United States Pharmacopoeia ・Food Chemical Codex 2. Regarding color description, refer to JIS Z 8102-2001 “Names of non-luminous object colors.” 28 (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 a matter specified in the composition standard. However, from the standpoint of efficacy and safety, the quantitation in feed is an important matter in order for feed manufacturers to practice quality control of the proper additive amount when the additive amount needs to be controlled. ［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 (the substance intended for analysis) as the blank sample, to confirm that there is no peak that 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 data such as a chromatograph, obtained in the studies, shall be attached to the test report because 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 and repeatability. Note that the spike recovery test shall be performed in accordance with the following conditions, in principle: ･ Sample: three kinds of compound feeds (choose those that correspond to the species of 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 ○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 and relative 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 conditions of 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 be tested. From the results of this test, the suitable storage conditions shall be discussed. ［Requirements］ The results of each of the ingredients for manufacturing and formulations shall be stated. Information about the ingredients, etc., tests, and other things shall include 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 always be recorded, as the purity may vary greatly depending on the lot. If differences among lots are comprehensibly described in original paper (test report) or annex, providing the 29 average value of three lots in the abstract will suffice. Tests shall be performed with samples from different lots. ○Method of analysis for the quantitation of the content: The method of analysis for the quantitation of the content shall be described, because the substance may decompose with time. ○Measurement items：Measure all items to be set in the composition standards at three or more time points including the start and ending times of the test. At other time points, observe the presence or absence of abnormal appearance, and measure the amount of active ingredients. For in-feed stability tests, ambient temperature storage tests, and humidity resistance tests, measure drying loss or moisture as well as items whose variations are anticipated, in particular, as necessary. ○Statistical analysis of test results: A statistical analysis shall be applied to a model that is thought to be the best for characterizing the relationship between the test period and the amount of the active ingredient. A regression analysis shall be used to calculate the 90% confidence interval for the population mean. ○Test results: The tests results shall be described in a properly arranged manner and using tables, as there are many test items. i. Ambient temperature storage tests The actual storage shall be conducted in an ambient temperature (1–30℃) to assess the stability of the substance under normal conditions. Storage tests shall be conducted using samples from at least three different lots of the ingredient, for manufacturing and for the formulation respectively. Put an appropriate amount of the objected substance in the packaging containers that are regularly used and store 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 (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. When shortening the test period, an effective period can be set based on the indicated standard. If there is a provision such as “ to % relative to the indicated value” as in the case of the enzymatic activity unit of an enzyme formulation, describe the transitions after the start of the test and percentages relative to the indicated value in an easy-to-understand manner. Moreover, provide a statement taking into account actual field conditions (storage period, temperature, etc.) of the enzyme formulation. ［Example 1］(A case where the objected substance is a white powder) Table 2 Ingredient for manufacturing Ambient temperature storage tests Test conditions: air temperature 25℃、humidity 40% (for 24 months), in a 20 kg paper bag Lot No. Parameter At the start 3 mos. 6 mos. 9 mos. 12 mos. 24 mos. A Appearance white powder white powder white powder white powder white powder white mud1 Identification test fit fit fit fit fit fit Purity test fit fit fit fit fit fit Amount of active ingredient (g) 214.6 212.1 210.4 208.3 205.4 207.9 Loss on drying 5.2 4.9 3.7 5.0 6.3 4.7 30 (g) Amount of moisture (g) 32.3 30.0 29.4 35.1 33.8 35.3 B Appearance white powder white powder white powder white powder white powder white powder …(omitted)… The numerical values are the average values of lot . Note 1: “White mud” indicates a white paste condition. Although the appearance of the substance turned to a white mud (paste condition) after 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. [Example 2] (in the case of an enzyme formulation) Table Formulation Ambient temperature storage tests Test conditions: Ambient temperature of 25℃, humidity of 40% (for 24 months) and storage in a 20 kg-capacity paper bag. Items At the start 3 months 6 months 9 months 12 months 24 months Measured values (unit: ) 3200 3100 3100 3000 2900 2800 Transitions after the test started 100% 97% 97% 94% 91% 88% Ratio relative to indicated value (3000) (standard: 85 to 170% of indicated value) 107% 103% 103% 100% 97% 93% The numerical values are the average values for lot . Note: If neither a variation among lots nor in-lot variation has not been described in the original paper (test report), attach such information as an annex. If a large variation is observed, describe it as necessary. The test results after months showed that … and indicated stability lasting for months. The storage period of the formulation is about months ( months from manufacture until import and months until it is added to feedstuffs domestically). The temperature management methods during distribution: refrigeration during transportation from (country) to Japan and … (temperature management method) during domestic distribution. Thus, because it is unlikely that a formulation less than the enzymatic activity unit (85 to 170% of indicated value) is distributed, it seems unnecessary to set provisions for a valid (effective) period or a storage temperature. 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 substance in such high temperatures, and ensure that no adverse changes are caused 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 the objected 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 (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). 31 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 the objected 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 that temperature and without the lid on the Petri dishes, for each of the following periods to assess their stability over the corresponding period: 0, 1, 2, 3 and 6 months (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). With regard to the level of relative humidity, more than two levels of humidity are set within the range in which 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 the said 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 the 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 the corresponding period: 0, 1, 2, 3 and 6 months (can be extended or shortened depending on the conditions or the physical properties under which the substance will be used as a feed additive). After each period of time, check if any adverse changes have been caused in its quality as a feed additive. ［Example]］(A case where the objected substance is a white powder) Table 3 Ingredient for manufacturing Light resistance test Test conditions：air temperature 25℃; humidity 50% (for 6 months); kept under a 500-lux fluorescent light Lot No. Parameter At the start 1 months 2 months 3 months 6 months A Appearance white powder white powder white powder white powder white powder Identification test fit fit fit fit fit Purity test fit fit fit fit fit Amount of active ingredient (g) 210.6 205.2 204.6 202.3 200.4 B Appearance 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 ingredient 32 decreased after 6 months of the substance’s exposure to the light. According to the paper by xx et al. (xxxx), the amount of the active ingredient will decrease to below the limit of quantitation 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 temperature and high humidity (especially in the summer season). Therefore, the stability of the objected 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 the ingredients for the manufacturing and the formulation. Put an appropriate amount of the objected 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 the corresponding storage period: 0, 1, 3 and 6 months (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, or with other feed additives, shall be tested. The objected substance (formulation) is added to at least three kinds of typically 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 to assess their stability over the corresponding storage period: 0, 0.5, 1, 2 and 3 months (and 6 months when needed). The method used for the quantitation of the objected substance in the 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 interlaboratory bias) 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 and with 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℃, 33 humidity 50%) for 3 months. (Data are the ratio to the content at the start of the test expressed in a 100-point scale.) Sample Run At the start 0.5 months 1 months 2 months 3 months Feed I 1 100 99.8 98.3 97.9 97.1 2 100 99.2 98.3 96.9 96.2 Average 100 99.5 98.3 97.4 96.6 Feed II 1 100 98.8 97.5 97.6 95.1 …(omitted)… For all the tested feeds, the residue rates remained at 95% and more. It can therefore be concluded that the objected substance is highly stable, even in feeds, for up to 3 months. 34 3 Items Concerning Efficacy The following items concerning the efficacy of the objected substance shall be assessed. a. Efficacy as a feed additive: which of the three effects specified in the Feed Safety Act is attained. b. Most effective livestock and the administration stage (time of feeding) c. Most effective dose (additive amount) and usage method The outline of “tests for checking the effect of growth promotion or feed efficacy” is shown below. Regarding “tests for a substance intended to prevent feed quality deterioration” and “tests for a substance intended to supplement the nutritional components and other effective ingredients of feed,” refer to the notification about the notification of the “Establishment of the Standard for the Evaluation of Feed Additives” and the following. (1) Basic tests to prove efficacy i. In vitro test Clarify or estimate the efficacy of the objected substance in a test tube and compare the efficacy with that of other feed additives having similar efficacy. 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, etc. (2) Field Application tests to prove efficacy The numbers of repeats and facilities for test animals (tests concerning live microbial agents) Cattle 15 cattle or more per group (1 cow/bull x 5 repetitions x 3 facilities, or 1 cow/bull x 5 repetitions x 1 facility at 3 times during different periods) or more Pig 60 pigs or more per group (4 pigs x 5 repetitions x 3 facilities, or 4 pigs x 5 repetitions x 1 facility at 3 times during different periods) or more Chicken 300 chickens or more per group (20 chickens x 5 repetitions x 3 facilities, or 20 A test to assess whether the expected efficacy (effectiveness) can be brought about under the conditions of the actual use of the objected substance. Therefore, be sure to include a statement taking into account feeding conditions (feed composition, breed of targeted livestock, etc.) in Japan. Any livestock for which the efficacy is not confirmed cannot be designated as targeted livestock. The table below shows provisions concerning test design. For antibiotics, synthetic antimicrobials and organic acids, test results at two or more (multiple) domestic facilities need to be included. For live microbial agents, test results at one or more domestic facilities need to be included. 35 chickens x 5 repetitions x 1 facility at 3 times during different periods) or more Farmed aquatic animal 180 farmed aquatic animals or more per group (30 farmed aquatic animals x 2 repetitions x 3 facilities, or 30 farmed aquatic animals x 2 repetitions x 1 facility at 3 times during different periods) or more The numbers of repeats and facilities for test animals (tests concerning an enzyme) Cattle 5 cattle or more per group (1 cow/bull x 5 repetitions x 1 facility) or more Pig 20 pigs per group (4 pigs x 5 repetitions x 1 facility) or more Chicken 100 chickens per group (20 chickens x 5 repetitions x 1 facility) or more Farmed aquatic animal 60 farmed aquatic animals per group (30 animals x 2 repetitions x 1 facility) or more The numbers of repeats and facilities for test animals (tests concerning substances other than live microbial agents/enzymes) Cattle 1 cow/bull or more per group The number of repeats shall be set in such a manner that the degree of freedom for repetitive measurement errors is at least 10 or more, or 20 or more if possible. 3 facilities or more Pig 4 pigs or more per group The number of repeats shall be set in such a manner that the degree of the freedom for repetitive measurement errors is at least 10 or more, or 20 or more if possible. 3 facilities or more Chicken 20 chickens or more per group The number of repeats shall be set in such a manner that the degree of the freedom for repetitive measurement errors is at least 10 or more, or 20 or more if possible. 3 facilities or more Farmed aquatic animal 30 farmed aquatic animals per group 2 repetitions or more per facility 3 facilities or more [Requirements] ○Testing laboratory and period, testing place and conditions: Describe which testing laboratory 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, such as the number of animals in a group. ○Method of administration and dose: Describe the additive amount of the objected 36 substance contained in the sample feeds (mg/kg-feed), the method and period of the administration, as well as the daily intake amount of the objected substance per unit of body weight consumed by the test animals (mg/kg-BW/day; calculated from the body weight of the test animal and the daily consumption of feed). In principle, arrange three different 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 is equal to the planned application period of the objected substance. Also provide the details of feedstuffs for a negative control group and a positive control group. ○Statistical analysis: Describe what types of dispersion analysis and multiple comparison test were performed to check the significance (effectiveness) of the administration of the objected substance (describe these item by item). Describe the above while referring to the examples in the next page and《Reference 8》. ○Test results and observations: Describe the body weight (live weight gain), the intake amount of feeds, feed efficiency, etc., for each of the different dose groups separately. The efficacy of the administration of the objected substance shall be assessed using an analysis of variance for each testing laboratory, in principle. Consolidate the results for concluding the assessment with a statement confirming the significant 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. As a summary of the tests concerning efficacy, estimate the optimum additive amount based on the results from the tests and describe it. When submitting the results of a test performed overseas, be sure to include a statement taking into account feeding conditions (feed composition, breed of targeted livestock, etc.) in Japan. [Example 1] [Method] A field application test to prove the efficacy was conducted at xx laboratory in the U.S. Broilers (Ross〇〇; male, 1 day old; x in number; 40 grams in average body weight) reared in an indoor chicken coop were fed with a basal diet (negative control group), a feedstuff made by adding 200 mg/kg feed of xx to the basal diet (positive control group) and a feedstuff made by adding the objected substance to the basal diet in quantities of 80, 100, and 150 mg/kg feed (the daily intake amounts of the objected substance per unit of body weight: 5.0, 6.3 and 9.4 mg/kgBW/day) for 42 consecutive days, which is the actual scheduled duration for using the objected substance (40 broilers per group; 8 repetitions). The consumption of water and feeds by the test animals was discretionary. A feedstuff deficient in xx was used as the basal diet. The concentration of the xx in the basal diet was mg/kg. This test was performed in the U.S., and the feedstuff used in the test is more deficient in xx than feedstuffs commonly used in Japan. However, given the action mechanism of the objected substance, the deficiency in xx is unlikely to affect the efficacy of the objected substance. Moreover, Ross〇〇 is a breed commonly raised in Japan. Based on this, the objected substance will be effective under feeding conditions in Japan, too. 37 〈Statistical analysis〉 Regarding the feed conversion ratio and live weight gain of each group, a dispersion analysis (two-way ANOVA) and multiple comparisons by a 〇〇test were performed. [Results] The test results are shown in the form below. (Appendix Form 1) Regarding the feed conversion ratio and live weight gain, these increased more significantly in the group fed with a feedstuff containing 150 mg/kg feed of the objected substance than in the negative control group (p > 0.05). No significant difference from the positive control group was observed (p > 0.05). [Example 2] [Method] In xx laboratory in the U.S., a field application test for validating the efficacy of the objected substance was performed. Broilers (Ross〇〇; male; 1 day old; x in number; average body weight of 40 g) raised in an indoor chicken coop were classified into the test groups shown below. The feeding period was set at 42 days, which is the actual scheduled duration for using the objected substance. The consumption of water and feed by the test animals was discretionary. Test group Feed Negative control group Basal diet (feed deficient in xx, concentration in feed: mg/kg feed) Positive control group Feed made by adding an adequate amount of xx to the basal diet (concentration of xx in feed: mg/kg feed) 80xx group Feed made by adding the objected substance in quantities of 80 enzymatic activity unit/kg feed (ranging from enzymatic activity unit/kgBW/day [calculate based on the body weight at the end of the test] to enzymatic activity unit/kgBW/day [calculate based on the body weight at the start of the test]) to the basal diet 100xx group Feed made by adding the objected substance in quantities of 100 enzymatic activity unit/kg feed (ranging from enzymatic activity unit/kgBW/day to enzymatic activity unit/kgBW/day) to the basal diet 150xx group Feed made by adding the objected substance in quantities of 150 enzymatic activity unit/kg feed (ranging from enzymatic activity unit/kgBW/day to enzymatic activity unit/kgBW/day) to the basal diet Because the objected substance is anticipated to be added to feed deficient in xx, the basal diet was assumed to be deficient in xx. Because …, the objected substance is expected to produce a similar effect when it is added to feedstuffs commonly used in Japan, too. Ross〇〇 is a breed commonly raised in Japan. [Results] The results are shown in Appendix Form 1 below. Regarding the feed conversion ratio and live weight gain, … [Example 3] [Method] At xx University in France, cows (Holstein; females; to days since lactation started) 38 were fed with a basal diet, a feedstuff made by adding ●● in quantities of 1000 mg/kg feed (50 mg/kgBW/day) to the basal diet and a feedstuff made by adding the objected substance in quantities of 1000 mg/kg feed (49 mg/kgBW/day) to the basal diet, for 10 weeks. Their milk was sampled once a week. [Statistical analysis] A statistical analysis was performed regarding dietary intake, live weight gain, and each component in the milk by means of MIXED procedure (SAS Institute, 1999-2000) and by using and (procedure names, selected analytical methods, settings, etc.). [Results] … 39 [Reference 8] Points to note when describing the test results The council will deliberate about targeted livestock for which the efficacy has been proved, the period of feeding, concentrations, etc., by taking into account the submitted documents (test data, etc.). Therefore, test results that demonstrate the conditions in which the objected substance produces effects shall be described in an organized manner. Any livestock animals for which the efficacy of the objected substance has not been proved cannot be designated as a target animal. Regarding statistical analysis: When describing the presence or absence of a significant difference, describe the method of verification and p values. It should be noted that the LSD method is not to be used for 4 or more groups of targeted livestock. For each value of the test results, state a standard deviation (SD) (Appendix Form 1). A statistical method must be described with information that enables the reproduction of the analytical results. (When statistical software was used, state the analytical method, procedure, and other items set by the operator, in addition to the software name and version.) (Amount added to feed) 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. [Example] In the case of providing a feedstuff made by adding the objected substance in quantities of 100 mg/kg feed to a basal diet (daily dietary intake at 2.89 kg/day and livestock animal body weight at 93 kgBW) 100 mg/kg × 2.89 kg/day ÷ 93 kgBW → 3.11 mg/kgBW/day 40 [Reference 9] Phytase test In the case of phytase, check whether the following have been included in the efficacy test: a. Phosphorous digestibility (examples: phytic phosphorus digestibility and apparent digestibility) b. Accumulation of phosphorus in bones (in the case of layer chickens, a description of the layer performance is acceptable.) c. Live weight gain and feed conversion ratio In principle, use the terms “non-phytic phosphorus” and “phytic phosphorus” (describe digestible phosphorus and available phosphorus as “non-phytic phosphorus” and non-digestible phosphorus as “phytic phosphorus.”). With regard to phytase, data on effectiveness and safety for layer chickens can be extrapolated to quails as long as adequate considerations are provided. In the cases of an effect test and a feeding test using targeted livestock, state the considerations. Aside from these, if scientific and reasonable considerations for efficacy and safety have been stated, the council can deliberate on whether such livestock is acceptable (can be included) as targeted livestock. 41 Appendix Form 1 Tests concerning efficacy (Livestock) Document No. Testing laboratory, Testing place and period Test animal Test group assignment Test results Notes*4 Species No. of animals in a group Test group Dose of test substance*1 (mg/kg Feed) No. of repeats Total number of animals Feeding period *3 Average live weight gain (g) (％) Average intake amount of feed (actual no.) Average feed conversion ratio*3 (g/g) (%) Survival rate (%) Pathological test findings xx laboratory Fr. xx xx, xxxx To xx xx, xxxx Ross308 40 Negative control group Positive control group Group with additive 0 200 (additive amount: xx) 80 100 150 8 320 per test group (1600 in total) 42 days 2701±270a 2881±288b 2732±273a 2780±278a 2881±288b 100 107 101 103 107 4320±432c 4382±438c 4311±431c 4403±440c 4382±438c 1.60±0.16e 1.52±1.5f 1.58±1.5e 1.58±1.5e 1.52±1.5f 100 95 99 99 95 100 100 100 100 100 Pathological test was not performed because neither a test animal with abnormal health condition nor a dead animal was observed. …(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. (two-way ANOVA, ○○test, p<0.05) 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. 42 [Reference 10] Conditions to be complied with in conducting animal tests “The notification of the Standard Concerning the Performance of Animal Tests for Feed Additive Assessments*1” sets out the conditions to be complied with in conducting tests concerning the safety and persistence of feed additives. This standard was established 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 the amendment of specifications and standards, etc. The standards are intended for the following 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, etc. Residue tests using targeted livestock, etc. Based on these standards, keep the relevant records and documents as appropriate for at least 5 years after the designation of feed additive or the setting of code or standard. We may check the contents of your procedure, so please make sure that it can be referenced as necessary beforehand. 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 and Development), 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 the Food and Agricultural Materials inspection Center (FAMIC) will conduct inspections of test facilities, according to the “Notification on the Establishment of Inspection Guidelines based on the Standards Concerning the Performance of Animal Tests for Feed Additive Assessments*2” (feed additive GLP). In addition to facilities conforming to the feed additive GLP, the submittal of the results of a test performed at a facility conforming to the pharmaceutical product GLP is also possible. *1 “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) *2 “Establishment of Inspection Guidelines based on the Standards Concerning the Performance of Animal Tests for Feed Additive Assessments” (Ministry of Agriculture, Forestry and Fisheries, Fisheries Agency Director-General Notification No. Genchiku-A-3441 issued on January 16, 1990) 43 4 Items Concerning Residue Residue tests using targeted livestock, 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, and not be discharged in the excreta. In the residue tests, the objected substances shall be administered to livestock animals and the amounts of the objected substance that remain in each 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, the residue 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) 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 confirmed that the objected substance ingested by the animal has been completely discharged in the excreta and does not remain in the animal’s body through the in vivo kinetics test, the residue test may be omitted. [Requirements] ○Testing laboratory and period, testing place and conditions: Describe which testing laboratory 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, such as 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 administration, as well as the daily intake amount of the objected substance per unit of body weight consumed by the test animal (mg/kg-BW/day; calculated from the body weight of the test animal and the daily consumption of feed). The minimum dose for the test shall be set as equal to the maximum level of the dose in the actual use, and the dose shall be administered several dozens of times higher than the said minimum dose in 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 well in cases where the persistence of the metabolite needs to be examined. Describe the information about the metabolites (name, whether they have bioactive effects, etc.). In order to discuss the persistence of the substance in livestock bodies, the method for quantitating both the substance remaining in the body and the substance excreted to outside 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 mean value) ≤ 0.1. These specifications need to be described. However, note that the limit of detection must be equal to, or lower than, the residue limit of the objected substance in foods if the said residue limit is set at below 0.05 mg/kg. 44 ○Results of the analysis: If the results indicate that the amount of residue is smaller than the limit of quantitation, describe as “< limit of quantitation.” [Example] A case of an application to weaned cows [Method] A field application test to assess the persistence of the objected substance was conducted using cows at the xx laboratory in xx prefecture. The cows reared in the indoor cow house of the laboratory (strain: xx; ten pens each of males and females; 2 years old; average body weight: 450 kg) were given feeds continuously with the objected substance added at the following different concentrations: 100, 300 and 1,000 mg/kg (each concentration corresponding to the following daily intake amounts of the objected substance per unit of body weight: 0.3, 0.9 and 3.0 mg/kg-BW/day) for four weeks. (The consumption of water and 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 no non-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 the milk and muscles (skeletal muscles) was below the limit of quantitation even in the samples from the cows that had consumed feedstuff containing 1,000 mg/kg of additive. The maximum residue, 8 mg/kg, was detected in the liver and kidneys of the cows that had consumed feedstuff containing 1,000 mg/kg of additive. In the fat in the abdominal area, residue was identified in the cows which had consumed feed containing 300 mg/kg or more of the additive. However, the amount of residue was equal to or less than the limit of quantitation in all parts of the bodies of the cows that had consumed feedstuff containing 100 mg/kg of additive, which is the maximum additive amount at which the efficacy of the objected substance is visible. Table 5 Animal residue test of the objected substance in cows Concentration of the objected substance in the feed (mg/kg) Analyzed body part Muscle (skeletal muscle) Liver Kidneys Fat (abdominal area) Milk 100 <2 <2 <2 <2 <1 300 <2 <2 <2 10～15 <1 1,000 <2 5～7 5～8 40～50 <1 45 Limit of quantitation: 1μg/kg for milk and 2μg/kg for other parts 46 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 obtain the 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) (i) Single dose toxicity test A test to assess the toxic effects from both a quality and a quantity aspect by administering a single dose of the objected substance to the test animals and estimating the median lethal 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 testing laboratory 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 in weeks, 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 and specify the dose. Conduct an exploratory test to get a whole picture of the toxicity and approximate 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 the approximate lethal dose. Observe the level of intoxication and when symptoms are detected, as well as any transitions, reversibility, etc. Record any kinds of signs of toxicity that are evident via gross observation, as well as the when symptoms are detected, for every single test animal, for all the involved animals, and provide them in Appendix Form 2. [Example] A case of a single dose toxicity test in mice [Method] A single dose 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 body weight: 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 the start of test, then every 12 hours every day. 47 [Results] In the group administered with 1,200 mg/kgBW, one female died 8 days after administration. The dead rat had shown symptoms of kyphosis and decreased appetite on the 7th day after administration. Macroscopy during autopsy indicated enlargement of the stomach (gastric dilatation). A histopathological examination revealed atrophy of the gastric mucosa. These proved that the death was caused by a gastrointestinal tract disturbance due to a high administration dosage and was not directly caused by the objected substance. In the group administered by 2,000 mg/kgBW, 1 male and 2 females died 2 days after administration. All of them had shown symptoms of kyphosis, lethargy, and closed eyelids from the time immediately after administration. Gross pathological findings via autopsy indicated that the male had edema of the abdominal cavity. In one of the dead females, dilation of the uterus was observed, and in the other, a blood clot was observed in the abdominal cavity. Based on these results, the LD50 is estimated to be 2,000 mg/kg-BW or more. The details of the results are summarized in the form below (Appendix Form 2). [Reference 11] Points to note when recording safety test results Describe all of the adverse events that occurred during the test in the appendix and discuss safety. In particular, regardless of whether they are below NOAEL or not, if there are any abnormal values compared with the standard lot, discuss safety in relation to livestock on the basis of such values (show reference values as the basis for judgments). In the event of any deaths, provide the cause(s) and record them in the abstract and appendix. 48 Appendix Form 2 Single dose toxicity test Document No. …(omitted)… Testing laboratory and testing period xx laboratory (conforming to OECD-GLP for the year xxxx) 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 (mg/kg) 2,000 or more Approximate lethal dose (mg/kg-BW) Group administered 1,200 mg/kgBW Kyphosis and decreased appetite Group administered 2,000 mg/kgBW Kyphosis, lethargy, and closed eyelids …(omitted)… Period of occurrence, prosperity and decline of the signs of toxicity, and period of death Dead cases 1,200 mg/kgBW 1 female Day 8 (Gastrointestinal tract disturbance due to a high administration dosage) 2,000 mg/kgBW 2 females and 1 male Day 2 …(omitted)… Symptoms of toxicity 1,200 mg/kgBW 1 male Kyphosis and decreased appetite were observed from the time immediately after administration until 7 days after administration (subjects later died). …(omitted)… Notes 49 (ii) Repeated dose toxicity test (short term) A test to estimate the dose that will evoke apparent toxic changes and to clarify the details of toxic changes by administering the objected substance to the test animals continuously for 3 or more months (iii) Repeated dose 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 testing laboratory 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, body weight at the beginning of test, etc., of the test animals. It is desirable that testing begins as early as possible (to administer the feed) - preferably by the time they reach 8 weeks. ○Method of administration and dose: Add the objected substance to feed or water, in principle, and administer the feed by a continuous oral gavage. Arrange at least three different dose groups, including a dose that causes no effect in the animals and another dose that will cause a certain sign of toxicity. If solvents are not used, a non-treated group is the control. When a solvent is used, a group fed the solvent is the control. Note that 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 the whole 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 [Method] A long-term, repeated-dose toxicity test in mice was conducted at xx laboratory in xx Prefecture. The mice (strain: xx; 16 males and 16 females; 5 weeks old; average body weight: 20.7 g) were fed for 12 months. The objected substance was added to feed at the following concentrations: 100, 200, 500, 1,000, and 2,000 mg/kg feed. The body weight (BW)-based daily intakes of the objected substance were 8, 16, 40, 80, and 160 mg/kgBW. The consumption of the water and feed by the test animals was discretionary. After the test period, clinical tests and pathological tests were conducted. In cases of death, autopsies were performed. [Statistical analysis] With respect to the live weight gain of each group, a dispersion analysis by and multiple comparisons by test were performed. With regard to the results of other inspections, dispersion analysis by means of was performed. As for items showing a significant difference, multiple comparisons by test were performed. [Results] The details of the results are summarized in the form below (Appendix Form 3). One female died in a group administered 1,000 mg/kg feed and 1 male and 3 females died in a group administered 2,000 mg/kg feed. Among these, the female administered 1.000 mg/kg feed had tumors in the lungs. A histological examination found xx, not the administration of the objected substance, as the cause of the tumors. In one of the three dead females in the 2,000 mg/kg feed group, symptoms of decreased appetite and akinesis were observed two 50 days before its death. A dissection examination was performed, which provided no noticeable findings in the gross observation. The death was therefore deemed unrelated to the objected substance. Autopsies were also performed for the remaining deaths, and a macrography found xx, which was estimated to be the cause of the deaths. With regard to the amount of feed intake, the higher dose group (1,000 mg/kg feed or higher) showed constant values and did not indicate a significant difference in terms of live weight gain (p > 0.05), either. In a blood biochemical inspection, increases in AST and ALT in blood plasma were observed in the group administered 2,000 mg/kg feed. This was probably due to the xx generated during the metabolic process of . A hematological inspection did not reveal any significant variable items (p > 0.05). Based on these results, the NOAEL is estimated to be 1,000 mg/kg feed (80 mg/kgBW/day). 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 No. of animals per group 32 (16/16) Method of administration Mixed feeding Purity of the test substance 99.8% Test group and dose (mg/kg-feed; mg/kg-BW/day) 0 (Control group) 100 mg/kg feed (8 mg/kgBW/day) …(omitted)… General symptoms and death rate ・General symptoms Male: Bleeding on Day 57 (bite wound), recovered a few days later Male: Loss of fur observed on Day 112 …(omitted)… ・General symptoms Male: Grooming behavior increased on Day 48. Female: Piloerection was observed on Day 100 …(omitted)… Average live weight gain (g/day) 0.6 0.6 Feed Average intake of feed (g/day) 0.8 0.78 Feed efficiency 0.75 0.77 Total dose administered of the test substance (mg/animal) 0 58 Clinical test findings* Hematological test Red blood cell count (x 104/mm3) MCV (fl) MCH (pg) …(omitted)… Blood biochemical test Glucose (mg/dl) AST (I.U./L) ALT (I.U./L) Total bilirubin (mg/dl) Urine test pH Pathological test findings* Gross observation Stomach: dilation Kidney tubule: inflammation Absolute weight of the organs (g) Liver Heart Relative weight of the organs (%) Liver Heart Histological test Gastric mucosa: atrophied NOAEL and toxic dose NOAEL: 1,000 mg/kg feed (80 mg/kgBW/day) Notes 51 * Only clinical inspection findings and pathological examination findings indicating a significant difference are provided here. * Test data listed in superscript indicates that the difference between the data is significant (●●, ○○ test, p<0.05). 52 ii. Special toxicity tests (subject to the feed additive GLP) (i) 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 testing laboratory 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, body weight 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 water and administer the feed/water by a continuous oral gavage. Arrange at least three different dose groups to assess the dose-response relationship. The maximum dose shall 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 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 the whole 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 [Method] A transgenerational reproductive toxicity test in mice was conducted at the xx laboratory in xx prefecture. The mice (Generation: p; strain: xx; 30 females; 6 weeks old; average body weight: 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 30 mice prepared for the test, 22 mice became pregnant. These 22 pregnant females were used for the test. [Statistical analysis] Dispersion analyses were performed by means of ●●, and multiple comparisons by means of ○○ test concerning average values were performed for items showing a significant difference. [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<0.05). 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 obtained in 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 differences were 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 and 53 clinical 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 substance causes a transgenerational effect, promotes live weight gain and affects the reproductive potential. 54 Appendix Form 4 Transgenerational reproductive toxicity test 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 No. of animals per group 30 (female) Method of administration Mixed feeding Purity of the test substance 99.8% Generation P Feeding period: 300 days F1 Feeding period: days F2 Feeding period: days Test group and dose (mg/kg-feed; mg/kg-BW/day) 0 100 500 … (omitted)… …(omitted)… …(omitted)… G eneral feeding param eters General symptoms - - Death rate 0 0 0 Average live weight gain (g) 1.2 1.8 2.3 Average live weight gain (g) 2.6 2.7 3.1 Average feed efficiency 0.46 0.67 0.74 Findings - - R eproductive param eters Period of the estrous cycle before copulation, normality (Female) 25 25 25 No. of copulations 14 16 15 Rate of copulation 88 100 94 No. of pregnancies 10 13 11 Rate of pregnancy 63 81 73 No. of live births 82 90 88 Average body weight of the newborns 0.5 0.7 0.8 No. of stillborns 0 0 0 Birth rate 100 100 100 Average litter size 6 10 10 Average live weight gain (at 21 days of age; g) 10.2 11.2 10.8 Surviving rate of the babies at 21 days of age 100 100 100 Sex distribution (%) 64 64 73 Findings - - Notes Note: “-” indicates no abnormality was observed. * Test data listed in superscript indicates that the difference between the data is significant (●●, ○○ test, p<0.05). 55 (ii) Developmental toxicity test A test to assess the effects of the substance on the birth of fetuses, especially on their teratogenicity, by administering the objected substance to pregnant animals in the organogenetic period of the fetus. [Requirements] ○Testing laboratory and period, testing place and conditions: Describe which testing laboratory 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 the test, etc. of the test animals. Also, describe the information about the test, such as the number 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 the dose response relationship. The maximum dose shall be set within the extent of the confinement that the physicochemical properties define, as equal to the dose that invokes a certain sign of toxicity in the mother animals, such as an inhibition of the body weight gain. The minimum dose shall be set as the dose that causes no damage in both the mother and fetus. If solvents are not used, a non-treated group is the control. When a solvent is used, the group fed with the solvent is the control. The solvent shall not have an 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 so that the level of toxicity can be fully understood. Describe the details of the observation results in Appendix Form 5. [Example] A case of a developmental toxicity test in mice [Method] 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 body weight: 21.3 g) were administered the objected substance on the tenth day of pregnancy by an 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 male and female, 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 dead case 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). 56 Appendix Form 5 Developmental toxicity test 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 No. of animals per group 80 (40/40) Method of administration Oral gavage Purity of the test substance 99.8% Test group and dose (mg/kg-feed; mg/kg-BW/day) 0 (Control group) …(omitted)… 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 Im plantation findings 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 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 of newborns Notes Note: “-” indicates no abnormality was observed. 57 (iii) 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 testing laboratory 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 the test, etc., of the test animals. Also, describe the information about the test, such as the number 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, in principle, and administer these continuously via an oral route. Arrange at least three different dose groups to assess the dose-response relationship. The maximum dose shall be set as equal to the dose that is expected to cause an apparent effect on the frequency of tumor occurrence, and thereby on the lifespan of the test animals. Note that 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 the whole 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 [Method] 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 the death rate. Based on the above, it can be concluded that the objected substance does not have an oncogenicity effect. The details of the results are summarized in the form below (Appendix Form 6). 58 Appendix Form 6 Carcinogenicity test 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 No. of animals per group 100 (50/50) Method of administration Mixed feeding Purity of the test substance 99.8% Test group and dose (mg/kg-feed; mg/kg-BW/day) 0 (Control group) …(omitted)… Cumulative death rate 0 Average live weight gain (g/day) 1.5 Average intake of feed (g/day) 2.3 General symptoms …(omitted)… Absolute weight of the organs (g) Body 22.1 Brain 0.42 Heart 0.12 Lungs 0.15 Kidney 0.27 (left) 0.13 (right) 0.14 Liver 0.95 Relative weight of the organs (%) (100 x organ weight/body weight) Brain 1.9 Heart 0.54 Lungs 0.68 Kidney 1.22 (left) 0.59 (right) 0.63 Liver 4.3 Histopathological findings …(omitted)… Incidence of tumors (and incidence of a specific tumor) 0 ( 0 ) Findings of other tests 185th day: dead (1 m.) 241st day: dead (1 fe.) Note 59 (iv) Mutagenicity test A test to assess the mutagenicity of the objected substance, the manufacture of which is scheduled, 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. Summarize the results of each mutagenicity test in an appendix form as shown below. [Example] Appendix Form 7: Results of each mutagenicity test Classification Test Target Dosage Result Reference in vitro Reverse mutation test Salmonella typhimurium TA98, TA100, TA102, TA1535 Escherichia coli WP2 uvrA 0-5000 µg/plate (+/-S9) Negative Reference material X Chromosome aberration test Cells derived from Chinese hamster ovary (CHL cells) 0-5,000 µg/mL (+/-S9)12 h treatment Negative Reference material X Chromosome aberration test Cells derived from Chinese hamster ovary (CHL cells) 0-1,000 µg/mL (+/ S9) Positive (structural anomaly) Reference material X in vivo Micronucleus test Mice (strain xx) 500-2,000 mg/kgBW (body weight) Forced oral dosage (gavage administration) Negative Reference material X (iv- 1) Reverse mutation test A test for checking the presence/absence of gene mutation inducibility (effect on 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 the groups administered a known mutagenic agent. For the positive control groups, both the substance requiring the presence of the S9 mix and the substance not requiring the S9 mix are prepared. Conduct the test using suitable metabolic activation methods (S9 mix) and observe the results. 60 ○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 〈Method〉 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/plate as 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; with and without the presence of the S9 mix metabolic activation. The negative control and positive control groups (three plates each) were included as well. After culturing, the number of reverse mutation colonies was measured for each plate. [Results] The results are summarized in the table below (Table 6). Table 6: Number of reverse mutation colonies of Salmonella typhimurium S9mix Concentration (μg/plate) Salmonella typhimurium … (om itted)… TA98 TA100 TA102 TA1535 (－) 0*1 13 160 …(omitted)… 1.6 15 155 8.0 14 152 40.0 13 162 200.0 15 161 1,000.0 14 158 5,000.0 11 169 Positive control 12 163 (＋) 0*1 …(omitted)… …(omitted)… (*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：MItomycine(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 reverse mutation 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 have mutagenicity. 61 (iv- 2) Chromosome aberration test Chromosome aberrations caused by damage to the DNA and the proteins can be detected by a microscopic observation in the metaphase of the cell division. [Requirements] ○Test cells: Chinese hamster ovary cells (CHO), Chinese hamster lung cells (V79), Chinese hamster lung cells (CHL/IC), human-derived cells (TK6), other cell strains, or primary cultured cells containing peripheral blood lymphocyte from humans or of mammals other than humans are to be used. ○Doses used in test: Arrange at least three different dose groups with the maximum dose equivalent to the concentration that causes a cell (cyto)toxicity of 55±5%. In cases where cell cytotoxicity is not identified, a concentration equivalent to 10 mM or 2 mg/mL, whichever is lower, shall be the limit. As cytotoxicity indicators, the relative cell population doubling (RPD) index or the relative increase cell count (RICC) is to be used for cell strains, and the mitotic index (MI) is to be used for primary cultured cells (first-stage culture cell). A concentration that brings about a cytotoxicity of 55±5% means a concentration at which these indicators make up 40 to 50% of the negative control simultaneously. 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: In cases of 300 or more metaphase cells per unit dose, observe the incidence of chromosomally aberrant cells and the incidence of polyploids, and record these values. [Example] (Test 1) [Method] A test was conducted to check the presence/absence of chromosome aberrations using the CHL cells of a Chinese hamster. CHL cells were disseminated on 10 mm dishes at a concentration of 5×104 cells/mL, 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 for metabolic activation for 6 hours on the third day from the dissemination, and were cultured for a further 18 hours. Four dose levels of the objected substance were arranged (500, 1,000, and 2,000 μg/mL), with the 2,000 μg/mL as the maximum dose. The number of cells was counted, and PRD for each dosage was calculated. [Results] As shown in Table 7, the concentration at which RPD made up 40 to 50% of the negative control was 1,000 µg/mL, in both the presence and the absence of the metabolic activation system of a continuous treatment method and a short-time treatment method. In test 2, the maximum concentration was set at 1,000 µg/mL. 62 Table 7 Results of the cytostatic test S9mix Treatment period (h) Observations Additive concentration of the objected substance (μg/mL) 0 *1 500 1,000 2,000 (－) 6 Number of increased cells (×104 cells) 72 48 30 …(omitted)… RPD (% negative control) 100 66.7 41.7 (＋) Number of increased cells (×104 cells) 81 58 38 RPD (% negative control) 100 71.6 47.0 …(omitted)… RPD: Relative cell population doubling index (Test 2) [Method] With the maximum dosage of the objected substance being set at 1,000 µg/mL, cells were cultured in the same manner as in test 1. After culturing, cells were checked for any chromosome structural abnormalities (e.g. chromatid gaps and chromosome gaps). [Results] As shown in Table 8, neither chromosome structural abnormalities nor aberrant cells were observed in any case. Table 8 Incidence of chromosome aberrations S9mix Treatment period (h) No. of cells observed (cells) Concentration (μg/dish) Incidence of structural chromosome aberrations (%) Judgment (*8) gap (*2) ctb (*3) cte (*4) csb (*5) cse (*6) f (*7) Total (-) 6 300 0(*１) 0 0 0 0 0 0 0 300 500 1 0 0 0 0 0 1 300 1,000 0 0 0 0 0 0 0 300 Positive control 0 1 0 0 0 1 2 (+) 6 300 0(*１) 0 0 0 0 0 0 0 …(omitted)… …(omitted)… 63 S9mix Treatment period (h) No. of cells observed (cells) Concentration (µg/mL) Incidence of chromosomally aberrant cells (%) Polypoid Judgment (*8) Chromatid gap (-) (+) (-) 6 300 0(*1) 0 0 0.0 300 500 1 0 0.0 300 1,000 0 0 0.0 300 Positive control 0 1 0.0 (+) 6 300 0(*!) 0 0 0.0 …(omitted)… …(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. 64 (iv- 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: Carry out administration taking into account any anticipated paths of exposure to humans and use a method in which the target tissues are appropriately exposed. Administer a single dose, as well as a repeated dose 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 administration, kill all the test animals and collect the bone marrow from each of them to prepare smears. In principle, search for micronuclei in at least 4,000 immature erythrocytes (also referred to as ‘polychromatophilic erythrocytes’ or ‘reticulocytes’) per test animal. At the same time, examine the incidence of polychromatophilic erythrocytes to all the erythrocytes. [Example] [Method] 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). Three different 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 4,000 immature erythrocyte cells were examined per test mouse to detect the micronuclei. [Statistical analysis] A 〇〇 test was performed to compare the incidences of polychromatophilic erythrocytes. [Results] The results are summarized in the table below (Table 9). The incidence of micronucleated cells was calculated (by the number of micronuclei/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 (p<0.05). Also, there were no significant differences between the treated groups and the positive control group (p>0.05). Based on the above, it can be concluded that the objected substance does not have inducibility of micronuclei. 65 Table 9 Incidence of micronucleated erythrocytes Dose (mg/kg-BW) Treatment period (h) No. of cells observed (cells) No. of polychromatophilic erythrocytes (cells) No. of orthochromatic erythrocytes (cells) No. of micronuclei (cells) Incidence of micronuclei (%) 0 (controls) 24 4,000 3,796 204 2.0 0.05 48 4,000 3,828 172 3.2 0.08 …(omitted)… 66 (v) Other tests In addition to the above test examples, the following tests can be used to check each mutagenicity. Describe these as necessary. (v- 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 (v- 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 (v- 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 (v- 4) Other tests ･Somatic recombination and gene exchange test using fermentum ･Sperm morphology aberration test in mice iii. Pharmacological test Test conducted to assess the pharmacological effect of the objected substance when it is anticipated. 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, for example) than the inhibition of the productivity in livestock, caused by the particular pathogenicity organisms in their juvenile period, cannot be designated as feed additives. iv. In vivo kinetics tests 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 testing laboratory 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 target animals of the objected substance, and add rats and rabbits, 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 to the method of analysis so that the test substance, etc., can be quantitated in the body tissues and/or in the excreta. ○Method of analysis: Employ an adequate method capable for the analysis of the in vivo kinetics. ○Test results and observations: The items to be observed are described in detail later. ([Reference 12] Tests for Analyzing In Vivo Kinetics) 67 [Example] A case of an in vivo kinetics test in pigs [Method] A field application test was conducted to prove the efficacy of the substance at xx laboratory in xx Prefecture. Weaned piglets reared in an indoor pig house (strain: xx; 12 males and 12 females; 14 days old; average body weight: 11.7 kg) were administered a single dose of the objected substance at 100 mg/kg feed ( mg/kgBW) via a single oral administration (gavage). 12C of the objected substance was replaced with 14C, and the dynamic states of the radio isotopes were traced inside the body for a three-day period from the time of the administration. [Results] The results of the quantitative analysis of the discharged 14C in the excreta are summarized in 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 is low, 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 the total administered 14C) Elapsed time (hrs.) 3 day total 24 48 72 Urine 0.48 0.66 0.32 1.46 Feces 77.29 2.34 1.91 81.54 Daily total 77.77 3.00 2.23 83.00 A quantitative analysis was also performed on the objected substance contained in the excreta. (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 The ratio of 14C 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 is considered 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. 68 [Reference 12] 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, the objected substance is labeled by a radioactive material such as 14C: that is, the isotope-labeled compound produced by replacing a part of 12C of the objected substance with 14C, is administered to animals and the radioactive materials are traced 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 the digestive 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, and an 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 is performed of the distribution of those substances in the muscles, fat, liver, kidneys, and the other organs and anatomies, and its time-dependent changes, and the biological 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 that are involved in the metabolization is conducted for the analysis.) When a difference in the production rate of the main metabolites is found between animal species, it is desirable to conduct further similar tests in other species of animals. 69 (2) Feeding tests using targeted livestock, etc. (Subject to the feed additive GLP) Based on the targeted livestock and the effective dose, both of which are supposed to act on the efficacy of the objected substance as a feed additive, conduct a test to administer the objected substance to the target animal practically and continuously, and assess the effects on the targeted livestock. [Requirements] ○Testing laboratory and period, testing place and conditions: Describe which testing laboratory 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 when used 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 objected substance contained in the sample feeds (mg/kg-feed), the method and period of the administration, as well as the daily intake amount of the objected substance per unit of body weight ingested by the test animals (mg/kg-BW/day; calculated from the body weight of the test animal and its daily consumption of feed). Arrange at least two dose groups including a dose equivalent to the maximum of the optimum dose range, and another dose of approximately 10 times said dose (except for the control groups). 〇Method of analysis: If a statistical analysis was carried out, please refer to “Reference 8” and provide the necessary information. ○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 group separately. When any abnormalities are found in these parameters, perform a hematological test, biochemical test, pathological test, etc., on an as-needed basis. Refer to the Appendix Form 8 for the details of the items in the observation. [Example] A case of an application to weaned piglets [Method] A safety test using the targeted livestock was conducted at xx laboratory in xx Prefecture. Pigs reared in an indoor pig house (strain: xx; 15 males and 15 females; 30 days old; average body weight: 8.2 kg) were given feed continuously for four weeks, which was the intended period for actual use. The objected substance was added to the feed at the following different concentrations: 80 mg/kg as (the optimum dose, 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 (two times the optimum dose, equivalent to 0.8 mg/kg-BW/day); 400 mg/kg feed (five times the optimum dose, equivalent to 2.0 mg/kg-BW/day); and 800 mg/kg feed (ten times the optimum dose, equivalent to 4.0 mg/kg-BW/day). The consumption of water and feed by the test animals was discretionary. [Statistical analysis] For each value, a dispersion analysis was performed. For items that indicated a significant difference, multiple comparison by means of a 〇〇 test was performed. [Results] The results are summarized in the table below (Appendix Form 8). When the additive was added at the optimum dose (80 mg/kg feed), a significant increase in the live weight gain was observed (p<0.01) compared with the non-additive group. Although the increase in both 70 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 this group 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 a mild gastric erosion, which was cured in a week or so. After the cure, the live weight gain increased. The other pig did not show any noticeable symptoms. 71 Appendix Form 8 Feeding test on targeted livestock, etc. Document No. Testing laboratory, Testing place and period Test animal Test group assignment Test results Notes Species No. of animals in a group (males/females) Test group Dose of the test substance (mg/kg feed) No. of repeats Total no. of animals Feeding period Average live weight gain (actual no.) (%) Average intake amount of feed (actual no.) Average feed demand rate (actual no.) (%) Surviving rate (%) Hematological test findings Blood biochemical findings Pathological test findings xx laboratory Fr. xx xx, xxxx To xx xx, xxxx xx breed 30 (15/15) Control group Additive group 0 80 160 2 60 per test group (300 in total) 4 weeks 0.41±0.04a 0.55±0.05b 0.62±0.06c 100 134 151 0.71±0.07d 0.61±0.06de 0.60±0.06e 1.73±0.17f 1.11±0.11h 0.97±0.09i 100 64 56 100 100 100 …(omitted)… …(omitted)… …(omitted)… Note: Test data listed in superscript indicates that the difference between the data is significant. (●●, ○○test, p<0.05) 72 (3) Tests concerning the emergence of resistant bacteria Perform quality and quantity assessments on the items concerning the emergence of drug-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 natural environment by way of the excreta, etc., of livestock animals. Source: Appendix 2-1 to the “Ministerial Ordinance on the Specifications and Standards of Feed 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 the physicochemical 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 the pertinent section are for reference purposes only and do not constitute the elements of 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 the formula 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 a chemically pure substance. (3) The following signs are used for the main measurement units. meter m centimeter cm millimeter mm micrometer µm nanometer nm square centimeter cm2 liter L milliliter mL microliter µL ton t(1,000kg) kilogram kg gram g milligram mg microgram µg [Reference 13] 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,” are listed in the Ministerial Ordinance on the Specifications and Standards of Feed and Feed Additives 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 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 the Specifications 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) ・List of the Specifications and Standards of Feed Additives (13th Edition) 73 kilopascal kPa mole mol micromole µmol 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) inin100g 100 mL of solution; % v/v for the content of the substance (mL) in 100 mL of solution; % 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 potency of the discussed antibiotics and the enzyme per liter unit of the discussed enzyme are used, 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” indicates a 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 in a water bath” indicates heating in a boiling water bath or a steam bath at approximately 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 0.90–1.10 g 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 and shall be rounded off to two decimal points. (11) The term “reduced pressure” indicates a pressure not exceeding 2.0 kPa, unless otherwise 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 not stating 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 each solution has a concentration corresponding to 3 ml, 10 ml and 100 mL of solution, all of 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. 74 (15) The tests of the feed additives shall be conducted at an ordinary temperature, and the observations shall be made immediately after the operation, unless otherwise specified. When examining temperature-sensitive matters, the conditions at the standard temperature shall be examined. (16) The term “white” used in the item of physicochemical properties indicates a white or practically white color. Similarly, the term “colorless” indicates a colorless or practically colorless item. Unless otherwise specified, the test of the color tone is performed by placing 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 a colorless test tube measuring 15 mm as the inside diameter, and is observed in front of 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 white background. Only a black background shall be used for testing the fluorescence of a liquid feed additive. (17) The term “odorless” used in the item of physicochemical properties indicates an odorless or practically odorless item. Unless otherwise specified, the test of odor shall be 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 are defined 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 of vigorous shaking repeated at 5-minute intervals. Term Volume of solvent required for dissolving 1 g or 1 mL of the solute Very soluble < 1 mL Freely soluble 1mL ≤ < 10 mL Soluble 10mL ≤ < 30 mL Sparingly soluble 30mL ≤ < 100 mL Slightly soluble 100mL ≤ < 1,000 mL Very slightly soluble 1,000mL ≤ < 10,000 mL Practically insoluble, or insoluble 10,000mL ≤ (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 a clear solution or mixture. A fraction of fibers or dust should be considered as within the allowance. (20) The identification test is the test to identify feed additives or the main ingredients of feed 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 each article. 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 are used or are predicted to be added, this test shall be performed on those substances. 75 (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 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 to make 20 ml of solution. Add 1 ml of diluted nitric acid (1 → 3), 0.5 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 “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 “very slightly 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 stand for 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 that the difference of the masses measured before and after an additional 1 hour of drying or ignition is not more than 0.10% of the preceding mass of the dried substance or ignited 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. 76 (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. (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 in conjunction with the test samples or standard substances. Similarly, the word “ignite” indicates igniting under the same conditions as are specified in the section of the igniting loss of each article. (26) For the content of an ingredient determined by the quantitative method specified in each 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 a content equal to 90–110% of the labeled content” indicates that the substance is prepared 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 and methods specified in each article when the same level of or a higher accuracy and precision 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 all parts 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 sealed container is specified, it may be replaced by a tightly sealed container or by a hermetically sealed container. (30) A “tightly sealed container” indicates a container that is capable of protecting the contained feed additives from extraneous solids, liquids or moisture, from the loss of the contents, and from efflorescence, deliquescence, or evaporation under the ordinary or customary conditions of handling and storage. Where a tightly sealed container 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. 77 Ⅳ Exemplary Abstract After referring to “Chapter III: Items to be Described in the Required Document (Abstract)” and following the format shown in subsequent pages, please create an abstract. The appendix is intended to organize the test results summary, so please arrange items appropriately as necessary. The exemplary abstract exhibited on subsequent pages contains blank spaces that are marked with “…(omitted) …” where descriptions of the required items are omitted to simplify the explanation. In an actual abstract prepared for submission, all of the required items should be described. If any items are omitted, the reasons and grounds must be presented. Insufficient descriptions in the submitted documents, such as in the abstract, will require that more time be spent on the document checks by the secretariat, which may result in a delay in deliberation by the council and designation for the feed additive. Even if the deliberation 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. When preparing the abstract, attach the cited test reports, academic papers, etc. with a summary in Japanese, number the documents, and submit them as attachments to the abstract. Additionally, please refer to the “Chapter VI: Checklist” which summarizes points of caution for the wording and descriptions of the required items, and make use of it during the preparation of the abstract. The checklist shall also be submitted together with the abstract after each of the items has been checked. 78 Cover Abstract of the Test Results, etc., of the Objected Substance (xxxx) mmmm, dd, yyyy Feed Additive Business Operator 79 General name …(omitted)… Chemical name …(omitted)… Trade name …(omitted)… Intended use and dosage …(omitted)… Chemical structure …(omitted)… List of Items to be Described in the Abstract (page) 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……………………………………… 81 (2) Status of authorization and use in foreign countries……………………… 81 (3) Status of authorization and use as veterinary drugs and importation…… 81 (4) Comparison to related substances…………………………………………… 81 2. Items Concerning Standards (1) Name……………………………………………………………………………… 81 i. General name………………………………………………………………… 81 ii. Chemical name……………………………………………………………… 81 iii. Trade name…………………………………………………………………… 81 (2) Chemical structure……………………………………………………………… 82 (3) Manufacturing process………………………………………………………… 82 (4) Biological and physicochemical properties…………………………………… 82 i. Physical and chemical properties…………………………………………… 82 ii. Identification tests…………………………………………………………… 82 iii. Purity tests…………………………………………………………………… 83 iv. Content and quantitative method…………………………………………… 83 (5) Quantitation in feeds…………..………………………………………………… 83 (6) Changes with time……………………………………………………………… 83 i. Ambient temperature storage tests ………………………………………… 84 ii. Heat resistance test…………………………………………………………… 84 iii. Humidity resistance test……………………………………………………… 85 iv. Light resistance test………………………………………………………… 85 v. Accelerated test……………………………………………………………… 85 vi. In-feed stability test………………………………………………………… 85 3. Items Concerning Efficacy (1) Basic tests to prove effectiveness……………………………………………… 85 i. In vitro test……………………………………………………………………… 85 ii. In vivo test……………………………………………………………………… 85 (2) Basic tests to prove efficacy…………………………………………………… 85 4. Items Concerning Residue Residue test in targeted livestock, etc.…………………………………………… 86 5. Items Concerning Safety (1) Toxicity tests……………………………………………………………………… 87 i. General toxicity test…………………………………………………………… 87 a. Single dose toxicity test…………………………………………………… 87 b. Repeated dose toxicity test (short term)………………………………… 88 c. Repeated dose toxicity test (long term)………………………………… 88 ii. Special toxicity tests………………………………………………………… 88 a. Transgenerational reproduction toxicity test……………………………… 88 b. Developmental toxicity test………………………………………………… 88 c. Carcinogenicity test………………………………………………………… 88 80 d. Mutagenicity test…………………………………………………………… 88 e. Other tests…………………………………………………………………… 89 iii. Pharmacological test………………………………………………………… 90 iv. Test concerning the in vivo kinetics and metabolism……………………… 90 (2) Feeding test using targeted livestock, etc.…………………………………… 91 (3) Test concerning the emergence of resistant bacteria………………………… 91 (4) Others...………………………………………………………………………… 91 81 Document No. Item Specific item Examined item and summary of results 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 (development) (2) Status of its authorization and use as a feed additive in foreign countries, etc. (3) Status of its manufacturing and distribution authorization, and its importation as a veterinary medicinal product (4) Comparison to related substances (generic substances having the same effect) (1) Origin or background of the discovery (development) … (omitted) … (2) Status of the substance’s authorization and use as a feed additive in foreign countries, etc. … (omitted) … (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) … 2．Items Concerning Standards (1) Name i. General name ii. Chemical name iii. Trade name (1) Name i. General name … (omitted) … ii. Chemical name … (omitted) … iii. Trade name … (omitted) … 82 (2)Chemical structure (3) Manufacturing process (4) Biological and physicochemical properties i. Physical and chemical properties ii. Identification test (2) Chemical structure Definition … (omitted) … Potency … (omitted) … Structural formula, molecular formula, molecular weight, … (omitted) … (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 the unrefined 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 and physicochemical properties i. 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 practically insoluble in benzene. … (omitted) … ii. Identification test This substance shows an IR absorption peak at near 1,750 cm-1 of the wavenumber in the 83 iii. Purity test iv. Content and quantitative method (5) Quantitation in feed (6) Changes with time infrared absorption spectrum by the potassium bromide tablet method. The aqueous solution of this substance (1→10) shows a quantitative reaction with xx salt. … (omitted) … iii. 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 is practically clear. b. Chloride limit: When conducting a chloride limit test with 1.0 g (0.95–1.04 g) of this substance, 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 and quantitative method Content: This material contains 99.5% or more of the objected substance (describe the chemical 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 of formalin 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. 1 mL of 0.5 mol/L sodium hydroxide solution = equivalent weight in mg, chemical formula of the objected substance … (omitted) … (5) Quantitation in 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 and ether (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 the test 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 time 84 i. Ambient temperature storage tests ii. Heat resistance i. Ambient temperature storage tests Pack the substance in a typical package (such as a plastic bag), then store the package in an indoor warehouse for 24 months at 25℃, 50% humidity to assess the stability. The observed changes 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 At the start 3 mos. 6 mos. 9 mos. 12 mos. 24 mos. A Appearance white powder white powder white powder white powder white powder white mud1 Identification test fit fit fit fit fit fit Purity test fit fit fit fit fit fit Amount of active ingredient (g) 214.6 212.1 210.4 208.3 205.4 207.9 Drying loss(g) 5.2 4.9 3.7 5.0 6.3 4.7 Amount of moisture (g) 32.3 30.0 29.4 35.1 33.8 35.3 B Appearance white powder white powder white powder white powder white powder white powder (omitted) Note 1: “White mud” indicates a white paste condition. Although the appearance of the substance turned to a white mud (paste condition) after a period 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 test 85 test iii. Humidity resistance test iv. Light resistance test v. Accelerated test vi. In-feed stability test … (omitted) … iii. Humidity resistance test … (omitted) … iv. Light resistance test … (omitted) … v. Accelerated test … (omitted) … vi. In-feed stability test … (omitted) … 3. Items Concerning Efficacy (1) Basic tests to prove the efficacy i. In vitro test ii. In vivo test (2) Field application tests to prove efficacy (1) Basic tests to prove the efficacy i. In vitro test … (omitted) … ii. In vivo test … (omitted) … (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 the objected 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 no non exposure period. After four weeks, all of the test animals were slaughtered and subjected to a dissection examination. The test animals were given feeds continuously with the objected substance added at the 86 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 actual use. [Results] The test results are shown in the form below. (Appendix Form 2) The live weight gain significantly increased at the concentration 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. … (omitted) … 4．Items Concerning Residue 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 of males 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 slaughtered and 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%, and the 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 the milk 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 87 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 and more of the additive amount. However, the amount of residue was equal to or less than the limit 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 efficacy of the objected substance. Table 3 Animal residue test of the objected substance in cows Concentration of the objected substance in the feed (mg/kg) Analyzed body part Muscles (skeletal muscles) Liver Kidneys Fat (abdominal area) Milk 100 <2 <2 <2 <2 <1 300 <2 <2 <2 10–15 <1 1,000 <2 5–7 5–8 40–50 <1 Limit of quantitation: 1µg/kg for milk and 2µg/kg for other parts … (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 of males 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 found dead. 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. 88 ii. Repeated dose toxicity test (short term) iii. Repeated toxicity test (long term) ii. Special toxicity tests a. Transgenerational reproductive toxicity test b. Developmental toxicity test c. Carcinogenicity test d. Mutagenicity test The details of the results are summarized in the form below (Appendix Form 4) … (omitted) … 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) … (omitted) … b. Developmental toxicity test … (omitted) … c. Carcinogenicity test … (omitted) … 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/plate as 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 and without 89 e. Other tests the presence of the S9 mix for metabolic activation, along with negative control and positive control groups, at 37℃ for 3 days. After the culture, the revertant colonies were counted for each individual plate. [Results] The results are summarized in the table below (Table 5). Table 5 Number of revertant colonies of Salmonella typhimurium S9mix Concentration (µg/plate) Salmonella typhimurium (o m itt ed ) TA98 TA100 TA102 TA1535 (-) 0*1 13 160 (omitted) 1.6 15 155 8.0 14 152 40.0 13 162 200.0 15 161 1,000.0 14 158 5,000.0 11 169 Positive control 12 163 (+) 0*1 (omitted) (omitted) (*1) Negative control 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 the S9 mix for metabolic activation. Based on the above results, it can be concluded that the objected substance does not have mutagenicity. … (omitted) … e. Other tests Other tests were not conducted. 90 iii. Pharmacological test iv. In vivo kinetics tests iii. Pharmacological test The pharmacological test was omitted because the objected substance does not have any pharmacological effects. iv. In vivo kinetics tests 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 state of 14C inside the bodies. Information about the tested individual mouse, cow dog and human is 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 substance at 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 was measured at 0.01, compared to that of the objected substance being 1.0. Almost all of the objected substance (approximately 98%) was metabolized in the liver and excreted as metabolites. Similar data was obtained in the cow: 98% of the objected substance was metabolized 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) Animal species Test sample Elapsed time (hrs.) 3 days total 24 48 72 Mouse Urine 0.48 0.66 0.32 1.46 Feces 89.29 4.34 3.91 97.54 Cow Urine 0.61 0.52 0.10 1.23 Feces 89.44 5.78 2.98 98.20 (omitted) Scintigraphy for the analysis of the dynamic state of the radioactive substance inside the body, as well as an infrared spectroscopy and nuclear magnetic resonance spectroscopy for the 91 (2) Feeding test using targeted livestock (3) Test concerning the emergence of resistant bacteria (4) Other tests identification of the chemical structure were used. The details of the method are as follows. … (omitted) … (2) Feeding test using targeted livestock 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 of males and females in a group Dose and method of administration: Continuous administration was given by mixing the objected substance in the feeds for 3 months. Consumption of the water and feeds by the test animals was discretionary. The dose was 50–800 mg/kg (with 80 mg/kg as the optimum dose). … (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 an increase 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 when compared 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 a mild gastric erosion, which was cured in a week or so. After the cure, the live weight gain increased. … (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. 92 List of the original papers used for the preparation of the Abstract No. of original paper Title of original paper 1 (omitted) 2 (omitted) 3 (omitted) 4 (omitted) 5 (omitted) (omitted) 93 Title of Original Paper 1 *The original papers (attachments), etc., cited in the abstract are exhibited on the next page and the following pages. １ 94 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 this handbook does not necessarily guarantee the substance’s successful designation, even if the intended substance is a chemical substance. If the intended substance is an antibiotic or a live microbial agent, significantly different documents are required from those needed for chemical substances. Please contact the secretariat or the Japan Scientific Feed Association with any questions. In closing, we hope that this handbook will come in useful for those who are preparing such documents. We would like to thank the Japan Scientific Feed Association and many other groups and individuals for their support and advice during the preparation of this 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 95 Ⅵ Checklist For a smooth deliberation, refer to this checklist when preparing the abstract. Efficacy and safety Check Items to be checked Is the font size 12pt or over? Check the examination item list to see whether all items are included. (When omitting the part of the data, provide the reason for the omission after the item name. Data may not be ommi tted for each item.) Has the test design been described after referring to the examples in the handbook? Have the values of concentrations in the feeds been converted into the daily intake amount per unit of body weight? Have the values of the test results been summarized in an appendix? Have the results of an efficacy test been described by taking into account feeding conditions (feed composition, the breed of the targeted livestock, etc.) in Japan? Have any adverse events that occurred during a test been described in an appendix? Have the tests that are subject to the “feed additive GLP” been performed in accordance with these principles? Have you carried out tests using an appropriate statistical method and stated their results? [Examples] A significant difference of … was observed. (xx test, p<0.05) Note: The LSD method is not to be used for 4 or more groups of target animals. Make sure that all technical terms written in English are translated into the correct Japanese terms. Have Japanese summaries of the original papers been attached? Have the parts which have been cited from an original paper (attached material) been indicated by underlining, fluor escent marker, or the like? Are the title and company name indicated on the front and back of the file? Is a table of contents with page numbers included? 96 Standard Check Items to be checked Is the font size 12pt or over? Have Comparison Table 1, Comparison Table 2, the analysis results, etc. been provided according to the document composition outlined in “II-2 Outline of the Required Documents” in the handbook? Have you checked whether all the items in “1. Origin or Background of the Discovery …” and “2. Items Concerning S tandards 000229049.pdf (pmda.go.jp)” have been provided in the examination item list? (When omitting the part of t he data, provide the reason for the omission after the item name. Data may not be ommitted for each item.) Have you carried out tests using an appropriate statistical method and stated their results? [Examples] A significant difference of … was observed. (xx test, p<0.05) Note: The LSD method is not to be used for 4 or more groups of target animals. Have the reasons for setting standards been organized? [Example 1] The standards were set by referring to the standards for existing feed additives. [Example 2] The standards were set by referring to the company standards. Have the purity levels (enzymatic activity unit, titer, etc.) and values proposed in the purity test been organized? [Example 1] The values were proposed according to the company standard. [Example 2] These values were set based on quality management analysis results, due to…reason. Have “lead” and “arsenic” been set as purity test items? Is the number of digits appropriate? When citing an analytical method included in a ministerial ordinance, in principle, please use the existing description forms. When proposing a new analytical method, describe it so that the significant figures necessary for judgment can be o btained. Are the descriptions of colors based on JIS Z 8102-2001 (“Names of non-luminous object colors”)? Are the employed test methods and reagents commonly used in Japan? Have all the raw materials of each formulation been listed? (When a substance other than a feed additive, a raw material for feed, a diluent, or a food additive has been used, p rovide an explanation.) [Example] Substance xx is listed in the Pharmaceutical Excipient Standard of Japan as xx agent. In (country), the use of the substance in food and feed as xx agent is permitted. Have the parts which have been cited from an original paper (attached material) been indicated by underlining, fluorescent marker, or the like? Are the title and company name indicated on the front and back of the file? Is a table of contents with page numbers included? https://www.pmda.go.jp/files/000229049.pdf https://www.pmda.go.jp/files/000229049.pdf 97 Date (day/month/year) Feed Additive: Overview 1. Name of the substance: (Corresponding/not corresponding to a poison, dangerous substance, or toxic agent) Trade name: Name of the applicant (business operator) 2. Necessity of the feed additive (Describe what benefits will be obtained from designation of the substance as a feed additive and how the current situation will improve. Additionally, explain whether its effectiveness is equivalent to or higher than existing feed additives.) 3. Intended uses of the feed additive (If the feed additive does not fall under any of the categories, describe it as “other” and explain specifically.) 4. Amount of the feed additive added to feed Target feed (*Note) Amount of the feed additive added to the feed *Note: Describe the feed to which the feed additive is added (target feed), the type of livestock, and their growth stages (examples: “feed for cows” “feed for broilers,” “feed for pigs whose weights are generally kg or less,” etc.) in detail. 5. Indicate the documents which have collected by putting a “○” in the examination item list corresponding to the objective of the feed additive. If you have omitted any items, please provide the reason for the omission. 6. Whether findings or test data relating to residue in livestock products are present/absent (Such findings and test data may be required during a hearing of the Health, Labour and Welfare Ministry, and may be required for items other than the main components.) Uses specified by relevant ordinances of the Ministry of Agriculture, Forestry and Fisheries Categories Prevention of feed quality deterioration Antioxidant, fungicide, binder, emulsifier, adjuster Supplement of a nutrient component or other effective ingredient in feed Amino acid, vitamin, mineral, pigment Promotion of the effectiveness of a nutrient component contained in feed Synthetic antimicrobial, antibiotic, flavoring agent, taste component, enzyme, live microbial agents, organic acid February 2017 version 98 Point of contact: Department in Charge of Feed Additives, Feed Safety Standard Group, Animal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries Phone: 03-3502-8111 (ext. 4546) feed additive@maff.go.jp (Up to about 5 MB; a ZIP file is unacceptable.) mailto:feed additive@maff.go.jp 99 Date (day/month/year) Revision of Composition Standards for Feed Additives: Overview 1. Name of the substance 2. Name of the business operator 3. Outline of standard revision (1)Reason and outline 【Example】 ・One of the provisions requires the addition of in the process of manufacturing the ingredients for manufacturing, but as it has become difficult to obtain , we wish to use xx as an alternative. xx is included in the list of diluents. (2)Specific revised item 【Example】 ・Standard for ingredient manufacturing method Current standard: The ingredient is manufactured by adding ○○ to and by condensing and drying the mixture. Proposed revision: The ingredient is manufactured by adding ○○ or xx to and by drying the mixture. 4. List of documents to be submitted: 【Example】 ・Abstract ・Original papers cited when preparing the abstract ・Test results concerning the revision March 2017 version Point of contact: Department in Charge of Feed Additives, Feed Safety Standard Group, Animal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries Phone: 03-3502-8111 (ext. 4546) feed additive@maff.go.jp (Up to about 5 MB; a ZIP file is unacceptable.) mailto:feed additive@maff.go.jp