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e-CFR data is current as of August 12, 2020

Title 9Chapter ISubchapter EPart 113 → Subject Group


Title 9: Animals and Animal Products
PART 113—STANDARD REQUIREMENTS


Standard Procedures

§113.25   Culture media for detection of bacteria and fungi.

(a) Ingredients for which standards are prescribed in the United States Pharmacopeia, or elsewhere in this part, shall conform to such standards. In lieu of preparing the media from the individual ingredients, they may be made from dehydrated mixtures which, when rehydrated with purified water, have the same or equivalent composition as such media and have growth-promoting buffering, and oxygen tension-controlling properties equal to or better than such media. The formulas for the composition of the culture media prescribed in §§113.26 and 113.27 are set forth in the United States Pharmacopeia, 19th Edition.

(b) The licensee shall test each quantity of medium prepared at one time from individual ingredients and the first quantity prepared from each lot of commercial dehydrated medium for growth-promoting qualities. If any portion of a lot of commercial dehydrated medium is held for 90 days or longer after being so tested, it shall be retested before use. Two or more strains of micro-organisms that are exacting in their nutritive requirements shall be used. More than one dilution shall be used to demonstrate the adequacy of the medium to support the growth of a minimum number of micro-organisms.

(c) The sterility of the medium shall be confirmed by incubating an adequate number of test vessels and examining each for growth. Additional control may be used by incubation of representative uninoculated test vessels for the required incubation period during each test.

(d) A determination shall be made by the licensee for each biological product of the ratio of inoculum to medium which shall result in sufficient dilution of such product to prevent bacteriostatic and fungistatic activity. The determination may be made by tests on a representative biological product for each group of comparable products containing identical preservatives at equal or lower concentrations. Inhibitors or neutralizers of preservatives, approved by the Administrator, may be considered in determining the proper ratio.

[35 FR 16039, Oct. 13, 1970, as amended at 37 FR 2430, Feb. 1, 1972; 41 FR 27715, July 6, 1976; 56 FR 66784, Dec. 26, 1991]

§113.26   Detection of viable bacteria and fungi except in live vaccine.

Each serial and subserial of biological product except live vaccines shall be tested as prescribed in this section unless otherwise specified by the Administrator. When cell lines, primary cells, or ingredients of animal origin used in the preparation of a biological product are required to be free of viable bacteria and fungi, they shall also be tested as prescribed in this section.

(a) The media to be used shall be as follows:

(1) Fluid Thioglycollate Medium with 0.5 percent beef extract shall be used to test for bacteria in biological products containing clostridial toxoids, bacterins, and bacterin-toxoids.

(2) Fluid Thioglycollate Medium with or without 0.5 percent beef extract shall be used to test for bacteria in biological products other than clostridial toxoids, bacterins, and bacterin-toxoids.

(3) Soybean-Casein Digest Medium shall be used to test biological products for fungi; provided, that Fluid Thioglycollate Medium without beef extract shall be substituted when testing biological products containing mercurial preservatives.

(b) Test procedure:

(1) Ten test vessels shall be used for each of two media selected in accordance with paragraph (a)(1), (a)(2), or (a)(3) of this section. Each test vessel shall contain sufficient medium to negate the bacteriostatic or fungistatic activity in the inoculum as determined in §113.25(d).

(2) Inoculum:

(i) When completed product is tested, 10 final container samples from each serial and each subserial shall be tested. One ml from each sample shall be inoculated into a corresponding individual test vessel of culture medium: Provided, That, if each final container sample contains less than 2 ml, one-half of the contents shall be used as inoculum for each test vessel.

(ii) When cell lines, primary cells, or ingredients of animal origin are tested, at least a 20 ml test sample from each lot shall be tested. One ml shall be inoculated into each test vessel of medium.

(3) Incubation shall be for an observation period of 14 days at 30 °to 35 °C. to test for bacteria and 14 days at 20 °to 25 °C. to test for fungi.

(4) If the inoculum renders the medium turbid so that the absence of growth cannot be determined by visual examination, subcultures shall be made on the seventh to eleventh day from biological products prepared from clostridial toxoids, bacterins, and bacterin-toxoids and the third to seventh day for other biological products. Portions of the turbid medium in amounts of not less than 1.0 ml. shall be transferred to 20 to 25 ml. of fresh medium, and incubated the balance of the 14-day period.

(c) Examine the contents of all test vessels for macroscopic microbial growth during the incubation period. When demonstrated by adequate controls to be invalid, the test may be repeated. For each set of test vessels representing a serial or subserial in a valid test, the following rules shall apply:

(1) If no growth is found in any test vessel, the serial or subserial meets the requirements of the test.

(2) If growth is found in any test vessel, one retest to rule out faulty technique may be conducted using 20 unopened final container samples.

(3) If growth is found in any test vessel of the final test, the serial, subserial, or ingredients to be used in the preparation of a biological product, as the case may be, is unsatisfactory.

[35 FR 16039, Oct. 13, 1970, as amended at 37 FR 2430, Feb. 1, 1972; 39 FR 21042, June 18, 1974; 40 FR 758, Jan. 3, 1975; 40 FR 14084, Mar. 28, 1975; 56 FR 66784, Dec. 26, 1991]

§113.27   Detection of extraneous viable bacteria and fungi in live vaccines.

Unless otherwise specified by the Administrator or elsewhere exempted in this part, each serial and subserial of live vaccine and each lot of Master Seed Virus and Master Seed Bacteria shall be tested for extraneous viable bacteria and fungi as prescribed in this section. A Master Seed found unsatisfactory shall not be used in vaccine production and a serial found unsatisfactory shall not be released.

(a) Live viral vaccines. Each serial and subserial of live viral vaccine shall be tested for purity as prescribed in this paragraph. However, products of chicken embryo origin recommended for administration other than by parenteral injection may be tested as provided in paragraph (e) of this section.

(1) Soybean Casein Digest Medium shall be used.

(2) Ten final container samples from each serial and subserial shall be tested.

(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and desiccated vaccine shall be rehydrated as recommended on the label with accompanying diluent or with sterile purified water.

(4) To test for bacteria, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 120 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in §113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 30 °to 35 °C for 14 days.

(5) To test for fungi, place 0.2 ml of vaccine from each final container sample into a corresponding individual vessel containing at least 40 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in §113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 20 °to 25 °C for 14 days.

(6) Examine the contents of all test vessels macroscopically for microbial growth at the end of the incubation period. If growth in a vessel cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.

(7) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:

(i) If growth is found in 2 or 3 test vessels of the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final container samples.

(ii) If no growth is found in 9 or 10 of the test vessels in the initial test, or 19 or 20 vessels in the retest, the serial or subserial meets the requirements of the test.

(iii) If growth is found in four or more test vessels in the initial test, or two or more in a retest, the serial or subserial is unsatisfactory.

(b) Live bacterial vaccines. Each serial or subserial of live bacterial vaccine shall be tested for purity as prescribed in this paragraph.

(1) Soybean Casein Digest Medium and Fluid Thioglycollate Medium shall be used.

(2) Ten final container samples from each serial and subserial shall be tested.

(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and desiccated vaccine shall be rehydrated as recommended on the label with accompanying diluent or with sterile purified water. Product recommended for mass vaccination shall be rehydrated at the rate of 30 ml sterile purified water per 1,000 doses.

(4) To test for extraneous bacteria, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 40 ml of Fluid Thioglycollate Medium. Additional medium shall be used if the determination required in §113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 30 °to 35 °C for 14 days.

(5) To test for extraneous fungi, place 0.2 ml of vaccine from each final container into a corresponding individual vessel containing at least 40 ml of Soybean Casein Digest Medium. Additional medium shall be used if the determination required in §113.25(d) indicates the need for a greater dilution of the product. Incubation shall be at 20 °to 25 °C for 14 days.

(6) Examine the contents of all test vessels macroscopically for atypical microbial growth at the end of the incubation period. If growth of extraneous microorganisms cannot be reliably determined by visual examination, judgment shall be confirmed by subculturing, microscopic examination, or both.

(7) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:

(i) If extraneous growth is found in 2 or 3 test vessels of the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final container samples.

(ii) If no extraneous growth is found in 9 or 10 test vessels in the initial test, or 19 or 20 vessels in the retest, the serial or subserial meets the requirements of the test.

(iii) If extraneous growth is found in 4 or more test vessels in the initial test, or 2 or more in a retest, the serial or subserial is unsatisfactory.

(c) Master Seed Virus. Not less than 4 ml of each lot of Master Seed Virus shall be tested. Frozen liquid Master Seed Virus shall be thawed, and desiccated Master Seed Virus shall be rehydrated with Soybean Casein Digest Medium immediately prior to starting the test.

(1) To test for bacteria, place 0.2 ml of the sample of Master Seed Virus into 10 individual vessels each containing at least 120 ml of Soybean Casein Digest Medium. Incubation shall be at 30 °to 35 °C for 14 days.

(2) To test for fungi, place 0.2 ml of the sample of Master Seed Virus into 10 individual vessels each containing at least 40 ml of Soybean Casein Digest Medium. Incubation shall be at 20 °to 25 °C for 14 days.

(3) Examine the contents of all test vessels macroscopically for microbial growth at the end of the incubation period. If growth in a vessel cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.

(4) For each set of test vessels representing a lot of Master Seed Virus tested according to these procedures, the following rules shall apply:

(i) If growth is found in any test vessel of the initial test, one retest to rule out faulty technique may be conducted using a new sample of Master Seed Virus.

(ii) If growth is found in any test vessel of the final test, the lot of Master Seed Virus is unsatisfactory.

(d) Master Seed Bacteria. Not less than 4 ml of each lot of Master Seed Bacteria shall be tested. Frozen liquid Master Seed Bacteria shall be thawed, and desiccated Master Seed Bacteria shall be rehydrated with sterile purified water immediately prior to starting the test.

(1) To test for extraneous bacteria, place 0.2 ml of the sample of Master Seed Bacteria into 10 individual vessels each containing at least 40 ml of Fluid Thioglycollate Medium. Incubation shall be at 30 °to 35 °C for 14 days.

(2) To test for extraneous fungi, place 0.2 ml of the sample of Master Seed Bacteria into 10 individual vessels each containing at least 40 ml of Soybean Casein Digest Medium. Incubation shall be at 20 °to 25 °C for 14 days.

(3) Examine the contents of all test vessels macroscopically for atypical microbial growth at the end of the incubation period. If growth of extraneous microorganisms cannot be reliably determined by visual examination, judgment shall be confirmed by subcultures, microscopic examination, or both.

(4) For each set of test vessels representing a lot of Master Seed Bacteria tested according to these procedures, the following rules shall apply:

(i) If extraneous growth is found in any test vessel of the initial test, one retest to rule out faulty technique may be conducted using a new sample of Master Seed Bacteria.

(ii) If extraneous growth is found in any test vessel of the final test, the lot of Master Seed Bacteria is unsatisfactory.

(e) Live viral vaccines of chicken embryo origin recommended for administration other than by parenteral injection, which were not tested or have not been found free of bacteria and fungi by the procedures prescribed in paragraph (a) of this section, may be tested according to the procedures prescribed in this paragraph.

(1) Brain Heart Infusion Agar shall be used with 500 Kinetic (Kersey) units of penicillinase per ml of medium added just prior to pouring the plates.

(2) Ten final containers from each serial and each subserial shall be tested.

(3) Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and lyophilized vaccine shall be rehydrated to the quantity recommended on the label using the accompanying sterile diluent or sterile purified water. Product recommended for mass vaccination shall be rehydrated at the rate of 30 ml sterile purified water per 1,000 doses.

(4) From each container sample, each of 2 plates shall be inoculated with vaccine equal to 10 doses if the vaccine is recommended for poultry or 1 dose if the vaccine is recommended for other animals. Twenty ml of medium shall be added to each plate. One plate shall be incubated at 30 °to 35 °for 7 days and the other plate shall be incubated at 20 °to 25 °C for 14 days.

(5) Colony counts shall be made for each plate at the end of the incubation period. An average colony count for the 10 samples representing the serial or subserial shall be made for each incubation condition.

(6) For each set of test vessels representing a serial or subserial tested according to these procedures, the following rules shall apply:

(i) If the average count at either incubation condition exceeds 1 colony per dose for vaccines recommended for poultry, or 10 colonies per dose for vaccines recommended for other animals in the initial test, 1 retest to rule out faulty technique may be conducted using 20 unopened final containers.

(ii) If the average count at either incubation condition of the final test for a serial or subserial exceeds 1 colony per dose for vaccines recommended for poultry, or 10 colonies per dose for vaccines recommended for other animals, the serial or subserial is unsatisfactory.

[48 FR 28430, June 22, 1983, as amended at 56 FR 66784, Dec. 26, 1991]

§113.28   Detection of mycoplasma contamination.

The heart infusion test, using heart infusion broth and heart infusion agar, provided in this section shall be conducted when a test for mycoplasma contamination is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.

(a) Media additives provided in this paragraph shall be prepared as follows:

(1) DPN-Cysteine Solution:

(i) Use Nicotinamide adenine dinucleotide (oxidized) and L-Cysteine hydrochloride.

(ii) Prepare 1 gram/100 milliliters (ml) purified water (1 percent solution) of each. Mix the solutions together; the cysteine reduces the DPN. Filter sterilize, dispense in appropriate amounts and store frozen at −20 degrees centigrade.

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

(b) Heart infusion broth shall be prepared as provided in this paragraph.

(1) Dissolve in 970 ml of purified water, 25 grams of heart infusion broth, 10 grams of proteose peptone No. 3, and either 5 grams of yeast autolysate or 5 ml of fresh yeast extract.

(2) Add the following:

1 percent tetrazolium chloride (ml)5.5
1 percent thallium acetate (ml)25
Penicillin (units)500,000
Inactivated horse serum (ml)100

(3) Adjust pH to 7.9 with NaOH, filter sterilize, and dispense 100 ml aliquots into 125 ml flasks and store until needed.

(4) Add 2 ml of DPN-Cysteine solution to each 100 ml of broth on day of use.

(c) Heart Infusion Agar shall be prepared as provided in this paragraph.

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

Heart infusion agar (g)25
Heart-infusion broth (g)10
Proteose peptone No. 3 (g)10
1 pct thallium acetate (ml)25

(2) Cool the medium and adjust pH to 7.9 with NaOH.

(3) Autoclave the medium.

(4) Cool the medium 30 minutes in a 56 °C waterbath.

(5) Dissolve 5 grams of yeast autolysate in 100 ml of distilled water, filter sterilize, and add to the medium.

(6) Add to the medium:

126 ml of inactivated horse serum

21 ml of DPN-Cysteine solution

525,000 units of Penicillin.

Dispense 10 ml aliquots into 60 × 15 mm disposable culture dishes or petri dishes.

(d) The test procedure provided in this paragraph shall be followed when conducting the mycoplasma detection test.

(1) Preparation of inoculum. Immediately prior to starting the test, frozen liquid vaccine shall be thawed, and lyophilized vaccine shall be rehydrated to the volume recommended on the label with mycoplasma medium. In the case of a lyophilized biological product, e.g., 1,000 dose vial of poultry vaccine to be administered via the drinking water, the vaccine shall be rehydrated to 30 ml with mycoplasma medium. In the case of a cell line or a sample of primary cells, the inoculum shall consist of the resuspended cells. Control tests shall be established as provided in paragraph (d)(4) of this section.

(2) Inoculation of plate. Plate 0.1 ml of inoculum on an agar plate and make a short, continuous streak across the plate with a pipet. Tilt the plate to allow the inoculum to flow over the surface.

(3) Inoculation of flask of medium. Transfer 1 ml of the inoculum into a flask containing 100 ml mycoplasma medium and mix thoroughly. Incubate the flask at 33 to 37 °C for 14 days during which time, one of four agar plates shall be streaked with 0.1 ml of material from the incubating flask of inoculated medium on the 3d day, one on the 7th day, one on the 10th day, and one on the 14th day post-inoculation.

(4) Control tests shall be conducted simultaneously with the detection test using techniques provided in paragraphs (d)(2) and (3) of this section, except the inoculum for the positive control test shall be selected mycoplasma cultures and the negative control test shall be uninoculated medium from the same lot used in the detection test.

(5) All plates shall be incubated in a high humidity, 4-6 percent 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.

(e) Interpretation of test results.

(1) If growth appears on at least one of the plates in the positive control test and does not appear on any of the plates in the negative control test, the test is valid.

(2) If mycoplasma colonies are found on any of the plates inoculated with material being tested, the results are positive for mycoplasma contamination.

[38 FR 29887, Oct. 30, 1973, as amended at 41 FR 6752, Feb. 13, 1976; 41 FR 32882, Aug. 6, 1976]

§113.29   Determination of moisture content in desiccated biological products.

Methods provided in this section must be used when a determination of moisture content in desiccated biological products is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product. Firms currently using methods other than those provided in this section for determining the moisture content in desiccated biological products have until November 5, 2004 to update their Outlines of Production to be in compliance with this requirement.

(a) Final container samples of completed product shall be tested. The weight loss of the sample due to drying in a vacuum oven shall be determined. All procedures should be performed in an environment with a relative humidity less than 45 percent. The equipment necessary to perform the test is as follows:

(1) Cylindrical weighing bottles with airtight glass stoppers.

(2) Vacuum oven equipped with validated thermometer and thermostat. A suitable air-drying device should be attached to the inlet valve.

(3) Balance, accurate to 0.1 mg (rated precision ±0.01mg).

(4) Desiccator jar equipped with phosphorous pentoxide, silica gel, or equivalent.

(5) Desiccated vaccine in original sealed vial. Sample and control should be kept at room temperature in their original airtight containers until use.

(b) Test procedure:

(1) Thoroughly cleaned and labeled sample-weighing bottles with stoppers should be allowed to dry at 60 ±3 °C under vacuum at less than 2.5 kPa.

(i) Transfer hot bottles and stoppers into the desiccator and allow to cool to room temperature.

(ii) After bottles have cooled, insert stoppers and weigh and record the weights of the bottles as “A.”

(iii) Return weighing bottles to the desiccator.

(2) Remove the sample container seal.

(i) Using a spatula, break up the sample plug and transfer the required amount of sample to the previously tared weighing bottle.

(ii) Insert the stopper and weigh and record the weights of the weighing bottles as “B.”

(3) Place the weighing bottle with the stopper at an angle in the vacuum oven. Set the vacuum to <2.5 kPa and the temperature to 60 ±3 °C.

(4) After a minimum of 3 hours of drying time, turn off the vacuum pump and allow dry air to bleed into the oven until the pressure inside the oven is equalized with the prevailing atmospheric pressure.

(5) While the bottle is still warm, replace the stopper in its normal position and transfer the weighing bottle to the desiccator.

(i) Allow a minimum of 2 hours for the weighing bottle to cool to room temperature or for its weight to reach equilibrium.

(ii) Weigh, and record the weight as “C.”

(6) Calculate the percentage of moisture in the original sample as follows:

(B−C)/(B−A) × (100) = Percentage of residual moisture, where:

A = tare weight of weighing bottle

B−A = weight of sample before drying

B−C = weight of sample after drying

(7) The results are considered satisfactory if the percentage of residual moisture is less than or equal to the manufacturer's specification.

[68 FR 57608, Oct. 6, 2003]

§113.30   Detection of Salmonella contamination.

The test for detection of Salmonella contamination provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.

(a) Samples shall be collected from the bulk suspension before bacteriostatic or bactericidal agents have been added. When tissue culture products are to be tested, 1 ml of tissue extract used as the source of cells or 1 ml of the minced tissue per se shall be tested.

(b) Five ml of the liquid vaccine suspension shall be used to inoculate each 100 ml of liquid broth medium (tryptose and either selenite F or tetrathionate). The inoculated media shall be incubated 18-24 hours at 35-37 °C.

(c) Transfers shall be made to either MacConkey agar or Salmonella-Shigella agar, incubated for 18-24 hours and examined.

(d) If no growth typical of Salmonella is noted, the plates shall be incubated an additional 18-24 hours and again examined.

(e) If suspicious colonies are observed, further subculture on suitable media shall be made for positive identification. If Salmonella is found, the bulk suspension is unsatisfactory.

[38 FR 29888, Oct. 30, 1973]

§113.31   Detection of avian lymphoid leukosis.

The complement-fixation test for detection of avian lymphoid leukosis provided in this section shall be conducted on all biological products containing virus which has been propagated in substrates of chicken origin: Provided, An inactivated viral product shall be exempt from this requirement if the licensee can demonstrate to Animal and Plant Health Inspection Service that the agent used to inactivate the vaccine virus would also inactivate lymphoid leukosis virus.

(a) Propagation of contaminating lymphoid leukosis viruses, if present, shall be done in chick embryo cell cultures.

(1) Each vaccine virus, cytopathic to chick embryo fibroblast cells, shall be effectively neutralized, inactivated, or separated so that minimal amounts of lymphoid leukosis virus can be propagated on cell culture during the 21-day growth period. If a vaccine virus cannot be effectively neutralized, inactivated, or separated, a sample of another vaccine prepared the same week from material harvested from each source flock (or other sampling procedure acceptable to Animal and Plant Health Inspection Service) used for the preparation of the questionable vaccine virus that cannot be neutralized, inactivated, or separated shall be tested each week during the preparation of such questionable vaccine.

(2) When cell cultures are tested, 5 ml of the final cell suspension as prepared for seeding of production cell cultures shall be used as inoculum. When vaccines are tested, the equivalent of 200 doses of Newcastle disease vaccine or 500 doses of other vaccines for use in poultry, or one dose of vaccine for use in other animals shall be used as inoculum. Control cultures shall be prepared from the same cell suspension as the cultures for testing the vaccine.

(3) Uninoculated chick embryo fibroblast cell cultures shall act as negative controls. One set of chick fibroblast cultures inoculated with subgroup A virus and another set inoculated with subgroup B virus shall act as positive controls, A and B respectively.

(4) The cell cultures shall be propagated at 35-37 °C for at least 21 days. They shall be passed when necessary to maintain viability and samples harvested from each passage shall be tested for group specific antigen.

(b) The microtiter complement-fixation test shall be performed using either the 50 percent or the 100 percent hemolytic end point technique to determine complement unitage. Five 50 percent hemolytic units or two 100 percent hemolytic units of complement shall be used for each test.

(1) All test materials, including positive and negative controls, shall be stored at −60 °C or colder until used in the test. Before use, each sample shall be thawed and frozen three times to disrupt intact cells and release the group specific antigen.

(2) The antiserum used in the microtiter complement-fixation test shall be a standard reagent supplied or approved by the Animal and Plant Health Inspection Service. Four units of antiserum shall be used for each test.

(3) Presence of complement-fixing activity in the harvested samples (from passages) at the 1:4 or higher dilution, in the absence of anticomplementary activity, shall be considered a positive test unless the activity can definitely be established to be caused by something other than lymphoid leukosis virus, subgroups A and/or B. Activity at the 1:2 dilution shall be considered suspicious and the sample further subcultured to determine presence or absence of the group specific antigen.

(4) Biological products or primary cells which are found contaminated with lymphoid leukosis viruses are unsatisfactory. Source flocks from which contaminated material was obtained are also unsatisfactory.

[38 FR 29888, Oct. 30, 1973, as amended at 38 FR 32917, Nov. 29, 1973; 39 FR 21042, June 18, 1974; 56 FR 66784, Dec. 26, 1991]

§113.32   Detection of Brucella contamination.

The test for detection of Brucella contamination provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in a filed Outline of Production for the product.

(a) One ml of the minced tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of antibiotics, diluent and stabilizer, shall be inoculated onto each of three tryptose agar plates and incubated in a 10 percent CO2 atmosphere at a temperature of 35-37 °C for at least 7 days.

(b) If colonies are identified as Brucella, the biological product is unsatisfactory.

(c) If colonies suspicious of Brucella are observed but cannot be identified as a Brucella species, either

(1) The biological product shall be regarded as unsatisfactory and destroyed; or

(2) Further subculture or other procedures shall be carried out until a positive identification can be made.

[38 FR 29888, Oct. 30, 1973]

§113.33   Mouse safety tests.

One of the mouse safety tests provided in this section shall be conducted when such test is prescribed in a Standard Requirement or in the filed Outline of Production for a biological product recommended for animals other than poultry: Provided, That if the inherent nature of one or more ingredients makes the biological product lethal or toxic for mice but not lethal or toxic for the animals for which it is recommended, the licensee shall demonstrate the safety of such product by an acceptable test written into such Outline of Production.

(a) Final container samples of completed product from live virus vaccines shall be tested for safety using young adult mice in accordance with the test provided in this paragraph.

(1) Vaccine prepared for use as recommended on the label shall be tested by inoculating eight mice intraperitoneally or subcutaneously with 0.5 mL (the inoculation volume may be divided among more than one injection site), and the animals observed for 7 days.

(2) If unfavorable reactions attributable to the product occur in any of the mice during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

(b) Bulk or final container samples of completed product from liquid products, such as but not limited to antiserums and bacterins, shall be tested for safety in accordance with the test provided in this paragraph.

(1) Unless otherwise prescribed in the Standard Requirement or approved in a filed Outline of Production for the product, a 0.5 ml dose shall be injected intraperitoneally or subcutaneously into eight mice and the animals observed for 7 days.

(2) If unfavorable reactions attributable to the product occur in any of the mice during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

[38 FR 34727, Dec. 18, 1973, as amended at 39 FR 16857, May 10, 1974; 72 FR 72564, Dec. 21, 2007]

§113.34   Detection of hemagglutinating viruses.

The test for detection of hemagglutinating viruses provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.

(a) Final container samples of completed product rehydrated as recommended on the label shall be used as inoculum: Provided, That poultry vaccines distributed without diluent shall be rehydrated with 30 ml of sterile distilled water per 1,000 doses and used as inoculum. When one or more fractions are to be used in combination with Newcastle Disease Vaccine, test samples shall be collected from bulk suspensions of each prior to mixing with the Newcastle Disease Vaccine.

(b) Each of ten 9- to 10-day-old embryonating eggs from Newcastle disease susceptible flocks shall be inoculated into the allantoic cavity with 0.2 ml of the undiluted inoculum.

(1) Test five uninoculated embryos of the same age and from the same flock as those used for the test as negative controls.

(2) Test an allantoic fluid sample of Newcastle disease virus as a positive control.

(c) Three to five days post-inoculation, a sample of allantoic fluid from each egg shall be tested separately by a rapid plate test for hemagglutinating activity using a 0.5 percent suspension of fresh chicken red blood cells.

(d) If the results are inconclusive, one or two blind passages shall be made using fluids from each of the original test eggs. Fluids from dead and live embryos may be pooled separately for inoculum in these passages.

(e) If hemagglutinating activity attributable to the product is observed, the serial is unsatisfactory.

[38 FR 29889, Oct. 30, 1973]

§113.35   Detection of viricidal activity.

The test for detection of viricidal activity provided in this section shall be conducted when such a test is prescribed in an applicable standard requirement or in the filed Outline of Production for each inactivated liquid biological product used as diluent for a desiccated live virus vaccine in a combination package.

(a) Bulk or final container samples of completed product from each serial shall be tested.

(b) The product shall be tested with each virus fraction for which it is to be used as a diluent. If the vaccine to be rehydrated contains more than one virus fraction, the test shall be conducted with each fraction after neutralization of the other fraction(s), and/or dilution of the vaccine beyond the titer range of the other fraction(s), or the test shall be conducted using representative single-fraction desiccated vaccines which are prepared by the licensee and which are licensed. Provided, That the Administrator may authorize licensees to prepare and use unlicensed single-fraction vaccines for this purpose.

(c) Test procedure: (1) Rehydrate at least two vials of the vaccine with the liquid product under test according to label recommendations and pool the contents.

(2) Rehydrate at least two vials of the vaccine with the same volume of sterile purified water and pool the contents.

(3) Neutralize to remove other fractions, if necessary.

(4) Hold the two pools of vaccine at room temperature (20 °to 25 °C) for 2 hours. The holding period shall begin when rehydration is completed.

(5) Titrate the virus(es) in each pool of vaccine as provided in the filed Outline of Production or an applicable standard requirement.

(6) Compare respective titers.

(d) If the titer of the vaccine virus(es) rehydrated with the product under test is more than 0.7 log10 below the titer of the vaccine virus(es) rehydrated with sterile purified water, the product is unsatisfactory for use as diluent.

(e) If the product is unsatisfactory in the first test, one retest to rule out faulty techniques may be conducted using four vials of the vaccine for each pool and the acceptability of the product judged by the results of the second test.

(f) Liquid products found to be unsatisfactory for use as diluent by this test are not prohibited from release as separate licensed products if labeled as prescribed in §112.7(g).

[44 FR 25412, May 1, 1979, as amended at 56 FR 66784, Dec. 26, 1991; 64 FR 43044, Aug. 9, 1999]

§113.36   Detection of pathogens by the chicken inoculation test.

The test for detection of extraneous pathogens provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.

(a) The biological product to be tested shall be prepared for use as recommended on the label, or in the case of desiccated vaccine to be used in poultry, rehydrated with sterile distilled water at the rate of 30 ml per 1,000 doses.

(b) At least 25 healthy susceptible young chickens, properly identified and obtained from the same source and hatch, shall be immunized at least 14 days prior to being put on test. The immunizing agent shall be the same as the product to be tested but from a serial previously tested and found satisfactory.

(c) At least 20 of the previously immunized birds shall be inoculated with 10 label doses of the vaccine being tested by each of the following routes: Subcutaneous, intratracheal, eye-drop, and comb scarification (1 cm2). Twenty birds may be used for each route or combination of routes.

(d) At least five birds shall be isolated as control birds.

(e) All birds shall be observed for 21 days for signs of septicemic diseases, respiratory diseases, or other pathologic conditions.

(f) If the controls remain healthy and unfavorable reactions attributable to the product occur in the vaccinates, the serial or subserial tested is unsatisfactory. If the controls do not remain healthy or if unfavorable reactions not attributable to the product occur in the vaccinates, or both, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial of subserial tested shall be considered unsatisfactory.

[38 FR 29889, Oct. 30, 1973, as amended at 39 FR 21042, June 18, 1974; 43 FR 7610, Feb. 24, 1978]

§113.37   Detection of pathogens by the chicken embryo inoculation test.

The test for detection of extraneous pathogens provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in the filed Outline of Production for the product.

(a) The biological product to be tested shall be prepared for use as recommended on the label, or in the case of desiccated vaccine to be used in poultry, rehydrated with sterile distilled water at the rate of 30 ml per 1,000 doses.

(b) One volume of the prepared vaccine shall be mixed with up to nine volumes of sterile heat-inactivated specific antiserum to neutralize the vaccine virus in the product. Each lot of antiserum shall be demonstrated by virus neutralization tests not to inhibit other viruses known to be possible contaminants.

(c) After neutralization, 0.2 ml of the vaccine-serum mixture shall be inoculated into each of at least 20 fully susceptible chicken embryos.

(1) Twenty embryos, 9 to 11 days old, shall be inoculated on the chorio-allantoic membrane (CAM) with 0.1 ml, and in the allantoic sac with 0.1 ml.

(2) Eggs shall be candled daily for 7 days. Deaths occurring during the first 24 hours shall be disregarded but at least 18 viable embryos shall survive 24 hours post-inoculation for a valid test. Examine all embryos and CAM's from embryos which die after the first day. When necessary, embryo subcultures shall be made to determine the cause of a death. The test shall be concluded on the seventh day post-inoculation and the surviving embryos (including CAM's) examined.

(d) If death and/or abnormality attributable to the inoculum occur, the serial is unsatisfactory: Provided, That, if there is a vaccine virus override, the test may be repeated, using a higher titered antiserum.

[38 FR 29889, Oct. 30, 1973, as amended at 39 FR 21042, June 18, 1974]

§113.38   Guinea pig safety test.

The guinea pig safety test provided in this section shall be conducted when prescribed in a Standard Requirement or approved Outline of Production for a biological product. When desiccated products are tested, final container samples of completed product prepared for administration in the manner recommended on the label shall be used. When liquid products are tested, either bulk or final container samples of completed product shall be used.

(a) Unless otherwise specified in the Standard Requirement or approved Outline of Production for the product, a 2 ml dose shall be injected either intramuscularly or subcutaneously into each of two guinea pigs and the animals observed for 7 days.

(b) If unfavorable reactions attributable to the product occur in either of the guinea pigs during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

[39 FR 16857, May 10, 1974; 39 FR 20368, June 10, 1974]

§113.39   Cat safety tests.

The safety tests provided in this section shall be conducted when prescribed in a standard requirement or in the filed Outline of Production for a biological product recommended for use in cats.

(a) The cat safety test provided in this paragraph shall be used when the Master Seed Virus is tested for safety.

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

(i) Throat swabs shall be collected from each cat and individually tested on susceptible cell cultures for the presence of the virus. Blood samples shall also be drawn and individual serum samples tested for antibody to the virus.

(ii) The cats shall be considered susceptible if swabs are negative for virus isolation and the serums are free of virus antibody at the 1:2 final dilution in a 50 percent plaque reduction test or other serum-neutralization test of equal sensitivity.

(iii) When determining susceptibility to a virus which does not lend itself to the methods in paragraphs (a)(1)(i) and (ii) of this section, a method acceptable to Animal and Plant Health Inspection Service shall be used.

(2) Each of at least 10 susceptible cats shall be administered a sample of the Master Seed Virus equivalent to the amount of virus to be used in one cat dose of the vaccine, by the method to be recommended on the label, and the cats observed each day for 14 days.

(3) If unfavorable reactions attributable to the virus occur in any of the cats during the observation period, the Master Seed Virus is unsatisfactory. If unfavorable reactions occur which are not attributable to the Master Seed Virus, the test shall be declared a No Test and repeated: Provided, That, if not repeated, the Master Seed Virus shall be unsatisfactory.

(b) The cat safety test provided in this paragraph shall be used when a serial of vaccine is tested for safety before release.

(1) Each of two healthy cats shall be administered 10 cat doses by the method recommended on the label and the cats observed each day for 14 days.

(2) If unfavorable reactions attributable to the biological product occur during the observation period, the serial is unsatisfactory. If unfavorable reactions occur which are not attributable to the product, the test shall be declared a No Test and repeated: Provided, That, if not repeated, the serial shall be unsatisfactory.

[44 FR 58898, Oct. 12, 1979, as amended at 56 FR 66784, Dec. 26, 1991]

§113.40   Dog safety tests.

The safety tests provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a biological product recommended for use in dogs. Serials which are not found to be satisfactory when tested pursuant to the procedures in this section may not be released for shipment.

(a) The dog safety test provided in this paragraph shall be used when the Master Seed Virus is tested for safety.

(1) The test animals shall be determined to be susceptible to the virus under test by a method acceptable to the Animal and Plant Health Inspection Service.

(2) Each of at least 10 susceptible dogs shall be administered a sample of the Master Seed Virus equivalent to the amount of virus to be used in one dog dose of the vaccine, by the method recommended on the label, and the dog shall be observed each day for 14 days.

(3) If unfavorable reactions attributable to the virus occur in any of the dogs during the observation period, the Master Seed Virus is unsatisfactory. If unfavorable reactions occur which are not attributable to the Master Seed Virus, the test shall be declared a No Test and may be repeated: Provided: That, if the test is not repeated, the Master Seed Virus shall be considered unsatisfactory.

(b) The dog safety test provided in this paragraph shall be used when a serial of vaccine is tested for safety before release.

(1) Each of two healthy dogs shall be administered 10 dog doses by the method recommended on the label and the dogs shall be observed each day for 14 days.

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

[60 FR 14358, Mar. 17, 1995]

§113.41   Calf safety test.

The calf safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.

(a) Test procedure. Each of two calves shall be injected with the equivalent of 10 doses of vaccine administered in the manner recommended on the label and observed each day for 21 days.

(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the calves during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated: Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

[39 FR 27428, July 29, 1974]

§113.42   Detection of lymphocytic choriomeningitis contamination.

The test for detection of lymphocytic choriomeningitis (LCM) virus provided in this section shall be conducted when such a test is prescribed in an applicable Standard Requirement or in a filed Outline of Production. Vaccine virus may be neutralized with specific antiserum when necessary.

(a) Each of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days.

(b) If any of the mice show swelling in the injected footpad or if more than one becomes systemically abnormal, the material being tested is unsatisfactory.

[42 FR 6794, Feb. 4, 1977]

§113.43   Detection of chlamydial agents.

The test for chlamydial agents provided in this section shall be conducted when such a test is prescribed in an applicable standard requirement or in a filed Outline of Production.

(a) The yolk sac of 6-day-old chicken embryos shall be injected. Three groups of 10 embryos shall be used sequentially.

(1) The inoculum for each embryo in the first group shall consist of 0.5 ml of a mixture of equal parts of the seed virus with phosphate buffered saline that may contain Streptomycin, Vancomycin, Kanamycin, or a combination thereof. Not more than 2 mg/ml of each antibiotic shall be used.

(2) On the 10th day postinoculation, the yolk sac of viable embryos shall be harvested, pooled, homogenized as a 20 percent suspension in phosphate buffered saline antibiotic diluent, and 0.5 ml of the mixture injected into the second group of chicken embryos. This process shall be repeated for the injection of the third group of embryos using the yolk sacs of viable embryos from the second group.

(3) For each of the three passages, embryo deaths occurring within 48 hours of injection shall be disregarded, except that if more than three such deaths occur at any passage, that passage shall be repeated.

(b) If one or more embryo deaths occur at any passage after 48 hours postinjection, the yolk sacs from each of the dead embryos shall be subcultured into 10 additional embryos. If one or more embryo deaths again occur due to chlamydial agents, the Master Seed Virus is unsatisfactory for use to produce vaccine.

[44 FR 58899, Oct. 12, 1979]

§113.44   Swine safety test.

The swine safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.

(a) Test procedure. (1) Inject each of two swine of the minimum age for which the product is recommended with the equivalent of two doses of bacterial vaccine or 10 doses of viral vaccine.

(2) Administer vaccine in the manner recommended on the label.

(3) Observe swine each day for 21 days.

(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the swine during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated; Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

[48 FR 33476, July 22, 1983]

§113.45   Sheep safety test.

The sheep safety test provided in this section shall be conducted when prescribed in a Standard Requirement or in the filed Outline of Production for a product.

(a) Test procedure. (1) Inject each of two sheep of the minimum age for which the product is recommended with the equivalent of two doses of bacterial vaccine or 10 doses of viral vaccine.

(2) Administer vaccine in the manner recommended on the label.

(3) Observe sheep each day for 21 days.

(b) Interpretation. If unfavorable reactions attributable to the product occur in either of the sheep during the observation period, the serial or subserial is unsatisfactory. If unfavorable reactions which are not attributable to the product occur, the test shall be declared a No Test and may be repeated; Provided, That, if the test is not repeated, the serial or subserial shall be declared unsatisfactory.

[48 FR 33476, July 22, 1983]

§113.46   Detection of cytopathogenic and/or hemadsorbing agents.

The tests for detection of cytopathogenic and/or hemadsorbing agents provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in the filed Outline of Production for a product.

(a) Test for cytopathogenic agents. One or more monolayers that are at least 6 cm2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.

(1) Stain each monolayer with a suitable cytological stain.

(2) Examine the entire area of each stained monolayer for evidence of inclusion bodies, abnormal number of giant cells, or other cytopathology indicative of cell abnormalities attributable to an extraneous agent.

(b) Test for hemadsorbing agents. One or more monolayers that are at least 6 cm2 and at least 7 days from the last subculture shall be tested as provided in this paragraph.

(1) Wash the monolayer with several changes of phosphate buffered saline.

(2) Add an appropriate volume of a 0.2 percent red blood cell suspension to uniformly cover the surface of the monolayer of cultured cells. Suspensions of washed guinea pig and chicken red blood cells shall be used. These suspensions may be mixed before addition to the monolayer or they may be added separately to individual monolayers.

(3) Incubate the monolayer at 4 °C for 30 minutes, wash with phosphate buffered saline, and examine for hemadsorption.

(4) If no hemadsorption is apparent, repeat step (b)(2) of this section and incubate the monolayers at 20-25 °C for 30 minutes, wash with phosphate buffered saline, and examine again for hemadsorption. If desired, separate monolayers may be used for each incubation temperature.

(c) If specific cytopathology or hemadsorption attributable to an extraneous agent is found, the material under test is unsatisfactory and shall not be used to prepare biological products. If an extraneous agent is suspected because of cytopathology or hemadsorption and cannot be eliminated as a possibility by additional testing, the material under test is unsatisfactory.

[50 FR 441, Jan. 4, 1985, as amended at 58 FR 50252, Sept. 27, 1993]

§113.47   Detection of extraneous viruses by the fluorescent antibody technique.

The test for detection of extraneous viruses by the fluorescent antibody technique provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in a filed Outline of Production for a product.

(a) Monolayer cultures of cells (monolayers), at least 7 days after the last subculturing, shall be processed and stained with the appropriate antiviral fluorochrome-conjugated antibody as specified in paragraph (b) of this section.

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

(i) At the time of the last subculturing, one group of test monolayers shall be inoculated with approximately 100-300 FAID50 of the specific virus being tested for as positive controls.

(ii) One group of monolayers shall be the “material under test.”

(iii) One group of monolayers, that are of the same type of cells as the test monolayers and that have been tested as prescribed in §§113.51 or 113.52 (whichever is applicable), shall be prepared as negative controls.

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

(b) The antiviral fluorochrome-conjugated antibodies to be used shall depend on the type of cells required to be tested for extraneous viruses as specified in an applicable Standard Requirement or in a filed Outline of Production. Antiviral fluorochrome-conjugated antibodies specific for the extraneous viruses shall be applied to each respective type of cell in accordance with the following list. Under certain circumstances, additional tests may need to be conducted, as determined by the Administrator. When a specific antiviral fluorochrome-conjugated antibody is used in testing for the listed extraneous viruses specified in more than one cell type, it need only be applied to the most susceptible cell type.

(1) All cells shall be tested for:

(i) Bovine virus diarrhea virus;

(ii) Reovirus; and

(iii) Rabies virus.

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

(i) Bluetongue virus;

(ii) Bovine adenoviruses;

(iii) Bovine parvovirus; and

(iv) Bovine respiratory syncytial virus.

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

(i) Canine coronavirus;

(ii) Canine distemper virus; and

(iii) Canine parvovirus.

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

(i) Equine herpesvirus; and

(ii) Equine viral arteritis virus.

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

(i) Feline infectious peritonitis virus; and

(ii) Feline panleukopenia virus.

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

(i) Porcine adenovirus;

(ii) Porcine parvovirus;

(iii) transmissible gastroenteritis virus; and

(iv) Porcine hemagglutinating encephalitis virus.

(7) Firms that do not have rabies virus on premises either for research or production purposes are exempt from having to produce positive rabies virus control monolayers. Fixed positive rabies virus control monolayers will be provided by the National Veterinary Services Laboratories.

(c) After staining, each group of monolayers shall be examined for the presence of specific fluorescence attributable to the presence of extraneous viruses.

(1) If the material under test shows any evidence of specific viral fluorescence, it is unsatisfactory and may not be used; Provided, That, if specific fluorescence attributable to the virus being tested for is absent in the positive control monolayers, the test is a No Test and may be repeated.

(2) If the fluorescence of the monolayers inoculated with the specific virus as positive controls is equivocal, or if the negative monolayers show equivocal fluorescence indicating possible viral contamination, or both, the test shall be declared a No Test, and may be repeated; Provided, That, if the test is not repeated, the material under test shall be regarded as unsatisfactory for use in the production of biologics.

[60 FR 24548, May 9, 1995]

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