(1) Selection shall be made as follows:

(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.

(1) Vaccines:

(2) Bacterins and bacterin-toxoids:

(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:

(5) 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.

(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.

(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.

(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:

(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:

(4) If the average is equal to or greater than the required minimum, the following shall apply to bacterial vaccines:

(5) Exceptions. When a product is evaluated in terms other than log₁₀ 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.

(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.

(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.

(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:

(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:

(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.

(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:

(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.

(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.

(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:

(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:

(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:

(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:

(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:

(1) DPN-Cysteine Solution:

(2) Inactivated horse serum—horse serum which has been inactivated at 56 °C for 30 minutes.

(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:

(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.

(1) Dissolve in 900 ml of purified water by boiling the following:

(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:

(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 CO₂ 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.

(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.

(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.

(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.

(2) Remove the sample container seal.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(1) The test animals shall be determined to be susceptible to the virus under test as follows:

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(1) Three groups of one or more monolayers shall be required for each specific virus prescribed in paragraph (b) of this section.

(2) Each group of monolayers shall have a total area of at least 6 cm².

(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.

(1) All cells shall be tested for:

(2) Bovine, caprine, and ovine cells shall, in addition, be tested for:

(3) Canine cells shall, in addition, be tested for:

(4) Equine cells shall, in addition, be tested for:

(5) Feline cells shall, in addition, be tested for:

(6) Porcine cells shall, in addition, be tested for:

(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.

(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.

(1) The test monolayer shall be maintained using the medium (with additives) and under conditions similar to those used to prepare biological products.

(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:

(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.

(1) At least four monolayers with a total area of at least 6 cm² shall be grown to at least 80 percent confluency.

(2) The monolayers shall be removed from their media, processed, stained, and examined.

(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.

(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 cm². 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:

(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 cm²) of:

(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 cm². 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:

(1) Monolayers at least 75 cm² 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 cm² 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 cm². 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:

(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 cm² 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).

(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.

(1) Cytopathogenic and/or hemadsorbing agents as prescribed in § 113.46;

(2) Extraneous agents by the fluorescent antibody technique as prescribed in § 113.47.

(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:

(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:

(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.

(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.

(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.

(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:

(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.

(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.

(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 LD₅₀ 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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(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.

(1) Safety test. Samples of completed product from each serial or first subserial shall be tested for safety.

(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.

(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.

(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.

(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.

(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).

(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.

(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.

(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).

(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 LD₅₀ 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:

(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.

(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 LD₅₀ 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:

(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.

(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 canicola organisms, using a dose of 10-10,000 hamster LD₅₀ 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, test is valid and the results shall be evaluated according to the following table:

(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.

(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 grippotyphosa organisms, using a dose of 10-10,000 hamster LD₅₀ 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:

(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.

(1) Each of at least 8 but not more than 10 guinea pigs, each weighing 300 to 500 grams, shall be injected subcutaneously with a guinea pig dose. A second guinea pig dose shall be injected 21 to 23 days after the first dose. Each guinea pig dose shall be one-fifth of the dose recommended on the label for a calf.

(2) Clostridium chauvoei challenge material, available upon request from Animal and Plant Health Inspection Service, shall be used for challenge 14 to 15 days following the last injection of the product. Each of eight vaccinates and each of five additional nonvaccinated guinea pigs for controls shall be injected intramuscularly with approximately 100 LD₅₀ of challenge material. This dose shall be determined by statistical analysis of results of titrations of the challenge material. The vaccinates and controls shall be observed for 3 days postchallenge and all deaths recorded.

(3) For a valid test, at least 80 percent of the controls shall die within the 3 day post-challenge observation period. If this requirement is met, the results of the potency test shall be evaluated according to the following table:

(1) Each of at least 8 but not more than 10 guinea pigs, each weighing 300 to 500 grams, shall be injected subcutaneously with a guinea pig dose. A second guinea pig dose shall be injected 21 to 23 days after the first dose. Each guinea pig dose shall be one-fifth of the dose recommended on the label for a calf.

(2) Clostridium haemolyticum challenge material, available upon request from Animal and Plant Health Inspection Service, shall be used for challenge 14 to 15 days following the last injection of the product. Each of eight vaccinates and each of five additional nonvaccinated guinea pigs for controls shall be injected intramuscularly with approximately 100 LD₅₀ of challenge material. This dose shall be determined by statistical analysis of results of titrations of the challenge material. The vaccinates and controls shall be observed for 3 days postchallenge and all deaths recorded.

(3) For a valid test, at least 80 percent of the controls shall die within the 3 day post-challenge observation period. If this requirement is met, the results of the potency test shall be evaluated according to the following table:

(1) When used in this test, the following words and terms shall mean:

(2) Each of at least eight rabbits of a strain acceptable to the Animal and Plant Health Inspection Service, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the recommended cattle dose. Provided, That, if the product is recommended only for sheep, half of the recommended sheep dose shall be used. A second dose shall be given not less than 20 days nor more than 23 days after the first dose.

(3) Fourteen to seventeen days after the second dose, all surviving rabbits shall be bled, and the serum tested for antitoxin content.

(4) The antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows:

(5) Test Interpretation shall be as follows:

(1) When used in this test, the following words and terms shall mean:

(2) Each of at least eight rabbits of a strain acceptable to the Animal and Plant Health Inspection Service, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the recommended cattle dose: Provided, That, if the product is recommended only for sheep, half of the recommended sheep dose shall be used. A second dose shall be given not less than 20 days nor more than 23 days after the first dose.

(3) Fourteen to seventeen days after the second dose, all surviving rabbits shall be bled, and the serum tested for antitoxin content.

(4) The antitoxin content of the rabbit serums shall be determined by the serum neutralization test as follows:

(5) Test Interpretation shall be as follows:

(1) Each of the vaccinates shall be injected subcutaneously with the dose recommended on the label for mink. Twenty-one to twenty-eight days post-injection, the vaccinates and the controls shall be challenged intraperitoneally with botulinum Type C toxin which has been titrated in mice to provide for a 10^4.0 mouse MLD dose. The titration technique shall include inoculation of the mice intraperitoneally.

(2) The vaccinates and controls shall be observed for 7 days post-challenge and signs of botulism and deaths noted. For a valid test, the controls shall die of botulism. If the test is valid and 80 percent of the vaccinates do not remain free of botulism, the serial is unsatisfactory.

(1) When used in this test, the following words and terms shall mean:

(2) Each of at least eight rabbits of a strain acceptable to APHIS, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the largest recommended dose for any species indicated on the product label. A second equivalent dose shall be given not less than 20 days nor more than 23 days after the first does.

(3) Fourteen to seventeen days after the second dose, all surviving rabbits shall be bled and the serum tested for antitoxin content.

(4) The antitoxin content of the rabbit serums shall be determined as follows:

(5) Test Interpretation shall be as follows:

(1) When used in this test, the following words and terms shall mean:

(2) Each of at least eight rabbits of a strain acceptable to APHIS, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the largest recommended dose for any species indicated on the product label. A second equivalent dose shall be given not less than 20 days nor more than 23 days after the first dose.

(3) Fourteen to seventeen days after the second dose, all surviving rabbits shall be bled, and the serum tested for antitoxin content.

(4) The antitoxin content of the rabbit serums shall be determined as follows:

(5) Test Interpretation shall be as follows:

(1) More than one microorganism isolated from the same herd may be used as seed.

(2) Under normal circumstances, microorganisms from one herd must not be used to prepare an autogenous biologic for another herd. The Administrator, however, may authorize preparation of an autogenous biologic for use in herds adjacent to the herd of origin, when adjacent herds are considered to be at risk. To request authorization to prepare a product for use in herds adjacent to the herd of origin, the establishment seeking authorization must submit to the Administrator (in c/o the Director, Center for Veterinary Biologics, Inspection and Compliance, 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010) the following information. (If any of the data are unavailable, the applicant for authorization should indicate that such data are unavailable and why.)

(3) The Administrator may authorize preparation of an autogenous biologic for use in herds which are not adjacent to the herd of origin, but which he or she considers to be at risk of infection with the same microorganism(s). Except as provided below, the same information which is required for preparation of such product for use in herds adjacent to the herd of origin must be submitted to the Administrator (in c/o the Director, Center for Veterinary Biologics, Inspection and Compliance, 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010) for authorization to prepare a product for use in herds not adjacent to the herd of origin. Because the recipient herd involved may not be known when autogenous biologics are to be used in other geographic areas, the following data may be used in place of the data required in paragraphs (a)(2)(viii) and (a)(2)(ix) of this section.

(4) Under normal circumstances, microorganism(s) used for the production of autogenous biologics may not be older than 15 months from the date of isolation, or 12 months from the date of harvest of the first serial of product produced from the microorganism(s), whichever comes first. The Administrator, however, may authorize production of additional serials from microorganism(s) older than the above stated time periods, Provided, That, the person requesting such authorization submits the following supporting information to the address listed in paragraph (a)(3):

(1) Microorganisms used to prepare autogenous biologics shall not be maintained in the licensed establishment beyond the time authorized for use in production.

(2) The expiration date of the autogenous biologic shall not exceed 18 months from the date of harvest.

(1) Final container samples of completed product from the first serial or subserial of an autogenous biologic produced from an isolate shall be tested for purity as prescribed in § 113.26, and for safety as prescribed in § 113.33(b) or § 113.38 except that:

(2) Each serial or subserial of autogenous bacterial product other than the first serial or subserial produced from an isolate shall meet the applicable general requirements prescribed in § 113.100 and the special requirements prescribed in this section. Each serial or subserial of autogenous viral product other than the first serial or subserial produced from an isolate shall meet the applicable general requirements prescribed in § 113.200 and the special requirements prescribed in this section. A serial or subserial found unsatisfactory by any prescribed test shall not be released.

(1) Six weeks after injection, all surviving guinea pigs shall be bled and equal portions of serum, but not less than 0.5 ml from each, shall be pooled. For a valid test, the pool shall contain the serum from at least eight animals.

(2) The antitoxin titer of the pooled serum shall be determined in antitoxin units (A.U.) per ml using an enzyme-linked immunosorbent assay method acceptable to the Animal and Plant Health Inspection Service.

(3) If the antitoxin titer of the serum pool is at least 2.0 A.U. per ml, the serial is satisfactory. If the antitoxin titer of the serum pool is less than 2.0 A.U. per ml, the serial may be retested by the following procedure: Provided, That, if the serial is not retested, it shall be declared unsatisfactory.

(4) For serials in which the serum pool contains less than 2.0 A.U. per ml, the individual serum that constituted the pool may be tested by the enzyme-linked immunosorbent assay. If at least 80 percent of the individual serums have an antitoxin titer of at least 2.0 A.U. per ml, the serial is satisfactory. If less than 80 percent of the individual serums have an antitoxin titer of at least 2.0 A.U. per ml, the serial may be retested in 10 guinea pigs using the procedure described in (c)(1) and (2) above. The antitoxin titer of the pooled serum from the guinea pigs used in the retest shall be averaged with the antitoxin level of the pooled serum from the initial test. If the average of the two pools is at least 2.0 A.U. per ml, the serial is satisfactory. If the average of the two pools is less than 2.0 A.U. per ml, the serial is unsatisfactory and shall not be retested further.

(1) Each rabbit shall be given a series of not more than three intramuscular injections at 7 day intervals (1.0 ml, 2.0 ml, 3.0 ml) and observed from 7-14 days following the third injection. At the end of the observation period, a blood sample shall be taken from each rabbit.

(2) The sample of serum from each rabbit, if the five rabbit individual test is conducted or a pooled sample of equal quantities of serum from the rabbits if the eight rabbit pooled serum test is conducted, shall be tested to determine the staphylococcus alpha antitoxin units per ml as provided in paragraphs (c)(3), (4), (5), (6), (7), and (8) of this section.

(3) Inactivate rabbit serum 56 °C for 30 minutes.

(4) Make serial twofold dilutions of the serum samples and conduct the test, using 1 ml of the serial dilutions. Appropriate controls should be included for accurate interpretations.

(5) Add 1 ml of the standardized toxin containing the established “Lh” dose. The “Lh” dose is the amount of toxin which when mixed with one unit of standard antitoxin produces a 50 percent hemolysis of rabbit red blood cells.

(6) Incubate toxin-antitoxin mixture at room temperature for 30 minutes and add 1 ml of a 1.5 percent suspension of washed freshly drawn rabbit red blood cells suspended in normal saline to each tube. Mix and incubate the combined product in a 37 °C water bath for 1 hour. Refrigerate at 5 °C overnight.

(7) Read the hemolysis produced and establish the 50 percent end point. The 50 percent end point of hemolysis should be established by determining the size of the button produced by the unlysed red blood cells.

(8) Determine the units of antitoxin per 1 ml of serum.

(9) If the individual samples from four of the five rabbits in the individual serum test or the pooled samples from the eight rabbits in the pooled serum test do not contain three alpha antitoxin units per ml, the serial is unsatisfactory.

(1) Vaccinates. Each of not more than 21 turkeys shall be vaccinated with the dose and by the route recommended on the label. A second dose shall be given after 3 weeks and the turkeys observed for an additional 2-week prechallenge period.

(2) Unvaccinated controls. Each of not more than 11 turkeys shall be held as controls.

(3) Challenge. Not less than 14 days after the second dose, each of 20 vaccinates, and each of 10 unvaccinated controls shall be challenged intramuscularly with virulent Pasteurella multocida, Strain P-1662, Type 4 (Little and Lyons classification) and observed daily for a 14-day postchallenge period. Only dead birds shall be considered in evaluating the product.

(4) Validity requirements. Eight or more unvaccinated controls must die for the test to be valid. If this requirement is met, the potency test results are evaluated according to stage one of the following table. The test is a No Test and may be repeated if the validity requirement is not met, but the serial is unsatisfactory if the test is not repeated.

(5) The serial shall pass or fail based on the stage one results of the potency test. However, the second stage may be conducted if seven or eight vaccinates die in stage one, but the serial is unsatisfactory if the second stage is not conducted.

(6) The second stage shall be conducted in a manner identical to the first stage. The serial shall be evaluated according to stage two of the table. On the basis of accumulated results from the data of both stage tests, a serial shall either pass or fail the second stage.

(1) Vaccinates. Each of not more than 21 chickens shall be injected with the dose and by the route recommended on the label. A second dose shall be injected after 3 weeks and the chickens observed for an additional 2 week prechallenge period.

(2) Unvaccinated controls. Each of not more than 11 chickens shall be held as controls.

(3) Challenge. Not less than 14 days after the second injection, each of 20 vaccinates, and each of 10 unvaccinated controls shall be challenged intramuscularly with a minimum of 250 colony-forming units of virulent Pasteurella multocida, Strain X-73, Type 1 (Little and Lyons classification) and observed daily for a 14-day postchallenge period. Only dead birds shall be considered in evaluating the product.

(4) Validity requirements. Eight or more unvaccinated controls must die for the test to be valid. If these requirement are met, the potency test results are evaluated according to stage one of the following table. The test is a No Test and may be repeated if the validity requirements are not met, but the serial is unsatisfactory if the test is not repeated.

(5) The serial shall pass or fail based on the stage one results of the potency test. However, the second stage may be conducted if seven or eight vaccinates die in stage one, but the serial is unsatisfactory if the second stage is not conducted.

(6) The second stage shall be conducted in a manner identical to the first stage. The serial shall be evaluated according to stage two of the table. On the basis of accumulated results from the data of both stage tests, a serial shall either pass or fail the second stage.

(1) Vaccinates. Each of not more than 21 turkeys shall be injected with the dose and by the route recommended on the label. A second dose shall be injected after 3 weeks and the turkeys observed for an additional 2 week prechallenge period.

(2) Unvaccinated controls. Each of not more than 11 turkeys shall be held as controls.

(3) Challenge. Not less than 14 days after the second injection, each of 20 vaccinates, and each of 10 unvaccinated controls shall be challenged intramuscularly with a minimum of 150 colony-forming units of virulent Pasteurella multocida, Strain P-1059, Type 3 (Little and Lyons Classification) and observed daily for a 14-day postchallenge period. Only dead birds shall be considered in evaluating the product.

(4) Validity requirements. Eight or more unvaccinated controls must die for the test to be valid. If these requirements are met, the potency test results are evaluated according to stage one of the following table. The test is a No Test and may be repeated if the validity requirements are not met, but the serial is unsatisfactory if the test is not repeated.

(5) The serial shall pass or fail based on the stage one results of the potency test. However, the second stage may be conducted if seven or eight vaccinates die in stage one, but the serial is unsatisfactory if the second stage is not conducted.

(6) The second stage shall be conducted in a manner identical to the first stage. The serial shall be evaluated according to stage two of the table. On the basis of accumulated results from the data of both stage tests, a serial shall either pass or fail the second stage.

(1) The ability of the bacterin being tested (Unknown) to protect mice shall be compared with a Standard Reference Bacterin (Standard) which is either supplied by or acceptable to Animal and Plant Health Inspection Service.

(2) At least three threefold dilutions shall be made with the Standard and the same threefold dilutions shall be made for each Unknown. Dilutions shall be made with physiological saline solution.

(3) For each dilution of the Standard and each dilution of an Unknown, a group of at least 20 mice, each weighing 16 to 22 grams, shall be used. Each mouse in each group shall be injected subcutaneously with one mouse dose of the appropriate dilution.

(4) Each of 20 injected mice from each group shall be challenged subcutaneously 14 to 21 days after being injected. A dose containing at least 100 mouse LD₅₀ of a suitable culture of Erysipelothrix rhusiopathiae shall be used. All survivors in each group of mice shall be recorded 10 days postchallenge.

(5) Test for valid assay: At least two dilutions of the Standard shall protect more than 0 percent and two dilutions shall protect less than 100 percent of the mice injected. The lowest dilution of the Standard shall protect more than 50 percent of the mice. The highest dilution of the Standard shall protect less than 50 percent of the mice.

(6) The relative potency (RP) of the Unknown is determined by comparing the 50 percent endpoint dilution (highest bacterin dilution protecting 50 percent of the mice) of the Unknown with that of the standard by the following formula:

(7) If the RP of the Unknown is less than 0.6, the serial being tested is unsatisfactory.

(8) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the lowest dilution does not exceed 50 percent protection, that serial may be retested in a manner identical to the initial test: Provided, That, if the Unknown is not retested or if the protection provided by the lowest dilution of the Standard exceeds the protection provided by the lowest dilution of the Unknown by six mice or more; or, if the total number of mice protected by the Standard exceeds the total number of mice protected by the Unknown by eight mice or more, the serial is unsatisfactory.

(9) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the highest dilution exceeds 50 percent protection, the Unknown is satisfactory without additional testing.

(10) If the RP is less than 0.6, the serial may be retested by conducting two independent replicate tests in a manner identical to the initial test. The average of the RP values obtained in the retests shall be determined. If the average RP is less than 0.6, the serial is unsatisfactory without further testing. If the average RP obtained in the retests is equal to or greater than 0.6, the following shall apply:

(1) The ability of the bacterin being tested (Unknown) to protect mice shall be compared with a Standard Reference Bacterin (Standard) which is either supplied by or acceptable to Animal and Plant Health Inspection Service.

(2) At least three tenfold dilutions shall be made with the Standard and the same tenfold dilutions shall be made for each Unknown. The dilutions shall be made in Phosphate Buffered Saline.

(3) For each dilution of the Standard and each dilution of an Unknown, a group of at least 20 mice, each weighing 16-22 grams, shall be used. Each mouse in a group shall be injected intraperitoneally with one mouse dose of the appropriate dilution. Each mouse shall be revaccinated on day 14, using the same schedule.

(4) Each of 20 vaccinated mice per group shall be challenged intraperitoneally 7-10 days after the second vaccination with a 0.25 ml dose containing 100-10,000 mouse LD₅₀ as determined by titration, of a suitable culture of Salmonella typhimurium. All survivors in each group of mice shall be recorded 14 days postchallenge.

(5) Test for valid assay: At least two dilutions of the Standard shall protect more than 0 percent and two dilutions shall protect less than 100 percent of the mice injected. The lowest dilution of the Standard shall protect more than 50 percent of the mice. The highest dilution of the Standard shall protect less than 50 percent of the mice.

(6) The relative potency (RP) of the Unknown is determined by comparing the 50 percent endpoint dilution (highest bacterin dilution protecting 50 percent of the mice) of the Unknown with that of the Standard by the following formula:

(7) If the RP of the Unknown is less than 0.30, the serial being tested is unsatisfactory.

(8) If the 50 percent endpoint of an Unknown cannot be calculated because the lowest dilution does not exceed 50 percent protection, that serial may be retested in a manner identical to the initial test; Provided, That, if the Unknown is not retested or if the protection provided by the lowest dilution of the Unknown by six mice or more; or, if the total number of mice protected by the Standard exceeds the total number of mice protected by the Unknown by eight mice or more, the serial being tested is unsatisfactory.

(9) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the highest dilution exceeds 50 percent protection, the Unknown is satisfactory without additional testing.

(10) If the RP is less than the minimum required in paragraph (c)(7) of this section, the serial may be retested by conducting two independent replicate tests in a manner identical to the initial test. The average of the RP values obtained in the retests shall be determined. If the average RP is less than the required minimum, the serial is unsatisfactory. If the average RP obtained in the retests is equal to or greater than the required minimum, the following shall apply:

(1) The ability of the bacterin being tested (Unknown) to protect mice shall be compared with a Standard Reference Bacterin (Standard) which is either supplied by or acceptable to Animal and Plant Health Inspection Service.

(2) At least three fivefold dilutions shall be made with the Standard and the same fivefold dilutions shall be made for each Unknown. The dilutions will be made in Phosphate Buffered Saline.

(3) For each dilution of the Standard and each dilution of each Unknown, a group of at least 20 mice, each weighing 16-22 grams, shall be used. Each mouse in a group shall be injected intraperitoneally with one mouse dose of the appropriate dilution. Each mouse shall be revaccinated on day 14, using the same schedule.

(4) Each of 20 injected mice per group shall be challenged intraperitoneally 10-12 days after the second vaccination with a 0.2 ml dose containing 100-10,000 mouse LD₅₀, as determined by titration, of a suitable culture of Pasteurella multocida. All survivors in each group of mice shall be recorded 10 days postchallenge.

(5) Test for valid assay: At least two dilutions of the Standard shall protect more than 0 percent and two dilutions shall protect less than 100 percent of the mice injected. The lowest dilution of the Standard shall protect more than 50 percent of the mice. The highest dilution of the Standard shall protect less than 50 percent of the mice.

(6) The relative potency (RP) of the Unknown is determined by comparing the 50 percent endpoint dilution (highest bacterin dilution protecting 50 percent of the mice) of the Unknown with that of the Standard by the following formula:

(7) If the RP of the Unknown is less than 0.50, the serial being tested is unsatisfactory.

(8) If the 50 percent endpoint of an Unknown cannot be calculated because the lowest dilution does not exceed 50 percent protection, that serial may be retested in a manner identical to the initial test: Provided, That, if the Unknown is not retested or if the protection provided by the lowest dilution of the Standard exceeds the protection provided by the lowest dilution of the Unknown by six mice or more; or, if the total number of mice protected by the Standard exceeds the total number of mice protected by the Unknown by eight mice or more, the serial being tested is unsatisfactory.

(9) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the highest dilution exceeds 50 percent protection, the Unknown is satisfactory without additional testing.

(10) If the RP is less than the minimum required in paragraph (c)(7) of this section, the serial may be retested by conducting two independent replicate tests in a manner identical to the initial test. The average of the RP values obtained in the retests shall be determined. If the average RP is less than the required minimum, the serial is unsatisfactory. If the average RP obtained in the retests is equal to or greater than the required minimum, the following shall apply:

(1) The ability of the bacterin being tested (Unknown) to protect mice shall be compared with a Standard Reference Bacterin (Standard) which is either supplied by or acceptable to Veterinary Services.

(2) At least three fivefold dilutions shall be made with the Standard and the same fivefold dilution shall be made for each Unknown. The dilutions shall be made in Phosphate-Buffered Saline.

(3) For each dilution of the Standard and each dilution of an Unknown, a group of at least 20 mice, each weighing 16 to 22 grams, shall be used. Each mouse in a group shall be injected intraperitoneally with one mouse dose of the appropriate dilution. Each mouse shall be revaccinated on day 14, using the same schedule.

(4) Each of 20 vaccinated mice per group shall be challenged intraperitoneally 7 to 10 days after the second vaccination with a 0.25 ml dose containing 10-1,000 mouse LD₅₀ as determined by titration of a suitable culture of Salmonella choleraesuis. All survivors in each group of mice shall be recorded 14 days postchallenge.

(5) Test for valid assay: At least two dilutions of the Standard shall protect more than 0 percent and two dilutions shall protect less than 100 percent of the mice injected. The lowest dilution of the Standard shall protect more than 50 percent of the mice. The highest dilution of the Standard shall protect less than 50 percent of the mice.

(6) The relative potency (RP) of the Unknown is determined by comparing the 50 percent endpoint dilution (highest bacterin dilution protecting 50 percent of the mice) of the Unknown with that of the Standard by the following formula:

(7) If the RP of the Unknown is less than 0.50, the serial being tested is unsatisfactory.

(8) If the 50 percent endpoint of an Unknown cannot be calculated because the lowest dilution does not exceed 50 percent protection, that serial may be retested in a manner identical to the initial test; Provided, That, if the Unknown is not retested or if the protection provided by the lowest dilution of the Standard exceeds the protection provided by the lowest dilution of the Unknown by six mice or more; or, if the total number of mice protected by the Standard exceeds the total number of mice protected by the Unknown by eight mice or more, the serial being tested is unsatisfactory.

(9) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the highest dilution exceeds 50 percent protection, the Unknown is satisfactory without additional testing.

(10) If the RP is less than the minimum required in paragraph (c)(7) of this section, the serial may be retested by conducting two independent replicate tests in a manner identical to the initial test. The average of the RP values obtained in the retests shall be determined. If the average RP is less than the required minimum, the serial is unsatisfactory. If the average RP obtained in the retests is equal to or greater than the required minimum, the following shall apply:

(1) The ability of the bacterin being tested (Unknown) to protect mice shall be compared with a Standard Reference Bacterin (Standard) which is either supplied by or acceptable to Veterinary Services.

(2) At least three tenfold dilutions shall be made with the Standard and the same tenfold dilutions shall be made for each Unknown. The dilutions shall be made in Phosphate-Buffered Saline.

(3) For each dilution of the Standard and each dilution of an Unknown, a group of at least 20 mice, each weighing 16 to 22 grams, shall be used. Each mouse in a group shall be injected intraperitoneally with one mouse dose of the appropriate dilution. Each mouse shall be revaccinated on day 14, using the same schedule.

(4) Each of 20 vaccinated mice per group shall be challenged intraperitoneally 7 to 10 days after the second vaccination with a 0.25 ml dose containing 1,000-100,000 mouse LD₅₀ as determined by titration of a suitable culture of Salmonella dublin. All survivors in each group of mice shall be recorded 14 days postchallenge.

(5) Test for valid assay: At least two dilutions of the Standard shall protect more than 0 percent and two dilutions shall protect less than 100 percent of the mice injected. The lowest dilution of the Standard shall protect more than 50 percent of the mice. The highest dilution of the Standard shall protect less than 50 percent of the mice.

(6) The relative potency (RP) of the Unknown is determined by comparing the 50 percent endpoint dilution (highest bacterin dilution protecting 50 percent of the mice) of the Unknown with that of the Standard by the following formula:

(7) If the RP of the Unknown is less than 0.30, the serial being tested is unsatisfactory.

(8) If the 50 percent endpoint of an Unknown cannot be calculated because the lowest dilution does not exceed 50 percent protection, that serial may be retested in a manner identical to the initial test; Provided, That, if the Unknown is not retested or if the protection provided by the lowest dilution of the Standard exceeds the protection provided by the lowest dilution of the Unknown by six mice or more; or, if the total number of mice protected by the Standard exceeds the total number of mice protected by the Unknown by eight mice or more, the serial being tested is unsatisfactory.

(9) If the 50 percent endpoint of an Unknown in a valid test cannot be calculated because the highest dilution exceeds 50 percent protection, the Unknown is satisfactory without additional testing.

(10) If the RP is less than the minimum required in paragraph (c)(7) of this section, the serial may be retested by conducting two independent replicate tests in a manner identical to the initial test. The average of the RP values obtained in the retests shall be determined. If the average RP is less than the required minimum, the serial is unsatisfactory. If the average RP obtained in the retests is equal to or greater than the required minimum, the following shall apply:

(1) Bacteria and fungi. Final container samples of completed product from each serial shall be tested as prescribed in § 113.26.

(2) Avian origin vaccine. Bulk pooled material or final container samples from each serial shall also be tested for:

(3) Mycoplasma. If the licensee cannot demonstrate that the agent used to kill the vaccine virus would also kill mycoplasma, each serial of the vaccine shall be tested for mycoplasma as prescribed in § 113.28, prior to adding the killing agent. Material found to contain mycoplasma is unsatisfactory for use.

(4) Extraneous viruses. Each lot of Master Seed Virus used to prepare killed virus vaccine recommended for animals other than poultry shall meet the requirements for extraneous viruses as prescribed in § 113.55.

(1) Vaccination. Each of the five vaccinates shall be injected with one dose of vaccine as recommended on the label and observed each day for 14 days.

(2) Challenge. At least 2 weeks after the last inoculation, the five vaccinates and the five controls shall be challenged with virulent mink enteritis virus and observed each day for 12 days. Fecal material shall be collected on one day between days 4-8 (inclusive) postchallenge from each test animal that remains free of enteric signs and tested for the presence of mink enteritis virus by cell culture with fluorescent antibody examination.

(3) Interpretation. A serial is satisfactory if at least 80 percent of the vaccinates remain free of enteric signs and do not shed virus in the feces, while at least 80 percent of the controls develop clinical signs of mink enteritis or shed virus in the feces. If at least 80 percent of the vaccinates remain free of enteric signs and do not shed virus in the feces, while less than 80 percent of the controls develop clinical signs of mink enteritis or shed virus in the feces, the test is considered a No Test and may be repeated: Provided, That, if at least 80 percent of the vaccinates do not remain well and free of detectable virus in the feces, the serial is unsatisfactory.

(1) Ten or more chickens shall be vaccinated as recommended on the label and kept isolated under observation for at least 14 days.

(2) After at least 14 days post-vaccination, the vaccinates and at least 10 unvaccinated chickens that have been kept isolated as controls shall be challenged with a virulent strain of Newcastle disease virus supplied by or approved by Veterinary Services and the vaccinates observed each day for 14 days.

(3) If at least 90 percent of the controls do not show typical signs of Newcastle disease or die, the test is a No Test and may be repeated. If at least 90 percent of the vaccinates do not remain normal, the serial is unsatisfactory.

(1) The vaccine shall be a tissue extract representing at least 10 percent weight to volume suspension of wart tissue; and

(2) The vaccine shall be limited to use in the prevention of warts in cattle. Labeling recommendations shall be in accordance with § 112.7(h).

(1) Each of at least ten 6 to 12 hour old chickens shall be injected subcutaneously with 0.5 ml of the product and the chickens observed each day for 10 days.

(2) If unfavorable reactions attributable to the product occur in the chickens during the observation period, the serial is unsatisfactory. If unfavorable reactions not attributable to the product occur, the test is a No Test and may be repeated: Provided, That, if the test is not repeated, the serial is unsatisfactory.

(1) For this test, a guinea pig dose shall be one-half the amount recommended on the label for a horse and shall be administered as recommended for a horse. Each of 10 healthy guinea pigs (vaccinates) shall be injected with two guinea pig doses with an interval of 14 to 21 days between doses. Two additional guinea pigs from the same source shall be held as controls.

(2) Fourteen to 21 days after the second injection, serum samples from each vaccinate and each control shall be tested by a plaque reduction, serum neutralization test using Vero 76 cells.

(3) If the control serum samples show a titer of 1:4 or greater for any fraction, the test is a No Test for that fraction and may be repeated: Provided, That, if four or more of the vaccinate serum samples show a titer of less than 1:40 for the Eastern type fraction, less than 1:40 for the Western type fraction, or less than 1:4 for the Venezuelan type fraction, the serial or subserial is unsatisfactory without further testing.

(4) If two or three of the vaccinate serum samples show a titer of less than 1:40 for the Eastern type fraction, less than 1:40 for the Western type fraction, or less than 1:4 for the Venezuelan type fraction, the second stage of the test may be used for the relevant fraction(s): Provided, That, if a fraction is found acceptable by the first stage of the test, the second stage need not be conducted for that fraction.

(5) If the second stage is used and four or more of the vaccinate serum samples show a titer of less than 1:40 for the Eastern type fraction or the Western type fraction, or less than 1:4 for the Venezuelan type fraction, the serial or subserial is unsatisfactory.

(6) The results shall be evaluated according to the following table:

(1) The prechallenge part of the potency test prescribed in paragraph (b) of this section shall constitute a safety test. If any of the vaccinates develop clinical signs of disease or die due to causes attributable to the product, the serial is unsatisfactory.

(2) An inactivation test for viable avian encephalomyelitis (AE) virus shall be conducted on each serial. The test shall be conducted using susceptible chicken embryos: Provided, That, if a non-embryo adapted virus is used for vaccine production, the test shall be conducted in susceptible chickens.

(1) At least 28 days post-injection, the vaccinates and the controls shall be challenged intramuscularly with a virulent AE virus and the chickens observed each day for 21 days.

(2) If at least 80 percent of the controls do not show clinical signs of or die from AE infection, the test is a No Test and may be repeated.

(3) If at least 80 percent of the vaccinates do not remain normal, the serial is unsatisfactory.

(1) Each lot of Master Seed Virus propagated in tissue or cells of avian origin shall also be tested for extraneous pathogens by procedures prescribed in § 113.37.

(2) Each lot of Master Seed Virus propagated in primary cell cultures of mouse or hamster origin or brain tissues of mouse origin shall be tested for lymphocytic choriomeningitis (LCM) virus by the procedure prescribed in § 113.42. If LCM virus is detected, the Master Seed Virus is unsatisfactory.

(1) The preinactivation virus titer must be established as soon as possible after harvest by at least five separate virus titrations. A mean relative potency value of the vaccine to be used in the host animal potency test must be established by at least five replicate potency tests conducted in accordance with the standard NIH test for potency in chapter 37 of “Laboratory Techniques in Rabies,” Fourth Edition (1996), edited by F.X. Meslin, M.M. Kaplan, and H. Koprowski, World Health Organization, Geneva, Switzerland (ISBN 92 4 154479 1). The provisions of chapter 37 of “Laboratory Techniques in Rabies,” Fourth Edition (1996), are the minimum standards for achieving compliance with this section and are incorporated by reference. These provisions state that the challenge virus standard to be used as the challenge in the NIH test and the reference vaccine for the test are available from the national control authority. In the United States, that authority is the Animal and Plant Health Inspection Service's Center for Veterinary Biologics Laboratory, located at 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010; phone (515) 337-6100; fax (515) 337-6120. This incorporation by reference was approved by the Director of the Federal Register in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies may be obtained from the World Health Organization Publications Center USA, 49 Sheridan Avenue, Albany, NY 12210. Copies may be inspected at the Animal and Plant Health Inspection Service, Center for Veterinary Biologics, Policy, Evaluation, and Licensing, 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010, or at the National Archives and Records Administration (NARA). For information on the availability of this material at NARA, call 202-741-6030, or go to: http://www.archives.gov/federal_register/code_of_federal_regulations/ibr_locations.html.

(2) The dose of vaccine to be used in the immunogenicity test shall be no more than the amount which, on the basis of The NIH Test For Potency, has been diluted to the proposed minimum acceptable potency value.¹

(3) Test animals shall be uniform and have no neutralizing antibodies to rabies as determined by serum-neutralization (SN) tests.

(4) An alternative to challenging all surviving test animals in accordance with paragraph (b)(3)(iv) of this section may be used when the test animals are of species other than carnivores. Vaccinates shall be challenged at 1 year postvaccination. These shall include five vaccinates with the lowest SN titers at the 270th-day bleeding, five vaccinates with the lowest SN titers at the 365th-day bleeding, and all vaccinates with SN titers below 1:10 by the mouse SN test or below 1:16 by the rapid-fluorescent-focus-inhibition test at any bleeding. At least five SN-negative controls of each species shall be challenged at the same time as the vaccinates. All SN titers shall be titrated to an endpoint. All of the challenged vaccinates must remain well for a period of 90 days, and at least 80 percent of the controls must die of rabies for a satisfactory test without further challenge. If one or more of the vaccinates die from rabies, all the remaining vaccines, regardless of titer, along with the five controls shall be challenged. The cumulative results from the two challenges shall be evaluated for acceptance as specified in paragraph (b)(3)(v) of this section.

(5) An Outline of Production change shall be made before authority for use a new lot of Master Seed Virus shall be granted by Animal and Plant Health Inspection Service.

(1) Purity test. Primary cell cultures of hamster origin or brain tissues of mouse origin used in vaccine production shall be tested for LCM virus as prescribed in § 113.42. Hamster origin cells shall be disrupted and undiluted cell fluids from each lot shall be tested. Where mouse brains are used in production, at least five mice which have not been injected with rabies virus shall be sacrificed and a 10 percent suspension of brain material shall be prepared and tested.

(2) Safety tests. Bulk samples from each serial shall be tested for virus inactivation and safety as follows:

(3) Potency test. Bulk or final container samples of completed product from each serial must be tested for potency by tests conducted in accordance with the standard NIH test for potency in Chapter 37 of “Laboratory Techniques in Rabies,” Fourth Edition (1996), which is incorporated by reference at paragraph (b)(1) of this section. The relative potency of each serial must be at least equal to that used in an approved host animal immunogenicity test.

(1) Safety. Vaccinates used in the potency test in paragraph (d)(2) of this section shall be observed each day during the prechallenge period. If unfavorable reactions occur, including oral lesions, which are attributable to the vaccine, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the vaccine, the test is inconclusive and may be repeated. If the test is not repeated, the serial is unsatisfactory.

(2) Potency. Bulk or final container samples of completed product shall be treated for potency as follows:

(1) Safety. Vaccinates used in the potency test in paragraph (d)(2) of this section shall be observed each day during the prechallenge period. If unfavorable reactions attributable to the vaccine occur, the serial is unsatisfactory. If unfavorable reactions which are not attributable to the vaccine occur, the test is a No Test and may be repeated. If the test is not repeated, the serial is unsatisfactory.

(2) Potency. Bulk or final container samples of completed product from each serial shall be tested for potency using the two-stage potency test provided in this paragraph.