Animal and Plant Health Inspection Service, USDA § 113.3

§ 112.9 9 CFR Ch. I (1–1–21 Edition)

subject to the applicable provisions in issued in accordance with § 104.4, with this paragraph. the exception of products imported (1) After leaving the licensed estab- under § 104.4(d), shall be labeled as pro- lishment, a biological product shall not vided in this section. be bottled, repackaged, relabeled, or (a) The label shall identify the prod- otherwise altered in any way while in uct and the name and address of the the United States; and manufacturer and shall provide in- (2) An exported biological product structions for proper use of the prod- shall not be returned to the United uct, including all warnings and cau- States: Provided, That, in the case of a tions needed by the permittee to safely biological product exported in labeled use the product. 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 iden- PART 113—STANDARD tified as to contents with an approved 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. 113.3 Sampling of biological products. containers identified with an approved 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. 113.10 Testing of bulk material for export or tainers identified with an approved for further manufacture. label that contains the words ‘‘For Ex- 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 contamination. A biological product imported for re- 113.29 Determination of moisture content in search and evaluation under a permit desiccated biological products.

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113.30 Detection of Salmonella contamina- 113.107 Clostridium Haemolyticum tion. Bacterin. 113.31 Detection of avian lymphoid leukosis. 113.108 Clostridium Novyi Bacterin-Toxoid. 113.32 Detection of Brucella contamination. 113.109 Clostridium Sordellii Bacterin-Tox- 113.33 Mouse safety tests. oid. 113.34 Detection of hemagglutinating vi- 113.110 Clostridium Botulinum Type C ruses. Bacterin-Toxoid. 113.35 Detection of viricidal activity. 113.111 Clostridium Perfringens Type C Tox- 113.36 Detection of pathogens by the chick- oid and Bacterin-Toxoid. en inoculation test. 113.112 Clostridium Perfringens Type D Tox- 113.37 Detection of pathogens by the chick- oid and Bacterin-Toxoid. en embryo inoculation test. 113.113 Autogenous biologics. 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. 113.51 Requirements for primary cells used KILLED VIRUS VACCINES 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. 705

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113.310 Bovine Rhinotracheitis Vaccine. regulations are conducted, National 113.311 Bovine Virus Diarrhea Vaccine. Veterinary Services Laboratories, U.S. 113.312 Rabies Vaccine, Live Virus. Department of Agriculture, may pro- 113.313 Measles Vaccine. 113.314 Feline Calicivirus Vaccine. vide testing aids, when available, to li- 113.315 Feline Rhinotracheitis Vaccine. censees, permittees, and applicants for 113.316 Canine Parainfluenza Vaccine. licenses and permits. Such aids shall be 113.317 Parvovirus Vaccine (Canine). as follows: 113.318 Pseudorabies Vaccine. (a) Supplemental Assay Method 113.319–113.324 [Reserved] (SAM) is a technical bulletin con- 113.325 Avian Encephalomyelitis Vaccine. taining detailed instructions for con- 113.326 Avian Pox Vaccine. 113.327 Bronchitis Vaccine. ducting a test. Such instructions shall 113.328 Fowl Laryngotracheitis Vaccine. be in accordance with the procedures 113.329 Newcastle Disease Vaccine. currently being followed at National 113.330 Marek’s Disease Vaccines. Veterinary Services Laboratories and 113.331 Bursal Disease Vaccine. as improved, proven procedures are de- 113.332 Tenosynovitis Vaccine. veloped, shall be revised and reissued DIAGNOSTICS AND REAGENTS prior to application. (b) Standard Reference Preparation 113.400–113.405 [Reserved] is a serum, virus, bacterial culture, or 113.406 Tuberculin, Intradermic. 113.407 Pullorum antigen. antigen to be used in test systems for 113.408 Avian mycoplasma antigen. direct comparison with serials of bio- 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 specified in the Standard Requirements passive transfer. 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 products. § 113.1 Compliance. Each licensee and permittee shall The regulations in this part apply to furnish representative samples of each each serial or subserial of a licensed bi- serial or subserial of a biological prod- ological product manufactured in a li- uct manufactured in the United States censed establishment and to each serial or imported into the United States as or subserial of a biological product in prescribed in this section. Additional each shipment imported for distribu- samples may be purchased in the open tion and sale. market by a Animal and Plant Health Inspection Service representative. § 113.2 Testing aids. (a) Either an employee of the Depart- To better ensure consistent and re- ment of Agriculture, of the licensee, or producible test results when Standard of the permittee, as designated by the Requirement tests prescribed in the Administrator shall select prerelease

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samples of biological product in the (i) Six multiple-dose samples of number prescribed in paragraph (b) of Brucella Abortus Vaccine; this section. Each sample shall be (ii) Twelve samples of all other live marked for identification by the person bacterial vaccines; making the selection after which they (iii) Two samples of Coccidiosis Vac- shall be packaged by the licensee or cine; permittee, as the case may be, and for- (iv) Eighteen samples of Rabies Vac- warded to National Veterinary Serv- cine, Modified Live Virus; ices Laboratories; except that an em- (v) Sixteen samples of all other vac- ployee of the Department may forward cines consisting of live microorga- or deliver the samples to National Vet- nisms; erinary Services Laboratories if such (vi) Thirty single-dose or 14 multiple- action deemed advisable by the Admin- dose samples of Equine istrator. Encephalomyelitis Vaccine, Killed (1) Selection shall be made as fol- Virus; lows: (vii) Twenty-two single-dose or 14 (i) Nonviable liquid biological prod- multiple-dose samples of Rabies Vac- ucts—either bulk or final container cine, Killed Virus; samples of completed product shall be (viii) Sixteen single-dose or 12 mul- selected for purity, safety, or potency tiple-dose samples of all other vaccines tests. Biological product in final con- consisting of killed microorganisms. tainer shall be selected to test for via- (2) Bacterins and bacterin-toxoids: ble bacteria and fungi. (i) Twelve samples of single-fraction 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 (4) Antitoxins: sampled at the end of the filling oper- (i) Fourteen single-dose or 12 mul- ation. tiple dose samples of Tetanus Anti- (iv) Representative samples of each toxin; serial or subserial in each shipment of (ii) Twelve samples of all other imported biological products shall be antitoxins. selected. (5) Toxoids: (2) Comparable samples shall be used (i) Eighteen single-dose or 12 mul- by Animal and Plant Health Inspection tiple dose samples of all toxoids. Service, the licensee, and the per- (6) Antigens: Twelve samples of poul- mittee for similar tests. try antigens or 20 samples of tuber- (3) When bulk samples of completed culin or four samples of all other diag- product in liquid form are to be tested nostic antigens. as prescribed in paragraph (a)(1) of this (7) Diagnostic test kits: Two samples of section, the number of such samples diagnostic test kits. The licensee or from each serial and the minimum permittee will hold one of these se- quantity of product to be provided in lected samples at the storage tempera- each sample shall be stated in the filed ture recommended on the label while Outline of Production. awaiting a request by the animal and (b) Unless otherwise prescribed by Plant Health Inspection Service to sub- the Administrator, the number of final mit the additional sample. If submis- container samples to be selected from sion is not requested by the Animal each serial and subserial shall be: and Plant Health Inspection Service, (1) Vaccines: the additional sample may be returned

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to the serial inventory after the serial with a minimum individual volume of 1 is released. In the case of diagnostic ml shall be submitted as follows: test kits in which final packaging con- (1) Ten samples of Bacterial Master sists of multiple microtiter test plates Seeds. or strips, the licensee or permittee may (2) Thirteen samples of viral Master submit a specified number of test Seeds or nonviral Master Seeds requir- plates or strips along with all other ing cell culture propagation. For Mas- test reagents as prescribed in a filed ter Seeds isolated or passed in a cell Outline of Production and retain a line different from the species of in- similar amount as a second sample for tended use, an additional 2 samples are submission upon request. When the ini- required for each additional species. tial sample is not representative of For Master Seeds grown in cell culture final packaging by the licensee of per- and intended for use in more than one mittee, e.g., does not consist of all the species, an additional 2 samples are re- microtiter test plates or strips, the sec- quired for each additional species. ond sample is not eligible to be re- (3) Thirty-six samples of at least 1 ml turned to serial inventory after the se- each or six samples of at least 1 ml rial is released. each, one sample of at least 20 ml, and (8) Autogenous biologics: With the ex- one sample of at least 10 ml of Master ception of the first serial or subserial, Cell Stocks. In the case of Master Cell 10 samples must be selected and sub- Stocks which are persistently infected mitted to the Animal and Plant Health with a virus, an additional four samples 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

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other test reagents as prescribed in a duction or the Standard Requirements filed Outline of Production; for the product shall be submitted to (2) Be adequate in quantity for appro- Animal and Plant Health Inspection priate examination and testing; Service. Blank forms shall be furnished (3) Be truly representative and in upon request to Animal and Plant final containers; Health Inspection Service. (4) Be held in a special compartment (d) When the initial or any subse- set aside by the licensee or permittee quent test is declared a No Test, the for holding these samples under refrig- reasons shall be reported in the test eration at the storage temperature rec- records, the results shall not be consid- ommended on the labels for 6 months ered as final, and the test may be re- 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 inconclusive, the reasons shall be reported in [38 FR 29886, Oct. 30, 1973, as amended at 40 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 inconclusive or No Test and the biologi- § 113.4 Exemptions to tests. cal product is not further tested, the (a) The test methods and procedures test designation of unsatisfactory is contained in all applicable Standard the final conclusion. Requirements shall be complied with (e) When new test methods are devel- unless otherwise exempted by the Ad- oped and approved by Animal and ministrator and provided that such ex- Plant Health Inspection Service, bio- emption is noted in the filed Outline of logical products tested thereafter shall Production for the product. be evaluated by such methods, and if (b) Test methods and procedures by not found to be satisfactory when so which the biological products shall be tested shall not be released. evaluated shall be designated in the Outline of Production for such prod- (Approved by the Office of Management and Budget under control number 0579–0059) ucts. [34 FR 18004, Nov. 4, 1969, as amended at 39 [38 FR 29887, Oct. 30, 1973, as amended at 56 FR 25463, July 11, 1974; 40 FR 45420, Oct. 2, FR 66784, Dec. 26, 1991] 1975; 40 FR 46093, Oct. 6, 1975; 41 FR 6751, Feb. 13, 1976; 48 FR 57473, Dec. 30, 1983; 56 FR 66784, § 113.5 General testing. Dec. 26, 1991; 79 FR 55969, Sept. 18, 2014] (a) No biological product shall be released prior to the completion of tests § 113.6 Animal and Plant Health In- prescribed in a filed Outline of Produc- spection Service testing. tion or Standard Requirements for the A biological product shall with rea- product to establish the product to be sonable certainty yield the results in- pure, safe, potent, and efficacious. tended when used as recommended or (b) Tests of biological products shall suggested in its labeling or proposed la- be observed by a competent employee beling prior to the expiration date. of the manufacturer during all critical (a) The Administrator is authorized periods. A critical period shall be the to cause a biological product, manufac- time when certain specified reactions tured in the United States or imported must occur in required tests to prop- into the United States, to be examined erly evaluate the results. and tested for purity, safety, potency, (c) Records of all tests shall be kept or efficacy; in which case, the licensee in accordance with part 116 of this or permittee shall withhold such prod- chapter. Results of all required tests uct from the market until a determina- prescribed in the filed Outline of Pro- tion has been made.

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(b) The final results of each test con- from a required animal potency test for ducted by the licensee and Animal and release when an evaluation can, with Plant Health Inspection Service shall reasonable certainty, be made by: be considered in evaluating a biological (1) Subjecting the master seed to the product. A serial or subserial which has applicable requirements prescribed in been found unsatisfactory by a re- §§ 113.64, 113.100, 113.200, and 113.300; quired test prescribed in a filed Outline (2) Testing the Master Seed for of Production or Standard Require- immunogenicity in a manner accept- ment is not in compliance with the reg- able to the Animal and Plant Health ulations and shall not be released for Inspection Service (APHIS); market. (3) Establishing satisfactory potency [34 FR 18004, Nov. 7, 1969, as amended at 40 for the product in accordance with the FR 45420, Oct. 2, 1975; 40 FR 53378, Nov. 18, following provisions: 1975; 41 FR 6751, Feb. 13, 1976; 56 FR 66784, (i) Potency for live products may be Dec. 26, 1991] determined by log10 virus titer or determining the live bacterial count § 113.7 Multiple fractions. based on the protective dose used in (a) When a biological product con- the Master Seed immunogenicity test tains more than one immunogenic frac- plus an adequate overage for adverse tion, the completed product shall be conditions and test error; and evaluated by tests applicable to each (ii) Potency for inactivated products fraction. may be determined using tests for rel- (b) When similar potency tests are re- ative antigen content by comparing quired for more than one fraction of a the antigen content of the test serial combination biological product, dif- to a reference preparation using a par- ferent animals must be used to evalu- allel line immunoassay or equivalent ate each fraction except when written method which measures linearity, Standard Requirements or outlines of specificity, and reproducibility in a production make provisions and set manner acceptable to APHIS. forth conditions for use of the same (b) In the case of live products, each animals for testing different fractions. serial and subserial of desiccated prod- (c) When the same safety test is re- uct derived from an approved Master quired for more than one fraction, re- Seed and bulk or final container sam- quirements are fulfilled by satisfactory ples of each serial of completed liquid results from one test of the completed product derived from an approved Mas- product. 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.

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(3) If the average is equal to or great- using immunoassays which do not sat- er than the required minimum, the fol- isfy this requirement shall have 2 years lowing shall apply to live virus vac- from the effective date of the final rule cines: to update their filed Outlines of Pro- (i) If the difference between the aver- duction to be in compliance with this age titer obtained in the retests and requirement unless granted an exten- 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

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retests 2 and 3 (tests 3 and 4) as follows: the data are acceptable to APHIS, the if either retest (test 3 or 4) shows that potency test may be repeated by the potency is less than the required min- firm, subject to the provisions specified imum potency, the serial is unsatisfac- in paragraphs (c)(4) (i) and (ii) and tory. If either retest 2 or retest 3 (tests (c)(5) (i) and (ii) of this section, and 3 or 4) is an equivocal test, or in the confirmatory testing by APHIS. 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 retested a minimum of two times (tests of the reference to determine when the 2 and 3) but not more than three times potency starts to decline and for tak- (tests 2, 3, and 4) with disposition as ing the appropriate steps to requalify a specified in paragraphs (c)(5) (i) and (ii) reference with declining potency either of this section; Provided, That, if the by testing a Qualifying Serial in host serial is not retested or the other pro- animals or by providing other evidence visions of this section are not satisfied, of immunogenicity, e.g., antibody the serial shall be deemed unsatisfac- titers or laboratory animal test data tory. previously correlated to host animal (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 ex- tended 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.

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(e) Final container samples of com- reevaluation of the product is made pleted product derived from Master and the problem is resolved. Seed found immunogenic in accordance [49 FR 22625, May 31, 1984, as amended at 56 with paragraph (a) of this section and FR 66784, 66786, Dec. 26, 1991; 62 FR 19038, Apr. found satisfactory in accordance with 18, 1997; 72 FR 72564, Dec. 21, 2007; 79 FR 31021, paragraphs (b) and (c) of this section May 30, 2014] may also be subjected to an animal potency test by Animal and Plant Health § 113.9 New potency test. Inspection Service as provided in this A potency test written into the filed paragraph. Products shall be used ac- Outline of Production for a product cording to label directions including shall be considered confidential infor- dose(s) and route of administration. mation by Animal and Plant Health In- (1) A one stage test using 20 vac- spection Service until at least two ad- cinates and 5 controls or a two stage ditional product licenses are issued for test using 10 vaccinates and 5 controls the product or unless use of the test is for each stage shall be used. The cri- authorized by the licensee, in which 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 are prescribed in the United States

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Pharmacopeia, or elsewhere in this § 113.26 Detection of viable bacteria part, shall conform to such standards. and fungi except in live vaccine. In lieu of preparing the media from the Each serial and subserial of biologi- individual ingredients, they may be cal product except live vaccines shall made from dehydrated mixtures which, be tested as prescribed in this section when rehydrated with purified water, unless otherwise specified by the Ad- have the same or equivalent composi- ministrator. When cell lines, primary tion as such media and have growth- cells, or ingredients of animal origin promoting buffering, and oxygen ten- used in the preparation of a biological sion-controlling properties equal to or product are required to be free of via- better than such media. The formulas ble bacteria and fungi, they shall also for the composition of the culture be tested as prescribed in this section. media prescribed in §§ 113.26 and 113.27 (a) The media to be used shall be as are set forth in the United States Phar- follows: macopeia, 19th Edition. (1) Fluid Thioglycollate Medium with (b) The licensee shall test each quan- 0.5 percent beef extract shall be used to tity of medium prepared at one time test for bacteria in biological products from individual ingredients and the containing clostridial toxoids, first quantity prepared from each lot of bacterins, and bacterin-toxoids. commercial dehydrated medium for (2) Fluid Thioglycollate Medium with growth-promoting qualities. If any por- or without 0.5 percent beef extract tion of a lot of commercial dehydrated shall be used to test for bacteria in bio- medium is held for 90 days or longer logical products other than clostridial after being so tested, it shall be re- toxoids, bacterins, and bacterin-tox- tested before use. Two or more strains oids. of micro-organisms that are exacting (3) Soybean-Casein Digest Medium in their nutritive requirements shall be shall be used to test biological prod- used. More than one dilution shall be ucts for fungi; provided, that Fluid used to demonstrate the adequacy of Thioglycollate Medium without beef the medium to support the growth of a extract shall be substituted when test- minimum number of micro-organisms. ing biological products containing mer- (c) The sterility of the medium shall curial preservatives. be confirmed by incubating an ade- (b) Test procedure: quate number of test vessels and exam- (1) Ten test vessels shall be used for ining each for growth. Additional con- each of two media selected in accord- trol may be used by incubation of rep- ance with paragraph (a)(1), (a)(2), or resentative uninoculated test vessels (a)(3) of this section. Each test vessel for the required incubation period dur- shall contain sufficient medium to ne- ing each test. gate the bacteriostatic or fungistatic 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.

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(3) Incubation shall be for an observa- tested for purity as prescribed in this tion period of 14 days at 30 °to 35 °C. to paragraph. However, products of chick- test for bacteria and 14 days at 20 °to 25 en embryo origin recommended for ad- °C. to test for fungi. ministration other than by parenteral (4) If the inoculum renders the me- injection may be tested as provided in dium turbid so that the absence of paragraph (e) of this section. growth cannot be determined by visual (1) Soybean Casein Digest Medium examination, subcultures shall be made shall be used. 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.

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

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(1) To test for extraneous bacteria, (4) From each container sample, each place 0.2 ml of the sample of Master of 2 plates shall be inoculated with vac- Seed Bacteria into 10 individual vessels cine equal to 10 doses if the vaccine is each containing at least 40 ml of Fluid recommended for poultry or 1 dose if Thioglycollate Medium. Incubation the vaccine is recommended for other shall be at 30 °to 35 °C for 14 days. animals. Twenty ml of medium shall be (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 injection, which were not tested or have not [48 FR 28430, June 22, 1983, as amended at 56 been found free of bacteria and fungi by FR 66784, Dec. 26, 1991] 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 this paragraph. The heart infusion test, using heart (1) Brain Heart Infusion Agar shall be infusion broth and heart infusion agar, used with 500 Kinetic (Kersey) units of provided in this section shall be con- penicillinase per ml of medium added ducted when a test for mycoplasma just prior to pouring the plates. contamination is prescribed in an ap- (2) Ten final containers from each se- plicable Standard Requirement or in rial and each subserial shall be tested. the filed Outline of Production for the (3) Immediately prior to starting the product. test, frozen liquid vaccine shall be (a) Media additives provided in this thawed, and lyophilized vaccine shall paragraph shall be prepared as follows: be rehydrated to the quantity rec- (1) DPN-Cysteine Solution: ommended on the label using the ac- (i) Use Nicotinamide adenine companying sterile diluent or sterile dinucleotide (oxidized) and L-Cysteine purified water. Product recommended hydrochloride. for mass vaccination shall be re- (ii) Prepare 1 gram/100 milliliters hydrated at the rate of 30 ml sterile pu- (ml) purified water (1 percent solution) rified water per 1,000 doses. of each. Mix the solutions together; the

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cysteine reduces the DPN. Filter steri- to be administered via the drinking lize, dispense in appropriate amounts water, the vaccine shall be rehydrated and store frozen at ¥20 degrees centi- to 30 ml with mycoplasma medium. In grade. the case of a cell line or a sample of (2) Inactivated horse serum—horse primary cells, the inoculum shall con- serum which has been inactivated at 56 sist of the resuspended cells. Control ° C for 30 minutes. tests shall be established as provided in (b) Heart infusion broth shall be pre- paragraph (d)(4) of this section. pared as provided in this paragraph. (2) Inoculation of plate. Plate 0.1 ml (1) Dissolve in 970 ml of purified 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 needed. shall be streaked with 0.1 ml of mate- (4) Add 2 ml of DPN-Cysteine solu- rial from the incubating flask of inocu- tion to each 100 ml of broth on day of lated medium on the 3d day, one on the use. 7th day, one on the 10th day, and one (c) Heart Infusion Agar shall be pre- on the 14th day post-inoculation. pared as provided in this paragraph. (4) Control tests shall be conducted (1) Dissolve in 900 ml of purified simultaneously with the detection test water by boiling the following: using techniques provided in para- Heart infusion agar (g) ...... 25 graphs (d)(2) and (3) of this section, ex- Heart-infusion broth (g) ...... 10 cept the inoculum for the positive con- Proteose peptone No. 3 (g) ...... 10 1 pct thallium acetate (ml) ...... 25 trol test shall be selected mycoplasma (2) Cool the medium and adjust pH to cultures and the negative control test 7.9 with NaOH. shall be uninoculated medium from the (3) Autoclave the medium. same lot used in the detection test. (4) Cool the medium 30 minutes in a (5) All plates shall be incubated in a 56 °C waterbath. high humidity, 4–6 percent CO2 atmos- (5) Dissolve 5 grams of yeast autoly- phere at 33 °to 37 °C for 10–14 days and sate in 100 ml of distilled water, filter examined with a stereoscopic micro- sterilize, and add to the medium. scope at 35x to 100x or with a regular (6) Add to the medium: microscope at 100x. 126 ml of inactivated horse serum (e) Interpretation of test results. 21 ml of DPN-Cysteine solution (1) If growth appears on at least one 525,000 units of Penicillin. of the plates in the positive control Dispense 10 ml aliquots into 60 × 15 mm dis- test and does not appear on any of the posable culture dishes or petri dishes. plates in the negative control test, the (d) The test procedure provided in test is valid. this paragraph shall be followed when (2) If mycoplasma colonies are found conducting the mycoplasma detection on any of the plates inoculated with test. material being tested, the results are (1) Preparation of inoculum. Imme- positive for mycoplasma contamina- diately prior to starting the test, fro- tion. zen liquid vaccine shall be thawed, and lyophilized vaccine shall be rehydrated [38 FR 29887, Oct. 30, 1973, as amended at 41 to the volume recommended on the FR 6752, Feb. 13, 1976; 41 FR 32882, Aug. 6, label with mycoplasma medium. In the 1976] case of a lyophilized biological product, e.g., 1,000 dose vial of poultry vaccine

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§ 113.29 Determination of moisture (ii) Insert the stopper and weigh and content in desiccated biological record the weights of the weighing bot- products. tles as ‘‘B.’’ Methods provided in this section (3) Place the weighing bottle with the must be used when a determination of stopper at an angle in the vacuum moisture content in desiccated biologi- oven. Set the vacuum to <2.5 kPa and cal products is prescribed in an appli- the temperature to 60 ±3 °C. cable Standard Requirement or in the (4) After a minimum of 3 hours of filed Outline of Production for the drying time, turn off the vacuum pump 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 (B¥C)/(B¥A) × (100) = Percentage of re- airtight glass stoppers. sidual moisture, where: (2) Vacuum oven equipped with vali- A = tare weight of weighing bottle dated thermometer and thermostat. A B¥A = weight of sample before drying suitable air-drying device should be at- B¥C = weight of sample after drying tached to the inlet valve. (7) The results are considered satis- (3) Balance, accurate to 0.1 mg (rated factory if the percentage of residual precision ±0.01mg). moisture is less than or equal to the (4) Desiccator jar equipped with phos- manufacturer’s specification. phorous pentoxide, silica gel, or equivalent. [68 FR 57608, Oct. 6, 2003] (5) Desiccated vaccine in original sealed vial. Sample and control should § 113.30 Detection of Salmonella con- be kept at room temperature in their tamination. original airtight containers until use. The test for detection of Salmonella (b) Test procedure: contamination provided in this section (1) Thoroughly cleaned and labeled shall be conducted when such a test is sample-weighing bottles with stoppers prescribed in an applicable Standard should be allowed to dry at 60 ±3 °C Requirement or in the filed Outline of under vacuum at less than 2.5 kPa. Production for the product. (i) Transfer hot bottles and stoppers (a) Samples shall be collected from into the desiccator and allow to cool to the bulk suspension before room temperature. bacteriostatic or bactericidal agents (ii) After bottles have cooled, insert have been added. When tissue culture stoppers and weigh and record the products are to be tested, 1 ml of tissue weights of the bottles as ‘‘A.’’ extract used as the source of cells or 1 (iii) Return weighing bottles to the ml of the minced tissue per se shall be desiccator. tested. (2) Remove the sample container (b) Five ml of the liquid vaccine sus- seal. pension shall be used to inoculate each (i) Using a spatula, break up the sam- 100 ml of liquid broth medium (tryptose ple plug and transfer the required and either selenite F or tetrathionate). amount of sample to the previously The inoculated media shall be incu- tared weighing bottle. bated 18–24 hours at 35–37 °C.

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(c) Transfers shall be made to either 500 doses of other vaccines for use in MacConkey agar or Salmonella- poultry, or one dose of vaccine for use Shigella agar, incubated for 18–24 hours in other animals shall be used as and examined. inoculum. Control cultures shall be (d) If no growth typical of Salmonella prepared from the same cell suspension is noted, the plates shall be incubated as the cultures for testing the vaccine. an additional 18–24 hours and again ex- (3) Uninoculated chick embryo fibro- amined. blast cell cultures shall act as negative (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 li- percent hemolytic units or two 100 per- censee can demonstrate to Animal and cent hemolytic units of complement Plant Health Inspection Service that shall be used for each test. the agent used to inactivate the vac- (1) All test materials, including posi- cine virus would also inactivate lymph- tive and negative controls, shall be oid leukosis virus. ¥ ° (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 an- (3) Presence of complement-fixing ac- other vaccine prepared the same week tivity in the harvested samples (from from material harvested from each passages) at the 1:4 or higher dilution, source flock (or other sampling proce- in the absence of anticomplementary dure acceptable to Animal and Plant activity, shall be considered a positive Health Inspection Service) used for the test unless the activity can definitely preparation of the questionable vaccine be established to be caused by some- virus that cannot be neutralized, inac- thing other than lymphoid leukosis tivated, or separated shall be tested virus, subgroups A and/or B. Activity each week during the preparation of at the 1:2 dilution shall be considered such questionable vaccine. suspicious and the sample further sub- (2) When cell cultures are tested, 5 ml cultured to determine presence or ab- of the final cell suspension as prepared sence of the group specific antigen. for seeding of production cell cultures (4) Biological products or primary shall be used as inoculum. When vac- cells which are found contaminated cines are tested, the equivalent of 200 with lymphoid leukosis viruses are un- doses of Newcastle disease vaccine or satisfactory. Source flocks from which

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contaminated material was obtained (1) Vaccine prepared for use as rec- are also unsatisfactory. ommended on the label shall be tested by inoculating eight mice [38 FR 29888, Oct. 30, 1973, as amended at 38 intraperitoneally or subcutaneously FR 32917, Nov. 29, 1973; 39 FR 21042, June 18, 1974; 56 FR 66784, Dec. 26, 1991] with 0.5 mL (the inoculation volume may be divided among more than one § 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

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inoculum: Provided, That poultry vac- or dilution of the vaccine beyond the cines distributed without diluent shall titer range of the other fraction(s), or be rehydrated with 30 ml of sterile dis- the test shall be conducted using rep- tilled water per 1,000 doses and used as resentative single-fraction desiccated inoculum. When one or more fractions vaccines which are prepared by the li- are to be used in combination with censee and which are licensed. Pro- 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- ° ° tion, a sample of allantoic fluid from room temperature (20 to 25 C) for 2 each egg shall be tested separately by a hours. The holding period shall begin rapid plate test for hemagglutinating when rehydration is completed. 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 requirement or in the filed Outline of (f) Liquid products found to be unsat- Production for each inactivated liquid isfactory for use as diluent by this test biological product used as diluent for a are not prohibited from release as sepa- desiccated live virus vaccine in a com- rate licensed products if labeled as pre- bination package. scribed in § 112.7(g). (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

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Requirement or in the filed Outline of of desiccated vaccine to be used in Production for the product. poultry, rehydrated with sterile dis- (a) The biological product to be test- tilled water at the rate of 30 ml per ed shall be prepared for use as rec- 1,000 doses. ommended on the label, or in the case (b) One volume of the prepared vac- of desiccated vaccine to be used in cine shall be mixed with up to nine vol- 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,

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a 2 ml dose shall be injected either (3) If unfavorable reactions attrib- intramuscularly or subcutaneously utable to the virus occur in any of the into each of two guinea pigs and the cats during the observation period, the animals observed for 7 days. Master Seed Virus is unsatisfactory. If (b) If unfavorable reactions attrib- unfavorable reactions occur which are utable to the product occur in either of not attributable to the Master Seed the guinea pigs during the observation Virus, the test shall be declared a No period, the serial or subserial is unsat- Test and repeated: Provided, That, if isfactory. If unfavorable reactions not repeated, the Master Seed Virus which are not attributable to the prod- shall be unsatisfactory. uct occur, the test shall be declared a (b) The cat safety test provided in No Test and may be repeated: Provided, this paragraph shall be used when a se- That, if the test is not repeated, the serial of vaccine is tested for safety be- rial or subserial shall be declared un- fore release. satisfactory. (1) Each of two healthy cats shall be administered 10 cat doses by the meth- [39 FR 16857, May 10, 1974; 39 FR 20368, June od recommended on the label and the 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 The safety tests provided in this sec- during the observation period, the se- tion shall be conducted when pre- rial is unsatisfactory. If unfavorable scribed in a standard requirement or in reactions occur which are not attrib- the filed Outline of Production for a bi- utable to the product, the test shall be ological product recommended for use declared a No Test and repeated: Pro- in cats. vided, That, if not repeated, the serial (a) The cat safety test provided in shall be unsatisfactory. 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

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

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Virus is unsatisfactory for use to peated, the serial or subserial shall be produce vaccine. declared unsatisfactory. [44 FR 58899, Oct. 12, 1979] [48 FR 33476, July 22, 1983]

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

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not be used to prepare biological prod- after subculturing shall be stained at ucts. If an extraneous agent is sus- the same time as the test monolayers pected because of cytopathology or and negative controls fixed at least 7 hemadsorption and cannot be elimi- days after subculturing. nated as a possibility by additional (b) The antiviral fluorochrome-con- testing, the material under test is un- jugated antibodies to be used shall de- satisfactory. pend on the type of cells required to be tested for extraneous viruses as speci- [50 FR 441, Jan. 4, 1985, as amended at 58 FR 50252, Sept. 27, 1993] fied in an applicable Standard Require- ment or in a filed Outline of Produc- § 113.47 Detection of extraneous vi- tion. Antiviral fluorochrome-con- ruses by the fluorescent antibody jugated antibodies specific for the ex- technique. traneous 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

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

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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 by dissolving 1 gram of peptone and Type C Toxoid and Bacterin-Toxoid. 0.25 grams of sodium chloride in each Clostridium Perfringens Type C Tox- 100 ml of distilled water; adjusting the oid and Clostridium Perfringens Type C pH to 7.2; autoclaving at 250 °F for 25 Bacterin-Toxoid shall be produced from minutes; and storing at 4 °C until used. a culture of Clostridium perfringens (2) Each of at least eight rabbits of a Type C which has been inactivated and strain acceptable to APHIS, each is nontoxic. Each serial 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 largest recommended dose potency as prescribed in this section. for any species indicated on the prod- Any serial found unsatisfactory by a uct label. A second equivalent dose prescribed test shall not be released. shall be given not less than 20 days nor (a) Purity test. Final container sam- more than 23 days after the first does. ples of completed product from each se- (3) Fourteen to seventeen days after rial and each subserial shall be tested the second dose, all surviving rabbits for viable bacteria and fungi as pro- shall be bled and the serum tested for vided in § 113.26. antitoxin content. (b) Safety test. Bulk or final container (i) At least seven rabbits are required samples of completed product from to make an acceptable serum pool. each serial shall be tested for safety as (ii) Equal quantities of serum from provided in § 113.33(b). each rabbit shall be combined and test- (c) Potency test. Bulk or final con- ed as a single pooled serum. tainer samples of completed product (iii) If less than seven rabbits are from each serial shall be tested for po- available, the test is a No Test and tency using the Beta toxin-neutraliza- shall be repeated: Provided, That, if the tion test provided in this paragraph. test is not repeated, the serial shall be (1) When used in this test, the fol- declared unsatisfactory. lowing words and terms shall mean: (4) The antitoxin content of the rab- (i) International antitoxin unit. (I.U.) bit serums shall be determined as fol- That quantity of Beta Antitoxin which lows: reacts with L and L + doses of Stand- 0 (i) Make a dilution of Standard Anti- ard Toxin according to their defini- toxin to contain 10 International Units tions. of antitoxin per ml. (ii) L 0 dose. The largest quantity of 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 quantity of Toxin to contain 10 L ∂ doses per ml. toxin which can be mixed with one unit (iii) Combine 10 International Units of Standard Antitoxin and cause death of Standard Antitoxin with 10 L 0 doses in at least 80 percent of injected mice. of diluted Standard Toxin and combine (iv) Standard antitoxin. The Beta 10 International Units of Standard Antitoxin preparation which has been Antitoxin with 10 L∂ doses of diluted standardized as to antitoxin unitage on Standard Toxin. the basis of the International Clos- (iv) Combine 1 ml of undiluted serum tridium perfringens Beta Antitoxin with 10 L0 doses of diluted Standard Standard and which is either supplied Toxin. by or acceptable to Animal and Plant (v) Neutralize all toxin-antitoxin Health Inspection Service. The anti- mixtures at room temperature for 1 toxin unit value shall be stated on the hour and hold in ice water until injec- label. tions of mice can be made. (v) Standard toxin. The Beta toxin (vi) Five Swiss white mice, each preparation which is supplied by or is weighing 16–20 grams, shall be used for acceptable to Animal and Plant Health each toxin-antitoxin mixture. A dose of Inspection Service. 0.2 ml shall be injected intravenously (vi) Diluent. The solution used to into each mouse. Conclude the test 24 make proper dilutions prescribed in hours post-injection and record all this test. Such solutions shall be made 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 pre- served 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 codel oflfederallregulations/ 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 Parainfluenza3 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 un- used 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- SOURCE: 39 FR 16869, May 10, 1974, unless other type of study acceptable to otherwise noted. APHIS, to demonstrate functionality of product antibody. § 114.1 Applicability. (c) Potency. Bulk or final container Unless exempted by regulation or samples of completed product from otherwise authorized by the Adminis- each serial shall be tested for IgG con- trator, all biological products prepared, tent as provided in this paragraph. sold, bartered or exchanged, shipped or Samples of the test serial and of an IgG delivered for shipment in or from the Reference Product established in ac- United States, the District of Colum- cordance with paragraph (a) of this sec- bia, any Territory of the United States, tion shall be concurrently tested for or any place under the jurisdiction of IgG content by the RID method re- the United States shall be prepared in ferred to in paragraph (a)(5) of this sec- accordance with the regulations in this tion. Five IgG measurements shall be part. The licensee or permittee shall made on each. If the IgG level per dose adopt and enforce all necessary meas- of the test serial does not meet or ex- ures and shall comply with all direc- ceed that of the reference, one com- tions the Administrator prescribes for plete retest, involving five IgG meas- carrying out such regulations. urements on both the reference and two samples of the test serial, may be [52 FR 11026, Apr. 7, 1987, as amended at 56 conducted. If, upon retest, the average FR 66784, Dec. 26, 1991] IgG level per dose of the two samples of the test serial does not meet or exceed § 114.2 Products not prepared under li- that of the reference, or if a retest is cense. not conducted, the serial is unsatisfac- (a) When an establishment license is tory. issued, if biological products which [61 FR 51777, Oct. 4, 1996] were not prepared in compliance with the regulations are in the establish- PART 114—PRODUCTION REQUIRE- ment, such products shall not be MENTS FOR BIOLOGICAL PROD- shipped or delivered for shipment or otherwise dealt with as having been UCTS prepared under such regulations. Sec. (b) Except as provided in 9 CFR part 114.1 Applicability. 103, a biological product shall not be 114.2 Products not prepared under license. prepared in a licensed establishment 114.3 Separation of establishments. unless the person to whom the estab- 114.4 Identification of biological products. lishment license is issued holds an un- 114.5 Micro-organisms used as seed. expired, unsuspended, and unrevoked 114.6 Mixing biological products. product license issued by the Adminis- 114.7 Personnel at licensed establishments. trator to prepare such biological prod- 114.8 Outline of Production required. 114.9 Outline of Production guidelines. uct, or unless the products prepared 114.10 Antibiotics as preservatives. are subject to the provisions of § 107.2 114.11 Storage and handling. of this subchapter. 114.12 Expiration date required for a serial. (c) A biological product produced in a 114.13 Determination of the dating period of USDA-licensed establishment shall be a product. produced under a U.S. Veterinary Bio- 114.14 Extension of expiration date for a se- logical Product License or a license rial or subserial. 114.15 Disposal of unsatisfactory products granted by a State under § 107.2 (re- and byproducts. ferred to as a State biological product 114.16 Producing subsidiaries. license and the products prepared pur- 114.17 Rebottling of biological products. suant thereto as State-licensed biologi- 114.18 Reprocessing of biological products. cal products, including autogenous bio- AUTHORITY: 21 U.S.C. 151–159; 7 CFR 2.22, logics), but not under both a U.S. Vet- 2.80, and 371.4. erinary Biological Product License and

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