Subchapter E—Viruses, Serums, Toxins, and Analogous Products; Organisms and Vectors

SUBCHAPTER E—VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS

PART 101—DEFINITIONS immunostimulants, certain cytokines, antigenic or immunizing components Sec. of live organisms, and diagnostic com- 101.1 Applicability. ponents, that are of natural or syn- 101.2 Administrative terminology. thetic origin, or that are derived from 101.3 Biological products and related terms. synthesizing or altering various sub- 101.4 Labeling terminology. stances or components of substances 101.5 Testing terminology. such as microorganisms, genes or ge- 101.6 Cell cultures. netic sequences, carbohydrates, pro- 101.7 Seed organisms. teins, antigens, allergens, or anti- AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, bodies. 2.80, and 371.4. (1) A product’s intended use shall be SOURCE: 38 FR 8426, Apr. 2, 1973, unless oth- determined through an objective stand- erwise noted. ard and not a subjective one, and would be dependent on factors such as rep- § 101.1 Applicability. resentations, claims (either oral or When used in parts 101 through 117 of written), packaging, labeling, or ap- this subchapter, the meaning of the pearance. words and phrases listed shall be as de- (2) The term analogous products shall fined in this part. include: (i) Substances, at any stage of pro- § 101.2 Administrative terminology. duction, shipment, distribution, or The following administrative words sale, which are intended for use in the and phrases shall mean: treatment of animals and which are Adjacent herd. Adjacent herds are similar in function to biological prod- herds physically contiguous to the herd ucts in that they act, or are intended of origin; there are no herds between to act, through the stimulation, sup- an adjacent herd and the herd of origin. plementation, enhancement, or modu- Administrator. The Administrator, lation of the immune system or im- Animal and Plant Health Inspection mune response; or Service, or any person authorized to (ii) Substances, at any stage of pro- act for the Administrator. duction, shipment, distribution, or Animal and Plant Health Inspection sale, which are intended for use in the Service. The agency in the Department treatment of animals through the de- of Agriculture responsible for admin- tection or measurement of antigens, istering the Virus-Serum-Toxin Act. antibodies, nucleic acids, or immunity; Biological products. The term biologi- or cal products, also referred to in this (iii) Substances, at any stage of pro- subchapter as biologics, biologicals, or duction, shipment, distribution, or products, shall mean all viruses, se- sale, which resemble or are represented rums, toxins (excluding substances as biological products intended for use that are selectively toxic to microorga- in the treatment of animals through nisms, e.g., antibiotics), or analogous appearance, packaging, labeling, products at any stage of production, claims (either oral or written), rep- shipment, distribution, or sale, which resentations, or through any other are intended for use in the treatment means. of animals and which act primarily (3) The term treatment shall mean the through the direct stimulation, sup- prevention, diagnosis, management, or plementation, enhancement, or modu- cure of diseases of animals. lation of the immune system or im- Department. The U.S. Department of mune response. The term ‘‘biological Agriculture. products’’ includes but is not limited Distributor. A person who sells, dis- to vaccines, bacterins, allergens, anti- tributes, or otherwise places in chan- bodies, antitoxins, toxoids, nels of trade, one or more biological

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products he does not produce or im- Licensed establishment. An establish- port. ment operated by a person holding an Division. A marketing unit estab- unexpired, unsuspended, and unrevoked lished by the licensee which may be U.S. Veterinary Biologics Establish- named on labels, advertisements and ment License. promotional material in addition to Licensee. A person to whom an estab- the name and address of the producer. lishment license and at least one prod- Domestic animals. All animals, other uct license has been issued. than man, including poultry. Microorganisms. Microscopic or sub- Establishment. One or more premises microscopic organisms, which are designated on the establishment li- sometimes referred to as organisms, cense. which may introduce or disseminate Guidelines. Guidelines establish prin- disease of animals. ciples or practices related to test pro- Nonadjacent herd. Nonadjacent herds cedures, manufacturing practices, are all herds other than the herd of ori- product standards, scientific protocols, gin and other than herds adjacent to labeling, and other technical or policy the herd of origin. Herds adjacent to considerations. Guidelines contain pro- the herd of origin but in a different cedures or standards of general applica- State from the herd of origin are also bility that are usually not regulatory considered nonadjacent herds. in nature, but that are related to mat- Permittee. A person who resides in the ters that fall under the Virus-Serum- United States or operates a business Toxin Act. Guidelines issued by the establishment within the United agency include Veterinary Biologics States, to whom a permit to import bi- Licensing Considerations, Memoranda, ological products has been issued. Notices, and Supplemental Assay Person. Any individual, firm, partner- Methods. ship, corporation, company, associa- Herd. Any group of animals, includ- tion, educational institution, State or ing birds, fish, and reptiles, maintained local governmental agency, or other at a common location (e.g. lot, farm or organized group of any of the fore- ranch) for any purpose. The herd (or going, or any agent, officer, or em- flock) includes all animals subse- ployee of any thereof. quently housed at the common loca- Premises. All buildings, appur- tion. If the principal animals of a group tenances, and equipment used to are moved to a different location, the produce and store biological products group is still considered the same herd. located within a particular land area Herd of origin. The herd from which shown on building plans or drawings the microorganism used as seed for furnished by the applicant or the li- production of an autogenous biologic is censee and designated by an address isolated. Offspring and excess breeding adequate for identification. stock (not the principal animals) Prepare or preparation. Sometimes removed or sold from one group of ani- ferred to as manufacture or produce, mals to another have changed herds means the steps and procedures used in and are no longer considered part of the processing, testing, packaging, la- the herd they originated from. Groups beling, and storing of a biological prod- of animals under the same ownership uct. but at different locations are separate Regulations. The provisions in parts herds. 101 through 118 of this subchapter. Inspection. An examination made by Research investigator or research spon- an inspector to determine the fitness of sor. A person who has requested au- animals, establishments, facilities, and thorization to ship an experimental bi- procedures used in connection with the ological product for the purpose of preparation, testing, and distribution evaluating such product, or has been of biological products and the examina- granted such authorization. tion or testing of biological products. Secretary. The Secretary of Agri- Inspector. Any officer or employee of culture of the United States or any of- Animal and Plant Health Inspection ficer or employee of the Department to Service who is authorized by the Ad- whom authority has heretofore been ministrator to do inspection work. delegated, or to whom authority may

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hereafter be delegated, to act in his unrevoked product license for such stead. product. Subsidiary. A corporation in which a (b) Experimental biological product. A corporate licensee owns in excess of 50 biological product which is being eval- percent of the voting stock. uated to substantiate an application Veterinary Services. Veterinary Serv- for a product license or permit. ices unit of Animal and Plant Health (c) Completed product. A biological Inspection Service of the Department. product in bulk or final container pro- Virus-Serum-Toxin Act. The Act of duced in compliance with the regula- March 4, 1913, 37 Stat. 832–833; as tions to final form and composition. amended December 23, 1985, Public Law (d) Finished product. A completed 99–198, 99 Stat. 1654–1655; and as further product which has been bottled, sealed, amended September 28, 1988, Public packaged, and labeled as required by Law 100–449, 102 Stat. 1868; 21 U.S.C. the regulations. 151–159. (e) Released product. A finished prod- U.S. Veterinary Biological Product Li- uct released for marketing after all re- cense. A document, sometimes referred quirements have been satisfactorily to as a product license, which is issued complied with. pursuant to part 102 of this subchapter (f) Fraction. A specific antigen, its to the holder of an establishment li- antibodies, or its antitoxin which con- cense, as a part of and ancillary to the stitutes a component of a biological establishment license, and which au- product. thorizes production of a specified bio- (g) Diluent. A liquid used to re- logical product in the designated li- hydrate a desiccated product or a liq- censed establishment. uid used to dilute another substance. U.S. Veterinary Biological Product Per- (h) Serial. The total quantity of com- mit. A document, sometimes referred to pleted product which has been thor- as a permit, issued to a person author- oughly mixed in a single container and izing the importation of specified bio- identified by a serial number: Provided, logical products subject to restrictions That, when all or part of a serial of liq- and controls as provided in the regula- uid biological product is packaged as tions. diluent for all or part of a serial of des- U.S. Veterinary Biologics Establishment iccated product, the resulting combina- License. A document referred to as an tion packages shall be considered a se- establishment license, which is issued rial of the multiple fraction product. pursuant to part 102 of this subchapter, (i) Subserial. Each of two or more authorizing the use of designated prem- properly identified portions of a serial ises for production of biological prod- which are further processed at different ucts specified in one or more unex- times or under different conditions pired, unsuspended, and unrevoked such as, but not limited to, being des- product license(s). iccated in different size final containers and/or at different times. [38 FR 8426, Apr. 2, 1973; 38 FR 9221, Apr. 12, (j) Outline of production. A detailed 1973, as amended at 40 FR 46093, Oct. 6, 1975; 41 FR 44358, Oct. 8, 1976; 49 FR 22624, May 31, protocol of methods of manufacture to 1984; 52 FR 30131, Aug. 13, 1987; 56 FR 66782, be followed in the preparation of a bio- 66783, Dec. 26, 1991; 57 FR 38756, Aug. 27, 1992; logical product and which may some- 62 FR 31328, June 9, 1997; 64 FR 43044, Aug. 9, times be referred to as an outline. 1999] (k) Product Code Number. A number assigned by Animal and Plant Health § 101.3 Biological products and related Inspection Service to each type of li- terms. censed biological product. When used in conjunction with or in (l) Harvest date. Unless otherwise reference to a biological product, the specified in a filed Outline of Produc- following terms shall mean: tion, the harvest date shall be the date (a) Licensed biological product. A bio- blood or tissues are collected for pro- logical product prepared within a li- duction or the date cultures of living censed establishment by a person hold- microorganisms are removed from pro- ing an unexpired, unsuspended, and duction incubators.

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(m) Bacterin. An inactivated bacterial ucts, which also may be marketed indi- product consisting of an antigenic sus- vidually unless otherwise restricted. pension of organisms or particulate [38 FR 8426, Apr. 2, 1973, as amended at 42 FR parts of organisms, representing a 63770, Dec. 20, 1977; 50 FR 24903, June 14, 1985; whole culture or a concentrate thereof, 56 FR 66782, Dec. 26, 1991; 60 FR 14354, Mar. 17, with or without the unevaluated 1995; 81 FR 59433, Aug. 30, 2016] growth products, which has been inactivated as demonstrated by acceptable § 101.4 Labeling terminology. tests written into the filed Outline of Terms pertaining to identification Production for the product. and packaging of biological products (n) Toxoid. An inactivated bacterial shall mean: product which consists of a sterile, an- (a) Label. All written, graphic, or tigenic toxin or toxic growth product, printed matter: which has resulted from the growth of (1) Upon or attached to a final con- bacterial organisms in a culture me- tainer of a biological product; (2) Appearing upon any immediate dium from which the bacterial cells carton or box used to package such have been removed, which has been in- final container; and activated without appreciable loss of (3) Appearing on any accompanying antigenicity as measured by suitable enclosures (leaflets, inserts, or circu- tests, and which is nontoxic as dem- lars) on which required information or onstrated by acceptable tests written directions as to the use of the biologi- into the filed Outline of Production. cal product shall be found. (o) Bacterin-toxoid. An inactivated (b) Labeling. All labels and other bacterial product which is either: written, printed, or graphic matter ac- (1) A suspension of organisms, rep- companying the final container. resenting a whole culture or a con- (c) Final container. The unit, bottle, centrate thereof, with the toxic growth vial, ampule, tube, or other receptacle products from the culture which has into which any biological product is been inactivated without appreciable filled for distribution and sale. loss of antigenicity as measured by (d) True name. The name entered on suitable tests, the inactivation of orga- the product license or permit at the nisms and toxins being demonstrated time of issuance to differentiate the bi- by acceptable tests written into the ological product from others: Provided, filed Outline of Production: Provided, That, the principal part of such name That it shall contain cellular antigens shall be emphasized on such license or and shall stimulate the development of permit by being more prominently let- antitoxin; or tered than descriptive terms which (2) A combination product in which may be necessary to complete the dif- one or more toxoids or bacterin-toxoids ferentiation. (e) Serial number. Numbers or num- is combined with one or more bacterins bers and letters used to identify and or one or more bacterin-toxoids. distinguish one serial from others. (p) Bacterial extract. An inactivated (f) Expiration date. A date designating bacterial product which consists of the the end of the period during which a bi- sterile, nontoxic, antigenic derivatives ological product, when properly stored extracted from bacterial organisms or and handled, can be expected with rea- from culture medium in which bac- sonable certainty, to be efficacious. terial organisms have grown. (g) Label number. A number assigned (q) Combination package. Biological by Animal and Plant Health Inspection product consisting of two or more li- Service to each label or sketch sub- censed biological products. Each com- mitted for review. pleted product in final container is (h) Master label. The finished carton, packaged together and mixed prior to container, or enclosure label for the administration. A combination pack- smallest size final container that is au- age is issued a separate U.S. Veteri- thorized for a biological product, that nary Biological Product License and serves as the Master template label ap- assigned a product code number to dis- plicable to all other size containers or tinguish it from its component prod- cartons of the same product that is

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marketed by a licensee, subsidiary, di- (h) Dose. The amount of a biological vision, or distributor. product recommended on the label to be given to one animal at one time. [38 FR 8426, Apr. 2, 1973, as amended at 42 FR (i) Vaccinate. An animal which has 63770, Dec. 20, 1977; 56 FR 66782, Dec. 26, 1991; 61 FR 29464, June 11, 1996] been inoculated, injected, or otherwise administered a biological product § 101.5 Testing terminology. being evaluated. (j) Control animal. An animal, which Terms used when evaluating biologi- may be referred to as a control, used in cal products shall mean: a test procedure for purposes of com- (a) Standard Requirement. Test meth- parison or to add validity to the re- ods, procedures, and criteria estab- sults. lished by Animal and Plant Health In- (k) Day. Time elapsing between any spection Service for evaluating biologi- regular working hour of one day and cal products to be pure, safe, potent, any regular working hour of the fol- and efficacious, and not to be worth- lowing day. less, contaminated, dangerous, or (l) Test results. Terms used to des- harmful under the Act. ignate testing results are as follows: (b) Log. Logarithm computed to the (1) No Test. Designation used when a base 10. deficiency in the test system has ren- (c) Pure or purity. Quality of a bio- dered a test unsuitable for drawing a logical product prepared to a final form valid conclusion. relatively free of extraneous micro-or- (2) Satisfactory. Designation is a final ganisms and extraneous material (or- conclusion given to a valid test with ganic or inorganic) as determined by results that meet the release criteria test methods or procedures established stated in the filed Outline of Produc- by Animal and Plant Health Inspection tion or Standard Requirement. Service in Standard Requirements or (3) Unsatisfactory. Designation is a in the approved Outline of Production final conclusion given to a valid test for such product, but free of extraneous with results that do not meet the re- microorganisms or material which in lease criteria stated in the filed Out- the opinion of the Administrator ad- line of Production or Standard Re- versely affects the safety, potency, or quirement. efficacy of such product. (4) Inconclusive. Designation used for an initial test when a sequential test (d) Safe or safety. Freedom from prop- design established in the filed Outline erties causing undue local or systemic of Production or Standard Require- reactions when used as recommended ment allows further testing if a valid or suggested by the manufacturer. initial test is not satisfactory. (e) Sterile or sterility. Freedom from (m) Healthy. Apparently normal in all viable contaminating microorganisms vital functions and free of signs of dis- as demonstrated by procedures pre- ease. scribed in part 113 of this subchapter, (n) Unfavorable reactions. Overt ad- Standard Requirements, and approved verse changes which occur in healthy Outlines of Production. test animals subsequent to initiation (f) Potent or potency. Relative of a test and manifested during the ob- strength of a biological product as de- servation period prescribed in the test termined by test methods or proce- protocol which are attributable either dures as established by Animal and to the biological product being tested Plant Health Inspection Service in or to factors unrelated to such product Standard Requirements or in the ap- as determined by the responsible indi- proved Outline of Production for such vidual conducting the test. product. (o) Master reference. A Master Ref- (g) Efficacious or efficacy. Specific erence is a reference whose potency is ability or capacity of the biological correlated, directly or indirectly, to product to effect the result for which it host animal immunogenicity. The Mas- is offered when used under the condi- ter Reference may be used as the work- tions recommended by the manufac- ing reference in in vitro tests for rel- turer. ative potency. The Master Reference

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may also be used to establish the rel- in the currently filed Outline of Pro- ative potency of a serial of product duction. used in requalification studies and to (r) Immunogenicity. The ability of a establish the relative potency of work- biological product to elicit an immune ing references. The preparation of a response in animals as determined by Master Reference as described in a filed test methods or procedures acceptable Outline of Production may be: to the Animal and Plant Health Inspec- (1) A completed serial of vaccine or tion Service. bacterin prepared in accordance with a filed Outline of Production; [38 FR 8426, Apr. 2, 1973, as amended at 40 FR (2) A purified preparation of a protec- 45419, Oct. 2, 1975; 41 FR 6751, Feb. 13, 1976; 43 tive immunogen or antigen; or FR 3701, Jan. 27, 1978; 56 FR 66782, 66783 Dec. (3) A nonadjuvanted harvested cul- 26, 1991; 62 FR 19037, Apr. 18, 1997; 79 FR 55969, ture of microorganisms. Sept. 18, 2014] (p) Working reference. A Working Ref- § 101.6 Cell cultures. erence is the reference preparation that is used in the in vitro test for the When used in conjunction with or in release of serials of product. Working reference to cell cultures, which may References may be: be referred to as tissue cultures, the (1) Master References; or following terms shall mean: (2) Serials of product that have been (a) Batches of primary cells. A pool of prepared and qualified, in a manner ac- original cells derived from normal tis- ceptable to Animal and Plant Health sue up to and including the 10th sub- Inspection Service for use as reference culture. preparations. (b) Cell line. A pool of cells which are (q) Qualifying serial. (1) A serial of bi- 11 or more subcultures from the tissue ological product used to test for of origin. immunogenicity when the Master or (c) Subculture. Each flask to flask Working Reference is a purified antigen or nonadjuvanted harvest material. transfer or passage regardless of the Qualifying serials shall be produced in number of cell replications. accordance with the filed Outline of (d) Master Cell Stock (MCS). The sup- Production, tested for immunogenicity ply of cells of a specific passage level in accordance with methods deemed from which cells for production of bio- appropriate by the Animal and Plant logics originate. Health Inspection Service, and have a [38 FR 8426, Apr. 2, 1973, as amended at 40 FR geometric mean relative potency, when 45419, Oct. 2, 1975; 49 FR 22624, May 31, 1984] compared to the Master Reference, of not greater than 1.0 as established by: § 101.7 Seed organisms. independent parallel line assays with five or more replicates; or other valid When used in conjunction with or in assay methods for determining relative reference to seed organisms, the fol- antigen content which demonstrate lowing shall mean: linearity, specificity, and reproduc- (a) Master Seed. An organism at a spe- ibility at least equivalent to the par- cific passage level which has been se- allel line assay and are acceptable to lected and permanently stored by the the Animal and Plant Health Inspec- producer from which all other seed pas- tion Service. sages are derived within permitted lev- (2) Qualifying serials used to re- els. qualify or extend the dating period of a (b) Working Seed. An organism at a Master Reference shall be determined passage level between Master Seed and to be immunogenic in accordance with Production Seed. methods deemed appropriate by the (c) Production Seed. An organism at a Animal and Plant Health Inspection specified passage level which is used Service as provided in paragraph (a)(1) without further propagation for initi- of this section, and, in addition, shall ating preparation of a fraction. be within their permitted dating period and have been prepared in accordance [49 FR 22625, May 31, 1984] with the production method described

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PART 102—LICENSES FOR nished upon request to Animal and BIOLOGICAL PRODUCTS Plant Health Inspection Service. (2) When a person conducts more Sec. than one establishment, a separate ap- 102.1 Licenses issued by the Administrator. plication shall be made for each estab- 102.2 Licenses required. lishment. 102.3 License applications. (3) Whenever subsidiaries are to oper- 102.4 U.S. Veterinary Biologics Establish- ate in an establishment for which li- ment License. 102.5 U.S. Veterinary Biological Product Li- cense application is made, the appli- cense. cant shall apply for permission for such 102.6 Conditional licenses. subsidiaries to operate in the establish- AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, ment and furnish therewith a complete 2.80, and 371.4. statement regarding the relationship between the applicant and the subsidi- § 102.1 Licenses issued by the Adminis- aries. trator. (4) Facilities documents, prepared as Each establishment qualified to pre- prescribed in part 108 of this sub- pare biological products under the chapter, shall accompany the applica- Virus-Serum-Toxin Act shall hold an tion for license unless previously filed unexpired and unrevoked U.S. Veteri- with Animal and Plant Health Inspec- nary Biologics Establishment License tion Service. issued by the Administrator and a U.S. (5) Each application for a U.S. Veteri- Veterinary Biological Product License nary Biologics Establishment License for each product prepared in such es- shall be accompanied by an application tablishment unless the product is sub- for one or more U.S. Veterinary Bio- ject to the provisions of 9 CFR parts logical Product Licenses and the sup- 103 or 106 of this subchapter. porting documents required by para- [60 FR 48021, Sept. 18, 1995] graph (b)(2) of this section. (6) A new application shall be made § 102.2 Licenses required. when a change of ownership, operation, (a) Every person who prepares bio- or location of an establishment occurs; logical products subject to the Virus- or prior to the expiration of a U.S. Vet- Serum-Toxin Act shall hold an unex- erinary Biologics Establishment Li- pired, unsuspended, and unrevoked U.S. cense issued for an interim period of Veterinary Biologics Establishment Li- time. cense and at least one unexpired, un- (b) U.S. Veterinary Biological Product suspended, and unrevoked U.S. Veteri- License. (1) The licensee of each estab- nary Biological Product License issued lishment or applicant for an establish- by the Administrator to prepare a bio- ment license shall make written appli- logical product. cation to the Administrator for a U.S. (b) An applicant who applies for an Veterinary Biological Product License establishment license must also apply for each biological product to be pre- for at least one product license. An es- pared in the licensed establishment. tablishment license will not be issued (2) Each application for a U.S. Veteri- without a license authorizing the pro- nary Biological Product License shall duction of a biological product in the be supported by: establishment. (i) At least two copies of an Outline [52 FR 11026, Apr. 7, 1987, as amended at 56 of Production prepared in accordance FR 66783, Dec. 26, 1991; 61 FR 52873, Oct. 9, with §§ 114.8 and 114.9 of this sub- 1996] chapter; and (ii) At least three copies of test re- § 102.3 License applications. ports and research data sufficient to (a) U.S. Veterinary Biologics Establish- establish purity, safety, potency, and ment License. (1) The operator of each efficacy of the product; and establishment of the kind specified in (iii) Legends prepared as prescribed § 102.2 shall make written application in § 108.5 of this subchapter designating to the Administrator for a license. which facilities are to be used in the Blank forms of application will be fur- preparation of each fraction; and

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(iv) Labels in finished form or Service that the biological products sketches prepared as prescribed in which are licensed to be prepared § 112.5 of this subchapter, together with therein shall not be so advertised as to information regarding all claims to be mislead or deceive the purchasers and made on labels and in advertising mat- that the packages or containers in ter to be used in connection with or re- which the same are to be marketed lated to the biological product. shall not bear any statement, design, (Approved by the Office of Management and or device which is false or misleading Budget under control number 0579–0013) in any particular. [39 FR 37763, Oct. 24, 1974, as amended at 48 (c) U.S. Veterinary Biologics Estab- FR 57472, Dec. 30, 1983; 49 FR 21043, May 18, lishment Licenses shall be numbered. 1984; 50 FR 50763, Dec. 12, 1985; 56 FR 66783, (d) Two or more licenses may bear Dec. 26, 1991; 75 FR 20772, Apr. 21, 2010] the same number when they are issued § 102.4 U.S. Veterinary Biologics Estab- for establishments under the same lishment License. ownership or control, provided a serial (a) Before a U.S. Veterinary Biologics letter is added to one or more to iden- Establishment License will be issued tify each license and the product pro- by the Administrator for any establish- duced thereunder. ment, an inspection shall be made to (e) When a U.S. Veterinary Biologics determine whether the condition, Establishment License is issued for an equipment, facilities, and the like, of establishment, it shall not apply to the establishment, and the methods more than one person at the same loca- used to prepare biological products are tion, except that subsidiaries of the li- in conformity with the requirements in censee, when named in the license, may the regulations. operate thereunder at the establish- (b) A license shall not be issued un- ment named. The licensee with its sub- less: sidiaries will be held responsible for all (1) In the opinion of the Adminis- operations conducted in the licensed trator, the condition of the establish- establishment. ment, including its facilities, and the (f) When a licensee no longer holds at methods of preparation of biological least one unexpired, unsuspended, or products are such as reasonably to as- unrevoked product license authorizing sure that the products shall accomplish the preparation of a biological product, the purpose for which they are in- or is in the process of obtaining a prod- tended; and (2) The Administrator is satisfied on uct license, the establishment license the basis of information before him shall no longer be valid and shall be re- that: turned to the Administrator. In the (i) The establishment shall be oper- case where an establishment license ated in compliance with the Act and expires or is suspended or revoked, any applicable regulations and be under the product license authorizing prepara- supervision of person(s) competent in tion of a product at such establishment the preparation of biological products; shall be invalid indefinitely or for as and long as the suspension is in effect. (ii) The applicant, or the person hav- (g) Any license issued under this part ing the responsibility for producing bi- to establishments in which biological ological products in the establishment, products are prepared shall be issued or both, is qualified by education and on condition that the licensee permit experience, and has demonstrated fit- the inspection of such establishments, ness to produce such products in com- products, product preparation, and all pliance with the Act and regulations relevant records as provided in part 115 issued pursuant thereto; Provided, of this subchapter. Failure to permit That, previous violations of the Act, or inspection may result in the license such regulations or both shall be rel- being suspended or revoked. evant to the Administrator’s determination of fitness. (h) The provisions of paragraph (b) of (3) Written assurance is filed with this section shall also be applicable to, Animal and Plant Health Inspection

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and be considered by, the Adminis- (3) When requested by the Adminis- trator in connection with each applica- trator, a licensee shall submit a list of tion for an additional product license. licensed biological products prepared (Approved by the Office of Management and in the licensed establishment. Budget under control number 0579–0013) (d) Where the Administrator determines that the protection of domestic [39 FR 37762, Oct. 24, 1974; 39 FR 38364, Nov. 1, 1974, as amended at 41 FR 44359, Oct. 8, 1976; animals or the public health, interest, 48 FR 57472, Dec. 30, 1983; 52 FR 11026, Apr. 7, or safety, or both, necessitates restric- 1987; 52 FR 30131, Aug. 13, 1987; 56 FR 66783, tions on the use of a product, the prod- Dec. 26, 1991; 60 FR 48021, Sept. 18, 1995; 61 FR uct shall be subject to such additional 52873, Oct. 9, 1996; 62 FR 13294, Mar. 20, 1997] restrictions as are prescribed on the license. Such restrictions may include, § 102.5 U.S. Veterinary Biological Product License. but are not limited to, limits on distribution of the product or provisions (a) Authorization to produce each bi- that the biological product is re- ological product shall be specified on a stricted to use by veterinarians, or U.S. Veterinary Biological Product Li- under the supervision of veterinarians, cense, issued by the Administrator, and supplementary to the U.S. Veterinary or both. Biologics Establishment License (e) Any person may request that the named therein. distribution and use of a veterinary bi- (b) The following shall appear on the ological product be restricted if the re- U.S. Veterinary Biological Product Li- striction pertains to the protection of cense: domestic animals or the public health, (1) The U.S. Veterinary Biologics Es- interest, or safety, or both. All re- tablishment License Number for the es- quests must be sent, in writing, to the tablishment from which the product is Director, Center for Veterinary Bio- released for marketing. logics, Policy, Evaluation, and Licens- (2) The true name of the product. ing, 1920 Dayton Avenue, P.O. Box 844, (3) The product code number for the Ames, IA 50010. Requests must specify product. the restriction(s) being requested and (4) The date of issuance. must explain why the restrictions are (5) Any restrictions designated by the needed. Copies of any supporting docu- Administrator under paragraph (e) of ments, such as scientific literature, this section. published or unpublished articles, or (6) When necessary to comply with data from tests, should be attached to § 102.6 of this part, a termination date the request. When a decision is reached and a brief description of requirements regarding the request, the person sub- to be met for reissuance. mitting the request will be sent writ- (c) The following provisions shall ten notification of such decision. apply to all licensed biological products: (Approved by the Office of Management and (1) Licensed biological products shall Budget under control number 0579–0013) be prepared as required by the regula- [39 FR 37763, Oct. 24, 1974, as amended at 48 tions and in accordance with a filed FR 57472, Dec. 30, 1983; 50 FR 50764, Dec. 12, Outline of Production as prescribed in 1985; 52 FR 11026, Apr. 7, 1987; 56 FR 66783, §§ 114.8 and 114.9 of this subchapter. No Dec. 26, 1991; 57 FR 38760, Aug. 27, 1992; 59 FR change shall be made in the prepara- 67616, Dec. 30, 1994; 62 FR 13294, Mar. 20, 1997; tion of a biological product without 64 FR 43044, Aug. 9, 1999; 75 FR 20772, Apr. 21, prior approval of the Administrator. 2010] (2) In addition to restrictions im- § 102.6 Conditional licenses. posed by the Administrator pursuant to paragraph (e) of this section, bio- In order to meet an emergency condi- logical products may be subject to re- tion, limited market, local situation, strictions which are imposed by any or other special circumstance, includ- State or other jurisdiction pertaining ing production solely for intrastate use to the distribution and use of such under a State-operated program, the products, based on local disease condi- Administrator may, in response to an tions. application submitted as specified in

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§ 102.3(b) of this part, issue a condi- Upon application therefor, the Admin- tional U.S. Veterinary Biological Prod- istrator may authorize the preparation uct License to an establishment under of experimental products on the prem- an expedited procedure which assures ises of a licensed establishment if he purity and safety, and a reasonable ex- determines that such preparation will pectation of efficacy. Preparation of not result in contamination of the li- products under a conditional license censed products. Each request for per- shall be in compliance with all applica- mission to prepare an experimental bi- ble regulations and standards and may ological product on licensed premises be restricted as follows: shall indicate the nature of the unli- (a) The preparation may be limited censed product, designate facilities to to a predetermined time period which shall be established at the time of be used, and specify precautions which issuance and specified on the license. will be taken to prevent contamination Prior to termination of the license, the of licensed products. Such requests licensee may request reissuance. Such shall be submitted to the Adminis- requests shall be substantiated with trator. Research facilities that are en- data and information obtained since tirely separate and apart from facili- the license was issued. After consid- ties used for the preparation of licensed ering all data and information avail- biological products will not be consid- able, the Administrator shall either re- ered a part of the licensed premises for issue the U.S. Veterinary Biological purposes of this section. Product License or allow it to termi- (Approved by the Office of Management and nate. Budget under control number 0579–0013) (b) Distribution may be limited to the extent necessary to assure that the [30 FR 11848, Sept. 16, 1965, as amended at 48 product will meet the basic criteria for FR 57473, Dec. 30, 1983; 56 FR 66783, Dec. 26, issuance of the conditional license. 1991] (c) Labeling for the product may be required to contain information on the § 103.2 Disposition of animals adminis- conditional status of the license. tered experimental biological products or live organisms. [52 FR 11026, Apr. 7, 1987, as amended at 60 FR 48021; Sept. 18, 1995] Safeguards as herein provided shall be established by the research investi- PART 103—EXPERIMENTAL PRO- gator or research sponsor to control disposition of all animals administered DUCTION, DISTRIBUTION, AND experimental biological products or EVALUATION OF BIOLOGICAL live organisms. PRODUCTS PRIOR TO LICENSING (a) Surviving test animals (including challenged control animals) shall not Sec. 103.1 Preparation of experimental biological be removed from the premises on which products. the tests are conducted for at least 14 103.2 Disposition of animals administered days after administration of an experi- experimental biological products or live mental biological product or live orga- organisms. nisms: Provided, however, That this 103.3 Shipment of experimental biological holding period may be increased or de- products. creased as permitted or requested by AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, the Administrator following review of 2.80, and 371.4. all relevant information or data avail- § 103.1 Preparation of experimental bi- able. ological products. (b) All animals administered experi- Except as otherwise provided in this mental biological products which are section, experimental biological prod- to be slaughtered at establishments ucts which are neither composed of nor subject to the Federal Meat Inspection prepared with organisms or antigens Act, as amended and extended (21 used in biologicals already licensed, U.S.C. 601 et. seq.) are subject to the ap- shall not be prepared in the production plicable requirements of § 309.16 of this facilities of a licensed establishment. title (Meat Inspection Regulations).

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(c) Except as otherwise provided in product for experimental study and this paragraph, the research investi- evaluation shall be accompanied by the gator or research sponsor shall main- following: tain adequate records relative to the (a) One copy of a permit or letter of disposition of each animal adminis- permission from the proper State or tered experimental biological products. foreign animal health authorities of These records shall be maintained for a each State or foreign country involved. minimum period of two years from the (b) Two copies of a tentative list of date that an experimental product was the names of the proposed recipients administered to such animal, and shall and quantity of experimental product show the name and address of the that is to be shipped to each individual. owner; number, species, class and loca- In the event of subsequent changes, ad- tion of the animals; and if sold, the ditional information shall be furnished name and address of the consignee, when such facts are known; buyer, commission, firm or abattoir: (c) Two copies of a description of the Provided, however, That a research investigator or research sponsor may be product, recommendations for use, and exempted from these recordkeeping re- results of preliminary research work; quirements by the Administrator on (d) A copy of the labels or label the basis of acceptable data dem- sketches which show the name or iden- onstrating that use of the experimental tification of the product and bear the biological product will not result in the statement ‘‘Notice! For experimental presence of any unwholesome condition use only-Not For Sale’’ or equivalent. in the edible parts of animals subse- Such statement shall appear on final quently presented for slaughter. container labels, except that it may appear on the carton in the case of very (Approved by the Office of Management and small final container labels and label- Budget under control number 0579–0059) ing for diagnostic test kits. The U.S. [30 FR 11848, Sept. 16, 1965, as amended at 48 Veterinary License legend shall not ap- FR 57473, Dec. 30, 1983; 56 FR 66783, Dec. 26, pear on such labels; and 1991; 66 FR 21063, Apr. 27, 2001] (e) Two copies of a proposed general § 103.3 Shipment of experimental bio- plan covering the methods and proce- logical products. dures for evaluating the product and Except as provided in this section, no for maintaining records of the quan- person shall ship or deliver for ship- tities of experimental product pre- ment in or from the United States, the pared, shipped and used. At the conclu- District of Columbia, or any Territory sion of field studies, results shall be ob- of the United States any unlicensed bi- tained, summarized, and submitted to ological product for experimental use the Animal and Plant Health Inspec- in animals. For the benefit of license tion Service. applicants and to permit and encourage (f) Data acceptable to the Adminis- research, a person may be authorized trator demonstrating that use of the by the Administrator to ship unli- experimental biological product in censed biological products for the pur- meat animals is not likely to result in pose of evaluating such experimental the presence of any unwholesome con- products by treating limited numbers dition in the edible parts of animals of animals, Provided, that, the Admin- subsequently presented for slaughter. istrator determines that the conditions (g) A statement from the research in- under which the experiment is to be vestigator or research sponsor agreeing conducted are adequate to prevent the to furnish, upon the Administrator’s spread of disease and approves the pro- request, additional information con- cedures set forth in the request for cerning each group of meat animals in- such authorization. Special restric- volved prior to movement of these ani- tions or tests may be imposed, espe- mals from the premises where the test cially in the case of products con- is to be conducted. Such information taining live organisms, when they are shall include the owner’s name and ad- deemed necessary or advisable by the dress; number, species, class and loca- Administrator. A request for author- tion of animals involved; date ship- ization to ship an unlicensed biological ment is anticipated; along with name

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and address of consignee, buyer, com- § 104.2 Permit authorized. mission firm or abattoir. (a) Animal and Plant Health Inspec- (h) Any information the Adminis- tion Service is authorized to issue trator may require in order to assess three types of permits for importing bi- the product’s impact on the environ- ological products. They shall be: ment. (1) U.S. Veterinary Biological Prod- [26 FR 7726, Aug. 18, 1961, as amended at 30 uct Permit for Research and Evalua- FR 11848, Sept. 16, 1965; 52 FR 30131, Aug. 13, tion; 1987; 56 FR 66783, Dec. 26, 1991; 75 FR 20772, (2) U.S. Veterinary Biological Prod- Apr. 21, 2010; 81 FR 59433, Aug. 30, 2016] uct Permit for Distribution and Sale; or PART 104—PERMITS FOR (3) U.S. Veterinary Biological Prod- BIOLOGICAL PRODUCTS uct Permit for Transit Shipment Only. (b) A permit shall not be issued for a Sec. biological product from countries 104.1 Permit required. known to have exotic diseases, includ- 104.2 Permit authorized. ing but not limited to foot-and-mouth 104.3 Permit application. disease, rinderpest, highly pathogenic 104.4 Products for research and evaluation. avian influenzaswine vesicular disease, 104.5 Products for distribution and sale. Newcastle disease, and African swine 104.6 Products for transit shipment only. fever, if in the opinion of the Adminis- 104.7 Product permit. trator, such products may endanger 104.8 Illegal shipments. the livestock or poultry of this coun- AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, try. 2.80, and 371.4. (c) A permit shall not be issued until an inspector has determined the condi- SOURCE: 38 FR 32916, Nov. 29, 1973, unless otherwise noted. tion of the equipment and facilities of the producer, of the applicant, or of § 104.1 Permit required. both if such a determination is considered necessary by the Administrator. Unless otherwise authorized or di- (d) A permit shall not be issued for a rected by the Administrator, each per- biological product prepared in the mit to import a biological product into United States, exported, and presented the United States shall be issued in ac- for reentry except as provided in cordance with the regulations in this § 104.4(d). part. (a) No biological product shall be [38 FR 32916, Nov. 29, 1973, as amended at 56 brought into the United States unless a FR 66783, Dec. 26, 1991; 56 FR 66783, Dec. 26, 1991; 78 FR 19085, Mar. 29, 2013] permit has been issued for such product. A separate U.S. Veterinary Bio- § 104.3 Permit application. logical Product Permit shall be required for each shipment of biological (a) Each person desiring to import a product to be imported: Provided, That, biological product shall make written application to Animal and Plant a permit shall also be required for each Health Inspection Service for a permit. transit shipment of biological products Application forms are available on the moved through the United States. Internet at (http://www.aphis.usda.gov/ (b) Each person importing biological animallhealth/ vetlbiologics/ products shall hold an unexpired, un- vblforms.shtml) and application for a suspended, and unrevoked permit permit to import a veterinary biologic issued by Animal and Plant Health In- for research and evaluation or transit spection Service. Such person shall re- shipment may be made on the Internet side within the United States, or oper- at (http://www.aphis.usda.gov/ ate a business establishment within animallhealth/permits/ the United States, or both. vetlbiolpermits.shtml). [38 FR 32916, Nov. 29, 1973, as amended at 56 (b) The application shall specify the FR 66783, Dec. 26, 1991; 56 FR 66783, Dec. 26, type of permit required, the port of 1991] entry at which the product shall be

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cleared through Customs, the esti- product will not endanger the livestock mated quantity involved, and the an- or poultry of this country. ticipated date on which the importation shall be made. (Approved by the Office of Management and Budget under control number 0579–0013) (Approved by the Office of Management and [38 FR 32916, Nov. 29, 1973, as amended at 48 Budget under control number 0579–0013) FR 57473, Dec. 30, 1983; 52 FR 30131, Aug. 13, [38 FR 32916, Nov. 29, 1973, as amended at 48 1987; 56 FR 66783, Dec. 26, 1991] FR 57473, Dec. 30, 1983; 56 FR 66783, Dec. 26, 1991; 75 FR 20772, Apr. 21, 2010] § 104.5 Products for distribution and sale. § 104.4 Products for research and eval- An application for a U.S. Veterinary uation. Biological Product Permit to import a (a) An application for a U.S. Veteri- biological product for Distribution and nary Biological Product Permit to im- Sale shall be accompanied by support a biological product for research porting material necessary to satisfy and evaluation shall be accompanied the requirements provided in this sec- by a brief description of such product, tion. methods of propagating antigens in- (a) A permit shall not be issued un- cluding composition of medium, spe- less the conditions under which the bi- cies of animals or cell cultures in- ological product is to be prepared or volved, degree of inactivation or at- the methods to be used are such as to tenuation, recommendations for use, reasonably insure that the product is and the proposed plan of evaluation. pure, safe, potent, and efficacious. The applicant shall also provide any information the Administrator may re- (1) Two copies of blueprints of the quire in order to assess the product’s producing foreign establishment shall impact on the environment. be submitted with the application un- (b)(1) A permit to import a biological less satisfactory plans are on file with product for research and evaluation Animal and Plant Health Inspection shall not be issued unless the scientific Service from a previous application. capabilities of the investigator are de- The production facilities to be used for termined to be adequate to safeguard each product prepared at the establish- domestic animals and protect public ment shall be designated. health, interest, or safety from any (2) The manufacturer shall submit deleterious effects which might result written authorization for properly ac- from use of such product. Special re- credited inspectors to inspect without strictions or tests may be specified as previous notification, and at such part of the permit when they are times as may be demanded by the deemed necessary or advisable by the aforesaid inspectors, all parts of the es- Administrator. tablishment in which biological prod- (2) No person shall ship a product im- ucts shall be prepared, all processes of ported under this section for research preparation, and all records relative to and evaluation anywhere in or from such preparation. the United States unless authorized by (3) The manufacturer shall furnish the Administrator in accordance with written assurance that a biological the provisions of § 103.3 of this sub- product to be imported for Distribution chapter. and Sale shall be prepared under the (c) A biological product shall not be supervision of a person competent by imported for Research and Evaluation education and experience to handle all which is not packaged and labeled in matters pertaining to the preparation accordance with § 112.9 of this sub- of such product and that each biologi- chapter. cal product shall be prepared in accord- (d) When a licensed product has been ance with the regulations applicable to exported from the United States, a per- the product or in a manner acceptable mit may be issued to the producer for to the Administrator so as to carry out a small quantity of such product for in the purposes of the Act. vitro Research and Evaluation tests: (4) The methods to be used in the Provided, That, the importation of such preparation of each biological product

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shall be written into an approved Out- § 104.6 Products for transit shipment line of Production prepared in accord- only. ance with the applicable provisions of An application for a permit for Tran- part 114 of this subchapter. Two copies sit Shipment Only shall be required of such Outlines of Production shall be when a biological product is being submitted to Animal and Plant Health shipped from one foreign country to Inspection Service and be approved be- another foreign country by way of the fore the permit is issued. United States. The shipment shall (5) Data shall be furnished by the ap- move under a permit subject to the fol- plicant which establishes that the lowing restrictions: product involved complies with the (a) The shipment shall be confined to provisions of the Act and the regula- the carrier at all times when such ship- tions issued pursuant thereto. When ment is to transit the United States on deemed necessary to obtain required the same carrier on which it arrived. If information, Animal and Plant Health the shipment is to be transferred to a Inspection Service may require that carrier other than the one on which it the product be tested under field condi- shall arrive into the United States, a tions within or outside the United schedule of arrival and departure of States as the occasion demands. each shipment shall be furnished by the permittee to Animal and Plant (b) The permittee shall furnish the Health Inspection Service prior to ar- following: rival in the United States. (1) Adequate facilities for storing all (b) The permittee shall be responsible imported biological products. An in- to Animal and Plant Health Inspection spection of such facilities shall be Service for handling, storing, and for- made by inspectors before a permit is warding of the biological product. Ani- issued and additional inspections shall mal and Plant Health Inspection Serv- be made at any time subsequent to the ice shall be notified of all shipments importation of the biological products received and forwarded by the per- if deemed necessary by the Adminis- mittee and an accurate accounting trator; shall be made. (2) Information regarding all claims (Approved by the Office of Management and to be made on labels and advertising Budget under control number 0579–0013) matter used in connection with or related to the biological product to be [38 FR 32916, Nov. 29, 1973, as amended at 48 imported; FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, 1991; 61 FR 52873, Oct. 9, 1996] (3) Mounted copies of final container labels, carton labels, and enclosures to § 104.7 Product permit. be used with the imported product as (a) A permit shall be numbered and provided in part 112 of this subchapter; dated. and (b) The purpose for which the product (4) Samples of each serial from each is imported shall be specified on the shipment of biological products im- permit as for Research and Evaluation, ported or offered for importation. Such Distribution and Sale, or Transit Ship- samples shall be collected, examined, ment Only. and tested in a manner specified by the (c) A permit shall not be used after Administrator. The biological products the date specified. being sampled shall not be further distributed by the permittee until re- [38 FR 32916, Nov. 29, 1973, as amended at 56 leased by Animal and Plant Health In- FR 66783, Dec. 26, 1991; 62 FR 13294, Mar. 20, 1997] spection Service. (Approved by the Office of Management and § 104.8 Illegal shipments. Budget under control number 0579–0013) (a) Biological products which are pre- [38 FR 32916, Nov. 29, 1973, as amended at 48 sented for importation without a per- FR 57473, Dec. 30, 1983; 49 FR 21044, May 18, mit having been issued shall be re- 1984; 56 FR 66783, Dec. 26, 1991; 75 FR 20772, turned to the country of origin at the Apr. 21, 2010] expense of the importer or in lieu

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thereof, destroyed by Department per- (4) The licensee, permittee, or the sonnel. foreign manufacturer has failed to (b) Biological products for Distribu- maintain and make available for in- tion and Sale presented for importa- spection records in connection with the tion under a permit and found to be development and preparation of prod- worthless, contaminated, dangerous, or uct, has failed to provide complete and harmful shall, within a period of 30 accurate information when requested, days after such finding, be returned to or has failed to provide complete and the country of origin at the expense of accurate information in the Outline of the importer or in lieu thereof, de- Production or in reports and records; stroyed by Department personnel: Pro- (5) The licensee or permittee has vio- vided, That such product shall not be lated or failed to comply with any pro- returned to the country of origin while vision of the Virus-Serum-Toxin Act or bearing a U.S. permit number on the the regulations in this subchapter; label. (6) The license or permit is otherwise used to facilitate or effect the prepara- PART 105—SUSPENSION, REVOCA- tion, sale, barter, exchange, shipment, or importation, contrary to the Virus- TION, OR TERMINATION OF BIO- Serum-Toxin Act, of any worthless, LOGICAL LICENSES OR PERMITS contaminated, dangerous, or harmful biological product. Sec. (b) In case of willfulness or where the 105.1 Suspension or revocation. public health, interest, or safety so re- 105.2 Notification of infractions. 105.3 Notices re: worthless, contaminated, quired the Secretary may, without dangerous, or harmful biological prod- hearing, informally suspend such es- ucts. tablishment license, product license, or 105.4 Termination of licenses and permits permit upon the grounds set forth in for inactivity. paragraph (a) of this section pending AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, determination of formal proceedings 2.80, and 371.4. under part 123 of this subchapter for suspension or revocation of the license § 105.1 Suspension or revocation. or permit. (a) An establishment license, product [38 FR 23512, Aug. 31, 1973, as amended at 41 license, or permit issued under the FR 44359, Oct. 8, 1976; 61 FR 52874, Oct. 9, 1996; Virus-Serum-Toxin Act may be for- 64 FR 43044, Aug. 9, 1999] mally suspended or revoked after opportunity for hearing has been ac- § 105.2 Notification of infractions. corded the licensee or permittee as pro- If an infraction of a requirement of a vided in part 123 of this subchapter if product license is brought to the atten- the Secretary is satisfied that the li- tion of the licensee by written notifica- cense or permit is being used to facili- tion thereof by Animal and Plant tate or effect the preparation, sale, Health Inspection Service, a subse- barter, exchange, shipment, or impor- quent violation of similar nature oc- tation contrary to said Act of any curring with the same licensed biologi- worthless, contaminated, dangerous, or cal product within 6 months of the said harmful biological product. Such use written notification shall be may be found to exist if: primafacie evidence of willful violation (1) The construction of the establish- and the license for the product shall be ment in which the biological product is subject to suspension or revocation prepared is defective, or the establish- under the provisions of § 105.1(b). ment is not conducted as required by [42 FR 31430, June 21, 1977, as amended at 56 the regulations in parts 101 through 118 FR 66783, Dec. 26, 1991] of this subchapter; (2) The methods of preparation of the § 105.3 Notices re: worthless, contami- product are faulty, or the product con- nated, dangerous, or harmful bio- tains impurities or lacks potency; logical products. (3) The product is so labeled or adver- (a) If at any time it appears that the tised as to mislead or deceive the pur- preparation, sale, barter, exchange, chaser in any particular; shipment, or importation, as provided

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in the Virus-Serum-Toxin Act, of any tion Service pursuant to § 116.5 of this biological product by any person hold- subchapter. ing a license or permit may be dangerous in the treatment of domestic (Approved by the Office of Management and Budget under control number 0579–0318) animals, the Secretary may without hearing notify the licensee or per- [38 FR 23512, Aug. 31, 1973, as amended at 56 mittee, and pending determination of FR 66783, Dec. 26, 1991; 72 FR 17798, Apr. 10, formal proceedings instituted under 2007] part 123 of this subchapter for suspen- § 105.4 Termination of licenses and sion or revocation of the license or per- permits for inactivity. mit insofar as it authorizes the manufacture or importation of the par- (a) If a biological product has not ticular product, no person so notified been prepared by a licensee, or im- shall thereafter so prepare, sell, barter, ported by a permittee for a period of 5 exchange, ship, deliver for shipment, or years or more, the Administrator may import such product. require the licensee to show intent to (b) If a serial of biological product is resume production, or the permittee to found to be unsatisfactory according to show intent to resume importation, applicable Standard Requirements, the within 6 months of notification. If the Administrator may notify the licensee licensee does not resume preparation, to stop distribution and sale of the se- or the permittee does not resume im- rial. portation, within 6 months of notifica- (c) When notified to stop distribution tion, or within a mutually agreeable and sale of a serial or subserial of a period, the product license, or permit, veterinary biological product under the may be terminated by the Adminis- provisions of paragraph (a) or (b) of trator. this section, veterinary biologics li- (b) When a license or permit is termi- censees or permittees shall: nated, the licensee or permittee shall (1) Stop the preparation, distribu- continue to be subject to the applicable tion, sale, barter, exchange, shipment, records provisions of § 116.8. or importation of the affected serial(s) [61 FR 52874, Oct. 9, 1996] or subserial(s) of any veterinary biological product pending further instructions from APHIS. PART 106—EXEMPTION FOR BIO- (2) Immediately, but no later than 2 LOGICAL PRODUCTS USED IN DE- days, send stop distribution and sale PARTMENT PROGRAMS OR notifications to any wholesalers, job- UNDER DEPARTMENT CONTROL bers, dealers, foreign consignees, or OR SUPERVISION other persons known to have any such veterinary biological product in their AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, possession, which instruct them to stop 2.80, and 371.4. the preparation, distribution, sale, barter, exchange, shipment, or importa- § 106.1 Biological products; exemption. tion of any such veterinary biological product. All notifications shall be doc- The Administrator may exempt any umented in writing by the licensee or biological product from one or more of permittee. the requirements of this subchapter if (3) Account for the remaining quan- he determines that such product will tity of each serial(s) or subserial(s) of be used by the Department or under any such veterinary biological product the supervision or control of the De- at each location in the distribution partment in the prevention, control or channel known to the manufacturer (li- eradication of animal diseases in con- censee) or importer (permittee). nection with (a) an official USDA program; or (b) an emergency animal dis- (4) When required by the Adminis- ease situation, or (c) a USDA experi- trator, submit complete and accurate reports of all notifications concerning mental use of the product. stop distribution and sale actions to [45 FR 65184, Oct. 2, 1980, as amended at 56 FR the Animal and Plant Health Inspec- 66783, Dec. 26, 1991]

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PART 107—EXEMPTIONS FROM nary diagnosis of the medical condition PREPARATION PURSUANT TO AN of the animal(s). This means that the UNSUSPENDED AND UNREVOKED veterinarian has recently seen and is personally acquainted with the keeping LICENSE and care of the animal(s), and/or by medically appropriate and timely vis- Sec. its to the premises where the animal(s) 107.1 Veterinary practitioners and animal owners. are kept; and when 107.2 Products under State license. (iii) The practicing veterinarian is readily available for followup in case of AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, adverse reactions or failure of the regi- 2.80, and 371.4. men. § 107.1 Veterinary practitioners and (2) All steps in the preparation of animal owners. product being prepared under the ex- Products prepared as provided in emption in paragraph (a)(1) of this sec- paragraphs (a) and (b) of this section tion must be performed at the facilities and facilities in which such products that the veterinarian utilizes for the are prepared, shall be exempt from day-to-day activities associated with preparation pursuant to unsuspended the treatment of animals in the course and unrevoked establishment and prod- of his/her State-licensed professional uct licenses. Persons exempt from li- practice of veterinary medicine. A vet- censure under this part shipping prod- erinary assistant employed by the vet- ucts which contain live organisms shall erinary practitioner and working at provide any information the Adminis- the veterinary practice’s facility under trator may require prior to shipment, the veterinarian’s direct supervision or at any other time deemed necessary, may perform the steps in the prepara- in order to assess the products’ safety tion of product. Such preparation may and effect on the environment. The not be consigned to any other party or shipment or delivery for shipment any- sub-contracted to a commercial labora- where in or from the United States of tory/manufacturing facility. any exempted product which is worth- (3) Veterinarians preparing products less, contaminated, dangerous, or subject to the exemption for products harmful is prohibited, and any person under this section shall maintain and shipping such product, or delivering make available for inspection by Ani- such product for shipment, shall be mal and Plant Health Inspection Serv- subject to sanctions under the Act. ice representatives or other Federal (a)(1) Products prepared by a veteri- employees designated by the Secretary nary practitioner (veterinarian) solely such records as are necessary to estab- for administration to animals in the lish that a valid veterinarian-client-pa- course of a State licensed professional tient relationship exists and that there practice of veterinary medicine by is a valid basis for the exemption under such veterinarian under a veterinarian- this section. client-patient relationship and facili- (b) Products prepared by a person ties in which such products are pre- solely for administration to animals pared shall be exempt from licensing owned by that person shall be exempt under the Act and regulations. Such a from the requirement that preparation relationship is considered to exist be pursuant to an unsuspended and when: unrevoked license. (i) The veterinarian has assumed the [52 FR 30131, Aug. 13, 1987, as amended at 56 responsibility for making medical FR 66783, Dec. 26, 1991; 80 FR 26821, May 11, judgments regarding the health of the 2015] animal(s) and the need for medical treatment, and the client (owner or § 107.2 Products under State license. other caretaker) has agreed to follow (a) The Administrator shall exempt the instructions of the veterinarian; from the requirement of preparation and when pursuant to an unsuspended and (ii) There is sufficient knowledge of unrevoked USDA establishment and the animal(s) by the veterinarian to product license, any biological product initiate at least a general or prelimi- prepared solely for distribution within

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the State of production pursuant to a cluded under the exemptions in this license granted by such State under a section. program determined by the Adminis- [52 FR 30131, Aug. 13, 1987, as amended at 56 trator to be consistent with the intent FR 66783, Dec. 26, 1991] of the Act to prohibit the preparation, sale, barter, exchange, or shipment of PART 108—FACILITY REQUIREMENTS worthless, contaminated, dangerous, or FOR LICENSED ESTABLISHMENTS harmful biological products. (b) A request for exemption under Sec. this section must be made by the ap- 108.1 Applicability. propriate State authority and shall in- 108.2 Plot plans, blueprints, and legends re- clude information demonstrating that: quired. (1) The State has the authority to li- 108.3 Preparation of plot plans. 108.4 Preparation of blueprints. cense viruses, serums, toxins, and anal- 108.5 Preparation of legends. ogous products and establishments 108.6 Revision of plot plans, blueprints, and that produce such products; and legends. (2) The State has the authority to re- 108.7 Filing of plot plans, blueprints, and view the purity, safety, potency, and legends. 108.8 Construction of buildings. efficacy of such products prior to re- 108.9 Dressing rooms and other facilities. lease to the market; and 108.10 Outer premises and stables. (3) The State has the authority to re- 108.11 Water quality requirements. view product test results to assure AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, compliance with applicable standards 2.80, and 371.4. of purity, safety, and potency prior to SOURCE: 39 FR 16854, May 10, 1974, unless release to the market; and otherwise noted. (4) The State has the authority to deal effectively with violations of § 108.1 Applicability. State law regulating viruses, serums, Unless otherwise authorized by the toxins, and analogous products; and Administrator, all buildings, appur- (5) The State effectively exercises the tenances, and equipment used in the authority specified in paragraphs (b)(1) preparation of biological products shall through (4) of this section consistent be in compliance with the regulations with the intent of the Act prohibiting in this part. Each land area on which the preparation, sale, barter, exchange, such buildings and appurtenances are or shipment of worthless, contami- located shall be identified by an ad- nated, dangerous, or harmful viruses, dress which shall appear on the estab- serums, toxins, or analogous products. lishment license. (c) Each product to be exempted and [39 FR 16854, May 10, 1974, as amended at 56 each establishment preparing such FR 66783, Dec. 26, 1991] product shall be identified by the State and the State shall give written notifi- § 108.2 Plot plans, blueprints, and legends required. cation to the Administrator of each such product and establishment. The Each applicant for an establishment State shall also give written notice to license shall prepare a plot plan show- the Administrator of each new license ing all buildings for each particular issued and of each license terminated. land area, blueprints for each building used in the preparation of biological (d) In order to determine whether a products and legends containing a brief State exercises its authority with re- description of all activities in each spect to biological products and estab- room or area. lishments and whether its laws and regulations are being achieved, the Ad- § 108.3 Preparation of plot plans. ministrator, in cooperation with proper Plot plans shall show all of the build- State authorities, may conduct an on- ings on a particular land area, whether site evaluation of the State’s program or not they are all used for the prepa- which may include inspection of estab- ration and initial shipping of biological lishments and/or products to be in- products: Provided, That, when a great

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number of buildings are on the same (d) Identify the floors if the drawing premises, only those surrounding the is not for all floors in a multiple-story buildings used for preparation and ini- building and identify activities on each tial shipping of biological products floor. shall be shown. The presence of the re- (e) Identify all rooms by letters or mainder of the buildings may be ac- numbers. counted for by a single statement de- (f) Show the location of important noting the total number of such build- stationary equipment by a suitable ings not used for the preparation or code which will be further identified on shipping of biological products. legends. (a) Reduce the entire premises to any (g) Explain on the blueprint or on the standard scale on one sheet of paper legend, by a statement or listing, which meets any of the American which rooms are equipped with water standard trimmed sizes. Indicate the outlets, drains, and lighting. Show the scale used. location of doors and windows. (b) Clearly mark the boundaries of (h) Show compass points. the licensed premises and indicate (i) Show building number. what marking denotes the boundaries. (j) Show date of preparation. Such boundaries shall coincide with (k) Apply signature of responsible of- some readily apparent perimeter line. ficial of firm. Identify all fences, walls, or streets. (c) Show buildings as reduced dimen- § 108.5 Preparation of legends. sional drawings in the proper scale dis- A brief description of the activities tance relationship with each other. performed in each room or area shall (d) Number, letter, or otherwise iden- be prepared as provided in this section tify all buildings so that they may be and shall be referred to as a legend. correlated with the respective blue- Legends shall be provided for each plot prints and legends. plan and each blueprint or drawing. All (e) Describe on the plot plan the use pages of the legends shall be numbered, of immediate adjacent properties such identified with corresponding plot plan as, residential area, pasture, box fac- or blueprint, and submitted in booklet tory, or the like. form either stapled together or clipped (f) Show compass points. into a suitable folder. (g) Show date of preparation. (a) Plot plan legends shall show the (h) Apply signature of responsible of- following: ficial of the firm. (1) Number of each building and the functions performed in each: Provided, § 108.4 Preparation of blueprints. That if it is a multiple-story building (a) Blueprints, drawn to any suitable in which biological products are pre- scale, on regular blueprint paper or a pared or handled, briefly describe func- good grade of white paper of any one of tions performed on each floor. the American standard trimmed sizes (2) A practical and nontechnical de- shall be acceptable: Provided, That the scription of construction materials same scale shall be used for future revi- used throughout those buildings used sions unless the entire blueprint is re- entirely or partially for production and vised. Indicate the scale used. handling of biological products. (b) Use a single sheet of paper for (b) Blueprint legends shall show the each floor of all buildings in which bio- following: logical products are prepared. Illus- (1) A listing of all rooms by identi- trate in detail the areas in each build- fying letters or numbers and the frac- ing utilized for such preparation. tions prepared in each. Exceptions may (c) If only a portion of a floor is used be listed for general purpose areas or in the preparation of a biological prod- rooms. Functions performed in each uct, the blueprint shall illustrate the area and room shall be described, entire floor in essentially the same de- whether the licensed or unlicensed tail throughout. All functions or ac- products. In rooms where products are tivities performed in the remainder of exposed to the surroundings, a descrip- the floor shall be indicated. tion of decontamination procedures

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and other precautions against cross appropriate comments for correction contamination shall be included. and resubmission. Acceptable submis- (2) A listing of the coded stationary sions shall be stamped as filed and the equipment. date noted. One stamped copy shall be (3) A general listing of other essen- returned and two copies retained for tial biological equipment such as mills, Animal and Plant Health Inspection centrifuges, mixing tanks, bottling and Service files. sealing equipment, and the like, which are not regarded as stationary but are [39 FR 16854, May 10, 1974, as amended at 56 maintained in certain rooms. FR 66783, Dec. 26, 1991; 75 FR 20772, Apr. 21, 2010] [39 FR 16854, May 10, 1974, as amended at 40 FR 51413, Nov. 5, 1975; 50 FR 50764, Dec. 12, § 108.8 Construction of buildings. 1985] (a) The floors, walls, ceilings, parti- § 108.6 Revision of plot plans, blue- tions, posts, doors, and all other parts prints, and legends. of all structures, rooms, or facilities Preliminary drawings may be sub- used for the preparation of biological mitted to Animal and Plant Health In- products or ingredients of biological spection Service for comment prior to products at licensed establishments construction of new facilities or when shall be of such material, construction, remodeling is anticipated, old facilities and finish as may be readily and thor- are to be torn down, or other changes oughly cleaned. affecting the workflow are to be made. (b) All rooms used in connection with The licensee shall: the preparation of biological products (a) Prepare revised plot plans, blue- shall be so constructed and arranged as prints, or legends and submit to Ani- to prevent cross-contamination of such mal and Plant Health Inspection Serv- biological products. Halls or walkways ice for review and filing when changes shall be provided for the movement of have been completed. Also prepare a personnel or materials to each biologi- statement to accompany each revision cal products preparation area without to identify, by date of the superseded going through another such area. item, what is being superseded. (c) Rooms or compartments separate (b) Prepare a drawing of the revised from the remainder of the establish- rooms, unit, or section to the same ment shall be provided at licensed es- scale as the blueprint on file which tablishments for preparing, handling, shall be stamped and applied to the ex- and storing virulent or dangerous isting blueprint. If changes are numer- microorganisms and products. ous, prepare a new blueprint. (d) All rooms and compartments at (c) Drawings of new buildings may be licensed establishments shall have an added to existing plot plans. Indicate adequate air handling system to supply the distance from surrounding build- proper ventilation sufficient to insure ings and boundary lines. sanitary and hygienic conditions for (d) Any change prescribed in this sec- the protection of the products and per- tion shall necessitate a change in one sonnel. or more pages of the respective leg- (e) The supply of hot and cold water ends. The revised pages shall carry the at licensed establishments shall be same numbers as superseded pages. ample and clean. Adequate facilities [39 FR 16854, May 10, 1974, as amended at 56 shall be provided for the distribution of FR 66783, Dec. 26, 1991] water in each establishment and for the washing of all containers, machin- § 108.7 Filing of plot plans, blueprints, ery, instruments, other equipment, and and legends. animals used in the preparation of a bi- Two copies of all plot plans, blue- ological product. prints, and legends, including revi- (f) There shall be an efficient drain- sions, shall be submitted to Animal age and plumbing system for each li- and Plant Health Inspection Service censed establishment and premises for review and filing. When the re- thereof, and all drains and gutters viewer takes exception to a submitted shall be properly installed with ap- item, such item shall be returned with proved traps and vents.

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§ 108.9 Dressing rooms and other fa- Plant Health Inspection Service for cilities. each licensed establishment. Each licensed establishment shall [39 FR 16854, May 10, 1974, as amended at 56 have dressing rooms, toilet facilities, FR 66783, Dec. 26, 1991] and lavatory accommodations, including hot and cold running water, soap, PART 109—STERILIZATION AND towels, and the like. They shall be in PASTEURIZATION AT LICENSED sufficient number, ample in size, con- ESTABLISHMENTS veniently located, properly ventilated, and meeting all requirements as to Sec. sanitary construction and equipment. 109.1 Equipment and the like. (a) These rooms and facilities shall 109.2 Sterilizers. be separate from rooms or compart- 109.3 Pasteurizers. ments in which biological products are AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, prepared, handled, or stored. 2.80, and 371.4. (b) These rooms and facilities shall be so located in the establishment as to § 109.1 Equipment and the like. be readily accessible to all persons (a) All containers, instruments, and without having to enter or pass other apparatus and equipment, before through biological products prepara- being used in preparing, handling, or tion areas. storing biological products, at a licensed establishment, except as other- § 108.10 Outer premises and stables. wise prescribed herein, shall be thor- (a) The outer premises of licensed es- oughly sterilized by live steam at a tablishments, embracing docks, drive- temperature of at least 120 °C. for not ways, approaches, yards, pens, chutes, less than one-half hour, or by dry heat and alleys shall be drained properly at a temperature of at least 160 °C. for and kept in a clean and orderly condi- not less than one hour. If for any rea- tion. No nuisance shall be allowed in son such methods of sterilization are any licensed establishment or on its impracticable, then a process known to premises. be equally efficacious in destroying (b) Stables or other premises for ani- microorganisms and their spores may mals used in the production or testing be substituted after approval by the of biological products at licensed es- Administrator. tablishments shall be properly venti- (b) Instruments which are found to be lated and lighted, appropriately damaged by exposure to the degree of drained and guttered, and kept in sani- heat prescribed in this section, after tary condition. having been thoroughly cleaned, may (c) Every practical precaution shall be sterilized by boiling for not less be taken to keep licensed establish- than 15 minutes. ments free of flies, rats, mice, and [23 FR 10051, Dec. 23, 1958, as amended at 34 other vermin. The accumulation, on FR 18119, Nov. 11, 1969; 56 FR 66783, Dec. 26, the premises of an establishment, of 1991] any material in which flies or other vermin may breed is forbidden. Suit- § 109.2 Sterilizers. able arrangements, in keeping with the Steam and dry-heat sterilizers used local health practices, shall be made in connection with the processing of bi- for the disposal of all refuse. ological products at licensed establishments shall be equipped with auto- § 108.11 Water quality requirements. matic temperature recording gauges: A certification from the appropriate Provided, That other record keeping water pollution control agency, that systems may be used when approved by the establishment is in compliance the Administrator. When gauges are with applicable water quality control used, they shall be periodically stand- standards, pursuant to section 401 of ardized to assure accuracy. Charts and the Federal Water Pollution Control other temperature records made during Act, as amended (86 Stat. 877; 33 U.S.C. production shall be available at all 1341), shall be filed with Animal and times charts and records shall be kept

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in accordance with part 116 of this AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, chapter. 2.80, and 371.4. SOURCE: 38 FR 12094, May 9, 1973, unless [35 FR 16039, Oct. 13, 1970, as amended at 56 otherwise noted. FR 66783, Dec. 26, 1991] § 112.1 General. § 109.3 Pasteurizers. (a) Unless otherwise authorized or di- All pasteurizing equipment shall rected by the Administrator, each bio- meet the requirements in paragraphs logical product prepared at a licensed (a), (b), and (c) of this section and be establishment, or imported, shall be acceptable to Animal and Plant Health packaged and labeled as prescribed in Inspection Service. this part before it is removed from the (a) Metal serum containers shall be licensed establishment or presented for used in licensed establishments. During importation: Provided, That biological the heating process, each container products to be imported for research shall be surrounded by a separate and evaluation shall be subject to water jacket or equivalent so that the packaging and labeling requirements entire container, including its lid, is in § 112.9. Provided further, That, unless heated to the required temperature. otherwise exempted, all preparation, Each serum container shall be equipped including packaging and labeling, of bi- with a motor-driven agitator and a sep- ological products shall only be per- arate automatic recording thermom- formed in a licensed establishment eter. under an approved Outline of Produc- (b) Each water bath shall have an tion. automatic temperature control to (b) No person shall apply or affix to limit the temperature of the water to a or include with, or cause to be applied maximum of 62 °C., an automatic re- or affixed to or included with, any car- cording thermometer, an indicating ton or final container of a biological thermometer set in a fixed position, product, any label, stamp, mark or and circulating mechanism adequate to statement that is false or misleading in insure equal temperatures throughout any particular, is not in compliance the bath. The heating unit for the bath with the regulations, or is not ap- shall be separated from the serum con- proved by APHIS. tainer and the water jacket. (c) No person shall alter, mark or re- (c) Accurate thermometers at li- move any approved labeling affixed to censed establishments shall be used at or included with any biological product frequent intervals to check tempera- prior to selling or otherwise distrib- tures of the serum as registered by re- uting such product. In addition, no per- cording thermometers. son shall mark any carton, other container, or final container of a biologi- [35 FR 16039, Oct. 13, 1970, as amended at 56 cal product so as to falsify the labeling, FR 66783, Dec. 26, 1991] make it misleading, or cause it to be il- legible. PART 112—PACKAGING AND (d) Labels that are stamped, printed LABELING or glued directly on cartons, other containers, or final containers shall be leg- Sec. ible throughout the dating period. Bio- 112.1 General. logical products bearing labels, which 112.2 Final container label, carton label, have been altered, mutilated, de- and enclosure. stroyed, obliterated or removed, shall 112.3 Diluent labels. be withheld from the market. 112.4 Subsidiaries, divisions, distributors, and permittees. [38 FR 12094, May 9, 1973, as amended at 59 112.5 Review and approval of labeling. FR 43445, Aug. 24, 1994] 112.6 Packaging biological products. 112.7 Special additional requirements. § 112.2 Final container label, carton label, and enclosure. 112.8 For export only. 112.9 Biological products imported for re- (a) Unless otherwise provided, final search and evaluation. container labels, carton labels, and en- 112.10 Special packaging and labeling. closures (inserts, circulars, or leaflets)

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shall include the information specified (PCN) assigned by the Department, in this section. which shall be shown only as ‘‘VLN/ (1) The complete true name of the bi- PCN’’ and ‘‘VPN/PCN,’’ respectively, ological product which name shall be except that: identical with that shown in the prod- (i) Only the VLN or VPN is required uct license under which such product is on container labels of interchangeable prepared or the permit under which it (non-critical) components of diagnostic is imported, shall be prominently let- kits and container labels for individual tered and placed giving equal emphasis products packaged together for co-ad- to each word composing it. Descriptive ministration. terms used in the true name on the (ii) The PCN may be used in lieu of product license or permit shall also ap- the true name of the kit on small con- pear. Abbreviations of the descriptive tainer labels for critical components of terms may be used on the final con- diagnostic kits. tainer label if complete descriptive (iii) Container labels for individually terms appear on the carton label and licensed biological products, when mar- enclosure. The following exceptions are keted as components of combination applicable to small final containers, packages, must include a statement re- and containers of interchangeable re- ferring the consumer to the carton or agents included in diagnostic test kits: enclosure for the PCN of the combina- (i) For small final containers, an ab- tion package. breviated true name of the biological (4) Storage temperature rec- product, which shall be identical with ommendation for the biological prod- that shown in the product license uct stated as 2 to 8 °C or 35 to 46 °F, or under which the product is prepared or both. the permit under which it is imported, (5) Full instructions for the proper may be used: Provided, That the com- use of the product, including indica- plete true name of the product must tions for use, target species, minimum appear on the carton label and enclo- age of administration, route of admin- sures; istration, vaccination schedule, prod- (ii) In addition to the true name of uct license restriction(s) that bear on the kit, the functional and/or chemical product use, warnings, cautions, and name of the reagent must appear on la- any other vital information for the beling for small final containers of re- product’s use; except that in the case agents included in diagnostic kits: Pro- of limited space on final container la- vided, That the true name is not re- bels, a statement as to where such in- quired on labeling for small final con- formation is to be found, such as ‘‘See tainers of interchangeable (non-crit- enclosure for complete directions,’’ ical) components of diagnostic kits. ‘‘Full directions on carton,’’ or com- (2) For biological product prepared in parable statement. the United States or in a foreign coun- (6) In the case of a multiple-dose try, the name and address of the pro- final container, a warning to use entire ducer (licensee, or subsidiary) or per- contents when first opened: Provided, mittee and of the foreign producer, and That a diagnostic or a desensitizing an appropriate consumer contact tele- antigen packaged in a multiple-dose phone number: Provided, That in the final container is exempt. case of a biological product exported (7) The following warning state- from the United States in labeled final ments, or equivalent statements, shall containers, a consumer contact tele- appear on the labeling as applicable: phone number is not required; however, (i) Products other than diagnostic small single dose containers marketed kits: ‘‘Do not mix with other products, in the United States must include con- except as specified on this label.’’ tact telephone information on carton (ii) Injectable products and other and enclosures. products containing hazardous compo- (3) The United States Veterinary Bio- nents: ‘‘In case of human exposure, logics Establishment License Number contact a physician.’’ (VLN) or the United States Veterinary (iii) Products containing viable orga- Biological Product Permit Number nisms: ‘‘Inactivate unused contents be- (VPN), and the Product Code Number fore disposal.’’

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(8) In the case of a biological product container of biological product. The recommended for use in domestic ani- number of final containers of diluent, if mals, the edible portion of which may any, and the quantity in each shall be used for food purposes, a with- also be stated on each carton label. holding statement of not less than 21 (12) An indications statement to days to read: ‘‘Do not vaccinate within read, ‘‘This product has been shown to (insert number) days before slaughter’’ be effective for the vaccination of or ‘‘Do not vaccinate food-producing healthy (insert name of species) ll animals within (insert number) days weeks of age or older against ll.’’ before slaughter’’: That Provided, Provided, That in the case of very small longer periods shall be stated when final container labels or carton, a deemed necessary by the Adminis- statement as to where such informa- trator. Very small final container labels are exempted from this require- tion is to be found, such as ‘‘See enclo- ment. sure for complete directions,’’ ‘‘Full di- (9) The following information shall rections on carton,’’ or comparable appear on the final container label and statement. carton label, if any, but need not ap- (b) Labels may also include any other pear on the enclosure: statement which is not false or mis- (i) A permitted expiration date; leading and may include factual state- (ii) The number of doses where appli- ments regarding variable response of cable; different animals when vaccinated as (iii) The recoverable quantity of the directed but may not include dis- content of each final container stated claimers of merchantability, fitness for in cubic centimeters (cc.) or milliliters the purpose offered, or responsibility (ml.) or units. for the product. (iv) A serial number by which the (c) Labels of biological products pre- product can be identified with the man- pared at licensed establishments or im- ufacturer’s records of preparation: Pro- ported shall not include any statement, vided, That when a liquid antigenic design, or device, which overshadows fraction is to be used instead of a water the true name of the product as li- diluent for one or more desiccated an- censed or which is false or misleading tigenic fractions in a combination in any particular or which may other- package, a hyphenated serial number wise deceive the purchaser. composed of a serial number for the desiccated fraction and the serial num- (d) Carton labels and enclosures shall ber for the liquid fraction shall be used be subject to paragraph (d)(1), (d)(2), on the carton. and (d)(3) of this section. (v) A statement similar to ‘‘For more (1) The statement, ‘‘Restricted to use information regarding efficacy and by or under the direction of a veteri- safety data, go to narian’’ or ‘‘Restricted to use by a vet- productdata.aphis.usda.gov. erinarian,’’ shall be used on all carton (10) In the case of a product that con- labels and enclosures when such re- tains a preservative that is added dur- striction is prescribed on the product ing the production process and is not license. reduced to undetectable levels in the (2) If the licensee states on the car- completed product through the produc- ton labels and enclosures of a product tion process, the statement ‘‘Contains that its sales are restricted to veteri- [name of preservative] as a preserva- narians, then the entire production of tive’’ or an equivalent statement must that particular product in the licensed appear on cartons and enclosures, if establishment shall be so restricted by used. If cartons are not used, such in- the licensee. formation must appear on the final (3) The statement ‘‘For use in ani- container label. (11) The number of final containers of mals only’’ may appear on the labeling biological product and the number of as appropriate for a product to indicate doses in each final container shall be that the product is recommended spe- stated on each carton label for all car- cifically for animals and not for hu- tons containing more than one final mans.

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(e) When label requirements of a for- face of the tray cover may contain in- eign country differ from the require- formation suitable for an enclosure. ments as prescribed in this part, spe- (Approved by the Office of Management and cial labels may be approved by APHIS Budget under control number 0579–0013) for use on biological products to be exported to such country upon receipt of [38 FR 12094, May 9, 1973, as amended at 39 FR 16856, May 10, 1974; 41 FR 44359, Oct. 8, written authorization, acceptable to 1976; 42 FR 11825, Mar. 1, 1977; 42 FR 29854, APHIS, from regulatory officials of the June 10, 1977; 42 FR 41850, Aug. 19, 1977; 48 FR importing country, provided that: 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, 1991; (1) If the labeling contains claims or 80 FR 39674, July 10, 2015; 81 FR 59433, Aug. indications for use not supported by 30, 2016; 81 FR 78500, Nov. 8, 2016] data on file with APHIS, the special labels for export shall not bear the VLN. § 112.3 Diluent labels. (2) All other labels for export shall Each final container of diluent, other bear the VLN unless the importing than a liquid biological product, pack- country provides documentation that aged with desiccated biological prod- the VLN is specifically prohibited. ucts shall bear a label that includes the When laws, regulations, or other re- following: quirements of foreign countries require (a) The name—Sterile Diluent. exporters of biological products pre- (b) True name of the biological prod- pared in a licensed establishment to uct with which the diluent is packaged, furnish official certification that such except that when the firm packages all products have been prepared in accord- desiccated biological products with the ance with the Virus-Serum-Toxin Act same diluent, or two or more types of and regulations issued pursuant to the diluent are used, and the licensees’ Act, such certification may be made by methods of identification and storage APHIS. insure that all products are packaged with the correct type of diluent, labels (f) Multiple-dose final containers of affixed to the containers of diluent are liquid biological product and carton exempt from this provision. tray covers showing required labeling (c) The recoverable quantity of con- information are subject to the require- tents in cubic centimeters (cc) or milli- ments in this paragraphs. liters (ml). (1) If a carton label or an enclosure is (d) A serial number by which the dil- required to complete the labeling for a uent can be identified with the manu- multiple-dose final container of liquid facturer’s records of preparation; biological product, only one final con- (e) Name and address of the licensee tainer, with a container of diluent if or the permittee; applicable, shall be packaged in each (f) In the case of a diluent with which carton: That if the multiple- Provided, a desiccated biological product is to dose final container is fully labeled come in contact while the diluent is in without a carton label or enclosure, its original container; and, two or more final containers, and a (1) Is in a multiple-dose container, a corresponding number of diluent con- positive warning that all of the biologi- tainers, may be packaged in a single cal product shall be used at the time carton which shall be considered a the container is first opened; and/or shipping box. Labels or stickers for (2) The biological product is com- shipping boxes shall not contain false posed of viable or dangerous organisms or misleading information, but need or viruses, the notice, ‘‘Inactivate un- not be submitted to APHIS for ap- used contents before disposal.’’ proval. (g) The establishment license number (2) When required labeling informa- or the permit number, as the case may tion is shown on a carton tray cover, it be, in one of the forms provided in must be printed on the outside face of § 112.2(a)(3). such tray cover where it may be read without opening the carton. The inside [38 FR 12094, May 9, 1973; 38 FR 13476, May 22, 1973, and amended at 39 FR 16856, May 10, 1974; 81 FR 59434, Aug. 30, 2016]

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§ 112.4 Subsidiaries, divisions, dis- of such product. The manufacturer tributors, and permittees. shall be identified by name and address Labels used by subsidiaries, divi- with the term ‘‘manufactured by,’’ sions, distributors, and permittees ‘‘produced by,’’ or an equivalent term shall be affixed by the licensee in a li- prominently placed in connection censed establishment where the prod- therewith. Reference to the permittee uct is produced. Such labels shall com- shall be made by name, address, and ply with requirements for their review, permit number with the term ‘‘im- approval, and filing as provided in the ported by,’’ ‘‘produced for,’’ or an regulations. equivalent term prominently placed in (a) Subsidiaries. Labels to be used on connection therewith. a licensed biological product prepared [50 FR 46417, Nov. 8, 1985, as amended at 59 by a subsidiary operating in a licensed FR 43445, Aug. 24, 1994] establishment shall be submitted in accordance with § 112.5. Only labels ap- § 112.5 Review and approval of label- proved for use on such product shall be ing. used by the subsidiary. (b) Divisions. Labels to be used on a Labels used with biological products licensed biological product prepared in prepared at licensed establishments or a licensed establishment for distribu- imported for general distribution and tion by a division or marketing unit of sale must be submitted to the Animal the licensee shall be submitted in ac- and Plant Health Inspection Service cordance with § 112.5. The name, ad- for review for compliance with the reg- dress, and license number of the li- ulations and approval in writing prior censee shall be prominently placed on to use, except as provided in paragraph such labels. The relationship of the di- (d) of this section and under the master vision or marketing unit to the li- label system provided in paragraph (e) censee shall appear prominently on the of this section. label by use of the term ‘‘division of’’ (a) Transmittal forms, available on or equivalent. the APHIS Web page at http:// (c) Distributors. The name and address www.aphis.usda.gov/animalhealth/cvb/ of the distributor or any statement, de- forms, shall be used with each submis- sign, or device shall not be placed on sion of sketches (including proofs) and the labels or containers of a licensed labels. Separate forms shall be used for biological product in a manner which each biological product but only one could be false or misleading or which copy of the form shall be used for all could indicate that the distributor is sketches and labels submitted at the the manufacturer of such product or same time for the same biological operating under the license number product. shown on the label. The manufacturer (b) A data summary, available on the shall be identified by name, address, Internet at productdata.aphis.usda.gov, and license number with the term shall be used with each submission of ‘‘manufactured by,’’ ‘‘produced by,’’ or efficacy and safety data in support of a an equivalent term prominently placed label claim. Manufacturers will submit in connection therewith. The name and the efficacy and safety data informa- address of the distributor may be tion with either the efficacy and safety placed on labels or containers if the studies or at the time of label submis- term ‘‘distributor,’’ or ‘‘distributed sion. This information will be posted at by,’’ or an equivalent term is promi- productdata.aphis.usda.gov to allow nently placed in connection therewith. public disclosure of product perform- (d) Permittees. The name and address ance. of the permittee and any statement, (c) Sketches may be submitted for design, or device shall not be placed on comment to Animal and Plant Health the labels or containers of a biological Inspection Service by the licensee or product imported for sale and distribu- permittee before preparing the finished tion in accordance with § 104.5 in a label. Such sketches shall be returned manner which could be false or mis- to the licensee or permittee with com- leading or which could falsely indicate ments, if any. Failure of the reviewer that the permittee is the manufacturer to take exception to a sketch shall not

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constitute approval of a finished label (e) Labels and sketches submitted subsequently prepared. shall be prepared in the number and (d)(1) Labels must be submitted to manner prescribed in this paragraph. the Animal and Plant Health Inspec- (1) Copies required: tion Service for review and written ap- (i) For label sketches, submit two proval. Only labels which are approved copies of each sketch of a final con- as provided in paragraph (e) of this sec- tainer label, carton label, and enclo- tion may be used. When changes are sure. Sketches must be legible, and made in approved labels, the new labels must include all information specified shall be subject to review and approval in § 112.2. One copy of each sketch will before use: Provided, That certain be returned with applicable comments, minor changes may be made in labels and one copy will be held on file by for products with approved labels or APHIS for no more than one year after master labels, and the revised labels processing, until replaced by a finished may be used prior to review by APHIS, label: Provided, That sketches sub- with the provision that a new label or mitted in support of an application for master label bearing these changes is a license or permit shall be held as long submitted to APHIS for review and as the application is considered active. written approval within 60 days of label (ii) For master label sketches, sub- use, and that such minor changes do mit for each product two copies of each not render the product mislabeled or sketch of an enclosure, label for the the label false and misleading in any smallest size final container, and car- particular. ton label; Provided, That labels for (2) Minor label changes that may be larger size containers and/or cartons made under the provision for products that are identical, except for physical with approved labels or master labels dimensions, need not be submitted. One are: copy of each master label sketch will (i) Changes in the physical dimen- be returned with applicable comments, sions of the label provided that such and one copy will be held on file by change does not affect the legibility of APHIS for one year after processing, the label; until replaced by a finished master (ii) Changes in the color of label print label that is submitted according to or background, provided that such paragraph (e)(1)(iii) of this section: Pro- changes do not affect the legibility of vided, That master label sketches sub- the label; mitted in support of an application for license or permit shall be held as long (iii) The addition or deletion of a as the application is considered active. Trade Mark (TM) or Registered (R) (iii) For finished labels, submit two symbol; copies of each finished final container (iv) The correction of typographical label, carton label, and enclosure: Pro- errors; vided, That when an enclosure is to be (v) Adding, changing, deleting, or used with more than one product, one repositioning label control numbers, extra copy shall be submitted for each universal product codes, or other in- additional product. One copy of each ventory control numbers; finished label will be retained by (vi) Revising or updating logos; APHIS. One copy will be stamped and (vii) Changing the telephone contact returned to the licensee or permittee. number; Labels to which exceptions are taken (viii) Adding, changing, or deleting shall be marked as sketches and han- an email and/or Web site address; dled under paragraph (e)(1)(i) of this (ix) Changing the establishment li- section. cense or permit number assigned by (iv) For finished master labels, sub- APHIS, and/or changing the name and/ mit for each product two copies each of or address of the manufacturer or per- the enclosure and the labels for the mittee, provided that such changes are smallest size final container and car- identical to information on the current ton. Labels for larger sizes of con- establishment license or permit; and tainers or cartons of the same product (x) Adding or changing the name and/ that are identical, except for physical or address of a distributor. dimensions, need not be submitted.

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Such labels become eligible for use the name and code number of the prod- concurrent with the approval of the ap- uct affected. propriate finished master label, pro- (ii)(A) Designation of the specimen as vided that the marketing of larger size a label or master label: sketch, final final containers is approved in the filed container label, carton label, or enclo- Outline of Production, and the appro- sure. priate larger sizes of containers or car- (B) If two final container labels or tons are identified on the label mount- multiple parts are on one sheet, each ing sheet. When a master label enclo- shall be named, and the label or part sure is to be used with more than one being revised shall be designated. product, one extra copy for each addi- (iii) Size of package (dose, ml., cc., or tional product shall be submitted. One units) for which the labels or enclo- copy of each finished master label will sures are to be used. be retained by APHIS. One copy will be (4) To appear on the bottom of each stamped and returned to the licensee page in the lower left hand corner, if or permittee. Master labels to which applicable: exception are taken will be marked as (i) The dose size(s) to which the mas- sketches and handled under paragraph ter label applies. (e)(1)(ii) of this section. (ii) The APHIS assigned number for (2) Mounting: the label or sketch to be replaced. (i) Each label or sketch shall be se- (iii) The APHIS assigned number for curely fastened to a separate sheet of the label to be used as a reference for heavy bond paper (81⁄2″ × 11″) in such a reviewing the submitted label. manner that all information is available for review. (f) Special requirements for foreign (ii) Two-or three-part cartons, in- language labels: cluding ‘‘sleeves,’’ shall be considered (1) An accurate English translation as one label. All parts shall be sub- must accompany each foreign language mitted together. label submitted for approval. A state- (iii)(A) When two final containers are ment affirming the accuracy of the packaged together in a combination translation must also be included. package, the labels for each shall be (2) Foreign language portion of a bi- mounted on the same sheet of paper lingual label shall be a true translation and shall be treated as one label. For of the English portion. Reference to ad- diagnostic test kits, the labels for use ditional information on the enclosure on the individual reagent containers to shall not be made unless that enclosure be included in the kit shall be mounted is also bilingual. together on a single sheet of paper, if (g) When a request is received from possible; if necessary, a second sheet of Animal and Plant Health Inspection paper may be used. The carton label Service, the licensee or permittee shall and enclosure shall be mounted on sep- submit a list of all approved labels cur- arate individual sheets. rently being used. Each label listed (B) If either final container label is shall be identified as to: also used alone or in another combina- (1) Name and product code number as tion package, sets of separate labels for it appears on the product license or each biological product with which it is permit for the product; and used shall be submitted for review. (2) Where applicable, the size of the (iv) When the same final container package (doses, ml., cc., or units) on label is applied by different methods which the label shall be used; and such as paper or screen printing, one of (3) Label number and date assigned; each shall be mounted on the same and sheet of paper as one submission. (4) Name of licensee or subsidiary ap- (3) To appear on the top of each page: pearing on the label as the producer. (i)(A) Name and product code number (h) At the time of an inspection, or of the biological product as it appears when requested by APHIS, licensees or on the product license or permit. permittees shall make all labels and (B) Extra copies of enclosures to be master labels, including labels ap- used with another product shall bear proved for use but exempted from filing

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under the master label system, avail- tiple-dose final containers as specified able for review by authorized inspec- in the filed Outline of Production. tors. Such labels shall be identical to Poultry products for manual adminis- the approved label or master label ex- tration to individual birds shall not except for physical dimensions, reference ceed 1,000 doses in each final container. to recoverable volume or doses and/or Diluent need not be packaged with the certain minor differences permitted in final container(s) of the product, but accordance with paragraph (d) of this the licensee shall provide the required section. number of containers of diluent as (Approved by the Office of Management and specified in the filed Outline of Produc- Budget under control number 0579–0013) tion. The following requirements apply [38 FR 12094, May 9, 1973, as amended at 48 to cartons containing more than one FR 57473, Dec. 30, 1983; 49 FR 21044, May 18, final container of poultry product: 1984; 56 FR 66783, Dec. 26, 1991; 59 FR 43445, (1) They shall be sealed prior to leav- Aug. 24, 1994; 61 FR 29464, June 11, 1996; 61 FR ing the licensed establishment. 33175, June 26, 1996; 64 FR 43044, Aug. 9, 1999; (2) The contents may not be repack- 75 FR 20772, Apr. 21, 2010; 80 FR 39675, July 10, 2015; 81 FR 47005, July 20, 2016; 81 FR 59435, aged. Aug. 30, 2016] (3) The contents of such cartons may not be sold in fractional units. § 112.6 Packaging biological products. (4) The following statement must ap- (a) Multiple-dose final containers of a pear in a prominent place on the car- biological product with final container ton label: ‘‘Federal regulations pro- labeling including all information re- hibit the repackaging or sale of the quired under the regulations may be contents of this carton in fractional packaged one or more per carton with units. Do not accept if seal is broken.’’ a container(s) of the proper volume of (d) Diluent for the following products diluent, if required, for that dose as need not be packaged with the final specified in the filed Outline of Produc- container(s) of the product, but the li- tion: Provided, That cartons containing censee shall provide the consumer with more than one final container of prod- the required number of containers of uct must comply with the conditions the proper amount of diluent as speci- set forth in paragraphs (c)(1) through fied in the filed Outline of Production: (4) of this section. Multiple-dose final (1) Marek’s Disease Vaccine. containers of a product that require a (2) Poultry vaccines administered to carton or enclosure in order to provide individual birds using automatic vacci- all information required under the reg- nating equipment. ulations shall be packaged one con- (e) Final containers of biological tainer per carton with the proper vol- product prepared at a licensed estab- ume of diluent, if required, for that lishment, or imported, in cartons or dose as specified in the filed Outline of Production. other containers shall not be removed (b) Single-dose final containers of a from such cartons or containers for product need not be packaged one per sale or distribution, unless each final carton. For single-dose products which container bears, or is packaged in a require a diluent for administration, carton with, complete and approved la- the number of containers of the proper beling which is affixed to or included amount of diluent specified in the filed with each container by the licensed es- Outline of Production for the number tablishment producing the product or of doses contained in the carton shall by the producer in the case of imported be included in each carton. product: Provided, That this paragraph (c) Poultry products for mass adminis not intended to apply to licensed istration (including but not limited to veterinary practitioners administering administration through drinking water or dispensing biological products in the and spray) and products used in auto- course of their practice under a veteri- matic vaccinating systems (including nary-client-patient-relationship as but not limited to pneumatic beak that term is used in § 107.1. injectors and automated needle (f) Labels which are affixed to or in- injectors) may be packaged in mul- cluded with a biological product shall

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not be removed or altered in any man- the possible hazard to human health ner. should be considered and State Public Health Officials should be consulted for [47 FR 8761, Mar. 2, 1982, as amended at 48 FR 12691, Mar. 28, 1983; 59 FR 43445, Aug. 24, 1994; specific recommendations’’ shall be 64 FR 43044, Aug. 9, 1999; 81 FR 59435, Aug. 30, prominently placed on all carton labels 2016] and on enclosures, if used. (5) That vaccine be administered to § 112.7 Special additional require- animals at 3 months of age or older, ments. with a repeat dose 1 year later. The label requirements in this sec- (6) Subsequent revaccination as de- tion are additional to those prescribed termined from the results of duration elsewhere in this part. of immunity studies conducted as pre- (a) In the case of biological products scribed in § 113.312, paragraph (b) or (c), containing live Newcastle Disease or both. virus, a caution statement indicating (e) Labeling for all products for use that Newcastle Disease can cause in- in mammals must bear an appropriate flammation of the eyelids of humans, statement concerning use in pregnant and a warning to the user to avoid in- animals. fecting his eyes shall be included on (1) For bovine rhinotracheitis vaccine the enclosure. or bovine virus diarrhea vaccine con- (b) In the case of a biological product taining modified live virus, all labeling containing infectious bronchitis virus, except small final container labels all labels shall show the infectious shall bear the following statement: bronchitis virus type or types used in ‘‘Do not use in pregnant cows or in the product. Abbreviation is permitted. calves nursing pregnant cows.’’: Pro- (c) In the case of a biological product vided, That such vaccines which have containing inactivated rabies virus, carton labels, enclosures, and all but been shown to be safe for use in preg- very small final container labels shall nant cows may be excepted from this include a warning against freezing and label requirement by the Adminis- the recommendations provided in this trator. paragraph. (2) For other modified live and inac- (1) That vaccine be administered to tivated vaccine, labeling shall bear a animals at 3 months of age or older, statement appropriate to the level of with a repeat dose 1 year later. safety that has been demonstrated in (2) Subsequent revaccination as de- pregnant animals. termined from the results of duration (i) Products known to be unsafe in of immunity studies conducted as pre- pregnant animals shall include state- scribed in § 113.209, paragraph (b) or (c), ments such as ‘‘Do not use in pregnant or both. animals,’’ or ‘‘Unsafe for use in preg- (d) In the case of a biological product nant animals,’’ or an equivalent state- containing modified live rabies virus, ment acceptable to APHIS. the carton labels, enclosures, and all (ii) Products without safety docu- but very small final container labels mentation acceptable to APHIS, but shall include the recommendations not known to be unsafe, labeling shall provided in this paragraph. include the statement ‘‘This product (1) For low egg-passage (below the has not been tested in pregnant ani- 180th egg-passage level) the statement mals’’ or an equivalent statement ac- ‘‘For Use in Dogs Only! Not For Use in ceptable to APHIS. Any Other Animal!’’ (3) For modified live vaccines con- (2) For other vaccines containing taining agents with potential reproduc- modified live rabies virus, the state- tive effects but having acceptable preg- ment ‘‘For Use In (designate animal(s)) nant animal safety data on file with Only! Not For Use In Any Other Ani- APHIS, labeling still must bear the fol- mal!’’ lowing statement concerning residual (3) Intramuscular injection at one risk: ‘‘Fetal health risks associated site in the thigh shall be recommended. with the vaccination of pregnant ani- (4) The statement ‘‘In event of acci- mals with this vaccine cannot be un- dental exposure to the vaccine virus, equivocally determined during clinical

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trials conducted for licensure. Appro- and enclosures used with biological priate strategies to address the risks products containing modified live ca- associated with vaccine use in preg- nine hepatitis virus or modified live ca- nant animals should be discussed with nine adenovirus Type 2 shall bear the a veterinarian.’’ following statement: ‘‘Occasionally, (f) For biological products recom- transient corneal opacity may occur mending annual booster vaccinations, following the administration of this such recommendations must be sup- product.’’ ported by data acceptable to APHIS. In (l) All labels for autogenous biologics the absence of data that establish the must specify the name of the micro- need for booster vaccination, labeling organism(s) or antigen(s) that they must bear the following statement: contain, and shall bear the following ‘‘The need for annual booster vaccina- statement: ‘‘Potency and efficacy of tions has not been established for this autogenous biologics have not been es- product; consultation with a veteri- tablished. This product is prepared for narian is recommended.’’ use only by or under the direction of a (g) In the case of a liquid product au- veterinarian or approved specialist.’’ thorized in a filed Outline of Produc- (m) In the case of biological products tion to be used as a diluent in a com- containing Marek’s disease virus, all bination package, the carton labels and labels shall specify the Marek’s disease enclosures used for serials which are ei- virus serotype(s) used in the product. ther not tested for bactericidal or viri- (n) All labels for conditionally li- cidal activity or have been found un- censed products shall bear the fol- satisfactory by such test shall contain lowing statement: ‘‘This product li- the statement: ‘‘CAUTION: DO NOT cense is conditional; efficacy and po- USE AS DILUENT FOR LIVE VAC- tency have not been fully dem- CINES.’’ onstrated.’’ (h) In the case of wart vaccine, rec- (Approved by the Office of Management and ommendations shall be limited to use Budget under control number 0579–0013) in cattle. Indications for use shall be [38 FR 12094, May 9, 1973] for prophylactic use only, as an aid in the control of viral papillomas (warts). EDITORIAL NOTE: For FEDERAL REGISTER ci- tations affecting § 112.7, see the List of CFR All labels shall include a dosage rec- Sections Affected, which appears in the ommendation of at least 10 ml to be Finding Aids section of the printed volume given subcutaneously and the dose re- and at www.fdsys.gov. peated in 3 to 5 weeks. (i) All but very small final container § 112.8 For export only. labels for feline panleukopenia vac- The applicable regulations for pack- cines shall contain the following rec- aging and labeling a biological product ommendations for use: produced in the United States shall (1) Killed virus vaccines. Vaccinate apply to such biological product if ex- healthy cats with one dose, except that ported from the United States except if the animal is less than 12 weeks of as otherwise provided in this section. age, a second dose should be given no Only labels approved as provided in earlier than 16 weeks of age. § 112.5 shall be used. (2) Modified live virus vaccines. Vac- (a) Biological products which have cinate healthy cats with one dose, ex- been packaged and labeled for export cept that if the animal is less than 12 or which have been exported, shall be weeks of age, a second dose should be subject to the applicable provisions in given no earlier than16 weeks of age. this paragraph. (j) In the case of normal serum, anti- (1) After leaving the licensed estab- serum, or antiserum derivatives, the lishment, a biological product shall not type of preservative used shall be indi- be bottled, repackaged, relabeled, or cated on all labels. otherwise altered in any way while in (k) Unless acceptable data has been the United States; and filed with Animal and Plant Health In- (2) An exported biological product spection Service, to show that develop- shall not be returned to the United ment of corneal opacity is not associ- States: Provided, That, in the case of a ated with the product, carton labels biological product exported in labeled

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final containers, the Administrator (b) Labels on each product to be fur- may authorize by permit the importa- ther distributed in accordance with tion of a limited number for research § 103.3 shall bear the statement ‘‘No- and evaluation by the producing li- tice! For Experimental Use Only—Not censee; and for Sale!’’ (3) An exported biological product (c) The labeling shall contain any which is bottled, rebottled, or altered other information deemed necessary by in any way in a foreign country shall the Administrator and specified on the not bear a label which indicates by es- permit. tablishment license number that it has [50 FR 46417, Nov. 8, 1985, as amended at 56 been prepared in the United States. FR 66784, Dec. 26, 1991] (b) Desiccated and frozen liquid products, packaged and labeled as for do- § 112.10 Special packaging and label- mestic use, may be exported without ing. the diluent required for rehydration or A biological product, which requires dilution, as the case may be, if the la- special packaging and/or labeling not beling includes adequate instructions provided for in this part, shall be pack- for preparing the product for use and aged and/or labeled in accordance with the words ‘‘For Export Only’’. requirements written into the approved (c) Final containers of products, la- outline for such product. beled or unlabeled, may be exported in sealed shipping boxes, adequately identified as to contents with an approved PART 113—STANDARD label, and plainly marked ‘‘For Export REQUIREMENTS Only’’: Provided, That such products shall not be diverted to domestic use. APPLICABILITY (d) Completed inactivated liquid Sec. products, antiserums, and antitoxins, 113.1 Compliance. may be exported in large multiple-dose 113.2 Testing aids. containers identified with an approved 113.3 Sampling of biological products. 113.4 Exemptions to tests. label that contains the words ‘‘For Ex- 113.5 General testing. port Only’’ prominently displayed. 113.6 Animal and Plant Health Inspection (e) Concentrated inactivated liquid Service testing. product, completed except for dilution 113.7 Multiple fractions. to the proper strength for use, may be 113.8 In vitro tests for serial release. exported in large multiple-dose con- 113.9 New potency test. tainers identified with an approved 113.10 Testing of bulk material for export or label that contains the words ‘‘For Ex- for further manufacture. port Only’’ prominently displayed. STANDARD PROCEDURES [38 FR 12094, May 9, 1973, as amended at 39 113.25 Culture media for detection of bac- FR 19202, May 31, 1974; 40 FR 46093, Oct. 6, teria and fungi. 1975; 43 FR 11145, Mar. 17, 1978; 56 FR 66784, 113.26 Detection of viable bacteria and fungi Dec. 26, 1991] except in live vaccine. 113.27 Detection of extraneous viable bac- § 112.9 Biological products imported teria and fungi in live vaccines. for research and evaluation. 113.28 Detection of mycoplasma contamina- A biological product imported for re- tion. 113.29 Determination of moisture content in search and evaluation under a permit desiccated biological products. issued in accordance with § 104.4, with 113.30 Detection of Salmonella contamina- the exception of products imported tion. under § 104.4(d), shall be labeled as pro- 113.31 Detection of avian lymphoid leukosis. vided in this section. 113.32 Detection of Brucella contamination. (a) The label shall identify the prod- 113.33 Mouse safety tests. uct and the name and address of the 113.34 Detection of hemagglutinating vi- manufacturer and shall provide in- ruses. 113.35 Detection of viricidal activity. structions for proper use of the prod- 113.36 Detection of pathogens by the chick- uct, including all warnings and cau- en inoculation test. tions needed by the permittee to safely 113.37 Detection of pathogens by the chick- use the product. en embryo inoculation test.

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113.38 Guinea pig safety test. 113.114 Tetanus Toxoid. 113.39 Cat safety tests. 113.115 Staphylococcus Aureus Bacterin- 113.40 Dog safety tests. Toxoid. 113.41 Calf safety test. 113.116 Pasteurella Multocida Bacterin, 113.42 Detection of lymphocytic chorio- Avian Isolate, Type 4. meningitis contamination. 113.117 Pasteurella Multocida Bacterin, 113.43 Detection of chlamydial agents. Avian Isolate, Type 1. 113.44 Swine safety test. 113.118 Pasteurella Multocida Bacterin, 113.45 Sheep safety test. Avian Isolate, Type 3. 113.46 Detection of cytopathogenic and/or 113.119 Erysipelothrix Rhusiopathiae hemadsorbing agents. Bacterin. 113.47 Detection of extraneous viruses by 113.120 Salmonella Typhimurium Bacterin. the fluorescent antibody technique. 113.121 Pasteurella Multocida Bacterin. 113.122 Salmonella Choleraesuis Bacterin. INGREDIENT REQUIREMENTS 113.123 Salmonella Dublin Bacterin. 113.50 Ingredients of biological products. KILLED VIRUS VACCINES 113.51 Requirements for primary cells used for production of biologics. 113.200 General requirements for killed 113.52 Requirements for cell lines used for virus vaccines. production of biologics. 113.201–203 [Reserved] 113.53 Requirements for ingredients of ani- 113.204 Mink Enteritis Vaccine, Killed mal origin used for production of bio- Virus. logics. 113.205 Newcastle Disease Vaccine, Killed 113.54 Sterile diluent. Virus. 113.55 Detection of extraneous agents in 113.206 Wart Vaccine, Killed Virus. Master Seed Virus. 113.207 Encephalomyelitis Vaccine, Eastern, Western, and Venezuelan, Killed Virus. LIVE BACTERIAL VACCINES 113.208 Avian Encephalomyelitis Vaccine, 113.64 General requirements for live bac- Killed Virus. terial vaccines. 113.209 Rabies Vaccine, Killed Virus. 113.65 Brucella Abortus Vaccine. 113.210 Feline Calicivirus Vaccine, Killed 113.66 Anthrax Spore Vaccine—Nonencap- Virus. sulated. 113.211 [Reserved] 113.67 Erysipelothrix Rhusiopathiae Vac- 113.212 Bursal Disease Vaccine, Killed cine. Virus. 113.68 Pasteurella Haemolytica Vaccine, 113.213–113.214 [Reserved] Bovine. 113.215 Bovine Virus Diarrhea Vaccine, 113.69 Pasteurella Multocida Vaccine, Bo- Killed Virus. vine. 113.216 Bovine Rhinotracheitis Vaccine, 113.70 Pasteurella Multocida Vaccine, Avian Killed Virus. Isolate. 113.71 Chlamydia Psittaci Vaccine (Feline LIVE VIRUS VACCINES Pneumonitis), Live Chlamydia. 113.300 General requirements for live virus vaccines. INACTIVATED BACTERIAL PRODUCTS 113.301 Ovine Ecthyma Vaccine. 113.100 General requirements for inac- 113.302 Distemper Vaccine—Mink. tivated bacterial products. 113.303 Bluetongue Vaccine. 113.101 Leptospira Pomona Bacterin. 113.304 Feline Panleukopenia Vaccine. 113.102 Leptospira Icterohaemorrhagiae 113.305 Canine Hepatitis and Canine Bacterin. Adenovirus Type 2 Vaccine. 113.103 Leptospira Canicola Bacterin. 113.306 Canine Distemper Vaccine. 113.104 Leptospira Grippotyphosa Bacterin. 113.308 Encephalomyelitis Vaccine, Ven- 113.105 Leptospira Hardjo Bacterin. ezuelan. 113.106 Clostridium Chauvoei Bacterin. 113.309 Bovine Parainfluenza3 Vaccine. 113.107 Clostridium Haemolyticum 113.310 Bovine Rhinotracheitis Vaccine. Bacterin. 113.311 Bovine Virus Diarrhea Vaccine. 113.108 Clostridium Novyi Bacterin-Toxoid. 113.312 Rabies Vaccine, Live Virus. 113.109 Clostridium Sordellii Bacterin-Tox- 113.313 Measles Vaccine. oid. 113.314 Feline Calicivirus Vaccine. 113.110 Clostridium Botulinum Type C 113.315 Feline Rhinotracheitis Vaccine. Bacterin-Toxoid. 113.316 Canine Parainfluenza Vaccine. 113.111 Clostridium Perfringens Type C Tox- 113.317 Parvovirus Vaccine (Canine). oid and Bacterin-Toxoid. 113.318 Pseudorabies Vaccine. 113.112 Clostridium Perfringens Type D Tox- 113.319–113.324 [Reserved] oid and Bacterin-Toxoid. 113.325 Avian Encephalomyelitis Vaccine. 113.113 Autogenous biologics. 113.326 Avian Pox Vaccine.

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113.327 Bronchitis Vaccine. be in accordance with the procedures 113.328 Fowl Laryngotracheitis Vaccine. currently being followed at National 113.329 Newcastle Disease Vaccine. Veterinary Services Laboratories and 113.330 Marek’s Disease Vaccines. 113.331 Bursal Disease Vaccine. as improved, proven procedures are de- 113.332 Tenosynovitis Vaccine. veloped, shall be revised and reissued prior to application. DIAGNOSTICS AND REAGENTS (b) Standard Reference Preparation 113.400–113.405 [Reserved] is a serum, virus, bacterial culture, or 113.406 Tuberculin, Intradermic. antigen to be used in test systems for 113.407 Pullorum antigen. direct comparison with serials of bio- 113.408 Avian mycoplasma antigen. 113.409 Tuberculin—PPD Bovis, logical products under test. Intradermic. (c) Standard Test Reagent is a serum, antitoxin, fluorescent antibody con- ANTIBODY PRODUCTS jugate, toxin, virus, bacterial cultural, 113.450 General requirements for antibody or antigen to be used in test systems products. but not for direct comparison with se- 113.451 Tetanus Antitoxin. rials of biological products under test. 113.452 Erysipelothrix Rhusiopathiae Anti- (d) Seed cultures are small quantities body. 113.453 [Reserved] of standard organisms to be propagated 113.454 Clostridium Perfringens Type C by the recipient to establish a supply Antitoxin. for use. 113.455 Clostridium Perfringens Type D (e) Test Code Number is a number as- Antitoxin. signed by Animal and Plant Health In- 113.456–113.498 [Reserved] spection Service to each test procedure 113.499 Products for treatment of failure of passive transfer. specified in the Standard Requirements and in each filed Outline of Production AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, where such test is conducted to support 2.80, and 371.4. a request for release of a serial or sub- SOURCE: 34 FR 18004, Nov. 7, 1969, unless serial. otherwise noted. [39 FR 21041, June 18, 1974, as amended at 40 EDITORIAL NOTE: Nomenclature changes to FR 758, Jan. 3, 1975; 50 FR 21799, May 29, 1985; part 113 appear at 79 FR 55969, Sept. 18, 2014. 56 FR 66784, Dec. 26, 1991] APPLICABILITY § 113.3 Sampling of biological prod- § 113.1 Compliance. ucts. The regulations in this part apply to Each licensee and permittee shall each serial or subserial of a licensed bi- furnish representative samples of each ological product manufactured in a li- serial or subserial of a biological prod- censed establishment and to each serial uct manufactured in the United States or subserial of a biological product in or imported into the United States as each shipment imported for distribu- prescribed in this section. Additional tion and sale. samples may be purchased in the open market by a Animal and Plant Health § 113.2 Testing aids. Inspection Service representative. To better ensure consistent and re- (a) Either an employee of the Depart- producible test results when Standard ment of Agriculture, of the licensee, or Requirement tests prescribed in the of the permittee, as designated by the regulations are conducted, National Administrator shall select prerelease Veterinary Services Laboratories, U.S. samples of biological product in the Department of Agriculture, may pro- number prescribed in paragraph (b) of vide testing aids, when available, to li- this section. Each sample shall be censees, permittees, and applicants for marked for identification by the person licenses and permits. Such aids shall be making the selection after which they as follows: shall be packaged by the licensee or (a) Supplemental Assay Method permittee, as the case may be, and for- (SAM) is a technical bulletin con- warded to National Veterinary Serv- taining detailed instructions for con- ices Laboratories; except that an em- ducting a test. Such instructions shall ployee of the Department may forward

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or deliver the samples to National Vet- (vi) Thirty single-dose or 14 multiple- erinary Services Laboratories if such dose samples of Equine action deemed advisable by the Admin- Encephalomyelitis Vaccine, Killed istrator. Virus; (1) Selection shall be made as fol- (vii) Twenty-two single-dose or 14 lows: multiple-dose samples of Rabies Vac- (i) Nonviable liquid biological prod- cine, Killed Virus; ucts—either bulk or final container (viii) Sixteen single-dose or 12 mul- samples of completed product shall be tiple-dose samples of all other vaccines selected for purity, safety, or potency consisting of killed microorganisms. tests. Biological product in final con- (2) Bacterins and bacterin-toxoids: tainer shall be selected to test for via- (i) Twelve samples of single-fraction ble bacteria and fungi. products; (ii) Viable liquid biological products; (ii) Thirteen samples of two-fraction samples shall be in final containers and products; shall be randomly selected at the end (iii) Fourteen samples of products of the filling operation. Bulk con- consisting of 3 or more fractions. tainers of completed product may be (3) Antiserums: Twelve samples of sampled when authorized by the Ad- antiserum recommended for large ani- ministrator. mals or 14 samples of antiserum rec- (iii) Desiccated biological products; ommended for small animals or the samples shall be in final containers and number of reagent serum samples pre- shall be randomly selected if des- scribed in the filed Outline of Produc- iccated in the final container. Biologi- tion for the product. cal products desiccated in bulk shall be sampled at the end of the filling oper- (4) Antitoxins: ation. (i) Fourteen single-dose or 12 mul- (iv) Representative samples of each tiple dose samples of Tetanus Anti- serial or subserial in each shipment of toxin; imported biological products shall be (ii) Twelve samples of all other selected. antitoxins. (2) Comparable samples shall be used (5) Toxoids: by Animal and Plant Health Inspection (i) Eighteen single-dose or 12 mul- Service, the licensee, and the per- tiple dose samples of all toxoids. mittee for similar tests. (6) Antigens: Twelve samples of poul- (3) When bulk samples of completed try antigens or 20 samples of tuber- product in liquid form are to be tested culin or four samples of all other diag- as prescribed in paragraph (a)(1) of this nostic antigens. section, the number of such samples (7) Diagnostic test kits: Two samples of from each serial and the minimum diagnostic test kits. The licensee or quantity of product to be provided in permittee will hold one of these se- each sample shall be stated in the filed lected samples at the storage tempera- Outline of Production. ture recommended on the label while (b) Unless otherwise prescribed by awaiting a request by the animal and the Administrator, the number of final Plant Health Inspection Service to sub- container samples to be selected from mit the additional sample. If submis- each serial and subserial shall be: sion is not requested by the Animal (1) Vaccines: and Plant Health Inspection Service, (i) Six multiple-dose samples of the additional sample may be returned Brucella Abortus Vaccine; to the serial inventory after the serial (ii) Twelve samples of all other live is released. In the case of diagnostic bacterial vaccines; test kits in which final packaging con- (iii) Two samples of Coccidiosis Vac- sists of multiple microtiter test plates cine; or strips, the licensee or permittee may (iv) Eighteen samples of Rabies Vac- submit a specified number of test cine, Modified Live Virus; plates or strips along with all other (v) Sixteen samples of all other vac- test reagents as prescribed in a filed cines consisting of live microorga- Outline of Production and retain a nisms; similar amount as a second sample for

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submission upon request. When the ini- For Master Seeds grown in cell culture tial sample is not representative of and intended for use in more than one final packaging by the licensee of per- species, an additional 2 samples are re- mittee, e.g., does not consist of all the quired for each additional species. microtiter test plates or strips, the sec- (3) Thirty-six samples of at least 1 ml ond sample is not eligible to be re- each or six samples of at least 1 ml turned to serial inventory after the se- each, one sample of at least 20 ml, and rial is released. one sample of at least 10 ml of Master (8) Autogenous biologics: With the ex- Cell Stocks. In the case of Master Cell ception of the first serial or subserial, Stocks which are persistently infected 10 samples must be selected and sub- with a virus, an additional four sam- mitted to the Animal and Plant Health ples of at least 1 ml each are required. Inspection Service from each serial or If these persistently infected cell subserial of an autogenous biologic eli- stocks are intended for use in more gible to be shipped that consists of than one species, an additional two more than 50 containers. For first seri- samples of at least 1 ml each are re- als or subserials eligible for shipment quired for each additional species. consisting of more than 50 containers, (4) Four samples of the Master Cell 10 samples from each serial or subserial Stock + n (highest passage) cells. must be selected and held for submis- (d) Sterile diluent: A sample of Ster- sion to the Animal and Plant Health ile Diluent shall accompany each sam- Inspection Service upon request in ac- ple of product, other than Marek’s Dis- cordance with paragraph (e)(4) of this ease Vaccine, if such diluent is re- section. For serials or subserials of au- quired to rehydrate or dilute the prod- togenous biologic with 50 or fewer con- uct before use. The volume of diluent tainers, no samples, other than those shall be an appropriate amount to re- required by paragraph (e) of this sec- hydrate or dilute the product. Samples tion, are required. of Sterile Diluent prepared for use with (9) Miscellaneous: The number of sam- Marek’s Disease Vaccine shall be sub- ples from products not in the cat- mitted upon request from the Animal egories provided for in paragraphs and Plant Health Inspection Service. (b)(1) through (b)(8) of this section (e) Reserve samples shall be selected shall be prescribed in the filed Outline from each serial and subserial of bio- of Production for the product. logical product. Such samples shall be (c) Prelicensing and Outline of Pro- selected at random from final con- duction changes: Samples needed to tainers of completed product by an em- support a license application or a ployee of the Department, of the li- change in the Outline of Production for censee, or of the permittee, as des- a licensed product shall be submitted ignated by the administrator. Each only upon request from the animal and sample shall: Plant Health Inspection Service. Ex- (1) Consist of 5 single-dose packages, cept for miscellaneous products speci- 2 multiple-dose packages, or 2 diag- fied in paragraph (b)(9) of this section, nostic test kits, except that, in the the number of such samples shall be at case of diagnostic test kits in which least one and one-half times the num- final packaging consists of multiple ber prescribed for such product in para- microtiter test plates or strips, a sam- graph (b) of this section. Samples of ple may consist of a specified number Master Seeds and Master Cell Stocks of test plates or strips along with all with a minimum individual volume of 1 other test reagents as prescribed in a ml shall be submitted as follows: filed Outline of Production; (1) Ten samples of Bacterial Master (2) Be adequate in quantity for appro- Seeds. priate examination and testing; (2) Thirteen samples of viral Master (3) Be truly representative and in Seeds or nonviral Master Seeds requir- final containers; ing cell culture propagation. For Mas- (4) Be held in a special compartment ter Seeds isolated or passed in a cell set aside by the licensee or permittee line different from the species of infor holding these samples under refrig- tended use, an additional 2 samples are eration at the storage temperature rec- required for each additional species. ommended on the labels for 6 months

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after the expiration date stated on the peated. When a test is declared satis- labels. The samples that are stored in factory, the test designation is consid- this manner shall be delivered to the ered to be a final conclusion. When a Animal and Plant Health Inspection test is declared unsatisfactory, the test Service upon request. designation is considered to be a final (Approved by the Office of Management and conclusion. When the initial or any Budget under control number 0579–0013) subsequent test is declared inconclu- [38 FR 29886, Oct. 30, 1973, as amended at 40 sive, the reasons shall be reported in FR 758, Jan. 3, 1975; 40 FR 49768, Oct. 24, 1975; the test records, the result shall not be 41 FR 56627, Dec. 29, 1976; 48 FR 9506, Mar. 7, considered as final, and the test may be 1983; 48 FR 57473, Dec. 30, 1983; 50 FR 21799, repeated as established in the filed May 29, 1985; 56 FR 66784, Dec. 26, 1991; 60 FR Outline of Production or Standard Re- 14356, Mar. 17, 1995; 67 FR 15713, Apr. 3, 2002] quirement. If a test is designated in- § 113.4 Exemptions to tests. conclusive or No Test and the biologi- (a) The test methods and procedures cal product is not further tested, the contained in all applicable Standard test designation of unsatisfactory is Requirements shall be complied with the final conclusion. unless otherwise exempted by the Ad- (e) When new test methods are devel- ministrator and provided that such ex- oped and approved by Animal and emption is noted in the filed Outline of Plant Health Inspection Service, bio- Production for the product. logical products tested thereafter shall (b) Test methods and procedures by be evaluated by such methods, and if which the biological products shall be not found to be satisfactory when so evaluated shall be designated in the tested shall not be released. Outline of Production for such products. (Approved by the Office of Management and Budget under control number 0579–0059) [38 FR 29887, Oct. 30, 1973, as amended at 56 FR 66784, Dec. 26, 1991] [34 FR 18004, Nov. 4, 1969, as amended at 39 FR 25463, July 11, 1974; 40 FR 45420, Oct. 2, § 113.5 General testing. 1975; 40 FR 46093, Oct. 6, 1975; 41 FR 6751, Feb. 13, 1976; 48 FR 57473, Dec. 30, 1983; 56 FR 66784, (a) No biological product shall be re- Dec. 26, 1991; 79 FR 55969, Sept. 18, 2014] leased prior to the completion of tests prescribed in a filed Outline of Produc- § 113.6 Animal and Plant Health In- tion or Standard Requirements for the spection Service testing. product to establish the product to be pure, safe, potent, and efficacious. A biological product shall with rea- (b) Tests of biological products shall sonable certainty yield the results in- be observed by a competent employee tended when used as recommended or of the manufacturer during all critical suggested in its labeling or proposed la- periods. A critical period shall be the beling prior to the expiration date. time when certain specified reactions (a) The Administrator is authorized must occur in required tests to prop- to cause a biological product, manufac- erly evaluate the results. tured in the United States or imported (c) Records of all tests shall be kept into the United States, to be examined in accordance with part 116 of this and tested for purity, safety, potency, chapter. Results of all required tests or efficacy; in which case, the licensee prescribed in the filed Outline of Pro- or permittee shall withhold such prod- duction or the Standard Requirements uct from the market until a determina- for the product shall be submitted to tion has been made. Animal and Plant Health Inspection Service. Blank forms shall be furnished (b) The final results of each test con- upon request to Animal and Plant ducted by the licensee and Animal and Health Inspection Service. Plant Health Inspection Service shall (d) When the initial or any subse- be considered in evaluating a biological quent test is declared a No Test, the product. A serial or subserial which has reasons shall be reported in the test been found unsatisfactory by a re- records, the results shall not be consid- quired test prescribed in a filed Outline ered as final, and the test may be re-

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of Production or Standard Require- (2) Testing the Master Seed for ment is not in compliance with the reg- immunogenicity in a manner accept- ulations and shall not be released for able to the Animal and Plant Health market. Inspection Service (APHIS); (3) Establishing satisfactory potency [34 FR 18004, Nov. 7, 1969, as amended at 40 FR 45420, Oct. 2, 1975; 40 FR 53378, Nov. 18, for the product in accordance with the 1975; 41 FR 6751, Feb. 13, 1976; 56 FR 66784, following provisions: Dec. 26, 1991] (i) Potency for live products may be determined by log10 virus titer or de- § 113.7 Multiple fractions. termining the live bacterial count (a) When a biological product con- based on the protective dose used in tains more than one immunogenic frac- the Master Seed immunogenicity test tion, the completed product shall be plus an adequate overage for adverse evaluated by tests applicable to each conditions and test error; and fraction. (ii) Potency for inactivated products (b) When similar potency tests are re- may be determined using tests for rel- quired for more than one fraction of a ative antigen content by comparing combination biological product, dif- the antigen content of the test serial ferent animals must be used to evalu- to a reference preparation using a par- ate each fraction except when written allel line immunoassay or equivalent Standard Requirements or outlines of method which measures linearity, production make provisions and set specificity, and reproducibility in a forth conditions for use of the same manner acceptable to APHIS. animals for testing different fractions. (b) In the case of live products, each (c) When the same safety test is re- serial and subserial of desiccated prod- quired for more than one fraction, re- uct derived from an approved Master quirements are fulfilled by satisfactory Seed and bulk or final container sam- results from one test of the completed ples of each serial of completed liquid product. product derived from an approved Mas- ter Seed shall be evaluated by a test (d) When an inactivated fraction(s) is procedure acceptable to APHIS. On the used as a diluent for a live virus frac- basis of the results of the test, as com- tion(s), the inactivated fraction(s) may pared with the required minimum po- be tested separately and the live virus tency, each serial and subserial shall fraction(s) may be tested separately: either be released to the firm for mar- Provided, That, the viricidal test re- keting or withheld from the market. quirements prescribed in § 113.100 are The evaluation of such products shall complied with. be made in accordance with the fol- (e) Virus titrations for a multivirus lowing criteria: product shall be conducted by methods (1) If the initial test shows the count which will quantitate each virus. or titer to equal or exceed the required [34 FR 18004, Nov. 7, 1969, as amended at 40 minimum, the serial or subserial is sat- FR 46093, Oct. 6, 1975; 56 FR 66785, Dec. 26, isfactory without additional testing. 1991] (2) If the initial test shows the count or titer to be lower than the required § 113.8 In vitro tests for serial release. minimum, the serial or subserial may (a) Master Seed which has been es- be retested, using double the number of tablished as pure, safe, and samples. The average counts or titers immunogenic shall be used for pre- obtained in the retests shall be deter- paring seed for production as specified mined. If the average is less than the in the Standard Requirements or in the required minimum, the serial or sub- filed Outline of Production. The Ad- serial is unsatisfactory without further ministrator may exempt a product consideration. from a required animal potency test for (3) If the average is equal to or great- release when an evaluation can, with er than the required minimum, the fol- reasonable certainty, be made by: lowing shall apply to live virus vac- (1) Subjecting the master seed to the cines: applicable requirements prescribed in (i) If the difference between the aver- §§ 113.64, 113.100, 113.200, and 113.300; age titer obtained in the retests and

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the titer obtained in the initial test is sion by the Administrator based on a 10 0.7 or greater, the initial titer may be showing by the firm seeking the exten- considered a result of test system error sion that they have made a good faith and the serial or subserial considered effort with due diligence to achieve satisfactory for virus titer. compliance. On the basis of the results (ii) If the difference between the av- of such test procedures, each serial erage titer obtained in the retests and that meets the required minimum po- the titer obtained in the initial test is tency shall be released to the firm for less than 10 0.7, a new average shall be marketing; each serial not meeting the determined using the titers obtained in required minimum potency shall be all tests. If the new average is below withheld from the market. The evalua- the required minimum, the serial or tion of such products shall be made in subserial is unsatisfactory. accordance with the following criteria: (4) If the average is equal to or great- (1) A test that results in no valid er than the required minimum, the fol- lines is considered a ‘‘no test’’ and may lowing shall apply to bacterial vac- be repeated. cines: (2) An initial test (test 1) that results (i) If the average count obtained in in valid lines that are not parallel is the retests is at least three times the considered a valid equivocal test. Re- count obtained in the initial test, the lease of the serial may not be based on initial count may be considered a re- such test since the result cannot be sult of test system error and the serial termed ‘‘satisfactory’’ or ‘‘unsatisfac- or subserial considered satisfactory for tory.’’ bacterial count. (3) If the initial test (test 1) shows (ii) If the average count obtained in that potency equals or exceeds the re- the retests is less than three times the quired minimum potency, the serial is count obtained in the initial test, a satisfactory without additional test- new average shall be determined using ing. the counts obtained in all tests. If the (4) If the initial test (test 1) is an new average count is below the re- equivocal test due to lack of par- quired minimum, the serial or sub- allelism, the serial may be retested up serial is unsatisfactory. to three times (tests 2, 3, and 4) with (5) Exceptions. When a product is eval- disposition to be as specified in para- uated in terms other than log10 virus graphs (c)(4)(i) and (ii) of this section; titer or organism count, an appropriate Provided, That, if the serial is not re- difference between the average potency tested or the other provisions of this value obtained in the retests and the section are not satisfied, the serial potency value obtained in the initial shall be deemed unsatisfactory. test shall be established for use in (i) If: The first retest (test 2) fol- paragraphs (b)(3) and (b)(4) of this sec- lowing an initial equivocal test; the tion to evaluate such products and second retest (test 3) following two shall be specified in the product Stand- consecutive equivocal tests (tests 1 and ard Requirement or filed Outline of 2); or the third retest (test 4) following Production. three consecutive equivocal tests (tests (c) In the case of inactivated prod- 1, 2, and 3) shows that the potency ucts, bulk or final container samples of equals or exceeds the required min- completed product from each serial de- imum potency, the serial is satisfac- rived from an approved Master Seed, tory. shall be evaluated for relative antigen (ii) If the first retest (test 2) fol- content (potency) as compared with an lowing an initial equivocal test shows unexpired reference by a parallel line that potency is less than the required immunoassay or other procedure ac- minimum potency, disposition of the ceptable to APHIS. Firms currently serial will be based on the outcome of using immunoassays which do not sat- retests 2 and 3 (tests 3 and 4) as follows: isfy this requirement shall have 2 years if either retest (test 3 or 4) shows that from the effective date of the final rule potency is less than the required min- to update their filed Outlines of Pro- imum potency, the serial is unsatisfac- duction to be in compliance with this tory. If either retest 2 or retest 3 (tests requirement unless granted an exten- 3 or 4) is an equivocal test, or in the

704

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event that each retest (tests 2, 3, and 4) (d) Extending the dating of a reference. following an initial equivocal test is All determinations of relative antigen also an equivocal test, the accumulated content using parallel line test results shall be considered indic- immunoassays or equivalent methods ative of a lack of potency and release shall be conducted with an unexpired of the serial withheld. In which case, reference. The lot of reference used to the licensee may submit data con- determine antigenic content shall have firming the continued validity of the an initial dating period equal to the test system to APHIS for review and dating of the product or as supported approval. If the data are acceptable to by data acceptable to APHIS, except APHIS, the potency test may be re- that frozen references may have an ini- peated by the firm, subject to the pro- tial dating of up to 5 years, Provided, visions specified in paragraphs (i) and That the request for dating of the fro- (ii) and confirmatory testing by zen references beyond the dating of the APHIS. product is supported by preliminary (5) If the initial test (test 1) shows data acceptable to APHIS and includes that potency is less than the required provisions for monitoring the stability minimum potency, the serial may be of the reference to determine when the retested a minimum of two times (tests potency starts to decline and for tak- 2 and 3) but not more than three times ing the appropriate steps to requalify a (tests 2, 3, and 4) with disposition as reference with declining potency either specified in paragraphs (c)(5) (i) and (ii) by testing a Qualifying Serial in host of this section; Provided, That, if the animals or by providing other evidence serial is not retested or the other proof immunogenicity, e.g., antibody visions of this section are not satisfied, titers or laboratory animal test data the serial shall be deemed unsatisfac- previously correlated to host animal tory. (i) If two consecutive retests (tests 2 protection in a manner acceptable to and 3) show that potency of the serial APHIS. Prior to the expiration date, equals or exceeds the required min- such reference may be granted an ex- imum potency, the serial is satisfac- tension of dating, Provided, That its tory. If one of the two retests (test 2 or immunogenicity has been confirmed 3) shows that the potency is less than using a Qualifying Serial of product in the required minimum potency, the se- a manner acceptable to APHIS. The rial is unsatisfactory. dating period of the Master Reference (ii) If one of the retests (tests 2 or 3) and Working Reference may be ex- shows that the potency equals or extended by data acceptable to APHIS if ceeds the required minimum potency the minimum potency of the Master and the other retest (test 2 or 3) is an Reference is determined to be ade- equivocal test, a third retest (test 4) quately above the minimum level need- may be performed. If the third retest ed to provide protection in the host (test 4) shows that the potency of the animal. If a new Master Reference is serial equals or exceeds the required established, it shall be allowed an ini- minimum potency, the serial is deemed tial dating period equal to the dating satisfactory. If both retests (tests 2 and of the product or as supported by data 3) or if the third retest (test 4) is an acceptable to APHIS, except that fro- equivocal test, the accumulated test zen references may have an initial dat- results shall be considered indicative of ing period of 5 years, or as supported a lack of potency and release of the se- by data acceptable to APHIS. Prior to rial withheld, in which case the li- the expiration date, such reference censee may submit data confirming the may be granted an extension of dating continued validity of the test system by confirming its immunogenicity to APHIS for review and approval. If using a Qualifying Serial of product. the data are acceptable to APHIS, the (e) Final container samples of com- potency test may be repeated by the pleted product derived from Master firm, subject to the provisions specified Seed found immunogenic in accordance in paragraphs (c)(4) (i) and (ii) and with paragraph (a) of this section and (c)(5) (i) and (ii) of this section, and found satisfactory in accordance with confirmatory testing by APHIS. paragraphs (b) and (c) of this section

705

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may also be subjected to an animal po- § 113.9 New potency test. tency test by Animal and Plant Health A potency test written into the filed Inspection Service as provided in this Outline of Production for a product paragraph. Products shall be used ac- shall be considered confidential infor- cording to label directions including mation by Animal and Plant Health In- dose(s) and route of administration. spection Service until at least two ad- (1) A one stage test using 20 vac- ditional product licenses are issued for cinates and 5 controls or a two stage the product or unless use of the test is test using 10 vaccinates and 5 controls authorized by the licensee, in which for each stage shall be used. The cri- case, such potency test may be pub- teria used for judging the specific re- lished as part of the Standard Require- sponse in the controls and vaccinates ment for the product. shall be in accordance with the test (a) Until a potency test is published protocol used in the Master Seed as part of the Standard Requirement immunogenicity test. for the product, reference to such a (2) If at least 80 percent of the con- test shall be made in the filed Outline trols do not show specific responses to of Production and the test shall be con- challenge, the test is inconclusive and ducted. may be repeated. If a vaccinate shows (b) When a potency test has been pub- the specific responses to challenge ex- lished as part of the Standard Require- pected in the controls, the vaccinate ment, such test shall be conducted un- shall be listed as a failure. less the product is specifically exempt- (3) The results of the testing shall be ed as provided in § 113.4. evaluated according to the following [40 FR 14084, Mar. 28, 1975, as amended at 56 table: FR 66784, Dec. 26, 1991]

CUMULATIVE TOTALS § 113.10 Testing of bulk material for export or for further manufacture. Num- ber of Failures for Failures for When a product is prepared in a li- Stage ani- satisfactory unsatisfac- mals serials tory serials censed establishment for export in large multiple-dose containers as pro- 1 ...... 10 1 or less ...... 3 or more. vided in § 112.8(d) or (e) of this sub- 2 (or 1) ...... 20 4 or less ...... 5 or more. chapter or for further manufacturing purposes as provided in § 114.3(d) of this (4) When a serial has been found un- subchapter, samples of the bulk mate- satisfactory for potency by the test rial shall be subjected to all required provided in paragraphs (e)(1), (2), and tests prescribed in the filed Outline of (3) of this section, the serial shall be Production or Standard Requirements withheld from the market and the fol- for the product. Samples of con- lowing actions taken: centrated liquid product shall be di- (i) The Administrator shall require luted to a volume equal to the contents that at least two additional serials pre- of the sample times the concentration pared with the same Master Seed be factor prior to initiating potency tests. subjected to similar animal potency [49 FR 45846, Nov. 21, 1984] tests by Animal and Plant Health In- spection Service or the licensee or STANDARD PROCEDURES both. (ii) If another serial is found unsatis- § 113.25 Culture media for detection of factory for potency, the product shall bacteria and fungi. be removed from the market while a (a) Ingredients for which standards reevaluation of the product is made are prescribed in the United States and the problem is resolved. Pharmacopeia, or elsewhere in this part, shall conform to such standards. [49 FR 22625, May 31, 1984, as amended at 56 In lieu of preparing the media from the FR 66784, 66786, Dec. 26, 1991; 62 FR 19038, Apr. individual ingredients, they may be 18, 1997; 72 FR 72564, Dec. 21, 2007; 79 FR 31021, May 30, 2014] made from dehydrated mixtures which, when rehydrated with purified water,

706

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have the same or equivalent composi- cells, or ingredients of animal origin tion as such media and have growth- used in the preparation of a biological promoting buffering, and oxygen ten- product are required to be free of via- sion-controlling properties equal to or ble bacteria and fungi, they shall also better than such media. The formulas be tested as prescribed in this section. for the composition of the culture (a) The media to be used shall be as media prescribed in §§ 113.26 and 113.27 follows: are set forth in the United States Phar- (1) Fluid Thioglycollate Medium with macopeia, 19th Edition. 0.5 percent beef extract shall be used to (b) The licensee shall test each quan- test for bacteria in biological products tity of medium prepared at one time containing clostridial toxoids, from individual ingredients and the bacterins, and bacterin-toxoids. first quantity prepared from each lot of (2) Fluid Thioglycollate Medium with commercial dehydrated medium for or without 0.5 percent beef extract growth-promoting qualities. If any por- shall be used to test for bacteria in bio- tion of a lot of commercial dehydrated logical products other than clostridial medium is held for 90 days or longer toxoids, bacterins, and bacterin-tox- after being so tested, it shall be re- oids. tested before use. Two or more strains (3) Soybean-Casein Digest Medium of micro-organisms that are exacting shall be used to test biological prod- in their nutritive requirements shall be ucts for fungi; provided, that Fluid used. More than one dilution shall be Thioglycollate Medium without beef used to demonstrate the adequacy of extract shall be substituted when test- the medium to support the growth of a ing biological products containing mer- minimum number of micro-organisms. curial preservatives. (c) The sterility of the medium shall (b) Test procedure: be confirmed by incubating an ade- (1) Ten test vessels shall be used for quate number of test vessels and exam- each of two media selected in accord- ining each for growth. Additional con- ance with paragraph (a)(1), (a)(2), or trol may be used by incubation of rep- (a)(3) of this section. Each test vessel resentative uninoculated test vessels shall contain sufficient medium to ne- for the required incubation period dur- gate the bacteriostatic or fungistatic ing each test. activity in the inoculum as determined (d) A determination shall be made by in § 113.25(d). the licensee for each biological product (2) Inoculum: of the ratio of inoculum to medium (i) When completed product is tested, which shall result in sufficient dilution 10 final container samples from each of such product to prevent serial and each subserial shall be test- bacteriostatic and fungistatic activity. ed. One ml from each sample shall be The determination may be made by inoculated into a corresponding indi- tests on a representative biological vidual test vessel of culture medium: product for each group of comparable Provided, That, if each final container products containing identical preserva- sample contains less than 2 ml, one- tives at equal or lower concentrations. half of the contents shall be used as Inhibitors or neutralizers of preserva- inoculum for each test vessel. tives, approved by the Administrator, (ii) When cell lines, primary cells, or may be considered in determining the ingredients of animal origin are tested, proper ratio. at least a 20 ml test sample from each [35 FR 16039, Oct. 13, 1970, as amended at 37 lot shall be tested. One ml shall be in- FR 2430, Feb. 1, 1972; 41 FR 27715, July 6, 1976; oculated into each test vessel of me- 56 FR 66784, Dec. 26, 1991] dium. (3) Incubation shall be for an observa- § 113.26 Detection of viable bacteria tion period of 14 days at 30 °to 35 °C. to and fungi except in live vaccine. test for bacteria and 14 days at 20 °to 25 Each serial and subserial of biologi- °C. to test for fungi. cal product except live vaccines shall (4) If the inoculum renders the me- be tested as prescribed in this section dium turbid so that the absence of unless otherwise specified by the Ad- growth cannot be determined by visual ministrator. When cell lines, primary examination, subcultures shall be made

707

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on the seventh to eleventh day from bi- (2) Ten final container samples from ological products prepared from each serial and subserial shall be test- clostridial toxoids, bacterins, and ed. bacterin-toxoids and the third to sev- (3) Immediately prior to starting the enth day for other biological products. test, frozen liquid vaccine shall be Portions of the turbid medium in thawed, and desiccated vaccine shall be amounts of not less than 1.0 ml. shall rehydrated as recommended on the be transferred to 20 to 25 ml. of fresh label with accompanying diluent or medium, and incubated the balance of with sterile purified water. the 14-day period. (4) To test for bacteria, place 0.2 ml (c) Examine the contents of all test of vaccine from each final container vessels for macroscopic microbial into a corresponding individual vessel growth during the incubation period. containing at least 120 ml of Soybean When demonstrated by adequate con- Casein Digest Medium. Additional me- trols to be invalid, the test may be re- dium shall be used if the determination peated. For each set of test vessels rep- required in § 113.25(d) indicates the need resenting a serial or subserial in a for a greater dilution of the product. valid test, the following rules shall Incubation shall be at 30 °to 35 °C for 14 apply: days. (1) If no growth is found in any test (5) To test for fungi, place 0.2 ml of vessel, the serial or subserial meets the vaccine from each final container sam- requirements of the test. ple into a corresponding individual ves- (2) If growth is found in any test vessel containing at least 40 ml of Soy- sel, one retest to rule out faulty tech- bean Casein Digest Medium. Additional nique may be conducted using 20 un- medium shall be used if the determina- opened final container samples. tion required in § 113.25(d) indicates the (3) If growth is found in any test ves- need for a greater dilution of the prod- sel of the final test, the serial, sub- uct. Incubation shall be at 20 °to 25 °C serial, or ingredients to be used in the for 14 days. preparation of a biological product, as (6) Examine the contents of all test the case may be, is unsatisfactory. vessels macroscopically for microbial [35 FR 16039, Oct. 13, 1970, as amended at 37 growth at the end of the incubation pe- FR 2430, Feb. 1, 1972; 39 FR 21042, June 18, riod. If growth in a vessel cannot be re- 1974; 40 FR 758, Jan. 3, 1975; 40 FR 14084, Mar. liably determined by visual examina- 28, 1975; 56 FR 66784, Dec. 26, 1991] tion, judgment shall be confirmed by subcultures, microscopic examination, § 113.27 Detection of extraneous viable or both. bacteria and fungi in live vaccines. (7) For each set of test vessels rep- Unless otherwise specified by the Ad- resenting a serial or subserial tested ministrator or elsewhere exempted in according to these procedures, the fol- this part, each serial and subserial of lowing rules shall apply: live vaccine and each lot of Master (i) If growth is found in 2 or 3 test Seed Virus and Master Seed Bacteria vessels of the initial test, 1 retest to shall be tested for extraneous viable rule out faulty technique may be con- bacteria and fungi as prescribed in this ducted using 20 unopened final con- section. A Master Seed found unsatis- tainer samples. factory shall not be used in vaccine (ii) If no growth is found in 9 or 10 of production and a serial found unsatis- the test vessels in the initial test, or 19 factory shall not be released. or 20 vessels in the retest, the serial or (a) Live viral vaccines. Each serial and subserial meets the requirements of subserial of live viral vaccine shall be the test. tested for purity as prescribed in this (iii) If growth is found in four or paragraph. However, products of chick- more test vessels in the initial test, or en embryo origin recommended for ad- two or more in a retest, the serial or ministration other than by parenteral subserial is unsatisfactory. injection may be tested as provided in (b) Live bacterial vaccines. Each serial paragraph (e) of this section. or subserial of live bacterial vaccine (1) Soybean Casein Digest Medium shall be tested for purity as prescribed shall be used. in this paragraph.

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(1) Soybean Casein Digest Medium (iii) If extraneous growth is found in and Fluid Thioglycollate Medium shall 4 or more test vessels in the initial be used. test, or 2 or more in a retest, the serial (2) Ten final container samples from or subserial is unsatisfactory. each serial and subserial shall be test- (c) Master Seed Virus. Not less than 4 ed. ml of each lot of Master Seed Virus (3) Immediately prior to starting the shall be tested. Frozen liquid Master test, frozen liquid vaccine shall be Seed Virus shall be thawed, and des- thawed, and desiccated vaccine shall be iccated Master Seed Virus shall be re- rehydrated as recommended on the hydrated with Soybean Casein Digest label with accompanying diluent or Medium immediately prior to starting with sterile purified water. Product the test. recommended for mass vaccination (1) To test for bacteria, place 0.2 ml shall be rehydrated at the rate of 30 ml of the sample of Master Seed Virus into sterile purified water per 1,000 doses. 10 individual vessels each containing at (4) To test for extraneous bacteria, least 120 ml of Soybean Casein Digest place 0.2 ml of vaccine from each final Medium. Incubation shall be at 30 °to container into a corresponding indi- 35 °C for 14 days. vidual vessel containing at least 40 ml (2) To test for fungi, place 0.2 ml of of Fluid Thioglycollate Medium. Addi- the sample of Master Seed Virus into 10 tional medium shall be used if the de- individual vessels each containing at termination required in § 113.25(d) indi- least 40 ml of Soybean Casein Digest cates the need for a greater dilution of Medium. Incubation shall be at 20 °to the product. Incubation shall be at 30 25 °C for 14 days. °to 35 °C for 14 days. (3) Examine the contents of all test (5) To test for extraneous fungi, place vessels macroscopically for microbial 0.2 ml of vaccine from each final con- growth at the end of the incubation pe- tainer into a corresponding individual riod. If growth in a vessel cannot be re- vessel containing at least 40 ml of Soy- liably determined by visual examina- bean Casein Digest Medium. Additional tion, judgment shall be confirmed by medium shall be used if the determina- subcultures, microscopic examination, tion required in § 113.25(d) indicates the or both. need for a greater dilution of the prod- (4) For each set of test vessels rep- uct. Incubation shall be at 20 °to 25 °C resenting a lot of Master Seed Virus for 14 days. tested according to these procedures, (6) Examine the contents of all test the following rules shall apply: vessels macroscopically for atypical (i) If growth is found in any test ves- microbial growth at the end of the in- sel of the initial test, one retest to rule cubation period. If growth of extra- out faulty technique may be conducted neous microorganisms cannot be reli- using a new sample of Master Seed ably determined by visual examina- Virus. tion, judgment shall be confirmed by (ii) If growth is found in any test ves- subculturing, microscopic examina- sel of the final test, the lot of Master tion, or both. Seed Virus is unsatisfactory. (7) For each set of test vessels rep- (d) Master Seed Bacteria. Not less than resenting a serial or subserial tested 4 ml of each lot of Master Seed Bac- according to these procedures, the fol- teria shall be tested. Frozen liquid lowing rules shall apply: Master Seed Bacteria shall be thawed, (i) If extraneous growth is found in 2 and desiccated Master Seed Bacteria or 3 test vessels of the initial test, 1 shall be rehydrated with sterile puri- retest to rule out faulty technique may fied water immediately prior to start- be conducted using 20 unopened final ing the test. container samples. (1) To test for extraneous bacteria, (ii) If no extraneous growth is found place 0.2 ml of the sample of Master in 9 or 10 test vessels in the initial test, Seed Bacteria into 10 individual vessels or 19 or 20 vessels in the retest, the se- each containing at least 40 ml of Fluid rial or subserial meets the require- Thioglycollate Medium. Incubation ments of the test. shall be at 30 °to 35 °C for 14 days.

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(2) To test for extraneous fungi, place added to each plate. One plate shall be 0.2 ml of the sample of Master Seed incubated at 30 °to 35 °for 7 days and Bacteria into 10 individual vessels each the other plate shall be incubated at 20 containing at least 40 ml of Soybean °to 25 °C for 14 days. Casein Digest Medium. Incubation (5) Colony counts shall be made for shall be at 20 °to 25 °C for 14 days. each plate at the end of the incubation (3) Examine the contents of all test period. An average colony count for the vessels macroscopically for atypical 10 samples representing the serial or microbial growth at the end of the in- subserial shall be made for each incu- cubation period. If growth of extra- bation condition. neous microorganisms cannot be reli- (6) For each set of test vessels rep- ably determined by visual examina- resenting a serial or subserial tested tion, judgment shall be confirmed by according to these procedures, the fol- subcultures, microscopic examination, lowing rules shall apply: or both. (i) If the average count at either in- (4) For each set of test vessels rep- cubation condition exceeds 1 colony per resenting a lot of Master Seed Bacteria dose for vaccines recommended for tested according to these procedures, poultry, or 10 colonies per dose for vac- the following rules shall apply: cines recommended for other animals (i) If extraneous growth is found in in the initial test, 1 retest to rule out any test vessel of the initial test, one faulty technique may be conducted retest to rule out faulty technique may using 20 unopened final containers. be conducted using a new sample of (ii) If the average count at either in- Master Seed Bacteria. cubation condition of the final test for (ii) If extraneous growth is found in a serial or subserial exceeds 1 colony any test vessel of the final test, the lot per dose for vaccines recommended for of Master Seed Bacteria is unsatisfac- poultry, or 10 colonies per dose for vac- tory. cines recommended for other animals, (e) Live viral vaccines of chicken em- the serial or subserial is unsatisfac- bryo origin recommended for adminis- tory. tration other than by parenteral injec- [48 FR 28430, June 22, 1983, as amended at 56 tion, which were not tested or have not FR 66784, Dec. 26, 1991] been found free of bacteria and fungi by the procedures prescribed in paragraph § 113.28 Detection of mycoplasma con- (a) of this section, may be tested ac- tamination. cording to the procedures prescribed in The heart infusion test, using heart this paragraph. infusion broth and heart infusion agar, (1) Brain Heart Infusion Agar shall be provided in this section shall be con- used with 500 Kinetic (Kersey) units of ducted when a test for mycoplasma penicillinase per ml of medium added contamination is prescribed in an ap- just prior to pouring the plates. plicable Standard Requirement or in (2) Ten final containers from each se- the filed Outline of Production for the rial and each subserial shall be tested. product. (3) Immediately prior to starting the (a) Media additives provided in this test, frozen liquid vaccine shall be paragraph shall be prepared as follows: thawed, and lyophilized vaccine shall (1) DPN-Cysteine Solution: be rehydrated to the quantity rec- (i) Use Nicotinamide adenine ommended on the label using the ac- dinucleotide (oxidized) and L-Cysteine companying sterile diluent or sterile hydrochloride. purified water. Product recommended (ii) Prepare 1 gram/100 milliliters for mass vaccination shall be re- (ml) purified water (1 percent solution) hydrated at the rate of 30 ml sterile pu- of each. Mix the solutions together; the rified water per 1,000 doses. cysteine reduces the DPN. Filter steri- (4) From each container sample, each lize, dispense in appropriate amounts of 2 plates shall be inoculated with vac- and store frozen at ¥20 degrees centi- cine equal to 10 doses if the vaccine is grade. recommended for poultry or 1 dose if (2) Inactivated horse serum—horse the vaccine is recommended for other serum which has been inactivated at 56 animals. Twenty ml of medium shall be °C for 30 minutes.

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(b) Heart infusion broth shall be pre- tests shall be established as provided in pared as provided in this paragraph. paragraph (d)(4) of this section. (1) Dissolve in 970 ml of purified (2) Inoculation of plate. Plate 0.1 ml water, 25 grams of heart infusion broth, of inoculum on an agar plate and make 10 grams of proteose peptone No. 3, and a short, continuous streak across the either 5 grams of yeast autolysate or 5 plate with a pipet. Tilt the plate to ml of fresh yeast extract. allow the inoculum to flow over the (2) Add the following: surface. 1 percent tetrazolium chloride (ml) ...... 5.5 (3) Inoculation of flask of medium. 1 percent thallium acetate (ml) ...... 25 Transfer 1 ml of the inoculum into a Penicillin (units) ...... 500,000 Inactivated horse serum (ml) ...... 100 flask containing 100 ml mycoplasma medium and mix thoroughly. Incubate (3) Adjust pH to 7.9 with NaOH, filter the flask at 33 to 37 °C for 14 days dur- sterilize, and dispense 100 ml aliquots ing which time, one of four agar plates into 125 ml flasks and store until need- shall be streaked with 0.1 ml of mate- ed. rial from the incubating flask of inocu- (4) Add 2 ml of DPN-Cysteine solu- lated medium on the 3d day, one on the tion to each 100 ml of broth on day of 7th day, one on the 10th day, and one use. on the 14th day post-inoculation. (c) Heart Infusion Agar shall be pre- (4) Control tests shall be conducted pared as provided in this paragraph. simultaneously with the detection test (1) Dissolve in 900 ml of purified using techniques provided in para- water by boiling the following: graphs (d)(2) and (3) of this section, ex- Heart infusion agar (g) ...... 25 Heart-infusion broth (g) ...... 10 cept the inoculum for the positive con- Proteose peptone No. 3 (g) ...... 10 trol test shall be selected mycoplasma 1 pct thallium acetate (ml) ...... 25 cultures and the negative control test (2) Cool the medium and adjust pH to shall be uninoculated medium from the 7.9 with NaOH. same lot used in the detection test. (3) Autoclave the medium. (5) All plates shall be incubated in a (4) Cool the medium 30 minutes in a high humidity, 4–6 percent CO2 atmos- 56 °C waterbath. phere at 33 °to 37 °C for 10–14 days and (5) Dissolve 5 grams of yeast autoly- examined with a stereoscopic micro- sate in 100 ml of distilled water, filter scope at 35x to 100x or with a regular sterilize, and add to the medium. microscope at 100x. (6) Add to the medium: (e) Interpretation of test results. (1) If growth appears on at least one 126 ml of inactivated horse serum 21 ml of DPN-Cysteine solution of the plates in the positive control 525,000 units of Penicillin. test and does not appear on any of the Dispense 10 ml aliquots into 60 × 15 mm dis- plates in the negative control test, the posable culture dishes or petri dishes. test is valid. (d) The test procedure provided in (2) If mycoplasma colonies are found this paragraph shall be followed when on any of the plates inoculated with conducting the mycoplasma detection material being tested, the results are test. positive for mycoplasma contamina- (1) Preparation of inoculum. Imme- tion. diately prior to starting the test, fro- [38 FR 29887, Oct. 30, 1973, as amended at 41 zen liquid vaccine shall be thawed, and FR 6752, Feb. 13, 1976; 41 FR 32882, Aug. 6, lyophilized vaccine shall be rehydrated 1976] to the volume recommended on the label with mycoplasma medium. In the § 113.29 Determination of moisture case of a lyophilized biological product, content in desiccated biological e.g., 1,000 dose vial of poultry vaccine products. to be administered via the drinking Methods provided in this section water, the vaccine shall be rehydrated must be used when a determination of to 30 ml with mycoplasma medium. In moisture content in desiccated biologi- the case of a cell line or a sample of cal products is prescribed in an appli- primary cells, the inoculum shall con- cable Standard Requirement or in the sist of the resuspended cells. Control filed Outline of Production for the

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product. Firms currently using meth- and allow dry air to bleed into the oven ods other than those provided in this until the pressure inside the oven is section for determining the moisture equalized with the prevailing atmos- content in desiccated biological prod- pheric pressure. ucts have until November 5, 2004 to up- (5) While the bottle is still warm, re- date their Outlines of Production to be place the stopper in its normal position in compliance with this requirement. and transfer the weighing bottle to the (a) Final container samples of com- desiccator. pleted product shall be tested. The (i) Allow a minimum of 2 hours for weight loss of the sample due to drying the weighing bottle to cool to room in a vacuum oven shall be determined. temperature or for its weight to reach All procedures should be performed in equilibrium. an environment with a relative humid- (ii) Weigh, and record the weight as ity less than 45 percent. The equipment ‘‘C.’’ necessary to perform the test is as fol- (6) Calculate the percentage of mois- lows: ture in the original sample as follows: (1) Cylindrical weighing bottles with ¥ ¥ × airtight glass stoppers. (B C)/(B A) (100) = Percentage of re- (2) Vacuum oven equipped with vali- sidual moisture, where: dated thermometer and thermostat. A A = tare weight of weighing bottle suitable air-drying device should be at- B¥A = weight of sample before drying tached to the inlet valve. B¥C = weight of sample after drying (3) Balance, accurate to 0.1 mg (rated (7) The results are considered satis- precision ±0.01mg). factory if the percentage of residual (4) Desiccator jar equipped with phos- moisture is less than or equal to the phorous pentoxide, silica gel, or equiv- manufacturer’s specification. alent. (5) Desiccated vaccine in original [68 FR 57608, Oct. 6, 2003] sealed vial. Sample and control should be kept at room temperature in their § 113.30 Detection of Salmonella contamination. original airtight containers until use. (b) Test procedure: The test for detection of Salmonella (1) Thoroughly cleaned and labeled contamination provided in this section sample-weighing bottles with stoppers shall be conducted when such a test is should be allowed to dry at 60 ±3 °C prescribed in an applicable Standard under vacuum at less than 2.5 kPa. Requirement or in the filed Outline of (i) Transfer hot bottles and stoppers Production for the product. into the desiccator and allow to cool to (a) Samples shall be collected from room temperature. the bulk suspension before (ii) After bottles have cooled, insert bacteriostatic or bactericidal agents stoppers and weigh and record the have been added. When tissue culture weights of the bottles as ‘‘A.’’ products are to be tested, 1 ml of tissue (iii) Return weighing bottles to the extract used as the source of cells or 1 desiccator. ml of the minced tissue per se shall be (2) Remove the sample container tested. seal. (b) Five ml of the liquid vaccine sus- (i) Using a spatula, break up the sam- pension shall be used to inoculate each ple plug and transfer the required 100 ml of liquid broth medium (tryptose amount of sample to the previously and either selenite F or tetrathionate). tared weighing bottle. The inoculated media shall be incu- (ii) Insert the stopper and weigh and bated 18–24 hours at 35–37 °C. record the weights of the weighing bot- (c) Transfers shall be made to either tles as ‘‘B.’’ MacConkey agar or Salmonella- (3) Place the weighing bottle with the Shigella agar, incubated for 18–24 hours stopper at an angle in the vacuum and examined. oven. Set the vacuum to <2.5 kPa and (d) If no growth typical of Salmonella the temperature to 60 ±3 °C. is noted, the plates shall be incubated (4) After a minimum of 3 hours of an additional 18–24 hours and again ex- drying time, turn off the vacuum pump amined.

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(e) If suspicious colonies are ob- controls. One set of chick fibroblast served, further subculture on suitable cultures inoculated with subgroup A media shall be made for positive identi- virus and another set inoculated with fication. If Salmonella is found, the subgroup B virus shall act as positive bulk suspension is unsatisfactory. controls, A and B respectively. [38 FR 29888, Oct. 30, 1973] (4) The cell cultures shall be propagated at 35–37 °C for at least 21 days. § 113.31 Detection of avian lymphoid They shall be passed when necessary to leukosis. maintain viability and samples har- The complement-fixation test for de- vested from each passage shall be test- tection of avian lymphoid leukosis pro- ed for group specific antigen. vided in this section shall be conducted (b) The microtiter complement-fixa- on all biological products containing tion test shall be performed using ei- virus which has been propagated in ther the 50 percent or the 100 percent substrates of chicken origin: Provided, hemolytic end point technique to de- An inactivated viral product shall be termine complement unitage. Five 50 exempt from this requirement if the licensee can demonstrate to Animal and percent hemolytic units or two 100 per- Plant Health Inspection Service that cent hemolytic units of complement the agent used to inactivate the vac- shall be used for each test. cine virus would also inactivate lymph- (1) All test materials, including posi- oid leukosis virus. tive and negative controls, shall be (a) Propagation of contaminating stored at ¥60 °C or colder until used in lymphoid leukosis viruses, if present, the test. Before use, each sample shall shall be done in chick embryo cell cul- be thawed and frozen three times to tures. disrupt intact cells and release the (1) Each vaccine virus, cytopathic to group specific antigen. chick embryo fibroblast cells, shall be (2) The antiserum used in the effectively neutralized, inactivated, or microtiter complement-fixation test separated so that minimal amounts of shall be a standard reagent supplied or lymphoid leukosis virus can be propa- approved by the Animal and Plant gated on cell culture during the 21-day Health Inspection Service. Four units growth period. If a vaccine virus can- of antiserum shall be used for each not be effectively neutralized, inac- test. tivated, or separated, a sample of another vaccine prepared the same week (3) Presence of complement-fixing ac- from material harvested from each tivity in the harvested samples (from source flock (or other sampling proce- passages) at the 1:4 or higher dilution, dure acceptable to Animal and Plant in the absence of anticomplementary Health Inspection Service) used for the activity, shall be considered a positive preparation of the questionable vaccine test unless the activity can definitely virus that cannot be neutralized, inac- be established to be caused by some- tivated, or separated shall be tested thing other than lymphoid leukosis each week during the preparation of virus, subgroups A and/or B. Activity such questionable vaccine. at the 1:2 dilution shall be considered (2) When cell cultures are tested, 5 ml suspicious and the sample further sub- of the final cell suspension as prepared cultured to determine presence or ab- for seeding of production cell cultures sence of the group specific antigen. shall be used as inoculum. When vac- (4) Biological products or primary cines are tested, the equivalent of 200 cells which are found contaminated doses of Newcastle disease vaccine or with lymphoid leukosis viruses are un- 500 doses of other vaccines for use in satisfactory. Source flocks from which poultry, or one dose of vaccine for use contaminated material was obtained in other animals shall be used as are also unsatisfactory. inoculum. Control cultures shall be prepared from the same cell suspension [38 FR 29888, Oct. 30, 1973, as amended at 38 as the cultures for testing the vaccine. FR 32917, Nov. 29, 1973; 39 FR 21042, June 18, (3) Uninoculated chick embryo fibro- 1974; 56 FR 66784, Dec. 26, 1991] blast cell cultures shall act as negative

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§ 113.32 Detection of Brucella contami- injection site), and the animals ob- nation. served for 7 days. (2) If unfavorable reactions attrib- The test for detection of Brucella utable to the product occur in any of contamination provided in this section the mice during the observation period, shall be conducted when such a test is the serial or subserial is unsatisfac- prescribed in an applicable Standard tory. If unfavorable reactions which Requirement or in a filed Outline of are not attributable to the product Production for the product. occur, the test shall be declared a No (a) One ml of the minced tissue used Test and may be repeated: Provided, as the source of cells or 1 ml of the ex- That, if the test is not repeated, the se- tract of the tissue prior to the addition rial or subserial shall be declared un- of antibiotics, diluent and stabilizer, satisfactory. shall be inoculated onto each of three (b) Bulk or final container samples of tryptose agar plates and incubated in a completed product from liquid prod- 10 percent CO2 atmosphere at a tem- ucts, such as but not limited to perature of 35–37 °C for at least 7 days. antiserums and bacterins, shall be test- (b) If colonies are identified as ed for safety in accordance with the Brucella, the biological product is untest provided in this paragraph. satisfactory. (1) Unless otherwise prescribed in the (c) If colonies suspicious of Brucella Standard Requirement or approved in a are observed but cannot be identified filed Outline of Production for the as a Brucella species, either product, a 0.5 ml dose shall be injected (1) The biological product shall be re- intraperitoneally or subcutaneously garded as unsatisfactory and de- into eight mice and the animals ob- stroyed; or served for 7 days. (2) Further subculture or other proce- (2) If unfavorable reactions attrib- dures shall be carried out until a posi- utable to the product occur in any of tive identification can be made. the mice during the observation period, the serial or subserial is unsatisfac- [38 FR 29888, Oct. 30, 1973] tory. If unfavorable reactions which are not attributable to the product § 113.33 Mouse safety tests. occur, the test shall be declared a No One of the mouse safety tests pro- Test and may be repeated: Provided, vided in this section shall be conducted That, if the test is not repeated, the se- when such test is prescribed in a rial or subserial shall be declared un- Standard Requirement or in the filed satisfactory. Outline of Production for a biological [38 FR 34727, Dec. 18, 1973, as amended at 39 product recommended for animals FR 16857, May 10, 1974; 72 FR 72564, Dec. 21, other than poultry: Provided, That if 2007] the inherent nature of one or more ingredients makes the biological product § 113.34 Detection of hemagglutinating lethal or toxic for mice but not lethal viruses. or toxic for the animals for which it is The test for detection of recommended, the licensee shall dem- hemagglutinating viruses provided in onstrate the safety of such product by this section shall be conducted when an acceptable test written into such such a test is prescribed in an applica- Outline of Production. ble Standard Requirement or in the (a) Final container samples of com- filed Outline of Production for the pleted product from live virus vaccines product. shall be tested for safety using young (a) Final container samples of com- adult mice in accordance with the test pleted product rehydrated as rec- provided in this paragraph. ommended on the label shall be used as (1) Vaccine prepared for use as rec- inoculum: Provided, That poultry vac- ommended on the label shall be tested cines distributed without diluent shall by inoculating eight mice be rehydrated with 30 ml of sterile dis- intraperitoneally or subcutaneously tilled water per 1,000 doses and used as with 0.5 mL (the inoculation volume inoculum. When one or more fractions may be divided among more than one are to be used in combination with

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Newcastle Disease Vaccine, test sam- vided, That the Administrator may au- ples shall be collected from bulk sus- thorize licensees to prepare and use un- pensions of each prior to mixing with licensed single-fraction vaccines for the Newcastle Disease Vaccine. this purpose. (b) Each of ten 9- to 10-day-old (c) Test procedure: (1) Rehydrate at embryonating eggs from Newcastle dis- least two vials of the vaccine with the ease susceptible flocks shall be inocu- liquid product under test according to lated into the allantoic cavity with 0.2 label recommendations and pool the ml of the undiluted inoculum. contents. (1) Test five uninoculated embryos of (2) Rehydrate at least two vials of the same age and from the same flock the vaccine with the same volume of as those used for the test as negative sterile purified water and pool the con- controls. tents. (2) Test an allantoic fluid sample of (3) Neutralize to remove other frac- Newcastle disease virus as a positive tions, if necessary. control. (4) Hold the two pools of vaccine at (c) Three to five days post-inocula- room temperature (20 °to 25 °C) for 2 tion, a sample of allantoic fluid from hours. The holding period shall begin each egg shall be tested separately by a when rehydration is completed. rapid plate test for hemagglutinating activity using a 0.5 percent suspension (5) Titrate the virus(es) in each pool of fresh chicken red blood cells. of vaccine as provided in the filed Out- (d) If the results are inconclusive, line of Production or an applicable one or two blind passages shall be made standard requirement. using fluids from each of the original (6) Compare respective titers. test eggs. Fluids from dead and live (d) If the titer of the vaccine virus(es) embryos may be pooled separately for rehydrated with the product under test inoculum in these passages. is more than 0.7 log10 below the titer of (e) If hemagglutinating activity at- the vaccine virus(es) rehydrated with tributable to the product is observed, sterile purified water, the product is the serial is unsatisfactory. unsatisfactory for use as diluent. (e) If the product is unsatisfactory in [38 FR 29889, Oct. 30, 1973] the first test, one retest to rule out § 113.35 Detection of viricidal activity. faulty techniques may be conducted using four vials of the vaccine for each The test for detection of viricidal ac- pool and the acceptability of the prod- tivity provided in this section shall be uct judged by the results of the second conducted when such a test is pretest. scribed in an applicable standard re- (f) Liquid products found to be unsat- quirement or in the filed Outline of isfactory for use as diluent by this test Production for each inactivated liquid are not prohibited from release as sepa- biological product used as diluent for a rate licensed products if labeled as pre- desiccated live virus vaccine in a com- scribed in § 112.7(g). bination package. (a) Bulk or final container samples of [44 FR 25412, May 1, 1979, as amended at 56 completed product from each serial FR 66784, Dec. 26, 1991; 64 FR 43044, Aug. 9, shall be tested. 1999] (b) The product shall be tested with each virus fraction for which it is to be § 113.36 Detection of pathogens by the used as a diluent. If the vaccine to be chicken inoculation test. rehydrated contains more than one The test for detection of extraneous virus fraction, the test shall be con- pathogens provided in this section ducted with each fraction after neu- shall be conducted when such a test is tralization of the other fraction(s), and/ prescribed in an applicable Standard or dilution of the vaccine beyond the Requirement or in the filed Outline of titer range of the other fraction(s), or Production for the product. the test shall be conducted using rep- (a) The biological product to be test- resentative single-fraction desiccated ed shall be prepared for use as rec- vaccines which are prepared by the li- ommended on the label, or in the case censee and which are licensed. Pro- of desiccated vaccine to be used in

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poultry, rehydrated with sterile dis- umes of sterile heat-inactivated spe- tilled water at the rate of 30 ml per cific antiserum to neutralize the vac- 1,000 doses. cine virus in the product. Each lot of (b) At least 25 healthy susceptible antiserum shall be demonstrated by young chickens, properly identified and virus neutralization tests not to in- obtained from the same source and hibit other viruses known to be pos- hatch, shall be immunized at least 14 sible contaminants. days prior to being put on test. The im- (c) After neutralization, 0.2 ml of the munizing agent shall be the same as vaccine-serum mixture shall be inocu- the product to be tested but from a se- lated into each of at least 20 fully sus- rial previously tested and found satis- ceptible chicken embryos. factory. (1) Twenty embryos, 9 to 11 days old, (c) At least 20 of the previously im- shall be inoculated on the chorio- munized birds shall be inoculated with allantoic membrane (CAM) with 0.1 ml, 10 label doses of the vaccine being test- and in the allantoic sac with 0.1 ml. ed by each of the following routes: Sub- (2) Eggs shall be candled daily for 7 cutaneous, intratracheal, eye-drop, and days. Deaths occurring during the first comb scarification (1 cm 2). Twenty 24 hours shall be disregarded but at birds may be used for each route or least 18 viable embryos shall survive 24 combination of routes. hours post-inoculation for a valid test. (d) At least five birds shall be iso- Examine all embryos and CAM’s from lated as control birds. embryos which die after the first day. (e) All birds shall be observed for 21 When necessary, embryo subcultures days for signs of septicemic diseases, shall be made to determine the cause of respiratory diseases, or other a death. The test shall be concluded on pathologic conditions. the seventh day post-inoculation and (f) If the controls remain healthy and the surviving embryos (including unfavorable reactions attributable to CAM’s) examined. the product occur in the vaccinates, (d) If death and/or abnormality at- the serial or subserial tested is unsatis- tributable to the inoculum occur, the factory. If the controls do not remain serial is unsatisfactory: Provided, That, healthy or if unfavorable reactions not if there is a vaccine virus override, the attributable to the product occur in test may be repeated, using a higher the vaccinates, or both, the test shall titered antiserum. be declared a No Test and may be re- [38 FR 29889, Oct. 30, 1973, as amended at 39 peated: Provided, That, if the test is not FR 21042, June 18, 1974] repeated, the serial of subserial tested shall be considered unsatisfactory. § 113.38 Guinea pig safety test. [38 FR 29889, Oct. 30, 1973, as amended at 39 The guinea pig safety test provided FR 21042, June 18, 1974; 43 FR 7610, Feb. 24, in this section shall be conducted when 1978] prescribed in a Standard Requirement or approved Outline of Production for a § 113.37 Detection of pathogens by the biological product. When desiccated chicken embryo inoculation test. products are tested, final container The test for detection of extraneous samples of completed product prepared pathogens provided in this section for administration in the manner rec- shall be conducted when such a test is ommended on the label shall be used. prescribed in an applicable Standard When liquid products are tested, either Requirement or in the filed Outline of bulk or final container samples of com- Production for the product. pleted product shall be used. (a) The biological product to be test- (a) Unless otherwise specified in the ed shall be prepared for use as rec- Standard Requirement or approved ommended on the label, or in the case Outline of Production for the product, of desiccated vaccine to be used in a 2 ml dose shall be injected either poultry, rehydrated with sterile dis- intramuscularly or subcutaneously tilled water at the rate of 30 ml per into each of two guinea pigs and the 1,000 doses. animals observed for 7 days. (b) One volume of the prepared vac- (b) If unfavorable reactions attrib- cine shall be mixed with up to nine vol- utable to the product occur in either of

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the guinea pigs during the observation Test and repeated: Provided, That, if period, the serial or subserial is unsat- not repeated, the Master Seed Virus isfactory. If unfavorable reactions shall be unsatisfactory. which are not attributable to the prod- (b) The cat safety test provided in uct occur, the test shall be declared a this paragraph shall be used when a se- No Test and may be repeated: Provided, rial of vaccine is tested for safety be- That, if the test is not repeated, the se- fore release. rial or subserial shall be declared un- (1) Each of two healthy cats shall be satisfactory. administered 10 cat doses by the method recommended on the label and the [39 FR 16857, May 10, 1974; 39 FR 20368, June 10, 1974] cats observed each day for 14 days. (2) If unfavorable reactions attrib- § 113.39 Cat safety tests. utable to the biological product occur during the observation period, the se- The safety tests provided in this sec- rial is unsatisfactory. If unfavorable tion shall be conducted when pre- reactions occur which are not attrib- scribed in a standard requirement or in utable to the product, the test shall be the filed Outline of Production for a bi- declared a No Test and repeated: Pro- ological product recommended for use vided, That, if not repeated, the serial in cats. shall be unsatisfactory. (a) The cat safety test provided in this paragraph shall be used when the [44 FR 58898, Oct. 12, 1979, as amended at 56 Master Seed Virus is tested for safety. FR 66784, Dec. 26, 1991] (1) The test animals shall be determined to be susceptible to the virus § 113.40 Dog safety tests. under test as follows: The safety tests provided in this sec- (i) Throat swabs shall be collected tion shall be conducted when pre- from each cat and individually tested scribed in a Standard Requirement or on susceptible cell cultures for the in the filed Outline of Production for a presence of the virus. Blood samples biological product recommended for shall also be drawn and individual use in dogs. Serials which are not serum samples tested for antibody to found to be satisfactory when tested the virus. pursuant to the procedures in this sec- (ii) The cats shall be considered sus- tion may not be released for shipment. ceptible if swabs are negative for virus (a) The dog safety test provided in isolation and the serums are free of this paragraph shall be used when the virus antibody at the 1:2 final dilution Master Seed Virus is tested for safety. in a 50 percent plaque reduction test or (1) The test animals shall be deter- other serum-neutralization test of mined to be susceptible to the virus equal sensitivity. under test by a method acceptable to (iii) When determining susceptibility the Animal and Plant Health Inspec- to a virus which does not lend itself to tion Service. the methods in paragraphs (a)(1)(i) and (2) Each of at least 10 susceptible (ii) of this section, a method acceptable dogs shall be administered a sample of to Animal and Plant Health Inspection the Master Seed Virus equivalent to Service shall be used. the amount of virus to be used in one (2) Each of at least 10 susceptible cats dog dose of the vaccine, by the method shall be administered a sample of the recommended on the label, and the dog Master Seed Virus equivalent to the shall be observed each day for 14 days. amount of virus to be used in one cat (3) If unfavorable reactions attrib- dose of the vaccine, by the method to utable to the virus occur in any of the be recommended on the label, and the dogs during the observation period, the cats observed each day for 14 days. Master Seed Virus is unsatisfactory. If (3) If unfavorable reactions attrib- unfavorable reactions occur which are utable to the virus occur in any of the not attributable to the Master Seed cats during the observation period, the Virus, the test shall be declared a No Master Seed Virus is unsatisfactory. If Test and may be repeated: Provided: unfavorable reactions occur which are That, if the test is not repeated, the not attributable to the Master Seed Master Seed Virus shall be considered Virus, the test shall be declared a No unsatisfactory.

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(b) The dog safety test provided in 0.02 ml of the material being tested and this paragraph shall be used when a se- observed each day for 21 days. rial of vaccine is tested for safety be- (b) If any of the mice show swelling fore release. in the injected footpad or if more than (1) Each of two healthy dogs shall be one becomes systemically abnormal, administered 10 dog doses by the meth- the material being tested is unsatisfac- od recommended on the label and the tory. dogs shall be observed each day for 14 days. [42 FR 6794, Feb. 4, 1977] (2) If unfavorable reactions attributable to the biological product occur § 113.43 Detection of chlamydial during the observation period, the se- agents. rial is unsatisfactory. If unfavorable The test for chlamydial agents pro- reactions occur which are not attrib- vided in this section shall be conducted utable to the biological product, the when such a test is prescribed in an ap- test shall be declared a No Test and plicable standard requirement or in a may be repeated: Provided, That, if the filed Outline of Production. test is not repeated, the serial shall be (a) The yolk sac of 6-day-old chicken considered unsatisfactory. embryos shall be injected. Three [60 FR 14358, Mar. 17, 1995] groups of 10 embryos shall be used se- quentially. § 113.41 Calf safety test. (1) The inoculum for each embryo in The calf safety test provided in this the first group shall consist of 0.5 ml of section shall be conducted when pre- a mixture of equal parts of the seed scribed in a Standard Requirement or virus with phosphate buffered saline in the filed Outline of Production for a that may contain Streptomycin, product. Vancomycin, Kanamycin, or a com- (a) Test procedure. Each of two calves bination thereof. Not more than 2 mg/ shall be injected with the equivalent of ml of each antibiotic shall be used. 10 doses of vaccine administered in the (2) On the 10th day postinoculation, manner recommended on the label and the yolk sac of viable embryos shall be observed each day for 21 days. harvested, pooled, homogenized as a 20 (b) Interpretation. If unfavorable reac- percent suspension in phosphate tions attributable to the product occur in either of the calves during the obser- buffered saline antibiotic diluent, and vation period, the serial or subserial is 0.5 ml of the mixture injected into the unsatisfactory. If unfavorable reac- second group of chicken embryos. This tions which are not attributable to the process shall be repeated for the injec- product occur, the test shall be de- tion of the third group of embryos clared a No Test and may be repeated: using the yolk sacs of viable embryos Provided, That, if the test is not re- from the second group. peated, the serial or subserial shall be (3) For each of the three passages, declared unsatisfactory. embryo deaths occurring within 48 hours of injection shall be disregarded, [39 FR 27428, July 29, 1974] except that if more than three such § 113.42 Detection of lymphocytic cho- deaths occur at any passage, that pas- riomeningitis contamination. sage shall be repeated. The test for detection of lymphocytic (b) If one or more embryo deaths choriomeningitis (LCM) virus provided occur at any passage after 48 hours in this section shall be conducted when postinjection, the yolk sacs from each such a test is prescribed in an applica- of the dead embryos shall be subcul- ble Standard Requirement or in a filed tured into 10 additional embryos. If one Outline of Production. Vaccine virus or more embryo deaths again occur due may be neutralized with specific anti- to chlamydial agents, the Master Seed serum when necessary. Virus is unsatisfactory for use to (a) Each of at least 10 mice obtained produce vaccine. from a source free of LCM shall be injected in the footpad of a hindfoot with [44 FR 58899, Oct. 12, 1979]

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§ 113.44 Swine safety test. § 113.46 Detection of cytopathogenic and/or hemadsorbing agents. The swine safety test provided in this section shall be conducted when pre- The tests for detection of scribed in a Standard Requirement or cytopathogenic and/or hemadsorbing in the filed Outline of Production for a agents provided in this section shall be product. conducted when prescribed in an appli- (a) Test procedure. (1) Inject each of cable Standard Requirement or in the two swine of the minimum age for filed Outline of Production for a prod- which the product is recommended uct. with the equivalent of two doses of bac- (a) Test for cytopathogenic agents. One terial vaccine or 10 doses of viral vac- or more monolayers that are at least 6 2 cine. cm and at least 7 days from the last (2) Administer vaccine in the manner subculture shall be tested as provided recommended on the label. in this paragraph. (1) Stain each monolayer with a suit- (3) Observe swine each day for 21 able cytological stain. days. (2) Examine the entire area of each (b) Interpretation. If unfavorable reac- stained monolayer for evidence of in- tions attributable to the product occur clusion bodies, abnormal number of in either of the swine during the obser- giant cells, or other cytopathology in- vation period, the serial or subserial is dicative of cell abnormalities attrib- unsatisfactory. If unfavorable reac- utable to an extraneous agent. tions which are not attributable to the (b) Test for hemadsorbing agents. One product occur, the test shall be de- or more monolayers that are at least 6 clared a No Test and may be repeated; cm 2 and at least 7 days from the last Provided, That, if the test is not re- subculture shall be tested as provided peated, the serial or subserial shall be in this paragraph. declared unsatisfactory. (1) Wash the monolayer with several [48 FR 33476, July 22, 1983] changes of phosphate buffered saline. (2) Add an appropriate volume of a 0.2 § 113.45 Sheep safety test. percent red blood cell suspension to The sheep safety test provided in this uniformly cover the surface of the section shall be conducted when pre- monolayer of cultured cells. Suspen- scribed in a Standard Requirement or sions of washed guinea pig and chicken in the filed Outline of Production for a red blood cells shall be used. These sus- product. pensions may be mixed before addition (a) Test procedure. (1) Inject each of to the monolayer or they may be added two sheep of the minimum age for separately to individual monolayers. ° which the product is recommended (3) Incubate the monolayer at 4 C for with the equivalent of two doses of bac- 30 minutes, wash with phosphate terial vaccine or 10 doses of viral vac- buffered saline, and examine for cine. hemadsorption. (2) Administer vaccine in the manner (4) If no hemadsorption is apparent, recommended on the label. repeat step (b)(2) of this section and incubate the monolayers at 20–25 °C for 30 (3) Observe sheep each day for 21 minutes, wash with phosphate buffered days. saline, and examine again for (b) Interpretation. If unfavorable reac- hemadsorption. If desired, separate tions attributable to the product occur monolayers may be used for each incu- in either of the sheep during the obser- bation temperature. vation period, the serial or subserial is (c) If specific cytopathology or unsatisfactory. If unfavorable reac- hemadsorption attributable to an ex- tions which are not attributable to the traneous agent is found, the material product occur, the test shall be de- under test is unsatisfactory and shall clared a No Test and may be repeated; not be used to prepare biological prod- Provided, That, if the test is not re- ucts. If an extraneous agent is sus- peated, the serial or subserial shall be pected because of cytopathology or declared unsatisfactory. hemadsorption and cannot be elimi- [48 FR 33476, July 22, 1983] nated as a possibility by additional

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testing, the material under test is un- (b) The antiviral fluorochrome-con- satisfactory. jugated antibodies to be used shall de- pend on the type of cells required to be [50 FR 441, Jan. 4, 1985, as amended at 58 FR tested for extraneous viruses as speci- 50252, Sept. 27, 1993] fied in an applicable Standard Require- § 113.47 Detection of extraneous vi- ment or in a filed Outline of Produc- ruses by the fluorescent antibody tion. Antiviral fluorochrome-con- technique. jugated antibodies specific for the extraneous viruses shall be applied to The test for detection of extraneous each respective type of cell in accord- viruses by the fluorescent antibody ance with the following list. Under cer- technique provided in this section shall tain circumstances, additional tests be conducted when prescribed in an ap- may need to be conducted, as deter- plicable Standard Requirement or in a mined by the Administrator. When a filed Outline of Production for a prod- specific antiviral fluorochrome-con- uct. jugated antibody is used in testing for (a) Monolayer cultures of cells the listed extraneous viruses specified (monolayers), at least 7 days after the in more than one cell type, it need only last subculturing, shall be processed be applied to the most susceptible cell and stained with the appropriate type. antiviral fluorochrome-conjugated (1) All cells shall be tested for: antibody as specified in paragraph (b) (i) Bovine virus diarrhea virus; of this section. (ii) Reovirus; and (1) Three groups of one or more (iii) Rabies virus. monolayers shall be required for each (2) Bovine, caprine, and ovine cells specific virus prescribed in paragraph shall, in addition, be tested for: (b) of this section. (i) Bluetongue virus; (i) At the time of the last subcul- (ii) Bovine adenoviruses; turing, one group of test monolayers (iii) Bovine parvovirus; and shall be inoculated with approximately (iv) Bovine respiratory syncytial 100–300 FAID50 of the specific virus virus. being tested for as positive controls. (3) Canine cells shall, in addition, be (ii) One group of monolayers shall be tested for: the ‘‘material under test.’’ (i) Canine coronavirus; (iii) One group of monolayers, that (ii) Canine distemper virus; and are of the same type of cells as the test (iii) Canine parvovirus. monolayers and that have been tested (4) Equine cells shall, in addition, be as prescribed in §§ 113.51 or 113.52 tested for: (whichever is applicable), shall be pre- (i) Equine herpesvirus; and pared as negative controls. (ii) Equine viral arteritis virus. (2) Each group of monolayers shall (5) Feline cells shall, in addition, be have a total area of at least 6 cm 2. tested for: (3) Positive control monolayers may (i) Feline infectious peritonitis virus; be fixed (processed so as to arrest and growth and assure attachment of the (ii) Feline panleukopenia virus. monolayer to the surface of the vessel (6) Porcine cells shall, in addition, be in which they are grown) before 7 days tested for: after subculturing if fluorescence is en- (i) Porcine adenovirus; hanced by doing so, Provided, That a (ii) Porcine parvovirus; monolayer of the material under test is (iii) transmissible gastroenteritis also fixed at the same time as the posi- virus; and tive control and a monolayer of the (iv) Porcine hemagglutinating en- material under test is also fixed at cephalitis virus. least seven days after subculturing. (7) Firms that do not have rabies Monolayers that are fixed before 7 days virus on premises either for research or after subculturing shall be stained at production purposes are exempt from the same time as the test monolayers having to produce positive rabies virus and negative controls fixed at least 7 control monolayers. Fixed positive ra- days after subculturing. bies virus control monolayers will be

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provided by the National Veterinary mary cells found unsatisfactory by any Services Laboratories. prescribed test shall not be used. A se- (c) After staining, each group of rial of biological product shall not be monolayers shall be examined for the released if produced from primary cells presence of specific fluorescence attrib- that are found unsatisfactory by any utable to the presence of extraneous vi- prescribed test. ruses. (a) Final container samples of com- (1) If the material under test shows pleted product or samples of the final any evidence of specific viral fluores- pool of harvested material or samples cence, it is unsatisfactory and may not be used; Provided, That, if specific fluo- of each subculture of cells used to pre- rescence attributable to the virus being pare the biological product shall be tested for is absent in the positive con- shown free of mycoplasma as pre- trol monolayers, the test is a No Test scribed in § 113.28. The sample for test- and may be repeated. ing shall consist of at least 75 cm 2 of (2) If the fluorescence of the actively growing cells or the equiva- monolayers inoculated with the spe- lent in harvest fluids; Provided, That cific virus as positive controls is equiv- all sources of cells in the batch of pri- ocal, or if the negative monolayers mary cells are represented. show equivocal fluorescence indicating (b) Final container samples of com- possible viral contamination, or both, pleted product or samples of the final the test shall be declared a No Test, pool of harvested material or samples and may be repeated; Provided, That, if of each subculture of cells used to pre- the test is not repeated, the material pare the biological product shall be under test shall be regarded as unsatis- shown free of bacteria and fungi as pre- factory for use in the production of bio- scribed in § 113.26 or § 113.27 (whichever logics. is applicable). [60 FR 24548, May 9, 1995] (c) A monolayer at least 75 cm 2 from each batch of primary cells or each INGREDIENT REQUIREMENTS subculture of primary cells used to pre- § 113.50 Ingredients of biological prod- pare a biological product shall be ucts. shown free of extraneous agents as pre- All ingredients used in a licensed bio- scribed in this paragraph. logical product shall meet accepted (1) The test monolayer shall be main- standards of purity and quality; shall tained using the medium (with addi- be sufficiently nontoxic so that the tives) and under conditions similar to amount present in the recommended those used to prepare biological prod- dose of the product shall not be toxic ucts. to the recipient; and in the combina- (i) Monolayers of avian origin shall tions used shall not denature the spe- be maintained for at least 14 days and cific substances in the product below shall be subcultured at least once dur- the minimum acceptable potency with- ing the maintenance period. All but the in the dating period when stored at the last subculture shall result in a new recommended temperature. monolayer of at least 75 cm 2. The last [38 FR 29889, Oct. 30, 1973] subculture shall meet the minimum area requirement specified in §§ 113.46 § 113.51 Requirements for primary and 113.47. cells used for production of biologics. (ii) Monolayers not of avian origin shall be maintained for at least 28 days Primary cells used to prepare bio- and shall be subcultured at least twice logical products shall be derived from during the maintenance period. All but normal tissue of healthy animals. the last subculture shall result in a When prescribed in an applicable new monolayer of at least 75 cm 2. The Standard Requirement or in the filed Outline of Production, each batch of last subculture shall meet the min- primary cells used to prepare a biologi- imum area requirement specified in cal product shall be tested as pre- §§ 113.46 and 113.47. scribed in this section. A batch of pri-

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(2) Monolayers shall be examined reg- production, or other observable fea- ularly throughout the required mainte- tures. nance period for evidence of the pres- (b) The MCS shall be shown to be of ence of cytopathogenic agents. If evi- the same species of origin as that re- dence of a cytopathogenic agent is ported in paragraph (a)(1) of this sec- found, the batch of primary cells is un- tion by the following method: satisfactory. (1) At least four monolayers with a (3) At the conclusion of the required total area of at least 6 cm 2 shall be maintenance period, monolayers shall grown to at least 80 percent be tested for: confluency. (i) Cytopathogenic and/or (2) The monolayers shall be removed hemadsorbing agents as prescribed in from their media, processed, stained, § 113.46; and examined. (ii) Extraneous viruses by the fluo- (i) At least two monolayers shall be rescent antibody technique as pre- stained with an antispecies scribed in § 113.47. fluorchrome-conjugated antibody unrelated to the species of origin of the [50 FR 442, Jan. 4, 1985, as amended at 60 FR MCS. 24549, May 9, 1995] (ii) At least two monolayers shall be stained with an antispecies § 113.52 Requirements for cell lines used for production of biologics. fluorochrome-conjugated antibody specific to the species of origin of the When prescribed in an applicable MCS. Standard Requirement or in a filed (iii) All monolayers shall be exam- Outline of Production each cell line ined for evidence of specific fluores- used to prepare a biological product cence. shall be tested as prescribed in this sec- (3) If specific fluorescence is not tion. A cell line found unsatisfactory found in the monolayers stained with by any prescribed test shall not be the conjugate specific to the species of used. A serial of biological product origin of the MCS, the cell line is un- shall not be released if produced from a satisfactory and shall not be used for cell line that is found unsatisfactory vaccine production. by any prescribed test. (4) If nonspecific fluorescence is (a) General requirements. (1) A com- found in the monolayers stained with plete record of the cell line shall be conjugate from an unrelated species of kept, such as, but not limited to, the origin or other results make the test source, passage history, and medium results equivocal, the procedure shall used for propagation. be repeated until either specific fluo- (2) A Master Cell Stock (MCS) shall rescence is found only in the be established at a specified passage monolayers stained with conjugate spe- level for each cell line. The passage cific to the species of origin of the MCS level and identity of the MCS and the and not in the control monolayers or highest passage level (MCS + n) in- specific fluorescence cannot be identi- tended for use in the preparation of a fied and the MCS is declared unsatis- biological product shall be specified in factory. the Outline of Production for the prod- (5) Alternate tests to determine the uct. species of origin of the MCS may be (3) Sufficient 1.0 ml or larger aliquots used if approved by APHIS. of MCS and MCS + n shall be prepared, (c) The MCS and either each subcul- kept in a frozen state, and made avail- ture of cells used to prepare a biologi- able to Animal and Plant Health In- cal product or the final pool of har- spection Service (APHIS) upon request vested material (with or without the for performing the tests prescribed in stabilizer) or final container samples of this section. completed product for each serial of (4) Each lot of cells shall be mon- such product shall be shown to be free itored for the characteristics deter- of mycoplasma as prescribed in § 113.28. mined to be normal for the cell line, The sample for testing shall consist of such as, but not limited to, micro- at least 75 cm 2 of actively growing scopic appearance, growth rate, acid cells or the equivalent, in harvest

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fluids. The cells shall represent all pernatant into equal aliquots and dis- sources of cells in the batch. pense 1.0 ml onto each of at least one (d) The MCS and either each subcul- monolayer (at least 75 cm 2) of: ture used to prepare a biological prod- (i) Vero (African green monkey kid- uct or the final pool of harvested mate- ney) cell line; rial for each serial of such product or (ii) Embryonic cells, neonatal cells, final container samples of completed or a cell line of the same species of ori- product for each serial of such product gin as the MCS if different than pro- shall be tested for bacteria and fungi as vided in paragraph (f)(1)(i) of this sec- prescribed in § 113.26 or § 113.27 (which- tion; ever is applicable). If bacteria or fungi (iii) Embryonic cells, neonatal cells, are found in the MCS, the MCS shall or a cell line of the species for which not be used. If bacteria or fungi are the vaccine is recommended if different found in a subculture, the subculture than provided in paragraph (f)(1)(ii) of shall not be used. this section; and (e) A monolayer at least 75 cm 2 from (iv) Embryonic cells, neonatal cells, each MCS shall be shown free of extra- or a cell line of bovine origin if not neous agents as prescribed in this para- specified in paragraphs (f)(1)(ii), and graph. (iii) of this section. (1) The test monolayer shall be main- (2) The monolayers of cells specified tained for at least 21 days using the in paragraphs (f)(1)(i), (ii), (iii), and (iv) medium (with additives) intended for of this section shall be maintained for growth and maintenance and under at least 14 days after inoculation with conditions similar to those used to pre- the aliquot of disrupted MCS. pare biological products. Monolayers shall be subcultured at (2) Cells shall be subcultured at least least once during the maintenance pe- two times during the maintenance period. All but the last subculture shall riod. All but the last subculture shall result in a new monolayer of at least 75 result in at least one new monolayer of cm 2. The last subculture shall meet at least 75 cm 2. The last subculture the minimum area requirement speci- shall meet the minimum area require- fied in §§ 113.46 and 113.47. ment specified in §§ 113.46 and 113.47 and (3) Monolayers shall be examined reg- paragraph (f) of this section. ularly throughout the 14-day mainte- (3) Monolayers shall be examined reg- nance period for evidence of the pres- ularly throughout the 21-day mainte- ence of cytopathogenic agents. If evi- nance period for evidence of the pres- dence of a cytopathogenic agent is ence of cytopathogenic agents. If evi- found, the MCS is unsatisfactory. dence of a cytopathogenic agent is (4) At the conclusion of the 14-day found, the MCS is unsatisfactory. maintenance period, monolayers shall (4) At the conclusion of the 21-day be tested for: maintenance period, monolayers shall (i) Cytopathogenic and/or be tested for: hemadsorbing agents as prescribed in (i) Cytopathogenic and/or § 113.46; and hemadsorbing agents as prescribed in (ii) Extraneous viruses by the fluo- § 113.46; and rescent antibody technique as pre- (ii) Extraneous agents by the fluores- scribed in § 113.47. cent antibody technique as prescribed (g) The karyology of cells lines used in § 113.47. in the production of biologics shall be (f) At the conclusion of the 21-day examined as follows. A minimum of 50 maintenance period provided in para- mitotic cells shall be examined at both graph (e) of this section, at least one the MCS and MCS + n. The modal num- monolayer of at least 75 cm 2 shall also ber in the MCS + n shall not exceed be shown free of extraneous agents as plus or minus 15 percent of the modal prescribed in this paragraph. number of the MCS. Any marker chro- (1) Alternately freeze and thaw the mosomes present in the MCS shall per- monolayer(s) three times. Centrifuge sist at the MCS + n. If the modal num- the disrupted cells at no greater than ber exceeds the limits and/or the mark- 2,000 × g for no more than 15 minutes to er chromosomes do not persist remove cellular debris. Divide the su- (through the MCS + n passage level),

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the cell line shall not be used for vac- cell line and of primary cells or a cell cine production. line of the same species of origin as the (h) If direct or indirect evidence ex- ingredient shall be used in the test. ists that a cell line which is intended Cell lines used shall have been found for use in the preparation of a vaccine satisfactory when tested as prescribed may induce malignancies in the species in § 113.52 and primary cells used shall for which the product is intended, that have been found satisfactory when cell line shall be tested for tested as prescribed in § 113.51. tumorigenicity/oncogenicity by a (2) At least 3.75 ml or 15 percent of method acceptable to APHIS. the ingredient shall be used in the [50 FR 442, Jan. 4, 1985; 50 FR 3316, Jan. 24, growth medium for the preparation of 1985, as amended at 56 FR 66784, Dec. 26, 1991; at least 75 cm 2 test monolayers. The 60 FR 24549, May 9, 1995] ingredient shall also be used in the growth medium when monolayers are § 113.53 Requirements for ingredients subcultured. If the ingredient being of animal origin used for produc- tested is cytotoxic when tested in this tion of biologics. manner, other procedures may be used Each lot of ingredient of animal ori- if approved by APHIS. gin which is not subjected to heat ster- (3) The test monolayers shall be ilization or other sterilization methods maintained for at least 21 days. acceptable to Animal and Plant Health (4) Cells shall be subcultured at least Inspection Service (APHIS), such as, two times during the maintenance pe- but not limited to serum and albumin, riod. All but the last subculture shall used to prepare a biological product result in at least one new monolayer of shall be tested as prescribed in this sec- at least 75 cm 2. The last subculture tion by the licensee or a laboratory ac- shall meet the minimum area require- ceptable to VS. Results of all tests ments specified in §§ 113.46 and 113.47. shall be recorded by the testing labora- (5) Monolayers shall be examined reg- tory and made a part of the licensee’s ularly throughout the 21-day mainte- records. A lot of ingredient found un- nance period for evidence of satisfactory by any prescribed test cytopathogenic agents. If evidence of a shall not be used to prepare a biologi- cytopathogenic agent is found, the in- cal product. A serial of biological prod- gredient is unsatisfactory. uct shall not be released if produced using an ingredient that is found un- (6) At the conclusion of the 21-day satisfactory by any prescribed test. maintenance period, monolayers shall (a) Samples of each lot of ingredient be tested for: of animal origin which is not subjected (i) Cytopathogenic and/or to heat sterilization, used to prepare a hemadsorbing agents as prescribed in biological product shall be shown free § 113.46; and of mycoplasma by the method pre- (ii) Extraneous viruses by the fluo- scribed in § 113.28. rescent antibody technique as pre- (b) Samples of each lot of ingredient scribed in § 113.47. or animal origin which is not subjected (d) Each lot of porcine trypsin which to heat sterilization of other steriliza- has not been treated to inactivate portion methods acceptable to APHIS used cine parvovirus (PPV) in a manner ac- to prepare a biological product shall be ceptable to VS shall be tested for PPV shown free of bacteria and fungi as pre- as prescribed in this paragraph. scribed in § 113.26. (1) Not less than 5.0 grams of trypsin (c) Samples of each lot of ingredient shall be dissolved in a volume of suit- of animal origin, except porcine able diluent sufficient to fill a cen- trypsin, which is not subjected to heat trifuge angle head. After centrifuging sterilization or other viricidal proce- for 1 hour at 80,000 × g, the pellet mate- dure acceptable to APHIS used in the rial shall be reconstituted in distilled preparation of biological products shall water and inoculated into a flask con- be tested as prescribed in this para- taining 75 cm 2 of a 30 to 50 percent con- graph; fluent monolayer culture of primary (1) Monolayers at least 75 cm 2 of porcine cells or a porcine cell line of Vero (African green monkey kidney) proven equal PPV susceptibility. An

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additional flask of cells shall be held as prescribed in this section. A MSV a negative control. found unsatisfactory by any prescribed (2) The test and control monolayers test shall not be used. A serial of bio- shall be maintained for at least 14 days logical product shall not be released if and subcultured at least once during produced from a MSV that is found un- the maintenance period. satisfactory by any prescribed test. (3) At the end of the 14-day mainte- (a) At least a 1.0 ml aliquot per cell nance period, and 4 to 7 days after the culture of MSV shall be dispensed onto last subculturing, monolayers shall be monolayers (at least 75 cm 2 in area) of: tested for the presence of porcine parvovirus by the fluorescent antibody (1) Vero (African green monkey kid- technique as prescribed in § 113.47(c). ney) cell line; (e) A sample of serum from each (2) Embryonic cells, neonatal cells, donor horse used to produce a lot of or a cell line of the species for which equine serum used in the preparation the vaccine is recommended; and of biological products recommended for (3) Embryonic cells, neonatal cells, use in horses shall be tested at a lab- or a cell line of the species of cells in oratory approved by Animal and Plant which the MSV is presently being prop- Health Inspection Service using the agated if different than prescribed in Coggins test for equine infectious ane- paragraphs (a)(1) and (a)(2) of this sec- mia antibodies. If antibodies to equine tion. Cell lines used shall have been infectious anemia are found, the lot of found satisfactory when tested as pre- serum is unsatisfactory. scribed in § 113.52 and primary cells [50 FR 442, Jan. 4, 1985; 50 FR 3316, Jan. 24, used shall have been found satisfactory 1985, as amended at 56 FR 66784, Dec. 26, 1991; when tested as prescribed in § 113.51. If 60 FR 24549, May 9, 1995] the MSV is cytopathic for or causes § 113.54 Sterile diluent. hemadsorption in the cells in which it is to be tested, the MSV shall be neu- Sterile Diluent shall be supplied in a tralized with monospecific antiserum final container by the licensee when supplied or approved by Animal and such diluent is required for rehydration Plant Health Inspection Service or dilution of the vaccine. (a) Sterile Diluent may be distilled (APHIS) or counteracted by a method or deionized water or it may be a spe- approved by APHIS. cial liquid solution formulated in ac- (b) At least one monolayer of each cordance with an acceptable outline on cell type used in the test shall be main- file with Animal and Plant Health In- tained as an uninoculated control. spection Service. (c) Each monolayer shall be main- (b) Each quantity prepared at one tained at least 14 days. time in a single container and bottled (d) Cells shall be subcultured at least into final containers shall be des- once during the maintenance period. ignated as a serial. Each serial shall be All but the last subculture shall result given a number which shall be used in in at least one new monolayer at least records, test reports, and on the final 75 cm 2. The last subculture shall meet container label. the minimum area requirement speci- (c) Final container samples from fied in §§ 113.46 and 113.47. each serial shall be tested for bacteria and fungi in accordance with the test (e) Monolayers shall be examined provided in § 113.26. Any serial found to regularly throughout the 14-day main- be unsatisfactory shall not be released. tenance period for evidence of cytopathogenic agents. If evidence of a [39 FR 27428, July 29, 1974, as amended at 56 cytopathogenic agent is found, the FR 66784, Dec. 26, 1991] MSV is unsatisfactory. § 113.55 Detection of extraneous (f) At the conclusion of the 14-day agents in Master Seed Virus. maintenance period, monolayers shall Unless otherwise prescribed in a be tested for: Standard Requirement or in a filed (1) Cytopathogenic and/or Outline of Production, each Master hemadsorbing agents as prescribed in Seed Virus (MSV) shall be tested as § 113.46;

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(2) Extraneous agents by the fluores- (c) Identity test. At least one of the cent antibody technique as prescribed identity tests provided in this para- in § 113.47. graph shall be conducted for the Mas- ter Seed Bacteria and final container [50 FR 444, Jan. 4, 1985, as amended at 56 FR 66784, Dec. 26, 1991] samples from each serial or first subserial of completed biological product. LIVE BACTERIAL VACCINES A known positive control (reference) provided or approved by Animal and § 113.64 General requirements for live Plant Health Inspection Service shall bacterial vaccines. be included in such tests. When prescribed in an applicable (1) Fluorescent antibody test. The di- Standard Requirement or in the filed rect fluorescent antibody staining Outline of Production, a live bacterial technique shall be conducted using vaccine shall meet the requirements in suitable smears of the vaccine bac- this section. teria. Fluorescence typical for the bac- (a) Purity test. Final container sam- teria concerned shall be demonstrated. ples of completed product from each se- Fluorescence shall not occur in control rial and subserial, and samples of each smears treated with specific antiserum. lot of Master Seed Bacteria shall be (2) Tube agglutination test. A tube ag- tested for the presence of extraneous glutination test shall be conducted viable bacteria and fungi in accordance with a suitable suspension of the vac- with the test provided in § 113.27(b). cine bacteria using the constant anti- (b) Safety tests. (1) Samples of com- gen decreasing serum method with spe- pleted product from each serial or first cific antiserum. Agglutination typical subserial and samples of each lot of for the bacteria shall be demonstrated. Master Seed Bacteria shall be tested Agglutination shall not occur with for safety in young adult mice in ac- negative serum used as a control in cordance with the test provided in this test. § 113.33(b) unless: (3) Slide agglutination test. The rapid (i) The bacteria or agents in the vac- plate (slide) agglutination test shall be cine are inherently lethal for mice. conducted with suitable suspensions of (ii) The vaccine is recommended for the vaccine bacteria using the hanging poultry. drop, slide or plate method, with spe- (2) Samples of completed product cific antiserum. Agglutination typical from each serial or first subserial of for the bacteria shall be demonstrated live bacterial vaccine shall be tested by microscopic or macroscopic obser- for safety in one of the species for vation. Agglutination shall not occur which the product is recommended as with negative serum used as a control follows: in this test. (i) Live bacterial vaccine rec- (4) Characterization tests. Applicable ommended for use in dogs shall be test- biochemical and cultural characteris- ed as provided in § 113.40, except that tics shall be demonstrated as specified dogs shall be injected with the equiva- in the filed Outline of Production. lent of two doses of vaccine adminis- (d) Ingredient requirements. Ingredi- tered as recommended on the label. ents used for the growth and prepara- (ii) Live bacterial vaccine rection of Master Seed Bacteria and of ommended for use in cattle shall be live bacterial vaccine shall meet the tested as provided in § 113.41, except requirements provided in § 113.50. Ingre- that calves shall be injected with the dients of animal origin shall meet the equivalent of two doses of vaccine ad- applicable requirements provided in ministered as recommended on the § 113.53. label. (e) Moisture content. The maximum (iii) Live bacterial vaccine rec- percent moisture in desiccated vac- ommended for use in sheep shall be cines shall be stated in the filed Out- tested as provided in § 113.45. line of Production and shall be estab- (iv) Live bacterial vaccine rec- lished by the licensee as follows: ommended for use in swine shall be (1) Prelicensing. Data obtained by con- tested as provided in § 113.44. ducting accelerated stability tests and

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bacterial counts shall be acceptable on rays pass through the plate at a 45 a temporary basis. °angle. (2) Licensed products. Data shall be (ii) If the vaccine contains more than obtained by determining the percent 5 percent rough colonies or more than moisture and bacterial count at release 15 percent total undesirable colonies, and expiration on a minimum of 10 con- the serial or subserial is unsatisfac- secutive released serials. tory. If organisms or growth not char- (3) Final container samples of com- acteristic of Brucella abortus are found, pleted product from each serial and the serial or subserial is unsatisfac- subserial must be tested for moisture tory. The test may be repeated one content in accordance with the test time using double the number of sam- provided in § 113.29. ples: Provided, That, if the test is not [48 FR 33476, July 22, 1983, as amended at 54 repeated, the serial or subserial is un- FR 19352, May 5, 1989; 56 FR 66784, Dec. 26, satisfactory. 1991; 68 FR 57608, Oct. 6, 2003] (b) Bacterial count requirements for reduced dose vaccine. Each serial and each § 113.65 Brucella Abortus Vaccine. subserial shall be tested for potency. Brucella Abortus Vaccine shall be (1) Two final container vials of com- prepared as a desiccated live culture pleted product shall be tested for the bacterial vaccine from smooth colonial number of viable organisms per dose of forms of the Brucella abortus organism, rehydrated vaccine. A bacterial count identified as Strain 19. Each serial and per vial shall be made on tryptose agar subserial shall be tested for purity, po- plates from suitable dilutions using 1 tency, and moisture content. A serial percent peptone as a diluent. The in- or subserial found unsatisfactory by a oculated media shall be incubated at 35 prescribed test shall not be released. to 37 °C for 96 hours. (a) Purity tests. Each serial and sub- (2) If the average count of the two serial shall be tested for purity as pro- final container samples of freshly pre- vided in this paragraph. pared vaccine contains less than 3.0 or (1) Macroscopic and microscopic ex- more than 10.0 billion organisms per amination shall be made on bulk sam- dose, the serial or subserial is unsatis- ples from production containers. If or- factory. ganisms not typical of Brucella abortus (3) If the average count on the initial organisms are evident, the serial or test is less than the minimum or great- subserial is unsatisfactory. er than the maximum required in para- (2) Two final container vials of com- graph (b)(2) of this section, the serial pleted product shall be tested by or subserial may be retested one time inoculating one tube of Dextrose using four additional final container Andrades broth with gas tube and one vials. The average count of the retest tube of thioglycollate broth from each is determined. If the average count of vial. The inoculated media shall be in- the four vials retested is less than the cubated at 35 to 37 °C for 96 hours. If required minimum or greater than the growth not typical of Brucella abortus required maximum, the serial or sub- organisms is evident, the serial or subserial is unsatisfactory. If the average serial is unsatisfactory. count of the four vials retested is with- (3) Bacterial dissociation test. Final in the required limits described in container samples of completed prod- paragraph (b)(2) of this section, the fol- uct from each serial and subserial shall lowing shall apply: be tested for bacterial dissociation. (i) If the average count obtained in Smooth colonies are the desired form. the initial test is less than one-third or Rough colonies are undesirable ter- more than three times the average minal dissociation forms. Intermediate count obtained on the retest, the aver- and intermediate-to-rough are also un- age count of the initial test shall be desirable. considered the result of test system (i) The sample container shall be re- error and the serial or subserial is sat- hydrated and streaked on one potato isfactory. agar plate in such a manner as to (ii) If the average count obtained in produce confluent colonies. Artificial the initial test is one-third or more reflected light shall be used so that the than the average retest count or three

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times or less than the average retest first through the fifth passage from the count, a new average count shall be de- Master Seed. termined from the counts of all six (a) The Master Seed shall meet the vials. If the new average is less than applicable general requirements pre- the minimum or greater than the max- scribed in § 113.64 and the requirements imum required in paragraph (b)(2) of in this section. this section, the serial or subserial is (b) Each lot of Master Seed shall be unsatisfactory. tested for immunogenicity as follows: (4) If tested at any time within the (1) Forty-two susceptible guinea pigs expiration period, each dose of re- from the same source each weighing 400 hydrated vaccine must contain at least to 500 grams, shall be used as test ani- 3.0 billion viable organisms per dose. mals (30 vaccinates and 12 controls). (c) Bacterial count requirements for (2) An arithmetic mean spore count standard vaccine. Each serial and sub- of vaccine produced from the highest serial shall be tested for potency. passage of the Master Seed shall be es- (1) Two final container samples shall tablished before the immunogenicity be tested for the number of viable orga- test is conducted. The guinea pigs used nisms per milliliter of rehydrated vac- as vaccinates shall be injected as rec- cine. One bacterial count per vial shall ommended on the label with a pre- be made on tryptose agar plates from determined number of vaccine spores. suitable dilutions using 1 percent pep- To confirm the dosage, five replicate tone as a diluent. The inoculated media spore counts shall be conducted on a shall be incubated at 35 to 37 °C for 96 hours. sample of the vaccine dilution used. (2) If the average count of the two (3) Fourteen to fifteen days final container samples of freshly pre- postvaccination the vaccinates and pared vaccine does not contain at least controls shall each be challenged with 10 billion viable organisms per milli- not less than 4,500 guinea pig LD50 of a liter, the serial or subserial is unsatis- virulent suspension of Bacillus factory. anthracis furnished or approved by Ani- (3) If the initial bacterial count is mal and Plant Health Inspection Serv- less than 10 billion organisms per milli- ice and observed for 10 days. liter, the serial or subserial may be re- (4) If at least 10 of the 12 controls do tested one time using four samples. If not die from Bacillus anthracis within the average count of the four vials re- the 10-day postchallenge observation tested is less than the required min- period the test is invalid and may be imum, the serial or subserial is unsat- repeated. isfactory. (5) If at least 27 of 30 of the vac- (4) If tested at any time within the cinates do not survive the 10-day expiration period, each milliliter of re- postchallenge observation period, the hydrated vaccine does not contain at Master Seed is unsatisfactory. least 5 billion viable organisms per (6) An Outline of Production change milliliter, the serial or subserial is un- shall be made before authority for use satisfactory. of a new lot of Master Seed shall be granted by Animal and Plant Health [39 FR 16857, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974, and amended at 40 FR Inspection Service. 758, Jan. 3, 1975; 50 FR 23794, Jan. 6, 1985] (c) Test Requirements for Release. Each serial and subserial shall meet the ap- § 113.66 Anthrax Spore Vaccine—Non- plicable general requirements pre- encapsulated. scribed in 9 CFR 113.64 and the require- Anthrax Spore Vaccine—Nonencap- ments in this paragraph. Any serial or sulated shall be a live spore suspension subserial found unsatisfactory by a prepared from nonencapsulated prescribed test shall not be released. variants of Bacillus anthracis. Only (1) Safety test. Samples of completed Master Seed which has been estab- product from each serial or first sub- lished as pure, safe, and immunogenic serial shall be tested for safety in sheep shall be used for production. All serials or goats by the methods described in 9 of vaccine shall be prepared from the CFR 113.45(a).

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(2) Spore Count Requirements. Final swine shall be held separately from the container samples of completed prod- vaccinates. To confirm the dosage cal- uct shall be tested for spore count. culation, an arithmetic mean count Samples shall be diluted in tenfold shall be established by conducting five steps. Each dilution expected to yield replicate titrations on a sample of the 30 to 300 colonies per plate shall be bacterial vaccine dilution used. Only plated in triplicate on tryptose agar, plates containing between 30 and 300 inverted, and incubated at 35 to 70 °C colonies shall be considered in a valid for 24 hours to 28 hours. Each plate test. having uniformly distributed colonies (3) The vaccinates and controls shall shall be counted and an average count be examined and their average body determined. To be eligible for release, temperature determined prior to chal- each serial and each subserial shall lenge. Fourteen to twenty-one days have a spore count sufficiently greater postvaccination, the vaccinates and than that of the vaccine used in the controls shall be challenged with a vir- immunogenicity test to assure that ulent Erysipelothrix rhusiopathiae cul- when tested at any time within the ex- ture and observed for 7 days. The chal- piration period, each serial and sub- lenge culture and instructions for prep- serial shall have a spore count of at aration and use shall be obtained from least twice that used in the Animal and Plant Health Inspection immunogenicity test but not less than Service. 2,000,000 spores per dose. (4) A satisfactory challenge shall be [50 FR 23794, June 6, 1985, as amended at 56 evidenced in the controls by a high FR 66784, Dec. 26, 1991; 72 FR 72564, Dec. 21, body temperature or clinical signs in- 2007] cluding, but not limited to acute illness with hyperemia of the abdomen § 113.67 Erysipelothrix Rhusiopathiae and ears, possibly terminating in sud- Vaccine. den death; moribundity, with or with- Erysipelothrix Rhusiopathiae Vac- out metastatic skin lesions; depression cine shall be prepared as a desiccated with anorexia, stiffness, and/or joint live culture of an avirulent or modified involvement; or any combination of strain of Erysipelothrix rhusiopathiae. these symptoms and lesions. Only Master Seed which has been es- (5) If at least 80 percent of the con- tablished as pure, safe, and trols do not show characteristic signs immunogenic shall be used for vaccine during the observation period includ- production. ing, but not limited to a body tempera- (a) The Master Seed shall meet the ture of 105.6 °F or higher on at least 2 applicable requirements prescribed in consecutive days, the test shall be con- § 113.64 and the requirements in this sidered a No Test: Provided, That con- section. trol pigs which meet the criteria re- (b) Each lot of Master Seed used for quirements for susceptibility except vaccine production shall be tested for for high body temperature shall be con- immunogenicity. The selected bac- sidered susceptible if sacrificed and or- terial count from the lot of Master ganisms identified as Erysipelothrix Seed shall be established as follows: rhusiopathiae can be isolated from the (1) Thirty Erysipelothrix rhusiopathiae blood, spleen, or other organs. susceptible swine shall be used as test (6) To demonstrate immunity after animals (20 vaccinates and 10 controls) challenge, the vaccinates shall remain for each route of administration rec- free of clinical signs and the body tem- ommended on the label. perature shall not exceed 104.6 °F on 2 (2) An arithmetic mean count of the or more consecutive days. If at least 90 colony forming units from vaccine pro- percent of the vaccinates do not reduced from the highest passage of the main free from clinical signs and high Master Seed shall be established before body temperature throughout the ob- the immunogenicity test is conducted. servation period, the Master Seed is The 20 swine to be used as vaccinates unsatisfactory. shall be injected as recommended on (7) An Outline of Production change the label with a predetermined quan- shall be made before authority for use tity of vaccine bacteria. The 10 control of a new Master Seed shall be granted

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by Animal and Plant Health Inspection (1) Fifteen Pasteurella haemolytica Service. susceptible calves shall be used as test (c) Test requirements for release. Each animals (10 vaccinates and 5 controls) serial and subserial shall meet the ap- for each route of administration rec- plicable requirements in § 113.64 and the ommended on the label. requirements in this paragraph. Any (2) An arithmetic mean count of the serial or subserial found unsatisfactory colony forming units from vaccine pro- by a prescribed test shall not be reduced from the highest passage of the leased. Master Seed shall be established before (1) Safety test. Samples of completed the immunogenicity test is conducted. product from each serial or first sub- The 10 calves to be used as vaccinates serial shall be tested for safety in shall be injected as recommended on young adult mice as prescribed in the label with a predetermined quan- § 113.33(b) and in swine as prescribed in tity of vaccine bacteria. The five con- § 113.44. trol calves shall be held separately (2) Bacterial count requirements. Final from the vaccinates. To confirm the container samples of completed prod- dosage calculation, five replicate titra- uct from each serial and each subserial tions on a sample of the bacterial vac- shall be tested for bacterial count cine used. Only plates containing be- using the method used in paragraph tween 30 and 300 colonies shall be con- (b)(2) of this section. Two replicate ti- sidered a valid test. trations shall be conducted on each (3) The vaccinates and controls shall sample. To be eligible for release, each be examined and their average body serial and subserial shall have a bac- temperature determined prior to chal- terial count sufficiently greater than lenge. Fourteen to twenty-one days that of the vaccine used in the post vaccination, the vaccinates and immunogenicity test to assure that, controls shall each be challenged by when tested at any time within the ex- the respiratory route with a (virulent) piration period, each serial and sub- pneumonia producing Pasteurella serial shall have a bacterial count two haemolytica culture and observed for 4 times greater than that used in such to 7 days. The challenge culture and in- immunogenicity test. structions for preparation for use shall [50 FR 23795, June 6, 1985, as amended at 56 be furnished or approved by the Animal FR 66784, Dec. 26, 1991; 72 FR 72564, Dec. 21, and Plant Health Inspection Service. 2007] (4) A satisfactory challenge shall be evidenced in the controls by progres- § 113.68 Pasteurella Haemolytica Vac- sion of clinical signs consistent with cine, Bovine. respiratory system infection following Pasteurella Haemolytica Vaccine, challenge, including but not limited to Bovine, shall be prepared as a des- lacrimation, mucoid nasal exudates, iccated live culture bacterial vaccine expiratory dyspnea, tachypnea, pul- of an avirulent or modified strain of monary rales, and cough possibly ter- Pasteurella haemolytica, identified as minating in death; moribundity, de- serotype 1. Only Master Seed which has pression with anorexia, diarrhea with been established as pure, safe, and substantial weight loss; or any com- immunogenic shall be used for vaccine bination of these symptoms. production. All serials of vaccine shall (5) Lung lesion response to challenge be prepared from the first through the will be assessed in all calves. Lung le- fifth passage from the Master Seed. sions will be assessed at necropsy in (a) The Master Seed shall meet the calves that succumb to challenge. Sur- applicable general requirements pre- viving calves will be euthanized on day scribed in § 113.64 and the requirements 4 to 7 following challenge and lung le- in this section. sions assessed at necropsy. Lung lesion (b) Each lot of Master Seed used for scores will be used in the assessment of vaccine production shall be tested for the response to challenge exposure. If a immunogenicity. The immunogenicity significant difference in lung lesion of a selected bacterial count from the scores cannot be demonstrated between lot of Master Seed shall be established vaccinates and controls using a scoring as follows: system approved by the Animal and

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Plant Health Inspection Service, the (a) The Master Seed shall meet the Master Seed is unsatisfactory. applicable general requirements pre- (6) An Outline of Production change scribed in § 113.64 and the requirements must be made before authority for use in this section. of a new lot of Master Seed is granted (b) Each lot of Master Seed used for by the Animal and Plant Health In- vaccine production shall be tested for spection Service. immunogenicity. The immunogenicity (c) Test requirements for release. Each of a selected bacterial count from the serial and subserial shall meet the ap- lot of Master Seed shall be established plicable general requirements pre- as follows: scribed in §§ 113.8 and 113.64 and the re- (1) Fifteen Pasteurella multocida sus- quirements in this paragraph. Any se- ceptible calves shall be used as test rial or subserial found unsatisfactory animals (10 vaccinates and 5 controls) by a prescribed test shall not be re- for each route of administration rec- leased. ommended on the label. (1) Safety test. Samples of completed (2) An arithmetic mean count of the product from each serial or first sub- colony forming units from vaccine pro- serial shall be tested for safety in duced from the highest passage of the calves as provided in §§ 113.41(a) and Master Seed shall be established before 113.41(b) except, that the equivalent of the immunogenicity test is conducted. two doses of vaccine shall be used and The 10 calves to be used as vaccinates administered in the manner rec- shall be injected as recommended on ommended on the label. the label with a predetermined quan- (2) Bacterial count requirements. Final tity of vaccine bacteria. The five con- container samples of completed prod- trol calves shall be held separately uct shall be tested for bacterial count from the vaccinates. To confirm the using the method used in paragraph dosage calculation, arithmetic mean (b)(2) of this section. Two replicate ti- count shall be established by con- trations shall be conducted on each se- ducting five replicate titrations on a rial and subserial. Each sample shall be sample of the bacterial vaccine used. rehydrated with accompanying sterile Only plates containing between 30 and diluent to the volume indicated on the 300 colonies shall be considered a valid label. To be eligible for release, each test. serial and subserial shall have a bac- (3) The vaccinates and controls shall terial count sufficiently greater than be examined and their average body that of the vaccine used in the temperature determined prior to chal- immunogenicity test to assure that, lenge. Fourteen to twenty-one days when tested at any time within the ex- post vaccination, the vaccinates and piration period, each serial and sub- controls shall each be challenged by serial shall have a bacterial count at the respiratory route with a (virulent) least two times greater than that used pneumonia producing Pasteurella in the immunogenicity test. multocida culture and observed for 4 to [55 FR 35559, Aug. 31, 1990, as amended at 72 10 days. The challenge culture and in- FR 72564, Dec. 21, 2007] structions for preparation for use shall be furnished or approved by the Animal § 113.69 Pasteurella Multocida Vac- and Plant Health Inspection Service. cine, Bovine. (4) A satisfactory challenge shall be Pasteurella Multocida Vaccine, Bo- evidenced in the controls by progres- vine, shall be prepared as a desiccated sion of clinical signs consistent with live culture bacterial vaccine of an respiratory system infection following avirulent or modified strain of challenge, including but not limited to Pasteurella multocida, of bovine origin. acute illness with higher body tem- Only Master Seed which has been es- perature and respiration rate, tablished as pure, safe, and lacrimation, mucoid nasal exudate, ex- immunogenic shall be used for vaccine piratory dyspnea, tachypnea, pul- production. All serials of vaccine shall monary rales, and cough, possibly ter- be prepared from the first through the minating in death; moribundity, de- fifth passage from the Master Seed. pression with anorexia; diarrhea with

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substantial weight loss; or any com- times greater than that used in the bination of these symptoms. immunogenicity test. (5) Lung lesion response to challenge [55 FR 35560, Aug. 31, 1990, as amended at 72 will be assessed in all calves. Lung le- FR 72564, Dec. 21, 2007] sions will be assessed at necropsy in calves that succumb to challenge. Sur- § 113.70 Pasteurella Multocida Vac- viving calves will be euthanized on day cine, Avian Isolate. 4 to 10 following challenge and lung le- Pasteurella Multocida Vaccine, sions assessed at necropsy. Lung lesion Avian Isolate, shall be prepared as a scores will be used in the assessment of desiccated live culture of an avirulent the response to challenge exposure. If a or modified strain of Pasteurella significant difference in lung lesion multocida. Only Master Seed which has scores cannot be demonstrated between been established as pure, safe, and immunogenic shall be used for vaccine vaccinates and controls using a scoring production. system approved by the Animal and (a) The Master Seed shall meet the Plant Health Inspection Service, the applicable general requirements pre- Master Seed is unsatisfactory. scribed in § 113.64 and the requirements (6) An Outline of Production change in this section. must be made before authority for use (b) Each lot of Master Seed used for of a new lot of Master Seed is granted vaccine production shall be tested for by the Animal and Plant Health In- immunogenicity in each species and for spection Service. each serotype for which the Master (c) Test requirements for release. Each Seed is claimed to give protection. serial and subserial shall meet the ap- (1) Thirty Pasteurella multocida sus- plicable general requirements pre- ceptible birds shall be used as test ani- scribed in §§ 113.8 and 113.64 and the re- mals (20 vaccinates and 10 controls) for quirements in this paragraph. Any se- each bird species, route of administra- rial or subserial found unsatisfactory tion, and serotype for which protection by a prescribed test shall not be re- is claimed on the label. leased. (2) An arithmetic mean count of colony forming units from vaccine pro- (1) Safety Test. Samples of completed duced from the highest passage of Mas- product from each serial or first sub- ter Seed shall be established before the serial shall be tested for safety in immunogenicity test is conducted. The calves as provided in §§ 113.41(a) and 20 birds to be used as vaccinates shall 113.41(b), except that the equivalent of be inoculated, as recommended on the two doses of vaccine shall be used and label with a predetermined quantity of administered in the manner rec- vaccine bacteria. The 10 control birds ommended on the label. shall be held separately from the vac- (2) Bacterial count requirements. Final cinates. To confirm the dosage calcula- container samples of completed prod- tion, an arithmetic mean count shall uct shall be tested for bacterial count be established by conducting five rep- using the method used in paragraph licate titrations on a sample of the (b)(2) of this section. Two replicate ti- bacterial vaccine used. Only plates trations shall be conducted on each se- containing between 30 and 300 colonies rial and subserial. Each sample shall be shall be considered in a valid test. rehydrated with accompanying sterile (3) Not less than 14 days after vac- diluent to the volume indicated on the cination, each of 20 vaccinates and label. To be eligible for release, each each of 10 unvaccinated controls shall serial and subserial shall have a bac- be challenged intramuscularly or by terial count sufficiently greater than other methods acceptable to the Ani- that of the vaccine used in the mal and Plant Health Inspection Serv- ice with a virulent Pasteurella multocida immunogenicity test count per dose es- strain, for which protection is claimed, tablished to assure that, when tested and observed daily for a 14 day post- at any time within the expiration pe- challenge period. riod, each serial and subserial shall (4) Eight or more of the unvaccinated have a bacterial count at least two controls must die for the test to be

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valid. If at least 16 of 20 of the vac- § 113.71 Chlamydia Psittaci Vaccine cinates do not survive the 14-day (Feline Pneumonitis), Live postchallenge period, the Master Seed Chlamydia. is unsatisfactory at the selected bac- Chlamydia Psittaci Vaccine (Feline terial count. Pneumonitis), Live Chlamydia, shall be (c) Test requirements for release. Each prepared from chlamydia-bearing cell serial and subserial shall meet the ap- culture fluids or embryonated chicken plicable requirements in §§ 113.8 and eggs. Only Master Seed which has been 113.64 and the requirements in this established as pure, safe, and paragraph. Any serial or subserial immunogenic shall be used for vaccine found unsatisfactory by a prescribed production. All serials of vaccine shall test shall not be released. be prepared from the first through the (1) Safety test. Samples of completed fifth passage from the Master Seed. product from each serial or first sub- (a) The Master Seed shall meet the serial shall be tested for safety. applicable requirements prescribed in § 113.300 and the requirements in this (i) Ten birds of a species for which section. Master Seed propagated in the vaccine is recommended shall be chicken embryos shall be tested for given the equivalent of 10 doses each of pathogens by the chicken embryo test the vaccine and observed for 10 days. If prescribed in § 113.37. If found unsatis- the vaccine is recommended for more factory by any prescribed test, the than one species, only one species Master Seed shall not be used. needs to be tested. (b) Each lot of Master Seed used for (ii) If unfavorable reactions attrib- vaccine production shall be tested for utable to the vaccine occur during the immunogenicity. The immunogenicity observation period in two or more of of a selected dose from the lot of Mas- the test birds, the serial is unsatisfac- ter Seed shall be established as follows: tory. (1) Thirty feline pneumonitis suscep- (iii) If unfavorable reactions occur tible cats shall be used as test animals which are not attributable to the test (20 vaccinates and 10 controls). Blood vaccine, the test is a No Test and may samples shall be drawn and individual be repeated. If the results of the next serum samples tested. The cats shall be test are not satisfactory, or if the test considered suitable for use if all serums is not repeated, the serial shall be con- are negative for pneumonitis antibody sidered unsatisfactory. in a complement fixation test or other (2) Bacterial count requirements. Final test of equal sensitivity. container samples of completed prod- (2) A geometric mean titer of the uct shall be tested for bacterial count dried vaccine produced from the high- using the method used in paragraph est passage of the Master Seed shall be (b)(2) of this section. Two replicate ti- established before the immunogenicity trations shall be conducted on each se- test is conducted. The 20 cats used as rial and subserial. Each sample shall be vaccinates shall be administered a pre- rehydrated with accompanying sterile determined quantity of vaccine by the diluent to the volume indicated on the method to be recommended on the label. To be eligible for release, each label and the remaining 10 cats shall be serial and subserial shall have a bac- held as controls. To confirm the dosage terial count sufficiently greater than calculations, five replicate titrations shall be conducted on a sample of the that of the vaccine used in the vaccine dilution used. If two doses are immunogenicity test count per dose es- used, five replicate confirming titra- tablished to assure that, when tested tions shall be conducted on each dose. at any time within the expiration pe- (3) Fourteen or more days after the riod, each serial and subserial shall final dose of vaccine, the vaccinates have a bacterial count at least two and controls shall each be challenged times greater than that used in the intranasally with a minimum of 10,000 immunogenicity test. yolk sac LD50 of virulent feline pneu- [55 FR 35560, Aug. 31, 1990, as amended at 59 monitis furnished or approved by the FR 19633, Apr. 25, 1994; 64 FR 43044, Aug. 9, Animal and Plant Health Inspection 1999; 72 FR 72564, Dec. 21, 2007] Service and observed each day for 28

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days postchallenge. The rectal tem- INACTIVATED BACTERIAL PRODUCTS perature of each animal shall be taken and the presence or absence of clinical § 113.100 General requirements for in- signs noted and recorded each day. activated bacterial products. (i) If less than 8 of 10 controls show Unless otherwise prescribed in an ap- clinical signs of feline pneumonitis in- plicable Standard Requirement or in fection other than fever, the test is a the filed Outline of Production, an in- No Test and may be repeated. activated bacterial product shall meet (ii) If a significant difference in clin- the applicable requirements in this sec- ical signs other than fever or tion. chlamydia shedding cannot be dem- (a) Purity tests. (1) Final container onstrated between vaccinates and con- samples of completed product from trols using a scoring system approved each serial and each subserial shall be by the Animal and Plant Health In- tested for viable bacteria and fungi as spection Service, the Master Seed is provided in § 113.26. unsatisfactory. (2) Each lot of Master Seed Bacteria (4) An Outline of Production change shall be tested for the presence of ex- must be made before authority for use traneous viable bacteria and fungi in of a new lot of Master Seed is granted accordance with the test provided in § 113.27(d). by the Animal and Plant Health In- spection Service. (b) Safety tests. Bulk or final container samples of completed product (c) Test requirements for release: Ex- from each serial shall be tested for cept for § 113.300(a)(3)(ii), each serial safety in young adult mice in accord- and subserial shall meet the require- ance with the test provided in ments prescribed in § 113.300 and in this § 113.33(b) unless: paragraph. Final container samples of (1) The product contains material completed product shall be tested. Any which is inherently lethal for mice. In serial or subserial found unsatisfactory such instances, the guinea pig safety by a prescribed test shall not be retest provided in § 113.38 shall be con- leased. ducted in place of the mouse safety (1) The test for pathogens prescribed test. in § 113.37 shall be conducted on each (2) The product is recommended for serial or one subserial of avian origin poultry. In such instances, the product vaccine. shall be safety tested in poultry as de- (2) Chlamydia titer requirements. Final fined in the specific Standard Require- container samples of completed prod- ment or Outline of Production for the uct shall be tested for chlamydia titer product. using the titration method used in (3) The product is recommended for paragraph (b)(2) of this section. To be fish, other aquatic species, or reptiles. eligible for release, each serial and In such instances, the product shall be each subserial shall have a titer suffi- safety tested in fish, other aquatic spe- ciently greater than the titer of vac- cies, or reptiles as required by specific cine used in the immunogenicity test Standard Requirement or Outline of prescribed in paragraph (b) of this sec- Production for the product. tion to assure that when tested at any (c) Identity test. Methods of identi- time within the expiration period, each fication of Master Seed Bacteria to the serial and subserial shall have a titer genus and species level by laboratory 0.7 greater than that used in such tests shall be sufficient to distinguish immunogenicity test but not less than the bacteria from other similar bac- 2.5 ID50 per dose. teria according to criteria described in the most recent edition of ‘‘Bergey’s [55 FR 35561, Aug. 31, 1990, as amended at 56 Manual of Systematic Bacteriology’’ or FR 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, the American Society for Microbiology 2007] ‘‘Manual of Clinical Microbiology’’. If Master Seed Bacteria are referred to by serotype, serovar, subtype, pilus type, strain or other taxonomic subdivision

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below the species level, adequate test- § 113.101 Leptospira Pomona Bacterin. ing must be used to identify the bacteria to that level. Tests which may be Leptospira Pomona Bacterin shall be used to identify Master Seed Bacteria produced from a culture of Leptospira include, but are not limited to: pomona which has been inactivated and (1) Cultural characteristics, is nontoxic. Each serial of biological (2) Staining reaction, product containing Leptospira pomona (3) Biochemical reactivity, fraction shall meet the applicable re- (4) Fluorescent antibody tests, quirements in § 113.100 and shall be (5) Serologic tests, tested for purity, safety, and potency (6) Toxin typing, as prescribed in this section. A serial (7) Somatic or flagellar antigen char- found unsatisfactory by any prescribed acterization, and test shall not be released. (8) Restriction endonuclease analysis. (a) Purity test. Final container sam- (d) Ingredient requirements. Ingredi- ples of completed product from each se- ents used for the growth and prepara- rial and each subserial shall be tested tion of Master Seed Bacteria and of for viable bacteria and fungi as pro- final product shall meet the require- vided in § 113.26. ments provided in § 113.50. Ingredients of animal origin shall meet the appli- (b) Safety test. Bulk or final container cable requirements provided in § 113.53. samples of completed product from (e) Only serials tested for viricidal each serial shall be tested for safety as activity in accordance with the test provided in § 113.38. provided in § 113.35 and found satisfac- (c) Potency test. Bulk or final con- tory by such test shall be packaged as tainer samples of completed product diluent for desiccated fractions in com- shall be diluted with physiological sa- bination packages. line so that each 0.25 ml contains not (f) If formaldehyde is used as the in- more than 1/800th of the dose rec- activating agent, and the serial has not ommended on the label and shall be been found satisfactory by the viricidal tested for potency, using the two-stage activity test, bulk or final container test provided in this paragraph. samples of completed product from (1) Vaccinates. Inject each of at least each serial must be tested for residual 10 but not more than 12 young adult free formaldehyde content using the hamsters, each weighing 50 to 90 ferric chloride test. 1 Firms currently using tests for residual free formalde- grams, with 0.25 ml of the diluted hyde content other than the ferric bacterin either subcutaneously or chloride test have until July 14, 2004 to intramuscularly, in accordance with update their Outline of Production to the label recommendations for use. be in compliance with this require- (2) Controls. Retain at least 10 but not ment. more than 12 additional hamsters from (1) The residual free formaldehyde the same group as unvaccinated con- content of biological products controls. taining clostridial antigens must not (3) Challenge. From 14 to 18 days exceed 1.85 grams per liter (g/L). postvaccination, challenge each of 10 (2) The residual free formaldehyde vaccinates and each of 10 controls content of bacterins, bacterin-toxoids, intraperitoneally with a suspension of and toxoids, other than those con- virulent Leptospira pomona organisms, taining clostridial antigens, must not using a dose of 10–10,000 hamster LD exceed 0.74 grams per liter (g/L). 50 as determined by titration. [39 FR 16862, May 10, 1974. Redesignated at 55 (4) Post-challenge period. Observe the FR 35562, Aug. 31, 1990, as amended at 60 FR vaccinates and controls for 14 days 14355, Mar. 17, 1995; 68 FR 35283, June 13, 2003; 79 FR 31021, May 30, 2014] post-challenge and record all deaths. If eight or more controls die of lepto- spirosis, the test is valid and the re- 1 The procedures for performing the ferric chloride test for residual free formaldehyde sults shall be evaluated according to may be obtained from USDA, APHIS, Center the following table: for Veterinary Biologics-Laboratory, 1800 Dayton Road, P.O. Box 844, Ames, IA 50010.

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Cumu- Cumulative Cumulative (2) Controls. Retain at least 10 but not Number lative total dead total dead more than 12 additional hamsters from Stage of vac- number hamsters for hamsters for cinates of vac- satisfactory unsatisfactory the same group as unvaccinated con- cinates serial serial trols. 1 ...... 10 ...... 10 ...... 2 or less ..... 5 or more. (3) Challenge. From 14 to 18 days 2 ...... 10 ...... 20 ...... 5 or less ..... 6 or more. postvaccination, challenge each of 10 vaccinates and each of 10 controls (5) If three or four vaccinates die in intraperitoneally with a suspension of the first stage, the second stage shall virulent Leptospira icterohaemorrhagiae be conducted in a manner identical to organisms, using a dose of 10–10,000 the first stage. hamster LD50 as determined by titra- (6) If the second stage is used, each tion. serial shall be evaluated according to (4) Post-challenge period. Observe the the second part of the table. On the vaccinates and controls for 14 days basis of cumulative results, each serial post-challenge and record all deaths. If shall either pass or fail. eight or more controls die from lepto- spirosis, the test is valid and the re- [39 FR 16862, May 10, 1974, as amended at 40 FR 20067, May 8, 1975; 45 FR 40100, June 13, sults shall be evaluated according to 1980. Redesignated at 55 FR 35562, Aug. 31, the following table: 1990, as amended at 56 FR 66785, Dec. 26, 1991] Cumu- Cumulative Cumulative Number lative total dead total dead § 113.102 Leptospira Stage of vac- number hamsters for hamsters for cinates of vac- satisfactory unsatisfactory Icterohaemorrhagiae Bacterin. cinates serial serial Leptospira Icterohaemorrhagiae 1 ...... 10 ...... 10 ...... 2 or less ..... 5 or more. Bacterin shall be produced from a cul- 2 ...... 10 ...... 20 ...... 5 or less ..... 6 or more. ture of Leptospira icterohaemorrhagiae which has been inactivated and is (5) If three or four vaccinates die in nontoxic. Each serial of biological the first stage, the second stage shall product containing Leptospira be used. The second stage shall be con- icterohaemorrhagiae fraction shall meet ducted in a manner identical to the the applicable requirements in § 113.100 first stage. and be tested for purity, safety, and po- (6) If the second stage is used, each tency as prescribed in this section. A serial shall be evaluated according to serial found unsatisfactory by any pre- the second part of the table. On the scribed test shall not be released. basis of cumulative results, each serial (a) Purity test. Final container sam- shall either pass or fail. ples of completed product from each se- [39 FR 16862, May 10, 1974, as amended at 45 rial and each subserial shall be tested FR 40100, June 13, 1980. Redesignated at 55 for viable bacteria and fungi as pro- FR 35562, Aug. 31, 1990, as amended at 56 FR vided in § 113.26. 66785, Dec. 26, 1991] (b) Safety test. Bulk or final container samples of completed product from § 113.103 Leptospira Canicola each serial shall be tested for safety as Bacterin. provided in § 113.38. Leptospira Canicola Bacterin shall be (c) Potency test. Bulk or final con- produced from a culture of Leptospira tainer samples of completed product canicola which has been inactivated shall be diluted with physiological sa- and is nontoxic. Each serial of biologi- line so that each 0.25 ml contains not cal product containing Leptospira canic- more than 1/80th of the dose rec- ola fraction shall meet the applicable ommended on the label and shall be requirements in § 113.100 and shall be tested for potency, using the two-stage tested for purity, safety, and potency test provided in this paragraph. as prescribed in this section. Serials (1) Vaccinates. Inject each of at least found unsatisfactory by any prescribed 10 but not more than 12 young adult test shall not be released. hamsters, each weighing 50 to 90 (a) Purity test. Final container sam- grams, with 0.25 ml of the diluted ples of completed product from each se- bacterin either subcutaneously or rial and each subserial shall be tested intramuscularly, in accordance with for viable bacteria and fungi as pro- the label recommendations for use. vided in § 113.26.

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(b) Safety test. Bulk or final container § 113.104 Leptospira Grippotyphosa samples of completed product from Bacterin. each serial shall be tested for safety as Leptospira Grippotyphosa Bacterin provided in § 113.38. shall be produced from a culture of (c) Potency test. Bulk or final con- Leptospira grippotyphosa which has been tainer samples of completed product inactivated and is nontoxic. Each se- shall be diluted with physiological saline so that each 0.25 ml contains not rial of biological product containing more than 1/80th of the dose rec- Leptospira grippotyphosa fraction shall ommended on the label and shall be meet the applicable requirements in tested for potency, using the two-stage § 113.100 and shall be tested for purity, test provided in this paragraph. safety, and potency as prescribed in (1) Vaccinates. Inject each of at least this section. A serial found unsatisfac- 10 but not more than 12 young adult tory by any prescribed test shall not be hamsters, each weighing 50 to 90 released. grams, with 0.25 ml of the diluted (a) Purity test. Final container sam- bacterin either subcutaneously or ples of completed product from each se- intramuscularly, in accordance with rial and each subserial shall be tested the label recommendations for use. for viable bacteria and fungi as pro- (2) Controls. Retain at least 10 but not vided in § 113.26. more than 12 additional hamsters from (b) Safety test. Bulk or final container the same group as unvaccinated con- samples of completed product from trols. each serial shall be tested for safety as (3) Challenge. From 14 to 18 days provided in § 113.38. postvaccination, challenge each of 10 (c) Potency test. Bulk or final con- vaccinates and each of 10 controls tainer samples of completed product intraperitoneally with a suspension of shall be diluted with physiological sa- virulent Leptospira canicola organisms, line so that each 0.25 ml contains not using a dose of 10–10,000 hamster LD 50 more than 1/800th of the dose rec- as determined by titration. ommended on the label and shall be (4) Post-challenge period. Observe the vaccinates and controls for 14 days tested for potency, using the two-stage post-challenge and record all deaths. If test provided in this paragraph. eight or more controls die from lepto- (1) Vaccinates. Inject each of at least spirosis, test is valid and the results 10 but not more than 12 young adult shall be evaluated according to the fol- hamsters, each weighing 50 to 90 lowing table: grams, with 0.25 ml of the diluted bacterin either subcutaneously or Cumu- Cumulative Cumulative intramuscularly, in accordance with Number lative total dead total dead Stage of vac- number hamsters for hamsters for the label recommendations for use. cinates of vac- satisfactory unsatisfactory cinates serial serial (2) Controls. Retain at least 10 but not more than 12 additional hamsters from 1 ...... 10 ...... 10 ...... 2 or less ..... 5 or more. the same group as unvaccinated con- 2 ...... 10 ...... 20 ...... 5 or less ..... 6 or more. trols. (5) If three or four vaccinates die in (3) Challenge. From 14 to 18 days the first stage, the second stage shall postvaccination, challenge each of 10 be used. The second stage shall be con- vaccinates and each of 10 controls ducted in a manner identical to the intraperitoneally with a suspension of first stage. virulent Leptospira grippotyphosa orga- (6) If the second stage is used, each nisms, using a dose of 10–10,000 hamster serial shall be evaluated according to LD50 as determined by titration. the second part of the table. On the (4) Post-challenge period. Observe the basis of cumulative results, each serial vaccinates and controls for 14 days shall either pass or fail. post-challenge and record all deaths. If [39 FR 16862, May 10, 1974, as amended at 45 eight or more controls die of lepto- FR 40100, June 13, 1980. Redesignated at 55 spirosis, the test is valid and the re- FR 35562, Aug. 31, 1990, as amended at 56 FR sults shall be evaluated according to 66785, Dec. 26, 1991] the following table:

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CUMULATIVE TOTALS tridium chauvoei fraction shall meet the applicable requirements in § 113.100 and Number of Dead hamsters Dead hamsters Stage vaccinates for acceptance for rejection shall be tested for purity, safety, and potency as prescribed in this section. 1 ...... 10 ...... 2 or less ...... 5 or more. Serials found unsatisfactory by any 2 ...... 20 ...... 5 or less ...... 6 or more. prescribed test shall not be released. (a) Purity test. Final container sam- (5) If three or four vaccinates die in ples of completed product from each se- the first stage, the second stage shall rial and each subserial shall be tested be conducted in a manner identical to for viable bacteria and fungi as pro- the first stage. vided in § 113.26. (6) If the second stage is used, each (b) Safety test. Bulk or final container serial shall be evaluated according to samples of completed product from the second part of the table. On the each serial shall be tested for safety as basis of cumulative results, each serial provided in § 113.38. shall either pass or fail. (c) Potency test. Bulk or final con- [40 FR 17003, Apr. 16, 1975, as amended at 40 tainer samples of completed product FR 23989, June 4, 1975; 45 FR 40100, June 13, from each serial shall be tested for po- 1980. Redesignated at 55 FR 35562, Aug. 31, tency using the two-stage test provided 1990, as amended at 56 FR 66785, Dec. 26, 1991] in this paragraph. (1) Each of at least 8 but not more § 113.105 Leptospira Hardjo Bacterin. than 10 guinea pigs, each weighing 300 Leptospira Hardjo Bacterin shall be to 500 grams, shall be injected produced from a culture of Leptospira subcutaneously with a guinea pig dose. hardjo which has been inactivated and A second guinea pig dose shall be in- is nontoxic. Each serial of biological jected 21 to 23 days after the first dose. product containing Leptospira hardjo Each guinea pig dose shall be one-fifth fraction shall meet the applicable re- of the dose recommended on the label quirements in § 113.100 and shall be for a calf. tested for purity, safety, and potency (2) Clostridium chauvoei challenge ma- as prescribed in this section. A serial terial, available upon request from found unsatisfactory by any prescribed Animal and Plant Health Inspection test shall not be released. Service, shall be used for challenge 14 (a) Purity test. Final container sam- to 15 days following the last injection ples of completed product from each se- of the product. Each of eight vac- rial and each subserial shall be tested cinates and each of five additional non- for viable bacteria and fungi as pro- vaccinated guinea pigs for controls vided in § 113.26. shall be injected intramuscularly with (b) Safety test. Bulk or final container approximately 100 LD50 of challenge samples of completed product from material. This dose shall be determined each serial shall be tested for safety as by statistical analysis of results of ti- provided in § 113.38. trations of the challenge material. The (c) Potency test. Bulk or final con- vaccinates and controls shall be ob- tainer samples of completed product served for 3 days postchallenge and all from each serial shall be tested for po- deaths recorded. tency using the test written into the (3) For a valid test, at least 80 per- filed Outline of Production. cent of the controls shall die within [40 FR 17003, Apr. 16, 1975, as amended at 40 the 3 day post-challenge observation FR 20067, May 8, 1975. Redesignated at 55 FR period. If this requirement is met, the 35562, Aug. 31, 1990, as amended at 56 FR results of the potency test shall be 66785, Dec. 26, 1991] evaluated according to the following table: § 113.106 Clostridium Chauvoei Bacterin. Cumu- Cumulative Cumulative Number lative total number total number Clostridium Chauvoei Bacterin shall Stage of vac- number of deaths for of deaths for cinates of vac- a satisfac- an unsatisfac- be produced from a culture of Clos- cinates tory test tory test tridium chauvoei which has been inactivated and is nontoxic. Each serial of 1 ...... 8 ...... 8 ...... 1 or less ..... 3 or more. biological product containing Clos- 2 ...... 8 ...... 16 ...... 4 or less ..... 5 or more.

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The second stage shall be required only trations of the challenge material. The when exactly two animals die in the vaccinates and controls shall be ob- first stage. The second stage shall be served for 3 days postchallenge and all conducted in a manner identical to the deaths recorded. first stage. (3) For a valid test, at least 80 per- [39 FR 16862, May 10, 1974, as amended at 45 cent of the controls shall die within FR 40100, June 13, 1980. Redesignated at 55 the 3 day post-challenge observation FR 35562, Aug. 31, 1990 and amended at 56 FR period. If this requirement is met, the 66784, 66785, Dec. 26, 1991] results of the potency test shall be evaluated according to the following § 113.107 Clostridium Haemolyticum table: Bacterin. Cumu- Cumulative Cumulative Clostridium Haemolyticum Bacterin Number lative total number total number shall be produced from a culture of Stage of vac- number of deaths for of deaths for cinates of vac- a satisfac- an unsatisfac- Clostridium haemolyticum which has cinates tory test tory test been inactivated and is nontoxic. Each serial of biological product containing 1 ...... 8 ...... 8 ...... 1 or less ..... 3 or more. Clostridium haemolyticum fraction shall 2 ...... 8 ...... 16 ...... 4 or less ..... 5 or more. meet the applicable requirements in § 113.100 and shall be tested for purity, The second stage shall be required only safety, and potency as prescribed in when exactly two animals die in the this section. A serial found unsatisfac- first stage. The second stage shall be tory by any prescribed test shall not be conducted in a manner identical to the released. first stage. (a) Purity test. Final container sam- [39 FR 16862, May 10, 1974, as amended at 40 ples of completed product from each se- FR 20067, May 8, 1975; 45 FR 40100, June 13, rial and each subserial shall be tested 1980. Redesignated at 55 FR 35562, Aug. 31, for viable bacteria and fungi as pro- 1990, as amended at 56 FR 66784, 66785, Dec. vided in § 113.26. 26, 1991] (b) Safety test. Bulk or final container samples of completed product from § 113.108 Clostridium Novyi Bacterin- Toxoid. each serial shall be tested for safety as provided in § 113.38. Clostridium Novyi Bacterin-Toxoid (c) Potency test. Bulk or final con- shall be produced from a culture of tainer samples of completed product Clostridium novyi which has been inac- from each serial shall be tested for po- tivated and is nontoxic. Each serial of tency using the two-stage test provided biological product containing Clos- in this paragraph. tridium novyi fraction shall meet the (1) Each of at least 8 but not more applicable requirements in § 113.100 and than 10 guinea pigs, each weighing 300 shall be tested for purity, safety, and to 500 grams, shall be injected potency as prescribed in this section. A subcutaneously with a guinea pig dose. serial found unsatisfactory by any pre- A second guinea pig dose shall be in- scribed test shall not be released. jected 21 to 23 days after the first dose. (a) Purity test. Final container sam- Each guinea pig dose shall be one-fifth ples of completed product from each se- of the dose recommended on the label rial and each subserial shall be tested for a calf. for viable bacteria and fungi as pro- (2) Clostridium haemolyticum challenge vided in § 113.26. material, available upon request from (b) Safety test. Bulk or final container Animal and Plant Health Inspection samples of completed product from Service, shall be used for challenge 14 each serial shall be tested for safety as to 15 days following the last injection provided in § 113.38. of the product. Each of eight vac- (c) Potency test. Bulk or final con- cinates and each of five additional non- tainer samples of completed product vaccinated guinea pigs for controls from each serial shall be tested for po- shall be injected intramuscularly with tency using the Alpha toxin-neutraliza- approximately 100 LD50 of challenge tion test provided in this paragraph. material. This dose shall be determined (1) When used in this test, the fol- by statistical analysis of results of ti- lowing words and terms shall mean:

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(i) International antitoxin unit. (I.U.) be repeated: Provided, That, if the test That quantity of Alpha Antitoxin is not repeated, the serial shall be de- which reacts with Lo and L + doses of clared unsatisfactory. Standard Toxin according to their defi- (4) The antitoxin content of the rab- nitions. bit serums shall be determined by the (ii) Lo dose. The largest quantity of serum neutralization test as follows: toxin which can be mixed with one unit (i) Make a dilution of Standard Anti- of Standard Antitoxin and not cause toxin to contain 0.1 International Unit sickness or death in injected mice. of antitoxin per ml. (iii) L + dose. The smallest quantity (ii) Make a dilution of Standard of toxin which can be mixed with one Toxin in which 0.1 Lo dose is contained unit of Standard Antitoxin and cause in a volume of 1 ml or less. Make a sec- death in at least 80 percent of injected ond dilution of Standard Toxin in mice. which 0.1 L + dose is contained in a vol- (iv) Standard antitoxin. The Alpha ume of 1 ml or less. Antitoxin preparation which has been (iii) Combine 0.1 International Unit standardized as to antitoxin unitage on of Standard Antitoxin with 0.1 Lo dose the basis of the International Clos- of diluted Standard Toxin and combine tridium novyi Alpha Antitoxin Standard 0.1 International Unit of Standard and which is either supplied by or ac- Antitoxin with 0.1 L + dose of diluted ceptable to the Animal and Plant Standard Toxin. Each mixture is ad- Health Inspection Service. The anti- justed to a final volume of 2.0 ml with toxin unit value shall be stated on the diluent. label. (iv) Combine 0.1 Lo dose of diluted (v) Standard toxin. The Alpha toxin preparation which is supplied by or is Standard Toxin with a 0.2 ml volume of acceptable to the Animal and Plant undiluted serum. The mixture is ad- Health Inspection Service. justed to a final volume of 2.0 ml with diluent. (vi) Diluent. The solution used to make proper dilutions prescribed in (v) Neutralize all toxin-antitoxin this test. Such solutions shall be made mixtures at room temperature for 1 by dissolving 1 gram of peptone and hour and hold in ice water until injec- 0.25 gram of sodium chloride in each 100 tions of mice can be made. ml of distilled water; adjusting the pH (vi) Five Swiss white mice, each to 7.2; autoclaving at 121 °C for 25 min- weighing 16–20 grams, shall be used for utes; and storing at 4 °C until used. each toxin-antitoxin mixture. A dose of (2) Each of at least eight rabbits of a 0.2 ml shall be injected intravenously strain acceptable to the Animal and into each mouse. Conclude the test 72 Plant Health Inspection Service, each hours post injection and record all weighing 4–8 pounds, shall be injected deaths. subcutaneously with not more than (5) Test Interpretation shall be as fol- half of the recommended cattle dose. lows: Provided, That, if the product is rec- (i) If any mice inoculated with the ommended only for sheep, half of the mixture of 0.1 International Unit of recommended sheep dose shall be used. Standard Antitoxin and 0.1 Lo doses of A second dose shall be given not less Standard Toxin die, the results of the than 20 days nor more than 23 days serum neutralization test are a No Test after the first dose. and shall be repeated: Provided, That, if (3) Fourteen to seventeen days after the test is not repeated, the serial shall the second dose, all surviving rabbits be declared unsatisfactory. shall be bled, and the serum tested for (ii) If less than 80 percent of the mice antitoxin content. inoculated with the mixture of 0.1 (i) At least seven rabbits are required International Unit of Standard Anti- to make an acceptable serum pool. toxin and 0.1 L + doses of Standard (ii) Equal quantities of serum from Toxin die, the results of the serum neu- each rabbit shall be combined and test- tralization test are a No Test and shall ed as a single pooled serum. be repeated: Provided, That, if the test (iii) If less than seven rabbits are is not repeated, the serial shall be de- available, the test is invalid and shall clared unsatisfactory.

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(iii) If any mice inoculated with the ized as to antitoxin unitage on the mixture of 0.2 ml undiluted serum with basis of the International Clostridium 0.1 Lo dose of Standard Toxin die, the sordellii Antitoxin Standard and which serum is considered to contain less is either supplied by or acceptable to than 0.50 International Units per ml. the Animal and Plant Health Inspec- (iv) If the single pooled serum from tion Service. The antitoxin unit value seven or more rabbits contains less shall be stated on the label. than 0.5 International Unit per ml, the (v) Standard toxin. The toxin prepara- serial is unsatisfactory. tion which is supplied by or is acceptable to the Animal and Plant Health [39 FR 16862, May 10, 1974, as amended at 45 FR 40101, June 13, 1980. Redesignated at 55 Inspection Service. FR 35562, Aug. 31, 1990; 56 FR 37825, Aug. 9, (vi) Diluent. The solution used to 1991, as amended at 56 FR 66784, 66785, Dec. make proper dilutions prescribed in 26, 1991] this test. Such solutions shall be made by dissolving 1 gram of peptone and § 113.109 Clostridium Sordellii 0.25 gram of sodium chloride in each 100 Bacterin-Toxoid. ml of distilled water; adjusting the pH Clostridium Sordellii Bacterin-Tox- to 7.2; autoclaving at 121 °C for 25 min- oid shall be produced from a culture of utes; and storing at 4 °C until used. Clostridium sordellii which has been in- (2) Each of at least eight rabbits of a activated and is nontoxic. Each serial strain acceptable to the Animal and of biological product containing Clos- Plant Health Inspection Service, each tridium sordellii fraction shall meet the weighing 4–8 pounds, shall be injected applicable requirements in § 113.100 and subcutaneously with not more than shall be tested for purity, safety, and half of the recommended cattle dose: potency as prescribed in this section. A Provided, That, if the product is rec- serial found unsatisfactory by any pre- ommended only for sheep, half of the scribed test shall not be released. recommended sheep dose shall be used. (a) Purity test. Final container sam- A second dose shall be given not less ples of completed product from each se- than 20 days nor more than 23 days rial and each subserial shall be tested after the first dose. for viable bacteria and fungi as pro- (3) Fourteen to seventeen days after vided in § 113.26. the second dose, all surviving rabbits (b) Safety test. Bulk or final container shall be bled, and the serum tested for samples of completed product from antitoxin content. each serial shall be tested for safety as (i) At least seven rabbits are required provided in § 113.38. to make an acceptable serum pool. (c) Potency test. Bulk or final con- (ii) Equal quantities of serum from tainer samples of completed product each rabbit shall be combined and test- from each serial shall be tested for po- ed as a single pooled serum. tency using the toxin-neutralization (iii) If less than seven rabbits are test provided in this paragraph. available, the test is a No Test and (1) When used in this test, the fol- shall be repeated: Provided, That, if the lowing words and terms shall mean: test is not repeated, the serial shall be (i) International antitoxin unit. (I.U.) declared unsatisfactory. That quantity of antitoxin which re- (4) The antitoxin content of the rab- acts with Lo and L + doses of Standard bit serums shall be determined by the Toxin according to their definitions. serum neutralization test as follows: (ii) Lo dose. The largest quantity of (i) Make a dilution of Standard Anti- toxin which can be mixed with one unit toxin to contain 1.0 international unit of Standard Antitoxin and not cause of antitoxin per ml. sickness or death in injected mice. (ii) Make a dilution of Standard (iii) L + dose. The smallest quantity Toxin in which 1.0 Lo dose is contained of toxin which can be mixed with one in a volume of 1 ml or less. Make a sec- unit of Standard Antitoxin and cause ond dilution of Standard Toxin in death in at least 80 percent of injected which 1.0 L + dose is contained in a vol- mice. ume of 1 ml or less. (iv) Standard antitoxin. The antitoxin (iii) Combine 1.0 International Unit preparation which has been standard- Standard Antitoxin with 1.0 Lo dose of

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diluted Standard Toxin and combine 1.0 § 113.110 Clostridium Botulinum Type International Unit of Standard Anti- C Bacterin-Toxoid. toxin with 1.0 L + dose of diluted Clostridium Botulinum Type C Standard Toxin. Each mixture is ad- Bacterin-Toxoid shall be produced from justed to a final volume of 2.0 ml with a culture of Clostridium botulinum Type diluent. C which has been inactivated and is (iv) Combine 1.0 Lo dose of diluted nontoxic. Each serial of biological Standard Toxin with a 1.0 ml volume of product containing Clostridium botu- undiluted serum. This mixture is ad- linum Type C fraction shall meet the justed to a final volume of 2.0 ml with applicable requirements in § 113.100 and diluent. shall be tested for purity, safety, and (v) Neutralize all toxin-antitoxin potency as prescribed in this section. A mixtures at room temperature for 1 serial found unsatisfactory by any pre- hour and hold in ice water until injec- scribed test shall not be released. tions of mice can be made. (a) Purity test. Final container sam- (vi) Five Swiss white mice, each ples of completed product from each se- weighing 16–20 grams, shall be used for rial and each subserial shall be tested each toxin-antitoxin mixture. A dose of for viable bacteria and fungi as pro- 0.2 ml shall be injected intravenously vided in § 113.26. into each mouse. Conclude the test 72 (b) Safety test. Bulk or final container hours post injection and record all samples of completed product from deaths. each serial shall be tested for safety as (5) Test Interpretation shall be as fol- provided in § 113.33(b). lows: (c) Potency test. Bulk or final con- (i) If any mice inoculated with the tainer samples of completed product mixture of 1.0 International Unit of from each serial shall be tested for po- Standard Antitoxin and 1.0 Lo doses of tency, using susceptible mink as test Standard Toxin die, the results of the animals. At least five vaccinates and serum neutralization test are a No Test three unvaccinated controls of the and shall be repeated: Provided, That, if same source and approximately the the test is not repeated, the serial shall same age shall be used. be declared unsatisfactory. (1) Each of the vaccinates shall be in- (ii) If less than 80 percent of the mice jected subcutaneously with the dose inoculated with the mixture of 1.0 recommended on the label for mink. International Unit of Standard Anti- Twenty-one to twenty-eight days post- toxin and 1.0 L + doses of Standard injection, the vaccinates and the con- Toxin die, the results of the serum neu- trols shall be challenged tralization test are a No Test and shall intraperitoneally with botulinum Type be repeated: Provided, That, if the test C toxin which has been titrated in mice is not repeated, the serial shall be de- to provide for a 10 4.0 mouse MLD dose. clared unsatisfactory. The titration technique shall include (iii) If any mice inoculated with the inoculation of the mice mixture of 1.0 ml undiluted serum with intraperitoneally. 1.0 Lo dose of Standard Toxin die, the (2) The vaccinates and controls shall serum is considered to contain less be observed for 7 days post-challenge than 1.0 International Units per ml. and signs of botulism and deaths noted. (iv) If the single pooled serum from For a valid test, the controls shall die seven or more rabbits contains less of botulism. If the test is valid and 80 than 1.0 International Unit per ml, the percent of the vaccinates do not re- serial is unsatisfactory. main free of botulism, the serial is un- [39 FR 16862, May 10, 1974, as amended at 42 satisfactory. FR 61247, Dec. 2, 1977; 45 FR 40101, June 13, [39 FR 16862, May 10, 1974, as amended at 40 1980. Redesignated at 55 FR 35562, Aug. 31, FR 759, Jan. 3, 1975. Redesignated at 55 FR 1990; 56 FR 37826, Aug. 9, 1991; 56 FR 66784, 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991; 79 FR 55969, Sept. 18, 2014] 66785, Dec. 26, 1991]

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§ 113.111 Clostridium Perfringens this test. Such solutions shall be made Type C Toxoid and Bacterin-Toxoid. by dissolving 1 gram of peptone and Clostridium Perfringens Type C Tox- 0.25 grams of sodium chloride in each oid and Clostridium Perfringens Type C 100 ml of distilled water; adjusting the Bacterin-Toxoid shall be produced from pH to 7.2; autoclaving at 250 °F for 25 a culture of Clostridium perfringens minutes; and storing at 4 °C until used. Type C which has been inactivated and (2) Each of at least eight rabbits of a is nontoxic. Each serial shall meet the strain acceptable to APHIS, each applicable requirements in § 113.100 and weighing 4–8 pounds, shall be injected shall be tested for purity, safety, and subcutaneously with not more than potency as prescribed in this section. half of the largest recommended dose Any serial found unsatisfactory by a for any species indicated on the prod- prescribed test shall not be released. uct label. A second equivalent dose (a) Purity test. Final container sam- shall be given not less than 20 days nor ples of completed product from each se- more than 23 days after the first does. rial and each subserial shall be tested (3) Fourteen to seventeen days after for viable bacteria and fungi as pro- the second dose, all surviving rabbits vided in § 113.26. shall be bled and the serum tested for (b) Safety test. Bulk or final container antitoxin content. samples of completed product from (i) At least seven rabbits are required each serial shall be tested for safety as to make an acceptable serum pool. provided in § 113.33(b). (ii) Equal quantities of serum from (c) Potency test. Bulk or final con- each rabbit shall be combined and test- tainer samples of completed product ed as a single pooled serum. from each serial shall be tested for po- (iii) If less than seven rabbits are tency using the Beta toxin-neutraliza- available, the test is a No Test and tion test provided in this paragraph. shall be repeated: Provided, That, if the (1) When used in this test, the fol- test is not repeated, the serial shall be lowing words and terms shall mean: declared unsatisfactory. (i) International antitoxin unit. (I.U.) (4) The antitoxin content of the rab- That quantity of Beta Antitoxin which bit serums shall be determined as fol- + reacts with L 0 and L doses of lows: Standard Toxin according to their defi- (i) Make a dilution of Standard Anti- nitions. toxin to contain 10 International Units (ii) L 0 dose. The largest quantity of of antitoxin per ml. toxin which can be mixed with one unit (ii) Make one dilution of Standard of Standard Antitoxin and not cause Toxin to contain 10 L 0 doses per ml and sickness or death in injected mice. make a second dilution of Standard (iii) L + dose. The smallest quan- Toxin to contain 10 L ∂ doses per ml. tity of toxin which can be mixed with (iii) Combine 10 International Units one unit of Standard Antitoxin and of Standard Antitoxin with 10 L 0 doses cause death in at least 80 percent of in- of diluted Standard Toxin and combine jected mice. 10 International Units of Standard (iv) Standard antitoxin. The Beta Antitoxin with 10 L∂ doses of diluted Antitoxin preparation which has been Standard Toxin. standardized as to antitoxin unitage on (iv) Combine 1 ml of undiluted serum the basis of the International Clos- with 10 L0 doses of diluted Standard tridium perfringens Beta Antitoxin Toxin. Standard and which is either supplied (v) Neutralize all toxin-antitoxin by or acceptable to Animal and Plant mixtures at room temperature for 1 Health Inspection Service. The anti- hour and hold in ice water until injec- toxin unit value shall be stated on the tions of mice can be made. label. (vi) Five Swiss white mice, each (v) Standard toxin. The Beta toxin weighing 16–20 grams, shall be used for preparation which is supplied by or is each toxin-antitoxin mixture. A dose of acceptable to Animal and Plant Health 0.2 ml shall be injected intravenously Inspection Service. into each mouse. Conclude the test 24 (vi) Diluent. The solution used to hours post-injection and record all make proper dilutions prescribed in deaths.

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(5) Test Interpretation shall be as fol- (1) When used in this test, the follows: lowing words and terms shall mean: (i) If any mice inoculated with the (i) International antitoxin unit. (I.U.) mixture of 10 International Units of That quantity of Epsilon Antitoxin Standard Antitoxin and 10 L0 doses of which reacts with L0 and L∂ doses of Standard Toxin die, the results of the Standard Toxin according to their defi- test are a No Test and shall be re- nitions. peated: Provided, That, if the test is not (ii) L0 dose. The largest quantity of repeated, the serial shall be declared toxin which can be mixed with one- unsatisfactory. tenth unit of Standard Antitoxin and (ii) If less than 80 percent of the mice not cause sickness or death in injected inoculated with mixture of 10 Inter- mice. national Units of Standard Antitoxin (iii) L∂ dose. The smallest quantity of toxin which can be mixed with one- and 10 L∂ doses of Standard Toxin die, the results of the test are a No Test tenth unit of Standard Antitoxin and and shall be repeated: Provided, That, if cause death in at least 80 percent of in- the test is not repeated, the serial shall jected mice. be declared unsatisfactory. (iv) Standard antitoxin. The Epsilon (iii) If any mice inoculated with the Antitoxin preparation which has been standardized as to antitoxin unitage on mixture of serum with 10 L doses of 0 the basis of the International Clos- Standard Toxin die, the serum is con- tridium perfringens Epsilon Antitoxin sidered to contain less than 10 Inter- Standard and which is either supplied national Units per ml. and the serial is by or acceptable to Animal and Plant unsatisfactory Health Inspection Service. The anti- [39 FR 16862, May 10, 1974, as amended at 40 toxin unit value shall be stated on the FR 759, Jan. 3, 1975; 40 FR 41088, Sept. 5, 1975. label. Redesignated at 55 FR 35562, Aug. 31, 1990, as (v) Standard toxin. The Epsilon toxin amended at 56 FR 66784, 66785, Dec. 26, 1991; 62 preparation which is supplied by or is FR 31330, June 9, 1997; 79 FR 55969, Sept. 18, acceptable to Animal and Plant Health 2014] Inspection Service. § 113.112 Clostridium Perfringens (vi) Diluent. The solution used to Type D Toxoid and Bacterin-Toxoid. make proper dilutions prescribed in this test. Such solutions shall be made Clostridium Perfringens Type D Tox- by dissolving 1 gram of peptone and oid and Clostridium Perfringens Type 0.25 gram of sodium chloride in each 100 D Bacterin-Toxoid shall be produced ml of distilled water; adjusting the pH from a culture of Clostridium perfringens to 7.2; autoclaving at 250 °F for 25 min- Type D which has been inactivated and utes; and storing at 4 °C until used. is nontoxic. Each serial shall meet the (2) Each of at least eight rabbits of a applicable requirements in § 113.100 and strain acceptable to APHIS, each shall be tested for purity, safety, and weighing 4–8 pounds, shall be injected potency as prescribed in this section. subcutaneously with not more than Any serial found unsatisfactory by a half of the largest recommended dose prescribed test shall not be released. for any species indicated on the prod- (a) Purity test. Final container sam- uct label. A second equivalent dose ples of completed product from each se- shall be given not less than 20 days nor rial and each subserial shall be tested more than 23 days after the first dose. for viable bacteria and fungi as pro- (3) Fourteen to seventeen days after vided in § 113.26. the second dose, all surviving rabbits (b) Safety test. Bulk or final container shall be bled, and the serum tested for samples of completed product from antitoxin content. each serial shall be tested for safety as (i) At least seven rabbits are required provided in § 113.33(b). to make an acceptable serum pool. (c) Potency test. Bulk or final con- (ii) Equal quantities of serum from tainer samples of completed product each rabbit shall be combined and test- from each serial shall be tested for po- ed as a single pooled serum. tency using the Epsilon toxin-neutral- (iii) If less than seven rabbits are ization test provided in this paragraph. available, the test is a No Test and

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shall be repeated: Provided, That, if the national Units per ml, and the serial is test is not repeated, the serial shall be unsatisfactory. declared unsatisfactory. [39 FR 16865, May 10, 1974; 39 FR 20783, June (4) The antitoxin content of the rab- 14, 1974. Redesignated at 39 FR 25463, July 11, bit serums shall be determined as fol- 1974, and amended at 40 FR 759, Jan. 3, 1975; lows: 40 FR 41088, Sept. 5, 1975. Redesignated at 55 (i) Make a dilution of Standard Anti- FR 35562, Aug. 31, 1990, as amended at 56 FR toxin to contain 1 International Unit of 66784, 66785, Dec. 26, 1991; 62 FR 31331, June 9, 1997; 79 FR 55969, Sept. 18, 2014] antitoxin per ml. (ii) Make one dilution of Standard § 113.113 Autogenous biologics. Toxin to contain 10 Lo doses per ml and make a second dilution of Standard Autogenous biologics shall be prepared from cultures of microorganisms Toxin to contain 10 L∂ doses per ml. which have been inactivated and are (iii) Combine 1 International Unit of nontoxic. Such products shall be pre- Standard Antitoxin with 10 L doses of o pared only for use by or under the di- diluted Standard Toxin and Combine 1 rection of a veterinarian under a vet- International Unit of Standard Anti- erinarian-client-patient relationship, toxin with 10 L∂ doses of diluted Provided, That, such products may be Standard Toxin. prepared for use under the direction of (iv) Dilute 1 ml of serum with 1 ml of a person of appropriate expertise in diluent (1:2) and combine 1 ml of this specialized situations such as aqua- solution with 10 Lo doses of diluted culture, if approved by the Adminis- Standard Toxin. trator. (v) Neutralize all toxin-antitoxin Each serial of an autogenous biologic mixtures at room temperature for 1 shall meet the requirements in this hour and hold in ice water until injec- section, and if found unsatisfactory by tions of mice can be made. any prescribed test shall not be used. (vi) Five Swiss white mice, each (a) Seed requirements. The microorga- weighing 16–20 grams, shall be used for nisms used as seed to prepare autog- each toxin-antitoxin mixture. A dose of enous biologics shall be microorga- 0.2 ml shall be injected intravenously nisms which are isolated from sick or into each mouse. Conclude the test 24 dead animals in the herd of origin and hours post-injection and record all which there is reason to believe are the deaths. causative agent(s) of the current dis- (5) Test Interpretation shall be as fol- ease affecting such animals. lows: (1) More than one microorganism iso- (i) If any mice inoculated with the lated from the same herd may be used mixture of 1 International Unit of as seed. (2) Under normal circumstances, Standard Antitoxin and 10 Lo doses of Standard Toxin die, the results of the microorganisms from one herd must test are a No Test and shall be re- not be used to prepare an autogenous peated: Provided, That, if the test is not biologic for another herd. The Adminis- repeated, the serial shall be declared trator, however, may authorize prepa- unsatisfactory. ration of an autogenous biologic for use in herds adjacent to the herd of ori- (ii) If less than 80 percent of the mice gin, when adjacent herds are consid- inoculated with mixture of 1 Inter- ered to be at risk. To request author- national Unit of Standard Antitoxin ization to prepare a product for use in and 10 L∂ doses of Standard Toxin die, herds adjacent to the herd of origin, the results of the test area No Test and the establishment seeking authoriza- shall be repeated: Provided, That, if the tion must submit to the Administrator test is not repeated, the serial shall be (in c/o the Director, Center for Veteri- declared unsatisfactory. nary Biologics, Inspection and Compli- (iii) If any mice inoculated with the ance, 1920 Dayton Avenue, P.O. Box 844, mixture of serum with 10 Lo doses of Ames, IA 50010) the following informa- Standard Toxin die, the serum is con- tion. (If any of the data are unavail- sidered to contain less than 2 Inter- able, the applicant for authorization

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should indicate that such data are un- (ii) The geographic designations of available and why.) the area involved. (i) Name, address, and phone number (iii) A summary of the epidemiology of the owner of the herd of origin. of the disease situation that links the (ii) Attending veterinarian’s name, designated geographic areas with the address, and phone number. herd of origin. (iii) Animal species and number in In addition, an applicant for authoriza- herd of origin. tion under this paragraph (a)(3) shall (iv) Identification of microorga- provide written approval from the nism(s), at least to genus. State Veterinarian or other appro- (v) Diagnosis or clinical signs of the priate State Official in the State in disease observed. which the autogenous biologic is to be (vi) Name and address of the person used in nonadjacent herds. who isolated the microorganism(s) and (4) Under normal circumstances, the date of isolation. microorganism(s) used for the produc- (vii) Number of doses of autogenous tion of autogenous biologics may not biologic requested and vaccination be older than 15 months from the date schedule. of isolation, or 12 months from the date (viii) Each adjacent herd owner’s of harvest of the first serial of product name, address, and phone number. produced from the microorganism(s), (ix) Number of animals and species in whichever comes first. The Adminis- each adjacent herd. trator, however, may authorize production of additional serials from micro- (x) The attending veterinarian’s or organism(s) older than the above stat- approved specialist’s assessment of the ed time periods, Provided, That, the involvement of the adjacent herd(s) person requesting such authorization with the disease observed. submits the following supporting infor- The applicant shall give notice to the mation to the address listed in para- State Veterinarian or other appro- graph (a)(3): priate State Official in writing when an (i) The attending veterinarian’s or autogenous biologic is to be used in ad- approved specialist’s current assess- jacent herds. ment of the continued involvement of a (3) The Administrator may authorize herd with the originally isolated preparation of an autogenous biologic microorganism(s), including a sum- for use in herds which are not adjacent mary of the diagnostic work that has to the herd of origin, but which he or been done to support this assessment. she considers to be at risk of infection (ii) Evidence of satisfactory protec- with the same microorganism(s). Ex- tion from the previous use of the au- cept as provided below, the same infor- togenous biologic produced from the mation which is required for prepara- microorganisms involved. tion of such product for use in herds (iii) Any other information the Ad- adjacent to the herd of origin must be ministrator may require in order to de- submitted to the Administrator (in c/o termine the need to use the microorga- the Director, Center for Veterinary nism to make additional serials. Biologics, Inspection and Compliance, (b) Restrictions. Unless otherwise au- 1920 Dayton Avenue, P.O. Box 844, thorized by the Administrator, each se- Ames, IA 50010) for authorization to rial of an autogenous biologic shall be prepare a product for use in herds not subject to the following restrictions: adjacent to the herd of origin. Because (1) Microorganisms used to prepare the recipient herd involved may not be autogenous biologics shall not be main- known when autogenous biologics are tained in the licensed establishment to be used in other geographic areas, beyond the time authorized for use in the following data may be used in place production. of the data required in paragraphs (2) The expiration date of the autog- (a)(2)(viii) and (a)(2)(ix) of this section. enous biologic shall not exceed 18 (i) Names and addresses of practi- months from the date of harvest. tioners in the area in place of the (c) Testing requirements for autogenous name, address, and phone number of biologics. (1) Final container samples of the adjacent herd owner. completed product from the first serial

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or subserial of an autogenous biologic tainers in a serial or subserial is 50 or produced from an isolate shall be test- less, two final container samples from ed for purity as prescribed in § 113.26, each serial and subserial shall be tested and for safety as prescribed in as prescribed in § 113.26(b): Provided, § 113.33(b) or § 113.38 except that: That, 1 ml aliquots from each sample (i) When the number of final con- may be inoculated into five cor- tainers in a serial or subserial is 50 or responding individual test vessels of less, two final container samples from each of the test media required. each serial and subserial shall be tested (ii) Safety test. Bulk of final container as prescribed in § 113.26(b): Provided, samples of completed product from That, 1 ml aliquots from each sample each serial shall be tested for safety as may be inoculated into five cor- provided in § 113.33 (b) or § 113.38. responding individual test vessels of (iii) Identification. All microorga- each of the test media required. nisms used for the production of autog- (ii) Serials which are satisfactory enous biologics shall be identified as after the third day of observation of follows: Bacteria, fungi, and myco- purity test cultures and of safety test plasma shall be identified at least to animals may be released for shipment genus and species. Viruses shall be to the customer and the tests contin- identified at least to family. After 15 ued throughout the required period; months from the date of isolation, or 12 and months from the harvest date of the (iii) Serials released on the basis of first serial of autogenous product pro- satisfactory results of third day obser- duced from a microorganism, which- vations shall be immediately recalled ever comes first, characterization and if evidence of contamination occurs in identification shall be completed to test cultures or if any of the test ani- strain and/or serotype before such mals used to demonstrate product safe- microorganism may be used for produc- ty, sicken, or die during the observation. tion period. (iv) Test summaries must be sub- (iv) Antigenicity, or immunogenicity, mitted to the Administrator (in c/o the and potency. Persons seeking author- Director, Center for Veterinary Bio- ization to prepare additional serials of logics, Inspection and Compliance, 1920 autogenous biologics from microorga- Dayton Avenue, P.O. Box 844, Ames, IA nisms that are older than 24 months 50010) on a quarterly basis by the 21st from the date of isolation, shall be re- day of January, April, July, and Octo- quired to conduct the following addi- ber or more often as required by the tional tests: Administrator. (A) Completed product shall be tested (2) Each serial or subserial of autog- for antigenicity or immunogenicity in enous bacterial product other than the the species for which the product is first serial or subserial produced from recommended or in another animal an isolate shall meet the applicable species whose immunological response general requirements prescribed in has been shown in the scientific lit- § 113.100 and the special requirements erature to correlate with the response prescribed in this section. Each serial of the species for which the product is or subserial of autogenous viral prod- recommended. Such tests shall be con- uct other than the first serial or sub- ducted in accordance with a protocol serial produced from an isolate shall developed by the licensee and approved meet the applicable general require- by the Administrator and the results ments prescribed in § 113.200 and the submitted to the Director, Center for special requirements prescribed in this Veterinary Biologics, Policy, Evalua- section. A serial or subserial found un- tion, and Licensing, 1920 Dayton Ave- satisfactory by any prescribed test nue, P.O. Box 844, Ames, IA 50010 for re- shall not be released. view. Microorganisms not shown to be (i) Purity test. Final container sam- antigenic (that is, not shown to induce ples of completed product from each se- a significant serological response) or rial and subserial shall be tested for immunogenic by such approved tests viable bacteria and fungi as provided in shall not be used for the preparation of § 113.26. When the number of final con- such product.

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(B) Bulk or final container samples (c) Potency test. Bulk or final con- of completed product from each serial tainer samples of completed product of such autogenous biologics con- from each serial shall be tested for po- taining fractions for which standard re- tency. A group of 10 guinea pigs con- quirement potency test procedures sisting of an equal number of males have been established shall be tested and females weighing 450 to 550 grams for potency in accordance with applica- shall each be injected subcutaneously ble standard requirement potency tests with 0.4 of the largest dose rec- provided in 9 CFR part 113. If the cul- ommended on the product labels. ture of microorganisms used to produce (1) Six weeks after injection, all sur- such fractions is shown to be of a dif- viving guinea pigs shall be bled and ferent strain or serotype than the rea- equal portions of serum, but not less gent or challenge microorganisms used than 0.5 ml from each, shall be pooled. in the standard requirement potency For a valid test, the pool shall contain test, reagents or challenges of the same the serum from at least eight animals. strain or serotype as the microorga- (2) The antitoxin titer of the pooled nism used for production may be used. serum shall be determined in antitoxin (C) If no standard requirement po- units (A.U.) per ml using an enzyme- tency test procedures have been estab- linked immunosorbent assay method lished for a fraction(s) in the autog- acceptable to the Animal and Plant enous biologic, such fraction(s) of each Health Inspection Service. serial of product shall be tested for po- (3) If the antitoxin titer of the serum tency using a developmental potency test described in the filed outline of pool is at least 2.0 A.U. per ml, the se- production or shall at least be stand- rial is satisfactory. If the antitoxin ardized to contain an antigenic mass titer of the serum pool is less than 2.0 for such fraction(s) that has been A.U. per ml, the serial may be retested shown to be antigenic or immunogenic by the following procedure: Provided, in accordance with paragraph That, if the serial is not retested, it (c)(2)(iv)(A) of this section. shall be declared unsatisfactory. (4) For serials in which the serum [57 FR 38756, Aug. 27, 1992, as amended at 59 pool contains less than 2.0 A.U. per ml, FR 67616, Dec. 30, 1994; 64 FR 43044, Aug. 9, 1999; 67 FR 15714, Apr. 3, 2002; 75 FR 20773, the individual serum that constituted Apr. 21, 2010] the pool may be tested by the enzyme- linked immunosorbent assay. If at § 113.114 Tetanus Toxoid. least 80 percent of the individual se- Tetanus Toxoid shall be produced rums have an antitoxin titer of at least from a culture of Clostridium tetani 2.0 A.U. per ml, the serial is satisfac- which has been inactivated and is tory. If less than 80 percent of the indi- nontoxic. The toxoid may be either ab- vidual serums have an antitoxin titer sorbed, precipitated, or purified and of at least 2.0 A.U. per ml, the serial concentrated. Each serial of biological may be retested in 10 guinea pigs using product containing tetanus toxoid frac- the procedure described in (c)(1) and (2) tion shall meet the applicable require- above. The antitoxin titer of the pooled ments in § 113.100 and shall be tested for serum from the guinea pigs used in the purity, safety, and potency as pre- retest shall be averaged with the anti- scribed in this section. A serial or sub- toxin level of the pooled serum from serial found unsatisfactory by any pre- the initial test. If the average of the scribed test shall not be released. two pools is at least 2.0 A.U. per ml, (a) Purity test. Final container sam- the serial is satisfactory. If the average ples of completed product from each se- of the two pools is less than 2.0 A.U. rial and subserial shall be tested for per ml, the serial is unsatisfactory and viable bacteria and fungi as provided in shall not be retested further. § 113.26. [39 FR 16862, May 10, 1974, as amended at 46 (b) Safety test. Bulk or final container FR 23224, Apr. 24, 1981; 50 FR 24905, June 14, samples of completed product from 1985. Redesignated at 55 FR 35562, Aug. 31, each serial shall be tested for safety as 1990, as amended at 56 FR 37827, Aug. 9, 1991; provided in § 113.33(b). 56 FR 66785, Dec. 26, 1991]

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§ 113.115 Staphylococcus Aureus (4) Make serial twofold dilutions of Bacterin-Toxoid. the serum samples and conduct the Staphylococcus Aureus Bacterin- test, using 1 ml of the serial dilutions. Toxoid shall be prepared from toxoided Appropriate controls should be in- broth cultures of selected toxogenic cluded for accurate interpretations. strains of Staphylococcus aureus which (5) Add 1 ml of the standardized toxin has been inactivated and is nontoxic. containing the established ‘‘Lh’’ dose. Each serial of biological product con- The ‘‘Lh’’ dose is the amount of toxin taining Staphylococcus Aureus which when mixed with one unit of Bacterin-Toxoid shall meet the appli- standard antitoxin produces a 50 per- cable requirements in § 113.100 and shall cent hemolysis of rabbit red blood be tested for purity, safety, and po- cells. tency as prescribed in this section. A (6) Incubate toxin-antitoxin mixture serial found unsatisfactory by any pre- at room temperature for 30 minutes scribed test shall not be released. (a) Purity test. Final container sam- and add 1 ml of a 1.5 percent suspension ples of completed product from each se- of washed freshly drawn rabbit red rial shall be tested for viable bacteria blood cells suspended in normal saline and fungi as provided in § 113.26. to each tube. Mix and incubate the (b) Safety test. Bulk or final container combined product in a 37 °C water bath samples of completed product shall be for 1 hour. Refrigerate at 5 °C over- tested for safety as provided in night. § 113.33(b). Also, the rabbits used in the (7) Read the hemolysis produced and potency test provided in paragraph (c) establish the 50 percent end point. The of this section shall constitute an addi- 50 percent end point of hemolysis tional safety test. If unfavorable reac- should be established by determining tions attributable to the product occur the size of the button produced by the in any of the rabbits during the obser- unlysed red blood cells. vation period, the serial is unsatisfac- (8) Determine the units of antitoxin tory. per 1 ml of serum. (c) Potency test. Rabbits, each weigh- (9) If the individual samples from ing 2000–3000 grams, shall be used as four of the five rabbits in the indi- test animals. Either a five rabbit individual serum test or the pooled sam- vidual serum test or an eight rabbit pooled serum test shall be conducted. ples from the eight rabbits in the At the start of the test, individual se- pooled serum test do not contain three rums from the five rabbits or pooled se- alpha antitoxin units per ml, the serial rums from the eight rabbits shall con- is unsatisfactory. tain less than 0.2 alpha antitoxin units [39 FR 16862, May 10, 1974. Redesignated at 55 per ml. FR 35562, Aug. 31, 1990, as amended at 56 FR (1) Each rabbit shall be given a series 66785, Dec. 26, 1991] of not more than three intramuscular injections at 7 day intervals (1.0 ml, 2.0 § 113.116 Pasteurella Multocida ml, 3.0 ml) and observed from 7–14 days Bacterin, Avian Isolate, Type 4. following the third injection. At the Pasteurella Multocida Bacterin, end of the observation period, a blood Avian Isolate, Type 4 shall be prepared sample shall be taken from each rabbit. from cultures of Pasteurella multocida, (2) The sample of serum from each avian isolate, Type 4 (Little and Lyons rabbit, if the five rabbit individual test classification), which have been inac- is conducted or a pooled sample of equal quantities of serum from the rab- tivated, and are nontoxic. Each serial bits if the eight rabbit pooled serum of biological product containing test is conducted, shall be tested to de- Pasteurella Multocida Bacterin, Avian termine the staphylococcus alpha anti- Isolate, Type 4, shall meet the applica- toxin units per ml as provided in parable requirements in § 113.100 and shall graphs (c)(3), (4), (5), (6), (7), and (8) of be tested for purity, safety, and po- this section. tency, as prescribed in this section. A (3) Inactivate rabbit serum 56 °C for serial found unsatisfactory by any pre- 30 minutes. scribed test shall not be released.

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(a) Purity test. Final container sam- rial is unsatisfactory if the test is not ples of completed product shall be test- repeated. ed for viable bacteria and fungi as pro- Cummulative total number vided in 9 CFR 113.26. Cumu- of dead vaccinates Number lative lll (b) Safety test. Observation of the vac- Stage of vac- number for cinated turkeys during the cinates of vac- Satisfactory Unsatisfac- prechallenge period of the potency test cinates serial tory serial provided in paragraph (c) of this sec- 1 ...... 20 20 6 or less ..... 9 or more. tion shall constitute the safety test. If 2 ...... 20 40 15 or less ... 16 or more. unfavorable reactions that are attributable to the product occur, the serial (5) The serial shall pass or fail based is unsatisfactory. If unfavorable reac- on the stage one results of the potency tions that are not attributable to the test. However, the second stage may be product occur in one turkey, test re- conducted if seven or eight vaccinates sults shall be determined by observing die in stage one, but the serial is unsat- the remaining 20 turkeys. The test is a isfactory if the second stage is not con- No Test and may be repeated if unfa- ducted. vorable reactions that are not attrib- (6) The second stage shall be con- utable to the product occur in two or ducted in a manner identical to the more turkeys, but the serial is unsatis- first stage. The serial shall be evalu- factory if the test is not repeated. ated according to stage two of the (c) Potency test. Bulk or final con- table. On the basis of accumulated re- tainer samples of completed product sults from the data of both stage tests, shall be tested for potency of the Type a serial shall either pass or fail the sec- 4 strain, using the two-stage test pro- ond stage. vided in this paragraph. Turkeys at [47 FR 5795, Feb. 4, 1982; 47 FR 6817, Feb. 17, least 6 weeks old obtained from the 1982, as amended at 52 FR 9117, Mar. 23, 1987. same source and hatch shall be prop- Redesignated at 55 FR 35562, Aug. 31, 1990, as erly identified and used as provided in amended at 56 FR 66785, Dec. 26, 1991] this paragraph. (1) Vaccinates. Each of not more than § 113.117 Pasteurella Multocida 21 turkeys shall be vaccinated with the Bacterin, Avian Isolate, Type 1. dose and by the route recommended on Pasteurella Multocida Bacterin, the label. A second dose shall be given Avian Isolate, Type 1, shall be prepared after 3 weeks and the turkeys observed from cultures of Pasteurella multocida, for an additional 2-week prechallenge avian isolate, Type 1 (Little and Lyons period. classification), which have been inac- (2) Unvaccinated controls. Each of not tivated and are nontoxic. Each serial of more than 11 turkeys shall be held as biological product containing controls. Pasteurella Multocida Bacterin, Avian (3) Challenge. Not less than 14 days Isolate, Type 1, shall meet the applica- after the second dose, each of 20 vac- ble requirements in § 113.100 and shall cinates, and each of 10 unvaccinated be tested for purity, safety, and po- controls shall be challenged tency as prescribed in this section. A intramuscularly with virulent serial found unsatisfactory by any pre- Pasteurella multocida, Strain P–1662, scribed test shall not be released. Type 4 (Little and Lyons classification) (a) Purity test. Final container sam- and observed daily for a 14-day ples of completed product shall be test- postchallenge period. Only dead birds ed for viable bacteria and fungi as pro- shall be considered in evaluating the vided in § 113.26. product. (b) Safety test. Observation of the vac- (4) Validity requirements. Eight or cinated chickens during the more unvaccinated controls must die prechallenged period of the potency for the test to be valid. If this require- test provided in paragraph (c) of this ment is met, the potency test results section shall constitute the safety test. are evaluated according to stage one of If unfavorable reactions that are at- the following table. The test is a No tributable to the product occur, the se- Test and may be repeated if the valid- rial is unsatisfactory. If unfavorable ity requirement is not met, but the se- reactions that are not attributable to

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the product occur in one chicken, test test. However, the second stage may be results shall be determined by observ- conducted if seven or eight vaccinates ing the remaining 20 chickens. The test die in stage one, but the serial is unsatis a No Test and may be repeated if un- isfactory if the second stage is not con- favorable reactions that are not attrib- ducted. utable to the product occur in two or (6) The second stage shall be con- more chickens, but the serial is unsat- ducted in a manner identical to the isfactory if the test is not repeated. first stage. The serial shall be evalu- (c) Potency test. Bulk or final con- ated according to stage two of the tainer samples of completed product table. On the basis of accumulated re- shall be tested for potency of the Type sults from the data of both stage tests, 1 strain, using the two-stage test pro- a serial shall either pass or fail the sec- vided in this paragraph. Chickens, at ond stage. least 12 weeks of age, obtained from [39 FR 16866, May 10, 1974; 39 FR 20368, June the same source and hatch, shall be 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; properly identified and used as pro- 40 FR 23989, June 4, 1975; 47 FR 5195, Feb. 4, vided in this paragraph. 1982; 52 FR 9118, Mar. 23, 1987. Redesignated (1) Vaccinates. Each of not more than at 55 FR 35562, Aug. 31, 1990, as amended at 56 21 chickens shall be injected with the FR 66785, Dec. 26, 1991] dose and by the route recommended on the label. A second dose shall be in- § 113.118 Pasteurella Multocida jected after 3 weeks and the chickens Bacterin, Avian Isolate, Type 3. observed for an additional 2 week Pasteurella Multocida Bacterin, prechallenge period. Avian Isolate, Type 3, shall be prepared (2) Unvaccinated controls. Each of not from culture of Pasteurella multocida, more than 11 chickens shall be held as avian isolate, Type 3 (Little and Lyons controls. classification), which have been inac- (3) Challenge. Not less than 14 days tivated and are nontoxic. Each serial of after the second injection, each of 20 biological product containing vaccinates, and each of 10 unvaccinated Pasteurella Multocida Bacterin, Avian controls shall be challenged Isolate, Type 3, shall meet the applica- intramuscularly with a minimum of 250 ble requirements in § 113.100 and shall colony-forming units of virulent be tested for purity, safety, and po- Pasteurella multocida, Strain X–73, Type tency, as prescribed in this section. A 1 (Little and Lyons classification) and serial found unsatisfactory by any pre- observed daily for a 14-day scribed test shall not be released. postchallenge period. Only dead birds (a) Purity test. Final container sam- shall be considered in evaluating the ples of completed product shall be test- product. ed for viable bacteria and fungi as pro- (4) Validity requirements. Eight or vided in § 113.26. more unvaccinated controls must die (b) Safety test. Observation of the vac- for the test to be valid. If these re- cinated turkeys during the quirement are met, the potency test re- prechallenge period of the potency test sults are evaluated according to stage provided in paragraph (c) of this sec- one of the following table. The test is a tion shall constitute the safety test. If No Test and may be repeated if the va- unfavorable reactions that are attrib- lidity requirements are not met, but utable to the product occur, the serial the serial is unsatisfactory if the test is unsatisfactory. If unfavorable reac- is not repeated. tions that are not attributable to the product occur in one turkey, test re- Cummulative total number Cumu- of dead vaccinates sults shall be determined by observing Number lative lll Stage of vac- number for the remaining 20 turkeys. The test is a cinates of vac- Satisfactory Unsatisfac- No Test and may be repeated if unfa- cinates serial tory serial vorable reactions that are not attrib- 1 ...... 20 20 6 or less .... 9 or more. utable to the product occur in two or 2 ...... 20 40 15 or less .. 16 or more. more turkeys, but the serial is unsatisfactory if the test is not repeated. (5) The serial shall pass or fail based (c) Potency test. Bulk or final con- on the stage one results of the potency tainer samples of completed product

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shall be tested for potency of the Type sults from the data of both stage tests, 3 strain, using the two-stage test pro- a serial shall either pass or fail the sec- vided in this paragraph. Turkeys, at ond stage. least 6 weeks of age, obtained from the [39 FR 16862, May 10, 1974, as amended at 40 same source and hatch, shall be prop- FR 759, Jan. 3, 1975; 47 FR 5196, Feb. 4, 1982; erly identified and used as provided in 52 FR 9118, Mar. 23, 1987. Redesignated at 55 this paragraph. FR 35562, Aug. 31, 1990, as amended at 56 FR (1) Vaccinates. Each of not more than 66785, Dec. 26, 1991] 21 turkeys shall be injected with the dose and by the route recommended on § 113.119 Erysipelothrix Rhusiopathiae the label. A second dose shall be in- Bacterin. jected after 3 weeks and the turkeys Erysipelothrix Rhusiopathiae observed for an additional 2 week Bacterin shall be produced from a cul- prechallenge period. ture of Erysipelothrix rhusiopathiae (2) Unvaccinated controls. Each of not which has been inactivated and is more than 11 turkeys shall be held as nontoxic. Each serial of biological controls. product containing Erysipelothrix (3) Challenge. Not less than 14 days rhusiopathiae shall meet the applicable after the second injection, each of 20 requirements in § 113.100 and shall be vaccinates, and each of 10 unvaccinated tested for purity, safety, and potency controls shall be challenged as prescribed in this section. A serial intramuscularly with a minimum of 150 found unsatisfactory by any prescribed colony-forming units of virulent test shall not be released. Pasteurella multocida, Strain P–1059, (a) Purity test. Final container sam- Type 3 (Little and Lyons Classifica- ples of completed product from each se- tion) and observed daily for a 14-day rial and each subserial shall be tested postchallenge period. Only dead birds for viable bacteria and fungi as pro- shall be considered in evaluating the vided in § 113.26. product. (b) Safety test. Bulk or final container (4) Validity requirements. Eight or samples of completed product from more unvaccinated controls must die each serial shall be tested for safety as for the test to be valid. If these re- provided in § 113.38. quirements are met, the potency test (c) Potency test. Bulk or final con- results are evaluated according to tainer samples of completed product stage one of the following table. The from each serial shall be tested for po- test is a No Test and may be repeated tency using the mouse protection test if the validity requirements are not provided in this paragraph. A mouse met, but the serial is unsatisfactory if dose shall be 1⁄10 of the least dose rec- the test is not repeated. ommended on the label for swine. Such swine dose shall not be less than 1 ml. Cummulative total number Cumu- of dead vaccinates (1) The ability of the bacterin being Number lative lll Stage of vac- number for tested (Unknown) to protect mice shall cinates of vac- Satisfactory Unsatisfac- be compared with a Standard Reference cinates serial tory serial Bacterin (Standard) which is either 1 ...... 20 20 6 or less ..... 9 or more. supplied by or acceptable to Animal 2 ...... 20 40 15 or less ... 16 or more. and Plant Health Inspection Service. (2) At least three threefold dilutions (5) The serial shall pass or fail based shall be made with the Standard and on the stage one results of the potency the same threefold dilutions shall be test. However, the second stage may be made for each Unknown. Dilutions conducted if seven or eight vaccinates shall be made with physiological saline die in stage one, but the serial is unsat- solution. isfactory if the second stage is not con- (3) For each dilution of the Standard ducted. and each dilution of an Unknown, a (6) The second stage shall be con- group of at least 20 mice, each weigh- ducted in a manner identical to the ing 16 to 22 grams, shall be used. Each first stage. The serial shall be evalu- mouse in each group shall be injected ated according to stage two of the subcutaneously with one mouse dose of table. On the basis of accumulated re- the appropriate dilution.

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(4) Each of 20 injected mice from each ner identical to the initial test. The av- group shall be challenged erage of the RP values obtained in the subcutaneously 14 to 21 days after retests shall be determined. If the aver- being injected. A dose containing at age RP is less than 0.6, the serial is un- least 100 mouse LD50 of a suitable cul- satisfactory without further testing. If ture of Erysipelothrix rhusiopathiae shall the average RP obtained in the retests be used. All survivors in each group of is equal to or greater than 0.6, the fol- mice shall be recorded 10 days lowing shall apply: postchallenge. (i) If the RP obtained in the original (5) Test for valid assay: At least two test is one-third or less than the aver- dilutions of the Standard shall protect age RP obtained in the retests, the ini- more than 0 percent and two dilutions tial RP may be considered a result of shall protect less than 100 percent of test system error and the serial is sat- the mice injected. The lowest dilution isfactory for potency. of the Standard shall protect more (ii) If the RP value obtained in the than 50 percent of the mice. The high- original test is more than one-third the est dilution of the Standard shall pro- average RP obtained in the retests, a tect less than 50 percent of the mice. new average shall be determined using (6) The relative potency (RP) of the the RP values obtained in all tests. If Unknown is determined by comparing the new average is less than 0.6, the se- the 50 percent endpoint dilution (high- rial is unsatisfactory. est bacterin dilution protecting 50 percent of the mice) of the Unknown with [39 FR 16862, May 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; 40 FR 20067, May 8, 1975; that of the standard by the following 40 FR 51414, Nov. 5, 1975; 44 FR 71408, Dec. 11, formula: 1979; 50 FR 23795, June 6, 1985; 51 FR 23731, July 1, 1986. Redesignated at 55 FR 35562, reciprocal of 50 percent Aug. 31, 1990; 56 FR 66558, Dec. 24, 1991; 56 FR endpoint dilution of Unknown 66784, 66785, Dec. 26, 1991] RP = reciprocal of 50 percent § 113.120 Salmonella Typhimurium endpoint dilution of Standard Bacterin. (7) If the RP of the Unknown is less Salmonella Typhimurium Bacterin than 0.6, the serial being tested is un- shall be prepared from a culture of Sal- satisfactory. monella typhimurium which has been in- (8) If the 50 percent endpoint of an activated and is nontoxic. Each serial Unknown in a valid test cannot be cal- of biological product containing Sal- culated because the lowest dilution monella typhimurium fraction shall does not exceed 50 percent protection, meet the applicable requirements in that serial may be retested in a man- § 113.100 and shall be tested for purity, ner identical to the initial test: Pro- safety, and potency as prescribed in vided, That, if the Unknown is not re- this section. A serial found unsatisfac- tested or if the protection provided by tory by any prescribed test shall not be the lowest dilution of the Standard ex- released. ceeds the protection provided by the (a) Purity test. Final container sam- lowest dilution of the Unknown by six ples of completed product shall be test- mice or more; or, if the total number of ed for viable bacteria and fungi as pro- mice protected by the Standard ex- vided in § 113.26. ceeds the total number of mice pro- (b) Safety test. Bulk or final container tected by the Unknown by eight mice samples of completed product from or more, the serial is unsatisfactory. each serial shall be tested for safety as (9) If the 50 percent endpoint of an provided in § 113.33(b). Unknown in a valid test cannot be cal- (c) Potency test. Bulk or final con- culated because the highest dilution tainer samples of completed product exceeds 50 percent protection, the Un- from each serial shall be tested for po- known is satisfactory without addi- tency using the mouse test provided in tional testing. this paragraph. A mouse dose shall be (10) If the RP is less than 0.6, the se- 1⁄20 of the least dose recommended on rial may be retested by conducting two the label for other animals which shall independent replicate tests in a man- not be less than 2 ml.

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(1) The ability of the bacterin being retested in a manner identical to the tested (Unknown) to protect mice shall initial test; Provided, That, if the Un- be compared with a Standard Reference known is not retested or if the protec- Bacterin (Standard) which is either tion provided by the lowest dilution of supplied by or acceptable to Animal the Unknown by six mice or more; or, and Plant Health Inspection Service. if the total number of mice protected (2) At least three tenfold dilutions by the Standard exceeds the total num- shall be made with the Standard and ber of mice protected by the Unknown the same tenfold dilutions shall be by eight mice or more, the serial being made for each Unknown. The dilutions tested is unsatisfactory. shall be made in Phosphate Buffered (9) If the 50 percent endpoint of an Saline. Unknown in a valid test cannot be cal- (3) For each dilution of the Standard culated because the highest dilution and each dilution of an Unknown, a exceeds 50 percent protection, the Un- group of at least 20 mice, each weigh- known is satisfactory without addi- ing 16-22 grams, shall be used. Each tional testing. mouse in a group shall be injected (10) If the RP is less than the min- intraperitoneally with one mouse dose imum required in paragraph (c)(7) of of the appropriate dilution. Each this section, the serial may be retested mouse shall be revaccinated on day 14, by conducting two independent rep- using the same schedule. licate tests in a manner identical to (4) Each of 20 vaccinated mice per the initial test. The average of the RP group shall be challenged values obtained in the retests shall be intraperitoneally 7–10 days after the determined. If the average RP is less second vaccination with a 0.25 ml dose than the required minimum, the serial containing 100-10,000 mouse LD50 as de- is unsatisfactory. If the average RP ob- termined by titration, of a suitable cul- tained in the retests is equal to or ture of Salmonella typhimurium. All sur- greater than the required minimum, vivors in each group of mice shall be the following shall apply: recorded 14 days postchallenge. (5) Test for valid assay: At least two (i) If the RP obtained in the original dilutions of the Standard shall protect test is one-third or less than the aver- more than 0 percent and two dilutions age RP obtained in the retests, the ini- shall protect less than 100 percent of tial RP may be considered a result of the mice injected. The lowest dilution test system error and the serial is sat- of the Standard shall protect more isfactory. than 50 percent of the mice. The high- (ii) If the RP value obtained in the est dilution of the Standard shall pro- original test is more than one-third the tect less than 50 percent of the mice. average RP obtained in the retests, a (6) The relative potency (RP) of the new average shall be determined using Unknown is determined by comparing the RP values obtained in all tests. If the 50 percent endpoint dilution (high- the new average is less than the min- est bacterin dilution protecting 50 per- imum required in paragraph (c)(7) of cent of the mice) of the Unknown with this section, the serial is unsatisfac- that of the Standard by the following tory. formula: [40 FR 17003, Apr. 16, 1975, as amended at 42 FR 59487, Nov. 18, 1977; 48 FR 31008, July 6, reciprocal of 50 percent 1983. Redesignated at 55 FR 35562, Aug. 31, endpoint dilution of Unknown 1990, as amended at 56 FR 66784, 66785, Dec. RP = 26, 1991] reciprocal of 50 percent endpoint dilution of Standard § 113.121 Pasteurella Multocida (7) If the RP of the Unknown is less Bacterin. than 0.30, the serial being tested is un- Pasteurella Multocida Bacterin shall satisfactory. be prepared from a culture of (8) If the 50 percent endpoint of an Pasteurella multocida strains other than Unknown cannot be calculated because avian which have been inactivated and the lowest dilution does not exceed 50 are nontoxic. Each serial of biological percent protection, that serial may be product containing Pasteurella

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multocida fraction shall meet the appli- of the Standard shall protect more cable requirements in § 113.100 and shall than 50 percent of the mice. The high- be tested for purity, safety, and po- est dilution of the Standard shall pro- tency as prescribed in this section. A tect less than 50 percent of the mice. serial found unsatisfactory by any pre- (6) The relative potency (RP) of the scribed test shall not be released. Unknown is determined by comparing (a) Purity test. Final container sam- the 50 percent endpoint dilution (high- ples of completed product from each se- est bacterin dilution protecting 50 per- rial and each subserial shall be tested cent of the mice) of the Unknown with for viable bacteria and fungi as pro- that of the Standard by the following vided in § 113.26. formula: (b) Safety test. Bulk or final container samples of completed product from reciprocal of 50 percent each serial shall be tested for safety as endpoint dilution of Unknown RP = provided in § 113.33(b). The subcuta- reciprocal of 50 percent neous route is to be used. (c) Potency test. Bulk or final con- endpoint dilution of Standard tainer samples of completed product (7) If the RP of the Unknown is less from each serial shall be tested for po- than 0.50, the serial being tested is un- tency using the mouse test provided in satisfactory. this paragraph. A mouse dose shall be (8) If the 50 percent endpoint of an 1⁄20 of the least dose recommended on Unknown cannot be calculated because the label for other animals which shall the lowest dilution does not exceed 50 not be less than 2 ml. percent protection, that serial may be (1) The ability of the bacterin being retested in a manner identical to the tested (Unknown) to protect mice shall initial test: Provided, That, if the Un- be compared with a Standard Reference known is not retested or if the protec- Bacterin (Standard) which is either tion provided by the lowest dilution of supplied by or acceptable to Animal the Standard exceeds the protection and Plant Health Inspection Service. provided by the lowest dilution of the (2) At least three fivefold dilutions Unknown by six mice or more; or, if shall be made with the Standard and the total number of mice protected by the same fivefold dilutions shall be the Standard exceeds the total number made for each Unknown. The dilutions of mice protected by the Unknown by will be made in Phosphate Buffered Sa- eight mice or more, the serial being line. tested is unsatisfactory. (3) For each dilution of the Standard (9) If the 50 percent endpoint of an and each dilution of each Unknown, a Unknown in a valid test cannot be cal- group of at least 20 mice, each weigh- culated because the highest dilution ing 16–22 grams, shall be used. Each exceeds 50 percent protection, the Un- mouse in a group shall be injected known is satisfactory without addi- intraperitoneally with one mouse dose tional testing. of the appropriate dilution. Each (10) If the RP is less than the min- mouse shall be revaccinated on day 14, imum required in paragraph (c)(7) of using the same schedule. this section, the serial may be retested (4) Each of 20 injected mice per group by conducting two independent rep- shall be challenged intraperitoneally licate tests in a manner identical to 10–12 days after the second vaccination the initial test. The average of the RP with a 0.2 ml dose containing 100–10,000 values obtained in the retests shall be mouse LD50, as determined by titra- determined. If the average RP is less tion, of a suitable culture of Pasteurella than the required minimum, the serial multocida. All survivors in each group is unsatisfactory. If the average RP ob- of mice shall be recorded 10 days tained in the retests is equal to or postchallenge. greater than the required minimum, (5) Test for valid assay: At least two the following shall apply: dilutions of the Standard shall protect (i) If the RP obtained in the original more than 0 percent and two dilutions test is one-third or less than the aver- shall protect less than 100 percent of age RP obtained in the retests, the ini- the mice injected. The lowest dilution tial RP may be considered a result of

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test system error and the serial is sat- made for each Unknown. The dilutions isfactory. shall be made in Phosphate-Buffered (ii) If the RP value obtained in the Saline. original test is more than one-third the (3) For each dilution of the Standard average RP obtained in the retests, a and each dilution of an Unknown, a new average shall be determined using group of at least 20 mice, each weigh- the RP values obtained in all tests. If ing 16 to 22 grams, shall be used. Each the new average is less than the min- mouse in a group shall be injected imum required in paragraph (c)(7) of intraperitoneally with one mouse dose this section, the serial is unsatisfac- of the appropriate dilution. Each tory. mouse shall be revaccinated on day 14, [40 FR 17004, Apr. 16, 1975, as amended at 42 using the same schedule. FR 59487, Nov. 18, 1977; 48 FR 31008, July 6, (4) Each of 20 vaccinated mice per 1983. Redesignated at 55 FR 35562, Aug. 31, group shall be challenged 1990, as amended at 56 FR 66784, 66785, Dec. intraperitoneally 7 to 10 days after the 26, 1991] second vaccination with a 0.25 ml dose containing 10–1,000 mouse LD50 as de- § 113.122 Salmonella Choleraesuis termined by titration of a suitable cul- Bacterin. ture of Salmonella choleraesuis. All sur- Salmonella Choleraesuis Bacterin vivors in each group of mice shall be shall be prepared from a culture of Sal- recorded 14 days postchallenge. monella choleraesuis which has been in- (5) Test for valid assay: At least two activated and is nontoxic. Each serial dilutions of the Standard shall protect of biological product containing Sal- more than 0 percent and two dilutions monella choleraesuis fraction shall meet shall protect less than 100 percent of the applicable requirements in 9 CFR the mice injected. The lowest dilution 113.100 and shall be tested for purity, of the Standard shall protect more safety, and potency as prescribed in than 50 percent of the mice. The high- this section. A serial found unsatisfac- est dilution of the Standard shall pro- tory by any prescribed test shall not be tect less than 50 percent of the mice. released. (6) The relative potency (RP) of the (a) Purity test. Final container sam- Unknown is determined by comparing ples of completed product shall be test- the 50 percent endpoint dilution (high- ed for viable bacteria and fungi as pro- est bacterin dilution protecting 50 per- vided in 9 CFR 113.26. cent of the mice) of the Unknown with (b) Safety test. Bulk or final container that of the Standard by the following samples of completed product from formula: each serial shall be tested for safety as provided in 9 CFR 113.33(b). reciprocal of 50 percent The subcutaneous route shall be used endpoint dilution of Unknown RP = when the product is in combination reciprocal of 50 percent with Pasteurella Multocida Bacterin. (c) Potency test. Bulk or final con- endpoint dilution of Standard tainer samples of completed product (7) If the RP of the Unknown is less from each serial shall be tested for po- than 0.50, the serial being tested is un- tency using the mouse test provided in satisfactory. this paragraph. A mouse dose shall be (8) If the 50 percent endpoint of an 1⁄20 of the least dose recommended on Unknown cannot be calculated because the label for other animals which shall the lowest dilution does not exceed 50 not be less than 2 ml. percent protection, that serial may be (1) The ability of the bacterin being retested in a manner identical to the tested (Unknown) to protect mice shall initial test; Provided, That, if the Un- be compared with a Standard Reference known is not retested or if the protec- Bacterin (Standard) which is either tion provided by the lowest dilution of supplied by or acceptable to Veterinary the Standard exceeds the protection Services. provided by the lowest dilution of the (2) At least three fivefold dilutions Unknown by six mice or more; or, if shall be made with the Standard and the total number of mice protected by the same fivefold dilution shall be the Standard exceeds the total number

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of mice protected by the Unknown by (b) Safety test. Bulk or final container eight mice or more, the serial being samples of completed product from tested is unsatisfactory. each serial shall be tested for safety as (9) If the 50 percent endpoint of an provided in 9 CFR 113.33(b). Unknown in a valid test cannot be cal- (c) Potency test. Bulk or final con- culated because the highest dilution tainer samples of completed product exceeds 50 percent protection, the Un- from each serial shall be tested for po- known is satisfactory without addi- tency using the mouse test provided in tional testing. this paragraph. A mouse dose shall be (10) If the RP is less than the min- 1⁄20 of the least dose recommended on imum required in paragraph (c)(7) of the label for other animals which shall this section, the serial may be retested not be less than 2 ml. by conducting two independent rep- (1) The ability of the bacterin being licate tests in a manner identical to tested (Unknown) to protect mice shall the initial test. The average of the RP be compared with a Standard Reference values obtained in the retests shall be Bacterin (Standard) which is either determined. If the average RP is less supplied by or acceptable to Veterinary than the required minimum, the serial Services. is unsatisfactory. If the average RP ob- (2) At least three tenfold dilutions tained in the retests is equal to or shall be made with the Standard and greater than the required minimum, the same tenfold dilutions shall be the following shall apply: made for each Unknown. The dilutions (i) If the RP obtained in the original shall be made in Phosphate-Buffered test is one-third or less than the aver- Saline. age RP obtained in the retests, the ini- (3) For each dilution of the Standard tial RP may be considered a result of and each dilution of an Unknown, a test system error and the serial is sat- group of at least 20 mice, each weigh- isfactory. ing 16 to 22 grams, shall be used. Each (ii) If the RP value obtained in the mouse in a group shall be injected original test is more than one-third the intraperitoneally with one mouse dose average RP obtained in the retests, a of the appropriate dilution. Each new average shall be determined using mouse shall be revaccinated on day 14, the RP values obtained in all tests. If using the same schedule. the new average is less than the min- (4) Each of 20 vaccinated mice per imum required in paragraph (c)(7) of group shall be challenged this section, the serial is unsatisfac- intraperitoneally 7 to 10 days after the tory. second vaccination with a 0.25 ml dose [43 FR 25077, June 9, 1978, as amended at 48 containing 1,000–100,000 mouse LD50 as FR 31008, July 6, 1983. Redesignated at 55 FR determined by titration of a suitable 35562, Aug. 31, 1990, as amended at 56 FR culture of Salmonella dublin. All sur- 66785, Dec. 26, 1991] vivors in each group of mice shall be recorded 14 days postchallenge. § 113.123 Salmonella Dublin Bacterin. (5) Test for valid assay: At least two Salmonella Dublin Bacterin shall be dilutions of the Standard shall protect prepared from a culture of Salmonella more than 0 percent and two dilutions dublin which has been inactivated and shall protect less than 100 percent of is nontoxic. Each serial of biological the mice injected. The lowest dilution product containing Salmonella dublin of the Standard shall protect more fraction shall meet the applicable re- than 50 percent of the mice. The high- quirements in 9 CFR 113.100 and shall est dilution of the Standard shall pro- be tested for purity, safety, and po- tect less than 50 percent of the mice. tency as prescribed in this section. A (6) The relative potency (RP) of the serial found unsatisfactory by any pre- Unknown is determined by comparing scribed test shall not be released. the 50 percent endpoint dilution (high- (a) Purity test. Final container sam- est bacterin dilution protecting 50 per- ples of completed product shall be test- cent of the mice) of the Unknown with ed for viable bacteria and fungi as pro- that of the Standard by the following vided in 9 CFR 113.26. formula:

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this section, the serial is unsatisfac- reciprocal of 50 percent tory. endpoint dilution of Unknown RP = [43 FR 25077, June 9, 1978, as amended at 48 reciprocal of 50 percent FR 31009, July 6, 1983. Redesignated at 55 FR endpoint dilution of Standard 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991] (7) If the RP of the Unknown is less than 0.30, the serial being tested is un- KILLED VIRUS VACCINES satisfactory. (8) If the 50 percent endpoint of an § 113.200 General requirements for Unknown cannot be calculated because killed virus vaccines. the lowest dilution does not exceed 50 When prescribed in an applicable percent protection, that serial may be Standard Requirement or in the filed retested in a manner identical to the Outline of Production, a killed virus initial test; Provided, That, if the Un- vaccine shall meet the applicable re- known is not retested or if the protec- quirements in this section. tion provided by the lowest dilution of (a) Killing agent. The vaccine virus the Standard exceeds the protection shall be killed (inactivated) by an ap- provided by the lowest dilution of the propriate agent. The procedure in- Unknown by six mice or more; or, if volved may be referred to as inactiva- the total number of mice protected by tion. Suitable tests to assure complete the Standard exceeds the total number inactivation shall be written into the of mice protected by the Unknown by filed Outline of Production. eight mice or more, the serial being (b) Cell culture requirements. If cell tested is unsatisfactory. cultures are used in the preparation of (9) If the 50 percent endpoint of an the vaccine, primary cells shall meet Unknown in a valid test cannot be cal- the requirements in § 113.51 and cell culated because the highest dilution lines shall meet the requirements in exceeds 50 percent protection, the Un- § 113.52. known is satisfactory without addi- (c) Purity tests—(1) Bacteria and fungi. tional testing. Final container samples of completed (10) If the RP is less than the min- product from each serial shall be tested imum required in paragraph (c)(7) of as prescribed in § 113.26. this section, the serial may be retested (2) Avian origin vaccine. Bulk pooled by conducting two independent rep- material or final container samples licate tests in a manner identical to from each serial shall also be tested the initial test. The average of the RP for: values obtained in the retests shall be (i) Salmonella contamination as predetermined. If the average RP is less scribed in § 113.30; and than the required minimum, the serial (ii) Lymphoid leukosis virus contami- is unsatisfactory. If the average RP ob- nation as prescribed in § 113.31; and tained in the retests is equal to or (iii) Hemagglutinating viruses as pre- greater than the required minimum, scribed in § 113.34. the following shall apply: (3) Mycoplasma. If the licensee cannot (i) If the RP obtained in the original demonstrate that the agent used to kill test is one-third or less than the aver- the vaccine virus would also kill myco- age RP obtained in the retests, the ini- plasma, each serial of the vaccine shall tial RP may be considered a result of be tested for mycoplasma as prescribed test system error and the serial is sat- in § 113.28, prior to adding the killing isfactory. agent. Material found to contain myco- (ii) If the RP value obtained in the plasma is unsatisfactory for use. original test is more than one-third the (4) Extraneous viruses. Each lot of average RP obtained in the retests, a Master Seed Virus used to prepare new average shall be determined using killed virus vaccine recommended for the RP values obtained in all tests. If animals other than poultry shall meet the new average is less than the min- the requirements for extraneous vi- imum required in paragraph (c)(7) of ruses as prescribed in § 113.55.

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(d) Safety tests. Final container sam- able reactions not attributable to the ples of completed product from each se- vaccine occur, the test shall be de- rial shall be tested for safety in guinea clared a No Test and may be repeated: pigs as prescribed in § 113.38 and for Provided, That, if the test is not re- safety in mice as prescribed in § 113.33: peated, the serial is unsatisfactory. Provided, That, vaccines recommended (b) Potency test. Bulk or final con- for use only in poultry are exempt from tainer samples of completed product this requirement. shall be tested for potency using 10 (e) Viricidal activity test. Only serials mink enteritis susceptible mink (five tested for viricidal activity in accord- vaccinates and five controls) as fol- ance with the test provided in § 113.35 lows: and found satisfactory by such test (1) Vaccination. Each of the five vac- shall be packaged as diluent for des- cinates shall be injected with one dose iccated fractions in combination pack- of vaccine as recommended on the label ages. and observed each day for 14 days. (f) Formaldehyde content. If formaldehyde is used as the killing agent, the (2) Challenge. At least 2 weeks after residual free formaldehyde content the last inoculation, the five vac- must not exceed 0.74 grams per liter (g/ cinates and the five controls shall be L) as determined using the ferric chlo- challenged with virulent mink enter- ride test. 2 Firms currently using tests itis virus and observed each day for 12 for residual free formaldehyde content days. Fecal material shall be collected other than the ferric chloride test have on one day between days 4–8 (inclusive) until July 14, 2004 to update their Out- postchallenge from each test animal line of Production to be in compliance that remains free of enteric signs and with this requirement. tested for the presence of mink enteritis virus by cell culture with fluores- [39 FR 27428, July 29, 1974, as amended at 40 cent antibody examination. FR 23989, June 4, 1975; 43 FR 49528, Oct. 24, 1978. Redesignated at 55 FR 35562, Aug. 31, (3) Interpretation. A serial is satisfac- 1990; 68 FR 35283, June 13, 2003; 79 FR 31021, tory if at least 80 percent of the vac- May 30, 2014] cinates remain free of enteric signs and do not shed virus in the feces, while at §§ 113.201–113.203 [Reserved] least 80 percent of the controls develop clinical signs of mink enteritis or shed § 113.204 Mink Enteritis Vaccine, virus in the feces. If at least 80 percent Killed Virus. of the vaccinates remain free of enteric Mink Enteritis Vaccine, Killed Virus, signs and do not shed virus in the feces, shall be prepared from virus-bearing while less than 80 percent of the con- cell culture fluids or tissues obtained trols develop clinical signs of mink en- from mink that have developed mink teritis or shed virus in the feces, the enteritis following inoculation with test is considered a No Test and may be virulent mink enteritis virus. Each se- repeated: Provided, That, if at least 80 rial shall meet the applicable require- percent of the vaccinates do not re- ments prescribed in § 113.200 and special main well and free of detectable virus requirements prescribed in this sec- in the feces, the serial is unsatisfac- tion. Any serial found unsatisfactory tory. by a prescribed test shall not be released. [39 FR 27428, July 29, 1974. Redesignated at 55 (a) Safety test. Vaccinates used in the FR 35562, Aug. 31, 1990, as amended at 56 FR potency test in paragraph (b) of this 66786, Dec. 26, 1991; 60 FR 14361, Mar. 17, 1995] section shall be observed each day § 113.205 Newcastle Disease Vaccine, prior to challenge. If unfavorable reac- Killed Virus. tions attributable to the vaccine occur, the serial is unsatisfactory. If unfavor- Newcastle Disease Vaccine (Killed Virus) shall be prepared from virus- bearing tissues or fluids obtained from 2 The procedures for performing the ferric chloride test for residual free formaldehyde embryonated chicken eggs or cell cul- may be obtained from USDA, APHIS, Center tures. With the exception of for Veterinary Biologics-Laboratory, 1800 § 113.200(c)(2)(iii), each serial shall meet Dayton Road, P.O. Box 844, Ames, IA 50010. the applicable general requirements

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prescribed in § 113.200 and special re- (b) Safety. Bulk or final container quirements prescribed in this section. samples of completed product shall A serial found unsatisfactory by a pre- meet the requirements for safety as scribed test shall not be released. prescribed in §§ 113.33(b) and 113.38. (a) Safety test. The prechallenge part (c) Formaldehyde content. Bulk or of the potency test in paragraph (b) of final container samples of completed this section shall constitute a safety product shall meet the requirements test. If unfavorable reactions attrib- for formaldehyde content as prescribed utable to the product occur in any of in § 113.200(f). the vaccinates, the serial is unsatisfac- (d) Potency and efficacy. The efficacy tory. If unfavorable reactions which of wart vaccine has been demonstrated are not attributable to the product to the satisfaction of Veterinary Serv- occur, the test shall be declared a No ices as being a valuable biological Test and may be repeated: Provided, product. The inherent nature of the That, if the test is not repeated, the se- product precludes the possible develop- rial shall be declared unsatisfactory. ment of serial to serial potency tests (b) Potency test. A vaccination-chal- and none is required: Provided, That, lenge test shall be conducted using sus- (1) The vaccine shall be a tissue ex- ceptible chickens 2 to 6 weeks of age at tract representing at least 10 percent time of vaccination, properly identified weight to volume suspension of wart and obtained from the same source and tissue; and hatch. (2) The vaccine shall be limited to (1) Ten or more chickens shall be vac- use in the prevention of warts in cat- cinated as recommended on the label tle. Labeling recommendations shall be and kept isolated under observation for in accordance with § 112.7(h). at least 14 days. [40 FR 14084, Mar. 28, 1975, as amended at 40 (2) After at least 14 days post-vac- FR 23989, June 4, 1975; 40 FR 30803, July 23, cination, the vaccinates and at least 10 1975. Redesignated at 55 FR 35562, Aug. 31, unvaccinated chickens that have been 1990, as amended at 56 FR 66786, Dec. 26, 1991; kept isolated as controls shall be chal- 81 FR 59436, Aug. 30, 2016] lenged with a virulent strain of New- castle disease virus supplied by or ap- § 113.207 Encephalomyelitis Vaccine, Eastern, Western, and Venezuelan, proved by Veterinary Services and the Killed Virus. vaccinates observed each day for 14 days. Encephalomyelitis Vaccine, Eastern, (3) If at least 90 percent of the con- Western, and Venezuelan, Killed Virus, trols do not show typical signs of New- shall be prepared from virus-bearing castle disease or die, the test is a No cell culture fluids. Each serial or sub- Test and may be repeated. If at least 90 serial shall meet the requirements pre- percent of the vaccinates do not re- scribed in this section and the general main normal, the serial is unsatisfac- requirements prescribed in § 113.200, ex- tory. cept those in § 113.200(d). Any serial or subserial found unsatisfactory by a [39 FR 27428, July 29, 1974. Redesignated at 55 prescribed test shall not be released. FR 35562, Aug. 31, 1990, as amended at 56 FR (a) Safety test. Bulk samples of com- 66786, Dec. 26, 1991] pleted product from each serial shall be tested for encephalomyelitis virus in- § 113.206 Wart Vaccine, Killed Virus. activation. Wart Vaccine, Killed Virus, shall be (1) Each of at least ten 6 to 12 hour prepared from virus-bearing epidermal old chickens shall be injected tumors (warts) obtained from a bovine. subcutaneously with 0.5 ml of the prod- Each serial shall meet the require- uct and the chickens observed each day ments prescribed in this section and for 10 days. any serial found unsatisfactory by a (2) If unfavorable reactions attrib- prescribed test shall not be released. utable to the product occur in the (a) Purity. Final container samples of chickens during the observation period, completed product shall meet the re- the serial is unsatisfactory. If unfavor- quirements for purity as prescribed in able reactions not attributable to the § 113.200 (c)(1) and (3). product occur, the test is a No Test and

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may be repeated: Provided, That, if the (6) The results shall be evaluated ac- test is not repeated, the serial is unsat- cording to the following table: isfactory. (b) Potency test. Bulk or final con- CUMULATIVE TOTALS tainer samples of completed product Failures for Failures for from each serial shall be tested for po- Stage Vaccinates acceptance rejection tency in accordance with the two-stage test provided in this paragraph. For 1 ...... 10 ...... 1 or less ...... 4 or more. each fraction contained in the prod- 2 ...... 20 ...... 3 or less ...... Do. uct—Eastern type, Western type, or Venezuelan type—the serological inter- [39 FR 44714, Dec. 27, 1974, as amended at 40 pretations required in this test shall be FR 14084, Mar. 28, 1975; 42 FR 45284, Sept. 9, made independently. A serial or sub- 1977. Redesignated at 55 FR 35562, Aug. 31, serial found unsatisfactory for any of 1990, as amended at 56 FR 66786, Dec. 26, 1991; the fractions shall not be released. 61 FR 67930, Dec. 26, 1996] (1) For this test, a guinea pig dose § 113.208 Avian Encephalomyelitis shall be one-half the amount rec- Vaccine, Killed Virus. ommended on the label for a horse and shall be administered as recommended Avian Encephalomyelitis Vaccine for a horse. Each of 10 healthy guinea (Killed Virus) shall be prepared from pigs (vaccinates) shall be injected with virus-bearing tissues or fluids obtained two guinea pig doses with an interval from embryonated chicken eggs. Each of 14 to 21 days between doses. Two ad- serial shall meet the general require- ditional guinea pigs from the same ments prescribed in § 113.200 and the re- source shall be held as controls. quirements prescribed in this section. (2) Fourteen to 21 days after the sec- Any serial found unsatisfactory by a ond injection, serum samples from prescribed test shall not be released. each vaccinate and each control shall (a) Safety tests. (1) The prechallenge be tested by a plaque reduction, serum part of the potency test prescribed in neutralization test using Vero 76 cells. paragraph (b) of this section shall con- (3) If the control serum samples show stitute a safety test. If any of the vac- a titer of 1:4 or greater for any frac- cinates develop clinical signs of disease tion, the test is a No Test for that frac- or die due to causes attributable to the tion and may be repeated: Provided, product, the serial is unsatisfactory. That, if four or more of the vaccinate (2) An inactivation test for viable serum samples show a titer of less than avian encephalomyelitis (AE) virus 1:40 for the Eastern type fraction, less shall be conducted on each serial. The than 1:40 for the Western type fraction, test shall be conducted using suscep- or less than 1:4 for the Venezuelan type tible chicken embryos: Provided, That, fraction, the serial or subserial is un- if a non-embryo adapted virus is used satisfactory without further testing. for vaccine production, the test shall (4) If two or three of the vaccinate be conducted in susceptible chickens. serum samples show a titer of less than (i) Chicken Embryo Test. Each of 15 or 1:40 for the Eastern type fraction, less more AE susceptible 5 or 6 day old em- than 1:40 for the Western type fraction, bryos shall be injected in the yolk sac or less than 1:4 for the Venezuelan type with 0.2 ml of the vaccine. For a valid fraction, the second stage of the test test, at least 80 percent of the embryos may be used for the relevant frac- shall survive for 48 hours post-inoculation(s): Provided, That, if a fraction is tion (PI). Eleven to 13 days PI, all em- found acceptable by the first stage of bryos surviving the 48 hour PI period the test, the second stage need not be shall be examined for gross lesions of conducted for that fraction. AE; all these embryos shall be normal (5) If the second stage is used and or the serial is unsatisfactory. Concur- four or more of the vaccinate serum rently, five additional embryos from samples show a titer of less than 1:40 the same source shall be injected with for the Eastern type fraction or the live AE virus of the production strain Western type fraction, or less than 1:4 to serve as positive controls. At least 4 for the Venezuelan type fraction, the of the 5 embryos shall show evidence of serial or subserial is unsatisfactory. AE virus infection during the 11 to 13

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day PI period or the test shall be con- immunogenic shall be used for pre- sidered a No Test and repeated: Pro- paring the production seed virus for vided, That, if the test is not repeated, vaccine production. All serials of vac- the serial shall be declared unsatisfac- cine shall be prepared from the first tory. through the fifth passage from the (ii) Chicken test. Each of 10 or more Master Seed Virus. AE susceptible 7 day old chickens shall (a) The Master Seed Virus shall meet be injected intracerebrally with 0.1 ml the applicable requirements prescribed vaccine each. The chickens shall be ob- in § 113.200 and the requirements preserved each day for 28 days. If any scribed in this section. chickens show clinical signs of AE, the (1) Each lot of Master Seed Virus serial is unsatisfactory. Concurrently, propagated in tissue or cells of avian 5 additional chickens from the same origin shall also be tested for extra- source shall be injected intracerebrally neous pathogens by procedures pre- with live AE virus of the production scribed in § 113.37. strain to serve as positive controls. At (2) Each lot of Master Seed Virus least 4 of the 5 controls shall show evi- propagated in primary cell cultures of dence of AE virus infection during the mouse or hamster origin or brain tis- observation period or the test shall be sues of mouse origin shall be tested for a No Test and may be repeated: Pro- lymphocytic choriomeningitis (LCM) vided, That, if the test is not repeated, virus by the procedure prescribed in the serial shall be unsatisfactory. § 113.42. If LCM virus is detected, the (b) Potency test. Bulk or final container samples of completed product Master Seed Virus is unsatisfactory. from each serial or one subserial shall (b) The immunogenicity of vaccine be tested. Ten or more AE-susceptible prepared with virus at the highest pas- chickens (vaccinates), 4 weeks or older, sage from the Master Seed shall be es- properly identified and obtained from tablished in each species for which the the same source and hatch, shall be in- vaccine is recommended. Tests shall be jected as recommended on the label. At conducted in accordance with a pro- least 10 additional AE-susceptible tocol filed with Animal and Plant chickens, properly identified and ob- Health Inspection Service before initi- tained from the same source and hatch ation of the tests. The vaccine shall be shall be kept in isolation as controls. prepared using methods prescribed in (1) At least 28 days post-injection, the Outline of Production. If Rabies the vaccinates and the controls shall Vaccine is to be in combination with be challenged intramuscularly with a other fractions, the product to be test- virulent AE virus and the chickens ob- ed shall include all fractions to be test- served each day for 21 days. ed. (2) If at least 80 percent of the con- (1) The preinactivation virus titer trols do not show clinical signs of or must be established as soon as possible die from AE infection, the test is a No after harvest by at least five separate Test and may be repeated. virus titrations. A mean relative po- (3) If at least 80 percent of the vac- tency value of the vaccine to be used in cinates do not remain normal, the se- the host animal potency test must be rial is unsatisfactory. established by at least five replicate [39 FR 12958, Dec. 27, 1974, as amended at 40 potency tests conducted in accordance FR 41088, Sept. 5, 1975. Redesignated at 55 FR with the standard NIH test for potency 35562, Aug. 31, 1990, as amended at 56 FR in chapter 37 of ‘‘Laboratory Tech- 66786, Dec. 26, 1991] niques in Rabies,’’ Fourth Edition (1996), edited by F.X. Meslin, M.M. § 113.209 Rabies Vaccine, Killed Virus. Kaplan, and H. Koprowski, World Rabies Vaccine (Killed Virus) shall Health Organization, Geneva, Switzer- be prepared from virus-bearing cell cul- land (ISBN 92 4 154479 1). The provisions tures or nerve tissues obtained from of chapter 37 of ‘‘Laboratory Tech- animals that have developed rabies in- niques in Rabies,’’ Fourth Edition fection following injection with rabies (1996), are the minimum standards for virus. Only Master Seed Virus which achieving compliance with this section has been established as pure, safe, and and are incorporated by reference.

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These provisions state that the chal- tions, except as provided in (b)(4) of lenge virus standard to be used as the this section. The challenged animals challenge in the NIH test and the ref- shall be observed each day for 90 days erence vaccine for the test are avail- as prescribed in § 113.5(b). The brain of able from the national control author- each test animal that dies following ity. In the United States, that author- challenges shall be examined for rabies ity is the Animal and Plant Health In- by the fluorescent antibody test or spection Service’s Center for Veteri- other method acceptable to Animal and nary Biologics Laboratory, located at Plant Health Inspection Service. 1920 Dayton Avenue, P.O. Box 844, (v) Requirements for acceptance in Ames, IA 50010; phone (515) 337-6100; fax challenge tests shall be death due to (515) 337-6120. This incorporation by ref- rabies in at least 80 percent of the con- erence was approved by the Director of trols while at least 22 of 25 or 26 of 30 the Federal Register in accordance or a statistically equivalent number of with 5 U.S.C. 552(a) and 1 CFR part 51. the vaccinates remain well for a period Copies may be obtained from the World of 90 days. Health Organization Publications Cen- (4) An alternative to challenging all ter USA, 49 Sheridan Avenue, Albany, surviving test animals in accordance NY 12210. Copies may be inspected at with paragraph (b)(3)(iv) of this section the Animal and Plant Health Inspec- may be used when the test animals are tion Service, Center for Veterinary of species other than carnivores. Vac- Biologics, Policy, Evaluation, and Li- cinates shall be challenged at 1 year censing, 1920 Dayton Avenue, P.O. Box postvaccination. These shall include 844, Ames, IA 50010, or at the National five vaccinates with the lowest SN Archives and Records Administration titers at the 270th-day bleeding, five (NARA). For information on the avail- vaccinates with the lowest SN titers at ability of this material at NARA, call the 365th-day bleeding, and all vac- 202–741–6030, or go to: http:// cinates with SN titers below 1:10 by the www.archives.gov/federallregister/ mouse SN test or below 1:16 by the codeloflfederallregulations/ rapid-fluorescent-focus-inhibition test ibrllocations.html. at any bleeding. At least five SN-nega- (2) The dose of vaccine to be used in the immunogenicity test shall be no tive controls of each species shall be more than the amount which, on the challenged at the same time as the basis of The NIH Test For Potency, has vaccinates. All SN titers shall be been diluted to the proposed minimum titrated to an endpoint. All of the chal- acceptable potency value. 1 lenged vaccinates must remain well for (3) Test animals shall be uniform and a period of 90 days, and at least 80 per- have no neutralizing antibodies to ra- cent of the controls must die of rabies bies as determined by serum-neutral- for a satisfactory test without further ization (SN) tests. challenge. If one or more of the vac- (i) Twenty-five or more animals shall cinates die from rabies, all the remain- be used as vaccinates. Each shall be ad- ing vaccines, regardless of titer, along ministered a dose of vaccine at the pro- with the five controls shall be chal- posed minimum potency level and by lenged. The cumulative results from the method specified in the Outline of the two challenges shall be evaluated Production. for acceptance as specified in para- (ii) Ten or more additional animals graph (b)(3)(v) of this section. shall be held as controls. (5) An Outline of Production change (iii) On or about 30, 90, 180, 270, and shall be made before authority for use 365 days postvaccination, all test ani- a new lot of Master Seed Virus shall be mals shall be bled and individual serum granted by Animal and Plant Health samples tested for neutralizing anti- Inspection Service. bodies to rabies virus. (c) If more than 1 year duration of (iv) All surviving test animals shall immunity is to be claimed, a duration be challenged intramuscularly with of immunity test for the additional virulent rabies virus furnished or ap- time shall be conducted and inter- proved by Animal and Plant Health In- preted as prescribed in paragraph (b) of spection Service 1 year after vaccina- this section for the 1 year test. The

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test animals shall be monitored sero- in Chapter 37 of ‘‘Laboratory Tech- logically at least every 180 days. The niques in Rabies,’’ Fourth Edition time of challenge may be adjusted ac- (1996), which is incorporated by ref- cordingly. erence at paragraph (b)(1) of this sec- (d) Test requirements for release: tion. The relative potency of each se- Each serial and each subserial shall rial must be at least equal to that used meet the general requirements pre- in an approved host animal scribed in § 113.200 and special require- immunogenicity test. ments in this paragraph. [39 FR 44715, Dec. 27, 1974, as amended at 42 (1) Purity test. Primary cell cultures FR 6794, Feb. 4, 1977; 43 FR 49528, Oct. 24, 1978; of hamster origin or brain tissues of 50 FR 20090, May 14, 1985. Redesignated at 55 mouse origin used in vaccine produc- FR 35562, Aug. 31, 1990; 56 FR 66784, 66786, tion shall be tested for LCM virus as Dec. 26, 1991; 61 FR 31823, June 21, 1996; 64 FR prescribed in § 113.42. Hamster origin 45420, Aug. 20, 1999; 69 FR 18803, Apr. 9, 2004; cells shall be disrupted and undiluted 75 FR 20773, Apr. 21, 2010] cell fluids from each lot shall be tested. Where mouse brains are used in produc- § 113.210 Feline Calicivirus Vaccine, tion, at least five mice which have not Killed Virus. been injected with rabies virus shall be Feline Calicivirus Vaccine, Killed sacrificed and a 10 percent suspension Virus, shall be prepared from virus- of brain material shall be prepared and bearing cell culture fluids. Only Master tested. Seed which has been established as (2) Safety tests. Bulk samples from pure, safe, and immunogenic shall be each serial shall be tested for virus in- used for preparing seeds for vaccine activation and safety as follows: production. All serials of vaccine shall (i) At the end of the inactivation pe- be prepared from the first through the riod, each of 20 12 to 16 gram mice shall fifth passage from the Master Seed. be injected intracerebrally with 0.03 ml (a) The Master Seed shall meet the and two rabbits shall be injected into applicable general requirements pre- each cerebral hemisphere with 0.25 ml scribed in § 113.200. and observed each day for 21 days. The (b) The Master Seed shall be tested brains of animals dying between the for chlamydial agents as prescribed in fourth and 21st day post-injection shall § 113.43. be checked for rabies virus. Material (c) The immunogenicity of vaccine from each brain recovered shall be in- prepared from the Master Seed in ac- jected into each of five mice and the cordance with the Outline of Produc- mice observed each day for 14 days. The tion shall be established by a method fluorescent antibody test or serum neu- acceptable to Animal and Plant Health tralization test shall be used to con- Inspection Service. Vaccine used for firm the presence or absence or live ra- this test shall be at the highest passage bies virus. If live rabies virus is con- from the Master Seed and prepared at firmed, the serial is unsatisfactory un- the minimum preinactivation titer less reprocessed in accordance with specified in the Outline of Production. § 114.18. (d) Test requirements for release. Each (ii) A test for safety in three young serial and subserial shall meet the ap- seronegative animals of the most sus- plicable general requirements pre- ceptible species for which the vaccine scribed in § 113.200 and the special re- is recommended shall be conducted. quirements provided in this paragraph. Each shall in injected intramuscularly Any serial or subserial found unsatis- with one recommended dose of vaccine. factory by a prescribed test shall not If unfavorable reactions attributable to be released. the product occur during a 28 day ob- (1) Safety. Vaccinates used in the po- servation period, the serial is unsatis- tency test in paragraph (d)(2) of this factory. section shall be observed each day dur- (3) Potency test. Bulk or final con- ing the prechallenge period. If unfavor- tainer samples of completed product able reactions occur, including oral le- from each serial must be tested for po- sions, which are attributable to the tency by tests conducted in accordance vaccine, the serial is unsatisfactory. If with the standard NIH test for potency unfavorable reactions occur which are

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not attributable to the vaccine, the § 113.211 [Reserved] test is inconclusive and may be repeated. If the test is not repeated, the § 113.212 Bursal Disease Vaccine, serial is unsatisfactory. Killed Virus. (2) Potency. Bulk or final container Bursal Disease Vaccine, Killed Virus, samples of completed product shall be shall be prepared from virus-bearing treated for potency as follows: cell culture fluids or embryonated (i) Eight feline calicivirus susceptible chicken eggs. Only Master Seed which cats (five vaccinates and three con- has been established as pure, safe, and trols) shall be used as test animals. immunogenic shall be used for preparing seeds for vaccine production. All Throat and nasal swabs shall be col- serials shall be prepared from the first lected from each cat and individually through the fifth passage from the tested on susceptible cell cultures for Master Seed. the presence of feline calicivirus. Blood (a) The Master Seed shall meet the samples shall be drawn and individual applicable requirements prescribed in serum samples tested for neutralizing § 113.200. antibody. The cats shall be considered (b) Each lot of Master Seed shall be suitable for use if all swabs are nega- tested for pathogens by the chicken tive for virus isolation and all serums embryo inoculation test prescribed in are negative for calicivirus antibody at § 113.37, except that, if the test is a No the 1:2 final dilution in a 50 percent Test because of a vaccine virus over- plaque reduction test or other test of ride, the chicken inoculation test pre- equal sensitivity. scribed in § 113.36 may be conducted and (ii) The five cats used as vaccinates the virus judged accordingly. shall be administered one dose of vac- (c) The immunogenicity of vaccine cine by the method recommended on prepared in accordance with the Out- the label. If two doses are rec- line of Production shall be established ommended, the second dose shall be by a method acceptable to Animal and given after the interval recommended Plant Health Inspection Service. Vac- on the label. cine used for this test shall be at the highest passage from the Master Seed (iii) Fourteen or more days after the and prepared at the minimum final dose of vaccine, the vaccinates preinactivation titer specified in the and controls shall each be challenged Outline of Production. The test shall intranasally with virulent feline establish that the vaccine, when used calicivirus furnished or approved by as recommended on the label, is capa- Animal and Plant Health Inspection ble of inducing an immune response in Service and observed each day for 14 dams of sufficient magnitude to pro- days postchallenge. The rectal tem- vide significant protection to offspring. perature of each animal shall be taken (d) Test requirements for release. Each and the presence or absence of clinical serial and subserial shall meet the ap- signs, particularly lesions on the oral plicable general requirements pre- mucosa, noted and recorded each day. scribed in § 113.200 and the special re- (iv) If three of three controls do not quirements in this paragraph. Any se- show clinical signs of feline calicivirus rial or subserial found unsatisfactory infection other than fever, the test is by a prescribed test shall not be re- inconclusive and may be repeated. leased. (v) If a significant difference in clin- (1) Safety. Vaccinates used in the po- ical signs cannot be demonstrated be- tency test in paragraph (d)(2) of this tween vaccinates and controls using a section shall be observed each day dur- scoring system approved by Animal ing the prechallenge period. If unfavorable reactions attributable to the vac- and Plant Health Inspection Service cine occur, the serial is unsatisfactory. and prescribed in the Outline of Pro- If unfavorable reactions which are not duction, the serial is unsatisfactory. attributable to the vaccine occur, the [50 FR 433, Jan. 4, 1985. Redesignated at 55 test is a No Test and may be repeated. FR 35562, Aug. 31, 1990, as amended at 56 FR If the test is not repeated, the serial is 66784, 66786, Dec. 26, 1991] unsatisfactory.

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(2) Potency. Bulk or final container virus furnished or approved by Animal samples of completed product from and Plant Health Inspection Service. each serial shall be tested for potency (iv) Postchallenge period. Four days using the two-stage potency test pro- postchallenge, necropsy all chickens vided in this paragraph. and examine each for gross lesions of (i) Vaccinates. Inject each of 21 sus- bursal disease. For purposes of this ceptible chickens 14 to 28 days of age, test, gross lesions shall include properly identified and obtained from peribursal edema and/or edema and/or the same source and hatch, with one macroscopic hemorrhage in the bursal dose of vaccine by the route recommended on the label and observe for tissue. Vaccinated chickens showing at least 21 days. gross lesions shall be counted as fail- (ii) Controls. Retain at least 10 addi- ures. If at least 80 percent of the con- tional chickens from the same source trols do not have gross lesions of bursal and hatch as unvaccinated controls. disease in a stage of the test, that (iii) Challenge. Twenty-one to 28 days stage is considered inconclusive and postvaccination, challenge 20 vac- may be repeated. In a valid test, the re- cinates and 10 controls by eyedrop with sults shall be evaluated according to a virulent infectious bursal disease the following table:

Cumu- Cumulative total number of Number lative failures for— Stage of vac- number cinates of vac- Satisfactory Unsatisfac- cinates serial tory serial

1 ...... 20 20 3 or less ...... 6 or more. 2 ...... 20 40 8 or less ...... 9 or more.

(v) If four or five vaccinates show le- scribed in § 113.200 and the require- sions of bursal disease in the first ments of this section. stage, the second stage may be con- (b) The immunogenicity of vaccine ducted in a manner identical to the prepared from the Master Seed in ac- first stage. If the second stage is not cordance with the Outline of Produc- conducted, the serial is unsatisfactory. tion shall be established by a method (vi) If the second stage is used, each acceptable to the Animal and Plant serial shall be evaluated according to Health Inspection Service. Vaccine the second part of the table on the used for this test shall be at the high- basis of cumulative results. est passage from the Master Seed and [50 FR 434, Jan. 4, 1985. Redesignated at 55 at the minimum preinactivation titer FR 35562, Aug. 31, 1990, as amended at 56 FR provided in the Outline of Production. 66784, 66786, Dec. 26, 1991; 79 FR 55969, Sept. (c) Test requirements for release. Each 18, 2014] serial and subserial shall meet the applicable general requirements pre- §§ 113.213–113.214 [Reserved] scribed in § 113.200 and the special re- § 113.215 Bovine Virus Diarrhea Vac- quirements provided in this paragraph. cine, Killed Virus. Any serial or subserial found unsatis- Bovine Virus Diarrhea Vaccine, factory by a prescribed test shall not Killed Virus, shall be prepared from be released. virus-bearing cell culture fluids. Only (1) Safety. Vaccinates used in the po- Master Seed virus which has been es- tency test in paragraph (c)(2) of this tablished as pure, safe, and section shall be observed each day dur- immunogenic shall be used for pre- ing the prechallenge period. If unfavor- paring seed cultures for vaccine pro- able reactions occur, including res- duction. All serials of vaccine shall be piratory signs, which are attributable prepared from the first through the to the vaccine, the serial is unsatisfac- fifth passage from the Master Seed. tory. If unfavorable reactions occur (a) The Master Seed shall meet the which are not attributable to the vac- applicable general requirements pre- cine, the test is a No Test and may be

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repeated one time. If results of the sec- is a No Test and may be repeated one ond test are not satisfactory, or if the time. If two or more vaccinates show a test is not repeated, the serial is unsat- temperature of 104.0 °F for 2 or more isfactory. days and develop respiratory or clinical (2) Potency. Bulk or final container or other signs, the serial is unsatisfac- samples of completed product shall be tory. tested for potency using the method (vii) The prevaccination and described in this paragraph. postvaccination sera from a satisfac- (i) Eight bovine virus diarrhea sus- tory potency test shall be submitted to ceptible calves (five vaccinates and the National Veterinary Services Lab- three controls) shall be used as test oratories for confirmatory testing. animals. Individual serum samples [55 FR 35562, Aug. 31, 1990] shall be collected, inactivated, and individually tested for neutralizing anti- § 113.216 Bovine Rhinotracheitis Vac- body. cine, Killed Virus. (ii) A constant virus decreasing Infectious Bovine Rhinotracheitis serum neutralization test in cell cul- Vaccine, Killed Virus, shall be prepared ture using 50–300 TCID50 of virus shall from virus-bearing cell culture fluids. be used. Calves shall be considered sus- Only Master Seed virus which has been ceptible if there is no neutralization at established as pure, safe, and 1:2 final serum dilution. Other tests of immunogenic shall be used for pre- equal sensitivity approved by the Ani- paring seed cultures for vaccine pro- mal and Plant Health Inspection Serv- duction. All serials of vaccine shall be ice may be used. prepared from the first through the (iii) The five calves used as vac- fifth passage from the Master Seed. cinates shall be administered one dose (a) The Master Seed shall meet the of vaccine as recommended on the applicable general requirements pre- label. If two doses are recommended, scribed in § 113.200 and the require- the second dose shall be given accord- ments of this section. ing to the interval recommended on (b) The immunogenicity of vaccine the label. prepared in accordance with the Out- (iv) Fourteen days or more after the line of Production shall be established last vaccination, blood samples shall by a method acceptable to the Animal be drawn and the individual serum and Plant Health Inspection Service. samples inactivated and tested for bo- Vaccine used for this test shall be at vine virus diarrhea virus neutralizing the highest passage from the Master antibody by the same method used to Seed and at the minimum determine susceptibility. preinactivation titer provided in the (v) Test interpretation. If the controls Outline of Production. have not remained seronegative at 1:2, (c) Test requirements for release. Each the test is a No Test (NT) and may be serial and subserial shall meet the re- repeated. If at least four of the five quirements prescribed in § 113.200 and vaccinates in a valid test have not de- the special requirements provided in veloped 50 percent endpoint titers of 1:8 this paragraph. Any serial or subserial or greater, the serial is unsatisfactory, found unsatisfactory by a prescribed except as provided in paragraph test shall not be released. (c)(2)(vi) of this section. (1) Safety. Vaccinates used in the po- (vi) Virus Challenge Test. If the results tency test in paragraph (c)(2) of this of a valid serum neutralization test are section shall be observed each day dur- unsatisfactory, the vaccinates and con- ing the prechallenge period. If unfavor- trols may be challenged with virulent able reactions occur, which are attrib- bovine virus diarrhea virus furnished utable to the vaccine, the serial is un- or approved by the Animal and Plant satisfactory. If unfavorable reactions Health Inspection Service. The animals occur which are not attributable to the shall be observed for 14 days post-chal- vaccine, the test is a No Test and may lenge. If two of the three control calves be repeated one time. If the results of do not show a temperature rise to 104.5 the second test are not satisfactory, or °F and develop respiratory or clinical if the test is not repeated, the serial is signs of bovine virus diarrhea, the test unsatisfactory.

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(2) Potency. Bulk or final container the vaccinates shows a temperature of samples of completed product shall be 104.0 °F for 2 or more days or if more tested for potency using the method than one of the vaccinates develops described in this paragraph. respiratory or clinical or other signs, (i) Eight infectious bovine the serial is unsatisfactory. rhinotracheitis susceptible calves (five (vii) The prevaccination and vaccinates, three controls) shall be postvaccination sera from a satisfac- used as test animals. Individual serum tory potency test shall be submitted to samples shall be collected, inactivated, the National Veterinary Services Lab- and individually tested for neutralizing oratories for testing by the Animal and antibody. Plant Health Inspection Service. (ii) A constant virus decreasing [55 FR 35562, Aug. 31, 1990, as amended at 56 serum neutralization test in cell cul- FR 66786, Dec. 26, 1991] ture using 50–300 TCID50 of virus shall be used. Calves shall be considered sus- LIVE VIRUS VACCINES ceptible if there is no neutralization at 1:2 final serum dilution. Other tests of § 113.300 General requirements for live equal sensitivity acceptable to the Ani- virus vaccines. mal and Plant Health Inspection Serv- When prescribed in an applicable ice may be used. Standard Requirement or in the filed (iii) The five calves used as vac- Outline of Production, a live virus vaccinates shall be administered one dose cine shall meet the applicable require- of vaccine as recommended on the ments in this section. label. If two doses are recommended, (a) Purity tests—(1) Bacteria and fungi. the second dose shall be given accord- Final container samples of completed ing to the interval recommended on product and comparable samples of the label. each lot of Master Seed Virus shall be (iv) Fourteen or more days after the tested for bacteria and fungi in accord- last vaccination, blood samples shall ance with the test provided in § 113.27. be drawn and the individual serum (2) Mycoplasma. Final container sam- samples inactivated and tested for in- ples of completed product and com- fectious bovine rhinotracheitis virus parable samples of each lot of Master neutralizing antibody by the same Seed Virus shall be tested for myco- method used to determine suscepti- plasma in accordance with the test pro- bility. vided in § 113.28. (v) Test interpretation. If the three (3) Avian Origin Vaccine. Samples of controls have not remained each lot of Master Seed Virus and bulk seronegative at 1:2, the test is a No pooled material or final container sam- Test (NT) and may be repeated. If at ples from each serial shall also be test- least four of the five vaccinates in a ed for: valid test have not developed 50 per- (i) Salmonella contamination as pre- cent endpoint titers of 1:8, the serial is scribed in § 113.30; and unsatisfactory, except as provided in (ii) Lymphoid leukosis virus con- paragraph (c)(2)(vi) of this section. tamination as prescribed in § 113.31; and (vi) Virus Challenge Test. If the results (iii) Hemagglutinating viruses as pre- of a valid serum neutralization test are scribed in § 113.34. unsatisfactory, the vaccinates and con- (4) Extraneous viruses. Each lot of trols may be challenged with virulent Master Seed Virus used to prepare live infectious bovine rhinotracheitis virus virus vaccine recommended for animals furnished or approved by the Animal other than poultry shall meet the re- and Plant Health Inspection Service. quirements for extraneous viruses as The animals shall be observed each day prescribed in § 113.55 for 14 days post-challenge. If two of the (b) Safety tests. Samples of each lot of three control calves do not show a tem- Master Seed Virus and final container perature rise to 104.5 °F and develop samples of completed product from respiratory or other clinical signs of each serial or first subserial of live infectious bovine rhinotracheitis, the virus vaccine recommended for animals test is a No Test (NT) and may be re- other than poultry shall be tested for peated one time. If more than one of safety in at least one species for which

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the vaccine is intended using methods virus-bearing tissues obtained from prescribed in §§ 113.39, 113.40, 113.41, sheep that have developed ovine 113.44, and 113.45 or in a filed Outline of ecthyma following inoculation with Production. The mouse safety test pre- virulent ovine ecthyma virus. Ovine scribed in § 113.33(a) shall also be con- Ecthyma Vaccine is exempt from the ducted unless the virus or agent in the requirements prescribed in §§ 113.27 and vaccine is inherently lethal for mice. 113.300(a), (b), and (c). Each serial shall (c) Virus identity test. At least one of meet the moisture requirements in the virus identity tests provided in this § 113.300(e) and the special requirements paragraph or a suitable identity test prescribed in this section. Any serial prescribed in the filed Outline of Pro- found unsatisfactory by a prescribed duction shall be conducted on the Mas- test shall not be released. ter Seed Virus and final container samples from each serial or first subserial (a) Safety tests. (1) Bulk or final con- of biological product. tainer samples of completed product (1) Fluorescent antibody test. The fluo- from each serial shall be tested for rescent antibody test shall be con- safety as prescribed in § 113.38. ducted using virus inoculated cells and (2) The prechallenge period of the po- uninoculated control cells. Cells shall tency test shall constitute a safety be stained with fluorochrome con- test. If unfavorable reactions attrib- jugated specific antiserum. Fluores- utable to the vaccine occur in either of cence typical of the virus concerned the vaccinates during the observation shall be demonstrated in the inocu- period, the serial is unsatisfactory. lated cells. The control cells shall re- (b) Potency test. Final container sam- main free of such fluorescence. ples of completed product from each se- (2) Serum neutralization test. The rial and each subserial shall be tested serum neutralization test shall be con- for potency using susceptible lambs. ducted using the constant serum-de- The vaccine shall be prepared as rec- creasing virus method with specific ommended for use on the label. antiserum. For positive identification, (1) Each of two lambs (vaccinates) at least 100 ID of vaccine virus shall 50 shall be vaccinated by application of be neutralized by the antiserum. (d) Cell Culture Requirements. If cell the vaccine to a scarified area on the cultures are used in the preparation of medial surface of the thigh and ob- Master Seed Virus or of the vaccine, served each day for 14 days. primary cells shall meet the require- (2) The immunity of the two vac- ments prescribed in § 113.51, cell lines cinates and one or more unvaccinated shall meet the requirements prescribed lambs (controls) shall be challenged in in § 113.52, and ingredients of animal or- the same manner as for vaccination, igin shall meet the applicable require- using the opposite thigh. ments in § 113.53. (3) If typical signs of ovine ecthyma, (e) Moisture content. (1) The max- such as hyperemia, vesicles, and imum moisture content in desiccated pustules do not develop on the controls vaccines must be stated in the filed during the first 2 weeks following chal- Outline of Production. lenge and persist for approximately 30 (2) Final container samples of com- days, the test is a No Test and may be pleted product from each serial or sub- repeated. serial must be tested for moisture con- (4) If the vaccinates do not show a tent in accordance with the test pre- typical immune reaction, the serial is scribed in § 113.29. unsatisfactory: Provided, That, an ini- [39 FR 27430, July 29, 1974, as amended at 43 tial active reaction with hyperemia FR 49528, Oct. 24, 1978; 50 FR 1042, Jan. 9, 1985; which resolves progressively and dis- 54 FR 19352, May 5, 1989. Redesignated at 55 appears within 2 weeks, may be charac- FR 35562, Aug. 31, 1990; 60 FR 24549, May 9, 1995; 68 FR 57608, Oct. 6, 2003] terized as a typical immune reaction. [39 FR 27430, July 29, 1974. Redesignated at 55 § 113.301 Ovine Ecthyma Vaccine. FR 35562, Aug. 31, 1990, as amended at 56 FR Ovine Ecthyma Vaccine shall be pre- 66786, Dec. 26, 1991] pared from tissue culture fluids or

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§ 113.302 Distemper Vaccine—Mink. a predetermined quantity of vaccine Distemper Vaccine—Mink shall be virus and at least 5 additional mink prepared from virus-bearing cell cul- shall be held as unvaccinated controls. ture fluids. Only Master Seed Virus To confirm the dosage calculations, which has been established as pure, five replicate virus titrations shall be safe, and immunogenic shall be used conducted on a sample of the vaccine for preparing the production seed virus virus dilution used. for vaccine production. All serials of (3) At least twenty-one days post-in- vaccine shall be prepared from the first jection, the immunity of each of the through the fifth passage from the vaccinates and the controls shall be Master Seed Virus. challenged with the same size dose of (a) The Master Seed Virus shall meet virulent distemper virus and observed the applicable requirements prescribed each day for 21 days. in § 113.300 and the requirements pre- (i) If at least 80 percent of the con- scribed in this section. trols do not die or show severe signs of (b) The lot of Master Seed Virus shall distemper, the test is a No Test and be tested for extraneous viruses as fol- may be repeated. lows: (ii) If at least 19 of 20, 27 of 30, or 36 (1) To detect virulent canine dis- of 40 of the vaccinates do not survive temper virus, each of two distemper without showing clinical signs of dis- susceptible mink or ferrets shall be in- temper during the observation period, oculated with 1 ml of the Master Seed the Master Seed Virus is unsatisfac- Virus and observed each day for 21 tory. days. If undesirable reactions occur in (4) An Outline of Production change either test animal, the lot of Master shall be made before authority for use Seed Virus is unsatisfactory. of a new lot of Master Seed Virus shall (2) Master Seed Virus propagated in be authorized by Animal and Plant chicken embryos shall be tested for Health Inspection Service. pathogens by the chicken embryo test (d) Test requirements for release: prescribed in § 113.37 except lesions typ- Each serial and subserial shall meet ical of distemper virus may be dis- the general requirements prescribed in regarded. If found unsatisfactory, the § 113.300 and the requirements in this Master Seed Virus shall not be used. paragraph. Final container samples of (c) Each lot of Master Seed Virus completed product shall be tested. Any used for vaccine production shall be serial or subserial found unsatisfactory tested for immunogenicity. The se- by a prescribed test shall not be re- lected virus dose from the lot of Master leased. Seed Virus shall be established as fol- (1) Mink safety test. Each of 2 mink lows: shall be vaccinated with the equivalent (1) At least 25 distemper susceptible of 10 doses of vaccine rehydrated with mink shall be used as test animals. sterile diluent and administered in the Blood samples shall be drawn from manner recommended on the label. The these animals and individual serum mink shall be observed each day for 21 samples tested. The mink shall be con- days. If unfavorable reactions attrib- sidered susceptible if the results are utable to the product occur in either of negative at a 1:2 final serum dilution in the mink during the observation pe- a varying serum-constant virus neu- riod, the serial or subserial is unsatis- tralization test with less than 500 ID50 factory. If unfavorable reactions which of canine distemper virus. Other means are not attributable to the product of insuring susceptibility may be used occur, the test shall be declared a No if prior approval from Animal and Test and may be repeated: Provided, Plant Health Inspection Service is re- That if the test is not repeated, the se- ceived. rial or subserial shall be declared un- (2) A geometric mean titer of the satisfactory. dried vaccine produced from the high- (2) Potency Test. An in vitro potency est passage of the Master Seed Virus test shall be conducted. To be eligible shall be established before the for release, each serial and subserial immunogenicity test is conducted. At shall have a virus titer sufficiently least 20 mink shall be vaccinated with greater than the titer of vaccine virus

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used in the immunogenicity test pre- is conducted. The 20 lambs to be used scribed in paragraph (c) of this section as vaccinates shall be administered a to assure that, when tested at any time predetermined quantity of vaccine within the expiration period, each se- virus by the method recommended on rial and subserial shall have a virus the label. To confirm the virus dosage titer 10 0.7 greater than that used in administered, five replicate virus titra- such immunogenicity test when tested tions shall be conducted on a sample of by the method used in paragraph (c)(2) the vaccine used. of this section. (3) At least once during the period of [40 FR 53000, Nov. 14, 1975, as amended at 48 14 to 18 days postvaccination, indi- FR 33471, July 22, 1983. Redesignated at 55 FR vidual serum samples shall be collected 35562, Aug. 31, 1990, as amended at 56 FR from each of the vaccinates and tested 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, for virus neutralizing antibody using 2007] the 60 to 300 TCID50 of bluetongue virus. § 113.303 Bluetongue Vaccine. (4) Twenty-one to twenty-eight days Bluetongue Vaccine shall be prepared postvaccination the vaccinates and the from virus-bearing cell culture fluids. controls shall each be challenged with Only Master Seed which has been es- virulent bluetongue virus and observed tablished as pure, safe, and for 14 days. The rectal temperature of immunogenic shall be used for pre- each animal shall be taken and re- paring the seeds for vaccine produc- corded for 17 consecutive days begin- tion. All serials of vaccine shall be pre- ning 3 days prechallenge. The presence pared from the first through the tenth or absence of lesions or other clinical passage from the Master Seed. signs of bluetongue noted and recorded (a) The Master Seed shall meet the on each of 14 consecutive days applicable general requirements pre- postchallenge. scribed in § 113.300 and the require- (i) If at least four of the five controls ments in this section. do not show clinical signs of (b) Each lot of Master Seed shall be bluetongue and a temperature rise of 3 tested for transmissibility and rever- °F or higher over the prechallenge sion to virulence in sheep using a mean temperature, the test shall be method acceptable to Animal and Plant Health Inspection Service. If re- considered a No Test and may be reversion to virulence is demonstrated, peated. the Master Seed is unsatisfactory. (ii) If at least 19 of the 20 vaccinates (c) Each lot of Master Seed used for tested as prescribed in paragraph (c)(3) vaccine production shall be tested for of this section do not have bluetongue immunogenicity. The selected virus neutralizing antibody titers of 1:4 final dose from the lot of Master Seed shall serum dilution or higher, or if more be established as follows: than one of the vaccinates shows a (1) Twenty-five lambs, susceptible to temperature rise of 3 °F or higher than the bluetongue virus serotype con- its prechallenge mean temperature for tained in the vaccine, shall be used as 2 or more days, or if more than one of test animals (20 vaccinates and 5 con- the vaccinates exhibits clinical signs of trols). Blood samples shall be drawn bluetongue, the Master Seed is unsatis- from these animals and individual se- factory. rums tested. A lamb shall be consid- (5) An Outline of Production change ered susceptible if there is no neutral- shall be made before authority for use ization at a 1:2 final serum dilution in of a new lot of Master Seed shall be a constant virus varying serum neu- granted by Animal and Plant Health tralization test with 60 to 300 TCID50 of Inspection Service. bluetongue virus or another method ac- (d) Test requirements for release. Each ceptable to Animal and Plant Health serial and subserial shall meet the ap- Inspection Service. plicable general requirements pre- (2) A geometric mean titer of the vac- scribed in § 113.300 and the require- cine produced from the highest passage ments in this paragraph. Final con- from the Master Seed shall be estab- tainer samples of completed product lished before the immunogenicity test shall be tested. Any serial or subserial

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found unsatisfactory by a prescribed the Master Seed Virus is unsatisfac- test shall not be released. tory. (1) Safety test. The mouse safety test (2) To detect chlamydial agents, the prescribed in § 113.33(a) and the lamb Master Seed Virus shall be tested as safety test prescribed in § 113.45 shall be prescribed in § 113.43. conducted. (c) Each lot of Master Seed Virus (2) Virus titer requirements. Final con- used for vaccine production shall be tainer samples of completed product tested for immunogenicity. The se- shall be tested for virus titer using the lected virus dose from the lot of Master titration method used in paragraph Seed Virus shall be established as fol- (c)(2) of this section. To be eligible for lows: release, each serial and subserial shall (1) Twenty-five feline panleukopenia have a virus titer sufficiently greater susceptible cats shall be used as test than the titer of vaccine virus used in animals (20 vaccinates and 5 controls). the immunogenicity test prescribed in Blood samples drawn from each cat paragraph (c) of this section to assure shall be individually tested for neutral- that when tested at any time within izing antibody against feline the expiration period, each serial and panleukopenia virus to determine sus- subserial shall have a virus titer of ceptibility. 10 0.7 greater than that used in such (i) A constant virus-carrying serum immunogenicity test. neutralization test in tissue culture using 100 to 300 TCID50 of virus shall be [50 FR 23796, June 6, 1985. Redesignated at 55 used. FR 35562, Aug. 31, 1990, as amended at 56 FR (ii) Cats shall be considered suscep- 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, tible if there is no neutralization at a 2007] 1:2 final serum dilution. (2) A geometric mean titer of the § 113.304 Feline Panleukopenia Vac- cine. dried vaccine produced from the highest passage of the Master Seed Virus Feline Panleukopenia Vaccine shall shall be established before the be prepared from virus-bearing cell cul- immunogenicity test is conducted. The ture fluids. Only Master Seed Virus 20 cats used as vaccinates shall be in- which has been established as pure, jected with a predetermined quantity safe, and immunogenic shall be used of vaccine virus and the remaining five for preparing the production seed virus cats held as uninjected controls. To for vaccine production. All serials of confirm the dosage calculations, five vaccine shall be prepared from the first replicate virus titrations shall be con- through the fifth passage from the ducted on a sample of the vaccine virus Master Seed Virus. dilution used. (a) The Master Seed Virus shall meet (3) Fourteen days post-injection, the the applicable general requirements vaccinates and the controls shall be prescribed in § 113.300 and the require- challenged with virulent feline ments prescribed in this section. panleukopenia virus furnished by Ani- (b) The lot of Master Seed Virus shall mal and Plant Health Inspection Serv- be tested for other agents as follows: ice and the cats observed each day for (1) To detect virulent feline 14 days. panleukopenia virus or virulent mink (i) If at least 80 percent of the con- enteritis virus, each of two feline trols do not show clinical signs of fe- panleukopenia susceptible cats, as de- line panleukopenia during the observa- termined by the criteria prescribed in tion period, the test is a No Test and paragraph (c)(1) of this section, shall be may be repeated. Clinical signs of fe- injected subcutaneously with the line panleukopenia shall include a pro- equivalent of one cat dose each and the nounced leukopenia wherein the white cats observed each day for 21 days. If cell count drops to 4,000 or less per either or both cats show signs of dis- cubic mm, or the white cell count ease or reduced white blood cell counts drops to less than 25 percent of the nor- below 50 percent of the normal level es- mal level established by an average of tablished by an average of three or three or more counts taken prior to more counts taken prior to injection, challenge.

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(ii) If at least 19 of the 20 vaccinates § 113.305 Canine Hepatitis and Canine do not survive the observation period Adenovirus Type 2 Vaccine. without showing clinical signs of feline Canine Hepatitis Vaccine and Canine panleukopenia as described in para- Adenovirus Type 2 Vaccine shall be graph (c)(3)(i) of this section, the Mas- prepared from virus-bearing cell cul- ter Seed Virus is unsatisfactory. ture fluids. Only Master Seed Virus (4) An Outline of Production change which has been established as pure, shall be made before authority for use safe, and immunogenic shall be used in of a new lot of Master Seed Virus shall preparing the production seed virus for be granted by Animal and Plant Health vaccine production. All serials shall be Inspection Service. prepared from the first through the (d) Test requirements for release. Each fifth passage from the Master Seed serial and subserial shall meet the re- Virus. quirements prescribed in § 113.300 and (a) The Master Seed Virus shall meet in this paragraph. Final container sam- the applicable requirements prescribed ples of completed product shall be test- in § 113.300 except that the dog safety ed. Any serial or subserial found unsat- test prescribed in § 113.40(a) shall be isfactory by a prescribed test shall not conducted by the intravenous route. be released. (b) Each lot of Master Seed Virus (1) Safety test. The mouse safety test used for vaccine production shall be prescribed in § 113.33(a) and the cat tested for immunogenicity by one or safety test prescribed in § 113.39 shall be both of the following methods: conducted. (1) Immunogenicity for canine hepatitis. (i) Each of two healthy cats shall be Twenty-five canine hepatitis suscep- injected with 10 cat doses by the meth- tible dogs shall be used as test animals od recommended on the label and the (20 vaccinates and 5 controls). Blood cats observed each day for 14 days. samples shall be drawn from these ani- (ii) If unfavorable reactions attrib- mals and individual serum samples utable to the biological product occur tested. The dogs shall be considered during the observation period, the se- susceptible if the results are negative rial is unsatisfactory. If unfavorable at a 1:2 final serum dilution in a vary- reactions occur which are not attrib- ing serum-constant virus neutraliza- utable to the product, the test shall be tion test using 50 to 300 TCID50 of ca- declared a No Test and repeated: Pro- nine adenovirus. vided, That, if not repeated, the serial (i) A geometric mean titer of the shall be unsatisfactory. dried vaccine produced from the high- (2) Virus titer requirements. Final con- est passage of the Master Seed Virus tainer samples of completed product shall be established before the shall be tested for virus titer using the immunogenicity test is conducted. The titration method used in paragraph 20 dogs to be used as vaccinates shall (c)(2) of this section. To be eligible for be injected with a predetermined quan- release, each serial and each subserial tity of vaccine virus and the remaining shall have a virus titer sufficiently five dogs held as uninjected controls. greater than the titer of vaccine virus To confirm the dosage calculations, used in the immunogenicity test pre- five replicate virus titrations shall be scribed in paragraph (c) of this section conducted on a sample of the vaccine to assure that when tested at any time virus dilution used. within the expiration period, each se- (ii) Not less than 14 days rial and subserial shall have a virus postinjection, the vaccinates and the titer of 10 0.7 greater than that used in controls shall each be challenged intra- such immunogenicity test but not less venously with virulent infectious ca- 2.5 than 10 TCID50 per dose. nine hepatitis virus furnished or approved by the Animal and Plant Health [39 FR 44716, Dec. 27, 1974, as amended at 40 Inspection Service and observed each FR 53378, Nov. 18, 1975; 43 FR 25078, June 9, day for 14 days. 1978; 43 FR 41186, Sept. 15, 1978; 44 FR 58900, Oct. 12, 1979; 48 FR 33471, July 22, 1983. Redes- (A) If at least four of the five con- ignated at 55 FR 35562, Aug. 31, 1990, as trols do not show severe clinical signs amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 of canine hepatitis, the test is a No FR 72564, Dec. 21, 2007] Test and may be repeated.

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(B) If at least 19 of the 20 vaccinates shall be granted by the Animal and do not survive without showing clinical Plant Health Inspection Service. signs of infectious canine hepatitis dur- (c) Test requirements for release. Each ing the observation period, the Master serial and subserial shall meet the re- Seed Virus is unsatisfactory. quirements prescribed in § 113.300 and (2) Immunogenicity for canine in this paragraph. Final container sam- adenovirus Type 2. Thirty canine ples of completed product shall be test- adenovirus type 2 susceptible dogs ed. Any serial or subserial found unsat- shall be used as test animals (20 vac- isfactory by a prescribed test shall not cinates and 10 controls). Blood samples be released. shall be drawn from these animals and (1) Virus titer requirements. Final con- individual serum samples tested. The tainer samples of completed product dogs shall be considered susceptible if shall be tested for virus titer using the the results are negative at a 1:2 final titration method used in paragraph serum dilution in a varying serum-con- (b)(1)(i) and/or (b)(2)(i) of this section. stant virus neutralization test using 50 To be eligible for release, each serial and each subserial shall have a virus to 300 TCID of canine adenovirus. 50 titer sufficiently greater than the titer (i) A geometric mean titer of the of vaccine virus used in the dried vaccine produced from the high- immunogenicity test(s) prescribed in est passage of the Master Seed Virus paragraph (b) of this section to assure shall be established before the that when tested at any time within immunogenicity test is conducted. The the expiration period, each serial and 20 dogs to be used as vaccinates shall subserial shall have a virus titer of be injected with a predetermined quan- 10 0.7 greater than that used in such tity of vaccine virus and the remaining immunogenicity test(s) but not less 10 dogs held as uninjected controls. To 2.5 than 10 TCID50 dose. If both confirm the dosage calculations, five immunogenicity tests in paragraph (b) replicate virus titrations shall be con- of this section are conducted and a dif- ducted on a sample of the vaccine virus ferent amount of virus is used in each dilution used. test, the virus titer requirements shall (ii) Not less than 14 days be based on the higher of the two postinjection, the vaccinates and the amounts. controls shall be challenged by expo- (2) [Reserved] sure to a nebulized aerosol of virulent [60 FR 14361, Mar. 17, 1995, as amended at 72 canine adenovirus type 2 furnished or FR 72564, Dec. 21, 2007] approved by the Animal and Plant Health Inspection Service and observed § 113.306 Canine Distemper Vaccine. each day for 14 days postchallenge. The Canine Distemper Vaccine shall be rectal temperature of each animal prepared from virus-bearing cell cul- shall be taken and the presence of res- ture fluids or embryonated chicken piratory or other clinical signs of ca- eggs. Only Master Seed Virus which nine adenovirus type 2 noted and re- has been established as pure, safe, and corded each day. immunogenic shall be used for pre- (A) If at least 6 of 10 controls do not paring the production seed virus for show clinical signs of canine vaccine production. All serials of vac- adenovirus type 2 infection other than cine shall be prepared from the first fever, the test is a No Test and may be through the fifth passage from the repeated. Master Seed Virus. (B) If a significant difference in clin- (a) Master Seed Virus. The Master ical signs in a valid test cannot be Seed Virus shall meet the applicable demonstrated between vaccinates and requirements prescribed in § 113.300 and controls using a scoring system ap- the requirements prescribed in this sec- proved by the Animal and Plant Health tion. Inspection Service, the Master Seed (1) To detect ferret virulent canine Virus is unsatisfactory. distemper virus, each of five canine (iii) An Outline of Production change distemper susceptible ferrets shall be shall be made before authorization for injected with a sample of the Master use of a new lot of Master Seed Virus Seed Virus equivalent to the amount of

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virus to be used in one dog dose and ob- (4) An Outline of Production change served each day for 21 days. If undesir- shall be made before authorization for able reactions are observed during the use of a new lot of Master Seed Virus observation period, the lot of Master shall be granted by the Animal and Seed is unsatisfactory. Plant Health Inspection Service. (2) Master Seed Virus propagated in (c) Test requirements for release. Ex- tissues or cells of avian origin shall be cept for § 113.300(a)(3)(ii), each serial tested for pathogens by the chicken and subserial shall meet the require- embryo test prescribed in § 113.37. If ments prescribed in § 113.300 and in this found unsatisfactory, the Master Seed paragraph. Final container samples of Virus shall not be used. completed product shall be tested. Any (b) Each lot of Master Seed Virus serial or subserial found unsatisfactory used for vaccine production shall be by a prescribed test shall not be retested for immunogenicity. The se- leased. lected virus dose from the lot of Master (1) The test for pathogens prescribed Seed Virus shall be established as fol- in § 113.37 shall be conducted on each lows: serial or one subserial of avian origin (1) Twenty-five canine distemper sus- vaccine. ceptible dogs shall be used as test ani- (2) Virus titer requirements. Final con- mals (20 vaccinates and 5 controls). tainer samples of completed product Blood samples shall be drawn from shall be tested for virus titer using the these animals and individual serum titration method used in paragraph samples tested. The dogs shall be con- (b)(2) of this section. To be eligible for sidered susceptible if the results are release, each serial and subserial shall negative at a 1:2 final serum dilution in have a virus titer sufficiently greater a varying serum-constant virus neu- than the titer of vaccine virus used in tralization test using 50 to 300 TCID50 the immunogenicity test prescribed in of canine distemper virus. paragraph (b) of this section to assure (2) A geometric mean titer of the that when tested at any time within dried vaccine produced from the high- the expiration period, each serial and est passage of the Master Seed Virus subserial shall have a virus titer of shall be established before the 10 0.7 greater than that used in such immunogenicity test is conducted. The immunogenicity test but not less than 20 dogs used as vaccinates shall be in- 2.5 10 TCID50 per dose. jected with a predetermined quantity of vaccine virus and the remaining five [60 FR 14362, Mar. 17, 1995, as amended at 72 dogs held as uninjected controls. To FR 72564, Dec. 21, 2007] confirm the dosage calculations, five replicate virus titrations shall be con- § 113.308 Encephalomyelitis Vaccine, Venezuelan. ducted on a sample of the vaccine virus dilution used. Encephalomyelitis Vaccine, Ven- (3) At least 14 days post-injection, ezuelan, shall be prepared from virus- the vaccinates and the controls shall bearing cell culture fluids. Only Master each be challenged intracerebrally Seed which has been established as with virulent canine distemper virus pure, safe, and immunogenic shall be furnished or approved by the Animal used for preparing seeds for vaccine and Plant Health Inspection Service production. All serials of vaccine shall and observed each day for 21 days. be prepared from the first through the (i) If at least four of the five controls fifth passage from the Master Seed. do not die and the survivor, if any, does (a) The Master Seed shall meet the not show clinical signs of canine dis- applicable general requirements pre- temper the test is a No Test and may scribed in § 113.300 except (b), and the be repeated. requirements prescribed in this sec- (ii) If at least 19 of the 20 vaccinates tion. do not survive without showing clinical (b) Each lot of Master Seed shall be signs of canine distemper during the tested for immunogenicity. The se- observation period, the Master Seed lected virus dose from the lot of Master Virus is unsatisfactory. Seed shall be established as follows:

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(1) Tests conducted by the Depart- scribed in § 113.300 and special requirement have established that horses hav- ments in this paragraph. Any serial or ing Venezuelan equine subserial found unsatisfactory by a encephalomyelitis antibody titers of prescribed test shall not be released. 1:20 by the hemagglutination-inhibi- (1) Safety test. The mouse safety test tion (HI) method or 1:40 by the serum prescribed in § 113.33(b) shall be con- neutralization (SN) method were im- ducted. mune to challenge with virulent virus. (2) Virus titer requirements. Final con- The immunogenicity test is based on tainer samples of completed product the demonstration of a serological re- shall be tested for virus titer using the sponse of at least that magnitude fol- method in paragraph (b)(3) of this sec- lowing vaccination of serologically tion. To be eligible for release, each se- negative horses. rial and subserial shall have a virus (2) At least 22 horses (20 vaccinates titer sufficiently greater than the titer and 2 controls), susceptible to Ven- of the vaccine used in the ezuelan equine encephalomyelitis, immunogenicity test prescribed in shall be used as test animals. Blood paragraph (b) of this section to assure samples shall be taken from each horse that, when tested at any time within and the serums individually tested for the expiration period, each serial and neutralizing antibody. Horses shall be subserial shall have a virus titer of considered susceptible if there is no 10 0.7 greater than that used in the neutralization at a 1:2 final serum dilu- immunogenicity test, but not less than tion in a constant virus-varying serum 2.5 10 TCID50 per dose. neutralization test using 60 to 300 [50 FR 23797, June 6, 1985. Redesignated at 55 TCID50 of Venezuelan equine encephalomyelitis virus. FR 35562, Aug. 31, 1990, as amended at 56 FR (3) A geometric mean titer of the vac- 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007] cine produced from the highest passage of the Master Seed shall be established § 113.309 Bovine Parainfluenza 3 Vac- using a method acceptable to Veteri- cine. nary Services before the immunogenicity test is conducted. The Bovine Parainfluenza3 Vaccine shall 20 horses used as vaccinates shall be in- be produced from virus-bearing cell jected with a predetermined quantity culture fluids. Only Master Seed Virus of vaccine virus by the method to be which has been established as pure, recommended on the label. To confirm safe, and immunogenic shall be used the dosage administered, five replicate for preparing the production seed virus virus titrations shall be conducted on a for vaccine production. All serials of sample of the vaccine virus dilution vaccine shall be prepared from the first used. through the tenth passage from the (4) Twenty-one to twenty-eight days Master Seed Virus. postvaccination, blood samples shall be (a) The Master Seed Virus shall meet drawn from all test animals. For a the applicable general requirements valid test, the controls shall remain prescribed in § 113.300. seronegative at 1:2 final serum dilu- (b) Each lot of Master Seed Virus tion. In a valid test, if at least 19 of 20 shall meet the special requirements vaccinates do not have antibody titers prescribed in this section. of at least 1:20 in a hemagglutination- (c) Each lot of Master Seed Virus inhibition test or at least 1:40 in a used for vaccine production shall be serum neutralization test, the Master tested for immunogenicity. The se- Seed is unsatisfactory. lected virus dose from the lot of Master (5) An Outline of Production change Seed Virus shall be established as fol- shall be made before authority for use lows: of a new lot of Master Seed shall be (1) Twenty-five bovine parainfluenza, granted by Animal and Plant Health susceptible calves shall be used as test Inspection Service. animals (20 vaccinates and five con- (c) Test requirements for release. Each trols). Blood samples shall be drawn serial and subserial shall meet the ap- from these animals and individual se- plicable general requirements pre- rums tested. Also, nasal specimens

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shall be collected for virus isolation at- (6) Satisfactory Test Criteria: tempts. The calves shall be considered (i) All virus isolations attempts shall susceptible if: be by culture and at least one subcul- (i) The results are negative at a 1:2 ture in PI3 susceptible cells for a total final serum dilution in a varying serum of at least 14 days. constant virus neutralization test with (ii) Two to four weeks post-vaccina- less than 500 TCID50 of bovine tion, at least 19 of the 20 vaccinates parainfluenza3 virus; and shall have PI3 neutralizing antibody (ii) Shall be negative to bovine titers of at least 1:4 and all five con- parainfluenza3 virus isolation attempts trols shall be negative at 1:2 dilution. from the nasal specimens on the day of None of the post-vaccination serums injection. collected from the vaccinates on day 6 (2) A geometric mean titer of the ±2 days shall reveal serum neutraliza- dried vaccine produced from the high- tion antibody titers of 1:32 or greater est passage of the Master Seed Virus based upon final dilution. shall be established before the immunogenicity test is conducted. The (iii) Satisfactory resistance to chal- 20 calves to be used as vaccinates shall lenge by vaccinates shall be deter- be injected with a predetermined quan- mined by a significant difference be- tity of vaccine virus and the remaining tween virus isolation rates from vac- five calves held as uninjected controls. cinates and controls. The virus neu- To confirm the dosage calculation, five tralization titers of post-challenge se- replicate virus titrations shall be con- rums and respiratory symptoms and ducted on a sample of the vaccine virus temperatures from all animals shall be dilution used. considered in the evaluation of the test (3) The vaccinates and controls shall validity. be examined for clinical signs of res- (7) Designated animal alternates for piratory disease and the body tempera- test animals showing anamnestic anti- ture taken and recorded on each of the body responses (titers 1:32 or greater) first 14 consecutive days post-injection. on day 6 serums may be included in the The vaccinates shall be bled on day 6 ±2 study under the following provisions: days post-injection. (i) No more than five alternates shall (4) Three to four weeks post-vaccina- be allowed for the vaccinates and no tion, all calves shall be bled for serum more than two for the controls. antibodies and nasal specimens shall be (ii) Alternates shall be subject to all collected for PI3 virus isolation. On the requirements outlined for the animals same day, all vaccinates and controls for which they are alternates. shall be given acceptable challenge PI3 (iii) Antibody values from alternate 7.0 virus titrating at least 10 TCID50 per animals may be used only to replace ml and the animals observed for 14 values from up to and including five days. Two ml of the challenge virus vaccinates which develop antibody of shall be instilled in each nostril or 1:32 or greater by day 6 ±2 days post- shall be inhaled as an aerosol suspen- vaccination or up to and including two sion. Upon request, challenge virus and controls which develop antibody titers instructions shall be furnished by Ani- of 1:32 or greater by day 6 ±2 days post- mal and Plant Health Inspection Serv- challenge. ice. (5) Each animal shall be examined for (8) A sequential test procedure may clinical signs of respiratory disease and be used in lieu of the 20 calf require- the body temperature recorded on each ment. A beta value of .05 and a toler- of the 14 consecutive days of the post- ance level of .78 shall be required. challenge observation period. Each day (9) An Outline of Production change for at least the first 10 days post-chal- shall be made before authority for use lenge, nasal specimens for virus isola- of a new lot of Master Seed Virus shall tion attempts shall be taken. All ani- be granted by Animal and Plant Health mals shall be bled on day 6 ±2 days Inspection Service. post-challenge, and all animals shall be (d) Test requirements for release: bled at least once 14 to 28 days post- Each serial and subserial shall meet challenge for serum antibody studies. the applicable general requirements

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prescribed in § 113.300 and the require- tested for immunogenicity. The se- ments in this paragraph. Final con- lected virus dose from the lot of Master tainer samples of completed product Seed Virus shall be established as fol- shall be tested except as prescribed in lows: paragraph (d)(1) of this section. Any se- (1) Twenty-five infectious bovine rial or subserial found unsatisfactory rhinotracheitis susceptible calves shall by a prescribed test shall not be re- be used as test animals (20 vaccinates leased. and five controls). Blood samples shall (1) Purity test. The test for Brucella be drawn from these animals and indi- contamination prescribed in § 113.32 vidual serums tested. The calves shall shall be conducted on each batch of pri- be considered susceptible if the results mary cells intended for production use. are negative at a 1:2 final serum dilu- (2) Safety test. The mouse safety test tion by the virus plaque reduction prescribed in § 113.33(a) and the calf method. safety test prescribed in § 113.41 shall be (2) A geometric mean titer of the conducted. dried vaccine produced from the high- (3) Virus titer requirements. Final con- est passage of the Master Seed Virus tainer samples of completed product shall be established before the shall be tested for virus titer using the immunogenicity test is conducted. The titration method used in paragraph 20 calves to be used as vaccinates shall (c)(2) of this section. To be eligible for be injected with a predetermined quan- release, each serial and each subserial tity of vaccine virus and the remaining shall have a virus titer per dose suffi- five calves held as uninjected controls. ciently greater than the titer of vac- To confirm the dosage calculations, cine virus used in the immunogenicity five replicate virus titrations shall be test prescribed in paragraph (c) of this conducted on a sample of the vaccine section to assure that when tested at virus dilution used. any time within the expiration period, (3) At least once during a period of 14 each serial and subserial shall have a to 28 days post-vaccination, individual virus titer of 10 0.7 greater than that serum samples shall be collected for used in the immunogenicity test but virus-neutralization tests from each of 2.5 not less than 10 TCID50 per dose. the vaccinates. The test virus shall be 100 to 500 TCID bovine rhinotracheitis [39 FR 44719, Dec. 27, 1974, as amended at 40 50 FR 41089, Sept. 5, 1975; 43 FR 49529, Oct. 24, virus. Results shall be used in making 1978; 48 FR 33472, July 22, 1983. Redesignated a determination as prescribed in para- at 55 FR 35562, Aug. 31, 1990, as amended at 56 graph (c)(6) of this section. FR 66784, 66786, Dec. 26, 1991; 60 FR 14357, (4) The vaccinates and the controls Mar. 17, 1995; 72 FR 72564, Dec. 21, 2007] shall each be challenged with virulent infectious bovine rhinotracheitis virus § 113.310 Bovine Rhinotracheitis Vac- and observed for 14 days. The rectal cine. temperature of each animal shall be Bovine Rhinotracheitis Vaccine shall taken and the presence or absence of be prepared from virus-bearing cell cul- respiratory or other clinical signs of ture fluids. Only Master Seed Virus bovine rhinotracheitis noted and re- which has been established as pure, corded on each of the 14 consecutive safe, and immunogenic shall be used days. for preparing the production seed virus (5) If at least four of the five controls for vaccine production. All serials of do not show clinical signs of infectious vaccine shall be prepared from the first bovine rhinotracheitis and a marked through the tenth passage from the temperature rise to 104.5 °F. or higher Master Seed Virus. post-challenge, the test shall be consid- (a) The Master Seed Virus shall meet ered a No Test and may be repeated. the applicable general requirements (6) If less than 19 of the post-injection prescribed in § 113.300. serum samples tested as prescribed in (b) Each lot of Master Seed Virus paragraph (c)(3) of this section show shall meet the special requirements neutralization in all tubes of the 1:2 prescribed in this section. final serum dilution, or if more than (c) Each lot of Master Seed Virus one of the vaccinates show a tempera- used for vaccine production shall be ture of 103.5 °F. or higher for 2 or more

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days, or if more than one of the vac- § 113.311 Bovine Virus Diarrhea Vac- cinates exhibit respiratory or other cine. clinical signs of infectious bovine Bovine Virus Diarrhea Vaccine shall rhinotracheitis, or both, the Master be prepared from virus-bearing cell cul- Seed Virus is unsatisfactory. ture fluids. Only Master Seed Virus (7) A sequential test procedure may which has been established as pure, be used in lieu of the 20 calf require- safe, and immunogenic shall be used ment. A beta value of .05 and a toler- for preparing the production seed virus ance level of .78 shall be required. for vaccine production. All serials of (8) An outline of Production change vaccine shall be prepared from the first shall be made before authority for use through the tenth passage from the of a new lot of Master Seed Virus shall Master Seed Virus. be granted by Animal and Plant Health (a) The Master Seed Virus shall meet the applicable general requirements Inspection Service. prescribed in § 113.300. (d) Test requirements for release: (b) Each lot of Master Seed Virus Each serial and subserial shall meet shall meet the special requirements the applicable general requirements prescribed in this section. prescribed in § 113.300 and the require- (c) Each lot of Master Seed Virus ments in this paragraph. Final con- used for vaccine production shall be tainer samples of completed product tested for immunogenicity. The se- shall be tested except as prescribed in lected virus dose from the lot of Master paragraph (d)(1) of this section. Any se- Seed Virus shall be established as fol- rial or subserial found unsatisfactory lows: by a prescribed test shall not be re- (1) Twenty-five bovine virus diarrhea leased. susceptible calves shall be used as test (1) Purity test. The test for Brucella animals (20 vaccinates and five con- contamination prescribed in § 113.32 trols). Blood samples shall be drawn shall be conducted on each batch of pri- from these animals and individuals mary cells intended for production use. serum samples tested. The calves shall be considered susceptible to bovine (2) Safety test. The mouse safety test virus diarrhea virus infection if the re- prescribed in § 113.33(a) and the calf sults are negative at a 1:2 final serum safety test prescribed in § 113.41 shall be dilution in a varying serum-constant conducted. virus neutralization test with less than (3) Virus titer requirements. Final con- 500 TCID50 of bovine virus diarrhea tainer samples of completed product virus. shall be tested for virus titer using the (2) A geometric mean titer of the titration method used in paragraph dried vaccine produced from the high- (c)(2) of this section. To be eligible for est passage of the Master Seed Virus release, each serial and each subserial shall be established before the shall have a virus titer per dose suffi- immunogenicity test is conducted. The ciently greater than the titer of vac- 20 calves to be used as vaccinates shall cine virus used in the immunogenicity be injected with a predetermined quan- test prescribed in paragraph (c) of this tity of vaccine virus and the remaining section to assure that when tested at five calves held as uninjected controls. any time within the expiration period, To confirm the dosage calculations, each serial and subserial shall have a five replicate virus titrations shall be virus titer of 10 0.7 greater than that conducted on a sample of the vaccine used in the immunogenicity test but virus dilution used. not less than 10 2.5 TCID per dose. (3) At least once during a period 14 to 50 28 days post-vaccination, individual [39 FR 44720, Dec. 27, 1974, as amended at 40 serum samples shall be collected for FR 20067, May 8, 1975; 40 FR 23989, June 4, virus-neutralization tests from each of 1975; 40 FR 41089, Sept. 5, 1975; 43 FR 49529, the vaccinates. The test virus shall be Oct. 24, 1978; 48 FR 33472, July 22, 1983. Redes- less than 500 TCID50 of bovine virus di- ignated at 55 FR 35562, Aug. 31, 1990, as arrhea virus. The white cell count for amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 all vaccinates and controls shall be es- FR 72564, Dec. 21, 2007] tablished at least 3 days just before

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challenge. Results shall be used in ciently greater than the titer of vac- making a determination as prescribed cine virus used in the immunogenicity in paragraph (c)(5) of this section. test prescribed in paragraph (c) of this (4) The vaccinates and the controls section to assure that when tested at shall each be challenged with virulent any time within the expiration period, bovine virus diarrhea virus and ob- each serial and subserial shall have served for 14 consecutive days. The virus titer of 10 0.7 greater than that white cell count shall be determined used in the immunogenicity test but 2.5 daily on each animal from the second not less than 10 TCID 50 per dose. through the eighth day post-challenge. If leukopenia does not develop in at [39 FR 44721, Dec. 27, 1974, as amended at 40 least four of the five controls as com- FR 20067, May 8, 1975; 40 FR 41089, Sept. 5, 1975; 43 FR 49529, Oct. 24, 1978; 48 FR 33472, pared with the vaccinates, the test July 22, 1983. Redesignated at 55 FR 35562, shall be considered a No Test and may Aug. 31, 1990, as amended at 56 FR 66784, be repeated. 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007] (5) If less than 19 of the post-injection serum samples, tested as prescribed in § 113.312 Rabies Vaccine, Live Virus. paragraph (c)(3) of this section, show Rabies Vaccine shall be prepared neutralization in all tubes of the 1:8 di- from virus-bearing cell cultures or lution; or if more than one of the vac- embryonated chicken eggs. Only Mas- cinates exhibits respiratory or other ter Seed Virus which has been estab- clinical signs of bovine virus diarrhea lished as pure, safe and immunogenic post-challenge; or both, the Master shall be used for preparing the produc- Seed Virus is unsatisfactory. tion seed virus for vaccine production. (6) A sequential test procedure may All serials of vaccine shall be prepared be used in lieu of the 20 calf require- from the first through the fifth passage ment. A beta value of .05 and a toler- from the Master Seed Virus. ance level of .78 shall be required. (7) An Outline of Production change (a) The Master Seed Virus shall meet shall be made before authority for use the applicable general requirements of a new lot of Master Seed Virus shall prescribed in § 113.300. be granted by Animal and Plant Health (1) Each lot of Master Seed Virus Inspection Service. shall meet the special requirements (d) Test requirements for release: prescribed in this section. Each serial and subserial shall meet (2) Each lot of Master Seed Virus the applicable general requirements propagated in tissues or cells of avian prescribed in § 113.300 and the require- origin shall be tested for pathogens by ments in this paragraph. Final con- procedures prescribed in § 113.37. tainer samples of completed product (3) Each lot of Master Seed Virus shall be tested except as prescribed in propagated in primary cell cultures of paragraph (d)(1) of this section. Any se- mouse or hamster origin or brain tis- rial or subserial found unsatisfactory sues of mouse origin shall be tested for by a prescribed test shall not be re- lymphocytic choriomeningitis (LCM) leased. virus by the procedure prescribed in (1) Purity test. The test for Brucella § 113.42. If LCM virus is detected, the contamination prescribed in § 113.32 Master Seed Virus is unsatisfactory. shall be conducted on each batch of pri- (4) The Master Seed Virus shall be mary cells intended for production use. studied in each species of carnivore or (2) Safety test. The mouse safety test domesticated wild animal for which prescribed in § 113.33(a) and the calf the vaccine is specifically rec- safety test prescribed in § 113.41 shall be ommended to attempt to determine the conducted. fate of the vaccine virus. Results shall (3) Virus titer requirements. Final con- be considered in evaluating safety of tainer samples of completed product vaccine virus. shall be tested for virus titer using the (i) Obtain at least 10 unvaccinated titration method used in paragraph animals, negative at 1:2 final serum di- (c)(2) of this section. To be eligible for lution, of each species in which tests release, each serial and each subserial will be conducted. Divide each species shall have a virus titer per dose suffi- into two groups of five animals.

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(ii) For each species of animal, inject bies virus by the fluorescent antibody one group of five animals test and by mouse injection. intramuscularly. Infiltrate a major (v) If rabies is confirmed, the lot of nerve and the surrounding tissue in Master Seed Virus is unsatisfactory. each of the five animals in the other (b) The immunogenicity of vaccine group. Use 1.0 ml of high titer virus for prepared with virus at the highest pas- each method of administration. sage of the Master Seed shall be estab- (iii) Observe all animals for signs of lished in each species for which the rabies until scheduled time to sac- vaccine is recommended. Tests shall be rifice. If animals show definite symp- conducted in accordance with a pro- toms, sacrifice and check regional tocol filed with Animal and Plant lymph nodes, brain, salivary glands, Health Inspection Service before initi- and kidney for rabies virus by injection ation of the tests. The vaccine shall be of suckling mice (not more than 7 days prepared using methods prescribed in of age). Tissues may be held frozen at the Outline of Production. If Rabies ¥70 °C. until suckling mice are avail- Vaccine is to be in combination with able. Inject each mouse in one litter other fractions, the product tested intracerebrally with 0.02 ml of a ground shall include all fractions to be rec- tissue suspension from each organ. Ob- ommended. serve mice each day for 21 days. If any (1) A geometric mean virus titer of mice die, determine if the deaths were the dried vaccine produced from the due to rabies virus in the brain by a highest passage of the Master Seed fluorescent antibody test. Virus shall be established before the (iv) Sacrifice animals that do not immunogenicity test is conducted. To show signs of rabies according to the confirm the dosage calculations, five following schedule and check regional replicate virus titrations shall be con- lymph nodes, brain, salivary glands, ducted on a sample of the vaccine virus and kidney in suckling mice. dilution used. (2) The dose of vaccine to be used in Number of the immunogenicity test shall be no Route of injection Days after injection animals more than the amount of rehydrated Intramuscularly ...... 15, 20, 25, 30, 35 1 each day. vaccine which, on the basis of previous Intraneurally ...... 3, 6, 9, 15, 30 1 each day. titrations, has been diluted to the proposed minimum acceptable virus titer. (5) Each lot of Master Seed Virus (3) Test animals shall be uniform and shall be tested for safety in at least 10 have no neutralizing antibodies to ra- unvaccinated serologically negative bies as determined by serum-neutral- animals of each domestic species for ization (SN) tests. which the vaccine is recommended. (i) Twenty-five or more animals shall (i) Each group of 10 animals shall be be used as vaccinates. Each shall be in- divided into 2 groups of 5 animals. For jected intramuscularly at one site in each species, inject one group the thigh with a dose of vaccine at the intramuscularly with 10 doses of high proposed minimum virus titer as speci- titer virus. fied in the filed Outline of Production. (ii) Infiltrate a major nerve of each of (ii) Ten or more additional animals the animals in the other group of 5 shall be held as controls. with 10 doses of the same high titer (iii) On or about days 30, 90, 180, 270, virus. For all species except dogs and and 365 postvaccination, all animals cats, multiple injections along the cer- shall be bled and individual serums vical spine in the proximity to the tested for neutralizing antibodies to ra- nerve trunks emerging from the spinal bies virus. cord may be used: Provided, That a 1- (iv) All surviving test animals of dose volume shall be injected into each each species shall be challenged of four or more sites bilaterally. intramuscularly with virulent rabies (iii) Observe all animals each day for virus furnished or approved by Animal 90 days. and Plant Health Inspection Service 1 (iv) If any animals show clinical year after vaccination, except as pro- signs of rabies, sacrifice the animal and vided in paragraphs (b)(4), (b)(5), and check appropriate brain tissue for ra- (b)(6) of this section. The challenged

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animals shall be observed each day for time of challenge may be adjusted ac- 90 days as prescribed in § 113.5(b). The cordingly. brain of each test animal that dies fol- (d) Test requirements for release: lowing challenge shall be examined for Each serial and each subserial shall rabies by the fluorescent antibody test meet the general requirements pre- or other method acceptable to Animal scribed in § 113.300 and special require- and Plant Health Inspection Service. ments in this paragraph. (v) Requirements for acceptance in (1) Purity and safety tests. Final con- challenge tests shall be death due to tainer samples of completed product rabies in at least 80 percent of controls from each serial or one subserial shall while at least 22 of 25 or 26 of 30 or a be tested. statistically equivalent number of the (i) The test for pathogens, prescribed vaccinates remain well for a period of in § 113.37 shall be conducted on each 90 days. serial or one subserial of avian origin. (4) An alternative to challenging all If necessary, neutralize the rabies virus surviving test animals in accordance with specific rabies antiserum. with paragraph (b)(3)(iv) of this section (ii) A test for safety in three young may be used when the test animals are seronegative animals of the most sus- of species other than carnivores. Vac- ceptible species for which the vaccine cinates shall be challenged at 1 year is recommended shall be conducted. postvaccination. These shall include Each shall be injected intramuscularly five vaccinates with the lowest SN with 10 recommended doses of vaccine. titers at the 270th-day bleeding, five If unfavorable reactions attributable to vaccinates with the lowest SN titers at the product occur during a 28 day ob- the 365th-day bleeding, and all vac- servation period, the serial is unsatis- cinates with SN titers below 1:10 by the factory. mouse SN test or below 1:16 by the (iii) If primary cell cultures of ham- rapid-fluorescent-focus-inhibition test ster origin or of mouse origin are used at any bleeding. At least five SN-nega- vaccine production, they shall be test- tive controls of each species shall be ed for LCM virus as prescribed in challenged at the same time as the § 113.42. The cells shall be disrupted and vaccinates. All SN titers shall be undiluted cell fluids from each lot shall iterated to an endpoint. All of the chal- be tested. lenged vaccinates must remain well for (2) Virus titrations. Final container a period of 90 days, and at least 80 per- samples of completed product shall be cent of the controls must die of rabies tested for virus titer using the titra- for a satisfactory test without further tion method used in paragraph (b)(1) of challenge. If one or more of the vac- this section. To be eligible for release, cinates die from rabies, all the remain- each serial and each subserial shall ing vaccinates, regardless of titer, have a virus titer sufficiently higher along with the five controls shall be than the titer of the vaccine virus used challenged. The cumulative results in paragraph (b) of this section to as- from the two challenges shall be evalu- sure that, when tested at any time ated for acceptance as specified in within the expiration period, each se- paragraph (b)(3)(v) of this section. rial and subserial shall have a virus (5) An outline of Production change titer equal to or greater than that used shall be made before authority for use in the immunogenicity test. of a new lot of Master Virus shall be (3) Young adult mice, each weighing granted by Animal and Plant Health 14 to 16 grams, shall be used as test ani- Inspection Service. mals when the virus in vaccine pre- (c) If more than 1 year duration of pared with a low egg passage Flury immunity is to be claimed, a duration Strain or high cell passage Street Ala- of immunity test for the additional bama Dufferin Strain (HCP SAD) of ra- time shall be conducted and inter- bies virus is titrated. At least 10 mice preted as prescribed in paragraph (b) of for each dilution shall be used. this section for the 1 year test. The (i) At least 10 mice shall be used for test animals shall be monitored sero- each dilution. Each shall be injected logically at least every 180 days. The intracerebrally with 0.03 ml.

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(ii) The injected young adult mice (a) The Master Seed Virus shall meet shall be observed each day for 14 days the applicable general requirements except when testing vaccines made prescribed in § 113.300. Each lot of Mas- with HCP SAD strain of rabies virus, in ter Seed Virus shall meet the special which case, the mice shall be observed requirements prescribed in this sec- each day for 21 days. Deaths and paral- tion. ysis occurring subsequent to the fourth (b) To detect virulent canine dis- day post-injection shall be noted and temper virus, each of two canine dis- the LD50 titer calculated by the Reed temper susceptible ferrets shall be in- and Muench Method. jected with a sample of the Master (iii) Virus titer requirements for re- Seed Virus equivalent to the amount of lease and at expiration date shall be virus to be used in one dog dose and ob- determined for each vaccine on the served each day for 21 days. If undesir- basis of data available: Provided, That, able reactions occur in either ferret, the lowest titer permitted at expira- the lot of Master Seed Virus is unsatis- tion date when determined by this test factory. 3.0 shall be 10 LD50 per 0.03 ml. (c) Each lot of Master Seed Virus (4) Suckling mice, 6 days of age or used for vaccine production shall be younger, shall be used as test animals tested for immunogenicity. The se- when virus in vaccine prepared with a lected virus dose from the lot of Master high egg passage Flury Strain of rabies Seed Virus shall be established as fol- virus is titrated. lows: (i) Six to twelve mice shall be used (1) Twenty-five dogs, less than 12 for each dilution. Each shall be in- weeks of age and free of measles anti- jected intracerebrally with 0.02 ml. body, shall be used as test animals (20 (ii) The injected suckling mice shall vaccinates and five controls). Blood be observed each day for 21 days. samples shall be drawn from these ani- Deaths and paralysis occurring subse- mals and individual serum samples quent to the fourth day post-injection tested. The dogs shall be considered shall be noted and the LD50 titer cal- susceptible if the results are negative culated by the Reed and Muench Meth- at a 1:2 final serum dilution in a vary- od; and ing serum-constant virus neutraliza- (iii) Virus titer requirements for re- tion test with less than 500 ID50 of mea- lease and at expiration date shall be sles virus. determined for each vaccine on the (2) A geometric mean titer of the basis of data available: Provided, That, dried vaccine produced from the high- the lowest titer permitted at expira- est passage of the Master Seed Virus tion date when determined by this test shall be established before the 3.0 shall be 10 LD50 per 0.02 ml. immunogenicity test is conducted. Twenty dogs shall be vaccinated with a [39 FR 44721, Dec. 27, 1974, as amended at 40 predetermined quantity of vaccine FR 20067, May 8, 1975; 42 FR 6795, Feb. 4, 1977; 43 FR 49529, Oct. 24, 1978; 50 FR 20090, May 14, virus and the remaining five dogs held 1985; 50 FR 23797, June 6, 1985. Redesignated as unvaccinated controls. To confirm at 55 FR 35562, Aug. 31, 1990, as amended at 56 the dosage calculations, five replicate FR 66784, 66786, Dec. 26, 1991; 61 FR 31823, virus titrations shall be conducted on a June 21, 1996; 72 FR 72564, Dec. 21, 2007] sample of the vaccine virus dilution used. § 113.313 Measles Vaccine. (3) On the day of challenge, serum Measles Vaccine shall be prepared samples shall be obtained from each from virus-bearing cell culture fluids. vaccinate and individually tested for Only Master Seed Virus which has been antibody against canine distemper established as pure, safe, and virus. For a valid test, each vaccinate immunogenic shall be used for pre- shall be negative at a 1:4 final serum paring the production seed virus for dilution in varying serum-constant vaccine production. All serials of vac- virus neutralization test using less cine shall be prepared from the first than 500 ID50 of canine distemper virus. through the fifth passage from the (4) At least 21 days postinoculation, Master Seed Virus. the immunity of the vaccinates and

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controls shall be challenged by expo- the immunogenicity test but not less 2.5 sure to a uniform dose of aerosolized than 10 ID50 per dose. virulent canine distemper virus. All [40 FR 53001, Nov. 14, 1975, as amended at 43 test dogs shall be observed daily for 21 FR 49529, Oct. 24, 1978; 48 FR 33472, July 22, days postchallenge. 1983. Redesignated at 55 FR 35562, Aug. 31, (i) If at least 4 of the 5 controls do 1990, as amended at 56 FR 66784, 66786, Dec. not die or show signs of distemper, in- 26, 1991; 72 FR 72564, Dec. 21, 2007] ° cluding a temperature of 104.0 F. or § 113.314 Feline Calicivirus Vaccine. higher and at least 15 percent weight loss, the test is a No Test and may be Feline Calicivirus Vaccine shall be repeated. prepared from virus-bearing cell culture fluids. Only Master Seed Virus (ii) If at least 19 of the 20 vaccinates which has been established as pure, do not survive without showing a tem- safe, and immunogenic shall be used ° perature of 104.0 F. or higher and a for preparing the production seed virus weight loss exceeding 15 percent after for vaccine production. All serials of day 8 postchallenge, the Master Seed vaccine shall be prepared from the first Virus is unsatisfactory. through the fifth passage from the (5) When approved in advance by Ani- Master Seed Virus. mal and Plant Health Inspection Serv- (a) The Master Seed Virus shall meet ice, a sequential test procedure may be the applicable general requirements used in lieu of the 20 dog requirement. prescribed in § 113.300. A beta value of 0.05 and a tolerance (b) The Master Seed Virus shall be level of 0.78 shall be required. tested for chlamydial agents as pre- (6) An Outline of Production change scribed in § 113.43. shall be made before authority for use (c) Each lot of Master Seed Virus of a new lot of Master Seed Virus shall used for vaccine production shall be be granted by Animal and Plant Health tested for immunogenicity. The se- Inspection Service. lected virus dose from the lot of Master (d) Test requirements for release: Seed Virus shall be established as follows: Each serial and subserial shall meet (1) Thirty feline calicivirus suscep- the general requirements prescribed in tible cats shall be used as test animals § 113.300 and the requirements in this (20 vaccinates and 10 controls). Throat paragraph. Final container samples of swabs shall be collected from each cat completed product shall be tested. Any and individually tested on susceptible serial or subserial found unsatisfactory cell cultures for the presence of feline by a prescribed test shall not be re- calicivirus. Blood samples shall be leased. drawn and individual serum samples (1) Safety tests. The dog safety test tested. The cats shall be considered prescribed in § 113.40 and the mouse suitable for use if all swabs are nega- safety test prescribed in § 113.33(a) shall tive for virus isolation and if all se- be conducted. rums are negative for calicivirus anti- (2) Virus titer requirements. Final con- body at the 1:2 final dilution in a 50 tainer samples of completed product percent plaque reduction test or other shall be tested for virus titer using the SN test of equal sensitivity. titration method used in paragraph (2) A geometric mean titer of the (c)(2) of this section. To be eligible for dried vaccine produced from the high- release, each serial and each subserial est passage of the Master Seed Virus shall have a virus titer sufficiently shall be established before the greater than the titer of the vaccine immunogenicity test is conducted. The 20 cats used as vaccinates shall be ad- virus used in the immunogenicity test ministered a predetermined quantity of prescribed in paragraph (c) of this sec- vaccine virus by the method to be rection to assure that when tested at any ommended on the label and the re- time within the expiration period, each maining 10 cats shall be held as con- serial and subserial shall have a virus trols. To confirm the dosage calcula- titer of 10 0.7 greater than that used in tions, five replicate virus titrations shall be conducted on a sample of the

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vaccine virus dilution used. If two rial and subserial shall have a virus doses are used, five replicate con- titer of 10 0.7 greater than that used in firming titrations shall be conducted the immunogenicity test but not less 2.5 on each dose. than 10 TCID50 or plaque forming (3) Twenty-one or more days after units per dose. the final dose of vaccine, the vaccinates and controls shall each be chal- [44 FR 58899, Oct. 12, 1979; 44 FR 63083, Nov. 2, 1979, as amended at 48 FR 33472, July 22, 1983. lenged intranasally with a minimum of Redesignated at 55 FR 35562, Aug. 31, 1990, as 100,000 TCID50 or plaque forming units amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 of virulent feline calicivirus furnished FR 72564, Dec. 21, 2007] or approved by Animal and Plant Health Inspection Service and observed § 113.315 Feline Rhinotracheitis Vac- each day for 14 days postchallenge. The cine. rectal temperature of each animal Feline Rhinotracheitis Vaccine shall shall be taken and the presence or ab- be prepared from virus-bearing cell cul- sence of clinical signs, particularly le- ture fluids. Only Master Seed Virus sions on the oral mucosa, noted and re- which has been established as pure, corded each day. safe, and immunogenic shall be used (i) If less than 8 of 10 controls show for preparing the production seed virus clinical signs of feline calicivirus infec- for vaccine production. All serials of tion other than fever, the test is a No vaccine shall be prepared from the first Test and may be repeated. through the fifth passage from the (ii) If a significant difference in clin- Master Seed Virus. ical signs cannot be demonstrated between vaccinates and controls using a (a) The Master Seed Virus shall meet scoring system approved by Animal the applicable general requirements and Plant Health Inspection Service prescribed in § 113.300. and prescribed in the Outline of Pro- (b) The Master Seed Virus shall be duction, the Master Seed Virus is un- tested for chlamydial agents as pre- satisfactory. scribed in § 113.43. (4) An Outline of Production change (c) Each lot of Master Seed Virus shall be made before authority for use used for vaccine production shall be of a new lot of Master Seed Virus shall tested for immunogenicity. The se- be granted by Animal and Plant Health lected virus dose from the lot of Master Inspection Service. Seed Virus shall be established as fol- (d) Test requirements for release. Each lows: serial and subserial shall meet the re- (1) Thirty feline rhinotracheitis sus- quirements prescribed in § 113.300 and ceptible cats shall be used as test ani- in this paragraph. Final container sam- mals (20 vaccinates and 10 controls). ples of completed product shall be test- Throat swabs shall be collected from ed. Any serial or subserial found unsat- each cat and individually tested on sus- isfactory by a prescribed test shall not ceptible cell cultures for the presence be released. of feline rhinotracheitis virus. Blood (1) Safety test. The mouse safety test samples shall be drawn and individual prescribed in § 113.33(a) and the cat serum samples tested. The cats shall be safety test prescribed in § 113.39(b) shall considered suitable for use if all swabs be conducted. are negative for virus isolation and if (2) Virus titer requirements. Final con- all serums are negative for feline tainer samples of completed product rhinotracheitis virus antibody at the shall be tested for virus titer using the 1:2 final dilution in a 50 percent plaque titration method used in paragraph reduction test or other SN test of equal (c)(2) of this section. To be eligible for sensitivity. release, each serial and each subserial (2) A geometric mean titer of the shall have a virus titer sufficiently dried vaccine produced from the high- greater than the titer of vaccine virus est passage of the Master Seed Virus used in the immunogenicity test pre- shall be established before the scribed in paragraph (c) of this section immunogenicity test is conducted. The to assure that when tested at any time 20 cats used as vaccinates shall be ad- within the expiration period, each se- ministered a predetermined quantity of

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vaccine virus by the method to be rec- greater than the titer of vaccine virus ommended on the label and the re- used in the immunogenicity test pre- maining 10 cats shall be held as con- scribed in paragraph (c) of this section trols. To confirm the dosage calcula- to assure that when tested at any time tions, five replicate virus titrations within the expiration period, each se- shall be conducted on a sample of the rial and subserial shall have a virus vaccine virus dilution used. If two titer of 10 0.7 greater than that used in doses are used, five replicate con- the immunogenicity test but not less 2.5 firming titrations shall be conducted than 10 TCID50 or plaque forming on each dose. units per dose. (3) Twenty-one or more days after [44 FR 58899, Oct. 12, 1979, as amended at 48 the final dose of vaccine, the vac- FR 33472, July 22, 1983. Redesignated at 55 FR cinates and controls shall each be chal- 35562, Aug. 31, 1990, as amended at 56 FR lenged intranasally with a minimum of 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 100,000 TCID50 or plaque forming units 2007] of virulent feline rhinotracheitis virus furnished or approved by Animal and § 113.316 Canine Parainfluenza Vac- Plant Health Inspection Service and cine. observed each day for 14 days post- Canine Parainfluenza Vaccine shall challenge. The rectal temperature of be prepared from virus-bearing cell cul- each animal shall be taken and the ture fluids. Only Master Seed which presence of respiratory or other clin- has been established as pure, safe, and ical signs of feline rhinotracheitis immunogenic shall be used for pre- noted and recorded each day. paring seeds for vaccine production. All (i) If less than 8 of 10 controls show serials of vaccine shall be prepared clinical signs of feline rhinotracheitis from the first through the fifth passage infection other than fever, the test is a from the Master Seed. No Test and may be repeated. (a) The Master Seed shall meet the (ii) If a significant difference in clin- applicable general requirements pre- ical signs cannot be demonstrated be- scribed in § 113.300 and the require- tween vaccinates and controls using a ments in this section. scoring system approved by Veterinary (b) Each lot of Master Seed shall be Services and prescribed in the Outline tested for immunogenicity. The se- of Production, the Master Seed Virus is lected virus dose shall be established as unsatisfactory. follows: (4) An Outline of Production change (1) Twenty-five canine parainfluenza shall be made before authority for use susceptible dogs (20 vaccinates and 5 of a new lot of Master Seed Virus shall controls) shall be used as test animals. be granted by Animal and Plant Health Nasal swabs shall be collected from Inspection Service. each dog on the day the first dose of (d) Test requirements for release. Each vaccine is administered and individ- serial and subserial shall meet the re- ually tested on susceptible cell cul- quirements prescribed in § 113.300 and tures for the presence of canine in this paragraph. Final container sam- parainfluenza virus. Blood samples ples of completed product shall be test- shall also be drawn and individual ed. Any serial or subserial found unsat- serum samples tested for neutralizing isfactory by a prescribed test shall not antibody. Dogs shall be considered sus- be released. ceptible if all swabs are negative for (1) Safety test. The mouse safety test virus isolation and if all serums are prescribed in § 113.33(a) and the cat negative for canine parainfluenza anti- safety test prescribed in § 113.39(b) shall body at a 1:2 final dilution in a con- be conducted. stant virus-varying serum neutraliza- (2) Virus titer requirements. Final con- tion test using 50 to 300 TCID50 of ca- tainer samples of completed product nine parainfluenza virus. shall be tested for virus titer using the (2) A geometric mean titer of vaccine titration method used in paragraph produced at the highest passage from (c)(2) of this section. To be eligible for the Master Seed shall be established release, each serial and each subserial before the immunogenicity test is con- shall have a virus titer sufficiently ducted. The 20 dogs used as vaccinates

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shall be administered a predetermined (1) Virus titer requirements. Final con- quantity of vaccine virus. Five rep- tainer samples of completed product licate virus titrations shall be con- shall be tested for virus titer using the ducted on a sample of the vaccine virus titration method used in paragraph dilution used to confirm the dosage ad- (b)(2) of this section. To be eligible for ministered. If two doses are used, five release, each serial and each subserial replicate confirming titrations shall be shall have a virus titer sufficiently conducted on each dose. greater than the titer of vaccine virus (3) Three to 4 weeks after the final used in the immunogenicity test pre- dose of vaccine, all dogs shall be bled scribed in paragraph (b) of this section for serum antibodies and nasal swabs to assure that, when tested at any time shall be collected for canine within the expiration period, each se- parainfluenza virus isolation. On the rial and subserial shall have a virus same day, all vaccinates and controls titer at least 10 0.7 greater than that shall be challenged with canine used in the immunogenicity test but 2.5 parainfluenza virus furnished or ap- not less than 10 TCID50 per dose. proved by Animal and Plant Health In- (2) [Reserved] spection Service. [50 FR 436, Jan. 4, 1985. Redesignated at 55 (4) The rectal temperature of each FR 35562, Aug. 31, 1990, as amended at 56 FR dog shall be taken and the presence of 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, respiratory or other clinical signs of 2007] canine parainfluenza virus infection noted and recorded each day for 14 con- § 113.317 Parvovirus Vaccine (Canine). secutive days postchallenge. Nasal Parvovirus Vaccine recommended for swabs shall be collected from each dog use in dogs shall be prepared from each day for at least 10 consecutive virus-bearing cell culture fluids. Only days postchallenge. Individual swabs Master Seed which has been estab- shall be tested for virus isolation by lished as pure, safe, and immunogenic culture in canine parainfluenza virus shall be used for preparing seeds for susceptible cells for at least 7 days. Re- vaccine production. All serials of vac- sults shall be evaluated according to cine shall be prepared from the first the following criteria: through the fifth passage from the (i) If five of five controls have not re- Master Seed. mained seronegative at a final serum (a) The Master Seed shall meet the dilution of 1:2 during the prechallenge applicable general requirements pre- period, the test is a No Test and may scribed in § 113.300 and the require- be repeated. ments in this section. (ii) If more than one vaccinate shows (b) The Master Seed shall be tested febrile response, respiratory or other for reversion to virulence in dogs using clinical signs of canine parainfluenza a method acceptable to Animal and virus infection; or, if less than 19 of 20 Plant Health Inspection Service. If a vaccinates show serum neutralization significant increase in virulence is seen titers of 1:4 or greater; or, if there is within five backpassages, the Master not a significant reduction in virus iso- Seed is unsatisfactory. lation rate in vaccinates when com- (c) Each lot of Master Seed shall be pared with controls, the Master Seed is tested for immunogenicity. The se- unsatisfactory. lected virus dose shall be established as (5) An Outline of Production change follows: shall be made before authority for use (1) Twenty-five canine parvovirus of a new lot of Master Seed shall be susceptible dogs (20 vaccinates and 5 granted by Animal and Plant Health controls) shall be used as test animals. Inspection Service. Blood samples drawn from each dog (c) Test requirements for release. Each shall be individually tested for neutral- serial and subserial shall meet the ap- izing antibody against canine plicable general requirements pre- parvovirus to determine susceptibility. scribed in § 113.300 and the require- Dogs shall be considered susceptible if ments in this paragraph. Any serial or there is no neutralization at a 1:2 final subserial found unsatisfactory by a serum dilution in a constant virus- prescribed test shall not be released. varying serum neutralization test in

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cell culture using 50 to 300 TCID50 of ments in this paragraph. Any serial or canine parvovirus. subserial found unsatisfactory by a (2) A geometric mean titer of the vac- prescribed test shall not be released. cine produced at the highest passage (1) Virus titer requirements. Final con- from the Master Seed shall be estab- tainer samples of completed product lished before the immunogenicity test shall be tested for virus titer using the is conducted. The 20 dogs used as vac- titration method used in paragraph cinates shall be administered a pre- (c)(2) of this section. To be eligible for determined quantity of vaccine virus release, each serial and each subserial by the method recommended on the shall have a virus titer sufficiently label. To confirm the dosage calcula- greater than the titer of vaccine used tions, five replicate virus titrations in the immunogenicity test in para- shall be conducted on a sample of the graph (c) of this section to assure that, vaccine virus dilution used. If two when tested at any time within the ex- doses are used, five replicate con- piration period, each serial and sub- firming titrations shall be conducted serial shall have a virus titer of 10 0.7 on each dose. greater than that used in the (3) Fourteen days or more after the immunogenicity test, but not less than 2.5 final dose of vaccine the vaccinates and 10 ID50 per dose. the controls shall be challenged with virulent canine parvovirus furnished or [50 FR 436, Jan. 4, 1985. Redesignated at 55 approved by Animal and Plant Health FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, Inspection Service and the dogs ob- 2007] served each day for 14 days. Rectal temperature, blood lymphocyte count, § 113.318 Pseudorabies Vaccine. and feces for viral detection shall be taken from each dog each day for at Pseudorabies Vaccine shall be pre- least 10 days postchallenge and the pared from virus-bearing cell culture presence or absence of clinical signs fluids. Only Master Seed which has noted and recorded each day. been established as pure, safe, and (i) The immunogenicity of the Mas- immunogenic shall be used for pre- ter Seed shall be evaluated on the fol- paring seeds for vaccine production. All lowing criteria of infection: tempera- serials of vaccine shall be prepared ture ≥103.4 °F; lymphopenia of ≥50 per- from the first through the fifth passage cent of prechallenge normal; clinical from the Master Seed. signs such as diarrhea, mucus in feces, (a) The Master Seed shall meet the or blood in feces; and viral applicable general requirements pre- hemagglutinins at a level of ≥1:64 in a scribed in § 113.300 and the require- 1:5 dilution of feces or a test of equal ments in this section. sensitivity. If at least 80 percent of the (b) Each lot of Master Seed shall be controls do not show at least three of tested for immunogenicity. The se- the four criteria of infection during the lected virus dose shall be established as observation period, the test is a No follows: Test and may be repeated. (1) Twenty-five pseudorabies suscep- (ii) If at least 19 of the 20 vaccinates tible pigs (20 vaccinates and 5 controls) do not survive the observation period of the youngest age for which the vac- without showing more than one cri- cine is recommended, shall be used as terion of infection described in para- test animals. Blood samples shall be graph (c)(3)(i), of this section, the Mas- taken from each pig and the serums inter Seed is unsatisfactory. activated and individually tested for (4) An Outline of Production change neutralizing antibody against shall be made before authority for use pseudorabies virus. Pigs shall be con- of a new lot of Master Seed shall be sidered susceptible if there is no neu- granted by Animal and Plant Health tralization at a 1:2 final serum dilution Inspection Service. in a constant virus-varying serum neu- (d) Test requirements for release. Each tralization test using 50 to 300 TCID50 serial and subserial shall meet the ap- pseudorabies virus. plicable general requirements pre- (2) A geometric mean titer of the vac- scribed in § 113.300 and the require- cine produced at the highest passage

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from the Master Seed shall be estab- §§ 113.319–113.324 [Reserved] lished before the immunogenicity test is conducted. The 20 pigs used as vac- § 113.325 Avian Encephalomyelitis cinates shall be administered a pre- Vaccine. determined quantity of vaccine virus Avian Encephalomyelitis Vaccine by the method recommended on the shall be prepared from virus-bearing label. To confirm the dosage adminis- tissues or fluids from embryonated tered, five replicate virus titrations chicken eggs. Only Master Seed Virus shall be conducted on a sample of the which has been established as pure, vaccine virus dilution used. safe, and immunogenic in accordance (3) Fourteen to 28 days with the requirements in paragraphs postvaccination, the vaccinates and (a), (b), and (c) of this section shall be controls shall be challenged with viru- used for preparing the production seed lent pseudorabies virus furnished or ap- virus for vaccine production. All serials proved by Animal and Plant Health In- shall be prepared from the first spection Service and observed each day through the fifth passage from the for 14 days. Master Seed Virus. (i) If at least four of the five controls (a) The Master Seed Virus shall meet do not develop severe central nervous the applicable requirements prescribed system signs or die, the test is a No in § 113.300 and the requirements pre- Test and may be repeated. scribed in this section. (ii) If at least 19 of the 20 vaccinates (b) Each lot of Master Seed Virus in a valid test do not remain free of shall be tested for pathogens by the signs of pseudorabies, the Master Seed chicken embryo inoculation test pre- is unsatisfactory. scribed in § 113.37, except that, if the (4) An Outline of Production change test is a No Test because of a vaccine shall be made before authority for use virus override, the test may be re- of a new lot of Master Seed shall be peated and if the repeat test is incon- granted by Animal and Plant Health clusive for the same reason, the chick- Inspection Service. en inoculation test prescribed in § 113.36 (c) Test requirements for release. Each may be conducted and the virus judged serial and subserial shall meet the ap- accordingly. plicable general requirements pre- (c) Each lot of Master Seed Virus scribed in § 113.300 and the require- shall be tested for immunogenicity and ments in this paragraph. the selected virus dose to be used shall (2) Virus titer requirements. Final con- be established as follows: tainer samples of completed product (1) Avian encephalomyelitis suscep- shall be titrated by the method used in tible chickens, all of the same age paragraph (b)(2) of this section. To be (eight weeks or older) and from the eligible for release, each serial and sub- same source, shall be used. Twenty or more chickens shall be used as vac- serial shall have a virus titer suffi- cinates for each method of administra- ciently greater than the titer of the tion recommended on the label. Ten vaccine used in the immunogenicity additional chickens of the same age test prescribed in paragraph (b) of this and from the same source shall be held section to assure that, when tested at as unvaccinated controls. any time within the expiration period, (2) A geometric mean titer of the vac- each serial and subserial shall have a cine produced from the highest passage virus titer at least 10. 0.7 greater than of the Master Seed Virus shall be es- that used in the immunogenicity test, tablished before the immunogenicity 2.5 but not less than 10 TCID50 per dose. test is conducted. Each vaccinate shall [50 FR 437, Jan. 4, 1985. Redesignated at 55 receive a predetermined quantity of FR 35562, Aug. 31, 1990, as amended at 56 FR vaccine virus. Five replicate virus ti- 66784, 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, trations shall be conducted on an ali- 2007] quot of the vaccine virus to confirm the amount of virus administered to each chicken used in the test. At least

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three appropriate (not to exceed ten- (1) Final container samples from fold) dilutions shall be used and the each serial shall be tested for patho- test conducted as follows: gens by the chicken embryo inocula- (i) For each dilution, inoculate at tion test prescribed in § 113.37, except least 10 embryos, 5 or 6 days old, in the that, if the test is a No Test because of yolk sac with 0.2 ml each. Twenty simi- a vaccine virus override, the chicken lar embryos obtained from the same inoculation test prescribed in § 113.36 source shall be kept as uninoculated may be conducted and the vaccine negative controls. Disregard all deaths judged accordingly. during the first 48 hours post-inocula- (2) Safety test. Final container sam- tion. ples of completed product shall be test- (ii) Eggs for each dilution shall be ed for safety as follows: kept in separate containers and al- (i) At least 25 AE susceptible birds (6 lowed to hatch. Sufficient precaution to 10 weeks of age) shall be vaccinated shall be taken to assure that chickens with the equivalent of 10 doses by each from each dilution remain separated. of all routes recommended on the label To be a valid test, at least 75 percent of and be observed each day for 21 days. the uninoculated eggs shall hatch. (ii) If unfavorable reactions attrib- (iii) On the third day after normal utable to the biological product occur hatching time, count all unhatched during the observation period, the se- eggs and all dead, paralyzed and ataxic rial is unsatisfactory. If unfavorable chickens as positive evidence of viral reactions occur which are not attrib- infection. utable to the product, the test shall be (iv) A satisfactory titration shall declared a No Test and repeated, except have at least one dilution with between that, if the test is not repeated, the se- 50 and 100 percent positives and at least rial shall be unsatisfactory. one dilution with between 50 and 0 per- (3) Virus titer requirements. Final con- cent positives. tainer samples of completed product (v) Calculate the EID50 by the shall be tested for virus titer using the Spearman-Karber or Reed-Muench titration method used in paragraph method. (c)(2) of this section. To be eligible for (3) At least 21 days post-vaccination, release, each serial and each subserial the vaccinates and the controls shall shall have a virus titer sufficiently be challenged intracerebrally with a greater than the titer of vaccine virus virulent avian encephalomyelitis virus used in the immunogenicity test pre- and observed each day for 21 days. scribed in paragraph (c) of this section (4) If at least 80 percent of the con- to assure that when tested at any time trols do not show signs of avian within the expiration period, each se- encephalomyelitis or die, the test is a rial and subserial shall have a virus No Test and may be repeated. If at titer of 10 0.7 greater than that used in least 19 of 20, or 27 of 30, or 36 of 40 of such immunogenicity test but not less .5 the vaccinates in each group do not re- than 10 EID50 per dose. main free from clinical signs of avian [39 FR 44723, Dec. 27, 1974, as amended at 40 encephalomyelitis during the observa- FR 18405, Apr. 28, 1975; 40 FR 41089, Sept. 5, tion period, the Master Seed Virus is 1975; 42 FR 43617, Aug. 30, 1977; 48 FR 33473, unsatisfactory. July 22, 1983. Redesignated at 55 FR 35562, (5) An Outline of Production change Aug. 31, 1990, as amended at 56 FR 66784, shall be made before authority for use 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007; of a new lot of Master Seed Virus shall 79 FR 55969, Sept. 18, 2014] be granted by Animal and Plant Health Inspection Service. § 113.326 Avian Pox Vaccine. (d) After a lot of Master Seed Virus Fowl Pox Vaccine and Pigeon Pox has been established as prescribed in Vaccine shall be prepared from virus- paragraphs (a), (b), and (c) of this sec- bearing cell culture fluids or tion, each serial and subserial shall embryonated chicken eggs. Only Mas- meet the applicable requirements in ter Seed Virus which has been estab- § 113.300 and the requirements pre- lished as pure, safe, and immunogenic scribed in this paragraph. in accordance with the requirements in

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paragraphs (a), (b), and (c) of this sec- (iv) Calculate the EID50 by the tion shall be used for preparing the Spearman-Karber or Reed-Muench production seed virus for vaccine pro- method. duction. All serials shall be prepared (3) Fourteen to twenty-one days post- from the first through the fifth passage vaccination, all vaccinates and con- from the Master Seed Virus. trols shall be challenged by the wing (a) The Master Seed Virus shall meet web method and observed each day for the applicable requirements prescribed 10 days. If the wing web method was in § 113.300 except paragraph (c) of this used for vaccination, the opposite wing section and shall meet the require- shall be used for challenge. Challenge ments prescribed in this section. virus shall be provided or approved by (b) Each lot of Master Seed Virus Animal and Plant Health Inspection shall be tested for pathogens by the Service. chicken inoculation test prescribed in (4) If at least 90 percent of the con- § 113.36. trols do not develop fowl pox during (c) Each lot of Master Seed Virus the observation period, the test is a No shall be tested for immunogenicity and Test and may be repeated. If at least 19 the selected virus dose to be used shall of 20, or 27 of 30, or 36 of 40 of the vac- be established as follows: cinates in each group do not remain (1) Fowl pox susceptible birds all of free from clinical signs of fowl pox dur- the same age and from the same ing the observation period, the Master source, shall be used as test birds. Seed Virus is unsatisfactory. Twenty or more birds shall be used as (5) An Outline of Production change vaccinates for each method of administration recommended on the label. Ten shall be made before authority for use additional birds of the same age and of a new lot of Master Seed Virus shall from the same source as the vaccinates be granted by Animal and Plant Health shall be held as unvaccinated controls. Inspection Service. (2) A geometric mean titer of the (d) After a lot of Master Seed Virus dried vaccine produced from the high- has been established as prescribed in est passage of the Master Seed Virus paragraphs (a), (b), and (c) of this sec- shall be established before the tion, each serial and subserial shall immunogenicity test is conducted. meet the requirements in § 113.36, in Each vaccinate shall receive a pre- § 113.300 except paragraph (c), and in determined quantity of vaccine virus. this paragraph. Five replicate virus titrations shall be (1) Safety test. Final container sam- conducted on an aliquot of the vaccine ples of completed product from each se- virus to confirm the amount of virus rial shall be tested. Vaccines rec- administered to each bird used in the ommended for use in birds 10 days of test. At least three appropriate (not to age or younger shall be tested in ac- exceed tenfold) dilutions shall be used cordance with paragraphs (d)(1)(i), (ii), and the test conducted as follows: and (iii) of this section. (i) For each dilution, inoculate at (i) Each of 25 susceptible birds 5 days least five embryos, 9 to 11 days old, on of age or younger, properly identified the chorioallantoic membrane with at and obtained from the same source and least 0.2 ml each. Disregard all deaths hatch, shall be vaccinated with the during the first 24 hours post-inocula- equivalent of 10 doses of vaccine by tion. To be a valid test, at least four each of all routes recommended on the embryos in each dilution shall remain label and observed each day for 14 days. viable beyond 24 hours. Severe clinical signs or death shall be (ii) Examine the surviving embryos counted as failures. Two-stage sequen- for evidence of infection 5 to 7 days post-inoculation. tial testing may be conducted if the first test (which then becomes stage (iii) A satisfactory titration shall have at least one dilution with between one) has three failures. 50 and 100 percent positives and at least (ii) The results shall be evaluated ac- one dilution with between 50 and 0 per- cording to the following table: cent positives.

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CUMULATIVE TOTALS tablished as pure, safe, and immunogenic in accordance with the Failures for Failures for Stage Number of satisfactory unsatisfactory requirements in paragraphs (a), (b), and birds serials serials (c) of this section shall be used for preparing the production seed virus for 1 ...... 25 ...... 2 or less ...... 4 or more. 2 ...... 50 ...... 5 or less ...... 6 or more. vaccine production. All serials shall be prepared from the first through the (iii) If unfavorable reactions occur fifth passage from the Master Seed which are not attributable to the prod- Virus. uct, the test shall be declared a No (a) The Master Seed Virus shall meet Test and may be repeated or, in lieu the applicable requirements prescribed thereof, the serial declared unsatisfac- in § 113.300 and the requirements pre- tory. scribed in this section. (iv) Vaccines not recommended for (b) Each lot of Master Seed Virus use in birds 10 days of age or younger shall be tested for pathogens by the shall be tested for safety as follows: chicken embryo inoculation test pre- Each of twenty-five 3- to 5-week-old, scribed in § 113.37, except that, if the fowl-pox susceptible birds shall be vac- test is a No Test because of a vaccine cinated with the equivalent of 10 doses virus override, the test may be re- of vaccine by each of all routes rec- peated and if the repeat test is a No ommended on the label and observed Test for the same reason, the chicken each day for 14 days. If any of the birds inoculation test prescribed in § 113.36 show severe clinical signs of disease or may be conducted and the virus judged death during the observation period accordingly. due to causes attributable to the prod- (c) Each lot of Master Seed Virus uct, the serial is unsatisfactory. If unused for vaccine production shall be favorable reactions occur which are not tested for immunogenicity and the se- attributable to the product, the test lected virus dose to be used shall be es- shall be declared a No Test and may be tablished as follows: repeated or, in lieu thereof, the serial (1) Bronchitis susceptible chickens, declared unsatisfactory. all of the same age and from the same (2) Virus titer requirements. Final con- source, shall be used in the virus-recov- tainer samples of completed product ery test. For each method of adminis- shall be tested for virus titer using the tration recommended on the label for titration method used in paragraph each serotype against which protection (c)(2) of this section. To be eligible for is claimed, twenty or more chickens release, each serial and each subserial shall be used as vaccinates. Ten addi- shall have a virus titer sufficiently tional chickens for each serotype greater than the titer of vaccine virus against which protection is claimed used in the immunogenicity test pre- shall be held as unvaccinated controls. scribed in paragraph (c) of this section (2) A geometric mean titer of the to assure that when tested at any time dried vaccine produced from the high- within the expiration period, each se- est passage of the Master Seed Virus rial and subserial shall have a virus shall be established before the titer of 10 0.7 greater than that used in immunogenicity tests are conducted. such immunogenicity test but not less Each vaccinate shall receive a pre- 2.0 than 10 EID50 per dose. determined quantity of vaccine virus. Five replicate virus titrations shall be [39 FR 44724, Dec. 27, 1974, as amended at 40 FR 18406, Apr. 28, 1975; 40 FR 41089, Sept. 5, conducted on an aliquot of the vaccine 1975; 44 FR 33051, June 8, 1979; 48 FR 33473, virus to confirm the amount of virus July 22, 1983. Redesignated at 55 FR 35562, administered to each chicken used in Aug. 31, 1990, as amended at 56 FR 66784, such tests. At least three approved (not 66786, Dec. 26, 1991; 72 FR 72564, Dec. 21, 2007] to exceed tenfold) dilutions shall be used and the test conducted as follows; § 113.327 Bronchitis Vaccine. (i) For each dilution, inject at least Bronchitis Vaccine shall be prepared five embryos, 9 to 11 days old, in the from virus-bearing cell culture fluids allantoic cavity with 0.1 ml each. or embryonated chicken eggs. Only Deaths occurring during the first 24 Master Seed Virus which has been es- hours shall be disregarded, but at least

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four viable embyros in each dilution the test is a No Test and may be re- shall survive beyond 24 hours of a valid peated. test. After 5 to 8 days incubation, ex- (iv) If less than 90 percent of the vac- amine the surviving embryos for evi- cinates are negative for virus recovery, dence of infection. the Master Seed Virus is unsatisfac- (ii) A satisfactory titration shall tory. have at least one dilution with between (4) An Outline of Production change 50 and 100 percent positives and at least shall be made before authority for use one dilution with between 50 and 0 per- of a new lot of Master Seed Virus shall cent positives. be granted by Animal and Plant Health (iii) Calculate the EID50 by the Inspection Service. Spearman-Karber or Reed-Muench (d) After a lot of Master Seed Virus method. has been established as prescribed in (3) Twenty-one to twenty-eight days paragraphs (a), (b), and (c) of this sec- post-vaccination, all vaccinates and tion, each serial and subserial shall controls shall be challenged by eye- meet the applicable requirements in drop with virulent bronchitis virus. A § 113.300 and the requirements pre- separate set of vaccinates and controls scribed in this paragraph, except that, shall be used for each serotype against if the vaccine contains more than one which protection is claimed. Each chal- virus type, bulk samples taken from lenge virus shall be approved or pro- each type prior to mixing shall be used vided by Animal and Plant Health In- in the virus identity tests prescribed in spection Service and shall titer at least § 113.300(c). The additional require- 4.0 ments in this paragraph shall also be 10 EID50 per ml. (i) Tracheal swabs shall be taken met. once, 5 days post-challenge, from each (1) Final container samples from control and vaccinate. Each swab shall each serial shall be tested for patho- be placed in a test tube containing 3 ml gens by the chicken embryo inocula- of tryptose phosphate broth and anti- tion test prescribed in § 113.37, except biotics. The tube and swab shall be that, if the test is a No Test because of swirled thoroughly and if they are to a vaccine virus override, the chicken be stored, be immediately frozen and inoculation test prescribed in § 113.36 be stored at below ¥40 °C. pending egg may be conducted and the vaccine evaluation. For each chicken swab, at judged accordingly. least five chicken embryos 9 to 11 days (2) Safety test. Final container sam- old shall be inoculated in the allantoic ples of completed product shall be test- cavity with 0.2 ml each of broth from ed to determine safety for use in bron- each tube. chitis susceptible young chickens. (ii) All embryos surviving the third (i) Twenty-five susceptible chickens, day post-inoculation shall be used in 5 days of age or younger, properly iden- the evaluation, except that, if a swab is tified and obtained from the same not represented by at least four em- source and hatch, shall be vaccinated bryos, the test of that swab is invalid by the eye-drop method with the equiv- and the results a No Test. A tracheal alent of 10 doses of vaccine and ob- swab shall be positive for virus recov- served each day for 21 days post-vac- ery when any of the embryos in a valid cination. Severe respiratory signs or test show typical infectious bronchitis death shall be counted as failures. Two- virus lesions, such as but not limited stage sequential testing may be con- to, stunting, curling, kidney urates, ducted if the first test (which then be- clubbed down, or death during the 4 to comes stage one) has three failures. 7 day post-inoculation period. If less (ii) The results shall be evaluated ac- than 20 percent of the embryos which cording to the following table: survive the third day post-inoculation CUMULATIVE TOTALS die during the 4 to 7 day post-inoculation period and show no gross lesions Failures for Failures for Stage Number of satisfactory unsatisfactory typical of infectious bronchitis, they chickens serials serials may be disregarded. (iii) If less than 90 percent of the con- 1 ...... 25 ...... 2 or less ...... 4 or more. trols are positive for virus recovery, 2 ...... 50 ...... 5 or less ...... 6 or more.

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If unfavorable reactions occur which cell culture fluids or embryonated are not attributable to the product, the chicken eggs. Only Master Seed Virus test shall be declared a No Test and re- which has been established as pure, peated or, in lieu thereof, the serial de- safe, and immunogenic in accordance clared unsatisfactory. with the requirements in paragraphs (3) Virus titer requirements. Final con- (a), (b), and (c) of this section shall be tainer samples of completed product used for preparing the production seed shall be tested for virus titer using the virus for vaccine production. All serials procedure prescribed in paragraph shall be prepared from the first (c)(2) of this section and in this para- through the fifth passage from the graph. Master Seed Virus. (i) The Newcastle disease virus frac- (a) The Master Seed Virus shall meet tion of combined Newcastle-Bronchitis the applicable requirements prescribed Vaccines shall be neutralized prior to in § 113.300 and the requirements pre- titration of the bronchitis virus frac- scribed in this section. tion. Equal parts of heat-inactivated (b) Each lot of Master Seed Virus Newcastle disease antiserum shall be shall be tested for pathogens by the mixed with each appropriate serial ten- chicken embryo inoculation test pre- fold dilution of the vaccine. After inac- scribed in § 113.37, except that, if the tivation, embryos shall be injected test is a No Test because of vaccine with 0.2 ml each and results calculated virus override, the test may be re- as a 0.1 ml dose to allow for serum dilu- peated and if the repeat test is a No tion of the vaccine. The allantoic Test for the same reason, the chicken fluids, tested as prescribed in § 113.34 inoculation test prescribed in § 113.36 shall not show hemagglutinating activ- may be conducted and the virus judged ity in the lowest dilution used in the accordingly. Each lot shall also be test- titration. ed for safety as follows: (ii) Each bronchitis virus type shall (1) Each of at least ten 3 to 4 week be harvested separately and a sample old susceptible chickens obtained from of bulk harvested material shall be col- the same source and hatch as those lected prior to mixing with the other used in the immunogenicity test pre- virus type(s). Each sample shall con- scribed in paragraph (c) of this section tain not less than the minimum virus shall be injected intratracheally with titer stated in the filed Outline of Pro- 0.2 ml of the virus as used in the vac- duction. cine and the chickens observed each (iii) To be eligible for release, each day for 14 days. serial and each subserial shall have a virus titer sufficiently greater than the (2) If more than 20 percent of the titer of vaccine virus used in the chickens die during the observation pe- immunogenicity test prescribed in riod, the virus is unsatisfactory. paragraph (c) of this section to assure (c) Each lot of Master Seed Virus that when tested at any time within used for vaccine production shall be the expiration period, each serial and tested for immunogenicity and the se- subserial shall have a virus titer of lected virus dose to be used shall be es- 10 0.7 greater than that used in such tablished as follows: immunogenicity test but not less than (1) Fowl laryngotracheitis suscep- 2.0 tible chickens all of the same age and 10 EID50 per dose. from the same source shall be used. [39 FR 44724, Dec. 27, 1974, as amended at 40 Twenty or more chickens shall be used FR 18406, Apr. 28, 1975; 40 FR 41089, Sept. 5, as vaccinates for each method of ad- 1975; 42 FR 43617, Aug. 30, 1977; 48 FR 33473, July 22, 1983. Redesignated at 55 FR 35562, ministration recommended on the Aug. 31, 1990, as amended at 56 FR 66784, label. Ten additional chickens of the 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, 1999; same age and from the same source 72 FR 72564, Dec. 21, 2007] shall be held as unvaccinated controls. (2) A geometric mean titer of the § 113.328 Fowl Laryngotracheitis Vac- dried vaccine produced from the high- cine. est passage of the Master Seed Virus Fowl Laryngotracheitis Vaccine shall be established before the shall be prepared from virus-bearing immunogenicity test is conducted.

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Each vaccinate shall receive a pre- (d) After a lot of Master Seed Virus determined quantity of vaccine virus. has been established as prescribed in Five replicate virus titrations shall be paragraphs (a), (b), and (c) of this sec- conducted on an aliquot of the vaccine tion, each serial and subserial shall virus to confirm the amount of virus meet the applicable requirements in administered to each chicken used in § 113.300 and the requirements pre- the test. At least three appropriate scribed in this paragraph. (not to exceed tenfold) dilutions shall (1) Final container samples from be used for vaccine of chicken embryo each serial shall be tested for patho- origin and the test conducted as fol- gens by the chicken embryo inocula- lows: tion test prescribed in § 113.37, except (i) For each dilution, inject at least that, if the test is a No Test because of five embryos, 9 to 11 days old, on the a vaccine virus override, the chicken chorioallantoic membrane with 0.2 ml inoculation test prescribed in § 113.36 each. Disregard all deaths during the may be conducted and the vaccine first 24 hours post-injection. To be a judged accordingly. valid test, at least four embryos in (2) Safety test. Final container sam- each dilution shall remain viable be- ples of completed product from each se- yond 24 hours. rial of modified live virus vaccine shall (ii) Examine the surviving embryos be tested for safety as provided in this for evidence of infection 5 to 8 days paragraph. Live virus vaccine not pre- post-injection. pared with modified live virus shall be (iii) A satisfactory titration shall tested for safety as provided in the have at least one dilution with between filed Outline of Production. 50 and 100 percent positives and at least (i) Twenty-five 3 to 4 week old one dilution with between 50 and 0 per- laryngotracheitis susceptible chickens cent positives. shall be injected intratracheally with (iv) Calculate the EID50 by the 0.2 ml of vaccine rehydrated at the rate Spearman-Karber or Reed-Muench of 30 ml for 1,000 doses. Chickens shall method. be observed each day for 14 days. (3) Tissue culture origin vaccine may Deaths shall be counted as failures. be titrated by a tissue culture method Two-stage sequential testing may be approved by Animal and Plant Health conducted if the first test (which then Inspection Service and written into the becomes stage one) has five, six, or filed Outline of Productions. seven failures. (4) Ten to fourteen days post-vaccina- (ii) The results shall be evaluated action, all vaccinates and controls shall cording to the following table: be challenged intratracheally or in the orbital sinus with infectious fowl CUMULATIVE TOTALS laryngotracheitis virus and observed Failures for Failures for each day for 10 days. Challenge virus Number of Stage chickens satisfactory unsatisfactory shall be provided or approved by Ani- serials serials mal and Plant Health Inspection Serv- 1 ...... 25 ...... 4 or less ...... 8 or more. ice. 2 ...... 50 ...... 10 or less .... 11 or more. (5) If at least 80 percent of the controls do not die or show clinical signs (iii) If unfavorable reactions occur of fowl laryngotracheitis during the ob- which are not attributable to the prod- servation period, the test is a No Test uct, the test shall be declared a No and may be repeated. If at least 19 of Test and repeated or in lieu thereof, 20, 27 of 30, or 36 of 40 of the vaccinates the serial declared unsatisfactory. in each group do not remain free of (3) Virus titer requirements. Final con- clinical signs of fowl laryngotracheitis tainer samples of completed product during the observation period, the Mas- shall be tested for virus titer using the ter Seed Virus is unsatisfactory. titration method provided in para- (6) An Outline of Production change graphs (c)(2) or (3) of this section. To be shall be made before authority for use eligible for release, each serial and of a new lot of Master Seed Virus shall each subserial shall have a virus titer be granted by Animal and Plant Health sufficiently greater than the titer of Inspection Service. vaccine virus used in the

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immunogenicity test prescribed in additional chickens of the same age paragraph (c) of this section to assure and from the same source shall be held that when tested at any time within as unvaccinated controls. the expiration period, each serial and (2) A geometric mean titer of the subserial shall have a virus titer of dried vaccine produced from the high- 10 0.7 greater than that used in such est passage of the Master Seed Virus immunogenicity test but not less than shall be established before the 2.5 10 EID50 per dose for chicken embryo immunogenicity test is conducted. 2.0 2.5 origin vaccine and 10 EID50 or 10 Each vaccinate shall receive a pre- TCID50 per dose for tissue culture ori- determined quantity of vaccine virus. gin vaccine. Five replicate virus titrations shall be conducted on an aliquot of the vaccine [39 FR 44726, Dec. 27, 1974, as amended at 40 FR 18407, Apr. 28, 1975; 40 FR 41089, Sept. 5, virus to confirm the amount of virus 1975; 41 FR 44359, Oct. 8, 1976; 42 FR 43617, administered to each chicken used in Aug. 30, 1977; 48 FR 33473, July 22, 1983. Re- the test. At least three appropriate designated at 55 FR 35562, Aug. 31, 1990, as (not to exceed tenfold) dilutions shall amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 be used and the test conducted as fol- FR 72564, Dec. 21, 2007] lows: (i) For each dilution, inject at least § 113.329 Newcastle Disease Vaccine. five embryos, 9 to 11 days old, in the Newcastle Disease Vaccine shall be allantoic cavity with at least 0.1 ml prepared from virus-bearing cell cul- each. Disregard all deaths during the ture fluids or embryonated chicken first 24 hours post-injection. To be a eggs. Only Master Seed Virus which valid test, at least four embryos in has been established as pure, safe, and each dilution shall remain viable be- immunogenic in accordance with the yond 24 hours. requirements in paragraphs (a), (b), and (ii) Examine the surviving embryos (c) of this section shall be used for pre- for evidence of infection 5 to 7 days paring the production seed virus for post-injection. vaccine production. All serials shall be (iii) A satisfactory titration shall prepared from the first through the have at least one dilution with between fifth passage from the Master Seed 50 and 100 percent positives and at least Virus. one dilution with between 50 and 0 per- (a) The Master Seed Virus shall meet cent positives. the applicable requirements prescribed (iv) Calculate the EID50 by the in § 113.300, except § 113.34, and the re- Spearman-Karber or Reed-Muench quirements prescribed in this section. method. (b) Each lot of Master Seed Virus (3) Twenty to twenty-eight days shall be tested for pathogens by the postvaccination, all vaccinates and chicken embryo inoculation test pre- controls shall be challenged scribed in § 113.37, except that, if the intramuscularly with at least 10 4.0 test is a No Test because of a vaccine EID50 of virus per chicken and observed virus override, the test may be re- each day for 14 days. Challenge virus peated and if the repeat test is a No shall be provided or approved by Ani- Test for the same reason, the chicken mal and Plant Health Inspection Serv- inoculation test prescribed in § 113.36 ice. may be conducted and the virus judged (4) If at least 90 percent of the con- accordingly. trols do not develop clinical signs of (c) Each lot of Master Seed Virus Newcastle disease during the observa- used for vaccine production shall be tion period, the test is a No Test and tested for immunogenicity and the se- may be repeated. If at least 19 of 20, or lected virus dose to be used shall be es- 27 of 30, or 36 of 40 of the vaccinates in tablished as follows: each group do not remain free from (1) Newcastle Disease susceptible clinical signs of Newcastle disease dur- chickens, all of the same age and from ing the observation period, the Master the same source, shall be used. Twenty Seed Virus is unsatisfactory. or more chickens shall be used as vac- (5) A strain identity test acceptable cinates for each method of administra- to Animal and Plant Health Inspection tion recommended on the label. Ten Service shall be conducted.

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(6) An Outline of Production change younger shall be tested for safety as shall be made before authority for use follows: of a new lot of Master Seed Virus shall Each of twenty-five 3 to 5 week old be granted by Animal and Plant Health Newcastle disease susceptible chickens Inspection Service. shall be vaccinated as recommended on (d) After a lot of Master Seed Virus the label with the equivalent of ten has been established as prescribed in doses and observed each day for 21 paragraphs (a), (b), and (c) of this sec- days. If any of the birds show severe tion, each serial and subserial shall clinical signs of disease or death during meet the applicable requirements in the observation period due to causes § 113.300, except § 113.34, and the require- attributable to the product, the serial ments prescribed in this paragraph. is unsatisfactory. (1) Final container samples from (3) Virus titer requirements. Final con- each serial shall be tested for patho- tainer samples of completed product gens by the chicken embryo inocula- shall be tested for virus titer using the tion test prescribed in § 113.37, except titration method used in paragraph that, if the test is a No Test because of (c)(2) of this section. To be eligible for a vaccine virus override, the chicken release, each serial and each subserial inoculation test prescribed in § 113.36 shall have a virus titer per dose suffi- may be conducted and the vaccine ciently greater than the titer of vac- judged accordingly. cine virus used in the immunogenicity (2) Safety test: Final container sam- test prescribed in paragraph (c) of this ples of completed product from each se- section to assure that when tested at rial shall be tested to determine wheth- any time within the expiration period, er the vaccine is safe for use in suscep- each serial and subserial shall have a tible young chickens. Vaccines rec- virus titer of 10 0.7 greater than that ommended for use in chickens 10 days of age or younger shall be tested in ac- used in the immunogenicity test but 5.5 cordance with paragraphs (d)(2)(i), (ii), not less than 10 EID50 per dose. and (iii) of this section. [39 FR 44727, Dec. 27, 1974, as amended at 40 (i) Twenty-five susceptible chickens, FR 18407, Apr. 28, 1975; 40 FR 23721, June 2, 5 days of age or younger, properly iden- 1975; 40 FR 41090, Sept. 5, 1975; 42 FR 43618, tified and obtained from the same Aug. 30, 1977; 48 FR 33473, July 22, 1983. Re- source and hatch, shall be vaccinated designated at 55 FR 35562, Aug. 31, 1990, as by the eye drop method with the equiv- amended at 56 FR 66784, 66786, Dec. 26, 1991; 72 alent of 10 doses of vaccine and the FR 72564, Dec. 21, 2007] chickens observed each day for 21 days. § 113.330 Marek’s Disease Vaccines. Severe respiratory signs or death shall be counted as failures. Two-stage se- Marek’s disease vaccine shall be pre- quential testing may be conducted if pared from virus-bearing tissue culture the first test (which then becomes cells. Only Master Seed Virus which stage one) has 3 failures. has been established as pure, safe, and (ii) The results shall be evaluated ac- immunogenic shall be used for pre- cording to the following table: paring the production seed virus for vaccine production. CUMULATIVE TOTALS (a) The Master Seed Virus shall meet the applicable requirements prescribed Failures for Failures for Number of in § 113.300, and the requirements pre- Stage chickens satisfactory unsatisfactory serials serials scribed in this section. The identity 1 ...... 25 ...... 2 or less ...... 4 or more. test required in § 113.300(c) shall be con- 2 ...... 50 ...... 5 or less ...... 6 or more. ducted in a serotype-specific manner by a method acceptable to APHIS. (iii) If unfavorable reactions occur Each lot of Master Seed Virus shall which are not attributable to the prod- also be tested for pathogens by the uct, the test shall be declared a No chicken embryo inoculation test pre- Test and may be repeated. scribed in § 113.37, except that, if the (iv) Vaccines not recommended for test is a No Test because of a vaccine use in chickens 10 days of age or virus override, the chicken inoculation

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test prescribed in § 113.36 may be con- groups 1 or 4 is significantly (statis- ducted and the virus judged accord- tically) different from the average in ingly. group 3 at the end of the 120 days, the (b) Safety test. The Master Seed Virus lot of Master Seed Virus is unsatisfac- shall be nonpathogenic for chickens as tory. determined by the following procedure: (3) For tests involving in ovo inocula- (1) Specific pathogen free chickens or tion, hatchability results shall also be embryos, negative for Marek’s disease reported for each group. virus antibodies, and from the same (c) Immunogenicity. Each lot of Mas- source, shall be isolated into the fol- ter Seed Virus used for vaccine produc- lowing groups: tion shall be tested for (i) Group 1. At least 50 test subjects immunogenicity at the highest passage shall be inoculated with 10 times as level allowed for the product, and the much viable virus as will be contained virus dose to be used shall be estab- in one dose of vaccine, by the route lished as follows: recommended for vaccination. (1) Specific pathogen free chickens or (ii) Group 2. At least 50 test subjects embryos, negative for Marek’s disease shall be injected with a very virulent antibodies, and from the same source, Marek’s disease virus provided or ap- shall be isolated into the following proved by APHIS, at a dosage level groups: that will cause gross lesions of Marek’s (i) Group 1. A minimum of 35 test sub- disease in at least 80 per cent of the jects shall be inoculated with the vac- chickens within 50 days. cine, using the recommended route, at (iii) Group 3. Fifty uninoculated con- 1 day of age for chicks or 18 days of trols. For in ovo studies, this group embryonation for embryos. The dose should receive a sham inoculation of used shall be established by 5 replicate diluent. virus titrations conducted by a cell (iv) Group 4. For studies evaluating culture system or other titration Serotype 1 Master Seed Viruses, a method acceptable to APHIS. group of 50 uninoculated control chick- (ii) Group 2. A minimum of 35 nonvac- ens shall be housed in contact with the cinated test subjects shall be held as group 1 vaccinated chickens. challenge controls. (2) At least 40 chickens in each group (iii) Group 3. A minimum of 25 non- shall survive to 5 days of age. All vaccinated test subjects shall be held chickens that die shall be necropsied as nonchallenge controls. and examined for lesions of Marek’s (iv) Group 4. Except for studies evalu- disease and cause of death. The test ating vaccines which contain only a shall be judged according to the fol- Serotype 3 virus as the Marek’s disease lowing criteria: fraction, a minimum of 35 chicks shall (i) At 50 days of age, the remaining be vaccinated at 1 day of age with a li- chickens in group 2 shall be killed and censed Serotype 3 vaccine, in order to examined for gross lesions of Marek’s document the severity of the very viru- disease. If at least 80 percent of this lent challenge. group do not develop Marek’s disease, (2) At least 30 chickens in groups 1, 2, the test is a No Test and may be re- and 4, and at least 20 chickens in group peated. 3, shall survive to 5 days of age. All (ii) At 120 days of age, the remaining chickens in groups 1, 2, and 4 shall be chickens in groups 1, 3, and 4 shall be challenged at 5 days of age in the fol- weighed, killed, and necropsied. If less lowing manner: than 30 of the chickens in group 3 sur- (i) For studies evaluating vaccines vive the 120 day period, or if any of the which contain only a Serotype 3 virus chickens in group 3 have gross lesions as the Marek’s disease fraction, groups of Marek’s disease at necropsy, the test 1 and 2 shall be inoculated with a is declared a No Test. If less than 30 standard virulent challenge virus pro- chickens in groups 1 and 4 survive the vided or approved by APHIS. 120 day period; or if any of the chickens (ii) For all other Marek’s disease vac- in groups 1 and 4 have gross lesions of cines, groups 1, 2, and 4 shall be inocu- Marek’s disease at necropsy; or if the lated with a very virulent challenge average body weight of the chickens in virus provided or approved by APHIS.

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(3) All chickens shall be observed death determined, and the results re- until 7 weeks of age, necropsied, and corded. examined for grossly observable lesions (i) If at least 20 chickens do not sur- consistent with Marek’s disease. All vive the observation period, the test is chickens dying before the end of the 7 a No Test. week observation period shall be (ii) If lesions of any disease or cause necropsied and evaluated for gross le- of death are directly attributable to sions of Marek’s disease. Any chickens the vaccine, the serial is unsatisfac- not so examined shall be scored as posi- tory. tive for Marek’s disease. (iii) If less than 20 chicks survive the (4) For a valid test, at least 80 per- observation period and there are no cent of the chickens in group 2 must deaths or lesions attributable to the develop grossly observable lesions, vaccine, the test may be repeated one none of the chickens in group 3 shall time, Provided, that if the test is not develop grossly observable lesions, and repeated, the serial shall be declared (when included) greater than 20 percent unsatisfactory. of the chickens in group 4 must develop (3) Potency test. The samples shall be grossly observable lesions. titrated using a cell culture system or (5) For a valid test to be considered other titration method acceptable to satisfactory, at least 80 percent of the APHIS. For vaccines composed of more chickens in group 1 must remain free of than one Marek’s disease virus grossly observable lesions. The appro- serotype, each fraction shall be priate product claim resulting from a titrated in a serotype-specific manner. satisfactory test would be to aid in the (i) Samples of desiccated vaccine ° prevention of Marek’s disease, for vac- shall be incubated at 37 C for 3 days cines containing only a Serotype 3 before preparation for use in the po- virus as the Marek’s disease fraction, tency test. Samples of desiccated or or to aid in the prevention of very viru- frozen vaccine shall be reconstituted in lent Marek’s disease, for all other vac- diluent according to the label rec- cines. ommendations, and held in an ice bath ° ° (d) Test requirements for release. Each at 0 C to 4 C for 2 hours prior to use serial and subserial shall meet the ap- in the potency test. plicable requirements prescribed in (ii) For a serial or subserial to be eli- § 113.300. The identity test required in gible for release, each serotype contained in the vaccine shall have a virus § 113.300(c) shall be conducted in a titer per dose which is at least 3 times serotype-specific manner by a method greater than the number of plaque acceptable to APHIS. Final container forming units (pfu) used in the samples of completed product shall immunogenicity test prescribed in also meet the requirements in para- paragraph (c) of this section, but not graphs (d) (1), (2), and (3) of this sec- less than 1000 pfu per dose. tion. Any serial or subserial found un- (iii) When tested (without the pretest satisfactory by a prescribed test shall incubation of desiccated products) at not be released. any time within the expiration period, (1) Purity test. The chicken embryo each serotype contained in the vaccine inoculation test prescribed in § 113.37 shall have a virus titer per dose which shall be conducted, except that, if the is at least 2 times the number of pfu test is a No Test because of a vaccine used in the immunogenicity test, but virus override, the chicken inoculation not less than 750 pfu per dose. test prescribed in § 113.36 may be conducted and the virus judged accord- [61 FR 33841, July 1, 1996] ingly. (2) Safety test. At least 25 one-day-old, § 113.331 Bursal Disease Vaccine. specific pathogen free chickens shall be Bursal Disease Vaccine shall be pre- injected, by the subcutaneous route, pared from virus-bearing cell culture with the equivalent of 10 chicken doses fluids or embryonated chicken eggs. of virus (vaccine concentrated 10X). Only Master Seed Virus which has been The chickens shall be observed each established as pure, safe, and day for 21 days. Chickens dying during immunogenic in accordance with the the period shall be examined, cause of requirements in paragraphs (a), (b), and

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(c) of this section shall be used for previous reduction in size of the bursa paring the production seed virus for from normal. vaccine production. All serials shall be (2) Each of thirty-five 3- to 4-week- prepared from the first through the old bursal disease susceptible chickens fifth passage from the Master Seed (vaccinates) shall be vaccinated with Virus. approximately one minimum protec- (a) The Master Seed Virus shall meet tive dose of vaccine virus as deter- the applicable requirements prescribed mined in paragraph (c) of this section. in § 113.300 and the requirements pre- Each of 10 chickens of the same source scribed in this section. and hatch shall be administered at 2.0 (b) Each lot of Master Seed Virus least 10 EID50 of a virulent bursal shall be tested for pathogens by the disease virus by eye-drop, isolated, and chicken embryo inoculation test pre- used as positive controls. Also, each of scribed in § 113.37, except that, if the 20 additional chickens of the same test is a No Test because of a vaccine source and hatch shall be isolated and virus override, the chicken inoculation held as negative controls. test prescribed in § 113.36 may be con- (i) Three or four days ducted and the virus judged accord- postvaccination, 10 of the vaccinates, ingly. Each lot of Master Seed Virus the 10 positive controls, and 10 of the used in the preparation of modified live negative controls shall be necropsied virus vaccines shall also be nonpatho- and examined for gross lesions of genic to chickens as determined by the bursal disease. If any of the vaccinates following procedures: have such lesions, the Master Seed (1) Each of twenty-five 1-day-old Virus is unsatisfactory, except that, if bursal disease susceptible chickens any of the negative controls or less (vaccinates) shall be injected than 8 of the positive controls have subcutaneously with 10 times the rec- such lesions, the test is a No Test and ommended dose of vaccine virus and may be repeated. For purposes of this observed for 21 days. Fifteen chickens test, gross lesions shall include peri- of the same source and hatch shall be bursal edema and/or edema and/or mac- kept isolated as controls. roscopic hemorrhage in the bursal tis- (i) Seventeen days postvaccination, sue. each of five controls shall be adminis- (ii) Fourteen days post-vaccination, 2.0 tered at least 10 EID50 of a virulent the remaining vaccinates and negative bursal disease virus by eye-drop, iso- controls shall be necropsied and exam- lated, and used as positive controls. ined for obvious bursal atrophy. If any The remaining controls shall be used as of the vaccinates have such atrophy, negative controls. the Master Seed Virus is unsatisfac- (ii) If the vaccinates do not remain tory, except that, if any of the negative free of clinical signs of bursal disease, controls have such atrophy, the test is the Master Seed Virus is unsatisfac- a No Test and may be repeated. tory. If unfavorable reactions which (c) Each lot of Master Seed Virus are not attributable to the Master Seed shall be tested for immunogenicity and Virus occur in more than two of the the selected virus dose to be used shall vaccinates, the test shall be declared a be established as follows: No Test and may be repeated. (1) Bursal Disease susceptible chick- (iii) Twenty-one days ens, all of the same age (3 weeks or postvaccination, the vaccinates and younger) and from the same source, the controls shall be necropsied and ex- shall be used. Twenty or more chickens amined for gross lesions of bursal dis- shall be used as vaccinates for each ease. If more than two of the vac- method of administration rec- cinates have such lesions, the Master ommended on the label. Ten additional Seed Virus is unsatisfactory, except chickens of the same age and from the that, if any of the negative controls or same source shall be held as less than four of the positive controls unvaccinated controls. have such lesions, the test is a No Test (2) A geometric mean titer of the vac- and may be repeated. For purposes of cine produced from the highest passage this test, gross lesions shall include ob- of the Master Seed Virus shall be es- vious pathological processes and/or ob- tablished before the immunogenicity

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test is conducted. Each vaccinate shall tible chickens shall be vaccinated with receive a predetermined quantity of the equivalent of 10 doses by subcuta- vaccine virus. Five replicate virus ti- neous injection. trations shall be conducted on an ali- (B) For vaccines intended for drink- quot of the vaccine virus to confirm ing water administration, each of the amount of virus administered to twenty-five 4- to 5-week-old bursal dis- each chicken used in the test. At least ease susceptible chickens shall be vac- three appropriate (not to exceed ten- cinated orally with the equivalent of 10 fold) dilutions shall be used to conduct doses. the titrations by a method acceptable (C) Ten chickens of the same source to Animal and Plant Health Inspection and hatch shall be maintained in isola- Service. tion as negative controls. The vac- (3) When the test chickens are 28 to cinates and controls shall be observed 35 days of age but not less than 14 days each day for 21 days. postvaccination, each vaccinate and (ii) If unfavorable reactions which each control shall be challenged by are attributable to the biological prod- eye-drop with a virulent bursal disease uct occur during the observation pe- virus provided or approved by Animal riod, the serial is unsatisfactory. If un- and Plant Health Inspection Service. favorable reactions occur in more than (i) Three to five days postchallenge, one of the controls or if unfavorable re- all vaccinates and controls shall be actions which are not attributable to necropsied and examined for gross le- the biological product occur in more sions of bursal disease as described in than two of the vaccinates, the test paragraph (b)(2)(i) of this section. shall be declared a No Test and re- (ii) If at least 19 of 20, or 27 of 30, or peated, except that, if the test is not 36 of 40 vaccinates in each group are repeated, the serial shall be unsatisfac- not free from such lesions, the Master tory. Seed Virus is unsatisfactory, except (3) Virus titer requirements. Final con- that, if less than 90 percent of the container samples of completed product trols have such lesions, the test is a No shall be tested for virus titer using the Test and may be repeated. titration method used in paragraph (4) An Outline of Production change (c)(2) of this section. To be eligible for shall be made before authority for use release, each serial and each subserial of a new lot of Master Seed Virus shall shall have a virus titer sufficiently be granted by Animal and Plant Health greater than the titer of vaccine virus Inspection Service. used in the immunogenicity test pre- (d) After a lot of Master Seed Virus scribed in paragraph (c) of this section has been established as prescribed in to assure that when tested at any time paragraphs (a), (b), and (c) of this sec- within the expiration period, each se- tion, each serial and subserial shall rial and subserial shall have a virus meet the applicable requirements in titer of 10 0.7 times greater than that § 113.300 and the requirements pre- used in such immunogenicity test, but scribed in this paragraph. not less than 10 2.0 titration units (PFU (1) Tests for pathogens. Final con- or ID50’s) per dose. tainer samples from each serial shall [44 FR 60263, Oct. 19, 1979, as amended at 44 be tested for pathogens by the chicken FR 67087, Nov. 23, 1979; 48 FR 33473, July 22, embryo inoculation test prescribed in 1983. Redesignated at 55 FR 35562, Aug. 31, § 113.37, except that, if the test is a No 1990, as amended at 56 FR 66784, 66786, Dec. Test because of a vaccine virus over- 26, 1991; 64 FR 43045, Aug. 9, 1999; 72 FR 72564, ride, the chicken inoculation test pre- Dec. 21, 2007] scribed in § 113.36 may be conducted and the serial judged accordingly. § 113.332 Tenosynovitis Vaccine. (2) Safety tests. (i) Final container Tenosynovitis Vaccine shall be pre- samples of completed product from pared from virus-bearing cell culture each serial shall be tested to determine fluids or embryonated chicken eggs. whether the vaccine is safe as follows: Only Master Seed which has been es- (A) For vaccines intended for paren- tablished as pure, safe, and teral administration, each of twenty- immunogenic shall be used for pre- five 1-day-old bursal disease suscep- paring seeds for vaccine production. All

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serials of vaccine shall be prepared known tenosynovitis antigen shall be from the first through the fifth passage used in the test. A precipitin line shall from the Master Seed. form between the test antigen and the (a) The Master Seed shall meet the known antiserum in the center well applicable general requirements pre- which shows identity with the line scribed in § 113.300, except (a)(3)(ii) and formed between the antiserum and the (c), and the special requirements in known antigen, or the Master Seed is this section. unsatisfactory. (b) Each lot of Master Seed shall be (4) Safety using the following chick- tested for: en test: (1) Pathogens by the chicken inocula- (i) For vaccines intended for use in tion test prescribed in § 113.36. (2) Lymphoid leukosis virus contami- chickens less than 14 days of age, Mas- nation as follows: ter Seed equal to 10 label doses shall be (i) Each of at least 10 3-week-old or administered subcutaneously to each of older lymphoid leukosis free chickens 25 1-day-old tenosynovitis susceptible from the same source and hatch shall chickens. be injected intra-muscularly with an (ii) For vaccines intended for use amount of Master Seed equal to 100 only in chickens 14 days of age or label doses of vaccine. At least 15 older, Master Seed equal to 10 label chickens of the same source and hatch doses shall be administered shall be used as controls; 5 or more subcutaneously to each of 25 4-week-old shall be unvaccinated and serve as neg- or older tenosynovitis susceptible ative controls; 5 or more shall be in- chickens. jected with subgroup A lymphoid leu- (iii) The vaccinates shall be observed kosis virus; and 5 or more with sub- each day for 21 days. If unfavorable re- group B lymphoid leukosis virus. Each actions occur which are attributable to group of control chickens shall be held the vaccine, the Master Seed is unsat- isolated from each other and from the isfactory. If unfavorable reactions vaccinates. occur which are not attributable to the (ii) Twenty-one to 28 days vaccine, the test is a No Test and may postinoculation, blood samples shall be be repeated. taken from each chicken and the serum separated using a technique conducive (c) Each lot of Master Seed shall be to virus preservation. These serums tested for immunogenicity. The se- shall be used as inocula in the com- lected virus dose shall be established as plement fixation for avian lymphoid follows: leukosis (COFAL) test prescribed in (1) Tenosynovitis susceptible chick- § 113.31. ens, of the same age and from the same (iii) Serums from the vaccinates source shall be used as test birds. Vac- shall be tested separately, but serums cines intended for use in very young within each control group may be chickens shall be administered to pooled. A valid test shall have positive chickens of the youngest age for which COFAL reactions from each virus in- the vaccine is recommended. Vaccines oculated group and negative reactions intended for use in older chickens shall from the uninoculated controls. If any be administered to 4-week-old or older of the chickens injected with the Mas- chickens. Twenty or more vaccinates ter Seed have positive COFAL test re- shall be used for each method of admin- actions in a valid test, the Master Seed istration recommended on the label. is unsatisfactory. Ten or more chickens shall be held as (3) Identity using the following agar unvaccinated controls. gel immunodiffusion test. The undi- (2) A geometric mean titer of the vac- luted Master Seed may be used as test cine produced at the highest passage antigen or the Master Seed may be inoculated onto the chorioallantoic from the Master Seed shall be estab- membrane (CAM) of fully susceptible lished using a method acceptable to chicken embryos and the infected Animal and Plant Health Inspection CAMs ground and used as antigen. A Service before the immunogenicity known tenosynovitis antiserum and a test is conducted. A predetermined

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quantity of vaccine virus shall be ad- more than two vaccinates which are ministered to each vaccinate. Five rep- not attributable to the product, the licate virus titrations shall be con- test is a No Test and may be repeated. ducted on an aliquot of the vaccine If the test is not repeated, the serial is virus to confirm the dose. unsatisfactory. (3) Twenty-one to 28 days (3) Virus titer requirements. Final con- postvaccination, each vaccinate and tainer samples of completed product control shall be challenged by injecting shall be titrated by the method used in virulent virus furnished or approved by paragraph (c)(2) of this section. To be Animal and Plant Health Inspection eligible for release, each serial and sub- Service into one foot pad. The vac- serial shall have a virus titer suffi- cinates and controls shall be observed ciently greater than the titer of the each day for 14 days. If at least 90 per- vaccine virus used in the cent of the controls do not develop immunogenicity test prescribed in swelling and discoloration in the pha- paragraph (c) of this section to assure langeal joint area of the injected foot that, when tested at any time within pad typical of infection with the expiration period, each serial and tenosynovitis virus, the test is a No subserial shall have a virus titer 10 0.7 Test and may be repeated. If at least 19 times greater than that used in the of 20, 27 of 30, or 36 of 40 vaccinates do immunogenicity test, but not less than 2.0 not remain free from these signs, dis- 10 titration units (PFU or ID 50) per regarding transient swelling which sub- dose. sides within 5 days postchallenge, the (4) Identity. Bulk or final container Master Seed is unsatisfactory. samples of completed product from (4) An Outline of Production change each serial shall be tested for identity shall be made before authority for use as prescribed in paragraph (b)(3) of this of a new lot of Master Seed shall be section and shall meet the criteria granted by Animal and Plant Health stated therein. Inspection Service. [50 FR 438, Jan. 4, 1985. Redesignated at 55 (d) Test requirements for release. Each FR 35562, Aug. 31, 1990, as amended at 56 FR serial and subserial shall meet the ap- 66784, 66786, Dec. 26, 1991; 64 FR 43045, Aug. 9, plicable general requirements pre- 1999; 72 FR 72564, Dec. 21, 2007] scribed in § 113.300, except (c), and the requirements in this paragraph. DIAGNOSTICS AND REAGENTS (1) Purity. Final container samples of completed product from each serial §§ 113.400–113.405 [Reserved] shall be tested for pathogens by the chicken inoculation test prescribed in § 113.406 Tuberculin, Intradermic. § 113.36. Tuberculin, Intradermic, is a filtrate (2) Safety. (i) Final container samples produced from cultures of Pn, C, and of completed product from each serial Dt strains of Mycobacterium tuberculosis shall be safety tested as follows: (supplied by Animal and Plant Health (A) For vaccines intended for use in Inspection Service) which has been in- very young chickens, each of 25 1-day- activated and is non-toxic. Each serial old tenosynovitis susceptible chickens shall be tested for purity, safety, po- shall be vaccinated with the equivalent tency, and special chemical tests in ac- of 10 doses by one method rec- cordance with the conditions pre- ommended on the label. scribed for each test. A serial found un- (B) For vaccines intended for use in satisfactory by any prescribed test older chickens, each of 25 4-week-old or shall not be released. older tenosynovitis susceptible chick- (a) Purity test. Each serial shall be ens shall be vaccinated with the equiv- tested for purity as provided in this alent of 10 doses by one method rec- paragraph. ommended on the label. (1) Final container samples of com- (ii) The vaccinates shall be observed pleted product shall be tested for via- each day for 21 days. If unfavorable re- ble bacteria and fungi as prescribed in actions occur which are attributable to § 113.26. the product, the serial is unsatisfac- (2) A 20 ml sample shall be tory. If unfavorable reactions occur in centrifuged and the sediment examined

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microscopically for the presence of injected intradermally at one of the acidfast (Ziehl-Nielsen stain) or other test sites which has been randomly se- microorganisms (Gram stain). A serial lected for the dilution. which contains microorganisms is un- (4) The sensitivity of the tuberculins satisfactory for release. shall be determined 24 hours after in- (b) Safety test. Final container sam- jected by measuring the area of ery- ples of completed product from each se- thema. Measurements in millimeters rial shall be tested for safety. Two ma- shall be made anterior of the greatest ture guinea pigs shall be injected diameter and perpendicular to the first subcutaneously with 1 ml and observed measurement. The square millimeter for 10 days. If unfavorable reactions at- shall be calculated by multiplying the tributable to the product occur during two measurements. the observation period, the serial is un- (5) The total area of response for each satisfactory. If unfavorable reactions tuberculin tested shall be determined occur which are not attributable to the by adding the areas of erythema for product, the test shall be declared a No each dilution of each of the test ani- Test and repeated: Provided, That if the mals in a group. The sums of the areas test is not repeated, the serial shall be of erythema for all three dilutions of declared unsatisfactory. each tuberculin shall be added to give (c) Potency test. Bulk or final con- the total area of tuberculin response. tainer samples of completed product (6) The total tuberculin response area from each serial shall be subjected to a of the serial being tested shall be ex- comparison test using a Reference Tu- pressed as a percentage of the total tuberculin supplied by Animal and Plant berculin response area of the Reference Health Inspection Service. Test ani- Tuberculin. (The total response area of mals shall be 10 sensitized white female the serial divided by the total response guinea pigs from one source which area of the Reference Tuberculin times weigh 500–700 grams at the beginning of 100.) the test and which have not been used (7) If the total tuberculin response in a previous test. The comparison test area of the serial being tested does not shall be conducted in accordance with fall between 75 percent and 125 percent the procedures prescribed in para- of the total tuberculin response area of graphs (c)(1), (2), (3), (4), (5), (6), (7), and the Reference Tuberculin, the serial is (8) of this section. unsatisfactory. (1) The guinea pigs shall be sensitized (8) Two unsensitized guinea pigs are with a sterile heat-killed suspension of given 0.05 ml intradermal injections of equal amounts of strains Pn, C, and Dt 1:4 and 1:10 dilutions of both the serial of Mycobacterium tuberculosis. The heat- being tested and the Reference Tuber- killed sensitizing agent shall be in- culin as a control for nonspecific posi- jected in a volume of 0.5 ml per guinea tive reactions. If positive reactions are pig. The guinea pigs shall be considered observed with the Reference Tuber- sensitized for testing not less than 30 culin, the test is considered a ‘‘No days nor more than 120 days post-injec- Test’’ and repeated. If positive reaction. tions are observed with the serial being (2) The guinea pigs shall be prepared tested only, the serial is unsatisfac- for sensitivity testing at least 4 hours tory. prior to the injection of tuberculin. (d) Special chemical tests and require- The entire abdominal and flank areas ments. Final container samples of com- shall be clipped, a depilatory agent ap- pleted product from each serial shall be plied for 5–10 minutes, the area rinsed tested as follows: with warm water, and dried. (1) Hydrogen ion concentration. The (3) Dilutions of 1:100, 1:200, and 1:400 hydrogen ion concentration shall be de- shall be prepared with the Reference termined with a pH meter which has Tuberculin and the unknown tuber- been standardized with a pH 7.0 buffer culin. Three test sites on each side of just prior to use. The pH of the product and equidistant from the abdominal shall be 7.0 ±0.3. midline shall be chosen on each guinea (2) Total nitrogen determination. The pig. Using a tuberculin syringe and nitrogen content shall be determined needle, 0.05 ml of each dilution shall be by the Kjeldahl method on duplicate 15

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ml samples consisting of 5 ml from Antigen K shall be 1.0 ±0.2 percent as each of three vials. The total nitrogen determined by a colorimetric method. content of the product shall be 0.18 per- (2) The phenol content for Pullorum cent ±0.06 percent. Tube Antigen shall be 0.55 ±0.05 percent (3) Trichloroacetic acid precipitable ni- as determined by direct titration with trogen. The determination of precipi- a standardized bromide-bromate solu- table nitrogen by a final concentration tion. of 4 percent trichloroacetic acid shall (d) Sensitivity requirements. (1) Each be made by the Kjeldahl method on du- serial of antigen shall be compared plicate 15 ml samples, consisting of 5 with a reference antigen of known sen- ml from each of three vials. The sitivity using positive and negative trichloroacetic acid precipitable nitro- chicken serum. The manufacturers’ gen content shall be 0.047 percent ±0.01 recommendations for use on the ac- percent. companying label or package insert (4) Phenol determination. The phenol shall be followed. The recommended content shall be determined by direct time limit specified for each antigen titration with a standardized bromide- shall be carefully observed in the test. bromate solution. (A correction factor (2) A total of at least 12 serums shall of 0.04 should be subtracted from the be used. This shall include at least final value in the determination of phe- three definitely positive, at least three nol in tuberculin.) The phenol content weakly positive, and at least six nega- shall be 0.54 percent ±0.04 percent. tive serums. At least three positive (5) Clarity. The product shall be opti- chicken serums diluted with negative cally clear and free from any extra- chicken serum shall be used to further neous particles. assay comparative sensitivity between test and reference plate antigens. All [39 FR 16857, May 10, 1974. Redesignated at 39 test antigens shall agree closely with FR 25463, July 11, 1974. Redesignated at 55 FR the reference antigen. Tests in which 35561, Aug. 31, 1990, as amended at 56 FR variation of readings between the ref- 66784, Dec. 26, 1991] erence and test antigen would result in § 113.407 Pullorum antigen. a different National Poultry Improve- ment Plan classification shall be re- Pullorum Antigen shall be produced garded as unsatisfactory. No unsatis- from a culture of representative strains factory tests among the six or more of Salmonella pullorum which are of negative serums and not more than one known antigenic composition, high unsatisfactory test among the six or agglutinability, but are not sensitive more positive serums shall be per- to negative and nonspecific serum. mitted. All tests performed shall be in- Each serial shall be tested for purity, cluded for evaluation of the sensitivity density, preservative content, sensi- assay. In the event of an unsatisfactory tivity, homogeneity, and hydrogen ion test using positive serums, at least concentration. A serial found unsatis- three additional definitely positive and factory by any prescribed test shall not three additional weakly positive se- be released. rums shall be tested. If not more than (a) Purity test. Final container sam- one unsatisfactory test is obtained ples of completed product shall be test- with the additional serums, the anti- ed for viable bacteria and fungi as pre- gen shall be acceptable. scribed in § 113.26. In addition, each se- (e) Homogeneity requirement. Antigens rial shall be free from extraneous orga- shall show no evidence of nisms as determined by Gram staining autoagglutination or unusual appear- and microscopic examination. ance such as the presence of flakes, (b) Nephelometric determination of bac- specks, or a preponderance of filament terial density. The bacterial density forms. Microscopic examination shall shall be 80 ±15 times McFarland No. 1 be made in this determination. standard for stained antigen K’s and 50 (f) Hydrogen ion concentration. The ±10 times McFarland No. 1 standard for hydrogen ion concentration shall be de- tube antigen. termined with a pH meter which has (c) Preservative requirements. (1) The been standardized with a pH 4.0 buffer formalin content of Pullorum Stained just prior to use. The pH of Pullorum

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Stained Antigen K shall be 4.6 ±0.4. No been standardized with a pH buffer just pH level is specified for Pullorum Tube prior to use. The pH of Mycoplasma Antigen but after dilution as rec- Gallisepticum Antigen shall be 6.0±0.2. ommended for use, it shall have a pH of The pH of Mycoplasma Synoviae Anti- 8.2 to 8.5. gen and Mycoplasma Meleagridis Anti- [39 FR 16857, May 10, 1974. Redesignated at 39 gen shall be 7.0±0.2. FR 25463, July 11, 1974, and amended at 40 FR (e) Purity requirements. The antigen 760, Jan. 3, 1975. Redesignated at 55 FR 35561, shall be tested for viable bacteria and Aug. 31, 1990] fungi as prescribed in § 113.26. § 113.408 Avian mycoplasma antigen. (f) Sensitivity requirements. The reactivity of each antigen shall be tested Mycoplasma antigens shall be pre- by comparing the agglutination reac- pared from organisms, grown in broth tions of each serial of antigen with the cultures, that are inactivated and agglutination reactions of a standard standardized. Plate antigens shall be stained with a dye acceptable to Ani- reference antigen which is supplied by mal and Plant Health Inspection Serv- or acceptable to APHIS. A set con- ice (APHIS). Final container samples sisting of five known positive and five of completed product from each serial known negative serums shall be used. shall be tested for density, preservative The negative serums shall be tested content, homogeneity, hydrogen ion against the antigens undiluted and the concentration, purity, sensitivity, and positive serums shall be tested against specificity in accordance with the con- the antigens diluted 1:4 in buffer solu- ditions prescribed for each test. A se- tion formulated in the same manner as rial found unsatisfactory by any pre- the vehicle of the antigen being tested. scribed test shall not be released. If negative serums do not have nega- (a) Density requirements. A 2.5 ml sam- tive reactions in this test, the serial is ple of completed antigen shall be di- unsatisfactory. If the test antigen and luted with 2.5 ml of buffer solution for- the reference antigen do not have the mulated in the same manner as the ve- same agglutination reactions with at hicle of the antigen being tested in a least four of the five positive serums modified Hopkins tube and then used, the serial is unsatisfactory. × sedimented at 1,000 g in a refrigerated (1) The sensitivity of Mycoplasma centrifuge at 20 °C for 90 minutes. If Gallisepticum Antigen shall be tested the packed cell volume of the com- using a set of chicken and a set of tur- pleted antigen is not 1.2 percent (±0.4 key serums (the positive serums shall percent), the serial is unsatisfactory. (b) Preservative requirements. Preserv- have varying degrees of reactivity from atives shall be as specified in the Out- weakly positive to strongly positive). line of Production filed with APHIS in (2) The sensitivity of Mycoplasma accordance with 9 CFR 114.8. If phenol Synoviae Antigen shall be tested using is used, a direct titration with a stand- chicken serums. ardized bromide-bromate solution shall (3) The sensitivity of Mycoplasma be made. If the final concentration of Meleagridis Antigen shall be tested phenol is not 0.25 percent (±0.05 per- using turkey serums. cent), the serial is unsatisfactory. (g) Specificity requirements. Myco- (c) Homogeneity requirements. (1) Plate plasma Synoviae Antigen shall be ex- antigen shall be checked on a plate for amined for cross-agglutination with homogeneity and autoagglutination. If five Mycoplasma gallisepticum plate antigen is not homogeneous and antiserums (chicken origin); Myco- free of large visible particles (strands plasma Meleagridis Antigen shall be or clumps) or if it autoagglutinates, examined for cross-agglutination with the serial is unsatisfactory. five Mycoplasma gallisepticum (2) Stereo-microscopic examination antiserums (turkey origin) and five shall be used when necessary to evalu- antiserums (tur- ate a granular appearing antigen. Mycoplasma synoviae (d) Hydrogen ion concentration. The key origin). Tests shall be conducted hydrogen ion concentration shall be determined with a pH meter which has

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with undiluted antigen. If cross-agglu- (ii) Sensitize one group of guinea pigs tination occurs, the serial is unsatis- to M. avium. Inject each animal factory. intramuscularly with 0.5 ml of a sterile heat-killed suspension of M. avium [48 FR 33474, July 22, 1983. Redesignated at 55 Strain D–4 supplied by Animal and FR 35561, Aug. 31, 1990, as amended at 56 FR 66784, Dec. 26, 1991] Plant Health Inspection Service. (iii) Maintain an unsensitized group § 113.409 Tuberculin—PPD Bovis, as control animals. Intradermic. (3) Thirty-five days post-injection, Tuberculin—PPD Bovis, Intradermic the guinea pigs shall be used for tuber- is a purified protein derivative pro- culin testing. duced from cultures of Mycobacterium (4) The sensitized animals and con- bovis Strain AN–5 (supplied by Animal trols shall be prepared at least 4 hours and Plant Health Inspection Service), prior to injection of PPD tuberculin by which has been inactivated and is clipping the hair from the entire ab- nontoxic. Each serial shall be tested dominal and flank areas, applying a de- for purity, safety, potency, and special pilatory agent for 5 to 10 minutes, then chemical characteristics in accordance rinsing with warm water and drying. with the conditions prescribed for each (i) Select four sites on each guinea test. A serial found unsatisfactory by pig for injection of PPD tuberculin. any prescribed test shall not be re- Two sites shall be on each side of the leased. midline and spaced a sufficient dis- (a) Purity test. Each serial shall be tance from each other to avoid overlap- tested for viable bacteria and fungi as ping of skin reactions. prescribed in § 113.26. (ii) Prepare four dilutions of the Ref- (b) Safety test. Final container sam- erence PPD Tuberculin and each serial ples of completed product from each se- of PPD tuberculin being tested so as to rial shall be tested for safety as pre- contain 0.6, 1.2, 2.4, and 4.8 micrograms scribed in § 113.38. of protein per 0.1 ml dose. Each of the (c) Potency test. Bulk or final con- four dilutions of the same tuberculin tainer samples of completed product shall be randomly assigned a site on a from each serial shall be subjected to a guinea pig. comparison specificity test using a (iii) Inject one dose of each dilution Reference PPD Tuberculin supplied by at the assigned site using a tuberculin Animal and Plant Health Inspection syringe. Service. (5) Measurement of skin reactions. (1) Test animals. White female guinea Measure the area of erythema produced pigs from one source, which weigh 500 at each site on each guinea pig 24 hours to 700 grams at the beginning of the following injection of PPD tuberculin. test, and which have not been used in a Measurements in millimeters shall be previous test, shall be used in the spec- made anterior to posterior across the ificity test. Twenty-three guinea pigs greatest diameter and perpendicular to (10 sensitized with M. bovis, 10 sen- the first measurement. Calculate the sitized with M. avium and three area of erythema in square millimeters unsensitized) shall be required for each at each site by multiplying the two serial being tested, and 20 guinea pigs measurements. (10 sensitized with M. bovis and 10 sen- (6) Calculation of average response per sitized with M. avium) shall be required guinea pig. Obtain the total area of ery- for the Reference PPD Tuberculin. Al- thema for each guinea pig by adding lowance should be made for deaths dur- the areas of the four test sites. Add ing the sensitization period. these composite areas of erythema (2) Sensitization of guinea pigs. (i) Sen- from all guinea pigs with the same sensitize one group of guinea pigs to M. sitization and the same PPD tuberculin bovis. Inject each animal injection, then divide by the number of intramuscularly with 0.5 ml of a sterile animals in the group. The number ob- heat-killed suspension of M. bovis tained is the average response per guin- Strain AN–5 supplied by Animal and ea pig to the PPD tuberculin for the Plant Health Inspection Service. given type of sensitization.

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(7) Determination of specificity index. centration of 1.0 ±0.1 mg/ml. The Micro- The specificity index of a PPD tuber- kjeldahl Test for Nitrogen shall be culin is determined by subtracting the used. average response obtained on M. avium (2) Phenol content. Phenol content of sensitized guinea pigs from the average the final product shall be 0.50 percent response obtained on M. bovis sen- plus or minus 0.04 percent. A direct ti- sitized guinea pigs. tration with a standardized bromide- (8) Validity of bioassay. The bioassay bromate solution shall be conducted. test results obtained on serials tested concurrently in a single test series are [41 FR 8471, Feb. 27, 1976, as amended at 41 valid if the specificity index of the ref- FR 21760, May 28, 1976; 41 FR 32883, Aug. 6, erence PPD tuberculin is at least 400 1976. Redesignated at 55 FR 35561, Aug. 31, square millimeters. If the results are 1990, as amended at 56 FR 66784, Dec. 26, 1991] not valid, the bioassay test series must be repeated with a different set of sen- ANTIBODY PRODUCTS sitized guinea pigs. (9) Reactions in unsensitized guinea § 113.450 General requirements for antibody products. pigs. If a positive reaction (erythema) is observed in one or more of the 3 Unless otherwise prescribed in a unsensitized guinea pigs, the serial is Standard Requirement or in a filed unsatisfactory. Outline of Production, all antibody (10) Interpretation of specificity index. products shall meet the applicable re- When a bioassay is valid and reactions quirements of this section. are not observed in unsensitized guinea (a) Terminology. The following terms pigs, the following interpretation of in the regulations and standards con- the specificity index will be used for cerning antibody products shall mean: classifying each serial of PPD tuber- Antibody. An immunoglobulin mol- culin: ecule, having a precise glycoprotein Specificity index Classification structure, produced by certain cells of the B lymphocyte lineage in response 440 mm 2 or greater ...... Satisfactory. to antigenic stimulation, and func- Between 360 mm 2 and 440 mm 2 ...... Inconclusive. Less than 360 mm 2 ...... Unsatisfactory. tioning to specifically bind and influ- ence the antigens that induced its syn- (11) Second stage test. If a serial is thesis. classified as inconclusive, it can be de- IgG (Immunoglobulin G). One of the clared unsatisfactory or undergo a sec- several recognized classes of struc- ond stage test. The second stage shall turally related glycoproteins whose be conducted in a manner identical to representatives include all known anti- the first stage, except that bodies. unsensitized guinea pig controls are Monoclonal. Produced by, or derived not necessary. The results are evalu- from, the offspring of a single common ated by combining the results obtained progenitor cell. on all guinea pigs tested in stages one Failure of passive transfer. A condition and two. Calculate the average re- of neonates characterized by an abnor- sponse on the 20 M. bovis sensitized animals and on the 20 M. avium sensitized mally low concentration of circulating animals and determine the specificity maternal IgG. index. An inconclusive serial is satis- (b) Nomenclature. Antibody products factory after the second stage test, if shall be named as follows: its specificity index is 400 square milli- (1) Virus-specific products. The true meters or more, and unsatisfactory if name of a virus-specific product shall: its specificity index is less than 400 include the term ‘‘antibody,’’ specify square millimeters. the disease for which the product is in- (d) Special chemical tests and require- tended, and indicate the type of animal ments. Final container samples of com- that supplied the component anti- pleted product from each serial shall be bodies. If the antibodies are tested as follows: monoclonal, the term ‘‘monoclonal’’ (1) Protein concentration. The final shall be used. Example: ‘‘Duck Virus product shall contain a protein con- Hepatitis Antibody, Duck Origin.’’

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(2) Bacterium-specific products. The for infectious diseases. Records of all true name of a bacterium-specific prod- test results shall be maintained. An uct shall: include the term ‘‘antibody’’ animal which tests positive for an in- if the component antibodies are di- fectious disease shall not be used in the rected against a nontoxin antigen or manufacture of antibody products. the term ‘‘antitoxin’’ if the component Retests shall be conducted as deemed antibodies are directed against toxin, necessary by the Administrator. specify the organism against which the (i) Before first use, horses shall be product is intended, and indicate the tested as follows for: type of animal that supplied the com- (A) Equine infectious anemia (EIA) ponent antibodies. If the antibodies are at a laboratory approved by APHIS. monoclonal, the term ‘‘monoclonal’’ (B) Piroplasmosis, dourine, and glan- shall be used. Example: ‘‘Escherichia ders at the National Veterinary Serv- Coli Monoclonal Antibody, Murine Ori- ices Laboratories. gin.’’ (C) Brucellosis at a laboratory ap- (3) Failure of passive transfer products. proved by APHIS. Horses with standard The true name of a product for treat- agglutination titers of 1:50 or less can ment of failure of passive transfer shall be used for production. Horses with include the term ‘‘IgG’’ and indicate standard agglutination titers equal to the type of animal that supplied the or greater than 1:100 may be tested by component IgG. Example: ‘‘Bovine the Rivanol or card tests. Reactors to IgG.’’ these supplemental tests shall not be (4) Combination products. The true name of a product for treatment of fail- used for production. Nonreactors to the ure of passive transfer as well as for supplemental tests shall be retested the prevention and/or alleviation of a after 30 days. If the supplemental tests specific viral or bacterial disease shall are negative and the agglutination be named according to the nomen- titer has not increased, the animal clature prescribed above for virus-spe- may be used for production. Otherwise, cific or bacterium-specific products. the animal is unsatisfactory for this (c) Animals. All animals used in the purpose. production of antibody products shall (ii) Horses shall be retested annually be healthy. Their health status shall be for EIA and, if housed or pastured with determined by physical examination any other species, shall be retested an- by, or under the direct supervision of, a nually for brucellosis. licensed veterinarian and by tests for (iii) Before first use, cattle shall be infectious diseases. Such animals shall tested as follows for: be maintained at licensed establish- (A) Tuberculosis by an accredited ments: Provided, That cows maintained veterinarian: Provided, That cattle at at Grade A dairies (or the equivalent) Grade A dairies supplying only lacteal that are not injected with antigens for secretions need only be tested for tu- the purpose of stimulating the produc- berculosis in accordance with application of specific antibodies and that are ble Milk Ordinances or similar laws or used only for the purpose of supplying regulations. lacteal secretions are exempt from (B) Brucellosis at a laboratory ap- being maintained at a licensed estab- proved by APHIS. Cattle with standard lishment. agglutination titers of 1:50 or less can (1) No animal shall be used while be used for production. Cattle with showing clinical signs of disease. The standard agglutination titers equal to presence of minor localized injuries or or greater than 1:100 may be tested by lesions (contusions, lacerations, burns, the Rivanol or card tests. Reactors to etc.) without body temperature ele- these supplemental tests shall not be vation and without significant pain used for production. Nonreactors to the and distress shall not be construed as supplemental tests shall be retested clinical evidence of disease. after 30 days. If the supplemental tests (2) Before first use and on a regular are negative and the agglutination basis, all animals used in the manufac- titer has not increased, the animal ture of antibody products shall be indi- may be used for production; otherwise, vidually subjected to applicable tests the animal is unsatisfactory for this

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purpose. Cattle at Grade A dairies sup- onstrated to be at least as effective by plying only lacteal secretions need not data acceptable to APHIS and specified be tested individually for brucellosis if in the filed Outline of Production for a portion of their secretions contribute the product. These data are expected to to the herd milk pool tested as re- come from a study comparing the ef- quired by the brucellosis ring test. An fectiveness of the established and sub- animal of a herd testing positive by stitute procedures against a satisfac- this test shall not be used in produc- tory battery of potential contami- tion. nating microorganisms. (iv) Cattle shall be retested annually (1) Blood derivatives of equine origin for both tuberculosis and brucellosis. shall be heated at 58.0–59.0 °C for 60 Cattle at Grade A dairies supplying minutes, and blood derivatives of bo- only lacteal secretions need only be vine, porcine, or other origin shall be tested for tuberculosis in accordance heated at 58.0–59.0 °C for 30 minutes. In with applicable Milk Ordinances or lieu of heat treatment, blood deriva- similar laws or regulations. Cattle at tives of any origin may be treated with Grade A dairies supplying only lacteal at least 2.5 megarads of ionizing radi- secretions need not be tested individ- ation, with a maximum radiation dos- ually for brucellosis if a portion of age specified in the filed Outline of their secretions contribute to the herd Production for the product. milk pool tested as required by the (2) Lacteal secretions shall be heated brucellosis ring test. An animal of a as described in paragraph (e)(1) of this herd testing positive by this test shall section, or shall be pasteurized at ei- not be used in production. ther 72 °C for 15 seconds or 89 °C for 1 (v) For other species, appropriate second using appropriate equipment. In tests and the frequency with which lieu of the heat treatment regimens they are applied shall be specified in prescribed, lacteal secretions may be the filed Outline of Production for the treated with at least 2.5 megarads of product. ionizing radiation, with a maximum ra- (vi) If a positive result is obtained on diation dosage specified in the Outline any prescribed test, the positive ani- of Production for the product. mal(s) shall be removed from the herd (3) Egg material shall be heated at and the remaining animals retested. 58.0–59.0 °C for 30 minutes, or treated Production shall not be renewed until a with at least 2.5 megarads of ionizing negative herd test is obtained not less radiation, with a maximum radiation than 28 days following removal of the dosage specified in the filed Outline of positive animal(s). Production for the product. (vii) Negative animals shall be main- (4) Blood derivatives, lacteal secre- tained separate and apart from untest- tions, and egg material shall not con- ed or positive animals of any species. tain preservatives at the time of heat Production animals shall not be used treatment, and immediately after heat for any other purpose, such as testing, treatment shall be cooled to 7 °C or work, or recreation. lower. (d) Collection procedures. Blood, lac- (5) Licensees shall keep detailed teal secretions, and egg material shall records as to each batch treated and be collected as described in the filed each serial of product prepared for Outline of Production for the product. marketing. Recording charts shall bear (e) Ingredient handling and processing. full information concerning the mate- Blood derivatives (serum, plasma, etc.), rial treated and tests made of the lacteal secretions, and egg material equipment used for treatment. used in the production of antibody (f) Preservatives. Liquid antibody products shall be subjected to an ap- products, except those immediately propriate procedure for the inactiva- frozen following preparation and main- tion of potential contaminating micro- tained in a frozen state until time of organisms. The procedure shall be one use, shall contain at least one preserv- of those described below and specified ative from the following list, within in the filed Outline of Production for the range of concentration set forth: the product: Provided, That another (1) Phenol 0.25 to 0.55 percent, or procedure may be substituted if dem- (2) Cresol 0.10 to 0.30 percent, and/or

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(3) Thimerosal 0.01 to 0.03 percent, or considered a No Test and may be re- (4) Other preservative(s) specified in peated. If characteristic growth is ob- the filed Outline of Production for the served on any of the 10 plates con- product. taining samples of the serial, one (g) Antigens for hyperimmunization. If retest to rule out faulty technique may animals are hyperimmunized to gen- be conducted on samples from 20 final erate antibodies for a product for the containers. If characteristic growth is prevention and/or alleviation of a spe- observed on any of the retest plates, or cific infectious disease, and a USDA-li- if a retest is not initiated within 21 censed veterinary biological product is days of the completion of the original not employed for this purpose, the fol- test, the serial or subserial is unsatis- lowing shall apply: factory. (1) For each antigen, a Master Seed (ii) Salmonellae. One milliliter of each shall be established. rehydrated sample shall be pipetted (i) Bacterial Master Seeds shall be into a 100 × 15 mm petri dish and 10–15 tested for purity and identity as pre- ml of brilliant green agar at 45–50 °C scribed for live bacterial vaccines in added. The dish shall be manipulated § 113.64. to coat its entirety with the agar-sam- (ii) Viral Master Seeds shall be tested ple mixture and allowed to stand until for purity and identity as prescribed the mixture solidifies. The plate shall for live virus vaccines in § 113.300. then be incubated at 35 °C for 24 hours. (2) The maximum allowable passage A positive control plate and a negative level of the hyperimmunizing antigen control plate shall be prepared at the shall be the passage level of the anti- same time and in the same manner as gen used to generate product shown to the plates containing samples of the se- be efficacious and shall not exceed 10 rial. All plates shall be examined at the passages from the Master Seed. end of the incubation period. If char- (h) Purity tests. Final container sam- acteristic growth is observed on the ples of each serial and each subserial negative control plate, or no char- shall be tested for viable bacteria and acteristic growth is observed on the fungi as follows: positive control plate, the test shall be (1) Dried products for parenteral ad- considered a No Test and may be re- ministration and liquid products shall peated. If characteristic growth is ob- be tested as prescribed in § 113.26. served on any of the 10 plates con- (2) For dried products for oral admin- taining samples of the serial, one istration, 10 final container samples retest to rule out faulty technique may shall be reconstituted with sterile be conducted on samples from 20 final water at the volume recommended on containers. If characteristic growth is the label and tested for the following observed on any of the retest plates, or contaminants: if a retest is not initiated within 21 (i) Coliforms. One milliliter of each re- days of the completion of the original hydrated sample shall be pipetted into test, the serial or subserial is unsatis- a 100 × 15 mm petri dish and 10–15 ml of factory. violet red bile agar at 45–50 °C added. (iii) Fungi. One milliliter of each re- The plate shall be manipulated to coat hydrated sample shall be pipetted into its entirety with the agar-sample mix- a 100 × 15 mm petri dish and 10–15 ml of ture and allowed to stand until the appropriately acidified potato dextrose mixture solidifies. The plate shall then agar at 45–50 °C added. The plate shall be incubated at 35 °C for 24 hours. A be manipulated to coat its entirety positive control plate and a negative with the agar-sample mixture and al- control plate shall be prepared at the lowed to stand until the mixture solidi- same time and in the same manner as fies. The plate shall then be incubated the plates containing samples of the se- at 20–25 °C for 5 days. A positive con- rial. All plates shall be examined at the trol plate and a negative control plate end of the incubation period. If char- shall be prepared at the same time and acteristic growth is observed on the in the same manner as the plates con- negative control plate, or no char- taining samples of the serial. All plates acteristic growth is observed on the shall be examined at the end of the in- positive control plate, the test shall be cubation period. If growth is observed

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on the negative control plate, or no § 113.451 Tetanus Antitoxin. growth is observed on the positive con- Tetanus Antitoxin is a specific anti- trol plate, the test shall be considered body product containing antibodies di- a No Test and may be repeated. If rected against the toxin of Clostridium growth is observed on any of the 10 tetani. Each serial shall meet the appli- plates containing samples of the serial, cable general requirements provided in one retest to rule out faulty technique § 113.450 and paragraph (a) of this sec- may be conducted on samples from 20 tion, and be tested for potency as pro- final containers. If growth is observed vided in paragraph (b) of this section. on any of the retest plates, or if a Any serial found unsatisfactory by a retest is not initiated within 21 days of prescribed test shall not be released. the completion of the original test, the (a) General requirements. The amount serial or subserial is unsatisfactory. of antitoxin in a final container shall (iv) Total bacterial count. One milli- be the amount which is delivered from liter of each rehydrated sample, undi- such container when opened and inverted until the flow stops. A grad- luted or diluted as prescribed in the uated volumetric cylinder which con- Outline of Production, shall be pipetted × forms to the National Institute of into a 100 15 mm petri dish and 10–15 Standards and Technology require- ml of tryptone glucose extract agar at ments shall be used. The reading shall ° 45–50 C added. The plate shall be ma- be made at the bottom of the meniscus. nipulated to coat its entirety with the Volumes of 10 ml or less shall be re- agar-sample mixture and allowed to corded to the nearest 0.1 and volumes stand until the mixture solidifies. The over 10 ml shall be recorded to the plate shall then be incubated at 35 °C nearest ml. for 48 hours. A positive control plate (1) All final containers of Tetanus and a negative control plate shall be Antitoxin shall yield not less than the prepared at the same time and in the labeled unitage of antitoxin through- same manner as the plates containing out the dating period. The minimum samples of the serial. All plates shall package size permitted for marketing be examined at the end of the incuba- in the United States shall be a 1,500 tion period. If growth is observed on unit vial. the negative control plate, or no (2) The expiration date of Tetanus growth is observed on the positive con- Antitoxin shall be not more than 3 years after the date of a potency test trol plate, the test shall be considered which demonstrates that the recover- a No Test and may be repeated. If the able antitoxin from the final container average number of bacterial colonies provides at least 20 percent excess over on the 10 plates containing samples of the number of units claimed on the the serial exceeds that specified in the label or not more than 1 year after the filed Outline of Production for the date of a potency test which dem- product, one retest to rule out faulty onstrates that the recoverable anti- technique may be conducted on sam- toxin from the final container provides ples from 20 final containers. If the av- 10 to 19 percent excess over the number erage number of bacterial colonies on of units claimed on the label. the retest plates exceeds that specified (b) Potency test. Bulk or final con- in the filed Outline of Production for tainer samples of completed product the product, or if a retest is not initi- from each serial shall be assayed to ated within 21 days of the completion calculate the units of Tetanus Anti- of the original test, the serial or sub- toxin in each final container. A com- serial is unsatisfactory. parative toxin-antitoxin neutralization (i) Safety tests. Bulk or final con- test shall be conducted using a stand- tainer samples of each serial shall be ard antitoxin and a standard toxin. All tested as prescribed in § 113.33(b). Dried dilutions shall be made in M/15 phos- product shall be reconstituted as indi- phate buffered (pH) 7.4 physiological cated on the label and 0.5 ml injected saline with 0.2 percent gelatin. (1) One ml of the Standard Antitoxin per mouse. shall be diluted before use so the final [61 FR 51774, Oct. 4, 1996] volume contains 0.1 unit per ml. The

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dilution shall be held at 20° to 25 °C for creased muscle tonus, curvature of the 30 minutes prior to combination with a spine, asymmetry of the body outline test does of toxin. when the resting animal is viewed from (2) The Standard Toxin test dose is above, generalized spastic paralysis, that amount which when mixed with particularly of the extensor muscles, 0.1 unit of Standard Antitoxin, incu- inability to rise from a smooth surface bated at 20° to 25 °C for 1 hour, and in- when the animal is placed on its side, jected subcutaneously into a 340 to 380 or any combination of these signs. If gram guinea pig, results in death of the control guinea pigs do not respond that guinea pig within 60 to 120 hours in this manner, the entire test shall be with clinical signs of tetanus. The repeated. toxin shall be diluted so the test dose (7) Potency of an unknown antitoxin shall be in 2.0 ml. is determined by finding the mixture (3) A mixture of diluted Standard which will protect the test animal the Toxin and diluted Standard Antitoxin same as the Standard Toxin-Antitoxin shall be made so that 0.1 unit of anti- mixture. Test animals dying sooner toxin in 1 ml is combined with a test than the controls indicate the unit dose of toxin. This Standard Toxin- value selected in that dilution was not ° Antitoxin mixture shall be held at 20 present, whereas those living longer in- ° to 25 C for 1 hour before injections of dicate a greater unit value. guinea pigs are made. (4) A sample from each serial of anti- [39 FR 16859, May 10, 1974. Redesignated at 39 toxin shall be prepared as was the FR 25463, July 11, 1974, and amended at 40 FR Standard Toxin-Antitoxin mixture; ex- 760, Jan. 3, 1975; 40 FR 41996, Sept. 10, 1975; 43 cept the amount of antitoxin shall be FR 1479, Jan. 10, 1978; 50 FR 24905, June 14, 1985. Redesignated at 55 FR 35561, Aug. 31, based on an estimation of the expected 1990; 61 FR 51776, Oct. 4, 1996; 64 FR 43045, potency. When testing is done on bulk Aug. 9, 1999] material, the final container fill shall reflect the endpoint value plus 10 per- § 113.452 Erysipelothrix Rhusiopathiae cent overage for 1 year dating and 20 Antibody. percent overage for 3 year dating. Erysipelothrix Rhusiopathiae Anti- (5) Normal guinea pigs weighing body is a specific antibody product con- within a range of 340 to 380 grams shall taining antibodies directed against one be used. Pregnant guinea pigs must not or more somatic antigens of be used. Erysipelothrix rhusiopathiae. Each serial (i) Each of two guinea pigs (controls) shall be tested as provided in this sec- shall be injected subcutaneously with a tion. Any serial found unsatisfactory 3 ml dose of the Standard Toxin-Anti- by a prescribed test shall not be re- toxin mixture. Injections shall be made leased. in the same order that toxin is added to the dilutions of antitoxins. These (a) Each serial shall meet the appli- shall be observed parallel with the ti- cable general requirements provided in tration of one or more unknown § 113.450. antitoxins. (b) Potency test. Bulk or final con- (ii) Two guinea pigs shall be used as tainer samples of completed product test animals for each dilution of the from each serial shall be tested using unknown antitoxin. A 3.0 ml dose shall the two-stage test provided in this sec- be injected subcutaneously into each tion. animal. (1) In the first stage, each of 40 Swiss (6) Controls shall be observed until mice, each weighing 16 to 20 grams, they are down and are unable to rise or shall be injected subcutaneously with stand under their own power. At this 0.1 ml of product (dried product shall time they are euthanized and the time be rehydrated according to label direc- of death is recorded in hours. For a sat- tions). Twenty-four hours isfactory test, the controls must reach postinjection, the injected mice and 10 this point with clinical signs of tetanus additional mice designated controls within 24 hours of each other and with- shall be challenged subcutaneously in an overall time of 60 to 120 hours. with the same culture of Erysipelothrix The clinical signs to be observed are in- rhusiopathiae.

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(2) If less than eight of the 10 con- (1) When used in this test, the fol- trols die from erysipelas within 7 days lowing words and terms shall mean: post-challenge, the test is invalid. All (i) International antitoxin unit. (I.U.) dead mice shall be examined to deter- That quantity of Beta Antitoxin which mine if the cause of death was reacts with L0 and L∂ doses of Stand- Erysipelothrix rhusiopathiae infection. ard Toxin according to their defini- (3) The mice injected with product tions. shall be observed for 10 days (ii) L0dose. The largest quantity of postchallenge and all deaths recorded. toxin which can be mixed with one unit The second stage shall be required of Standard Antitoxin and not cause when 7–10 of the mice injected with sickness or death in injected mice. product die in the first stage. The sec- (iii) L∂dose. The smallest quantity of ond stage shall be conducted in a man- toxin which can be mixed with one unit ner identical to the first stage. of Standard Antitoxin and cause death (4) The results of the test shall be in at least 80 percent of injected mice. evaluated according to the following (iv) Standard antitoxin. The Beta table: Antitoxin preparation which has been standardized as to antitoxin unitage on Cumu- the basis of the International Clos- Cumu- lative lative tridium perfringens Beta Antitoxin total total num- Standard and which is either supplied Cumulative number of Number of vac- ber of by or acceptable to Animal and Plant Stage cinates number of vac- deaths deaths cinates for an Health Inspection Service. The anti- for a unsat- satis- toxin unit value shall be stated on the factory isfactory label. test test (v) Standard toxin. The Beta toxin 1 40 40 6 or 11 or preparation which is supplied by or is less more. acceptable to Animal and Plant Health 2 40 80 12 or 13 or less more. Inspection Service. (vi) Diluent. The solution used to make proper dilutions prescribed in [39 FR 16859, May 10, 1974. Redesignated at 39 this test. Such solution shall be made FR 25463, July 11, 1974, as amended at 40 FR 20067, May 8, 1975; 40 FR 23989, June 4, 1975. by dissolving 1 gram of peptone and Redesignated at 55 FR 35561, Aug. 31, 1990; 61 0.25 gram of sodium chloride in each 100 FR 51776, Oct. 4, 1996; 64 FR 43045, Aug. 9, ml of distilled water; adjusting the pH 1999] to 7.2; autoclaving at 250 °F. for 25 minutes; and storing at 4 °C. until used. § 113.453 [Reserved] (2) The antitoxin content of the test sample shall be determined as follows: § 113.454 Clostridium Perfringens (i) Make a dilution of Standard Anti- Type C Antitoxin. toxin to contain 10 International Units Clostridium Perfringens Type C Anti- of antitoxin per ml. toxin is a specific antibody product (ii) Make one dilution of Standard containing antibodies directed against Toxin to contain 10 L0 doses per ml and the toxin of Clostridium perfringens make a second dilution of Standard Type C. Each serial shall be tested as Toxin to contain 10 L∂ doses per ml. provided in this section. Any serial (iii) Dilute 1 ml of the test sample found unsatisfactory by a prescribed with 49 ml of diluent and combine 1 ml test shall not be released. of this dilution with 1 ml of the Stand- (a) Each serial shall meet the appli- ard Toxin diluted to contain 10 L0 cable general requirements provided in doses. § 113.450. (iv) Combine 10 International Units (b) Potency test. Bulk or final con- of Standard Antitoxin with 10 L0 doses tainer samples of completed product of diluted Standard Toxin and combine from each serial shall be tested using 10 International Units of Standard the toxin-neutralization test for Beta Antitoxin with 10 L∂ doses of diluted Antitoxin provided in this section. Standard Toxin. Dried products shall be rehydrated ac- (v) Neutralize all toxin-antitoxin cording to label directions. mixtures at room temperature for 1

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hour and hold in ice water until injec- (i) International antitoxin unit. (I.U.) tions of mice can be made. That quantity of Epsilon Antitoxin (vi) Five Swiss white mice, each which reacts with L0 and L∂ doses of weighing 16–20 grams, shall be used for Standard Toxin according to their defi- each toxin-antitoxin mixture. A dose of nitions.

0.2 ml shall be injected intravenously (ii) L0dose. The largest quantity of into each mouse. Conclude the test 24 toxin which can be mixed with one- hours post-injection and record all tenth unit of Standard Antitoxin and deaths. not cause sickness or death in injected (3) Test Interpretation. (i) If any mice mice. inoculated with the mixture of 10 (iii) L∂dose. The smallest quantity of International Units of Standard Anti- toxin which can be mixed with one- toxin and 10 L0 doses of Standard Toxin tenth unit of Standard Antitoxin and die, the results of the test are a No cause death in at least 80 percent of in- Test and shall be repeated: Provided, jected mice. That, if the test is not repeated, the se- (iv) Standard antitoxin. The Epsilon rial shall be declared unsatisfactory. Antitoxin preparation which has been (ii) If less than 80 percent of the mice standardized as to antitoxin unitage on inoculated with the mixture of 10 the basis of the International Clos- International Units of Standard Anti- tridium perfringens Epsilon Antitoxin toxin and 10 L∂ doses of Standard Standard and which is either supplied Toxin die, the results of the test are a by or acceptable to Animal and Plant No Test and shall be repeated: Provided, Health Inspection Service. The anti- That, if the test is not repeated, the se- toxin unit value shall be stated on the rial shall be declared unsatisfactory. label. (iii) If any mice inoculated with the (v) Standard toxin. The Epsilon toxin mixture of Clostridium Perfringens preparation which is supplied by or is Type C Antitoxin diluted 1:50 and 10 L0 acceptable to Animal and Plant Health doses of Standard Toxin die, the anti- Inspection Service. toxin is considered to contain less than (vi) Diluent. The solution used to 500 International Unit per ml and the make proper dilutions prescribed in serial is unsatisfactory. this test. Such solution shall be made [39 FR 16859, May 10, 1974. Redesignated at 39 by dissolving 1 gram of peptone and FR 25463, July 11, 1974. Redesignated at 55 FR 0.25 gram of sodium chloride in each 100 35561, Aug. 31, 1990, as amended at 56 FR ml of distilled water; adjusting the pH 66784, Dec. 26, 1991; 61 FR 51777, Oct. 4, 1996] to 7.2; autoclaving at 250 °F. for 25 min- ° § 113.455 Clostridium Perfringens utes; and storing at 4 C. until used. Type D Antitoxin. (2) The antitoxin content of the test sample shall be determined as follows: Clostridium Perfringens Type D Antitoxin is a specific antibody prod- (i) Make a dilution of Standard Anti- uct containing antibodies directed toxin to contain 1 International Unit of against the toxin of Clostridium antitoxin per ml. perfringens Type D. Each serial shall be (ii) Make one dilution of Standard tested as provided in this section. Any Toxin to contain 10 L0 doses per ml and serial found unsatisfactory by a pre- make a second dilution of Standard scribed test shall not be released. Toxin to contain 10 L∂ doses per ml. (a) Each serial shall meet the appli- (iii) Dilute 1 ml of the test sample cable general requirements provided in with 33 ml of diluent and combine 1 ml § 113.450. of this dilution with 1 ml of the Stand- (b) Potency test. Bulk or final con- ard Toxin diluted to contain 10 L0 tainer samples of completed product doses. from each serial shall be tested using (iv) Combine 1 International Unit of the toxin-neutralization test for Epsi- Standard Antitoxin with 10 L0 doses of lon Antitoxin provided in this section. Standard Toxin and combine 1 Inter- Dried products shall be rehydrated ac- national Unit of Standard Antitoxin cording to label directions. with 10 L∂ doses of Standard Toxin. (1) When used in this test, the fol- (v) Neutralize all toxin-antitoxin lowing words and terms shall mean: mixtures at room temperature for 1

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hour, and hold in ice water until injec- (a) Qualification of an IgG Reference tions of mice can be made. Product. An IgG Reference Product (ref- (vi) Five Swiss white mice, each erence) shall be a serial of product that weighing 16–20 grams, shall be used for is manufactured according to the filed each toxin-antitoxin mixture. A dose of Outline of Production, properly quali- 0.2 ml shall be injected intravenously fied, and used to assess the potency of into each mouse. Conclude the test 24 subsequent product serials, as de- hours post-injection and record all scribed in paragraph (c) below. The ref- deaths. erence shall be qualified as follows: (3) Test Interpretation. (i) If any (1) At least 20 newborn, colostrum-de- mice inoculated with the mixture of 1 prived animals of the species for which International Unit of Standard Anti- the product is recommended shall be toxin and 10 L0 doses of Standard Toxin randomly selected. die, the results of the test are a No (2) Blood samples shall be taken from Test and shall be repeated: Provided, each animal. That, if the test is not repeated, the se- (3) Each animal shall be administered rial shall be declared unsatisfactory. one dose of reference by the rec- (ii) If less than 80 percent of the mice ommended route and shall be observed inoculated with mixture of 1 Inter- for 24 hours. national Unit of Standard Antitoxin (i) Any adverse reactions shall be re- and 10 L∂ doses of Standard Toxin die, corded. the results of the test are a No Test (ii) The dosage of reference adminis- and shall be repeated: Provided, That, if tered to each animal shall be in accord- the test is not repeated, the serial shall ance with label directions. Label direc- be declared unsatisfactory. tions may indicate a single dosage re- (iii) If any mice inoculated with the gardless of weight, in which case the mixture of Clostridium Perfringens animals in the study shall be at or near Type D Antitoxin diluted 1:34 and 10 L0 the maximum weight for neonates of doses of Standard Toxin die, the anti- the species. toxin is considered to contain less than (4) After 24 hours, blood samples shall 34 International Units per ml and the be taken from each animal. serial is unsatisfactory. (5) Pretreatment and post treatment serum IgG concentrations shall be con- [39 FR 16859, May 10, 1974. Redesignated at 39 currently determined for each animal FR 25463, July 11, 1974, as amended at 40 FR 760, Jan. 3, 1975. Redesignated at 55 FR 35561, using a radial immunodiffusion (RID) Aug. 31, 1990, as amended at 56 FR 66784, Dec. method acceptable to APHIS and de- 26, 1991; 61 FR 51777, Oct. 4, 1996] scribed in the filed Outline of Produc- tion for the product. §§ 113.456–113.498 [Reserved] (6) Concurrently, using the same method, five IgG measurements shall § 113.499 Products for treatment of be made on an IgG Species Standard failure of passive transfer. supplied or approved by APHIS. The A product for the treatment of fail- IgG Species Standard shall be a prepa- ure of passive transfer (FPT) shall con- ration that contains IgG specific for tain a specified minimum quantity of the species in question at a concentra- IgG per dose and shall be recommended tion acceptable to APHIS. for use only in neonates of the same (7) For an IgG Reference Product to species as that of antibody origin. A be satisfactory, all animals used to product for oral administration shall qualify the reference must remain free not be recommended for use in animals of unfavorable product-related reac- more than 24 hours of age, while one tions and at least 90 percent of the for parenteral administration shall paired serum samples must reflect an only be recommended for use in neo- increase in IgG concentration natal animals. Each serial shall meet (posttreatment minus pretreatment the applicable general requirements concentration) equal to or greater than provided in § 113.450 and be tested for the IgG concentration of the IgG Spe- potency as provided in this section. cies Standard. Any serial found unsatisfactory by a (b) Antibody functionality. Prior to li- prescribed test shall not be released. censure, the prospective licensee shall

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perform a neutralization study, or an- § 114.1 Applicability. other type of study acceptable to Unless exempted by regulation or APHIS, to demonstrate functionality of product antibody. otherwise authorized by the Adminis- trator, all biological products prepared, (c) Potency. Bulk or final container samples of completed product from sold, bartered or exchanged, shipped or each serial shall be tested for IgG con- delivered for shipment in or from the tent as provided in this paragraph. United States, the District of Colum- Samples of the test serial and of an IgG bia, any Territory of the United States, Reference Product established in ac- or any place under the jurisdiction of cordance with paragraph (a) of this sec- the United States shall be prepared in tion shall be concurrently tested for accordance with the regulations in this IgG content by the RID method re- part. The licensee or permittee shall ferred to in paragraph (a)(5) of this sec- adopt and enforce all necessary meas- tion. Five IgG measurements shall be ures and shall comply with all direc- made on each. If the IgG level per dose tions the Administrator prescribes for of the test serial does not meet or ex- carrying out such regulations. ceed that of the reference, one complete retest, involving five IgG meas- [52 FR 11026, Apr. 7, 1987, as amended at 56 urements on both the reference and FR 66784, Dec. 26, 1991] two samples of the test serial, may be § 114.2 Products not prepared under li- conducted. If, upon retest, the average cense. IgG level per dose of the two samples of the test serial does not meet or exceed (a) When an establishment license is that of the reference, or if a retest is issued, if biological products which not conducted, the serial is unsatisfac- were not prepared in compliance with tory. the regulations are in the establishment, such products shall not be [61 FR 51777, Oct. 4, 1996] shipped or delivered for shipment or otherwise dealt with as having been PART 114—PRODUCTION REQUIRE- prepared under such regulations. MENTS FOR BIOLOGICAL PROD- (b) Except as provided in 9 CFR part UCTS 103, a biological product shall not be prepared in a licensed establishment Sec. unless the person to whom the estab- 114.1 Applicability. lishment license is issued holds an un- 114.2 Products not prepared under license. expired, unsuspended, and unrevoked 114.3 Separation of establishments. product license issued by the Adminis- 114.4 Identification of biological products. trator to prepare such biological prod- 114.5 Micro-organisms used as seed. uct, or unless the products prepared 114.6 Mixing biological products. 114.7 Personnel at licensed establishments. are subject to the provisions of § 107.2 114.8 Outline of Production required. of this subchapter. 114.9 Outline of Production guidelines. (c) A biological product produced in a 114.10 Antibiotics as preservatives. USDA-licensed establishment shall be 114.11 Storage and handling. produced under a U.S. Veterinary Bio- 114.12 Expiration date required. logical Product License or a license 114.13 Expiration date determination. granted by a State under § 107.2 (re- 114.14 Extension of expiration date for a se- ferred to as a State biological product rial or subserial. license and the products prepared pur- 114.15 Disposal of unsatisfactory products suant thereto as State-licensed biologi- and byproducts. 114.16 Producing subsidiaries. cal products, including autogenous bio- 114.17 Rebottling of biological products. logics), but not under both a U.S. Vet- 114.18 Reprocessing of biological products. erinary Biological Product License and

AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, 2.80, and 371.4.

SOURCE: 39 FR 16869, May 10, 1974, unless otherwise noted.

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a State biological product license. Be- that holds both a State and a U.S. Vet- fore a U.S. Veterinary Biological Prod- erinary Biological Products License for uct License (including a conditional li- autogenous biologics, the isolate shall cense) is issued, the licensee shall re- be designated by the licensee for use in linquish its State license for that prod- the production of an autogenous bio- uct: Provided, That autogenous biological product under either the State logics shall not be subject to this pro- product license, or the U.S. Veterinary vision when they are prepared in ac- Biological Product License: Provided, cordance with the provisions of para- That the isolate meets the require- graph (c)(5) of this section. ments of the respective regulatory au- (1) State-licensed biological products thority for an autogenous biologic. If, (including autogenous biologics) shall after producing the product pursuant only be distributed or shipped intra- to one license, the licensee elects to state, must not bear a U.S. Veterinary produce an autogenous biologic from Biologics Establishment License Num- the same isolate under provisions of ber, and must not otherwise be rep- the other license, the licensee may do resented in any manner as having met so only with the approval of the other the requirements for a U.S. Veterinary licensing authority. Biological Product license. Labeling of (ii) The true name of a State-licensed State- and USDA-licensed biological autogenous biologic shall specify the products produced in the same estab- State of licensure: e.g. lishment must be distinctly different ‘‘ lllll Autogenous Bacterin’’ in color and design. (2) All biological products in USDA- (State) licensed establishments, whether li- or lllll Autogenous Vaccine’’. censed by USDA or by the State, shall (State) be prepared only in locations indicated in legends filed in accordance with 9 [39 FR 16869, May 10, 1974, as amended at 60 CFR part 108. A description of each FR 48021, Sept. 21, 1995] State-licensed product must be filed § 114.3 Separation of establishments. with the Animal and Plant Health In- spection Service as part of the blue- (a) Each licensed establishment shall print legends and must be sufficient for be separate and distinct from any other Animal and Plant Health Inspection establishment in which a biological Service to determine any risk to the product is prepared. production of other products in the li- (b) No biological products authorized censed establishment and to determine to be prepared in a licensed establish- that adequate procedures are followed ment shall be prepared in whole or in to prevent contamination during pro- part by another licensed establishment duction. except as provided in paragraphs (c) (3) Records in such establishments and (d) of this section. must be maintained in accordance with (c) When a partially prepared biologi- §§ 116.1 and 116.2 of this subchapter and cal product cannot be completed at a shall include all products licensed by licensed establishment due to failure of the State or USDA. essential equipment, the Administrator (4) Reports prescribed in § 116.5 of this may authorize the use of similar equip- subchapter for USDA-licensed estab- ment at another licensed establish- lishments shall be submitted for all ment: Provided, That, such authoriza- veterinary biological products in the tion shall be limited to the duration of establishment. the emergency and to the phase of pro- (5) Under the following conditions, an duction affected by the equipment fail- autogenous biologic may be produced ure. in a USDA-licensed establishment (d) Partially prepared products or se- under either a State or U.S. Veterinary rials of completed products for further Biological Product License: manufacture may be moved from one (i) When a culture of microorga- licensed establishment to another li- nisms, isolated from a herd in a State, censed establishment, imported under is received at a USDA-licensed estab- the provisions of § 104.5, or moved from lishment that is in the same State but a licensed establishment for purpose of

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being exported under conditions pre- identification of the serial, shall be ap- scribed in an Outline of Production plied to identify it with the records of filed with Animal and Plant Health In- preparation and labeling. spection Service. Licensed products or products imported for distribution and § 114.7 Personnel at licensed establish- sale may be prepared and recommended ments. for final use, for further manufacturing (a) Each licensee shall designate a purposes, or both. All serials shall be person(s) to make all official contacts subject to the requirements for testing with Animal and Plant Health Inspec- and release specified in § 113.5 or § 113.10 tion Service on matters pertaining to and to the requirements for identifica- the preparation of biological products tion specified in § 114.4. under the Virus-Serum-Toxin Act. The licensee shall file three copies of bio- [39 FR 16869, May 10, 1974, as amended at 40 graphical summary with Animal and FR 46093, Oct. 6, 1975; 49 FR 45846, Nov. 21, Plant Health Inspection Service for 1984; 56 FR 66784, Dec. 26, 1991] such designated person and for each § 114.4 Identification of biological person responsible for any phase of products. preparation of a biological product. (b) All personnel employed in the Suitable tags or labels of a distinct preparation of biological products at a design shall be used for identifying all licensed establishment shall be com- ingredients used in the preparation of petent in good laboratory techniques biological products, all component through education or training, or both, parts to be combined to form a biologi- so as to consistently prepare high qual- cal product, all biological products ity products. while in the course of preparation and (c) All biological products prepared all completed biological products held at licensed establishments shall be pre- in storage at licensed establishments: pared and handled with due sanitary Provided, That, if such ingredients, precautions. Good sanitary measures components, or biological products are shall be practiced at all times by all not so identified, they shall be disposed personnel involved in such preparation of as provided in § 114.15. and handling of biological products. (1) The clothing worn by persons § 114.5 Micro-organisms used as seed. while preparing biological products Micro-organisms used in the prepara- shall be clean. All persons, imme- tion of biological products at licensed diately before entering laboratory establishments shall be free from the rooms of a licensed establishment, causative agents of other diseases or shall change their outer clothing or ef- conditions. A complete record of such fectively cover the same with gowns or micro-organisms shall be kept cur- other satisfactory clean garments. rently correct and a list submitted to (2) Unsanitary practices such as, but Animal and Plant Health Inspection not limited to, eating, smoking, or ex- Service upon request of the Adminis- pectorating on the floors or otherwise trator. creating a nuisance in any room, com- (Approved by the Office of Management and partment, or place in which biological Budget under control number 0579–0059) products are prepared, handled, or stored at licensed establishments are [39 FR 16869, May 10, 1974, as amended at 48 prohibited. FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, 1991] (Approved by the Office of Management and Budget under control number 0579–0013) § 114.6 Mixing biological products. [39 FR 16869, May 10, 1974, as amended at 48 Each biological product, when in liq- FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, uid form, shall be mixed thoroughly in 1991] a single container. During bottling operations, the product shall be con- § 114.8 Outline of Production required. stantly mixed sufficient to maintain An Outline of Production shall be on physical uniformity of the entire fill. A file with Animal and Plant Health In- serial number, with any other mark- spection Service for each licensed bio- ings that may be necessary for ready logical product or for each biological

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product authorized to be imported into (2) The Outline of Production to be the United States for Distribution and kept in the active file shall be des- Sale. Preparation of a biological prod- ignated. If more than one has been filed uct in a licensed establishment shall be for a product, only the Outline of Pro- in accordance with the Outline of Pro- duction currently being used shall be duction for such product filed with included. Animal and Plant Health Inspection (f) The Administrator may, upon the Service as provided in this section, but basis of information not available to subject to changes as may be required him at the time the current Outline of under § 114.8(f). Production for a biological product was (a) The Outline of Production shall filed, object to the methods or proce- be prepared as prescribed in § 114.9 and dures being used in the preparation of submitted to Animal and Plant Health such biological product and notify the Inspection Service for filing. When ob- licensee to modify the filed Outline of jectionable features, if any, are cor- Production to eliminate such objec- rected and no further exceptions are tions. If the licensee does not comply taken by Animal and Plant Health In- with the notice, the Administrator spection Service to an Outline of Pro- may, after affording opportunity for a duction for a biological product, such hearing to the licensee, suspend the Outline of Production shall be ap- product license for the biological prod- proved for filing. uct involved; in which case, the li- (b) Each page shall be stamped as censee shall not prepare such product filed on the date such action was taken until subsequent notice of withdrawal in the bottom right hand corner. Al- of the suspension is given to the li- though the filed outline may be re- censee. ferred to as an approved outline, ap- (Approved by the Office of Management and proval for filing constitutes no en- Budget under control number 0579–0013) dorsement by Animal and Plant Health [39 FR 16869, May 10, 1974, as amended at 48 Inspection Service of such biological FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, product or the methods and procedures 1991; 75 FR 20773, Apr. 21, 2010] used to prepare such biological product. § 114.9 Outline of Production guide- (c) One copy of the Outline of Produc- lines. tion shall be retained by the Animal Each Outline of Production shall be and Plant Health Inspection Service prepared in accordance with the appli- and one copy returned to the licensee cable directions provided in this sec- or permittee. tion. (d) Each licensee shall review each (a) General requirements. (1) All copies Outline of Production for accuracy and of each Outline of Production or spe- sufficiency not less frequently than cial outline or revised pages of either once a year. Revisions necessary to shall be prepared on heavy paper (8.5″ × bring an Outline of Production into 11″) of a type receptive to permanent compliance with the regulations shall stamp ink. be submitted to Animal and Plant (2) The name of the biological prod- Health Inspection Service. uct (or component), the establishment (e) When a list of licensed products to license number, and the date prepared be continued in production at a li- shall appear on a front cover page and censed establishment is requested by each page of the Outline of Production the Administrator in accordance with or special outline. The name of the li- § 102.5(d) of this subchapter, the licensee (or foreign manufacturer) shall censee shall supplement the list with appear on the front cover page. information for each product as fol- (3) The pages shall be numbered in lows: the upper center. At least 11⁄2 inch mar- (1) The Outline of Production cur- gin shall be left at the top of the first rently being used shall be identified as page and a 2 inch margin at the bottom to the date when last revised and filed of each page for the Animal and Plant with Animal and Plant Health Inspec- Health Inspection Service stamp. tion Service and the date of the last re- (4) Amended pages shall be numbered view made by the licensee. the same as those being superseded.

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They shall bear the date prepared and 4. Proportions of each micro-organism and refer to the date on the pages being su- strain. perseded. If one replacement page su- B. Identification methods used for each micro-organism and frequency with which persedes more than one page, the new these methods are applied. page shall indicate same, but if several C. Virulence and purity of cultures or anti- replacement pages are added to super- gen and the determination and maintenance sede one page, the page number fol- thereof. Range of subcultures or passages to lowed by letters shall be used. be used in production. (5) The last page of both copies of ei- D. Attenuation, if any, before use for pro- ther a new or a completely rewritten duction purposes. E. Character, size, and shape of containers Outline of Production and each page used for growing micro-organisms. revised separately shall be signed in F. Media used for stock, seed, and antigen the lower left corner by the authorized cultures (composition and reaction of). May representative of the licensee (or for- refer to a special outline by number. eign producer). Stamped or facsimile G. Preparation of the antigen or toxin and signatures are not acceptable. toxoid. Complete and full description of each (6) A summary of changes shall ap- step and its manner of accomplishment and number these steps in sequence. Include all pear on an attached page and refer to tests for each antigen, and the specifications each page, paragraph, or subparagraph for character, identity, virulence, concentra- being changed. tion, and standardization. (7) Transmittal forms shall be used III. Immunization of animals. A. Outline for the original and subsequent revi- fully with special attention given to the fol- sions. Transmittal forms are available lowing: on the Internet at (http:// 1. Character and dose of the antigen. 2. Method and frequency of injections. www.aphis.usda.gov/animallhealth/ 3. Time required for immunization or vetlbiologics/vblforms.shtml). hyperimmunization. (b) Special outline. An outline describ- 4. Preliminary bleedings and tests, if any, ing the preparation of a component of to ascertain quality of serum. a biological product or an operation 5. All other similar matters, including performed in the preparation of a bio- treatments between bleedings. logical product may be required if such B. Period of time elapsing between last injection and first bleeding; and between bleed- special outline could be referred to in ings. Outlines of Production to eliminate C. Technique of bleeding operations; vol- repetition. Each special outline shall ume of blood collected at each bleeding; and be identified by number and shall not period of rest. be used until accepted and filed by Ani- IV. Preparation of the biological product. A. mal and Plant Health Inspection Serv- Describe fully and show each step of prepara- ice. tion from the first bleeding to the completion of the preserved product in bulk con- (c) Outline of Production for anti- tainers prior to filling of final containers. serum, antitoxin, and normal serum B. Composition of the preservative and shall be written according to the fol- proportions used. Indicate at which step of lowing: production, and the method used in adding the preservative. OUTLINE GUIDE FOR PRODUCTION OF ANTI- C. Agglutination and complement-fixation SERUM AND ANTITOXIN AND NORMAL SERUM titers and the methods of their determina- License No. Name of Product Date tions. D. Disposition of unsatisfactory biological I. Serum animals. A. Species, conditions, products and infective materials not used in age, and general health. production. B. Examination, preparation, care, quar- E. Assembly of units to make a serial; vol- antine, tests, and treatment of animals be- ume of the average serial; and the volume of fore injections are started. the maximum serial. C. Holding, handling, exercising, and moni- V. Testing. Indicate the stages in the prepa- toring the condition of animals after injec- ration of the biological product at which tions are started. samples are collected. Refer to all applicable II. Antigens. A. Composition and character Standard Requirements. Outline all addi- of the antigen. tional tests in detail and state minimum re- 1. Micro-organisms. quirements for each satisfactory test. 2. Source and date of accession of each A. Purity. micro-organism. B. Safety. 3. Strains. C. Potency.

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D. Other tests. removal of micro-organisms or tissues for VI. Post preparatory steps. production purposes. A. Form and size of final containers in B. Minimum and maximum period of time which the product is to be distributed. elapsing from time of inoculation until har- B. Methods and techniques of filling final vest. containers. Volume of fill for each size final C. Technique of harvesting micro-orga- container. nisms or tissues (specify) for production pur- C. Collection, storage, and submission of poses. representative samples. Indicate at which D. Specifications for acceptable harvest steps in the production these samples are material. taken. E. Handling of discarded material not used D. Expiration date based on the earliest in production. date of harvest and the date of the last satis- F. Include any additional pertinent infor- factory potency test. mation. E. Use, dosage, and route of administration IV. Preparation of the product. Describe for each animal species for which it is rec- fully and show each step of preparation from ommended. harvest of antigen containing tissues or pro- F. Include any additional pertinent infor- duction cultures to the completion of the mation. finished product in final containers. In de- (d) Outline of Production for vaccines, scribing the preparation of the product, emphasize the following: bacterins, antigens, and toxoids shall be A. Method of inactivation, attenuation, or written according to the following: detoxification. B. Composition of preservative, adjuvant OUTLINE GUIDE FOR VACCINES, BACTERINS, or stabilizer, and proportions used stated in ANTIGENS, AND TOXOIDS such a manner that the concentration can be License No. Name of Product Date calculated; stage and method of addition. I. Composition, etc., of the product. A. Micro- C. Method and degree of concentration. organisms used. Give the isolation and pas- D. If product is standardized to give con- sage history. centration of antigen, show procedures and B. Source and date of accession of each calculations. micro-organism. E. 1. Assembly of units to make a serial (il- C. Strains. lustrate by example). D. Proportions of each strain. 2. Volume of average serial. II. Cultures. A. Brief description of methods 3. Volume of maximum serial. of identifying each micro-organism and the 4. Any other pertinent information. frequency with which these methods are ap- F. Volume of fill for each size vial. Type of plied. vial if unusual. B. Virulence and purity of cultures and the G. Method and technique of filling and determination and maintenance thereof. sealing of final containers. Range of subcultures or passages to be used H. Desiccation, including moisture control. in production. Give maximum percent moisture. C. Composition and reaction of media used I. Amount of antigenic material per dose for seed and production cultures. Include the or doses in final container. source of eggs, tissue, or cell cultures, and V. Testing. Indicate the stages in the prepa- the tests to determine that eggs, tissues, and ration of the biological product at which the cells are free of contamination. samples are collected. Refer to all applicable D. Character, size, and shape of containers Standard Requirements. Outline all addi- used for growing cultures. tional tests in detail and state the minimum E. Storage conditions of seed cultures. requirement for each satisfactory test. F. Methods of preparing suspensions for A. Purity. seeding or inoculation. B. Safety. G. Technique of inoculating (1) seed media; C. Potency. (2) production media. Titer or concentration D. Moisture, if desiccated. of inoculum, and the volume of medium for E. Any other tests. each size and type of culture container. VI. Post preparatory steps. A. Form and size H. Period of time and conditions for incu- of final containers in which the product is to bation and degree of temperature used for be distributed. each micro-organism or group of micro-orga- B. Collection, storage, and submission of nisms. representative samples. Indicate at which I. Character and amount of growth; obser- steps in the production these samples are vation as to contamination of growth. taken. J. Method of attenuation, if any, before C. Expiration date based on the earliest used for production purposes. date of harvest and the date of the last satis- III. Harvest. A. Handling and preparation of factory potency test. If applicable, give the cultures and media (including eggs) before date of lyophilization.

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D. Use, dosage, and route of administration OUTLINE GUIDE FOR DIAGNOSTIC TEST KITS for each animal species for which the bio- License No. Name of product Date logical product is recommended. (e) Outlines of Production for aller- Introduction genic extracts shall be written accord- Provide a brief description of the kit as fol- ing to the following: lows: 1. Principle of the test (ELISA, latex ag- OUTLINE GUIDE FOR ALLERGENIC EXTRACTS glutination, etc.). License No. Name of Product Date 2. Antigen or antibody detection test. 3. Sample(s) used for testing (serum, whole I. Composition of the product. A. Source and blood, tears, etc.). type of raw material. 4. List reagents, references, and equipment B. Weight/volume concentration. included. II. Preparation of the product. A. Describe 5. Identify materials obtained under split fully and show each step of preparation to manufacturing agreements. the completion of the finished product in 6. General description of test interpreta- true containers. In describing the preparations and their limitations, including fol- tion of the product, emphasize the following: lowup tests. 1. Method of extraction. 2. Composition of preservative, adjuvant or I. Antibody Components stabilizer, and proportions used; stage and method of addition. A. Production of polyclonal antibody com- 3. Method and degree of concentration. ponents. 4. Standardization of the product. 1. If purchased, list suppliers, criteria for 5. (a) Assembly of units to make a serial. acceptability, and describe all tests per- (b) Volume of average serial. formed after receipt to determine that speci- (c) Maximum serial. fications have been met. 6. Volume of fill for each size vial. 2. If produced in-house, describe the spe- 7. Method and technique of filling and seal- cies, age, weight, conditions, and general ing of final containers. health of all animals used in antiserum pro- 8. Amount material per dose or doses in duction. final container. a. Preinjection considerations: III. Testing. Indicate the stages in the prep- Describe the examination, preparation, aration of the biological product at which care, quarantine procedures, and treatments the samples are collected. Refer to all appli- administered before immunization(s). De- cable Standard Requirements. Outline all ad- scribe all tests used to determine suitability ditional tests in detail and state the min- for use. Describe the preparation of any imum requirement for each satisfactory test. standard negative serum(s) collected prior to A. Purity. immunization. B. Safety. b. Immunization of animals. C. Potency. i. Describe the character and dose of the D. Any other tests. antigen; if adjuvant is used provide details E. Include any additional pertinent infor- on its preparation. If commercial product is mation. used include its true name as shown on the IV. Post preparatory steps. A. Form and size label, the manufacturer, serial number, and of final containers in which the product is to expiration date. be distributed. ii. Describe the method and schedule for B. Collection, storage, and submission of immunizations. representative samples. Indicate at which iii. Describe the method for harvesting and steps in the production these samples are evaluating the immunization product, in- taken. cluding tests for acceptability. C. Expiration date based on the earliest iv. Provide number and intervals between date of harvest and the date of the last satis- harvests, volume obtained, and any other factory potency test. pertinent information. D. Use, dosage, and route of administration B. Production of Monoclonal Antibody for each animal species for which the bio- Components. logical product is recommended. 1. Hybridoma components: (f) Outlines of Production for diag- a. If hybridoma components are purchased, nostic test kits based on antigen-anti- list suppliers and criteria for acceptability; body reactions, and other diagnostics if tests are performed after receipt, describe fully. whose production methods are ame- b. If hybridomas are prepared inhouse, nable to description as described herein identify the antigen(s) used, describe the im- shall be written according to the fol- munization scheme, and the species of ani- lowing requirements: mal used.

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c. Identify the tissue of origin, and the pro- C. Describe the preparation and standard- cedures for harvesting, isolating, and identi- ization of the substrate(s). If purchased, list fying the immune cells. suppliers and criteria for acceptance. d. Describe the source, identity, and the D. Identify buffers, diluents, and other re- product secreted (light or heavy chain) by agents included in the kit. The preparation the parent Myeloma Cell Line. of these components may be described in this e. Summarize cloning and recloning proce- section or in filed Special Outlines. dures, including clone characterization and propagation, if appropriate. IV. Preparation of the Product f. If appropriate, describe procedures for establishing and maintaining seed lots. Fully describe methods used to standardize g. Describe any other pertinent tests or antigens, reference standards, positive con- procedures performed on the hybridoma cell trol serum, negative control serum, and line. standard reagents from production/purchase 2. Antibody production: to completion of finished product in final a. Describe the production method. If pro- containers, including the following: duced in cell culture, animal serum additives 1. Composition and quantity of preserva- must conform to 9 CFR 113.53. If produced in tive in each. animals, describe fully including husbandry 2. Method of filling, plating, or attaching practices and passage procedures. the antigen or antibody component to a solid b. Provide the criteria for acceptable phase. monoclonal antibody, including tests for purity. 3. Minimum and maximum acceptable fill c. Describe all tests or other methods used volumes for each final container of reagent to ensure uniformity between production included in the kit. lots of monoclonal antibody. Include all re- 4. The disposition of unsatisfactory mate- action conditions, equipment used, and reac- rial. tivity of the component. d. Describe all characterization procedures V. Testing and include the expected reactivity of all ref- Refer to all applicable standard require- erence monoclonal antibodies. ments. II. Antigen Preparation A. Purity. Describe all tests of the kit for purity or A. Identify the microorganism(s) or anti- specify the exemption as provided in 9 CFR gen being used. If previously approved Mas- 113.4. ter Seed virus, bacteria, or antigen derived B. Safety. therefrom is used, provide pertinent information on the testing performed, and details In vitro products are exempt from safety of dates of United States Department of Ag- tests. riculture confirmatory tests and approval, as C. Potency. appropriate. Provide details of tests used to determine B. Describe all propagation steps, includ- the relative reactivity of the kit including ing identification of cell cultures, media in- minimum requirements for a satisfactory gredients, cell culture conditions, and har- test. Reference standards and control serum vest methods. For antigen produced in eggs, used for this purpose should be identified by give the egg source, age, and route of inocu- unique codes or lot numbers. lation. If cell lines are being used, give dates of testing and approval as specified in 9 CFR VI. Postpreparatory Steps 113.52. C. Describe procedures used for extracting A. Describe the form and size of final con- and characterizing the antigen. tainers of each reagent/component included D. Describe the method used to standardize in the kit. the antigen. B. Describe the collection, storage, and E. If the antigen is purchased, identify the submission of representative samples. Refer supplier and describe the criteria for accept- to 9 CFR 113.3(b)(7). able material, including all tests performed C. Specify the expiration date. Refer to 9 by the producer and/or the recipient to deter- CFR 114.13. mine acceptability. D. Provide details of recommendations for use, including all limitations, qualifications, III. Preparation of Standard Reagents and interpretation of results. A. Describe the positive and negative controls included in the kit. If purchased, list suppliers and criteria for acceptance. B. Describe the preparation and standardization of the conjugate(s). If purchased, list suppliers and criteria for acceptance.

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E. Submit confidentiality statement iden- combination of penicillin and strepto- tifying specific parts of the outline con- mycin, or with a combination of poly- taining information, the release of which myxin B and neomycin. would cause harm to the submitter. (3) The maximum amount of each an- (Approved by the Office of Management and tibiotic in a combination shall be the Budget under control number 0579–0013) amount prescribed for such antibiotic [39 FR 16869, May 10, 1974, as amended at 48 in paragraph (b) of this section. FR 57473, Dec. 30, 1983; 56 FR 20124, May 2, (d) Antibiotics used in virus seed 1991; 56 FR 66784, Dec. 26, 1991; 75 FR 20773, stock purification are not restricted as Apr. 21, 2010] to kind or amounts provided carryover § 114.10 Antibiotics as preservatives. into the final product is controlled and specified in outlines of production. Antibiotics are authorized for use as preservatives for biological products if [39 FR 16869, May 10, 1974, as amended at 56 used within the limitations as to kinds FR 66784, Dec. 26, 1991] and amounts prescribed in this section. (a) When an antibiotic or combina- § 114.11 Storage and handling. tion of antibiotics, with or without a Biological products at licensed estab- fungistat is to be used in the prepara- lishments must be protected at all tion of a biological product, the kind(s) times against improper storage and and amount(s) of each shall be speci- handling. Completed product must be fied in the outline for such product in kept under refrigeration at 35 to 46 °F such a way that the concentration in (2 to 8 °C), unless the inherent nature the final product may be calculated. of the product makes storage at dif- Except as may be approved by the Ad- ferent temperatures advisable, in ministrator, only those individual anti- which case, the proper storage tem- biotics or combinations of antibiotics perature must be specified in the filed listed in paragraphs (b) and (c) of this Outline of Production. All biological section shall be used. products to be shipped or delivered (b) Permitted individual antibiotics: must be securely packed. (1) The antibiotic level of a specified individual antibiotic in one ml. of a bi- [81 FR 59436, Aug. 30, 2016] ological product, when prepared as recommended for use, shall not exceed the § 114.12 Expiration date required. amounts listed in this paragraph: Pro- Each serial or subserial of biological vided, That in the case a desiccated bio- product prepared in a licensed estab- logical product is to be used with an in- lishment shall be given an expiration definite quantity of water or other date determined in accordance with the menstruum, the determination shall be requirements provided in § 114.13 or based on 30 ml. per 1,000 dose vial or § 114.14. A licensed biological product equivalent. shall be considered worthless under the (2) Except as prescribed in paragraph Virus-Serum-Toxin Act subsequent to (c) of this section, only one antibiotic the expiration date appearing on the shall be used as a preservative in a bio- label. logical product. The kind and maximum amount per ml. of such anti- [41 FR 44687, Oct. 12, 1976] biotic shall be restricted to: § 114.13 Expiration date determina- Amphotericin B ...... 2.5 mcg. tion. Nystatin (Mycostatin) ...... 30.0 units Tetracyclines ...... 30.0 mcg. Unless otherwise provided for in a Penicillin ...... 30.0 units Standard Requirement of filed Outline Streptomycin ...... 30.0 mcg. Polymyxin B ...... 30.0 mcg. of Production, the expiration date for Neomycin ...... 30.0 mcg. each product shall be computed from Gentamicin ...... 30.0 mcg. the date of the initiation of the po- (c) Permitted combinations: tency test. Prior to licensure, stability (1) Penicillin and streptomycin. of each fraction shall be determined by (2) Either amphotericin B or nys- methods acceptable to Animal and tatin, but not both, may be used with Plant Health Inspection Service. Expi- one of the other antibiotics listed in ration dates based on this stability paragraph (b) of this section, or with a data shall be confirmed as follows:

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(a) Products consisting of viable orga- tory require it be removed from the nisms. Each serial shall be tested for market by the licensee. potency at release and at the approxi- (2) [Reserved] mate expiration date until a statistically valid stability record has been [50 FR 24903, June 14, 1985, as amended at 56 established. FR 66784, Dec. 26, 1991] (b) Nonviable biological products. Each § 114.15 Disposal of unsatisfactory serial presented in support of licensure products and byproducts. shall be tested for potency at release and at or after the dating requested. All biological products found to be (c) Subsequent changes in the dating unsatisfactory for marketing, all bio- period for a product may be granted, logical products which have become based on statistically valid data sub- worthless subsequent to the expiration mitted to support a revision of the Out- date, all refuse, other materials line of Production. deemed unsatisfactory for production purposes, all carcasses (part or whole) [50 FR 24903, June 14, 1985, as amended at 56 of production or test animals, and any FR 66784, Dec. 26, 1991] undesirable byproducts of manufacture § 114.14 Extension of expiration date shall be disposed of as may be required for a serial or subserial. by the Administrator. (a) Unless otherwise provided for in a [41 FR 44687, Oct. 12, 1976, as amended at 56 filed Outline of Production for the FR 66784, Dec. 26, 1991] product, the expiration date shall not be extended: § 114.16 Producing subsidiaries. (1) If all fractions of the product are A serial or subserial of a biological not evaluated for potency by tests des- product may be produced jointly by a ignated in the filed Outline of Produc- licensee and one or more subsidiaries, tion for such product in accordance or by two or more subsidiaries. The with § 113.4(b) of this subchapter. exact amount of each serial or sub- (2) For any serial or portion of any serial credited to each participating serial which has left licensed premises: producer shall be determined at the Provided, That product which has been time of labeling and packaging and shipped from one licensed premises to shall be noted in the records for such another licensed premises shall be ex- serial or subserial. empt from this requirement. (3) For a serial or portion of a serial [40 FR 46093, Oct. 6, 1975] if the expiration date has been extended previously, unless otherwise au- § 114.17 Rebottling of biological products. thorized in accordance with § 114.1. (b) An extension of the expiration The Administrator may authorize date may be granted by Animal and the rebottling of a completed product Plant Health Inspection Service if a re- in liquid form subject to the conditions quest from the licensee is substan- prescribed in this section. tiated by valid test data which dem- (a) All or part of a serial which has onstrate the potency of the product not left the licensed establishment meets or exceeds the requirements for may be aseptically returned to the release. The new expiration date shall mixing tank, thoroughly mixed, and re- be calculated from the date the latest bottled in new final containers. satisfactory potency test was initiated. (b) The rebottled product shall be The extension of the expiration date adequately identified by serial number shall not exceed the maximum dating or subserial number, as the case may allowed in the filed Outline of Produc- be. tion. (c) Required purity tests for final (1) Serials are approved for redating container samples of the product shall under the condition that Animal and be conducted on new samples selected Plant Health Inspection Service may from the rebottled product (serial or require the firm to retest the redated subserials). Rebottled product found to serial for potency during the extended be unsatisfactory by such tests shall dating period and if found unsatisfac- not be released.

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(d) New test samples from each serial hour during the day or night, and shall or subserial and copies of test reports be permitted to inspect, without pre- of all tests conducted on the rebottled vious notification, the entire premises product shall be submitted to Animal of the establishment, including all and Plant Health Inspection Service. buildings, compartments, and other (e) The licensee shall not release the places, all biological products, and or- rebottled product unless notified by ganisms and vectors in the establish- Animal and Plant Health Inspection ment, and all materials and equipment, Service that such product is eligible such as chemicals, instruments, appa- for release. Production records shall ratus, and the like, and the methods show the results of all tests conducted used in the manufacture of, and all and shall accurately reflect the actions records maintained relative to, biologi- taken. cal products produced at such establishment. [39 FR 16869, May 10, 1974, as amended at 56 (b) Each inspector will have in his or FR 66784, Dec. 26, 1991] her possession a numbered USDA badge § 114.18 Reprocessing of biological or identification card. Either shall be products. sufficient identification to entitle him/ her to admittance at all regular en- The Administrator may authorize a trances and to all parts of such estab- licensee to reprocess a serial of com- lishment and premises and to any place pleted product subject to the condi- at any time for the purpose of making tions prescribed in this section. an inspection pursuant to paragraph (a) Reprocessing shall not include (a) of this section. any method or procedure which would be deleterious to the product. [52 FR 30134, Aug. 13, 1987] (b) All appropriate tests for purity, safety, potency, and efficacy for the § 115.2 Inspections of biological products. product shall be conducted on the reprocessed product. A serial found un- (a) Any biological product, the con- satisfactory by a required test shall tainer of which bears a United States not be released. veterinary license number or a United (c) The reprocessed serial shall be States veterinary permit number or identified by a new serial number and other mark required by these regula- the records for the serial shall accu- tions, may be inspected at any time or rately reflect the action taken. place. If, as a result of such inspection, (d) Test samples of the reprocessed it appears that any such product is serial and test reports for all tests con- worthless, contaminated, dangerous, or ducted shall be submitted to Animal harmful, the Secretary shall give no- and Plant Health Inspection Service. tice to stop distribution and sale to the The licensee shall not release the serial manufacturer (licensee) or importer until notified by Animal and Plant (permittee) and may proceed against Health Inspection Service that the se- such product pursuant to the provi- rial is eligible for release. sions of part 118 of this subchapter. (b) When notified to stop distribution [50 FR 24904, June 15, 1985, as amended at 56 and sale of a serial or subserial of a FR 66784, Dec. 26, 1991] veterinary biological product by the Secretary, veterinary biologics licens- PART 115—INSPECTIONS ees or permittees shall: (1) Stop the preparation, distribu- Sec. tion, sale, barter, exchange, shipment, 115.1 Inspections of establishments. or importation of the affected serial(s) 115.2 Inspections of biological products. or subserial(s) of any such veterinary AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, biological product pending further in- 2.80, and 371.4. structions from APHIS. (2) Immediately, but no later than 2 § 115.1 Inspections of establishments. days, send stop distribution and sale (a) Any inspector shall be permitted notifications to any jobbers, whole- to enter any establishment where any salers, dealers, foreign consignees, or biological product is prepared, at any other persons known to have any such

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veterinary biological product in their eign establishment in which the prod- possession, which instruct them to stop ucts are prepared, detailed records of the preparation, distribution, sale, bar- information necessary to give a com- ter, exchange, shipment, or importa- plete accounting of all the activities tion of any such veterinary biological within each establishment. Such product. All notifications shall be doc- records shall include, but shall not be umented in writing by the licensee or limited to, the items enumerated in permittee. this part. (3) Account for the remaining quan- (1) Records shall be made concur- tity of each serial(s) or subserial(s) of rently with the performance of succes- any such veterinary biological product sive steps in the development and prep- at each location in the distribution aration of biological products, includ- channel known to the manufacturer (licensee) or importer (permittee). ing new products under development. (4) When required by the Adminis- Such records shall include the date and trator, submit complete and accurate where critical, the time that each es- reports of all notifications concerning sential step was taken, the identity stop distribution and sale actions to and quantity of ingredients added or the Animal and Plant Health Inspec- removed at each step, and any gain or tion Service pursuant to § 116.5 of this loss of product from the beginning to subchapter. the end of product preparation. (c) Unless and until the Secretary (2) Records shall be legible and indel- shall otherwise direct, no persons so ible; shall be as detailed as necessary notified shall thereafter sell, barter, or for a clear understanding of each step exchange any such product in any by one experienced in the preparation place under the jurisdiction of the of biological products; and shall be United States or ship or deliver for verified by initials or signature of the shipment any such product in or from person immediately responsible for the any State, Territory, or the District of action taken. Columbia. However, failure to receive (3) Records (other than disposition such notice shall not excuse any person records) required by this part shall be from compliance with the Virus- completed by the licensee or the for- Serum-Toxin Act. eign manufacturer, as the case may be, (Approved by the Office of Management and before any portion of a serial of any Budget under control number 0579–0318) product shall be marketed in the [72 FR 17798, Apr. 10, 2007] United States or exported. (b) In the case of imported products, PART 116—RECORDS AND REPORTS each permittee shall maintain at the permittee’s place of business detailed Sec. and accurate records that are relevant 116.1 Applicability and general consider- to each imported product and that in- ations. clude, but are not limited to, importa- 116.2 Inventory and disposition records. tion documents, sampling records, test 116.3 Label records. 116.4 Sterilization and pasteurization summaries, shipping records, and in- records. ventory and disposition records as re- 116.5 Reports. quired in § 116.2. 116.6 Animal records. (c) When authorized by the Adminis- 116.7 Test records. trator, the licensee, permittee, or for- 116.8 Completion and retention of records. eign manufacturer may maintain and AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, retain records required under this part 2.80, and 371.4. at an alternative location. Such au- § 116.1 Applicability and general con- thorization shall be confirmed by the siderations. filing of an addendum to the plot plan legend. The addendum shall list the lo- (a) Each licensee, permittee, and foreign manufacturer of biological prod- cation of the records and the condition ucts imported into the United States of their storage and shall permit the shall maintain, at the licensed or for-

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inspection of the records by APHIS in- § 116.4 Sterilization and pasteurization spectors, or foreign inspectors acting records. on behalf of APHIS. Records shall be made by means of (Approved by the Office of Management and automatic recording devices or an Budget under control number 0579–0013) equivalent accurate and reliable system. Such records shall be identified [39 FR 16872, May 10, 1974, as amended at 48 with the ingredients, equipment, or bi- FR 57473, Dec. 30, 1983; 61 FR 52874, Oct. 9, ological product subjected to steriliza- 1996; 66 FR 21064, Apr. 27, 2001] tion or pasteurization. § 116.2 Inventory and disposition (Approved by the Office of Management and records. Budget under control number 0579–0013) (a) Records shall show the quantity [39 FR 16872, May 10, 1974, as amended at 48 and location of each biological product FR 57473, Dec. 30, 1983; 61 FR 52874, Oct. 9, being prepared, in storage, and in dis- 1996; 66 FR 21064, Apr. 27, 2001] tribution channels. § 116.5 Reports. (b) Detailed disposition records, in a (a) When required by the Adminis- form satisfactory to the Adminis- trator, reports containing accurate and trator, shall be maintained by each li- complete information concerning bio- censee, each distributor, and each per- logical products, including but not lim- mittee showing the sale, shipment, or ited to, product development and prep- other disposition made of the biologi- aration, and market suspensions and cal products handled by such person. recalls, shall be prepared and sub- (Approved by the Office of Management and mitted to the Animal and Plant Health Budget under control number 0579–0013) Inspection Service by the licensee, permittee, or foreign manufacturer (whose [39 FR 16872, May 10, 1974, as amended at 48 products are being imported or offered FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, for importation). Unless otherwise au- 1991; 61 FR 52874, Oct. 9, 1996; 66 FR 21064, thorized by the Administrator, records Apr. 27, 2001] necessary to make such reports shall § 116.3 Label records. be maintained in each establishment. (b) If, at any time, there are indica- (a) Each licensee and permittee shall tions that raise questions regarding maintain a list of all approved labels the purity, safety, potency, or efficacy currently being used. Each label shall of a product, or if it appears that there be identified as to: may be a problem regarding the prepa- (1) Name and product code number as ration, testing, or distribution of a it appears on the product license or product, the licensee, permittee, or for- permit for the product; eign manufacturer must immediately (2) Where applicable, the size of the notify the Animal and Plant Health In- package (doses, ml, cc, or units) on spection Service concerning the cir- which the label shall be used; cumstances and the action taken, if (3) Label number and date assigned; any. Notification may be made by mail and to Director, Center for Veterinary Bio- logics, Inspection and Compliance, 1920 (4) Name of licensee or subsidiary ap- Dayton Avenue, P.O. Box 844, Ames, IA pearing on the label as the producer. 50010; by electronic mail to (b) All labels printed shall be ac- ([email protected]); by fax to (515) 337- counted for and an inventory main- 6120; or by telephone to (515) 337-6100. tained. (Approved by the Office of Management and Records shall include the disposition of Budget under control number 0579–0013) such labels including those not used in labeling a product. [61 FR 52874, Oct. 9, 1996, as amended at 64 FR 43045, Aug. 9, 1999; 75 FR 20773, Apr. 21, 2010] (Approved by the Office of Management and Budget under control number 0579–0013) § 116.6 Animal records. [39 FR 16872, May 10, 1974, as amended at 48 Complete records shall be kept for all FR 57473, Dec. 30, 1983; 61 FR 52874, Oct. 9, animals at a licensed establishment. 1996; 66 FR 21064, Apr. 27, 2001] Results of tests performed, antigens or

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treatment administered, maintenance 117.5 Segregation of animals. and production records, disposition 117.6 Removal of animals. records, necropsy records, if any, and AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, all other pertinent records shall be in- 2.80, and 371.4. cluded. SOURCE: 38 FR 15499, June 13, 1973, unless (Approved by the Office of Management and otherwise noted. Budget under control number 0579–0013) [39 FR 16872, May 10, 1974, as amended at 48 § 117.1 Applicability. FR 57473, Dec. 30, 1983; 61 FR 52874, Oct. 9, (a) All animals used in licensed es- 1996; 66 FR 21064, Apr. 27, 2001] tablishments in the preparation or § 116.7 Test records. testing of biological products shall meet the regulations in this subchapter Detailed records of all tests con- and special requirements as may be ducted on each serial and each sub- prescribed by the Administrator to pre- serial shall be maintained by the li- vent the preparation, sale, and dis- censee. Summaries of such tests shall tribution of worthless, contaminated, be prepared from such records and submitted to the Animal and Plant Health dangerous, or harmful biological prod- Inspection Service using APHIS Form ucts. 2008 or an acceptable equivalent form (b) Unless otherwise authorized or di- prior to release of the serial or sub- rected by the Administrator, animals serial. Blank forms for such summaries used in the preparation or testing of bi- shall be available from Animal and ological products shall be admitted to Plant Health Inspection Service upon and maintained at the licensed estab- request. lishment and ultimately disposed of in accordance with the regulations in this (Approved by the Office of Management and part, and with the Act of August 24, Budget under control number 0579–0013) 1966 (Pub. L. 89–544) as amended by the [39 FR 16872, May 10, 1974, as amended at 48 Animal Welfare Act of 1970 (Pub. L. 91– FR 57473, Dec. 30, 1983; 56 FR 66784, Dec. 26, 579) and the regulations in parts 1, 2, 1991; 61 FR 52874, Oct. 9, 1996] and 3 of this chapter. Personnel who supervise the care and welfare of such § 116.8 Completion and retention of records. animals shall be qualified by education, training, and experience to All records (other than disposition carry out the regulations in this part. records) required by this part shall be completed by the licensee, permittee, [38 FR 15499, June 13, 1973, as amended at 56 or foreign manufacturer before any FR 66784, Dec. 26, 1991] portion of a serial of any product may be marketed in the United States or § 117.2 Animal facilities. exported. All records shall be retained Animal facilities shall comply with at the licensed or foreign establish- the requirements provided in part 108 ment or permittee’s place of business of this chapter. for a period of two years after the expiration date of a product, or for such § 117.3 Admittance of animals. longer period as may be required by the (a) No animal which shows clinical Administrator. signs or other evidence of disease shall (Approved by the Office of Management and be admitted to the premises of licensed Budget under control number 0579–0013) establishments, except as provided in [61 FR 52874, Oct. 9, 1996] paragraphs (d) and (e) of this section. The health status of all animals offered PART 117—ANIMALS AT LICENSED for admission shall be determined by or under the direction of a veterinarian ESTABLISHMENTS prior to admission. If the determination cannot be made prior to admis- Sec. 117.1 Applicability. sion, the animals shall be kept sepa- 117.2 Animal facilities. rate from animals already on the prem- 117.3 Admittance of animals. ises and in a quarantine area to be pro- 117.4 Test animals. vided by the licensee for this purpose

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until the animal’s health status is de- logical product or otherwise, shall be termined. administered to a test animal during a (b) If special test requirements for test period which could interfere with a admittance of the animals are specified true evaluation of the biological prod- in the Outline of Production for the uct being tested. product to be produced, the animals (d) During the course of a test, ani- shall remain in the quarantine area mals that are injured or show clinical until such tests have been performed signs of illness or unfavorable reac- and the results obtained. Animals tions that are not due to the test may which do not meet the requirements be removed from the test and treated shall not be admitted to the production or humanely destroyed. If sufficient area or allowed to contact production animals do not remain for the test to animals. be evaluated, the test shall be declared (c) All animals admitted to the prem- inconclusive and may be repeated. ises of a licensed establishment shall be permanently identified either col- (e) Test animals that show clinical lectively or individually by the li- signs of illness that are due to the test censee with tags, marks, or other may be treated or humanely destroyed means acceptable to the Adminis- if the illness has progressed to a point trator. (defined in the filed Outline of Produc- (d) When an animal which has a dis- tion) when death is certain to occur ease is to be used to prepare a biologi- without therapeutic intervention. cal product for control of such disease, When interpreting the results of the the animal shall be admitted directly test, the animals that were treated or to the processing facilities in which the humanely destroyed because of illness product is to be prepared but shall not due to the test and the animals that be permitted contact with other ani- have died from illness due to the test mals on the premises. prior to being humanely destroyed (e) The Administrator may authorize shall be combined into a common sta- the maintenance of diagnostic facili- tistic of mortality due to the test. ties at the licensed establishment: Pro- vided, That safeguards proposed by the [38 FR 15499, June 13, 1973, as amended at 60 FR 43356, Aug. 21, 1995] licensee are adequate to prevent diseased or dead animals brought into § 117.5 Segregation of animals. such facilities from being a threat to biological products prepared in such es- Animals which have been infected tablishment or to other animals on the with or exposed to a dangerous, infec- premises used in the preparation of bio- tious, contagious, or communicable logical products. disease shall be kept effectively seg- regated at a licensed establishment [38 FR 15499, June 13, 1973, as amended at 56 until such time as they are humanely FR 66784, Dec. 26, 1991] destroyed or successfully treated and § 117.4 Test animals. removed as healthy animals. (a) All test animals shall be exam- § 117.6 Removal of animals. ined for clinical signs of illness, injury, or abnormal behavior prior to the start Production animals or ex-test ani- of a test and throughout the observa- mals which are no longer useful at the tion period specified in the test pro- licensed establishment may be re- tocol. moved from the premises of the li- (b) All animals used for test purposes censed establishment; provided, such shall be identified either collectively removal is accomplished in a manner or individually in a manner conducive as shall preclude the dissemination of to an accurate interpretation of the re- disease and in accordance with the fol- sults of the test. lowing conditions: (c) No test animals shall be given a (a) Meat-producing animals which re- biological product during the precondi- ceived a biological product containing tioning period which would affect its inactivated microorganisms and adju- eligibility according to the test re- vants within 21 days shall not be requirements. No treatment, with a bio- moved; or

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(b) Animals which received virulent which it is located when so detained, microorganisms within 30 days shall until released by such representative. not be removed; or [52 FR 30135, Aug. 13, 1987, as amended at 56 (c) Only animals that are in a FR 66784, Dec. 26, 1991] healthy condition as determined by a veterinarian shall be removed, except § 118.2 Method of detention; Notifica- as provided in paragraph (d) of this sections. tion. An authorized representative of the (d) Other animals that are injured or Administrator shall detain any biologi- otherwise unhealthy, except when af- cal product subject to detention under fected with a communicable disease, this part by: may be removed for immediate slaugh- (a) Giving oral notification to the ter to an abattoir operated in accord- owner of the biological product if such ance with the Federal Meat Inspection owner can be ascertained, and, if not, Act of March 4, 1907, 34 Stat. 1260, as to the agent representing the owner or amended by the Wholesome Meat Act to the immediate custodian of the bio- of 1967, 81 Stat. 585 (21 U.S.C. sec. 601 et logical product; and seq.): Provided, That such animals shall (b) Promptly furnishing the person so be properly marked for identification notified with a preliminary notice of and the inspector in charge of slaugh- detention which shall include identity ter operations is given due notice in and quantity of the product detained, advance. the location where detained, the reason (e) All animals on the premises shall for the detention, and the name of the be disposed of in accordance with the authorized representative of the Ad- provisions of the regulations in this ministrator. part and where specific provision is not (c) Within 48 hours after the deten- made therefor shall be disposed of as tion of any biological product, an au- required by the Administrator. thorized representative of the Adminis- trator shall, if the detention is to con- [38 FR 15499, June 13, 1973, as amended at 56 tinue, give written notification to the FR 66784, Dec. 26, 1991] owner of the biological product detained by furnishing a written state- PART 118—DETENTION; SEIZURE ment which shall include the identity AND CONDEMNATION and quantity of the product detained, the location where detained, specific Sec. description of the alleged noncompli- 118.1 Administrative detention. ance including reference to the provi- 118.2 Method of detention; Notifications. sions in the Act or the regulations 118.3 Movement of detained biological prod- which have resulted in the detention, ucts; Termination of detention. and the identity of the authorized rep- 118.4 Seizure and condemnation. resentative of the Administrator; or, if such owner cannot be ascertained and AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, notified within such period of time, 2.80, and 371.4. furnish such notice to the agent rep- SOURCE: 52 FR 30135, Aug. 13, 1987, unless resenting such owner, or the carrier or otherwise noted. other person having custody of the biological product detained. The notifica- § 118.1 Administrative detention. tion, with a copy of the preliminary Whenever any biological product notice of detention shall be served by which is prepared, sold, bartered, ex- either delivering the notification to changed, or shipped in violation of the the owner or to the agent or to such Act or regulations is found by any au- other person, or by certifying and mail- thorized representative of the Adminis- ing the notification, addressed to such trator upon any premises, it may be de- owner, agent, or other person, at the tained by such representative for a pe- last known residence or principal office riod not to exceed 20 days, pending ac- or place of business. tion under § 118.4, and shall not be [52 FR 30135, Aug. 13, 1987, as amended at 56 moved by any person from the place at FR 66784, Dec. 26, 1991]

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§ 118.3 Movement of detained biologi- States, but the product shall not be cal products; Termination of deten- sold contrary to the provisions of the tion. Act or the laws of the jurisdiction in Except as provided in paragraphs (a) which it is sold; Provided, That, upon and (b) of this section, no biological the execution and delivery of a good product detained in accordance with and sufficient bond conditioned that the provisions in this part shall be the product shall not be sold or other- moved by any person from the place at wise disposed of contrary to the provi- which such product is located when it sions of the Act or the laws or jurisdic- is detained. tion in which disposal is made, the (a) A detained biological product court may direct that such product be may be moved from the place at which delivered to the owner thereof subject it is located when so detained for the to such supervision by authorized rep- purpose of providing proper storage resentatives of the Administrator as is conditions if such movement has been necessary to ensure compliance with approved by an authorized representa- the applicable laws. When a decree of tive of the Administrator; Provided, condemnation is entered against the That, the biological product so moved product and it is released under bond, shall be detained by an authorized rep- or destroyed, court costs and fees, and resentative of the Administrator after storage and other proper expenses shall such movement. be awarded against the person, if any, (b) A detained biological product intervening as claimant of the product. may be moved from the place at which The proceedings in such libel cases it is detained on written notification shall conform, as nearly as may be by an authorized representative of the practicable, to the proceedings in ad- Administrator that the detention is miralty, except that either party may terminated; Provided, That, the condi- demand trial by jury of any issue of tions under which the detained biologi- fact joined in any case, and all such cal product may be moved will be spec- proceedings shall be at the suit of and ified in the written notification of the in the name of the United States. termination. The notification of termination shall be served by either person- [52 FR 30135, Aug. 13, 1987, as amended at 56 FR 66784, Dec. 26, 1991] ally delivering the notification, or by certifying and mailing the notification addressed to such person at the last PART 121—POSSESSION, USE, AND known residence or principal office or TRANSFER OF SELECT AGENTS place of business of the owner, agent, AND TOXINS or other person having custody of the biological product. Sec. 121.1 Definitions. [52 FR 30135, Aug. 13, 1987, as amended at 56 121.2 Purpose and scope. FR 66784, Dec. 26, 1991] 121.3 VS select agents and toxins. 121.4 Overlap select agents and toxins. § 118.4 Seizure and condemnation. 121.5 Exemptions for VS select agents and Any biological product which is pre- toxins. pared, sold, bartered, exchanged, or 121.6 Exemptions for overlap select agents shipped in violation of the Act or regu- and toxins. lations shall be liable to be proceeded 121.7 Registration and related security risk assessments. against and seized and condemned, at 121.8 Denial, revocation, or suspension of any time, on a libel of information in registration. any United States district court or 121.9 Responsible official. other proper court within the jurisdic- 121.10 Restricting access to select agents tion of which the product is found. If and toxins; security risk assessments. the product is condemned, it shall, 121.11 Security. after entry of the decree, be disposed of 121.12 Biosafety. by destruction or sale as the court may 121.13 Restricted experiments. 121.14 Incident response. direct, and the proceeds, if sold, less 121.15 Training. the court costs and fees, and storage 121.16 Transfers. and other proper expenses, shall be 121.17 Records. paid into the Treasury of the United 121.18 Inspections.

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121.19 Notification of theft, loss, or release. HHS select agent and/or toxin. A bio- 121.20 Administrative review. logical agent or toxin listed in 42 CFR AUTHORITY: 7 U.S.C. 8401; 7 CFR 2.22, 2.80, 73.3. and 371.4. Import. To move into, or the act of movement into, the territorial limits SOURCE: 70 FR 13284, Mar. 18, 2005, unless otherwise noted. of the United States. Information security. Protecting infor- § 121.1 Definitions. mation and information systems from Administrator. The Administrator, unauthorized access, use, disclosure, Animal and Plant Health Inspection disruption, modification, or destruc- Service, or any person authorized to tion in order to provide: act for the Administrator. (1) Integrity, which means guarding Animal and Plant Health Inspection against improper information modi- Service (APHIS). The Animal and Plant fication or destruction, and includes Health Inspection Service of the U.S. ensuring information authenticity; Department of Agriculture. (2) Confidentiality, which means pre- Attorney General. The Attorney Gen- serving authorized restrictions on ac- eral of the United States or any person cess and disclosure, including means authorized to act for the Attorney Gen- for protecting personal privacy and eral. proprietary information; and Biological agent. Any microorganism (3) Availability, which means ensuring (including, but not limited to, bacteria, timely and reliable access to and use of viruses, fungi, or protozoa), or infec- information. tious substance, or any naturally oc- Interstate. From one State into or curring, bioengineered, or synthesized through any other State, or within the component of any such microorganism District of Columbia, Guam, the Virgin or infectious substance, capable of Islands of the United States, or any causing: other territory or possession of the (1) Death, disease, or other biological United States. malfunction in a human, an animal, a Occupational exposure. Any reason- plant, or another living organism; ably anticipated skin, eye, mucous (2) Deterioration of food, water, membrane, parenteral contact, or res- equipment, supplies, or material of any piratory aerosol exposure to select kind; or agents or toxins that may result from (3) Deleterious alteration of the envi- the performance of an employee’s du- ronment. ties. Centers for Disease Control and Preven- Overlap select agent and/or toxin. A bi- tion (CDC). The Centers for Disease ological agent or toxin that is listed in Control and Prevention of the U.S. De- § 121.4 and 42 CFR 73.4. partment of Health and Human Serv- Permit. A written authorization by ices. the Administrator to import or move Diagnosis. The analysis of specimens interstate select agents or toxins, for the purpose of identifying or con- under conditions prescribed by the Ad- firming the presence or characteristics ministrator. of a select agent or toxin, provided Principal investigator. The one indi- that such analysis is directly related to vidual who is designated by the entity protecting the public health or safety, to direct a project or program and who animal health or animal products, or is responsible to the entity for the sci- plant health or plant products. entific and technical direction of that Entity. Any government agency (Fed- project or program. eral, State, or local), academic institu- Proficiency testing. The process of de- tion, corporation, company, partner- termining the competency of an indi- ship, society, association, firm, sole vidual or laboratory to perform a speci- proprietorship, or other legal entity. fied test or procedure. HHS Secretary. The Secretary of the Recombinant nucleic acids. (1) Mol- Department of Health and Human ecules that are constructed by joining Services or his or her designee, unless nucleic acid molecules and that can otherwise specified. replicate in a living cell; or

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(2) Molecules that result from the non-viable but allows the select agent replication of those described in para- to retain characteristics of interest for graph (1) of this definition. future use; or to render any nucleic Responsible official. The individual acids that can produce infectious forms designated by an entity with the au- of any select agent virus non-infectious thority and control to ensure compli- for future use. ance with the regulations in this part. Verification. The demonstration of ob- Security barrier. A physical structure taining established performance (e.g., that is designed to prevent entry by accuracy, precision, and the analytical unauthorized persons. sensitivity and specificity) specifica- Select agent and/or toxin. Unless other- tions for any procedure used for diag- wise specified, all of the biological nosis. agents or toxins listed in §§ 121.3 and Viability testing protocol. A protocol to 121.4. confirm the validated inactivation pro- Specimen. Samples of material from cedure by demonstrating the material humans, animals, plants, or the envi- is free of all viable select agent. ronment, or isolates or cultures from VS. The Veterinary Services Pro- such samples, for diagnosis, grams of the Animal and Plant Health verification, or proficiency testing. Inspection Service. State. Any of the several States of the VS select agent and/or toxin. A biologi- United States, the Commonwealth of cal agent or toxin listed in § 121.3. the Northern Mariana Islands, the Commonwealth of Puerto Rico, the [70 FR 13284, Mar. 18, 2005, as amended at 77 District of Columbia, Guam, the Virgin FR 61077, Oct. 5, 2012; 82 FR 6206, Jan. 19, Islands of the United States, or any 2017] other territory or possession of the United States. § 121.2 Purpose and scope. Synthetic nucleic acids. (1) Molecules This part implements the provisions that are chemically or by other means of the Agricultural Bioterrorism Pro- synthesized or amplified, including tection Act of 2002 setting forth the re- those that are chemically or otherwise quirements for possession, use, and modified but can base pair with natu- transfer of select agents and toxins. rally occurring nucleic acid molecules The biological agents and toxins listed (i.e., synthetic nucleic acids); or in this part have the potential to pose (2) Molecules that result from the a severe threat to public health and replication of those described in para- safety, to animal health, or to animal graph (1) of this definition. products. Overlap select agents and Toxin. The toxic material or product toxins are subject to regulation by of plants, animals, microorganisms (in- both APHIS and CDC. cluding, but not limited to, bacteria, viruses, fungi, or protozoa), or infec- § 121.3 VS select agents and toxins. tious substances, or a recombinant or synthesized molecule, whatever their (a) Except as provided in paragraphs origin and method of production, and (d) and (e) of this section, the Adminis- includes: trator has determined that the biologi- (1) Any poisonous substance or bio- cal agents and toxins listed in this sec- logical product that may be engineered tion have the potential to pose a severe as a result of biotechnology produced threat to animal health or to animal by a living organism; or products. The select agents and toxins (2) Any poisonous isomer or biologi- marked with an asterisk (*) are des- cal product, homolog, or derivative of ignated as Tier 1 select agents and tox- such a substance. ins and are subject to additional re- United States. All of the States. quirements as listed in this part. USDA. The U.S. Department of Agri- (b) VS select agents and toxins: Afri- culture. can horse sickness virus; African swine Validated inactivation procedure. A fever virus; Avian influenza virus; Clas- procedure, whose efficacy is confirmed sical swine fever virus; *Foot-and- by data generated from a viability test- mouth disease virus; Goat pox virus; ing protocol, to render a select agent Lumpy skin disease virus; Mycoplasma

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capricolum; Mycoplasma mycoides; New- (4) A select agent or regulated nu- castle disease virus; 1 Peste des petits cleic acids that can produce infectious ruminants virus; *Rinderpest virus; forms of any select agent virus that Sheep pox virus; Swine vesicular dis- has been subjected to a validated inac- ease virus. tivation procedure that is confirmed (c) Genetic elements, recombinant through a viability testing protocol. and/or synthetic nucleic acids, and re- Surrogate strains that are known to combinant and/or synthetic organisms: possess equivalent properties with re- (1) Nucleic acids that can produce inspect to inactivation can be used to fectious forms of any of the select validate an inactivation procedure; agent viruses listed in paragraph (b) of however, if there are known strain-to- 2 this section. strain variations in the resistance of a (2) Recombinant and/or synthetic nu- select agent to an inactivation proce- cleic acids that encode for the func- dure, then an inactivation procedure tional forms of any toxin listed in validated on a lesser resistant strain paragraph (b) of this section if the nucleic acids: must also be validated on the more re- (i) Can be expressed in vivo or in vitro; sistant strains. or (5) Material containing a select agent (ii) Are in a vector or recombinant that is subjected to a procedure that host genome and can be expressed in removes all viable select agent cells, vivo or in vitro. spores, or virus particles if the mate- (3) VS select agents and toxins listed rial is subjected to a viability testing in paragraph (b) of this section that protocol to ensure that the removal have been genetically modified. method has rendered the material free (d) VS select agents or toxins that of all viable select agent. meet any of the following criteria are (6) A select agent or regulated nu- excluded from the requirements of this cleic acids that can produce infectious part: forms of any select agent virus not sub- (1) Any VS select agent or toxin that jected to a validated inactivation pro- is in its naturally occurring environ- cedure or material containing a select ment, provided that the agent or toxin agent not subjected to a procedure that has not been intentionally introduced, removes all viable select agent cells, cultivated, collected, or otherwise ex- spores, or virus particles if the mate- tracted from its natural source. rial is determined by the Adminis- (2) Nonviable VS select agents or trator to be effectively inactivated or nontoxic VS toxins.3 effectively removed. To apply for a de- (3) A select agent or toxin that has termination an individual or entity been subjected to decontamination or a must submit a written request and sup- destruction procedure when intended porting scientific information to for waste disposal. APHIS. A written decision granting or denying the request will be issued. 1 A virulent Newcastle disease virus (avian (7) A VS select toxin identified in an paramyxovirus serotype 1) has an original food sample or clinical sample. intracerebral pathogenicity index in day-old chicks (Gallus gallus) of 0.7 or greater or has (8) Waste generated during the deliv- an amino acid sequence at the fusion (F) pro- ery of patient care by health care protein cleavage site that is consistent with vir- fessionals from a patient diagnosed ulent strains of Newcastle disease virus. A with an illness or condition associated failure to detect a cleavage site that is con- with a select agent, where that waste sistent with virulent strains does not con- is decontaminated or transferred for firm the absence of a virulent virus. 2 The importation and interstate move- destruction by complying with State ment of VS select agents or toxins listed in and Federal regulations within 7 cal- paragraphs (c)(1) through (c)(3) of this sec- endar days of the conclusion of patient tion may be subject to the permit require- care. ments under part 122 of this subchapter. (9) Any low pathogenic strains of 3 However, the importation and interstate avian influenza virus, avian movement of these nonviable select agents may be subject to the permit requirements paramyxovirus serotype-1 (APMV–1) vi- under part 122 of this subchapter. ruses which do not meet the criteria

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for Newcastle disease virus,4 including an attenuated strain of a select agent those identified as pigeon or a select toxin modified to be less po- paramyxovirus-12 5 isolated from a non- tent or toxic. The written request for poultry species, all subspecies Myco- reconsideration must state the facts plasma capricolum except subspecies and reasoning upon which the indi- capripneumoniae (contagious caprine vidual or entity relies to show the deci- pleuropneumonia), and all subspecies sion was incorrect. The Administrator Mycoplasma mycoides except subspecies will grant or deny the request for re- mycoides small colony (Mmm SC) (con- consideration as promptly as cir- tagious bovine pleuropneumonia), pro- cumstances allow and will state, in vided that the individual or entity can writing, the reasons for the decision. identify that the agent is within the (f) Any VS select agent or toxin exclusion category. seized by a Federal law enforcement (e) An attenuated strain of a select agency will be excluded from the reagent or a select toxin modified to be quirements of this part during the pe- less potent or toxic may be excluded riod between seizure of the agent or from the requirements of this part toxin and the transfer or destruction of based upon a determination by the Ad- such agent or toxin provided that: ministrator that the attenuated strain or modified toxin does not pose a se- (1) As soon as practicable, the Fed- vere threat to animal health or animal eral law enforcement agency transfers products. the seized agent or toxin to an entity (1) To apply for exclusion, an indi- eligible to receive such agent or toxin vidual or entity must submit a written or destroys the agent or toxin by a rec- request and supporting scientific infor- ognized sterilization or inactivation mation. A written decision granting or process. denying the request will be issued. An (2) The Federal law enforcement exclusion will be effective upon notifi- agency safeguards and secures the cation to the applicant. Exclusions will seized agent or toxin against theft, be listed on the National Select Agent loss, or release, and reports any theft, Registry Web site at http:// loss, or release of such agent or toxin. www.selectagents.gov/. (3) The Federal law enforcement (2) If an excluded attenuated strain agency reports the seizure of the select or modified toxin is subjected to any agent or toxin to APHIS or CDC. manipulation that restores or enhances (i) The seizure of any of the following its virulence or toxic activity, the re- VS select agents and toxins must be resulting select agent or toxin will be ported within 24 hours by telephone, subject to the requirements of this facsimile, or e-mail: African horse part. sickness virus, African swine fever (3) An individual or entity may make virus, avian influenza virus (highly a written request to the Administrator pathogenic), bovine spongiform for reconsideration of a decision deny- encephalopathy agent, classical swine ing an application for the exclusion of fever virus, foot-and-mouth disease virus, virulent Newcastle disease virus, 4 An APMV–1 virus isolated from poultry rinderpest virus, and swine vesicular which has an intracerebral pathogenicity disease virus. This report must be fol- index in day-old chicks (Gallus gallus) of 0.7 lowed by submission of APHIS/CDC or greater or has an amino acid sequence at Form 4 within 7 calendar days after the fusion (F) protein cleavage site that is seizure of the select agent or toxin. consistent with virulent strains of Newcastle disease virus. A failure to detect a cleavage (ii) For all other VS select agents or site that is consistent with virulent strains toxins, APHIS/CDC Form 4 must be does not confirm the absence of a virulent submitted within 7 calendar days after virus. seizure of the agent or toxin. 5 Pigeon paramyxovirus (PPMV–1) is a spe- (iii) A copy of APHIS/CDC Form 4 cies-adapted APMV–1 virus which is endemic must be maintained for 3 years. in pigeons and doves in the United States and can be identified through monoclonal (4) The Federal law enforcement antibody testing and demonstration of their agency reports the final disposition of characteristic amino acid signature at the the select agent or toxin by submission fusion gene cleavage site. of APHIS/CDC Form 4. A copy of the

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completed form must be maintained (1) Any overlap select agent or toxin for 3 years. that is in its naturally occurring environment, provided that the agent or [70 FR 13284, Mar. 18, 2005, as amended at 73 FR 61331, Oct. 16, 2008; 77 FR 61077, Oct. 5, toxin has not been intentionally intro- 2012; 79 FR 26830, May 12, 2014; 82 FR 6207, duced, cultivated, collected, or other- Jan. 19, 2017] wise extracted from its natural source. (2) Nonviable overlap select agents or § 121.4 Overlap select agents and tox- nontoxic overlap toxins.7 ins. (3) A select agent or toxin that has (a) Except as provided in paragraphs been subjected to decontamination or a (d) and (e) of this section, the Adminis- destruction procedure when intended trator has determined that the biologi- for waste disposal. cal agents and toxins listed in this sec- (4) A select agent or regulated nu- tion have the potential to pose a severe cleic acids that can produce infectious threat to public health and safety, to forms of any select agent virus that animal health, or to animal products. has been subjected to a validated inac- The select agents and toxins marked tivation procedure that is confirmed with an asterisk (*) are designated as through a viability testing protocol. Tier 1 select agents and toxins and are Surrogate strains that are known to subject to additional requirements as possess equivalent properties with re- listed in this part. spect to inactivation can be used to (b) Overlap select agents and toxins: validate an inactivation procedure; *Bacillus anthracis; Bacillus anthracis however, if there are known strain-to- (Pasteur strain); Brucella abortus; strain variations in the resistance of a Brucella melitensis; Brucella suis; select agent to an inactivation proce- *Burkholderia mallei; *Burkholderia dure, then an inactivation procedure pseudomallei; Hendra virus; Nipah virus; validated on a lesser resistant strain Rift Valley fever virus; Venezuelan must also be validated on the more re- equine encephalitis virus. sistant strains. (c) Genetic elements, recombinant (5) Material containing a select agent and/or synthetic nucleic acids, and re- that is subjected to a procedure that combinant and/or synthetic organisms: removes all viable select agent cells, (1) Nucleic acids that can produce in- spores, or virus particles if the mate- fectious forms of any of the overlap serial is subjected to a viability testing lect agent viruses listed in paragraph protocol to ensure that the removal (b) of this section. 6 method has rendered the material free (2) Recombinant and/or synthetic nu- of all viable select agent. cleic acids that encode for the toxic (6) A select agent or regulated nu- forms of any overlap toxin listed in cleic acids that can produce infectious paragraph (b) of this section if the nu- forms of any select agent virus not sub- cleic acids: jected to a validated inactivation pro- (i) Can be expressed in vivo or in vitro; cedure or material containing a select or agent not subjected to a procedure that (ii) Are in a vector or recombinant removes all viable select agent cells, host genome and can be expressed in spores, or virus particles if the mate- vivo or in vitro. rial is determined by the Adminis- (3) Overlap select agents and toxins trator or HHS Secretary to be effec- listed in paragraph (b) of this section tively inactivated or effectively re- that have been genetically modified. moved. To apply for a determination (d) Overlap select agents or toxins an individual or entity must submit a that meet any of the following criteria written request and supporting sci- are excluded from the requirements of entific information to APHIS or CDC. this part: A written decision granting or denying the request will be issued. 6 The importation and interstate movement of overlap select agents or toxins listed 7 However, the importation and interstate in paragraphs (c)(1) through (c)(3) of this sec- movement of these nonviable overlap select tion may be subject to the permit require- agents may be subject to the permit requirements under part 122 of this subchapter. ments under part 122 of this subchapter.

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(7) An overlap select toxin identified cumstances allow and will state, in in an original food sample or clinical writing, the reasons for the decision. sample. (f) Any overlap select agent or toxin (8) Waste generated during the deliv- seized by a Federal law enforcement ery of patient care by health care pro- agency will be excluded from the re- fessionals from a patient diagnosed quirements of this part during the pe- with an illness or condition associated riod between seizure of the agent or with a select agent, where that waste toxin and the transfer or destruction of is decontaminated or transferred for such agent or toxin provided that: destruction by complying with State (1) As soon as practicable, the Fed- and Federal regulations within 7 cal- eral law enforcement agency transfers endar days of the conclusion of patient the seized agent or toxin to an entity care. eligible to receive such agent or toxin (9) Any subtypes of Venezuelan or destroys the agent or toxin by a rec- equine encephalitis virus except for ognized sterilization or inactivation Subtypes IAB or IC, provided that the process. individual or entity can identify that (2) The Federal law enforcement the agent is within the exclusion cat- agency safeguards and secures the egory. seized agent or toxin against theft, (e) An attenuated strain of a select loss, or release, and reports any theft, agent or a select toxin modified to be loss, or release of such agent or toxin. less potent or toxic may be excluded (3) The Federal law enforcement from the requirements of this part agency reports the seizure of the over- based upon a determination by the Ad- lap select agent or toxin to APHIS or ministrator that the attenuated strain CDC. or modified toxin does not pose a se- (i) The seizure of any of the following vere threat to public health and safety, overlap select agents and toxins must animal health, or animal products. be reported within 24 hours by tele- (1) To apply for exclusion, an indi- phone, facsimile, or e-mail: Bacillus vidual or entity must submit a written anthracis, Burkholderia mallei, or request and supporting scientific infor- Burkholderia pseudomallei. This report mation. A written decision granting or must be followed by submission of denying the request will be issued. An APHIS/CDC Form 4 within 7 calendar exclusion will be effective upon notifi- days after seizure of the overlap select cation to the applicant. Exclusions will agent or toxin. be listed on the National Select Agent (ii) For all other overlap select Registry Web site at http:// agents or toxins, APHIS/CDC Form 4 www.selectagents.gov/. must be submitted within 7 calendar (2) If an excluded attenuated strain days after seizure of the agent or toxin. or modified toxin is subjected to any (iii) A copy of APHIS/CDC Form 4 manipulation that restores or enhances must be maintained for 3 years. its virulence or toxic activity, the re- (4) The Federal law enforcement sulting select agent or toxin will be agency reports the final disposition of subject to the requirements of this the overlap select agent or toxin by part. submission of APHIS/CDC Form 4. A (3) An individual or entity may make copy of the completed form must be a written request to the Administrator maintained for 3 years. or HHS Secretary for reconsideration [70 FR 13284, Mar. 18, 2005, as amended at 73 of a decision denying an application for FR 61331, Oct. 16, 2008; 77 FR 61078, Oct. 5, the exclusion of an attenuated strain of 2012; 79 FR 26830, May 12, 2014; 82 FR 6207, a select agent or a select toxin modi- Jan. 19, 2017] fied to be less potent or toxic. The written request for reconsideration § 121.5 Exemptions for VS select must state the facts and reasoning agents and toxins. upon which the individual or entity re- (a) Diagnostic laboratories and other lies to show the decision was incorrect. entities that possess, use, or transfer a The Administrator or HHS Secretary VS select agent or toxin that is con- will grant or deny the request for retained in a specimen presented for di- consideration as promptly as cir- agnosis or verification will be exempt

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from the requirements of this part for (3) The identification of the agent or such agent or toxin contained in the toxin, and its derivative, is reported to specimen, provided that: APHIS or CDC. To report the identi- (1) Unless directed otherwise by the fication of a select agent or toxin, Administrator, within 7 calendar days APHIS/CDC Form 4 must be submitted after identification of the select agent within 90 days of receipt of the agent or toxin, the select agent or toxin is or toxin. A copy of the completed form transferred in accordance with § 121.16 must be maintained for 3 years. or destroyed on-site by a recognized (c) Diagnostic reagents and vaccines sterilization or inactivation process; that are, bear, or contain VS select (2) The agent or toxin is secured agents or toxins that are produced at against theft, loss, or release during USDA diagnostic facilities will be ex- the period between identification of empt from the requirements of this the agent or toxin and transfer or depart. struction of such agent or toxin, and (d) Unless the Administrator by order any theft, loss, or release of such agent determines that additional regulation or toxin is reported; is necessary to protect animal health (3) Unless otherwise directed by the or animal products, products that are, Administrator, the clinical or diag- bear, or contain VS select agents or nostic specimens collected from a pa- toxins will be exempt from the require- tient infected with a select agent are ments of this part if the products have transferred in accordance with § 121.16 been cleared, approved, licensed, or or destroyed on-site by a recognized registered pursuant to: sterilization or inactivation process (1) The Federal Food, Drug, and Cos- within 7 calendar days after delivery of metic Act (21 U.S.C. 301 et seq.); patient care by heath care profes- (2) Section 351 of Public Health Serv- sionals has concluded; and ice Act (42 U.S.C. 262); (4) The identification of the agent or (3) The Virus-Serum-Toxin Act (21 toxin is reported to APHIS or CDC, the U.S.C. 151–159); or specimen provider, and to other appro- (4) The Federal Insecticide, Fun- priate authorities when required by gicide, and Rodenticide Act (7 U.S.C. Federal, State, or local law by tele- 131 et seq.). phone, facsimile, or email. This report (e) The Administrator may exempt must be followed by submission of from the requirements of this part an APHIS/CDC Form 4 to APHIS or CDC experimental product that is, bears, or within 7 calendar days after identifica- contains a VS select agent or toxin if tion. such product is being used in an inves- (b) Diagnostic laboratories and other tigation authorized by any Federal law entities that possess, use, or transfer a and the Administrator determines that VS select agent or toxin that is con- additional regulation under this part is tained in a specimen presented for pro- not necessary to protect animal health ficiency testing will be exempt from or animal products. To apply for an ex- the requirements of this part for such emption, an individual or entity must agent or toxin contained in the speci- submit APHIS/CDC Form 5. A written men, provided that: decision granting or denying the ex- (1) Unless directed otherwise by the emption will be issued. The applicant Administrator, within 90 calendar days must notify APHIS when an authoriza- of receipt, the agent or toxin is trans- tion for an investigation no longer ex- ferred in accordance with § 121.16 or de- ists. This exemption automatically ter- stroyed on-site by a recognized steri- minates when such authorization is no lization or inactivation process; longer in effect. (2) The agent or toxin is secured (f) In addition to the exemptions pro- against theft, loss, or release during vided in paragraphs (a) through (e) of the period between identification of this section, the Administrator may the agent or toxin and transfer or de- grant a specific exemption upon a struction of such agent or toxin, and showing of good cause and upon his or any theft, loss, or release of such agent her determination that such exemption or toxin is reported; and is consistent with protecting animal

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health or animal products. An indi- (4) The identification of the agent or vidual or entity may request in writing toxin is reported to APHIS or CDC, the an exemption from the requirements of specimen provider, and to other appro- this part. If granted, such exemptions priate authorities when required by are valid for a maximum of 3 years; Federal, State, or local law by tele- thereafter, an individual or entity phone, facsimile, or email. This report must request a new exemption. If a re- must be followed by submission of quest for exemption is denied, an indi- APHIS/CDC Form 4 to APHIS or CDC vidual or entity may request reconsid- within 7 calendar days after identifica- eration in writing to the Adminis- tion. trator. The request for reconsideration (b) Clinical or diagnostic laboratories must state all of the facts and reasons and other entities that possess, use, or upon which the individual or entity re- transfer an overlap select agent or lies to show that the exemption was toxin that is contained in a specimen wrongfully denied. The Administrator presented for proficiency testing will will grant or deny the request for re- be exempt from the requirements of consideration as promptly as cir- this part for such agent or toxin con- cumstances allow and will state, in tained in the specimen, provided that: writing, the reasons for the decision. (1) Unless directed otherwise by the [70 FR 13284, Mar. 18, 2005, as amended at 73 Administrator or the HHS Secretary, FR 61331, Oct. 16, 2008; 77 FR 61078, Oct. 5, within 90 days of receipt, the agent or 2012; 79 FR 26831, May 12, 2014; 82 FR 6208, toxin is transferred in accordance with Jan. 19, 2017] § 121.16 or 42 CFR 73.16 or destroyed on- site by a recognized sterilization or in- § 121.6 Exemptions for overlap select agents and toxins. activation process; (2) The agent or toxin is secured (a) Clinical or diagnostic laboratories against theft, loss, or release during and other entities that possess, use, or the period between identification of transfer an overlap select agent or the agent or toxin and transfer or de- toxin that is contained in a specimen struction of such agent or toxin, and presented for diagnosis or verification any theft, loss, or release of such agent will be exempt from the requirements or toxin is reported; and of this part for such agent or toxin con- (3) The identification of the agent or tained in the specimen, provided that: toxin, and its derivative, is reported to (1) Unless directed otherwise by the APHIS or CDC. To report the identi- Administrator, within 7 calendar days fication of an overlap select agent or after identification of the select agent toxin, APHIS/CDC Form 4 must be sub- or toxin, the select agent or toxin is mitted within 90 calendar days of re- transferred in accordance with § 121.16 ceipt of the agent or toxin. A copy of or destroyed on-site by a recognized the completed form must be main- sterilization or inactivation process; tained for 3 years. (2) The agent or toxin is secured against theft, loss, or release during (c) Unless the Administrator by order the period between identification of determines that additional regulation the agent or toxin and transfer or de- of a specific product is necessary to struction of such agent or toxin, and protect animal health or animal prod- any theft, loss, or release of such agent ucts, products that are, bear, or con- or toxin is reported; tain overlap select agents or toxins (3) Unless otherwise directed by the will be exempt from the requirements Administrator or HHS Secretary, the of this part if the products have been clinical or diagnostic specimens col- cleared, approved, licensed, or reg- lected from a patient infected with a istered pursuant to: select agent are transferred in accord- (1) The Federal Food, Drug, and Cos- ance with § 121.16 or destroyed on-site metic Act (21 U.S.C. 301 et seq.); by a recognized sterilization or inac- (2) Section 351 of Public Health Serv- tivation process within 7 calendar days ice Act (42 U.S.C. 262); after delivery of patient care by heath (3) The Virus-Serum-Toxin Act (21 care professionals has concluded; and U.S.C. 151–159); or

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(4) The Federal Insecticide, Fun- § 121.7 Registration and related secu- gicide, and Rodenticide Act (7 U.S.C. rity risk assessments. 131 et seq.). (a) Unless exempted under § 121.5, an (d) After consultation with the HHS individual or entity shall not possess, Secretary, the Administrator may ex- use, or transfer any VS select agent or empt from the requirements of this toxin without a certificate of registra- part an investigational product that is, tion issued by the Administrator. Un- bears, or contains an overlap select less exempted under § 121.6 or 42 CFR agent or toxin if such product is being 73.6, an individual or entity shall not used in an investigation authorized by possess, use, or transfer any overlap se- any Federal law and the Administrator lect agent or toxin without a certifi- determines that additional regulation cate of registration issued by the Ad- under this part is not necessary to pro- ministrator and the HHS Secretary. tect animal health or animal products. (b) As a condition of registration, (1) To apply for an exemption, an in- each entity is required to be in compli- dividual or entity must submit APHIS/ ance with the requirements of this part CDC Form 5. for select agents and toxins listed on the registration regardless of whether (2) The Administrator will make a the entity is in actual possession of the determination regarding an exemption select agent or toxin. With regard to within 14 calendar days after receipt of toxins, the entity registered for posses- the application and notification that sion, use, or transfer of a toxin must be the investigation has been authorized in compliance with the requirements of under a Federal law. A written decision this part regardless of the amount of granting or denying the exemption will toxins currently in its possession. be issued. (c) As a condition of registration, (3) The applicant must notify APHIS each entity must designate an indi- or CDC when an authorization for an vidual to be its responsible official. investigation no longer exists. This ex- While most registrants are likely to be emption automatically terminates entities, in the event that an indi- when such authorization is no longer in vidual applies for and is granted a cer- effect. tificate of registration, the individual (e) If it is necessary to respond to a will be considered the responsible offi- domestic or foreign agricultural emer- cial. gency involving an overlap select agent (d)(1) As a condition of registration, or toxin, the Administrator may ex- the following must be approved by the empt an individual or entity from the Administrator or the HHS Secretary requirements, in whole or in part, of based on a security risk assessment by this part for up to 30 calendar days. the Attorney General: The Administrator may extend the ex- (i) The individual or entity; emption once for an additional 30 days. (ii) The responsible official; and (f) Upon request of the Secretary of (iii) Unless otherwise exempted under Health and Human Services, the Ad- this section, any individual who owns or controls the entity. ministrator may exempt an individual (2) Federal, State, or local govern- or entity from the requirements, in mental agencies, including public ac- whole or in part, of this part for up to credited academic institutions, are ex- 30 calendar days if the Secretary of empt from the security risk assess- Health and Human Services has grant- ments for the entity and the individual ed an exemption for a public health who owns or controls such entity. emergency involving an overlap select (3) An individual will be deemed to agent or toxin. The Administrator may own or control an entity under the fol- extend the exemption once for an addi- lowing conditions: 8 tional 30 days. (i) For a private institution of higher [70 FR 13284, Mar. 18, 2005, as amended at 73 education, an individual will be deemed FR 61331, Oct. 16, 2008; 77 FR 61078, Oct. 5, 2012; 79 FR 26831, May 12, 2014; 82 FR 6208, 8 These conditions may apply to more than Jan. 19, 2017] one individual.

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to own or control the entity if the indi- (g) The issuance of a certificate of vidual is in a managerial or executive registration may be contingent upon capacity with regard to the entity’s se- inspection or submission of additional lect agents or toxins or with regard to information, such as the security plan, the individuals with access to the se- biosafety plan, incident response plan, lect agents or toxins possessed, used, or or any other documents required to be transferred by the entity. prepared under this part. (ii) For entities other than institu- (h) A certificate of registration will tions of higher education, an individual be valid for one physical location (a will be deemed to own or control the room, a building, or a group of build- entity if the individual: ings) where the responsible official will (A) Owns 50 percent or more of the be able to perform the responsibilities entity, or is a holder or owner of 50 per- required in this part, for specific select cent or more of its voting stock; or agents or toxins, and for specific ac- (B) Is in a managerial or executive tivities. capacity with regard to the entity’s se- (i) A certificate of registration may lect agents or toxins or with regard to be amended to reflect changes in cir- the individuals with access to the se- cumstances (e.g., replacement of the lect agents or toxins possessed, used, or responsible official or other personnel transferred by the entity. changes, changes in ownership or con- (4) An entity will be considered to be trol of the entity, changes in the ac- an institution of higher education if it tivities involving any select agents or is an institution of higher education as toxins, or the addition or removal of defined in section 101(a) of the Higher select agents or toxins). Education Act of 1965 (20 U.S.C. (1) Prior to any change, the respon- 1001(a)), or is an organization described sible official must apply for an amend- in 501(c)(3) of the Internal Revenue ment to a certificate of registration by Code of 1986, as amended (26 U.S.C. submitting the relevant page(s) of the 501(c)(3)). registration application. 9 (5) To obtain a security risk assess- (2) The responsible official will be no- ment, an individual or entity must sub- tified in writing if an application to mit the information necessary to con- amend a certificate of registration has duct a security risk assessment to the been approved. Approval of an amend- Attorney General. ment may be contingent upon an in- (e) To apply for a certificate of reg- spection or submission of additional in- istration for only VS select agents or formation, such as the security plan, toxins, or for VS and PPQ select agents biosafety plan, incident response plan, or toxins, an individual or entity must or any other documents required to be submit the information requested in prepared under this part. the registration application package (3) No change may be made without (APHIS/CDC Form 1) to APHIS. To such approval. apply for a certificate of registration (j) An entity must immediately no- for overlap select agents or toxins, tify APHIS or CDC if it loses the serv- overlap select agents or toxins and any ices of its responsible official. In the combination of PPQ or VS select event that an entity loses the services agents or toxins, or HHS select agents of its responsible official, an entity or toxins and any combination of PPQ may continue to possess or use select or VS select agents or toxins, an indi- agents or toxins only if it appoints as vidual or entity must submit the infor- the responsible official another indi- mation requested in the registration vidual who has been approved by the application package (APHIS/CDC Form Administrator or the HHS Secretary 1) to APHIS or CDC, but not both. following a security risk assessment by (f) Prior to the issuance of a certificate of registration, the responsible of- 9 ficial must promptly provide notifica- Depending on the change, a security risk assessment by the Attorney General may tion of any changes to the application also be required (e.g., replacement of the re- for registration by submitting the rel- sponsible official, changes in ownership or evant page(s) of the registration appli- control of the entity, new researchers or cation. graduate students, etc.)

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the Attorney General and who meets (3) Comply with all disposition in- the requirements of this part. structions issued by the Administrator (k) A certificate of registration will for each select agent or toxin covered be terminated upon the written request by the revocation or suspension. of the entity if the entity no longer (c) Denial of an application for reg- possesses or uses any select agents or istration and revocation of registration toxins and no longer wishes to be reg- may be appealed under § 121.20. How- istered. ever, any denial of an application for (l) A certificate of registration will registration or revocation of a certifi- be valid for a maximum of 3 years. cate of registration will remain in effect until a final agency decision has [70 FR 13284, Mar. 18, 2005, as amended at 73 been rendered. FR 61331, Oct. 16, 2008; 82 FR 6208, Jan. 19, 2017] [70 FR 13284, Mar. 18, 2005, as amended at 73 FR 61331, Oct. 16, 2008; 82 FR 6208, Jan. 19, § 121.8 Denial, revocation, or suspen- 2017] sion of registration. (a) An application may be denied or a § 121.9 Responsible official. certificate of registration revoked or (a) An individual or entity required suspended if: to register under this part must des- (1) The individual or entity, the re- ignate an individual to be the respon- sponsible official, or an individual who sible official. The responsible official owns or controls the entity is within must. any of the categories described in 18 (1) Be approved by the Administrator U.S.C. 175b; or the HHS Secretary following a secu- (2) The individual or entity, the re- rity risk assessment by the Attorney sponsible official, or an individual who General. owns or controls the entity is reason- (2) Be familiar with the requirements ably suspected by any Federal law en- of this part. forcement or intelligence agency of: (3) Have authority and responsibility (i) Committing a crime set forth in 18 to act on behalf of the entity. U.S.C. 2332b(g)(5); or (4) Ensure compliance with the re- (ii) Knowing involvement with an or- quirements of this part. ganization that engages in domestic or (5) Have a physical (and not merely a international terrorism (as defined in telephonic or audio/visual) presence at 18 U.S.C. 2331) or with any other orga- the registered entity to ensure that the nization that engages in intentional entity is in compliance with the select crimes of violence; or agent regulations and be able to re- (iii) Being an agent of a foreign spond in a timely manner to onsite in- power as defined in 50 U.S.C. 1801; cidents involving select agents and tox- (3) The individual or entity does not ins in accordance with the entity’s in- meet the requirements of this part; 10 cident response plan. or (6) Ensure that annual inspections (4) It is determined that such action are conducted for each registered space is necessary to protect animal health where select agents or toxins are or animal products. stored or used in order to determine (b) Upon revocation or suspension of compliance with the requirements of a certificate of registration, the indi- this part. The results of each inspec- vidual or entity must: tion must be documented, and any defi- (1) Immediately stop all use of each ciencies identified during an inspection select agent or toxin covered by the must be corrected and the corrections revocation or suspension order; documented. (2) Immediately safeguard and secure (7) Ensure that individuals are pro- each select agent or toxin covered by vided the contact information for the the revocation or suspension order USDA Office of Inspector General Hot- from theft, loss, or release; and line and the HHS Office of Inspector General Hotline so that they may 10 If registration is denied for this reason, anonymously report any biosafety/bio- we may provide technical assistance and containment or security concerns re- guidance. lated to select agents and toxins.

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(8) Investigate to determine the rea- identification. A copy of the completed son for any failure of a validated inac- form must be maintained for 3 years. tivation procedure or any failure to re- (2) To report the identification and move viable select agent from mate- final disposition of any other select rial. If the responsible official is unable agent or toxin, APHIS/CDC Form 4 to determine the cause of a deviation must be submitted within 7 calendar from a validated inactivation proce- days after identification. A copy of the dure or a viable select agent removal completed form must be maintained method; or receives any report of any for 3 years. inactivation failure after the move- (3) Less stringent reporting may be ment of material to another location, required during agricultural emer- the responsible official must report im- gencies or outbreaks, or in endemic mediately by telephone or email the areas. inactivation or viable agent removal (d) The responsible official must re- method failure to APHIS or CDC. port the identification and final dis- (9) Review, and revise as necessary, position of any select agent or toxin each of the entity’s validated inactiva- contained in a specimen presented for tion procedures or viable select agent proficiency testing. To report the iden- removal methods. The review must be tification and final disposition of a se- conducted annually or after any lect agent or toxin, APHIS/CDC Form 4 change in principal investigator, must be submitted within 90 calendar change in the validated inactivation days of receipt of the agent or toxin. A procedure or viable select agent re- copy of the completed form must be moval method, or failure of the vali- maintained for 3 years. dated inactivation procedure or viable [70 FR 13284, Mar. 18, 2005, as amended at 73 select agent removal method. The re- FR 61332, Oct. 16, 2008; 77 FR 61078, Oct. 5, view must be documented and training 2012; 79 FR 26831, May 12, 2014; 82 FR 6208, must be conducted if there are any Jan. 19, 2017] changes to the validated inactivation procedure, viable select agent removal § 121.10 Restricting access to select method, or viability testing protocol. agents and toxins; security risk as- (b) An entity may designate one or sessments. more individuals to serve as an alter- (a) An individual or entity required nate responsible official who acts for to register under this part may not the responsible official in his/her ab- provide an individual access to a select sence. These individuals must have the agent or toxin, and an individual may authority and control to ensure com- not access a select agent or toxin, un- pliance with the regulations when act- less the individual is approved by the ing as the responsible official. Administrator or the HHS Secretary (c) The responsible official must re- following a security risk assessment by port the identification and final dis- the Attorney General. position of any select agent or toxin (b) An individual will be deemed to contained in a specimen presented for have access at any point in time if the diagnosis or verification. individual has possession of a select (1) The identification of any of the agent or toxin (e.g., carries, uses, or following select agents or toxins must manipulates) or the ability to gain pos- be immediately reported by telephone, session of a select agent or toxin. facsimile, or email: African horse sick- (c) Each individual with access to se- ness virus, African swine fever virus, lect agents or toxins must have the ap- avian influenza virus (highly patho- propriate education, training, and/or genic), Bacillus anthracis, Burkholderia experience to handle or use such agents mallei, Burkholderia pseudomallei, clas- or toxins. sical swine fever virus, foot-and-mouth (d) To apply for access approval, each disease virus, virulent Newcastle dis- individual must submit the informa- ease virus, rinderpest virus, or swine tion necessary to conduct a security vesicular disease virus. The final dis- risk assessment to the Attorney Gen- position of the agent or toxin must be eral. reported by submission of APHIS/CDC (e) A person with valid approval from Form 4 within 7 calendar days after the HHS Secretary or Administrator to

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have access to select agents or toxins or toxins is terminated by the entity may request, through his or her Re- and the reasons therefore. sponsible Official, that the HHS Sec- [70 FR 13284, Mar. 18, 2005, as amended at 77 retary or Administrator provide their FR 61079, Oct. 5, 2012; 82 FR 6209, Jan. 19, approved access status to another reg- 2017] istered individual or entity for a specified period of time. A responsible offi- § 121.11 Security. cial must immediately notify the re- (a) An individual or entity required sponsible official of the visited entity to register under this part must de- if the person’s access to select agents velop and implement a written security and toxins has been terminated. plan. The security plan must be suffi- (f) An individual’s security risk as- cient to safeguard the select agent or sessment may be expedited upon writ- toxin against unauthorized access, ten request by the responsible official theft, loss, or release. and a showing of good cause (e.g., pub- (b) The security plan must be de- lic health or agricultural emergencies, signed according to a site-specific risk national security, or a short-term visit assessment and must provide graded by a prominent researcher). A written protection in accordance with the risk decision granting or denying the re- of the select agent or toxin, given its quest will be issued. intended use. A current security plan (g) An individual’s access approval must be submitted for initial registra- for VS select agents or toxins may be tion, renewal of registration, or when denied, limited, or revoked if: requested. (1) The individual is within any of (c) The security plan must: the categories described in 18 U.S.C. (1) Describe procedures for physical 175b; security, inventory control, and infor- (2) The individual is reasonably sus- mation systems control; pected by any Federal law enforcement (2) Contain provisions for the control or intelligence agency of committing a of access to select agents and toxins, crime set forth in 18 U.S.C. 2332b(g)(5); including the safeguarding of animals knowing involvement with an organi- (including arthropods) or plants inten- zation that engages in domestic or tionally or accidentally exposed to or international terrorism (as defined in infected with a select agent, against 18 U.S.C. 2331) or with any other orga- unauthorized access, theft, loss or re- nization that engages in intentional lease. crimes of violence; or being an agent of (3) Contain provisions for routine a foreign power as defined in 50 U.S.C. cleaning, maintenance, and repairs; 1801; or (4) Establish procedures for removing (3) It is determined that such action unauthorized or suspicious persons; is necessary to protect animal health (5) Describe procedures for addressing or animal products. loss or compromise of keys, keycards, (h) For overlap select agents or tox- passwords, combinations, etc. and pro- ins, an individual’s access approval will tocols for changing access permissions be denied or revoked if the individual or locks following staff changes; is within any of the categories de- (6) Contain procedures for reporting scribed in 18 U.S.C. 175b. An individ- unauthorized or suspicious persons or ual’s access approval may be denied, activities, loss or theft of select agents limited, or revoked for the reasons set or toxins, release of select agents or forth in paragraphs (f)(2) through (f)(3) toxins, or alteration of inventory of this section. records; (i) An individual may appeal the Ad- (7) Contain provisions for ensuring ministrator’s decision to deny, limit, that all individuals with access ap- or revoke access approval under proval from the Administrator or the § 121.20. HHS Secretary understand and comply (j) Access approval is valid for a max- with the security procedures. imum of 3 years. (8) Describe procedures for how the (k) The responsible official must im- responsible official will be informed of mediately notify APHIS or CDC when suspicious activity that may be crimi- an individual’s access to select agents nal in nature and related to the entity,

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its personnel, or its select agents or (1) Allow access only to individuals toxins; and describe procedures for how with access approval from the Adminis- the entity will notify the appropriate trator or the HHS Secretary; Federal, State, or local law enforce- (2) Allow individuals not approved for ment agencies of such activity. access by the Administrator or the (9) Contain provisions for informa- HHS Secretary to conduct routine tion security that: cleaning, maintenance, repairs, and (i) Ensure that all external connec- other activities not related to select tions to systems which manage secu- agents or toxins only when continu- rity for the registered space are iso- ously escorted by an approved indi- lated or have controls that permit only vidual if the potential to access to se- authorized and authenticated users; lect agents or toxins exists; (ii) Ensure that authorized and au- (3) Provide for the control of select thenticated users are only granted ac- agents and toxins by requiring freezers, cess to select agent and toxin related refrigerators, cabinets, and other con- information, files, equipment (e.g., tainers where select agents or toxins servers or mass storage devices), and are stored to be secured against unau- applications as necessary to fulfill thorized access (e.g., card access sys- their roles and responsibilities, and tem, lock boxes); that access is modified when the user’s (4) Inspect all suspicious packages before they are brought into or re- roles and responsibilities change or moved from an area where select when their access to select agents and agents or toxins are used or stored; toxins is suspended or revoked; (5) Establish a protocol for intra-en- (iii) Ensure that controls are in place tity transfers under the supervision of that are designed to prevent malicious an individual with access approval code (such as, but not limited to, com- from the Administrator or the HHS puter viruses, worms, spyware) from Secretary, including chain-of-custody compromising the confidentiality, in- documents and provisions for safe- tegrity, or availability of information guarding against theft, loss, or release; systems which manage access to spaces and registered under this part or records as (6) Require that individuals with ac- specified in § 121.17; cess approval from the Administrator (iv) Establish a robust configuration or the HHS Secretary refrain from management practice for information sharing with any other person their systems to include regular patching unique means of accessing a select and updates made to operating systems agent or toxin (e.g., keycards or pass- and individual applications; and words); (v) Establish procedures that provide (7) Require that individuals with ac- backup security measures in the event cess approval from the Administrator that access control systems, surveil- or the HHS Secretary immediately re- lance devices, and/or systems that port any of the following to the respon- manage the requirements of § 121.17 are sible official: rendered inoperable. (i) Any loss or compromise of keys, (10) Contain provisions and policies passwords, combinations, etc.; for shipping, receiving, and storage of (ii) Any suspicious persons or activi- select agents and toxins, including doc- ties; umented procedures for receiving, (iii) Any loss or theft of select agents monitoring, and shipping of all select or toxins; agents and toxins. These provisions (iv) Any release of a select agent or must provide that an entity will prop- toxin; erly secure containers on site and have (v) Any sign that inventory or use a written contingency plan for unex- records for select agents or toxins have pected shipments. been altered or otherwise com- (d) An individual or entity must ad- promised; and here to the following security require- (vi) Any loss of computer, hard drive ments or implement measures to or other data storage device containing achieve an equivalent or greater level information that could be used to gain of security: access to select agents or toxins; and

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(8) Separate areas where select (i) Procedures that will limit access agents and toxins are stored or used to a Tier 1 select agent or toxin to only from the public areas of the building. those individuals who are approved by (e) Entities must conduct complete the HHS Secretary or Administrator inventory audits of all affected select following a security risk assessment by agents and toxins in long-term storage the Attorney General, have had an en- when any of the following occur: tity-conducted pre-access suitability (1) Upon the physical relocation of a assessment, and are subject to the enti- collection or inventory of select agents ty’s procedures for ongoing suitability or toxins for those select agents or tox- assessment; ins in the collection or inventory; (ii) Procedures that limit access to (2) Upon the departure or arrival of a laboratory and storage facilities out- principal investigator for those select side of normal business hours to only agents and toxins under the control of those specifically approved by the re- that principal investigator; or sponsible official or designee; (3) In the event of a theft or loss of a (iii) Procedures for allowing visitors, select agent or toxin, all select agents their property, and vehicles at the and toxins under the control of that entry and exit points to the registered principal investigator. space, or at other designated points of (f) In addition to the requirements entry to the building, facility, or com- contained in paragraphs (c) and (d) of pound that are based on the entity’s this section, the security plan for an site-specific risk assessment; individual or entity possessing a Tier 1 (iv) A minimum of three security select agent or toxin must also: barriers where each security barrier (1) Describe procedures for con- adds to the delay in reaching secured ducting a pre-access suitability assess- areas where select agents and toxins ment of persons who will have access are used or stored. One of the security to a Tier 1 select agent or toxin; barriers must be monitored in such a (2) Describe procedures for how an way as to detect intentional and unin- entity’s responsible official will coordi- tentional circumventing of established nate their efforts with the entity’s access control measures under all con- safety and security professionals to en- ditions (day/night, severe weather, etc.) sure security of Tier 1 select agents The final barrier must limit access to and toxins and share, as appropriate, the select agent or toxin to personnel relevant information; and approved by the HHS Secretary or Ad- (3) Describe procedures for the ongo- ministrator, following a security risk ing assessment of the suitability of assessment by the Attorney General. personnel with access to a Tier 1 select (v) All registered space or areas that agent or toxin. The procedures must reasonably afford access to the reg- include: istered space must be protected by an (i) Self- and peer-reporting of inci- intrusion detection system (IDS) un- dents or conditions that could affect an less physically occupied; individual’s ability to safely have ac- (vi) Personnel monitoring the IDS cess to or work with select agents and must be capable of evaluating and in- toxins, or to safeguard select agents terpreting the alarm and alerting the and toxins from theft, loss, or release; designated security response force or (ii) The training of employees with law enforcement; access to Tier 1 select agents and tox- (vii) For powered access control sys- ins on entity policies and procedures tems, describe procedures to ensure for reporting, evaluation, and correc- that security is maintained in the tive actions concerning the assessment event of the failure of access control of personnel suitability; and systems due to power disruption affect- (iii) The ongoing suitability moni- ing registered space; toring of individuals with access to (viii) The entity must: Tier 1 select agents and toxins. (A) Determine that the response time (4) Entities with Tier 1 select agents for security forces or local police will and toxins must prescribe the following not exceed 15 minutes where the re- security enhancements: sponse time is measured from the time

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of an intrusion alarm, or report of a se- § 121.12 Biosafety. curity incident, to the arrival of the re- (a) An individual or entity required sponders at the first security barrier to register under this part must de- or; velop and implement a written bio- (B) Provide security barriers that are safety plan that is commensurate with sufficient to delay unauthorized access the risk of the select agent or toxin, until the response force arrives in given its intended use.11 The biosafety order to safeguard the select agents plan must contain sufficient informa- and toxins from theft, intentional re- tion and documentation to describe the lease, or unauthorized access. The re- biosafety and containment procedures sponse time is measured from the time for the select agent or toxin, including of an intrusion alarm, or report of a se- any animals (including arthropods) or curity incident, to the arrival of the re- plants intentionally or accidentally ex- sponders at the first security barrier. posed to or infected with a select (5) Entities that possess foot-and- agent. The current biosafety plan must mouth disease virus and rinderpest be submitted for initial registration, virus must have the following addi- renewal of registration, or when re- tional security requirements: quested. The biosafety plan must in- (i) A minimum of four barriers, one clude the following provisions: of which must be a perimeter security (1) The hazardous characteristics of fence or equivalent which is monitored each agent or toxin listed on the enti- 24 hours a day, 7 days a week (24/7) to ty’s registration and the biosafety risk detect the presence of unauthorized associated with laboratory procedures persons, vehicles, materials, or unau- related to the select agent or toxin; thorized activities; (2) Safeguards in place with associ- (ii) Onsite 24/7 armed security re- ated work practices to protect entity sponse force with roving patrol. Re- personnel, the public, and the environ- sponse time must not exceed 5 minutes ment from exposure to the select agent from the time of an intrusion alarm or or toxin including, but not limited to: report of a security incident; Personal protective equipment and (iii) CCTV surveillance with 24/7 mon- other safety equipment; containment itoring and recording; and equipment including, but not limited (iv) Transport vehicle with GPS to, biological safety cabinets, animal tracking designed to serve as a con- caging systems, and centrifuge safety tainment vehicle. containers; and engineering controls (g) In developing a security plan, an and other facility safeguards; individual or entity should consider (3) Written procedures for each vali- the document entitled, ‘‘Security Guid- dated method used for disinfection, de- ance for Select Agent or Toxin Facili- contamination, or destruction, as ap- ties.’’ This document is available on propriate, of all contaminated or pre- the National Select Agent Registry at sumptively contaminated materials in- http://www.selectagents.gov/. cluding, but not limited to: Cultures (h) The plan must be reviewed annu- and other materials related to the ally and revised as necessary. Drills or propagation of select agents or toxins, exercises must be conducted at least items related to the analysis of select annually to test and evaluate the effec- agents and toxins, personal protective tiveness of the plan. The plan must be equipment, animal caging systems and reviewed and revised, as necessary, bedding (if applicable), animal car- after any drill or exercise and after any casses or extracted tissues and fluids incident. Drills or exercises must be (if applicable), laboratory surfaces and documented to include how the drill or equipment, and effluent material; and exercise tested and evaluated the plan, (4) Procedures for the handling of se- any problems that were identified and lect agents and toxins in the same corrective action(s) taken, and the spaces with non-select agents and tox- names of registered entity personal ins to prevent unintentional contami- participants. nation. [70 FR 13284, Mar. 18, 2005, as amended at 77 FR 61079, Oct. 5, 2012; 79 FR 26831, May 12, 11 Technical assistance and guidance may 2014; 82 FR 6209, Jan. 19, 2017] be obtained by contacting APHIS.

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(b) The biosafety and containment (2) Experiments involving the delib- procedures must be sufficient to con- erate formation of synthetic or recom- tain the select agent or toxin (e.g., binant DNA containing genes for the physical structure and features of the biosynthesis of select toxins lethal for entity, and operational and procedural vertebrates at an LD[50] <100 ng/kg safeguards). body weight. (c) In developing a biosafety plan, an (b) The Administrator may revoke individual or entity should consider approval to conduct any of the experi- the following: ments in paragraph (a) of this section, (1) The CDC/NIH publication, ‘‘Bio- or revoke or suspend a certificate of safety in Microbiological and Bio- registration, if the individual or entity medical Laboratories.’’ This document fails to comply with the requirements is available on the National Select of this part. Agent Registry at http:// (c) To apply for approval to conduct www.selectagents.gov/. any of the experiments in paragraph (a) (2) The ‘‘NIH Guidelines for Research of this section, an individual or entity Involving Recombinant or Synthetic must submit a written request and sup- Nucleic Acid Molecules.’’ This docu- porting scientific information. A writ- ment is available on the Internet at ten decision granting or denying the http://www.selectagents.gov/. request will be issued. (d) The biosafety plan must include an occupational health program for in- [70 FR 13284, Mar. 18, 2005, as amended at 73 dividuals with access to Tier 1 select FR 61331, Oct. 16, 2008; 77 FR 61080, Oct. 5, 2012; 79 FR 26831, May 12, 2014] agents and toxins, and those individuals must be enrolled in the occupa- § 121.14 Incident response. 12 tional health program. (e) The plan must be reviewed annu- (a) An individual or entity required ally and revised as necessary. Drills or to register under this part must de- exercises must be conducted at least velop and implement a written inci- 13 annually to test and evaluate the effec- dent response plan based upon a site tiveness of the plan. The plan must be specific risk assessment. The incident reviewed and revised, as necessary, response plan must be coordinated with after any drill or exercise and after any any entity-wide plans, kept in the incident. Drills or exercises must be workplace, and available to employees documented to include how the drill or for review. The current incident re- exercise tested and evaluated the plan, sponse plan must be submitted for ini- any problems that were identified and tial registration, renewal of registra- corrective action(s) taken, and the tion, or when requested. names of registered entity personnel (b) The incident response plan must participants. fully describe the entity’s response procedures for the theft, loss, or release of [70 FR 13284, Mar. 18, 2005, as amended at 73 a select agent or toxin; inventory dis- FR 61331, Oct. 16, 2008; 77 FR 61080, Oct. 5, crepancies; security breaches (includ- 2012; 82 FR 6209, Jan. 19, 2017] ing information systems); severe § 121.13 Restricted experiments. weather and other natural disasters; workplace violence; bomb threats and (a) An individual or entity may not suspicious packages; and emergencies conduct, or possess products resulting such as fire, gas leak, explosion, power from, the following experiments unless outage, and other natural and man- approved by and conducted in accord- made events. ance with the conditions prescribed by (c) The response procedures must ac- the Administrator: count for hazards associated with the (1) Experiments that involve the de- select agent or toxin and appropriate liberate transfer of, or selection for, a drug resistance trait to select agents 12 that are not known to acquire the trait Nothing in this section is meant to su- persede or preempt incident response re- naturally, if such acquisition could quirements imposed by other statutes or reg- compromise the control of disease ulations. agents in humans, veterinary medicine, 13 Technical assistance and guidance may or agriculture. be obtained by contacting APHIS.

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actions to contain such select agent or tiveness of the plan. The plan must be toxin, including any animals (including reviewed and revised, as necessary, arthropods) or plants intentionally or after any drill or exercise and after any accidentally exposed to or infected incident. Drills or exercises must be with a select agent. documented to include how the drill or (d) The incident response plan must exercise tested and evaluated the plan, also contain the following information: any problems that were identified and (1) The name and contact informa- corrective action(s) taken, and the tion (e.g., home and work) for the indi- names of registered entity personnel vidual or entity (e.g., responsible offi- participants. cial, alternate responsible official(s), [70 FR 13284, Mar. 18, 2005, as amended at 73 biosafety officer, etc.); FR 61331, Oct. 16, 2008; 77 FR 61080, Oct. 5, (2) The name and contact informa- 2012; 82 FR 6209, Jan. 19, 2017] tion for the building owner and/or man- ager, where applicable; § 121.15 Training. (3) The name and contact informa- (a) An individual or entity required tion for tenant offices, where applica- to register under this part must pro- ble; vide information and training on bio- (4) The name and contact informa- containment, biosafety, security (in- tion for the physical security official cluding security awareness), and inci- for the building, where applicable; dent response to: (5) Personnel roles and lines of au- (1) Each individual with access ap- thority and communication; proval from the Administrator or HHS (6) Planning and coordination with Secretary. The training must address local emergency responders; the particular needs of the individual, (7) Procedures to be followed by em- the work they will do, and the risks ployees performing rescue or medical posed by the select agents or toxins. duties; The training must be accomplished (8) Emergency medical treatment prior to the individual’s entry into an and first aid; area where a select agent is handled or (9) A list of personal protective and stored, or within 12 months of the date emergency equipment, and their loca- the individual was approved by the Ad- tions; ministrator or the HHS Secretary for (10) Site security and control; access, whichever is earlier. (11) Procedures for emergency evacu- (2) Each individual not approved for ation, including type of evacuation, access to select agents and toxins by exit route assignments, safe distances, the Administrator or HHS Secretary and places of refuge; and before that individual enters areas (12) Decontamination procedures. under escort where select agents or (e) Entities with Tier 1 select agents toxins are handled or stored (e.g., lab- and toxins must have the following ad- oratories, growth chambers, animal ditional incident response policies or rooms, greenhouses, storage areas, procedures: shipping/receiving areas, production fa- (1) The incident response plan must cilities, etc.). Training for escorted fully describe the entity’s response pro- personnel must be based on the risk as- cedures for failure of intrusion detec- sociated with accessing areas where se- tion or alarm system; and lect agents and toxins are used and/or (2) The incident response plan must stored. The training must be accom- describe procedures for how the entity plished prior to the individual’s entry will notify the appropriate Federal, into where select agents or toxins are State, or local law enforcement agen- handled or stored (e.g., laboratories, cies of suspicious activity that may be growth chambers, animal rooms, green- criminal in nature and related to the houses, storage areas, shipping/receiv- entity, its personnel, or its select ing areas, production facilities, etc.). agents or toxins. (b) Entities with Tier 1 select agents (f) The plan must be reviewed annu- and toxins must conduct annual insider ally and revised as necessary. Drills or threat awareness briefings on how to exercises must be conducted at least identify and report suspicious behav- annually to test and evaluate the effec- iors.

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(c) Refresher training must be pro- (2) At the time of transfer, the recipi- vided annually for individuals with ac- ent has a certificate of registration cess approval from the HHS Secretary that includes the particular select or Administrator or at such time as agent or toxin to be transferred and the registered individual or entity sig- meets all of the requirements of this nificantly amends its security, incident part. response, or biosafety plans. (c) A select agent or toxin that is (d) The responsible official must en- contained in a specimen for proficiency sure a record of the training provided testing may be transferred without to each individual with access to select prior authorization from APHIS or agents and toxins and each escorted in- CDC provided that, at least 7 calendar dividual (e.g., laboratory workers, visi- days prior to the transfer, the sender tors, etc.) is maintained. The record reports to APHIS or CDC the select must include the name of the indi- agent or toxin to be transferred and vidual, the date of the training, a de- the name and address of the recipient. scription of the training provided, and (d) On a case-by-case basis, the Ad- the means used to verify that the em- ministrator may authorize a transfer ployee understood the training. of a select agent or toxin not otherwise (e) The responsible official must en- eligible for transfer under this part sure and document that individuals are under conditions prescribed by the Ad- provided the contact information of ministrator. the USDA Office of Inspector General (e) To obtain authorization for a Hotline and the HHS Office of Inspec- transfer, APHIS/CDC Form 2 must be tor General Hotline so that they may submitted. anonymously report any safety or se- (f) After authorization is provided by curity concerns related to select agents APHIS or CDC, the packaging of the and toxins. select agent(s) and toxin(s) is per- [77 FR 61081, Oct. 5, 2012, as amended at 82 FR formed by an individual approved by 6209, Jan. 19, 2017] the HHS Secretary or Administrator to have access to select agents and toxins § 121.16 Transfers. and is in compliance with all applica- (a) Except as provided in paragraphs ble laws concerning packaging. (c) and (d) of this section, a select (g) The sender must comply with all agent or toxin may only be transferred applicable laws governing shipping. to individuals or entities registered to (h) Transportation in commerce possess, use, or transfer that agent or starts when the select agent(s) or toxin. A select agent or toxin may only toxin(s) are packaged for shipment and be transferred under the conditions of ready for receipt by a courier trans- this section and must be authorized by porting select agent(s) or toxin(s) and APHIS or CDC prior to the transfer. 14 ends when the package is received by (b) A transfer may be authorized if: the intended recipient who is an indi- (1) The sender: vidual approved by the HHS Secretary (i) Has at the time of transfer a cer- or Administrator to have access to se- tificate of registration that covers the lect agents and toxins, following a se- particular select agent or toxin to be curity risk assessment by the Attorney transferred and meets all the require- General. ments of this part; (i) The recipient must submit a com- (ii) Meets the exemption require- pleted APHIS/CDC Form 2 within 2 ments for the particular select agent or business days of receipt of a select toxin to be transferred; or agent or toxin. (iii) Is transferring the select agent (j) The recipient must immediately or toxin from outside of the United notify APHIS or CDC if the select States and meets all import require- agent or toxin has not been received ments. within 48 hours after the expected delivery time or if the package con- 14 The requirements of this section do not taining the select agent or toxin has apply to transfers within a registered entity been damaged to the extent that a re- (i.e., the sender and the recipient are covered lease of the select agent or toxin may by the same certificate of registration). have occurred.

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(k) An authorization for a transfer (vii) For intra-entity transfers (send- shall be valid only for 30 calendar days er and the recipient are covered by the after issuance, except that such an au- same certificate of registration), the thorization becomes immediately null select agent, the quantity transferred, and void if any facts supporting the au- the date of transfer, the sender, and thorization change (e.g., change in the the recipient; and certificate of registration for the send- (viii) Records created under § 121.19 or er or recipient, change in the applica- 42 CFR 73.19 (Notification of theft, loss, tion for transfer). or release); (l) Transfer the amounts only after (2) An accurate, current accounting the transferor uses due diligence and of any animals or plants intentionally documents that the recipient has a le- or accidentally exposed to or infected gitimate need (e.g., prophylactic, pro- with a select agent (including number tective, bona fide research, or other and species, location, and appropriate peaceful purpose) to handle or use such disposition); toxins. Information to be documented (3) An accurate, current inventory includes, but is not limited, to the re- for each toxin held, including: cipient information, toxin and amount (i) The name and characteristics; transferred, and declaration that the (ii) The quantity acquired from an- recipient has legitimate purpose to other individual or entity (e.g., con- store and use such toxins. tainers, vials, tubes, etc.), date of ac- [70 FR 13284, Mar. 18, 2005, as amended at 73 quisition, and the source; FR 61331, Oct. 16, 2008; 77 FR 61081, Oct. 5, (iii) The initial and current quantity 2012; 82 FR 6210, Jan. 19, 2017] amount (e.g., milligrams, milliliters, grams, etc.); § 121.17 Records. (iv) The toxin used and purpose of (a) An individual or entity required use, quantity, date(s) of the use and by to register under this part must main- whom; tain complete records relating to the (v) Where stored (e.g., building, room, activities covered by this part. Such and freezer or other storage container); records must include: (vi) When moved from storage and by (1) An accurate, current inventory whom and when returned to storage for each select agent (including viral and by whom, including quantity genetic elements, recombinant and/or amount; synthetic nucleic acids, and organisms (vii) Records created under § 121.16 or containing recombinant and/or syn- 42 CFR 73.16 (Transfers); thetic nucleic acids) held in long-term (viii) For intra-entity transfers storage (placement in a system de- (sender and the recipient are covered signed to ensure viability for future by the same certificate of registra- use, such as in a freezer or lyophilized tion), the toxin, the quantity trans- materials), including: ferred, the date of transfer, the sender, (i) The name and characteristics and the recipient; (e.g., strain designation, GenBank Ac- (ix) Records created under § 121.19 or cession number, etc.); 42 CFR 73.19 (Notification of theft, loss, (ii) The quantity acquired from an- or release); other individual or entity (e.g., con- (x) If destroyed, the quantity of toxin tainers, vials, tubes, etc.), date of ac- destroyed, the date of such action, and quisition, and the source; by whom. (iii) Where stored (e.g., building, (4) A current list of all individuals room, and freezer or other storage con- that have been granted access approval tainer); by the Administrator or the HHS Sec- (iv) When moved from storage and by retary; whom and when returned to storage (5) Information about all entries into and by whom; areas containing select agents or tox- (v) The select agent used, purpose of ins, including the name of the indi- use, and, when applicable, final disposi- vidual, name of the escort (if application; ble), and the date and time of entry; (vi) Records created under § 121.16 or (6) Accurate, current records created 42 CFR 73.16 (Transfers); under § 121.9 or 42 CFR 73.9 (Responsible

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official), § 121.11 or 42 CFR 73.11 (Secu- cations, laboratory notebooks, institu- rity), § 121.12 or 42 CFR 73.12 (Bio- tional biosafety and/or animal use safety), § 121.14 or 42 CFR 73.14 (Incident committee minutes and approved pro- response), and § 121.15 or 42 CFR 73.15 tocols, and records associated with oc- (Training); cupational health and suitability pro- (7) A written explanation of any dis- grams. All records created under this crepancies; and part must be maintained for 3 years. (8) For select agents or material con- [70 FR 13284, Mar. 18, 2005, as amended at 77 taining select agents or regulated nu- FR 61081, Oct. 5, 2012; 82 FR 6210, Jan. 19, cleic acids that can produce infectious 2017] forms of any select agent virus that have been subjected to a validated in- § 121.18 Inspections. activation procedure or a procedure for (a) Without prior notification, APHIS removal of viable select agent: must be allowed to inspect any site at (i) A written description of the vali- which activities regulated under this dated inactivation procedure or viable part are conducted and must be al- select agent removal method used, in- lowed to inspect and copy any records cluding validation data; relating to the activities covered by (ii) A written description of the via- this part. bility testing protocol used; (b) Prior to issuing a certificate of (iii) A written description of the in- registration to an individual or entity, vestigation conducted by the entity re- APHIS may inspect and evaluate the sponsible official involving an inactiva- premises and records to ensure compli- tion or viable select agent removal ance with this part. failure and the corrective actions taken; § 121.19 Notification of theft, loss, or (iv) The name of each individual per- release. forming the validated inactivation or (a) An individual or entity must im- viable select agent removal method; mediately notify APHIS or CDC upon (v) The date(s) the validated inac- discovery of the theft or loss of a select tivation or viable select agent removal agent or toxin. Thefts or losses must be method was completed; reported even if the select agent or (vi) The location where the validated toxin is subsequently recovered or the inactivation or viable select agent re- responsible parties are identified. moval method was performed; and (1) The theft or loss of a select agent (vii) A certificate, signed by the prin- or toxin must be reported by telephone, cipal investigator, that includes the facsimile, or e-mail. The following in- date of inactivation or viable select formation must be provided: agent removal, the validated inactiva- (i) The name of the select agent or tion or viable select agent removal toxin and any identifying information method used, and the name of the prin- (e.g., strain or other characterization cipal investigator. A copy of the cer- information); tificate must accompany any transfer (ii) An estimate of the quantity sto- of inactivated or select agent removed len or lost; material. (iii) An estimate of the time during (b) The individual or entity must im- which the theft or loss occurred; plement a system to ensure that all (iv) The location (building, room) records and databases created under from which the theft or loss occurred; this part are accurate and legible, have and controlled access, and that their au- (v) The list of Federal, State, or local thenticity may be verified. law enforcement agencies to which the (c) The individual or entity must individual or entity reported, or in- promptly produce upon request any in- tends to report, the theft or loss. formation that is related to the re- (2) A completed APHIS/CDC Form 3 quirements of this part but is not oth- must be submitted within 7 calendar erwise contained in a record required days. to be kept by this section. The location (b) An individual or entity must im- of such information may include, but is mediately notify APHIS or CDC upon not limited to, biocontainment certifi- discovery of a release of a select agent

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or toxin causing occupational exposure § 122.1 Definitions. or a release of a select agent or toxin The following words, when used in outside of the primary barriers of the the regulations in this part 122, shall biocontainment area. be construed, respectively, to mean: (1) The release of a select agent or (a) Department. The U.S. Department toxin must be reported by telephone, of Agriculture. facsimile, or e-mail. The following information must be provided: (b) Secretary. ‘‘Secretary’’ means the (i) The name of the select agent or Secretary of Agriculture of the United toxin and any identifying information States, or any officer or employee of (e.g., strain or other characterization the Department to whom authority has information); heretofore been delegated, or to whom (ii) An estimate of the quantity re- authority may hereafter be delegated, leased; to act in his stead. (iii) The time and duration of the re- (c) Administrator. The Administrator, lease; Animal and Plant Health Inspection (iv) The environment into which the Service, United States Department of release occurred (e.g., in building or Agriculture, or any person authorized outside of building, waste system); to act for the Administrator. (v) The location (building, room) (d) Organisms. All cultures or collec- from which the release occurred; and tions of organisms or their derivatives, (vi) The number of individuals poten- which may introduce or disseminate tially exposed at the entity; any contagious or infectious disease of (vii) Actions taken to respond to the animals (including poultry). release; and (e) Vectors. All animals (including (viii) Hazards posed by the release. poultry) such as mice, pigeons, guinea (2) A completed APHIS/CDC Form 3 pigs, rats, ferrets, rabbits, chickens, must be submitted within 7 calendar dogs, and the like, which have been days. treated or inoculated with organisms, or which are diseased or infected with § 121.20 Administrative review. any contagious, infectious, or commu- (a) An individual or entity may ap- nicable disease of animals or poultry or peal a denial, revocation, or suspension which have been exposed to any such of registration under this part. The ap- disease. peal must be in writing, state the fac- (f) Permittee. A person who resides in tual basis for the appeal, and be sub- the United States or operates a busi- mitted to the Administrator within 30 ness establishment within the United calendar days of the decision. States, to whom a permit to import or (b) An individual may appeal a de- transport organisms or vectors has nial, limitation, or revocation of access been issued under the regulations. approval under this part. The appeal (g) Person. Any individual, firm, part- must be in writing, state the factual nership, corporation, company, society, basis for the appeal, and be submitted association, or other organized group to the Administrator within 180 cal- of any of the foregoing, or any agent, endar days of the decision. officer, or employee of any thereof. (c) The Administrator’s decision con- [31 FR 81, Jan. 5, 1966, as amended at 57 FR stitutes final agency action. 30899, July 13, 1992] [77 FR 61081, Oct. 5, 2012] § 122.2 Permits required. PART 122—ORGANISMS AND No organisms or vectors shall be im- VECTORS ported into the United States or trans- ported from one State or Territory or Sec. the District of Columbia to another 122.1 Definitions. State or Territory or the District of 122.2 Permits required. Columbia without a permit issued by 122.3 Application for permits. the Secretary and in compliance with 122.4 Suspension or revocation of permits. the terms thereof: Provided, That no AUTHORITY: 7 U.S.C. 8301–8317; 21 U.S.C. 151– permit shall be required under this sec- 158; 7 CFR 2.22, 2.80, and 371.4. tion for importation of organisms for

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which an import permit has been nicable disease of animals (including issued pursuant to part 102 of this sub- poultry). chapter or for transportation of orga- (b) In cases of wilfulness or where the nisms produced at establishments li- public health, interest or safety so re- censed under part 102 of this sub- quires, however, the Secretary may chapter. As a condition of issuance of without hearing informally suspend permits under this section, the per- such a permit upon the grounds set mittee shall agree in writing to observe forth in paragraph (a) of this section, the safeguards prescribed by the Ad- pending determination of formal pro- ministrator for public protection with ceedings under part 123 of this sub- respect to the particular importation chapter for suspension or revocation of or transportation. the permit. (Approved by the Office of Management and [23 FR 10065, Dec. 23, 1958. Redesignated at 31 Budget under control number 0579–0015) FR 81, Jan. 5, 1966, and amended at 57 FR [28 FR 7896, Aug. 2, 1963. Redesignated at 31 30899, July 13, 1992] FR 81, Jan. 5, 1966 and amended at 48 FR 57473, Dec. 30, 1983; 57 FR 30899, July 13, 1992; PART 123—RULES OF PRACTICE 59 FR 67134, Dec. 29, 1994] GOVERNING PROCEEDINGS § 122.3 Application for permits. UNDER THE VIRUS-SERUM-TOXIN The Secretary may issue, at his dis- ACT cretion, a permit as specified in § 122.2 when proper safeguards are set up as AUTHORITY: 7 U.S.C. 8301–8317; 21 U.S.C. 151– provided in § 122.2 to protect the public. 159; 7 CFR 2.22, 2.80, and 371.4. Application for such a permit shall be made in advance of shipment, and each § 123.1 Scope and applicability of rules permit shall specify the name and ad- of practice. dress of the consignee, the true name The Uniform Rules of Practice for and character of each of the organisms the Department of Agriculture promul- or vectors involved, and the use to gated in subpart H of part 1, subtitle A, which each will be put. title 7, Code of Federal Regulations, (Approved by the Office of Management and are the Rules of Practice applicable to Budget under control number 0579–0015) adjudicatory, administrative proceedings under the Virus-Serum-Toxin [23 FR 10065, Dec. 23, 1958. Redesignated at 31 FR 81, Jan. 5, 1966 and amended at 48 FR Act. 57473, Dec. 30, 1983; 59 FR 67134, Dec. 29, 1994] [42 FR 10960, Feb. 25, 1977] § 122.4 Suspension or revocation of permits. PART 124—PATENT TERM (a) Any permit for the importation or RESTORATION transportation of organisms or vectors issued under this part may be formally Subpart A—General Provisions suspended or revoked after opportunity Sec. for hearing has been accorded the per- 124.1 Scope. mittee, as provided in part 123 of this 124.2 Definitions. subchapter, if the Secretary finds that the permittee has failed to observe the Subpart B—Eligibility Assistance safeguards and instructions prescribed by the Administrator with respect to 124.10 APHIS liaison with PTO. the particular importation or transportation or that such importation or Subpart C—Regulatory Review Period transportation for any other reason 124.20 Patent term extension calculation. may result in the introduction or dis- 124.21 Regulatory review period determina- semination from a foreign country into tion. the United States, or from one State, 124.22 Revision of regulatory review period Territory or the District of Columbia determination. to another, of the contagion of any 124.23 Final action on regulatory review pe- contagious, infectious or commu- riod determination.

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Subpart D—Due Diligence Petitions Due diligence petition. A petition submitted under § 124.30 of this part. 124.30 Filing, format, and content of peti- Informal hearing. A hearing that is tions. not subject to the provisions of 5 U.S.C. 124.31 Applicant response to petition. 124.32 APHIS action on petition. 554, 556, and 557 and that is conducted 124.33 Standard of due diligence. as provided in 21 U.S.C. 321(x). License applicant. Any person who, in Subpart E—Due Diligence Hearing accordance with part 102 of this chapter, submits an application to the Ani- 124.40 Request for hearing. mal and Plant Health Inspection Serv- 124.41 Notice of hearing. ice of the U.S. Department of Agri- 124.42 Hearing procedure. culture for a U.S. Veterinary Biologi- 124.43 Administrative decision. cal Product License. AUTHORITY: 35 U.S.C. 156; 7 CFR 2.22, 2.80, Patent. A patent issued by the Patent and 371.4. and Trademark Office of the United SOURCE: 58 FR 11369, Feb. 25, 1993, unless States Department of Commerce. otherwise noted. Person. Any individual, firm, partner- ship, corporation, company, associa- Subpart A—General Provisions tion, educational institution, State or local government agency, or other or- § 124.1 Scope. ganized group of any of the foregoing, or any agent, officer, or employee of (a) This parts sets forth procedures any thereof. and requirements for APHIS review of PTO. The Patent and Trademark Of- applications for the extension of the fice of the United States Department of term of certain patents for veterinary Commerce. biological products pursuant to 35 U.S.C. 156—Extension of patent term. [58 FR 11369, Feb. 25, 1993, as amended at 68 Responsibilities of APHIS include: FR 6346, Feb. 7, 2003] (1) Assisting PTO in determining eligibility for patent term restoration; Subpart B—Eligibility Assistance (2) Determining the length of a product’s regulatory review period; § 124.10 APHIS liaison with PTO. (3) If petitioned, reviewing and ruling Upon receipt of a copy of an applica- on due diligence challenges to APHIS’s tion for extension of the term of a vet- regulatory review period determina- erinary biologic patent from PTO, tions; and APHIS will assist PTO in determining (4) Conducting hearings to review ini- whether a patent related to a biologi- tial APHIS findings on due diligence cal product is eligible for patent term challenges. extension by: (b) The regulations in this part are (a)(1) Verifying whether the product designed to be used in conjunction with was subject to a regulatory review pe- regulations issued by PTO concerning riod before its commercial marketing patent term extension which may be or use; found at 37 CFR 1.710 through 1.791. (2) Determining whether the permission for commercial marketing or use [58 FR 11369, Feb. 25, 1993, as amended at 64 of the product after the regulatory re- FR 43045, Aug. 9, 1999] view period was the first permitted commercial marketing or use of the § 124.2 Definitions. product under the provision of law Animal and Plant Health Inspection under which such regulatory review pe- Service (APHIS). The agency in the De- riod occurred, and, if so, whether it was partment of Agriculture responsible for the first permitted commercial mar- licensing veterinary biological prod- keting or use of the veterinary biologi- ucts under the Virus-Serum-Toxin Act. cal product for administration to a Applicant. Any person who submits food-producing animal; an application or an amendment or (3) Ascertaining whether the patent supplement to an application under 35 term restoration application was sub- U.S.C. 156 seeking extension of the mitted within 60 days after the product term of a patent. was approved for marketing or use; and

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(4) Providing such other information termined, APHIS will notify PTO in as may be necessary and relevant to writing of the determination, send a PTO’s determination of whether a pat- copy of the determination to the appli- ent related to a product is eligible for cant, and make a copy available for patent term restoration. public inspection in room 1141, South (b) APHIS will notify PTO of its find- Building, 14th Street and Independence ings in writing, send a copy of this no- Avenue SW., Washington, DC, between tification to the applicant, and make a 8 a.m. and 4:30 p.m., Monday through copy available for public inspection in Friday, except holidays. room 1141, South Building, 14th Street (b) APHIS will also publish a notice and Independence Avenue SW., Wash- of the regulatory review period deter- ington, DC, between 8 a.m. and 4:30 mination in the FEDERAL REGISTER. p.m., Monday through Friday, except The notice will include the following: holidays. (1) The name of the applicant; (2) The trade name and true name of Subpart C—Regulatory Review the product; Period (3) The number of the patent for which an extension of the term is § 124.20 Patent term extension calcula- sought; tion. (4) The approved indications or uses (a) As provided in 37 CFR 1.779 of for the product; PTO’s regulations, in order to deter- (5) The regulatory review period de- mine a product’s regulatory review pe- termination, including a statement of riod, APHIS will review the informa- the length of each phase of the review tion in each application to determine period and the dates used in calcu- the lengths of the following phases of lating each phase. the review period, and will then find their sum: § 124.22 Revision of regulatory review (1) The number of days in the period period determination. beginning on the date authorization to (a) Any interested person may re- prepare an experimental biological quest a revision of the regulatory re- product under the Virus-Serum-Toxin view period determination within the Act became effective and ending on the 30 day period beginning on its publica- date an application for a license was tion in the FEDERAL REGISTER. The re- initially submitted under the Virus- quest must be sent to the Director, Serum-Toxin Act; and Center for Veterinary Biologics, Pol- (2) The number of days in the period icy, Evaluation, and Licensing, 1920 beginning on the date an application Dayton Avenue, P.O. Box 844, Ames, IA for a license was initially submitted 50010. The request must specify the fol- for approval under the Virus-Serum- lowing: Toxin Act and ending on the date such (1) The identity of the product; license was issued. (2) The identity of the applicant for (b) A license application is ‘‘initially patent term restoration; submitted’’ on the date it contains suf- (3) The docket number of the FED- ficient information to allow APHIS to ERAL REGISTER notice announcing the commence review of the application. A regulatory review period determina- product license is issued on the date of tion; and the APHIS letter informing the appli- (4) The basis for the request for revi- cant of the issuance. The issuance of a sion, including any documentary evi- license releases the product for com- dence. mercial marketing or use. (b) If APHIS decides to revise its prior determination, APHIS will notify § 124.21 Regulatory review period de- PTO of the decision, and will send a termination. copy of notification to the applicant (a) Not later than 30 days after the and the person requesting the revision receipt of an application from PTO, (if different from the applicant) with a APHIS shall determine the regulatory request for comments within 10 days of review period. Once the regulatory re- notification. If no comment on the pro- view period for a product has been de- posed revision is received, APHIS will

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publish the revision in the FEDERAL review period and must set forth suffi- REGISTER, and include a statement giv- cient facts to merit an investigation by ing the reasons for the revision. If com- APHIS of whether the applicant acted ment is received, APHIS will make a with due diligence. final determination regarding the revi- (d) The petition must contain a cer- sion based on such comment and will tification that the petitioner has then publish the revision in the FED- served a true and complete copy of the ERAL REGISTER, giving reasons for its petition on interested parties by cer- determination. tified or registered mail (return receipt [59 FR 11369, Feb. 25, 1993, as amended at 59 requested) or by personal delivery. FR 67617, Dec. 30, 1994; 64 FR 43045, Aug. 9, 1999; 75 FR 20773, Apr. 21, 2010] § 124.31 Applicant response to petition. (a) The applicant may file with § 124.23 Final action on regulatory re- APHIS a written response to the peti- view period determination. tion no later than 20 days after the ap- APHIS will consider its regulatory plicant’s receipt of a copy of the peti- review period determination to be final tion. upon expiration of the 180-day period (b) The applicant’s response may for filing a due diligence petition under present additional facts and cir- § 124.30 unless it receives: cumstances to address the assertions in (a) New information from PTO the petition, but shall be limited to the records, or APHIS records, that affects issue of whether the applicant acted the regulatory review period deter- with due diligence during the regu- mination; latory review period. The applicant’s (b) A request under § 124.22 for revi- response may include documents that sion of the regulatory review period de- were not in the original patent term termination; extension application. (c) A due diligence petition filed (c) If the applicant does not respond under § 124.30; or to the petition, APHIS will decide the (d) A request for a hearing filed under matter on the basis of the information § 124.40. submitted in the patent term restora- [58 FR 11369, Feb. 25, 1993; 58 FR 29028, May tion application, the due diligence peti- 18, 1993] tion, and APHIS records.

Subpart D—Due Diligence § 124.32 APHIS action on petition. Petitions (a) Within 90 days after APHIS receives a petition filed under § 124.30, the § 124.30 Filing, format, and content of Under Secretary for Marketing and petitions. Regulatory Programs shall make a de- (a) Any interested person may file a termination under paragraphs (b) or (c) petition with APHIS, no later than 180 of this section or under § 124.33 whether days after the publication of a regu- the applicant acted with due diligence latory review period determination during the regulatory review period. under § 124.21, alleging that a license APHIS will publish its determination applicant did not act with due dili- in the FEDERAL REGISTER together with gence in seeking APHIS approval of the factual and legal basis for the deter- product during the regulatory review mination, notify PTO of the determina- period. tion in writing, and send copies of the (b) The petition must be filed with determination to PTO, the applicant, APHIS under the docket number of the and the petitioner. FEDERAL REGISTER notice of the agen- (b) APHIS may deny a due diligence cy’s regulatory review period deter- petition without considering the mer- mination. The petition must contain its of the petition if: any additional information required by (1) The petition is not filed in accord- this subpart. ance with § 124.30; (c) The petition must allege that the (2) The petition does not contain in- applicant failed to act with due dili- formation or allegations upon which gence sometime during the regulatory APHIS may reasonably determine that

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the applicant did not act with due dili- (3) Be delivered to the Director, Cen- gence during the applicable regulatory ter for Veterinary Biologics, Policy, review period; or Evaluation, and Licensing, 1920 Dayton (3) The petition fails to allege a suffi- Avenue, P.O. Box 844, Ames, IA 50010. cient total amount of time during (4) Contain a full statement of facts which the applicant did not exercise upon which the request for hearing is due diligence so that, even if the peti- based; tion were granted, the petition would (5) Contain the name, the address, not affect the maximum patent term and the principal place of business of extension which the applicant is enti- the person requesting the hearing; and tled to under 35 U.S.C. 156. (6) Contain a certification that the person requesting the hearing has [59 FR 11369, Feb. 25, 1993, as amended at 64 served a true and complete copy of the FR 43045, Aug. 9, 1999] request upon the petitioner of the due diligence determination and the appli- § 124.33 Standard of due diligence. cant for patent term extension by cer- (a) In determining the due diligence tified or registered mail (return receipt of an applicant, APHIS will examine requested) or by personal service. the facts and circumstances of the ap- (c) The request must state whether plicant’s actions during the regulatory the requesting party seeks a hearing review period to determine whether the not later than 30 days after the date applicant exhibited the degree of atten- APHIS receives the request, or, at the tion, continuous directed effort, and request of the person making the re- timeliness as may reasonably be ex- quest, not later than 60 days after such pected from, and are ordinarily exer- date. cised by, a person during a regulatory [58 FR 11369, Feb. 25, 1993, as amended at 59 review period. APHIS will take into FR 67617, Dec. 30, 1994; 64 FR 43045, Aug. 9, consideration all relevant factors, such 1999; 75 FR 20773, Apr. 21, 2010] as the amount of time between the approval of an experimental use permit § 124.41 Notice of hearing. and licensure of the veterinary biologi- No later than ten days before the cal product. hearing, APHIS will notify the request- (b) For purposes of this Part, the ac- ing party, the applicant, the petitioner, tions of the marketing applicant shall and any other interested person of the be imputed to the applicant for patent date, time, and location of the hearing. term restoration. The actions of an agent, attorney, contractor, employee, § 124.42 Hearing procedure. licensee, or predecessor in interest of (a) The presiding officer shall be ap- the marketing applicant shall be im- pointed by the Administrator of APHIS puted to the applicant for patent term from officers and employees of the De- restoration. partment who have not participated in any action of the Secretary which is Subpart E—Due Diligence Hearing the subject of the hearing and who are not directly responsible to an officer or § 124.40 Request for hearing. employee of the Department who has participated in any such action. (a) Any interested person may re- (b) Each party to the hearing shall quest, within 60 days beginning on the have the right at all times to be ad- date of publication of a due diligence vised and accompanied by an attorney. determination by APHIS in accordance (c) Before the hearing, each party to with § 124.32, that APHIS conduct an in- the hearing shall be given reasonable formal hearing on the due diligence de- notice of the matters to be considered termination. at the hearing, including a comprehen- (b) The request for a hearing must: sive statement of the basis for the ac- (1) Be in writing; tion taken or proposed by the Sec- (2) Contain the docket number of the retary which is the subject of the hear- FEDERAL REGISTER notice of APHIS’s ing and any general summary of the in- regulatory review period determina- formation which will be presented at tion; the hearing in support of such action.

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(d) At the hearing the parties to the practice adopted for the proceeding. hearing shall have the right to hear a APHIS will provide the requesting full and complete statement of the ac- party, the applicant, and the petitioner tion which is the subject of the hearing with an opportunity to participate as a together with the information and rea- party in the hearing. The standard of sons supporting such action, to con- due diligence set forth in § 124.33 will duct reasonable questioning, and to apply at the hearing. The party represent any oral and written informa- questing the due diligence hearing will tion relevant to such action. have the burden of proof at the hear- (e) The presiding officer in such hearing. ing shall prepare a written report of the hearing to which shall be attached § 124.43 Administrative decision. all written material presented at the Within 30 days after completion of hearing. The participants in the hear- the due diligence hearing, the Under ing shall be given the opportunity to Secretary for Marketing and Regu- review and correct or supplement the latory Programs, taking into consider- presiding officer’s report of the hear- ation the recommendation of the Ad- ing. ministrator, will affirm or revise the (f) The Secretary may require the determination made under § 124.32. hearing to be transcribed. A party to APHIS will publish the due diligence the hearing shall have the right to redetermination in the FEDERAL REG- have the hearing transcribed at his ex- ISTER, notify PTO of the redetermina- pense. Any transcription of a hearing tion, and send copies of the notice to shall be included in the presiding offi- PTO and the requesting party, the ap- cer’s report of the hearing. plicant, and the petitioner. (g) The due diligence hearing will be [59 FR 11369, Feb. 25, 1993, as amended at 64 conducted in accordance with rules of FR 43045, Aug. 9, 1999]

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