資料由法律人 LawPlayer整理提供·U.S. federal law / curated by LawPlayer from GPO govinfo & eCFR
The regulations in this part apply to each serial or subserial of a licensed biological product manufactured in a licensed establishment and to each serial or subserial of a biological product in each shipment imported for distribution and sale.
To better ensure consistent and reproducible test results when Standard Requirement tests prescribed in the regulations are conducted, National Veterinary Services Laboratories, U.S. Department of Agriculture, may provide testing aids, when available, to licensees, permittees, and applicants for licenses and permits. Such aids shall be as follows:
(a) Supplemental Assay Method (SAM) is a technical bulletin containing detailed instructions for conducting a test. Such instructions shall be in accordance with the procedures currently being followed at National Veterinary Services Laboratories and as improved, proven procedures are developed, shall be revised and reissued prior to application.
(b) Standard Reference Preparation is a serum, virus, bacterial culture, or antigen to be used in test systems for direct comparison with serials of biological products under test.
(c) Standard Test Reagent is a serum, antitoxin, fluorescent antibody conjugate, toxin, virus, bacterial cultural, or antigen to be used in test systems but not for direct comparison with serials of biological products under test.
(d) Seed cultures are small quantities of standard organisms to be propagated by the recipient to establish a supply for use.
(e) Test Code Number is a number assigned by Animal and Plant Health Inspection Service to each test procedure specified in the Standard Requirements and in each filed Outline of Production where such test is conducted to support a request for release of a serial or subserial.
Each licensee and permittee shall furnish representative samples of each serial or subserial of a biological product manufactured in the United States or imported into the United States as prescribed in this section. Additional samples may be purchased in the open market by a Animal and Plant Health Inspection Service representative.
(a) Either an employee of the Department of Agriculture, of the licensee, or of the permittee, as designated by the Administrator shall select prerelease samples of biological product in the number prescribed in paragraph (b) of this section. Each sample shall be marked for identification by the person making the selection after which they shall be packaged by the licensee or permittee, as the case may be, and forwarded to National Veterinary Services Laboratories; except that an employee of the Department may forward or deliver the samples to National Veterinary Services Laboratories if such action deemed advisable by the Administrator.
(1) Selection shall be made as follows:
(i) Nonviable liquid biological products—either bulk or final container samples of completed product shall be selected for purity, safety, or potency tests. Biological product in final container shall be selected to test for viable bacteria and fungi.
(ii) Viable liquid biological products; samples shall be in final containers and shall be randomly selected at the end of the filling operation. Bulk containers of completed product may be sampled when authorized by the Administrator.
(iii) Desiccated biological products; samples shall be in final containers and shall be randomly selected if desiccated in the final container. Biological products desiccated in bulk shall be sampled at the end of the filling operation.
(iv) Representative samples of each serial or subserial in each shipment of imported biological products shall be selected.
(2) Comparable samples shall be used by Animal and Plant Health Inspection Service, the licensee, and the permittee for similar tests.
(3) When bulk samples of completed product in liquid form are to be tested as prescribed in paragraph (a)(1) of this section, the number of such samples from each serial and the minimum quantity of product to be provided in each sample shall be stated in the filed Outline of Production.
(b) Unless otherwise prescribed by the Administrator, the number of final container samples to be selected from each serial and subserial shall be:
(1) Vaccines:
(i) Six multiple-dose samples of Brucella Abortus Vaccine;
(ii) Twelve samples of all other live bacterial vaccines;
(iii) Two samples of Coccidiosis Vaccine;
(iv) Eighteen samples of Rabies Vaccine, Modified Live Virus;
(v) Sixteen samples of all other vaccines consisting of live microorganisms;
(vi) Thirty single-dose or 14 multiple-dose samples of Equine Encephalomyelitis Vaccine, Killed Virus;
(vii) Twenty-two single-dose or 14 multiple-dose samples of Rabies Vaccine, Killed Virus;
(viii) Sixteen single-dose or 12 multiple-dose samples of all other vaccines consisting of killed microorganisms.
(2) Bacterins and bacterin-toxoids:
(i) Twelve samples of single-fraction products;
(ii) Thirteen samples of two-fraction products;
(iii) Fourteen samples of products consisting of 3 or more fractions.
(3) Antiserums: Twelve samples of antiserum recommended for large animals or 14 samples of antiserum recommended for small animals or the number of reagent serum samples prescribed in the filed Outline of Production for the product.
(4) Antitoxins:
(i) Fourteen single-dose or 12 multiple dose samples of Tetanus Antitoxin;
(ii) Twelve samples of all other antitoxins.
(5) Toxoids:
(i) Eighteen single-dose or 12 multiple dose samples of all toxoids.
(6) Antigens: Twelve samples of poultry antigens or 20 samples of tuberculin or four samples of all other diagnostic antigens.
(7) Diagnostic test kits: Two samples of diagnostic test kits. The licensee or permittee will hold one of these selected samples at the storage temperature recommended on the label while awaiting a request by the animal and Plant Health Inspection Service to submit the additional sample. If submission is not requested by the Animal and Plant Health Inspection Service, the additional sample may be returned to the serial inventory after the serial is released. In the case of diagnostic test kits in which final packaging consists of multiple microtiter test plates or strips, the licensee or permittee may submit a specified number of test plates or strips along with all other test reagents as prescribed in a filed Outline of Production and retain a similar amount as a second sample for submission upon request. When the initial sample is not representative of final packaging by the licensee of permittee, e.g., does not consist of all the microtiter test plates or strips, the second sample is not eligible to be returned to serial inventory after the serial is released.
(8) Autogenous biologics: With the exception of the first serial or subserial, 10 samples must be selected and submitted to the Animal and Plant Health Inspection Service from each serial or subserial of an autogenous biologic eligible to be shipped that consists of more than 50 containers. For first serials or subserials eligible for shipment consisting of more than 50 containers, 10 samples from each serial or subserial must be selected and held for submission to the Animal and Plant Health Inspection Service upon request in accordance with paragraph (e)(4) of this section. For serials or subserials of autogenous biologic with 50 or fewer containers, no samples, other than those required by paragraph (e) of this section, are required.
(9) Miscellaneous: The number of samples from products not in the categories provided for in paragraphs (b)(1) through (b)(8) of this section shall be prescribed in the filed Outline of Production for the product.
(c) Prelicensing and Outline of Production changes: Samples needed to support a license application or a change in the Outline of Production for a licensed product shall be submitted only upon request from the animal and Plant Health Inspection Service. Except for miscellaneous products specified in paragraph (b)(9) of this section, the number of such samples shall be at least one and one-half times the number prescribed for such product in paragraph (b) of this section. Samples of Master Seeds and Master Cell Stocks with a minimum individual volume of 1 ml shall be submitted as follows:
(1) Ten samples of Bacterial Master Seeds.
(2) Thirteen samples of viral Master Seeds or nonviral Master Seeds requiring cell culture propagation. For Master Seeds isolated or passed in a cell line different from the species of intended use, an additional 2 samples are required for each additional species. For Master Seeds grown in cell culture and intended for use in more than one species, an additional 2 samples are required for each additional species.
(3) Thirty-six samples of at least 1 ml each or six samples of at least 1 ml each, one sample of at least 20 ml, and one sample of at least 10 ml of Master Cell Stocks. In the case of Master Cell Stocks which are persistently infected with a virus, an additional four samples of at least 1 ml each are required. If these persistently infected cell stocks are intended for use in more than one species, an additional two samples of at least 1 ml each are required for each additional species.
(4) Four samples of the Master Cell Stock + n (highest passage) cells.
(d) Sterile diluent: A sample of Sterile Diluent shall accompany each sample of product, other than Marek's Disease Vaccine, if such diluent is required to rehydrate or dilute the product before use. The volume of diluent shall be an appropriate amount to rehydrate or dilute the product. Samples of Sterile Diluent prepared for use with Marek's Disease Vaccine shall be submitted upon request from the Animal and Plant Health Inspection Service.
(e) Reserve samples shall be selected from each serial and subserial of biological product. Such samples shall be selected at random from final containers of completed product by an employee of the Department, of the licensee, or of the permittee, as designated by the administrator. Each sample shall:
(1) Consist of 5 single-dose packages, 2 multiple-dose packages, or 2 diagnostic test kits, except that, in the case of diagnostic test kits in which final packaging consists of multiple microtiter test plates or strips, a sample may consist of a specified number of test plates or strips along with all other test reagents as prescribed in a filed Outline of Production;
(2) Be adequate in quantity for appropriate examination and testing;
(3) Be truly representative and in final containers;
(4) Be held in a special compartment set aside by the licensee or permittee for holding these samples under refrigeration at the storage temperature recommended on the labels for 6 months after the expiration date stated on the labels. The samples that are stored in this manner shall be delivered to the Animal and Plant Health Inspection Service upon request.
(Approved by the Office of Management and Budget under control number 0579-0013)
(a) The test methods and procedures contained in all applicable Standard Requirements shall be complied with unless otherwise exempted by the Administrator and provided that such exemption is noted in the filed Outline of Production for the product.
(b) Test methods and procedures by which the biological products shall be evaluated shall be designated in the Outline of Production for such products.
(a) No biological product shall be released prior to the completion of tests prescribed in a filed Outline of Production or Standard Requirements for the product to establish the product to be pure, safe, potent, and efficacious.
(b) Tests of biological products shall be observed by a competent employee of the manufacturer during all critical periods. A critical period shall be the time when certain specified reactions must occur in required tests to properly evaluate the results.
(c) Records of all tests shall be kept in accordance with part 116 of this chapter. Results of all required tests prescribed in the filed Outline of Production or the Standard Requirements for the product shall be submitted to Animal and Plant Health Inspection Service. Blank forms shall be furnished upon request to Animal and Plant Health Inspection Service.
(d) When the initial or any subsequent test is declared a No Test, the reasons shall be reported in the test records, the results shall not be considered as final, and the test may be repeated. When a test is declared satisfactory, the test designation is considered to be a final conclusion. When a test is declared unsatisfactory, the test designation is considered to be a final conclusion. When the initial or any subsequent test is declared inconclusive, the reasons shall be reported in the test records, the result shall not be considered as final, and the test may be repeated as established in the filed Outline of Production or Standard Requirement. If a test is designated inconclusive or No Test and the biological product is not further tested, the test designation of unsatisfactory is the final conclusion.
(e) When new test methods are developed and approved by Animal and Plant Health Inspection Service, biological products tested thereafter shall be evaluated by such methods, and if not found to be satisfactory when so tested shall not be released.
(Approved by the Office of Management and Budget under control number 0579-0059)
A biological product shall with reasonable certainty yield the results intended when used as recommended or suggested in its labeling or proposed labeling prior to the expiration date.
(a) The Administrator is authorized to cause a biological product, manufactured in the United States or imported into the United States, to be examined and tested for purity, safety, potency, or efficacy; in which case, the licensee or permittee shall withhold such product from the market until a determination has been made.
(b) The final results of each test conducted by the licensee and Animal and Plant Health Inspection Service shall be considered in evaluating a biological product. A serial or subserial which has been found unsatisfactory by a required test prescribed in a filed Outline of Production or Standard Requirement is not in compliance with the regulations and shall not be released for market.
(a) When a biological product contains more than one immunogenic fraction, the completed product shall be evaluated by tests applicable to each fraction.
(b) When similar potency tests are required for more than one fraction of a combination biological product, different animals must be used to evaluate each fraction except when written Standard Requirements or outlines of production make provisions and set forth conditions for use of the same animals for testing different fractions.
(c) When the same safety test is required for more than one fraction, requirements are fulfilled by satisfactory results from one test of the completed product.
(d) When an inactivated fraction(s) is used as a diluent for a live virus fraction(s), the inactivated fraction(s) may be tested separately and the live virus fraction(s) may be tested separately: Provided, That, the viricidal test requirements prescribed in § 113.100 are complied with.
(e) Virus titrations for a multivirus product shall be conducted by methods which will quantitate each virus.
(a) Master Seed which has been established as pure, safe, and immunogenic shall be used for preparing seed for production as specified in the Standard Requirements or in the filed Outline of Production. The Administrator may exempt a product from a required animal potency test for release when an evaluation can, with reasonable certainty, be made by:
(1) Subjecting the master seed to the applicable requirements prescribed in §§ 113.64, 113.100, 113.200, and 113.300;
(2) Testing the Master Seed for immunogenicity in a manner acceptable to the Animal and Plant Health Inspection Service (APHIS);
(3) Establishing satisfactory potency for the product in accordance with the following provisions:
(i) Potency for live products may be determined by log 10 virus titer or determining the live bacterial count based on the protective dose used in the Master Seed immunogenicity test plus an adequate overage for adverse conditions and test error; and
(ii) Potency for inactivated products may be determined using tests for relative antigen content by comparing the antigen content of the test serial to a reference preparation using a parallel line immunoassay or equivalent method which measures linearity, specificity, and reproducibility in a manner acceptable to APHIS.
(b) In the case of live products, each serial and subserial of desiccated product derived from an approved Master Seed and bulk or final container samples of each serial of completed liquid product derived from an approved Master Seed shall be evaluated by a test procedure acceptable to APHIS. On the basis of the results of the test, as compared with the required minimum potency, each serial and subserial shall either be released to the firm for marketing or withheld from the market. The evaluation of such products shall be made in accordance with the following criteria:
(1) If the initial test shows the count or titer to equal or exceed the required minimum, the serial or subserial is satisfactory without additional testing.
(2) If the initial test shows the count or titer to be lower than the required minimum, the serial or subserial may be retested, using double the number of samples. The average counts or titers obtained in the retests shall be determined. If the average is less than the required minimum, the serial or subserial is unsatisfactory without further consideration.
(3) If the average is equal to or greater than the required minimum, the following shall apply to live virus vaccines:
(i) If the difference between the average titer obtained in the retests and the titer obtained in the initial test is 10
0.7 or greater, the initial titer may be considered a result of test system error and the serial or subserial considered satisfactory for virus titer.
(ii) If the difference between the average titer obtained in the retests and the titer obtained in the initial test is less than 10
0.7 , a new average shall be determined using the titers obtained in all tests. If the new average is below the required minimum, the serial or subserial is unsatisfactory.
(4) If the average is equal to or greater than the required minimum, the following shall apply to bacterial vaccines:
(i) If the average count obtained in the retests is at least three times the count obtained in the initial test, the initial count may be considered a result of test system error and the serial or subserial considered satisfactory for bacterial count.
(ii) If the average count obtained in the retests is less than three times the count obtained in the initial test, a new average shall be determined using the counts obtained in all tests. If the new average count is below the required minimum, the serial or subserial is unsatisfactory.
(5) Exceptions. When a product is evaluated in terms other than log 10 virus titer or organism count, an appropriate difference between the average potency value obtained in the retests and the potency value obtained in the initial test shall be established for use in paragraphs (b)(3) and (b)(4) of this section to evaluate such products and shall be specified in the product Standard Requirement or filed Outline of Production.
(c) In the case of inactivated products, bulk or final container samples of completed product from each serial derived from an approved Master Seed, shall be evaluated for relative antigen content (potency) as compared with an unexpired reference by a parallel line immunoassay or other procedure acceptable to APHIS. Firms currently using immunoassays which do not satisfy this requirement shall have 2 years from the effective date of the final rule to update their filed Outlines of Production to be in compliance with this requirement unless granted an extension by the Administrator based on a showing by the firm seeking the extension that they have made a good faith effort with due diligence to achieve compliance. On the basis of the results of such test procedures, each serial that meets the required minimum potency shall be released to the firm for marketing; each serial not meeting the required minimum potency shall be withheld from the market. The evaluation of such products shall be made in accordance with the following criteria:
(1) A test that results in no valid lines is considered a “no test” and may be repeated.
(2) An initial test (test 1) that results in valid lines that are not parallel is considered a valid equivocal test. Release of the serial may not be based on such test since the result cannot be termed “satisfactory” or “unsatisfactory.”
(3) If the initial test (test 1) shows that potency equals or exceeds the required minimum potency, the serial is satisfactory without additional testing.
(4) If the initial test (test 1) is an equivocal test due to lack of parallelism, the serial may be retested up to three times (tests 2, 3, and 4) with disposition to be as specified in paragraphs (c)(4)(i) and (ii) of this section; Provided, That, if the serial is not retested or the other provisions of this section are not satisfied, the serial shall be deemed unsatisfactory.
(i) If: The first retest (test 2) following an initial equivocal test; the second retest (test 3) following two consecutive equivocal tests (tests 1 and 2); or the third retest (test 4) following three consecutive equivocal tests (tests 1, 2, and 3) shows that the potency equals or exceeds the required minimum potency, the serial is satisfactory.
(ii) If the first retest (test 2) following an initial equivocal test shows that potency is less than the required minimum potency, disposition of the serial will be based on the outcome of retests 2 and 3 (tests 3 and 4) as follows: if either retest (test 3 or 4) shows that potency is less than the required minimum potency, the serial is unsatisfactory. If either retest 2 or retest 3 (tests 3 or 4) is an equivocal test, or in the event that each retest (tests 2, 3, and 4) following an initial equivocal test is also an equivocal test, the accumulated test results shall be considered indicative of a lack of potency and release of the serial withheld. In which case, the licensee may submit data confirming the continued validity of the test system to APHIS for review and approval. If the data are acceptable to APHIS, the potency test may be repeated by the firm, subject to the provisions specified in paragraphs (i) and (ii) and confirmatory testing by APHIS.
(5) If the initial test (test 1) shows that potency is less than the required minimum potency, the serial may be retested a minimum of two times (tests 2 and 3) but not more than three times (tests 2, 3, and 4) with disposition as specified in paragraphs (c)(5) (i) and (ii) of this section; Provided, That, if the serial is not retested or the other provisions of this section are not satisfied, the serial shall be deemed unsatisfactory.
(i) If two consecutive retests (tests 2 and 3) show that potency of the serial equals or exceeds the required minimum potency, the serial is satisfactory. If one of the two retests (test 2 or 3) shows that the potency is less than the required minimum potency, the serial is unsatisfactory.
(ii) If one of the retests (tests 2 or 3) shows that the potency equals or exceeds the required minimum potency and the other retest (test 2 or 3) is an equivocal test, a third retest (test 4) may be performed. If the third retest (test 4) shows that the potency of the serial equals or exceeds the required minimum potency, the serial is deemed satisfactory. If both retests (tests 2 and 3) or if the third retest (test 4) is an equivocal test, the accumulated test results shall be considered indicative of a lack of potency and release of the serial withheld, in which case the licensee may submit data confirming the continued validity of the test system to APHIS for review and approval. If the data are acceptable to APHIS, the potency test may be repeated by the firm, subject to the provisions specified in paragraphs (c)(4) (i) and (ii) and (c)(5) (i) and (ii) of this section, and confirmatory testing by APHIS.
(d) Extending the dating of a reference. All determinations of relative antigen content using parallel line immunoassays or equivalent methods shall be conducted with an unexpired reference. The lot of reference used to determine antigenic content shall have an initial dating period equal to the dating of the product or as supported by data acceptable to APHIS, except that frozen references may have an initial dating of up to 5 years, Provided, That the request for dating of the frozen references beyond the dating of the product is supported by preliminary data acceptable to APHIS and includes provisions for monitoring the stability of the reference to determine when the potency starts to decline and for taking the appropriate steps to requalify a reference with declining potency either by testing a Qualifying Serial in host animals or by providing other evidence of immunogenicity, e.g., antibody titers or laboratory animal test data previously correlated to host animal protection in a manner acceptable to APHIS. Prior to the expiration date, such reference may be granted an extension of dating, Provided, That its immunogenicity has been confirmed using a Qualifying Serial of product in a manner acceptable to APHIS. The dating period of the Master Reference and Working Reference may be extended by data acceptable to APHIS if the minimum potency of the Master Reference is determined to be adequately above the minimum level needed to provide protection in the host animal. If a new Master Reference is established, it shall be allowed an initial dating period equal to the dating of the product or as supported by data acceptable to APHIS, except that frozen references may have an initial dating period of 5 years, or as supported by data acceptable to APHIS. Prior to the expiration date, such reference may be granted an extension of dating by confirming its immunogenicity using a Qualifying Serial of product.
(e) Final container samples of completed product derived from Master Seed found immunogenic in accordance with paragraph (a) of this section and found satisfactory in accordance with paragraphs (b) and (c) of this section may also be subjected to an animal potency test by Animal and Plant Health Inspection Service as provided in this paragraph. Products shall be used according to label directions including dose(s) and route of administration.
(1) A one stage test using 20 vaccinates and 5 controls or a two stage test using 10 vaccinates and 5 controls for each stage shall be used. The criteria used for judging the specific response in the controls and vaccinates shall be in accordance with the test protocol used in the Master Seed immunogenicity test.
(2) If at least 80 percent of the controls do not show specific responses to challenge, the test is inconclusive and may be repeated. If a vaccinate shows the specific responses to challenge expected in the controls, the vaccinate shall be listed as a failure.
(3) The results of the testing shall be evaluated according to the following table:
Cumulative totals
Stage
Number of animals
Failures for satisfactory serials
Failures for unsatisfactory serials
1
10
1 or less
3 or more.
2 (or 1)
20
4 or less
5 or more.
(4) When a serial has been found unsatisfactory for potency by the test provided in paragraphs (e)(1), (2), and (3) of this section, the serial shall be withheld from the market and the following actions taken:
(i) The Administrator shall require that at least two additional serials prepared with the same Master Seed be subjected to similar animal potency tests by Animal and Plant Health Inspection Service or the licensee or both.
(ii) If another serial is found unsatisfactory for potency, the product shall be removed from the market while a reevaluation of the product is made and the problem is resolved.
A potency test written into the filed Outline of Production for a product shall be considered confidential information by Animal and Plant Health Inspection Service until at least two additional product licenses are issued for the product or unless use of the test is authorized by the licensee, in which case, such potency test may be published as part of the Standard Requirement for the product.
(a) Until a potency test is published as part of the Standard Requirement for the product, reference to such a test shall be made in the filed Outline of Production and the test shall be conducted.
(b) When a potency test has been published as part of the Standard Requirement, such test shall be conducted unless the product is specifically exempted as provided in § 113.4.
When a product is prepared in a licensed establishment for export in large multiple-dose containers as provided in § 112.8(d) or (e) of this subchapter or for further manufacturing purposes as provided in § 114.3(d) of this subchapter, samples of the bulk material shall be subjected to all required tests prescribed in the filed Outline of Production or Standard Requirements for the product. Samples of concentrated liquid product shall be diluted to a volume equal to the contents of the sample times the concentration factor prior to initiating potency tests.
(a) Ingredients for which standards are prescribed in the United States Pharmacopeia, or elsewhere in this part, shall conform to such standards. In lieu of preparing the media from the individual ingredients, they may be made from dehydrated mixtures which, when rehydrated with purified water, have the same or equivalent composition as such media and have growth-promoting buffering, and oxygen tension-controlling properties equal to or better than such media. The formulas for the composition of the culture media prescribed in §§ 113.26 and 113.27 are set forth in the United States Pharmacopeia, 19th Edition.
(b) The licensee shall test each quantity of medium prepared at one time from individual ingredients and the first quantity prepared from each lot of commercial dehydrated medium for growth-promoting qualities. If any portion of a lot of commercial dehydrated medium is held for 90 days or longer after being so tested, it shall be retested before use. Two or more strains of micro-organisms that are exacting in their nutritive requirements shall be used. More than one dilution shall be used to demonstrate the adequacy of the medium to support the growth of a minimum number of micro-organisms.
(c) The sterility of the medium shall be confirmed by incubating an adequate number of test vessels and examining each for growth. Additional control may be used by incubation of representative uninoculated test vessels for the required incubation period during each test.
(d) A determination shall be made by the licensee for each biological product of the ratio of inoculum to medium which shall result in sufficient dilution of such product to prevent bacteriostatic and fungistatic activity. The determination may be made by tests on a representative biological product for each group of comparable products containing identical preservatives at equal or lower concentrations. Inhibitors or neutralizers of preservatives, approved by the Administrator, may be considered in determining the proper ratio.
Each serial and subserial of biological product except live vaccines shall be tested as prescribed in this section unless otherwise specified by the Administrator. When cell lines, primary cells, or ingredients of animal origin used in the preparation of a biological product are required to be free of viable bacteria and fungi, they shall also be tested as prescribed in this section.
(a) The media to be used shall be as follows:
(1) Fluid Thioglycollate Medium with 0.5 percent beef extract shall be used to test for bacteria in biological products containing clostridial toxoids, bacterins, and bacterin-toxoids.
(2) Fluid Thioglycollate Medium with or without 0.5 percent beef extract shall be used to test for bacteria in biological products other than clostridial toxoids, bacterins, and bacterin-toxoids.
(3) Soybean-Casein Digest Medium shall be used to test biological products for fungi; provided, that Fluid Thioglycollate Medium without beef extract shall be substituted when testing biological products containing mercurial preservatives.
(b) Test procedure:
(1) Ten test vessels shall be used for each of two media selected in accordance with paragraph (a)(1), (a)(2), or (a)(3) of this section. Each test vessel shall contain sufficient medium to negate the bacteriostatic or fungistatic activity in the inoculum as determined in § 113.25(d).
(2) Inoculum:
(i) When completed product is tested, 10 final container samples from each serial and each subserial shall be tested. One ml from each sample shall be inoculated into a corresponding individual test vessel of culture medium: Provided, That, if each final container sample contains less than 2 ml, one-half of the contents shall be used as inoculum for each test vessel.
(ii) When cell lines, primary cells, or ingredients of animal origin are tested, at least a 20 ml test sample from each lot shall be tested. One ml shall be inoculated into each test vessel of medium.
(3) Incubation shall be for an observation period of 14 days at 30 °to 35 °C. to test for bacteria and 14 days at 20 °to 25 °C. to test for fungi.
(4) If the inoculum renders the medium turbid so that the absence of growth cannot be determined by visual examination, subcultures shall be made on the seventh to eleventh day from biological products prepared from clostridial toxoids, bacterins, and bacterin-toxoids and the third to seventh day for other biological products. Portions of the turbid medium in amounts of not less than 1.0 ml. shall be transferred to 20 to 25 ml. of fresh medium, and incubated the balance of the 14-day period.
(c) Examine the contents of all test vessels for macroscopic microbial growth during the incubation period. When demonstrated by adequate controls to be invalid, the test may be repeated. For each set of test vessels representing a serial or subserial in a valid test, the following rules shall apply:
(1) If no growth is found in any test vessel, the serial or subserial meets the requirements of the test.
(2) If growth is found in any test vessel, one retest to rule out faulty technique may be conducted using 20 unopened final container samples.
(3) If growth is found in any test vessel of the final test, the serial, subserial, or ingredients to be used in the preparation of a biological product, as the case may be, is unsatisfactory.
Unless otherwise specified by the Administrator or elsewhere exempted in this part, each serial and subserial of live vaccine and each lot of Master Seed Virus and Master Seed Bacteria shall be tested for extraneous viable bacteria and fungi as prescribed in this section. A Master Seed found unsatisfactory shall not be used in vaccine production and a serial found unsatisfactory shall not be released.
(a) Live viral vaccines. Each serial and subserial of live viral vaccine shall be tested for purity as prescribed in this paragraph. However, products of chicken embryo origin recommended for administration other than by parenteral injection may be tested as provided in paragraph (e) of this section.
(1) Soybean Casein Digest Medium shall be used.
(2) Ten final container samples from each serial and subserial shall be tested.
(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and desiccated vaccine shall be rehydrated as recommended on the label with accompanying diluent or with sterile purified water.
(4) To test for bacteria, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 120 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in § 113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 30 °to 35 °C for 14 days.
(5) To test for fungi, place 0.2 ml of vaccine from each final container sample into a corresponding individual vessel containing at least 40 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in § 113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 20 °to 25 °C for 14 days.
(6) Examine the contents of all test vessels macroscopically for microbial growth at the end of the incubation period. If growth in a vessel cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.
(7) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:
(i) If growth is found in 2 or 3 test vessels of the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final container samples.
(ii) If no growth is found in 9 or 10 of the test vessels in the initial test, or 19 or 20 vessels in the retest, the serial or subserial meets the requirements of the test.
(iii) If growth is found in four or more test vessels in the initial test, or two or more in a retest, the serial or subserial is unsatisfactory.
(b) Live bacterial vaccines. Each serial or subserial of live bacterial vaccine shall be tested for purity as prescribed in this paragraph.
(1) Soybean Casein Digest Medium and Fluid Thioglycollate Medium shall be used.
(2) Ten final container samples from each serial and subserial shall be tested.
(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and desiccated vaccine shall be rehydrated as recommended on the label with accompanying diluent or with sterile purified water. Product recommended for mass vaccination shall be rehydrated at the rate of 30 ml sterile purified water per 1,000 doses.
(4) To test for extraneous bacteria, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 40 ml of Fluid Thioglycollate Medium. Additional medium shall be used if the determination required in § 113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 30 °to 35 °C for 14 days.
(5) To test for extraneous fungi, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 40 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in § 113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 20 °to 25 °C for 14 days.
(6) Examine the contents of all test vessels macroscopically for atypical microbial growth at the end of the incubation period. If growth of extraneous microorganisms cannot be reliably determined by visual examination, judgment shall be confirmed by subculturing, microscopic examination, or both.
(7) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:
(i) If extraneous growth is found in 2 or 3 test vessels of the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final container samples.
(ii) If no extraneous growth is found in 9 or 10 test vessels in the initial test, or 19 or 20 vessels in the retest, the serial or subserial meets the requirements of the test.
(iii) If extraneous growth is found in 4 or more test vessels in the initial test, or 2 or more in a retest, the serial or subserial is unsatisfactory.
(c) Master Seed Virus. Not less than 4 ml of each lot of Master Seed Virus shall be tested. Frozen liquid Master Seed Virus shall be thawed, and desiccated Master Seed Virus shall be rehydrated with Soybean Casein Digest Medium immediately prior to starting the test.
(1) To test for bacteria, place 0.2 ml of the sample of Master Seed Virus into 10 individual vessels each containing at least 120 ml of Soybean Casein Digest Medium. Incubation shall be at 30 °to 35 °C for 14 days.
(2) To test for fungi, place 0.2 ml of the sample of Master Seed Virus into 10 individual vessels each containing at least 40 ml of Soybean Casein Digest Medium. Incubation shall be at 20 °to 25 °C for 14 days.
(3) Examine the contents of all test vessels macroscopically for microbial growth at the end of the incubation period. If growth in a vessel cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.
(4) For each set of test vessels representing a lot of Master Seed Virus tested according to these procedures, the following rules shall apply:
(i) If growth is found in any test vessel of the initial test, one retest to rule out faulty technique may be conducted using a new sample of Master Seed Virus.
(ii) If growth is found in any test vessel of the final test, the lot of Master Seed Virus is unsatisfactory.
(d) Master Seed Bacteria. Not less than 4 ml of each lot of Master Seed Bacteria shall be tested. Frozen liquid Master Seed Bacteria shall be thawed, and desiccated Master Seed Bacteria shall be rehydrated with sterile purified water immediately prior to starting the test.
(1) To test for extraneous bacteria, place 0.2 ml of the sample of Master Seed Bacteria into 10 individual vessels each containing at least 40 ml of Fluid Thioglycollate Medium. Incubation shall be at 30 °to 35 °C for 14 days.
(2) To test for extraneous fungi, place 0.2 ml of the sample of Master Seed Bacteria into 10 individual vessels each containing at least 40 ml of Soybean Casein Digest Medium. Incubation shall be at 20 °to 25 °C for 14 days.
(3) Examine the contents of all test vessels macroscopically for atypical microbial growth at the end of the incubation period. If growth of extraneous microorganisms cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.
(4) For each set of test vessels representing a lot of Master Seed Bacteria tested according to these procedures, the following rules shall apply:
(i) If extraneous growth is found in any test vessel of the initial test, one retest to rule out faulty technique may be conducted using a new sample of Master Seed Bacteria.
(ii) If extraneous growth is found in any test vessel of the final test, the lot of Master Seed Bacteria is unsatisfactory.
(e) Live viral vaccines of chicken embryo origin recommended for administration other than by parenteral injection, which were not tested or have not been found free of bacteria and fungi by the procedures prescribed in paragraph (a) of this section, may be tested according to the procedures prescribed in this paragraph.
(1) Brain Heart Infusion Agar shall be used with 500 Kinetic (Kersey) units of penicillinase per ml of medium added just prior to pouring the plates.
(2) Ten final containers from each serial and each subserial shall be tested.
(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and lyophilized vaccine shall be rehydrated to the quantity recommended on the label using the accompanying sterile diluent or sterile purified water. Product recommended for mass vaccination shall be rehydrated at the rate of 30 ml sterile purified water per 1,000 doses.
(4) From each container sample, each of 2 plates shall be inoculated with vaccine equal to 10 doses if the vaccine is recommended for poultry or 1 dose if the vaccine is recommended for other animals. Twenty ml of medium shall be added to each plate. One plate shall be incubated at 30 °to 35 °for 7 days and the other plate shall be incubated at 20 °to 25 °C for 14 days.
(5) Colony counts shall be made for each plate at the end of the incubation period. An average colony count for the 10 samples representing the serial or subserial shall be made for each incubation condition.
(6) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:
(i) If the average count at either incubation condition exceeds 1 colony per dose for vaccines recommended for poultry, or 10 colonies per dose for vaccines recommended for other animals in the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final containers.
(ii) If the average count at either incubation condition of the final test for a serial or subserial exceeds 1 colony per dose for vaccines recommended for poultry, or 10 colonies per dose for vaccines recommended for other animals, the serial or subserial is unsatisfactory.
The heart infusion test, using heart infusion broth and heart infusion agar, provided in this section shall be conducted when a test for mycoplasma contamination is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.
(a) Media additives provided in this paragraph shall be prepared as follows:
(1) DPN-Cysteine Solution:
(i) Use Nicotinamide adenine dinucleotide (oxidized) and L-Cysteine hydrochloride.
(ii) Prepare 1 gram/100 milliliters (ml) purified water (1 percent solution) of each. Mix the solutions together; the cysteine reduces the DPN. Filter sterilize, dispense in appropriate amounts and store frozen at −20 degrees centigrade.
(2) Inactivated horse serum—horse serum which has been inactivated at 56 °C for 30 minutes.
(b) Heart infusion broth shall be prepared as provided in this paragraph.
(1) Dissolve in 970 ml of purified water, 25 grams of heart infusion broth, 10 grams of proteose peptone No. 3, and either 5 grams of yeast autolysate or 5 ml of fresh yeast extract.
(2) Add the following:
1 percent tetrazolium chloride (ml)
5.5
1 percent thallium acetate (ml)
25
Penicillin (units)
500,000
Inactivated horse serum (ml)
100
(3) Adjust pH to 7.9 with NaOH, filter sterilize, and dispense 100 ml aliquots into 125 ml flasks and store until needed.
(4) Add 2 ml of DPN-Cysteine solution to each 100 ml of broth on day of use.
(c) Heart Infusion Agar shall be prepared as provided in this paragraph.
(1) Dissolve in 900 ml of purified water by boiling the following:
Heart infusion agar (g)
25
Heart-infusion broth (g)
10
Proteose peptone No. 3 (g)
10
1 pct thallium acetate (ml)
25
(2) Cool the medium and adjust pH to 7.9 with NaOH.
(3) Autoclave the medium.
(4) Cool the medium 30 minutes in a 56 °C waterbath.
(5) Dissolve 5 grams of yeast autolysate in 100 ml of distilled water, filter sterilize, and add to the medium.
(6) Add to the medium:
126 ml of inactivated horse serum
21 ml of DPN-Cysteine solution
525,000 units of Penicillin.
Dispense 10 ml aliquots into 60 × 15 mm disposable culture dishes or petri dishes.
(d) The test procedure provided in this paragraph shall be followed when conducting the mycoplasma detection test.
(1) Preparation of inoculum. Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and lyophilized vaccine shall be rehydrated to the volume recommended on the label with mycoplasma medium. In the case of a lyophilized biological product, e.g., 1,000 dose vial of poultry vaccine to be administered via the drinking water, the vaccine shall be rehydrated to 30 ml with mycoplasma medium. In the case of a cell line or a sample of primary cells, the inoculum shall consist of the resuspended cells. Control tests shall be established as provided in paragraph (d)(4) of this section.
(2) Inoculation of plate. Plate 0.1 ml of inoculum on an agar plate and make a short, continuous streak across the plate with a pipet. Tilt the plate to allow the inoculum to flow over the surface.
(3) Inoculation of flask of medium. Transfer 1 ml of the inoculum into a flask containing 100 ml mycoplasma medium and mix thoroughly. Incubate the flask at 33 to 37 °C for 14 days during which time, one of four agar plates shall be streaked with 0.1 ml of material from the incubating flask of inoculated medium on the 3d day, one on the 7th day, one on the 10th day, and one on the 14th day post-inoculation.
(4) Control tests shall be conducted simultaneously with the detection test using techniques provided in paragraphs (d)(2) and (3) of this section, except the inoculum for the positive control test shall be selected mycoplasma cultures and the negative control test shall be uninoculated medium from the same lot used in the detection test.
(5) All plates shall be incubated in a high humidity, 4-6 percent CO 2 atmosphere at 33 °to 37 °C for 10-14 days and examined with a stereoscopic microscope at 35x to 100x or with a regular microscope at 100x.
(e) Interpretation of test results.
(1) If growth appears on at least one of the plates in the positive control test and does not appear on any of the plates in the negative control test, the test is valid.
(2) If mycoplasma colonies are found on any of the plates inoculated with material being tested, the results are positive for mycoplasma contamination.
Methods provided in this section must be used when a determination of moisture content in desiccated biological products is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product. Firms currently using methods other than those provided in this section for determining the moisture content in desiccated biological products have until November 5, 2004 to update their Outlines of Production to be in compliance with this requirement.
(a) Final container samples of completed product shall be tested. The weight loss of the sample due to drying in a vacuum oven shall be determined. All procedures should be performed in an environment with a relative humidity less than 45 percent. The equipment necessary to perform the test is as follows:
(1) Cylindrical weighing bottles with airtight glass stoppers.
(2) Vacuum oven equipped with validated thermometer and thermostat. A suitable air-drying device should be attached to the inlet valve.
(3) Balance, accurate to 0.1 mg (rated precision ±0.01mg).
(4) Desiccator jar equipped with phosphorous pentoxide, silica gel, or equivalent.
(5) Desiccated vaccine in original sealed vial. Sample and control should be kept at room temperature in their original airtight containers until use.
(b) Test procedure:
(1) Thoroughly cleaned and labeled sample-weighing bottles with stoppers should be allowed to dry at 60 ±3 °C under vacuum at less than 2.5 kPa.
(i) Transfer hot bottles and stoppers into the desiccator and allow to cool to room temperature.
(ii) After bottles have cooled, insert stoppers and weigh and record the weights of the bottles as “A.”
(iii) Return weighing bottles to the desiccator.
(2) Remove the sample container seal.
(i) Using a spatula, break up the sample plug and transfer the required amount of sample to the previously tared weighing bottle.
(ii) Insert the stopper and weigh and record the weights of the weighing bottles as “B.”
(3) Place the weighing bottle with the stopper at an angle in the vacuum oven. Set the vacuum to <2.5 kPa and the temperature to 60 ±3 °C.
(4) After a minimum of 3 hours of drying time, turn off the vacuum pump and allow dry air to bleed into the oven until the pressure inside the oven is equalized with the prevailing atmospheric pressure.
(5) While the bottle is still warm, replace the stopper in its normal position and transfer the weighing bottle to the desiccator.
(i) Allow a minimum of 2 hours for the weighing bottle to cool to room temperature or for its weight to reach equilibrium.
(ii) Weigh, and record the weight as “C.”
(6) Calculate the percentage of moisture in the original sample as follows:
(B−C)/(B−A) × (100) = Percentage of residual moisture, where:
A = tare weight of weighing bottle
B−A = weight of sample before drying
B−C = weight of sample after drying
(7) The results are considered satisfactory if the percentage of residual moisture is less than or equal to the manufacturer's specification.
The test for detection of Salmonella contamination provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.
(a) Samples shall be collected from the bulk suspension before bacteriostatic or bactericidal agents have been added. When tissue culture products are to be tested, 1 ml of tissue extract used as the source of cells or 1 ml of the minced tissue per se shall be tested.
(b) Five ml of the liquid vaccine suspension shall be used to inoculate each 100 ml of liquid broth medium (tryptose and either selenite F or tetrathionate). The inoculated media shall be incubated 18-24 hours at 35-37 °C.
(c) Transfers shall be made to either MacConkey agar or Salmonella-Shigella agar, incubated for 18-24 hours and examined.
(d) If no growth typical of Salmonella is noted, the plates shall be incubated an additional 18-24 hours and again examined.
(e) If suspicious colonies are observed, further subculture on suitable media shall be made for positive identification. If Salmonella is found, the bulk suspension is unsatisfactory.
The complement-fixation test for detection of avian lymphoid leukosis provided in this section shall be conducted on all biological products containing virus which has been propagated in substrates of chicken origin: Provided, An inactivated viral product shall be exempt from this requirement if the licensee can demonstrate to Animal and Plant Health Inspection Service that the agent used to inactivate the vaccine virus would also inactivate lymphoid leukosis virus.
(a) Propagation of contaminating lymphoid leukosis viruses, if present, shall be done in chick embryo cell cultures.
(1) Each vaccine virus, cytopathic to chick embryo fibroblast cells, shall be effectively neutralized, inactivated, or separated so that minimal amounts of lymphoid leukosis virus can be propagated on cell culture during the 21-day growth period. If a vaccine virus cannot be effectively neutralized, inactivated, or separated, a sample of another vaccine prepared the same week from material harvested from each source flock (or other sampling procedure acceptable to Animal and Plant Health Inspection Service) used for the preparation of the questionable vaccine virus that cannot be neutralized, inactivated, or separated shall be tested each week during the preparation of such questionable vaccine.
(2) When cell cultures are tested, 5 ml of the final cell suspension as prepared for seeding of production cell cultures shall be used as inoculum. When vaccines are tested, the equivalent of 200 doses of Newcastle disease vaccine or 500 doses of other vaccines for use in poultry, or one dose of vaccine for use in other animals shall be used as inoculum. Control cultures shall be prepared from the same cell suspension as the cultures for testing the vaccine.
(3) Uninoculated chick embryo fibroblast cell cultures shall act as negative controls. One set of chick fibroblast cultures inoculated with subgroup A virus and another set inoculated with subgroup B virus shall act as positive controls, A and B respectively.
(4) The cell cultures shall be propagated at 35-37 °C for at least 21 days. They shall be passed when necessary to maintain viability and samples harvested from each passage shall be tested for group specific antigen.
(b) The microtiter complement-fixation test shall be performed using either the 50 percent or the 100 percent hemolytic end point technique to determine complement unitage. Five 50 percent hemolytic units or two 100 percent hemolytic units of complement shall be used for each test.
(1) All test materials, including positive and negative controls, shall be stored at −60 °C or colder until used in the test. Before use, each sample shall be thawed and frozen three times to disrupt intact cells and release the group specific antigen.
(2) The antiserum used in the microtiter complement-fixation test shall be a standard reagent supplied or approved by the Animal and Plant Health Inspection Service. Four units of antiserum shall be used for each test.
(3) Presence of complement-fixing activity in the harvested samples (from passages) at the 1:4 or higher dilution, in the absence of anticomplementary activity, shall be considered a positive test unless the activity can definitely be established to be caused by something other than lymphoid leukosis virus, subgroups A and/or B. Activity at the 1:2 dilution shall be considered suspicious and the sample further subcultured to determine presence or absence of the group specific antigen.
(4) Biological products or primary cells which are found contaminated with lymphoid leukosis viruses are unsatisfactory. Source flocks from which contaminated material was obtained are also unsatisfactory.
The test for detection of Brucella contamination provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in a filed Outline of Production for the product.
(a) One ml of the minced tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of antibiotics, diluent and stabilizer, shall be inoculated onto each of three tryptose agar plates and incubated in a 10 percent CO 2 atmosphere at a temperature of 35-37 °C for at least 7 days.
(b) If colonies are identified as Brucella, the biological product is unsatisfactory.
(c) If colonies suspicious of Brucella are observed but cannot be identified as a Brucella species, either
(1) The biological product shall be regarded as unsatisfactory and destroyed; or
(2) Further subculture or other procedures shall be carried out until a positive identification can be made.
One of the mouse safety tests provided in this section shall be conducted when such test is prescribed in a Standard Requirement or in the filed Outline of Production for a biological product recommended for animals other than poultry: Provided, That if the inherent nature of one or more ingredients makes the biological product lethal or toxic for mice but not lethal or toxic for the animals for which it is recommended, the licensee shall demonstrate the safety of such product by an acceptable test written into such Outline of Production.
(a) Final container samples of completed product from live virus vaccines shall be tested for safety using young adult mice in accordance with the test provided in this paragraph.
(1) Vaccine prepared for use as recommended on the label shall be tested by inoculating eight mice intraperitoneally or subcutaneously with 0.5 mL (the inoculation volume may be divided among more than one injection site), and the animals observed for 7 days.
(2) If unfavorable reactions attributable to the product occur in any of the mice during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
(b) Bulk or final container samples of completed product from liquid products, such as but not limited to antiserums and bacterins, shall be tested for safety in accordance with the test provided in this paragraph.
(1) Unless otherwise prescribed in the Standard Requirement or approved in a filed Outline of Production for the product, a 0.5 ml dose shall be injected intraperitoneally or subcutaneously into eight mice and the animals observed for 7 days.
(2) If unfavorable reactions attributable to the product occur in any of the mice during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
The test for detection of hemagglutinating viruses provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.
(a) Final container samples of completed product rehydrated as recommended on the label shall be used as inoculum: Provided, That poultry vaccines distributed without diluent shall be rehydrated with 30 ml of sterile distilled water per 1,000 doses and used as inoculum. When one or more fractions are to be used in combination with Newcastle Disease Vaccine, test samples shall be collected from bulk suspensions of each prior to mixing with the Newcastle Disease Vaccine.
(b) Each of ten 9- to 10-day-old embryonating eggs from Newcastle disease susceptible flocks shall be inoculated into the allantoic cavity with 0.2 ml of the undiluted inoculum.
(1) Test five uninoculated embryos of the same age and from the same flock as those used for the test as negative controls.
(2) Test an allantoic fluid sample of Newcastle disease virus as a positive control.
(c) Three to five days post-inoculation, a sample of allantoic fluid from each egg shall be tested separately by a rapid plate test for hemagglutinating activity using a 0.5 percent suspension of fresh chicken red blood cells.
(d) If the results are inconclusive, one or two blind passages shall be made using fluids from each of the original test eggs. Fluids from dead and live embryos may be pooled separately for inoculum in these passages.
(e) If hemagglutinating activity attributable to the product is observed, the serial is unsatisfactory.
The test for detection of viricidal activity provided in this section shall be conducted when such a test is prescribed in an applicable standard requirement or in the filed Outline of Production for each inactivated liquid biological product used as diluent for a desiccated live virus vaccine in a combination package.
(a) Bulk or final container samples of completed product from each serial shall be tested.
(b) The product shall be tested with each virus fraction for which it is to be used as a diluent. If the vaccine to be rehydrated contains more than one virus fraction, the test shall be conducted with each fraction after neutralization of the other fraction(s), and/or dilution of the vaccine beyond the titer range of the other fraction(s), or the test shall be conducted using representative single-fraction desiccated vaccines which are prepared by the licensee and which are licensed. Provided, That the Administrator may authorize licensees to prepare and use unlicensed single-fraction vaccines for this purpose.
(c) Test procedure: (1) Rehydrate at least two vials of the vaccine with the liquid product under test according to label recommendations and pool the contents.
(2) Rehydrate at least two vials of the vaccine with the same volume of sterile purified water and pool the contents.
(3) Neutralize to remove other fractions, if necessary.
(4) Hold the two pools of vaccine at room temperature (20 °to 25 °C) for 2 hours. The holding period shall begin when rehydration is completed.
(5) Titrate the virus(es) in each pool of vaccine as provided in the filed Outline of Production or an applicable standard requirement.
(6) Compare respective titers.
(d) If the titer of the vaccine virus(es) rehydrated with the product under test is more than 0.7 log 10 below the titer of the vaccine virus(es) rehydrated with sterile purified water, the product is unsatisfactory for use as diluent.
(e) If the product is unsatisfactory in the first test, one retest to rule out faulty techniques may be conducted using four vials of the vaccine for each pool and the acceptability of the product judged by the results of the second test.
(f) Liquid products found to be unsatisfactory for use as diluent by this test are not prohibited from release as separate licensed products if labeled as prescribed in § 112.7(g).
The test for detection of extraneous pathogens provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.
(a) The biological product to be tested shall be prepared for use as recommended on the label, or in the case of desiccated vaccine to be used in poultry, rehydrated with sterile distilled water at the rate of 30 ml per 1,000 doses.
(b) At least 25 healthy susceptible young chickens, properly identified and obtained from the same source and hatch, shall be immunized at least 14 days prior to being put on test. The immunizing agent shall be the same as the product to be tested but from a serial previously tested and found satisfactory.
(c) At least 20 of the previously immunized birds shall be inoculated with 10 label doses of the vaccine being tested by each of the following routes: Subcutaneous, intratracheal, eye-drop, and comb scarification (1 cm
2 ). Twenty birds may be used for each route or combination of routes.
(d) At least five birds shall be isolated as control birds.
(e) All birds shall be observed for 21 days for signs of septicemic diseases, respiratory diseases, or other pathologic conditions.
(f) If the controls remain healthy and unfavorable reactions attributable to the product occur in the vaccinates, the serial or subserial tested is unsatisfactory. If the controls do not remain healthy or if unfavorable reactions not attributable to the product occur in the vaccinates, or both, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial of subserial tested shall be considered unsatisfactory.
The test for detection of extraneous pathogens provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.
(a) The biological product to be tested shall be prepared for use as recommended on the label, or in the case of desiccated vaccine to be used in poultry, rehydrated with sterile distilled water at the rate of 30 ml per 1,000 doses.
(b) One volume of the prepared vaccine shall be mixed with up to nine volumes of sterile heat-inactivated specific antiserum to neutralize the vaccine virus in the product. Each lot of antiserum shall be demonstrated by virus neutralization tests not to inhibit other viruses known to be possible contaminants.
(c) After neutralization, 0.2 ml of the vaccine-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos.
(1) Twenty embryos, 9 to 11 days old, shall be inoculated on the chorio-allantoic membrane (CAM) with 0.1 ml, and in the allantoic sac with 0.1 ml.
(2) Eggs shall be candled daily for 7 days. Deaths occurring during the first 24 hours shall be disregarded but at least 18 viable embryos shall survive 24 hours post-inoculation for a valid test. Examine all embryos and CAM's from embryos which die after the first day. When necessary, embryo subcultures shall be made to determine the cause of a death. The test shall be concluded on the seventh day post-inoculation and the surviving embryos (including CAM's) examined.
(d) If death and/or abnormality attributable to the inoculum occur, the serial is unsatisfactory: Provided, That, if there is a vaccine virus override, the test may be repeated, using a higher titered antiserum.
The guinea pig safety test provided in this section shall be conducted when prescribed in a Standard Requirement or approved Outline of Production for a biological product. When desiccated products are tested, final container samples of completed product prepared for administration in the manner recommended on the label shall be used. When liquid products are tested, either bulk or final container samples of completed product shall be used.
(a) Unless otherwise specified in the Standard Requirement or approved Outline of Production for the product, a 2 ml dose shall be injected either intramuscularly or subcutaneously into each of two guinea pigs and the animals observed for 7 days.
(b) If unfavorable reactions attributable to the product occur in either of the guinea pigs during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
The safety tests provided in this section shall be conducted when prescribed in a standard requirement or in the filed Outline of Production for a biological product recommended for use in cats.
(a) The cat safety test provided in this paragraph shall be used when the Master Seed Virus is tested for safety.
(1) The test animals shall be determined to be susceptible to the virus under test as follows:
(i) Throat swabs shall be collected from each cat and individually tested on susceptible cell cultures for the presence of the virus. Blood samples shall also be drawn and individual serum samples tested for antibody to the virus.
(ii) The cats shall be considered susceptible if swabs are negative for virus isolation and the serums are free of virus antibody at the 1:2 final dilution in a 50 percent plaque reduction test or other serum-neutralization test of equal sensitivity.
(iii) When determining susceptibility to a virus which does not lend itself to the methods in paragraphs (a)(1)(i) and (ii) of this section, a method acceptable to Animal and Plant Health Inspection Service shall be used.
(2) Each of at least 10 susceptible cats shall be administered a sample of the Master Seed Virus equivalent to the amount of virus to be used in one cat dose of the vaccine, by the method to be recommended on the label, and the cats observed each day for 14 days.
(3) If unfavorable reactions attributable to the virus occur in any of the cats during the observation period, the Master Seed Virus is unsatisfactory. If unfavorable reactions occur which are not attributable to the Master Seed Virus, the test shall be declared a No Test and repeated: Provided, That, if not repeated, the Master Seed Virus shall be unsatisfactory.
(b) The cat safety test provided in this paragraph shall be used when a serial of vaccine is tested for safety before release.
(1) Each of two healthy cats shall be administered 10 cat doses by the method recommended on the label and the cats observed each day for 14 days.
(2) If unfavorable reactions attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated: Provided, That, if not repeated, the serial shall be unsatisfactory.
The safety tests provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a biological product recommended for use in dogs. Serials which are not found to be satisfactory when tested pursuant to the procedures in this section may not be released for shipment.
(a) The dog safety test provided in this paragraph shall be used when the Master Seed Virus is tested for safety.
(1) The test animals shall be determined to be susceptible to the virus under test by a method acceptable to the Animal and Plant Health Inspection Service.
(2) Each of at least 10 susceptible dogs shall be administered a sample of the Master Seed Virus equivalent to the amount of virus to be used in one dog dose of the vaccine, by the method recommended on the label, and the dog shall be observed each day for 14 days.
(3) If unfavorable reactions attributable to the virus occur in any of the dogs during the observation period, the Master Seed Virus is unsatisfactory. If unfavorable reactions occur which are not attributable to the Master Seed Virus, the test shall be declared a No Test and may be repeated: Provided: That, if the test is not repeated, the Master Seed Virus shall be considered unsatisfactory.
(b) The dog safety test provided in this paragraph shall be used when a serial of vaccine is tested for safety before release.
(1) Each of two healthy dogs shall be administered 10 dog doses by the method recommended on the label and the dogs shall be observed each day for 14 days.
(2) If unfavorable reactions attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the biological product, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial shall be considered unsatisfactory.
The calf safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.
(a) Test procedure. Each of two calves shall be injected with the equivalent of 10 doses of vaccine administered in the manner recommended on the label and observed each day for 21 days.
(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the calves during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
The test for detection of lymphocytic choriomeningitis (LCM) virus provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in a filed Outline of Production. Vaccine virus may be neutralized with specific antiserum when necessary.
(a) Each of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days.
(b) If any of the mice show swelling in the injected footpad or if more than one becomes systemically abnormal, the material being tested is unsatisfactory.
The test for chlamydial agents provided in this section shall be conducted when such a test is prescribed in an applicable standard requirement or in a filed Outline of Production.
(a) The yolk sac of 6-day-old chicken embryos shall be injected. Three groups of 10 embryos shall be used sequentially.
(1) The inoculum for each embryo in the first group shall consist of 0.5 ml of a mixture of equal parts of the seed virus with phosphate buffered saline that may contain Streptomycin, Vancomycin, Kanamycin, or a combination thereof. Not more than 2 mg/ml of each antibiotic shall be used.
(2) On the 10th day postinoculation, the yolk sac of viable embryos shall be harvested, pooled, homogenized as a 20 percent suspension in phosphate buffered saline antibiotic diluent, and 0.5 ml of the mixture injected into the second group of chicken embryos. This process shall be repeated for the injection of the third group of embryos using the yolk sacs of viable embryos from the second group.
(3) For each of the three passages, embryo deaths occurring within 48 hours of injection shall be disregarded, except that if more than three such deaths occur at any passage, that passage shall be repeated.
(b) If one or more embryo deaths occur at any passage after 48 hours postinjection, the yolk sacs from each of the dead embryos shall be subcultured into 10 additional embryos. If one or more embryo deaths again occur due to chlamydial agents, the Master Seed Virus is unsatisfactory for use to produce vaccine.
The swine safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.
(a) Test procedure. (1) Inject each of two swine of the minimum age for which the product is recommended with the equivalent of two doses of bacterial vaccine or 10 doses of viral vaccine.
(2) Administer vaccine in the manner recommended on the label.
(3) Observe swine each day for 21 days.
(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the swine during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated; Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
The sheep safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.
(a) Test procedure. (1) Inject each of two sheep of the minimum age for which the product is recommended with the equivalent of two doses of bacterial vaccine or 10 doses of viral vaccine.
(2) Administer vaccine in the manner recommended on the label.
(3) Observe sheep each day for 21 days.
(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the sheep during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated; Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.
The tests for detection of cytopathogenic and/or hemadsorbing agents provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in the filed Outline of Production for a product.
(a) Test for cytopathogenic agents. One or more monolayers that are at least 6 cm
2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.
(1) Stain each monolayer with a suitable cytological stain.
(2) Examine the entire area of each stained monolayer for evidence of inclusion bodies, abnormal number of giant cells, or other cytopathology indicative of cell abnormalities attributable to an extraneous agent.
(b) Test for hemadsorbing agents. One or more monolayers that are at least 6 cm
2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.
(1) Wash the monolayer with several changes of phosphate buffered saline.
(2) Add an appropriate volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These suspensions may be mixed before addition to the monolayer or they may be added separately to individual monolayers.
(3) Incubate the monolayer at 4 °C for 30 minutes, wash with phosphate buffered saline, and examine for hemadsorption.
(4) If no hemadsorption is apparent, repeat step (b)(2) of this section and incubate the monolayers at 20-25 °C for 30 minutes, wash with phosphate buffered saline, and examine again for hemadsorption. If desired, separate monolayers may be used for each incubation temperature.
(c) If specific cytopathology or hemadsorption attributable to an extraneous agent is found, the material under test is unsatisfactory and shall not be used to prepare biological products. If an extraneous agent is suspected because of cytopathology or hemadsorption and cannot be eliminated as a possibility by additional testing, the material under test is unsatisfactory.
The test for detection of extraneous viruses by the fluorescent antibody technique provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in a filed Outline of Production for a product.
(a) Monolayer cultures of cells (monolayers), at least 7 days after the last subculturing, shall be processed and stained with the appropriate antiviral fluorochrome-conjugated antibody as specified in paragraph (b) of this section.
(1) Three groups of one or more monolayers shall be required for each specific virus prescribed in paragraph (b) of this section.
(i) At the time of the last subculturing, one group of test monolayers shall be inoculated with approximately 100-300 FAID 50 of the specific virus being tested for as positive controls.
(ii) One group of monolayers shall be the “material under test.”
(iii) One group of monolayers, that are of the same type of cells as the test monolayers and that have been tested as prescribed in §§ 113.51 or 113.52 (whichever is applicable), shall be prepared as negative controls.
(2) Each group of monolayers shall have a total area of at least 6 cm
2 .
(3) Positive control monolayers may be fixed (processed so as to arrest growth and assure attachment of the monolayer to the surface of the vessel in which they are grown) before 7 days after subculturing if fluorescence is enhanced by doing so, Provided, That a monolayer of the material under test is also fixed at the same time as the positive control and a monolayer of the material under test is also fixed at least seven days after subculturing. Monolayers that are fixed before 7 days after subculturing shall be stained at the same time as the test monolayers and negative controls fixed at least 7 days after subculturing.
(b) The antiviral fluorochrome-conjugated antibodies to be used shall depend on the type of cells required to be tested for extraneous viruses as specified in an applicable Standard Requirement or in a filed Outline of Production. Antiviral fluorochrome-conjugated antibodies specific for the extraneous viruses shall be applied to each respective type of cell in accordance with the following list. Under certain circumstances, additional tests may need to be conducted, as determined by the Administrator. When a specific antiviral fluorochrome-conjugated antibody is used in testing for the listed extraneous viruses specified in more than one cell type, it need only be applied to the most susceptible cell type.
(1) All cells shall be tested for:
(i) Bovine virus diarrhea virus;
(ii) Reovirus; and
(iii) Rabies virus.
(2) Bovine, caprine, and ovine cells shall, in addition, be tested for:
(i) Bluetongue virus;
(ii) Bovine adenoviruses;
(iii) Bovine parvovirus; and
(iv) Bovine respiratory syncytial virus.
(3) Canine cells shall, in addition, be tested for:
(i) Canine coronavirus;
(ii) Canine distemper virus; and
(iii) Canine parvovirus.
(4) Equine cells shall, in addition, be tested for:
(i) Equine herpesvirus; and
(ii) Equine viral arteritis virus.
(5) Feline cells shall, in addition, be tested for:
(i) Feline infectious peritonitis virus; and
(ii) Feline panleukopenia virus.
(6) Porcine cells shall, in addition, be tested for:
(i) Porcine adenovirus;
(ii) Porcine parvovirus;
(iii) transmissible gastroenteritis virus; and
(iv) Porcine hemagglutinating encephalitis virus.
(7) Firms that do not have rabies virus on premises either for research or production purposes are exempt from having to produce positive rabies virus control monolayers. Fixed positive rabies virus control monolayers will be provided by the National Veterinary Services Laboratories.
(c) After staining, each group of monolayers shall be examined for the presence of specific fluorescence attributable to the presence of extraneous viruses.
(1) If the material under test shows any evidence of specific viral fluorescence, it is unsatisfactory and may not be used; Provided, That, if specific fluorescence attributable to the virus being tested for is absent in the positive control monolayers, the test is a No Test and may be repeated.
(2) If the fluorescence of the monolayers inoculated with the specific virus as positive controls is equivocal, or if the negative monolayers show equivocal fluorescence indicating possible viral contamination, or both, the test shall be declared a No Test, and may be repeated; Provided, That, if the test is not repeated, the material under test shall be regarded as unsatisfactory for use in the production of biologics.
All ingredients used in a licensed biological product shall meet accepted standards of purity and quality; shall be sufficiently nontoxic so that the amount present in the recommended dose of the product shall not be toxic to the recipient; and in the combinations used shall not denature the specific substances in the product below the minimum acceptable potency within the dating period when stored at the recommended temperature.
Primary cells used to prepare biological products shall be derived from normal tissue of healthy animals. When prescribed in an applicable Standard Requirement or in the filed Outline of Production, each batch of primary cells used to prepare a biological product shall be tested as prescribed in this section. A batch of primary cells found unsatisfactory by any prescribed test shall not be used. A serial of biological product shall not be released if produced from primary cells that are found unsatisfactory by any prescribed test.
(a) Final container samples of completed product or samples of the final pool of harvested material or samples of each subculture of cells used to prepare the biological product shall be shown free of mycoplasma as prescribed in § 113.28. The sample for testing shall consist of at least 75 cm
2 of actively growing cells or the equivalent in harvest fluids; Provided, That all sources of cells in the batch of primary cells are represented.
(b) Final container samples of completed product or samples of the final pool of harvested material or samples of each subculture of cells used to prepare the biological product shall be shown free of bacteria and fungi as prescribed in § 113.26 or § 113.27 (whichever is applicable).
(c) A monolayer at least 75 cm
2 from each batch of primary cells or each subculture of primary cells used to prepare a biological product shall be shown free of extraneous agents as prescribed in this paragraph.
(1) The test monolayer shall be maintained using the medium (with additives) and under conditions similar to those used to prepare biological products.
(i) Monolayers of avian origin shall be maintained for at least 14 days and shall be subcultured at least once during the maintenance period. All but the last subculture shall result in a new monolayer of at least 75 cm
2 . The last subculture shall meet the minimum area requirement specified in §§ 113.46 and 113.47.
(ii) Monolayers not of avian origin shall be maintained for at least 28 days and shall be subcultured at least twice during the maintenance period. All but the last subculture shall result in a new monolayer of at least 75 cm
2 . The last subculture shall meet the minimum area requirement specified in §§ 113.46 and 113.47.
(2) Monolayers shall be examined regularly throughout the required maintenance period for evidence of the presence of cytopathogenic agents. If evidence of a cytopathogenic agent is found, the batch of primary cells is unsatisfactory.
(3) At the conclusion of the required maintenance period, monolayers shall be tested for:
(i) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46;
(ii) Extraneous viruses by the fluorescent antibody technique as prescribed in § 113.47.
When prescribed in an applicable Standard Requirement or in a filed Outline of Production each cell line used to prepare a biological product shall be tested as prescribed in this section. A cell line found unsatisfactory by any prescribed test shall not be used. A serial of biological product shall not be released if produced from a cell line that is found unsatisfactory by any prescribed test.
(a) General requirements. (1) A complete record of the cell line shall be kept, such as, but not limited to, the source, passage history, and medium used for propagation.
(2) A Master Cell Stock (MCS) shall be established at a specified passage level for each cell line. The passage level and identity of the MCS and the highest passage level (MCS + n) intended for use in the preparation of a biological product shall be specified in the Outline of Production for the product.
(3) Sufficient 1.0 ml or larger aliquots of MCS and MCS + n shall be prepared, kept in a frozen state, and made available to Animal and Plant Health Inspection Service (APHIS) upon request for performing the tests prescribed in this section.
(4) Each lot of cells shall be monitored for the characteristics determined to be normal for the cell line, such as, but not limited to, microscopic appearance, growth rate, acid production, or other observable features.
(b) The MCS shall be shown to be of the same species of origin as that reported in paragraph (a)(1) of this section by the following method:
(1) At least four monolayers with a total area of at least 6 cm
2 shall be grown to at least 80 percent confluency.
(2) The monolayers shall be removed from their media, processed, stained, and examined.
(i) At least two monolayers shall be stained with an antispecies fluorchrome-conjugated antibody unrelated to the species of origin of the MCS.
(ii) At least two monolayers shall be stained with an antispecies fluorochrome-conjugated antibody specific to the species of origin of the MCS.
(iii) All monolayers shall be examined for evidence of specific fluorescence.
(3) If specific fluorescence is not found in the monolayers stained with the conjugate specific to the species of origin of the MCS, the cell line is unsatisfactory and shall not be used for vaccine production.
(4) If nonspecific fluorescence is found in the monolayers stained with conjugate from an unrelated species of origin or other results make the test results equivocal, the procedure shall be repeated until either specific fluorescence is found only in the monolayers stained with conjugate specific to the species of origin of the MCS and not in the control monolayers or specific fluorescence cannot be identified and the MCS is declared unsatisfactory.
(5) Alternate tests to determine the species of origin of the MCS may be used if approved by APHIS.
(c) The MCS and either each subculture of cells used to prepare a biological product or the final pool of harvested material (with or without the stabilizer) or final container samples of completed product for each serial of such product shall be shown to be free of mycoplasma as prescribed in § 113.28. The sample for testing shall consist of at least 75 cm
2 of actively growing cells or the equivalent, in harvest fluids. The cells shall represent all sources of cells in the batch.
(d) The MCS and either each subculture used to prepare a biological product or the final pool of harvested material for each serial of such product or final container samples of completed product for each serial of such product shall be tested for bacteria and fungi as prescribed in § 113.26 or § 113.27 (whichever is applicable). If bacteria or fungi are found in the MCS, the MCS shall not be used. If bacteria or fungi are found in a subculture, the subculture shall not be used.
(e) A monolayer at least 75 cm
2 from each MCS shall be shown free of extraneous agents as prescribed in this paragraph.
(1) The test monolayer shall be maintained for at least 21 days using the medium (with additives) intended for growth and maintenance and under conditions similar to those used to prepare biological products.
(2) Cells shall be subcultured at least two times during the maintenance period. All but the last subculture shall result in at least one new monolayer of at least 75 cm
2 . The last subculture shall meet the minimum area requirement specified in §§ 113.46 and 113.47 and paragraph (f) of this section.
(3) Monolayers shall be examined regularly throughout the 21-day maintenance period for evidence of the presence of cytopathogenic agents. If evidence of a cytopathogenic agent is found, the MCS is unsatisfactory.
(4) At the conclusion of the 21-day maintenance period, monolayers shall be tested for:
(i) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46; and
(ii) Extraneous agents by the fluorescent antibody technique as prescribed in § 113.47.
(f) At the conclusion of the 21-day maintenance period provided in paragraph (e) of this section, at least one monolayer of at least 75 cm
2 shall also be shown free of extraneous agents as prescribed in this paragraph.
(1) Alternately freeze and thaw the monolayer(s) three times. Centrifuge the disrupted cells at no greater than 2,000 × g for no more than 15 minutes to remove cellular debris. Divide the supernatant into equal aliquots and dispense 1.0 ml onto each of at least one monolayer (at least 75 cm
2 ) of:
(i) Vero (African green monkey kidney) cell line;
(ii) Embryonic cells, neonatal cells, or a cell line of the same species of origin as the MCS if different than provided in paragraph (f)(1)(i) of this section;
(iii) Embryonic cells, neonatal cells, or a cell line of the species for which the vaccine is recommended if different than provided in paragraph (f)(1)(ii) of this section; and
(iv) Embryonic cells, neonatal cells, or a cell line of bovine origin if not specified in paragraphs (f)(1)(ii), and (iii) of this section.
(2) The monolayers of cells specified in paragraphs (f)(1)(i), (ii), (iii), and (iv) of this section shall be maintained for at least 14 days after inoculation with the aliquot of disrupted MCS. Monolayers shall be subcultured at least once during the maintenance period. All but the last subculture shall result in a new monolayer of at least 75 cm
2 . The last subculture shall meet the minimum area requirement specified in §§ 113.46 and 113.47.
(3) Monolayers shall be examined regularly throughout the 14-day maintenance period for evidence of the presence of cytopathogenic agents. If evidence of a cytopathogenic agent is found, the MCS is unsatisfactory.
(4) At the conclusion of the 14-day maintenance period, monolayers shall be tested for:
(i) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46; and
(ii) Extraneous viruses by the fluorescent antibody technique as prescribed in § 113.47.
(g) The karyology of cells lines used in the production of biologics shall be examined as follows. A minimum of 50 mitotic cells shall be examined at both the MCS and MCS + n. The modal number in the MCS + n shall not exceed plus or minus 15 percent of the modal number of the MCS. Any marker chromosomes present in the MCS shall persist at the MCS + n. If the modal number exceeds the limits and/or the marker chromosomes do not persist (through the MCS + n passage level), the cell line shall not be used for vaccine production.
(h) If direct or indirect evidence exists that a cell line which is intended for use in the preparation of a vaccine may induce malignancies in the species for which the product is intended, that cell line shall be tested for tumorigenicity/oncogenicity by a method acceptable to APHIS.
Each lot of ingredient of animal origin which is not subjected to heat sterilization or other sterilization methods acceptable to Animal and Plant Health Inspection Service (APHIS), such as, but not limited to serum and albumin, used to prepare a biological product shall be tested as prescribed in this section by the licensee or a laboratory acceptable to VS. Results of all tests shall be recorded by the testing laboratory and made a part of the licensee's records. A lot of ingredient found unsatisfactory by any prescribed test shall not be used to prepare a biological product. A serial of biological product shall not be released if produced using an ingredient that is found unsatisfactory by any prescribed test.
(a) Samples of each lot of ingredient of animal origin which is not subjected to heat sterilization, used to prepare a biological product shall be shown free of mycoplasma by the method prescribed in § 113.28.
(b) Samples of each lot of ingredient or animal origin which is not subjected to heat sterilization of other sterilization methods acceptable to APHIS used to prepare a biological product shall be shown free of bacteria and fungi as prescribed in § 113.26.
(c) Samples of each lot of ingredient of animal origin, except porcine trypsin, which is not subjected to heat sterilization or other viricidal procedure acceptable to APHIS used in the preparation of biological products shall be tested as prescribed in this paragraph;
(1) Monolayers at least 75 cm
2 of Vero (African green monkey kidney) cell line and of primary cells or a cell line of the same species of origin as the ingredient shall be used in the test. Cell lines used shall have been found satisfactory when tested as prescribed in § 113.52 and primary cells used shall have been found satisfactory when tested as prescribed in § 113.51.
(2) At least 3.75 ml or 15 percent of the ingredient shall be used in the growth medium for the preparation of at least 75 cm
2 test monolayers. The ingredient shall also be used in the growth medium when monolayers are subcultured. If the ingredient being tested is cytotoxic when tested in this manner, other procedures may be used if approved by APHIS.
(3) The test monolayers shall be maintained for at least 21 days.
(4) Cells shall be subcultured at least two times during the maintenance period. All but the last subculture shall result in at least one new monolayer of at least 75 cm
2 . The last subculture shall meet the minimum area requirements specified in §§ 113.46 and 113.47.
(5) Monolayers shall be examined regularly throughout the 21-day maintenance period for evidence of cytopathogenic agents. If evidence of a cytopathogenic agent is found, the ingredient is unsatisfactory.
(6) At the conclusion of the 21-day maintenance period, monolayers shall be tested for:
(i) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46; and
(ii) Extraneous viruses by the fluorescent antibody technique as prescribed in § 113.47.
(d) Each lot of porcine trypsin which has not been treated to inactivate porcine parvovirus (PPV) in a manner acceptable to VS shall be tested for PPV as prescribed in this paragraph.
(1) Not less than 5.0 grams of trypsin shall be dissolved in a volume of suitable diluent sufficient to fill a centrifuge angle head. After centrifuging for 1 hour at 80,000 × g, the pellet material shall be reconstituted in distilled water and inoculated into a flask containing 75 cm
2 of a 30 to 50 percent confluent monolayer culture of primary porcine cells or a porcine cell line of proven equal PPV susceptibility. An additional flask of cells shall be held as a negative control.
(2) The test and control monolayers shall be maintained for at least 14 days and subcultured at least once during the maintenance period.
(3) At the end of the 14-day maintenance period, and 4 to 7 days after the last subculturing, monolayers shall be tested for the presence of porcine parvovirus by the fluorescent antibody technique as prescribed in § 113.47(c).
(e) A sample of serum from each donor horse used to produce a lot of equine serum used in the preparation of biological products recommended for use in horses shall be tested at a laboratory approved by Animal and Plant Health Inspection Service using the Coggins test for equine infectious anemia antibodies. If antibodies to equine infectious anemia are found, the lot of serum is unsatisfactory.
Sterile Diluent shall be supplied in a final container by the licensee when such diluent is required for rehydration or dilution of the vaccine.
(a) Sterile Diluent may be distilled or deionized water or it may be a special liquid solution formulated in accordance with an acceptable outline on file with Animal and Plant Health Inspection Service.
(b) Each quantity prepared at one time in a single container and bottled into final containers shall be designated as a serial. Each serial shall be given a number which shall be used in records, test reports, and on the final container label.
(c) Final container samples from each serial shall be tested for bacteria and fungi in accordance with the test provided in § 113.26. Any serial found to be unsatisfactory shall not be released.
Unless otherwise prescribed in a Standard Requirement or in a filed Outline of Production, each Master Seed Virus (MSV) shall be tested as prescribed in this section. A MSV found unsatisfactory by any prescribed test shall not be used. A serial of biological product shall not be released if produced from a MSV that is found unsatisfactory by any prescribed test.
(a) At least a 1.0 ml aliquot per cell culture of MSV shall be dispensed onto monolayers (at least 75 cm
2 in area) of:
(1) Vero (African green monkey kidney) cell line;
(2) Embryonic cells, neonatal cells, or a cell line of the species for which the vaccine is recommended; and
(3) Embryonic cells, neonatal cells, or a cell line of the species of cells in which the MSV is presently being propagated if different than prescribed in paragraphs (a)(1) and (a)(2) of this section. Cell lines used shall have been found satisfactory when tested as prescribed in § 113.52 and primary cells used shall have been found satisfactory when tested as prescribed in § 113.51. If the MSV is cytopathic for or causes hemadsorption in the cells in which it is to be tested, the MSV shall be neutralized with monospecific antiserum supplied or approved by Animal and Plant Health Inspection Service (APHIS) or counteracted by a method approved by APHIS.
(b) At least one monolayer of each cell type used in the test shall be maintained as an uninoculated control.
(c) Each monolayer shall be maintained at least 14 days.
(d) Cells shall be subcultured at least once during the maintenance period. All but the last subculture shall result in at least one new monolayer at least 75 cm
2 . The last subculture shall meet the minimum area requirement specified in §§ 113.46 and 113.47.
(e) Monolayers shall be examined regularly throughout the 14-day maintenance period for evidence of cytopathogenic agents. If evidence of a cytopathogenic agent is found, the MSV is unsatisfactory.
(f) At the conclusion of the 14-day maintenance period, monolayers shall be tested for:
(1) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46;
(2) Extraneous agents by the fluorescent antibody technique as prescribed in § 113.47.
When prescribed in an applicable Standard Requirement or in the filed Outline of Production, a live bacterial vaccine shall meet the requirements in this section.
(a) Purity test. Final container samples of completed product from each serial and subserial, and samples of each lot of Master Seed Bacteria shall be tested for the presence of extraneous viable bacteria and fungi in accordance with the test provided in § 113.27(b).
(b) Safety tests. (1) Samples of completed product from each serial or first subserial and samples of each lot of Master Seed Bacteria shall be tested for safety in young adult mice in accordance with the test provided in § 113.33(b) unless:
(i) The bacteria or agents in the vaccine are inherently lethal for mice.
(ii) The vaccine is recommended for poultry.
(2) Samples of completed product from each serial or first subserial of live bacterial vaccine shall be tested for safety in one of the species for which the product is recommended as follows:
(i) Live bacterial vaccine recommended for use in dogs shall be tested as provided in § 113.40, except that dogs shall be injected with the equivalent of two doses of vaccine administered as recommended on the label.
(ii) Live bacterial vaccine recommended for use in cattle shall be tested as provided in § 113.41, except that calves shall be injected with the equivalent of two doses of vaccine administered as recommended on the label.
(iii) Live bacterial vaccine recommended for use in sheep shall be tested as provided in § 113.45.
(iv) Live bacterial vaccine recommended for use in swine shall be tested as provided in § 113.44.
(c) Identity test. At least one of the identity tests provided in this paragraph shall be conducted for the Master Seed Bacteria and final container samples from each serial or first subserial of completed biological product. A known positive control (reference) provided or approved by Animal and Plant Health Inspection Service shall be included in such tests.
(1) Fluorescent antibody test. The direct fluorescent antibody staining technique shall be conducted using suitable smears of the vaccine bacteria. Fluorescence typical for the bacteria concerned shall be demonstrated. Fluorescence shall not occur in control smears treated with specific antiserum.
(2) Tube agglutination test. A tube agglutination test shall be conducted with a suitable suspension of the vaccine bacteria using the constant antigen decreasing serum method with specific antiserum. Agglutination typical for the bacteria shall be demonstrated. Agglutination shall not occur with negative serum used as a control in this test.
(3) Slide agglutination test. The rapid plate (slide) agglutination test shall be conducted with suitable suspensions of the vaccine bacteria using the hanging drop, slide or plate method, with specific antiserum. Agglutination typical for the bacteria shall be demonstrated by microscopic or macroscopic observation. Agglutination shall not occur with negative serum used as a control in this test.
(4) Characterization tests. Applicable biochemical and cultural characteristics shall be demonstrated as specified in the filed Outline of Production.
(d) Ingredient requirements. Ingredients used for the growth and preparation of Master Seed Bacteria and of live bacterial vaccine shall meet the requirements provided in § 113.50. Ingredients of animal origin shall meet the applicable requirements provided in § 113.53.
(e) Moisture content. The maximum percent moisture in desiccated vaccines shall be stated in the filed Outline of Production and shall be established by the licensee as follows:
(1) Prelicensing. Data obtained by conducting accelerated stability tests and bacterial counts shall be acceptable on a temporary basis.
(2) Licensed products. Data shall be obtained by determining the percent moisture and bacterial count at release and expiration on a minimum of 10 consecutive released serials.
(3) Final container samples of completed product from each serial and subserial must be tested for moisture content in accordance with the test provided in § 113.29.
Brucella Abortus Vaccine shall be prepared as a desiccated live culture bacterial vaccine from smooth colonial forms of the Brucella abortus organism, identified as Strain 19. Each serial and subserial shall be tested for purity, potency, and moisture content. A serial or subserial found unsatisfactory by a prescribed test shall not be released.
(a) Purity tests. Each serial and subserial shall be tested for purity as provided in this paragraph.
(1) Macroscopic and microscopic examination shall be made on bulk samples from production containers. If organisms not typical of Brucella abortus organisms are evident, the serial or subserial is unsatisfactory.
(2) Two final container vials of completed product shall be tested by inoculating one tube of Dextrose Andrades broth with gas tube and one tube of thioglycollate broth from each vial. The inoculated media shall be incubated at 35 to 37 °C for 96 hours. If growth not typical of Brucella abortus organisms is evident, the serial or subserial is unsatisfactory.
(3) Bacterial dissociation test. Final container samples of completed product from each serial and subserial shall be tested for bacterial dissociation. Smooth colonies are the desired form. Rough colonies are undesirable terminal dissociation forms. Intermediate and intermediate-to-rough are also undesirable.
(i) The sample container shall be rehydrated and streaked on one potato agar plate in such a manner as to produce confluent colonies. Artificial reflected light shall be used so that the rays pass through the plate at a 45 °angle.
(ii) If the vaccine contains more than 5 percent rough colonies or more than 15 percent total undesirable colonies, the serial or subserial is unsatisfactory. If organisms or growth not characteristic of Brucella abortus are found, the serial or subserial is unsatisfactory. The test may be repeated one time using double the number of samples: Provided, That, if the test is not repeated, the serial or subserial is unsatisfactory.
(b) Bacterial count requirements for reduced dose vaccine. Each serial and each subserial shall be tested for potency.
(1) Two final container vials of completed product shall be tested for the number of viable organisms per dose of rehydrated vaccine. A bacterial count per vial shall be made on tryptose agar plates from suitable dilutions using 1 percent peptone as a diluent. The inoculated media shall be incubated at 35 to 37 °C for 96 hours.
(2) If the average count of the two final container samples of freshly prepared vaccine contains less than 3.0 or more than 10.0 billion organisms per dose, the serial or subserial is unsatisfactory.
(3) If the average count on the initial test is less than the minimum or greater than the maximum required in paragraph (b)(2) of this section, the serial or subserial may be retested one time using four additional final container vials. The average count of the retest is determined. If the average count of the four vials retested is less than the required minimum or greater than the required maximum, the serial or subserial is unsatisfactory. If the average count of the four vials retested is within the required limits described in paragraph (b)(2) of this section, the following shall apply:
(i) If the average count obtained in the initial test is less than one-third or more than three times the average count obtained on the retest, the average count of the initial test shall be considered the result of test system error and the serial or subserial is satisfactory.
(ii) If the average count obtained in the initial test is one-third or more than the average retest count or three times or less than the average retest count, a new average count shall be determined from the counts of all six vials. If the new average is less than the minimum or greater than the maximum required in paragraph (b)(2) of this section, the serial or subserial is unsatisfactory.
(4) If tested at any time within the expiration period, each dose of rehydrated vaccine must contain at least 3.0 billion viable organisms per dose.
(c) Bacterial count requirements for standard vaccine. Each serial and subserial shall be tested for potency.
(1) Two final container samples shall be tested for the number of viable organisms per milliliter of rehydrated vaccine. One bacterial count per vial shall be made on tryptose agar plates from suitable dilutions using 1 percent peptone as a diluent. The inoculated media shall be incubated at 35 to 37 °C for 96 hours.
(2) If the average count of the two final container samples of freshly prepared vaccine does not contain at least 10 billion viable organisms per milliliter, the serial or subserial is unsatisfactory.
(3) If the initial bacterial count is less than 10 billion organisms per milliliter, the serial or subserial may be retested one time using four samples. If the average count of the four vials retested is less than the required minimum, the serial or subserial is unsatisfactory.
(4) If tested at any time within the expiration period, each milliliter of rehydrated vaccine does not contain at least 5 billion viable organisms per milliliter, the serial or subserial is unsatisfactory.
Anthrax Spore Vaccine—Nonencapsulated shall be a live spore suspension prepared from nonencapsulated variants of Bacillus anthracis. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.
(a) The Master Seed shall meet the applicable general requirements prescribed in § 113.64 and the requirements in this section.
(b) Each lot of Master Seed shall be tested for immunogenicity as follows:
(1) Forty-two susceptible guinea pigs from the same source each weighing 400 to 500 grams, shall be used as test animals (30 vaccinates and 12 controls).
(2) An arithmetic mean spore count of vaccine produced from the highest passage of the Master Seed shall be established before the immunogenicity test is conducted. The guinea pigs used as vaccinates shall be injected as recommended on the label with a predetermined number of vaccine spores. To confirm the dosage, five replicate spore counts shall be conducted on a sample of the vaccine dilution used.
(3) Fourteen to fifteen days postvaccination the vaccinates and controls shall each be challenged with not less than 4,500 guinea pig LD 50 of a virulent suspension of Bacillus anthracis furnished or approved by Animal and Plant Health Inspection Service and observed for 10 days.
(4) If at least 10 of the 12 controls do not die from Bacillus anthracis within the 10-day postchallenge observation period the test is invalid and may be repeated.
(5) If at least 27 of 30 of the vaccinates do not survive the 10-day postchallenge observation period, the Master Seed is unsatisfactory.
(6) An Outline of Production change shall be made before authority for use of a new lot of Master Seed shall be granted by Animal and Plant Health Inspection Service.
(c) Test Requirements for Release. Each serial and subserial shall meet the applicable general requirements prescribed in 9 CFR 113.64 and the requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) Safety test. Samples of completed product from each serial or first subserial shall be tested for safety in sheep or goats by the methods described in 9 CFR 113.45(a).
(2) Spore Count Requirements. Final container samples of completed product shall be tested for spore count. Samples shall be diluted in tenfold steps. Each dilution expected to yield 30 to 300 colonies per plate shall be plated in triplicate on tryptose agar, inverted, and incubated at 35 to 70 °C for 24 hours to 28 hours. Each plate having uniformly distributed colonies shall be counted and an average count determined. To be eligible for release, each serial and each subserial shall have a spore count sufficiently greater than that of the vaccine used in the immunogenicity test to assure that when tested at any time within the expiration period, each serial and subserial shall have a spore count of at least twice that used in the immunogenicity test but not less than 2,000,000 spores per dose.
Erysipelothrix Rhusiopathiae Vaccine shall be prepared as a desiccated live culture of an avirulent or modified strain of Erysipelothrix rhusiopathiae. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for vaccine production.
(a) The Master Seed shall meet the applicable requirements prescribed in § 113.64 and the requirements in this section.
(b) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity. The selected bacterial count from the lot of Master Seed shall be established as follows:
(1) Thirty Erysipelothrix rhusiopathiae susceptible swine shall be used as test animals (20 vaccinates and 10 controls) for each route of administration recommended on the label.
(2) An arithmetic mean count of the colony forming units from vaccine produced from the highest passage of the Master Seed shall be established before the immunogenicity test is conducted. The 20 swine to be used as vaccinates shall be injected as recommended on the label with a predetermined quantity of vaccine bacteria. The 10 control swine shall be held separately from the vaccinates. To confirm the dosage calculation, an arithmetic mean count shall be established by conducting five replicate titrations on a sample of the bacterial vaccine dilution used. Only plates containing between 30 and 300 colonies shall be considered in a valid test.
(3) The vaccinates and controls shall be examined and their average body temperature determined prior to challenge. Fourteen to twenty-one days postvaccination, the vaccinates and controls shall be challenged with a virulent Erysipelothrix rhusiopathiae culture and observed for 7 days. The challenge culture and instructions for preparation and use shall be obtained from Animal and Plant Health Inspection Service.
(4) A satisfactory challenge shall be evidenced in the controls by a high body temperature or clinical signs including, but not limited to acute illness with hyperemia of the abdomen and ears, possibly terminating in sudden death; moribundity, with or without metastatic skin lesions; depression with anorexia, stiffness, and/or joint involvement; or any combination of these symptoms and lesions.
(5) If at least 80 percent of the controls do not show characteristic signs during the observation period including, but not limited to a body temperature of 105.6 °F or higher on at least 2 consecutive days, the test shall be considered a No Test: Provided, That control pigs which meet the criteria requirements for susceptibility except for high body temperature shall be considered susceptible if sacrificed and organisms identified as Erysipelothrix rhusiopathiae can be isolated from the blood, spleen, or other organs.
(6) To demonstrate immunity after challenge, the vaccinates shall remain free of clinical signs and the body temperature shall not exceed 104.6 °F on 2 or more consecutive days. If at least 90 percent of the vaccinates do not remain free from clinical signs and high body temperature throughout the observation period, the Master Seed is unsatisfactory.
(7) An Outline of Production change shall be made before authority for use of a new Master Seed shall be granted by Animal and Plant Health Inspection Service.
(c) Test requirements for release. Each serial and subserial shall meet the applicable requirements in § 113.64 and the requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) Safety test. Samples of completed product from each serial or first subserial shall be tested for safety in young adult mice as prescribed in § 113.33(b) and in swine as prescribed in § 113.44.
(2) Bacterial count requirements. Final container samples of completed product from each serial and each subserial shall be tested for bacterial count using the method used in paragraph (b)(2) of this section. Two replicate titrations shall be conducted on each sample. To be eligible for release, each serial and subserial shall have a bacterial count sufficiently greater than that of the vaccine used in the immunogenicity test to assure that, when tested at any time within the expiration period, each serial and subserial shall have a bacterial count two times greater than that used in such immunogenicity test.
Pasteurella Haemolytica Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent or modified strain of Pasteurella haemolytica, identified as serotype 1. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.
(a) The Master Seed shall meet the applicable general requirements prescribed in § 113.64 and the requirements in this section.
(b) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity. The immunogenicity of a selected bacterial count from the lot of Master Seed shall be established as follows:
(1) Fifteen Pasteurella haemolytica susceptible calves shall be used as test animals (10 vaccinates and 5 controls) for each route of administration recommended on the label.
(2) An arithmetic mean count of the colony forming units from vaccine produced from the highest passage of the Master Seed shall be established before the immunogenicity test is conducted. The 10 calves to be used as vaccinates shall be injected as recommended on the label with a predetermined quantity of vaccine bacteria. The five control calves shall be held separately from the vaccinates. To confirm the dosage calculation, five replicate titrations on a sample of the bacterial vaccine used. Only plates containing between 30 and 300 colonies shall be considered a valid test.
(3) The vaccinates and controls shall be examined and their average body temperature determined prior to challenge. Fourteen to twenty-one days post vaccination, the vaccinates and controls shall each be challenged by the respiratory route with a (virulent) pneumonia producing Pasteurella haemolytica culture and observed for 4 to 7 days. The challenge culture and instructions for preparation for use shall be furnished or approved by the Animal and Plant Health Inspection Service.
(4) A satisfactory challenge shall be evidenced in the controls by progression of clinical signs consistent with respiratory system infection following challenge, including but not limited to lacrimation, mucoid nasal exudates, expiratory dyspnea, tachypnea, pulmonary rales, and cough possibly terminating in death; moribundity, depression with anorexia, diarrhea with substantial weight loss; or any combination of these symptoms.
(5) Lung lesion response to challenge will be assessed in all calves. Lung lesions will be assessed at necropsy in calves that succumb to challenge. Surviving calves will be euthanized on day 4 to 7 following challenge and lung lesions assessed at necropsy. Lung lesion scores will be used in the assessment of the response to challenge exposure. If a significant difference in lung lesion scores cannot be demonstrated between vaccinates and controls using a scoring system approved by the Animal and Plant Health Inspection Service, the Master Seed is unsatisfactory.
(6) An Outline of Production change must be made before authority for use of a new lot of Master Seed is granted by the Animal and Plant Health Inspection Service.
(c) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §§ 113.8 and 113.64 and the requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) Safety test. Samples of completed product from each serial or first subserial shall be tested for safety in calves as provided in §§ 113.41(a) and 113.41(b) except, that the equivalent of two doses of vaccine shall be used and administered in the manner recommended on the label.
(2) Bacterial count requirements. Final container samples of completed product shall be tested for bacterial count using the method used in paragraph (b)(2) of this section. Two replicate titrations shall be conducted on each serial and subserial. Each sample shall be rehydrated with accompanying sterile diluent to the volume indicated on the label. To be eligible for release, each serial and subserial shall have a bacterial count sufficiently greater than that of the vaccine used in the immunogenicity test to assure that, when tested at any time within the expiration period, each serial and subserial shall have a bacterial count at least two times greater than that used in the immunogenicity test.
Pasteurella Multocida Vaccine, Bovine, shall be prepared as a desiccated live culture bacterial vaccine of an avirulent or modified strain of Pasteurella multocida, of bovine origin. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.
(a) The Master Seed shall meet the applicable general requirements prescribed in § 113.64 and the requirements in this section.
(b) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity. The immunogenicity of a selected bacterial count from the lot of Master Seed shall be established as follows:
(1) Fifteen Pasteurella multocida susceptible calves shall be used as test animals (10 vaccinates and 5 controls) for each route of administration recommended on the label.
(2) An arithmetic mean count of the colony forming units from vaccine produced from the highest passage of the Master Seed shall be established before the immunogenicity test is conducted. The 10 calves to be used as vaccinates shall be injected as recommended on the label with a predetermined quantity of vaccine bacteria. The five control calves shall be held separately from the vaccinates. To confirm the dosage calculation, arithmetic mean count shall be established by conducting five replicate titrations on a sample of the bacterial vaccine used. Only plates containing between 30 and 300 colonies shall be considered a valid test.
(3) The vaccinates and controls shall be examined and their average body temperature determined prior to challenge. Fourteen to twenty-one days post vaccination, the vaccinates and controls shall each be challenged by the respiratory route with a (virulent) pneumonia producing Pasteurella multocida culture and observed for 4 to 10 days. The challenge culture and instructions for preparation for use shall be furnished or approved by the Animal and Plant Health Inspection Service.
(4) A satisfactory challenge shall be evidenced in the controls by progression of clinical signs consistent with respiratory system infection following challenge, including but not limited to acute illness with higher body temperature and respiration rate, lacrimation, mucoid nasal exudate, expiratory dyspnea, tachypnea, pulmonary rales, and cough, possibly terminating in death; moribundity, depression with anorexia; diarrhea with substantial weight loss; or any combination of these symptoms.
(5) Lung lesion response to challenge will be assessed in all calves. Lung lesions will be assessed at necropsy in calves that succumb to challenge. Surviving calves will be euthanized on day 4 to 10 following challenge and lung lesions assessed at necropsy. Lung lesion scores will be used in the assessment of the response to challenge exposure. If a significant difference in lung lesion scores cannot be demonstrated between vaccinates and controls using a scoring system approved by the Animal and Plant Health Inspection Service, the Master Seed is unsatisfactory.
(6) An Outline of Production change must be made before authority for use of a new lot of Master Seed is granted by the Animal and Plant Health Inspection Service.
(c) Test requirements for release. Each serial and subserial shall meet the applicable general requirements prescribed in §§ 113.8 and 113.64 and the requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) Safety Test. Samples of completed product from each serial or first subserial shall be tested for safety in calves as provided in §§ 113.41(a) and 113.41(b), except that the equivalent of two doses of vaccine shall be used and administered in the manner recommended on the label.
(2) Bacterial count requirements. Final container samples of completed product shall be tested for bacterial count using the method used in paragraph (b)(2) of this section. Two replicate titrations shall be conducted on each serial and subserial. Each sample shall be rehydrated with accompanying sterile diluent to the volume indicated on the label. To be eligible for release, each serial and subserial shall have a bacterial count sufficiently greater than that of the vaccine used in the immunogenicity test count per dose established to assure that, when tested at any time within the expiration period, each serial and subserial shall have a bacterial count at least two times greater than that used in the immunogenicity test.
Pasteurella Multocida Vaccine, Avian Isolate, shall be prepared as a desiccated live culture of an avirulent or modified strain of Pasteurella multocida. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for vaccine production.
(a) The Master Seed shall meet the applicable general requirements prescribed in § 113.64 and the requirements in this section.
(b) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity in each species and for each serotype for which the Master Seed is claimed to give protection.
(1) Thirty Pasteurella multocida susceptible birds shall be used as test animals (20 vaccinates and 10 controls) for each bird species, route of administration, and serotype for which protection is claimed on the label.
(2) An arithmetic mean count of colony forming units from vaccine produced from the highest passage of Master Seed shall be established before the immunogenicity test is conducted. The 20 birds to be used as vaccinates shall be inoculated, as recommended on the label with a predetermined quantity of vaccine bacteria. The 10 control birds shall be held separately from the vaccinates. To confirm the dosage calculation, an arithmetic mean count shall be established by conducting five replicate titrations on a sample of the bacterial vaccine used. Only plates containing between 30 and 300 colonies shall be considered in a valid test.
(3) Not less than 14 days after vaccination, each of 20 vaccinates and each of 10 unvaccinated controls shall be challenged intramuscularly or by other methods acceptable to the Animal and Plant Health Inspection Service with a virulent Pasteurella multocida strain, for which protection is claimed, and observed daily for a 14 day post-challenge period.
(4) Eight or more of the unvaccinated controls must die for the test to be valid. If at least 16 of 20 of the vaccinates do not survive the 14-day postchallenge period, the Master Seed is unsatisfactory at the selected bacterial count.
(c) Test requirements for release. Each serial and subserial shall meet the applicable requirements in §§ 113.8 and 113.64 and the requirements in this paragraph. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) Safety test. Samples of completed product from each serial or first subserial shall be tested for safety.
(i) Ten birds of a species for which the vaccine is recommended shall be given the equivalent of 10 doses each of the vaccine and observed for 10 days. If the vaccine is recommended for more than one species, only one species needs to be tested.
(ii) If unfavorable reactions attributable to the vaccine occur during the observation period in two or more of the test birds, the serial is unsatisfactory.
(iii) If unfavorable reactions occur which are not attributable to the test vaccine, the test is a No Test and may be repeated. If the results of the next test are not satisfactory, or if the test is not repeated, the serial shall be considered unsatisfactory.
(2) Bacterial count requirements. Final container samples of completed product shall be tested for bacterial count using the method used in paragraph (b)(2) of this section. Two replicate titrations shall be conducted on each serial and subserial. Each sample shall be rehydrated with accompanying sterile diluent to the volume indicated on the label. To be eligible for release, each serial and subserial shall have a bacterial count sufficiently greater than that of the vaccine used in the immunogenicity test count per dose established to assure that, when tested at any time within the expiration period, each serial and subserial shall have a bacterial count at least two times greater than that used in the immunogenicity test.
Chlamydia Psittaci Vaccine (Feline Pneumonitis), Live Chlamydia, shall be prepared from chlamydia-bearing cell culture fluids or embryonated chicken eggs. Only Master Seed which has been established as pure, safe, and immunogenic shall be used for vaccine production. All serials of vaccine shall be prepared from the first through the fifth passage from the Master Seed.
(a) The Master Seed shall meet the applicable requirements prescribed in § 113.300 and the requirements in this section. Master Seed propagated in chicken embryos shall be tested for pathogens by the chicken embryo test prescribed in § 113.37. If found unsatisfactory by any prescribed test, the Master Seed shall not be used.
(b) Each lot of Master Seed used for vaccine production shall be tested for immunogenicity. The immunogenicity of a selected dose from the lot of Master Seed shall be established as follows:
(1) Thirty feline pneumonitis susceptible cats shall be used as test animals (20 vaccinates and 10 controls). Blood samples shall be drawn and individual serum samples tested. The cats shall be considered suitable for use if all serums are negative for pneumonitis antibody in a complement fixation test or other test of equal sensitivity.
(2) A geometric mean titer of the dried vaccine produced from the highest passage of the Master Seed shall be established before the immunogenicity test is conducted. The 20 cats used as vaccinates shall be administered a predetermined quantity of vaccine by the method to be recommended on the label and the remaining 10 cats shall be held as controls. To confirm the dosage calculations, five replicate titrations shall be conducted on a sample of the vaccine dilution used. If two doses are used, five replicate confirming titrations shall be conducted on each dose.
(3) Fourteen or more days after the final dose of vaccine, the vaccinates and controls shall each be challenged intranasally with a minimum of 10,000 yolk sac LD50 of virulent feline pneumonitis furnished or approved by the Animal and Plant Health Inspection Service and observed each day for 28 days postchallenge. The rectal temperature of each animal shall be taken and the presence or absence of clinical signs noted and recorded each day.
(i) If less than 8 of 10 controls show clinical signs of feline pneumonitis infection other than fever, the test is a No Test and may be repeated.
(ii) If a significant difference in clinical signs other than fever or chlamydia shedding cannot be demonstrated between vaccinates and controls using a scoring system approved by the Animal and Plant Health Inspection Service, the Master Seed is unsatisfactory.
(4) An Outline of Production change must be made before authority for use of a new lot of Master Seed is granted by the Animal and Plant Health Inspection Service.
(c) Test requirements for release: Except for § 113.300(a)(3)(ii), each serial and subserial shall meet the requirements prescribed in § 113.300 and in this paragraph. Final container samples of completed product shall be tested. Any serial or subserial found unsatisfactory by a prescribed test shall not be released.
(1) The test for pathogens prescribed in § 113.37 shall be conducted on each serial or one subserial of avian origin vaccine.
(2) Chlamydia titer requirements. Final container samples of completed product shall be tested for chlamydia titer using the titration method used in paragraph (b)(2) of this section. To be eligible for release, each serial and each subserial shall have a titer sufficiently greater than the titer of vaccine used in the immunogenicity test prescribed in paragraph (b) of this section to assure that when tested at any time within the expiration period, each serial and subserial shall have a titer 0.7 greater than that used in such immunogenicity test but not less than 2.5 ID50 per dose.
Unless otherwise prescribed in an applicable Standard Requirement or in the filed Outline of Production, an inactivated bacterial product shall meet the applicable requirements in this section.
(a) Purity tests. (1) Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in § 113.26.
(2) Each lot of Master Seed Bacteria shall be tested for the presence of extraneous viable bacteria and fungi in accordance with the test provided in § 113.27(d).
(b) Safety tests. Bulk or final container samples of completed product from each serial shall be tested for safety in young adult mice in accordance with the test provided in § 113.33(b) unless:
(1) The product contains material which is inherently lethal for mice. In such instances, the guinea pig safety test provided in § 113.38 shall be conducted in place of the mouse safety test.
(2) The product is recommended for poultry. In such instances, the product shall be safety tested in poultry as defined in the specific Standard Requirement or Outline of Production for the product.
(3) The product is recommended for fish, other aquatic species, or reptiles. In such instances, the product shall be safety tested in fish, other aquatic species, or reptiles as required by specific Standard Requirement or Outline of Production for the product.
(c) Identity test. Methods of identification of Master Seed Bacteria to the genus and species level by laboratory tests shall be sufficient to distinguish the bacteria from other similar bacteria according to criteria described in the most recent edition of “Bergey's Manual of Systematic Bacteriology” or the American Society for Microbiology “Manual of Clinical Microbiology”. If Master Seed Bacteria are referred to by serotype, serovar, subtype, pilus type, strain or other taxonomic subdivision below the species level, adequate testing must be used to identify the bacteria to that level. Tests which may be used to identify Master Seed Bacteria include, but are not limited to:
(1) Cultural characteristics,
(2) Staining reaction,
(3) Biochemical reactivity,
(4) Fluorescent antibody tests,
(5) Serologic tests,
(6) Toxin typing,
(7) Somatic or flagellar antigen characterization, and
(8) Restriction endonuclease analysis.
(d) Ingredient requirements. Ingredients used for the growth and preparation of Master Seed Bacteria and of final product shall meet the requirements provided in § 113.50. Ingredients of animal origin shall meet the applicable requirements provided in § 113.53.
(e) Only serials tested for viricidal activity in accordance with the test provided in § 113.35 and found satisfactory by such test shall be packaged as diluent for desiccated fractions in combination packages.
(f) If formaldehyde is used as the inactivating agent, and the serial has not been found satisfactory by the viricidal activity test, bulk or final container samples of completed product from each serial must be tested for residual free formaldehyde content using the ferric chloride test.
1
Firms currently using tests for residual free formaldehyde content other than the ferric chloride test have until July 14, 2004 to update their Outline of Production to be in compliance with this requirement.
1 The procedures for performing the ferric chloride test for residual free formaldehyde may be obtained from USDA, APHIS, Center for Veterinary Biologics-Laboratory, 1800 Dayton Road, P.O. Box 844, Ames, IA 50010.
(1) The residual free formaldehyde content of biological products containing clostridial antigens must not exceed 1.85 grams per liter (g/L).
(2) The residual free formaldehyde content of bacterins, bacterin-toxoids, and toxoids, other than those containing clostridial antigens, must not exceed 0.74 grams per liter (g/L).
Leptospira Pomona Bacterin shall be produced from a culture of Leptospira pomona which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira pomona fraction shall meet the applicable requirements in § 113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.
(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in § 113.26.
(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in § 113.38.
(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/800th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.
(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.
(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.
(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster LD 50 as determined by titration.
(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die of leptospirosis, the test is valid and the results shall be evaluated according to the following table:
Stage
Number of vaccinates
Cumulative number of vaccinates
Cumulative total dead hamsters for satisfactory serial
Cumulative total dead hamsters for unsatisfactory serial
1
10
10
2 or less
5 or more.
2
10
20
5 or less
6 or more.
(5) If three or four vaccinates die in the first stage, the second stage shall be conducted in a manner identical to the first stage.
(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.
Leptospira Icterohaemorrhagiae Bacterin shall be produced from a culture of Leptospira icterohaemorrhagiae which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira icterohaemorrhagiae fraction shall meet the applicable requirements in § 113.100 and be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.
(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in § 113.26.
(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in § 113.38.
(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/80th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.
(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.
(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.
(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira icterohaemorrhagiae organisms, using a dose of 10-10,000 hamster LD 50 as determined by titration.
(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die from leptospirosis, the test is valid and the results shall be evaluated according to the following table:
Stage
Number of vaccinates
Cumulative number of vaccinates
Cumulative total dead hamsters for satisfactory serial
Cumulative total dead hamsters for unsatisfactory serial
1
10
10
2 or less
5 or more.
2
10
20
5 or less
6 or more.
(5) If three or four vaccinates die in the first stage, the second stage shall be used. The second stage shall be conducted in a manner identical to the first stage.
(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.
STANDARD REQUIREMENTS (U.S.C.). Retrieved via LawPlayer, https://lawplayer.com/us/act/cfr-title-9-part-113
United States government works (U.S. Code, Code of Federal Regulations) are in the public domain under 17 U.S.C. § 105.
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