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Title 9Chapter ISubchapter EPart 113 → Subject Group


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


Antibody Products

§113.450   General requirements for antibody products.

Unless otherwise prescribed in a Standard Requirement or in a filed Outline of Production, all antibody products shall meet the applicable requirements of this section.

(a) Terminology. The following terms in the regulations and standards concerning antibody products shall mean:

Antibody. An immunoglobulin molecule, having a precise glycoprotein structure, produced by certain cells of the B lymphocyte lineage in response to antigenic stimulation, and functioning to specifically bind and influence the antigens that induced its synthesis.

IgG (Immunoglobulin G). One of the several recognized classes of structurally related glycoproteins whose representatives include all known antibodies.

Monoclonal. Produced by, or derived from, the offspring of a single common progenitor cell.

Failure of passive transfer. A condition of neonates characterized by an abnormally low concentration of circulating maternal IgG.

(b) Nomenclature. Antibody products shall be named as follows:

(1) Virus-specific products. The true name of a virus-specific product shall: include the term “antibody,” specify the disease for which the product is intended, and indicate the type of animal that supplied the component antibodies. If the antibodies are monoclonal, the term “monoclonal” shall be used. Example: “Duck Virus Hepatitis Antibody, Duck Origin.”

(2) Bacterium-specific products. The true name of a bacterium-specific product shall: include the term “antibody” if the component antibodies are directed against a nontoxin antigen or the term “antitoxin” if the component antibodies are directed against toxin, specify the organism against which the product is intended, and indicate the type of animal that supplied the component antibodies. If the antibodies are monoclonal, the term “monoclonal” shall be used. Example: “Escherichia Coli Monoclonal Antibody, Murine Origin.”

(3) Failure of passive transfer products. The true name of a product for treatment of failure of passive transfer shall include the term “IgG” and indicate the type of animal that supplied the component IgG. Example: “Bovine IgG.”

(4) Combination products. The true name of a product for treatment of failure of passive transfer as well as for the prevention and/or alleviation of a specific viral or bacterial disease shall be named according to the nomenclature prescribed above for virus-specific or bacterium-specific products.

(c) Animals. All animals used in the production of antibody products shall be healthy. Their health status shall be determined by physical examination by, or under the direct supervision of, a licensed veterinarian and by tests for infectious diseases. Such animals shall be maintained at licensed establishments: Provided, That cows maintained at Grade A dairies (or the equivalent) that are not injected with antigens for the purpose of stimulating the production of specific antibodies and that are used only for the purpose of supplying lacteal secretions are exempt from being maintained at a licensed establishment.

(1) No animal shall be used while showing clinical signs of disease. The presence of minor localized injuries or lesions (contusions, lacerations, burns, etc.) without body temperature elevation and without significant pain and distress shall not be construed as clinical evidence of disease.

(2) Before first use and on a regular basis, all animals used in the manufacture of antibody products shall be individually subjected to applicable tests for infectious diseases. Records of all test results shall be maintained. An animal which tests positive for an infectious disease shall not be used in the manufacture of antibody products. Retests shall be conducted as deemed necessary by the Administrator.

(i) Before first use, horses shall be tested as follows for:

(A) Equine infectious anemia (EIA) at a laboratory approved by APHIS.

(B) Piroplasmosis, dourine, and glanders at the National Veterinary Services Laboratories.

(C) Brucellosis at a laboratory approved by APHIS. Horses with standard agglutination titers of 1:50 or less can be used for production. Horses with standard agglutination titers equal to or greater than 1:100 may be tested by the Rivanol or card tests. Reactors to these supplemental tests shall not be used for production. Nonreactors to the supplemental tests shall be retested after 30 days. If the supplemental tests are negative and the agglutination titer has not increased, the animal may be used for production. Otherwise, the animal is unsatisfactory for this purpose.

(ii) Horses shall be retested annually for EIA and, if housed or pastured with any other species, shall be retested annually for brucellosis.

(iii) Before first use, cattle shall be tested as follows for:

(A) Tuberculosis by an accredited veterinarian: Provided, That cattle at Grade A dairies supplying only lacteal secretions need only be tested for tuberculosis in accordance with applicable Milk Ordinances or similar laws or regulations.

(B) Brucellosis at a laboratory approved by APHIS. Cattle with standard agglutination titers of 1:50 or less can be used for production. Cattle with standard agglutination titers equal to or greater than 1:100 may be tested by the Rivanol or card tests. Reactors to these supplemental tests shall not be used for production. Nonreactors to the supplemental tests shall be retested after 30 days. If the supplemental tests are negative and the agglutination titer has not increased, the animal may be used for production; otherwise, the animal is unsatisfactory for this purpose. Cattle at Grade A dairies supplying only lacteal secretions need not be tested individually for brucellosis if a portion of their secretions contribute to the herd milk pool tested as required by the brucellosis ring test. An animal of a herd testing positive by this test shall not be used in production.

(iv) Cattle shall be retested annually for both tuberculosis and brucellosis. Cattle at Grade A dairies supplying only lacteal secretions need only be tested for tuberculosis in accordance with applicable Milk Ordinances or similar laws or regulations. Cattle at Grade A dairies supplying only lacteal secretions need not be tested individually for brucellosis if a portion of their secretions contribute to the herd milk pool tested as required by the brucellosis ring test. An animal of a herd testing positive by this test shall not be used in production.

(v) For other species, appropriate tests and the frequency with which they are applied shall be specified in the filed Outline of Production for the product.

(vi) If a positive result is obtained on any prescribed test, the positive animal(s) shall be removed from the herd and the remaining animals retested. Production shall not be renewed until a negative herd test is obtained not less than 28 days following removal of the positive animal(s).

(vii) Negative animals shall be maintained separate and apart from untested or positive animals of any species. Production animals shall not be used for any other purpose, such as testing, work, or recreation.

(d) Collection procedures. Blood, lacteal secretions, and egg material shall be collected as described in the filed Outline of Production for the product.

(e) Ingredient handling and processing. Blood derivatives (serum, plasma, etc.), lacteal secretions, and egg material used in the production of antibody products shall be subjected to an appropriate procedure for the inactivation of potential contaminating microorganisms. The procedure shall be one of those described below and specified in the filed Outline of Production for the product: Provided, That another procedure may be substituted if demonstrated to be at least as effective by data acceptable to APHIS and specified in the filed Outline of Production for the product. These data are expected to come from a study comparing the effectiveness of the established and substitute procedures against a satisfactory battery of potential contaminating microorganisms.

(1) Blood derivatives of equine origin shall be heated at 58.0-59.0 °C for 60 minutes, and blood derivatives of bovine, porcine, or other origin shall be heated at 58.0-59.0 °C for 30 minutes. In lieu of heat treatment, blood derivatives of any origin may be treated with at least 2.5 megarads of ionizing radiation, with a maximum radiation dosage specified in the filed Outline of Production for the product.

(2) Lacteal secretions shall be heated as described in paragraph (e)(1) of this section, or shall be pasteurized at either 72 °C for 15 seconds or 89 °C for 1 second using appropriate equipment. In lieu of the heat treatment regimens prescribed, lacteal secretions may be treated with at least 2.5 megarads of ionizing radiation, with a maximum radiation dosage specified in the Outline of Production for the product.

(3) Egg material shall be heated at 58.0-59.0 °C for 30 minutes, or treated with at least 2.5 megarads of ionizing radiation, with a maximum radiation dosage specified in the filed Outline of Production for the product.

(4) Blood derivatives, lacteal secretions, and egg material shall not contain preservatives at the time of heat treatment, and immediately after heat treatment shall be cooled to 7 °C or lower.

(5) Licensees shall keep detailed records as to each batch treated and each serial of product prepared for marketing. Recording charts shall bear full information concerning the material treated and tests made of the equipment used for treatment.

(f) Preservatives. Liquid antibody products, except those immediately frozen following preparation and maintained in a frozen state until time of use, shall contain at least one preservative from the following list, within the range of concentration set forth:

(1) Phenol 0.25 to 0.55 percent, or

(2) Cresol 0.10 to 0.30 percent, and/or

(3) Thimerosal 0.01 to 0.03 percent, or

(4) Other preservative(s) specified in the filed Outline of Production for the product.

(g) Antigens for hyperimmunization. If animals are hyperimmunized to generate antibodies for a product for the prevention and/or alleviation of a specific infectious disease, and a USDA-licensed veterinary biological product is not employed for this purpose, the following shall apply:

(1) For each antigen, a Master Seed shall be established.

(i) Bacterial Master Seeds shall be tested for purity and identity as prescribed for live bacterial vaccines in §113.64.

(ii) Viral Master Seeds shall be tested for purity and identity as prescribed for live virus vaccines in §113.300.

(2) The maximum allowable passage level of the hyperimmunizing antigen shall be the passage level of the antigen used to generate product shown to be efficacious and shall not exceed 10 passages from the Master Seed.

(h) Purity tests. Final container samples of each serial and each subserial shall be tested for viable bacteria and fungi as follows:

(1) Dried products for parenteral administration and liquid products shall be tested as prescribed in §113.26.

(2) For dried products for oral administration, 10 final container samples shall be reconstituted with sterile water at the volume recommended on the label and tested for the following contaminants:

(i) Coliforms. One milliliter of each rehydrated sample shall be pipetted into a 100 × 15 mm petri dish and 10-15 ml of violet red bile agar at 45-50 °C added. The plate shall be manipulated to coat its entirety with the agar-sample mixture and allowed to stand until the mixture solidifies. The plate shall then be incubated at 35 °C for 24 hours. A positive control plate and a negative control plate shall be prepared at the same time and in the same manner as the plates containing samples of the serial. All plates shall be examined at the end of the incubation period. If characteristic growth is observed on the negative control plate, or no characteristic growth is observed on the positive control plate, the test shall be considered a No Test and may be repeated. If characteristic growth is observed on any of the 10 plates containing samples of the serial, one retest to rule out faulty technique may be conducted on samples from 20 final containers. If characteristic growth is observed on any of the retest plates, or if a retest is not initiated within 21 days of the completion of the original test, the serial or subserial is unsatisfactory.

(ii) Salmonellae. One milliliter of each rehydrated sample shall be pipetted into a 100 × 15 mm petri dish and 10-15 ml of brilliant green agar at 45-50 °C added. The dish shall be manipulated to coat its entirety with the agar-sample mixture and allowed to stand until the mixture solidifies. The plate shall then be incubated at 35 °C for 24 hours. A positive control plate and a negative control plate shall be prepared at the same time and in the same manner as the plates containing samples of the serial. All plates shall be examined at the end of the incubation period. If characteristic growth is observed on the negative control plate, or no characteristic growth is observed on the positive control plate, the test shall be considered a No Test and may be repeated. If characteristic growth is observed on any of the 10 plates containing samples of the serial, one retest to rule out faulty technique may be conducted on samples from 20 final containers. If characteristic growth is observed on any of the retest plates, or if a retest is not initiated within 21 days of the completion of the original test, the serial or subserial is unsatisfactory.

(iii) Fungi. One milliliter of each rehydrated sample shall be pipetted into a 100 × 15 mm petri dish and 10-15 ml of appropriately acidified potato dextrose agar at 45-50 °C added. The plate shall be manipulated to coat its entirety with the agar-sample mixture and allowed to stand until the mixture solidifies. The plate shall then be incubated at 20-25 °C for 5 days. A positive control plate and a negative control plate shall be prepared at the same time and in the same manner as the plates containing samples of the serial. All plates shall be examined at the end of the incubation period. If growth is observed on the negative control plate, or no growth is observed on the positive control plate, the test shall be considered a No Test and may be repeated. If growth is observed on any of the 10 plates containing samples of the serial, one retest to rule out faulty technique may be conducted on samples from 20 final containers. If growth is observed on any of the retest plates, or if a retest is not initiated within 21 days of the completion of the original test, the serial or subserial is unsatisfactory.

(iv) Total bacterial count. One milliliter of each rehydrated sample, undiluted or diluted as prescribed in the Outline of Production, shall be pipetted into a 100 × 15 mm petri dish and 10-15 ml of tryptone glucose extract agar at 45-50 °C added. The plate shall be manipulated to coat its entirety with the agar-sample mixture and allowed to stand until the mixture solidifies. The plate shall then be incubated at 35 °C for 48 hours. A positive control plate and a negative control plate shall be prepared at the same time and in the same manner as the plates containing samples of the serial. All plates shall be examined at the end of the incubation period. If growth is observed on the negative control plate, or no growth is observed on the positive control plate, the test shall be considered a No Test and may be repeated. If the average number of bacterial colonies on the 10 plates containing samples of the serial exceeds that specified in the filed Outline of Production for the product, one retest to rule out faulty technique may be conducted on samples from 20 final containers. If the average number of bacterial colonies on the retest plates exceeds that specified in the filed Outline of Production for the product, or if a retest is not initiated within 21 days of the completion of the original test, the serial or subserial is unsatisfactory.

(i) Safety tests. Bulk or final container samples of each serial shall be tested as prescribed in §113.33(b). Dried product shall be reconstituted as indicated on the label and 0.5 ml injected per mouse.

[61 FR 51774, Oct. 4, 1996]

§113.451   Tetanus Antitoxin.

Tetanus Antitoxin is a specific antibody product containing antibodies directed against the toxin of Clostridium tetani. Each serial shall meet the applicable general requirements provided in §113.450 and paragraph (a) of this section, and be tested for potency as provided in paragraph (b) of this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) General requirements. The amount of antitoxin in a final container shall be the amount which is delivered from such container when opened and inverted until the flow stops. A graduated volumetric cylinder which conforms to the National Institute of Standards and Technology requirements shall be used. The reading shall be made at the bottom of the meniscus. Volumes of 10 ml or less shall be recorded to the nearest 0.1 and volumes over 10 ml shall be recorded to the nearest ml.

(1) All final containers of Tetanus Antitoxin shall yield not less than the labeled unitage of antitoxin throughout the dating period. The minimum package size permitted for marketing in the United States shall be a 1,500 unit vial.

(2) The expiration date of Tetanus Antitoxin shall be not more than 3 years after the date of a potency test which demonstrates that the recoverable antitoxin from the final container provides at least 20 percent excess over the number of units claimed on the label or not more than 1 year after the date of a potency test which demonstrates that the recoverable antitoxin from the final container provides 10 to 19 percent excess over the number of units claimed on the label.

(b) Potency test. Bulk or final container samples of completed product from each serial shall be assayed to calculate the units of Tetanus Antitoxin in each final container. A comparative toxin-antitoxin neutralization test shall be conducted using a standard antitoxin and a standard toxin. All dilutions shall be made in M/15 phosphate buffered (pH) 7.4 physiological saline with 0.2 percent gelatin.

(1) One ml of the Standard Antitoxin shall be diluted before use so the final volume contains 0.1 unit per ml. The dilution shall be held at 20° to 25 °C for 30 minutes prior to combination with a test does of toxin.

(2) The Standard Toxin test dose is that amount which when mixed with 0.1 unit of Standard Antitoxin, incubated at 20° to 25 °C for 1 hour, and injected subcutaneously into a 340 to 380 gram guinea pig, results in death of that guinea pig within 60 to 120 hours with clinical signs of tetanus. The toxin shall be diluted so the test dose shall be in 2.0 ml.

(3) A mixture of diluted Standard Toxin and diluted Standard Antitoxin shall be made so that 0.1 unit of antitoxin in 1 ml is combined with a test dose of toxin. This Standard Toxin-Antitoxin mixture shall be held at 20° to 25 °C for 1 hour before injections of guinea pigs are made.

(4) A sample from each serial of antitoxin shall be prepared as was the Standard Toxin-Antitoxin mixture; except the amount of antitoxin shall be based on an estimation of the expected potency. When testing is done on bulk material, the final container fill shall reflect the endpoint value plus 10 percent overage for 1 year dating and 20 percent overage for 3 year dating.

(5) Normal guinea pigs weighing within a range of 340 to 380 grams shall be used. Pregnant guinea pigs must not be used.

(i) Each of two guinea pigs (controls) shall be injected subcutaneously with a 3 ml dose of the Standard Toxin-Antitoxin mixture. Injections shall be made in the same order that toxin is added to the dilutions of antitoxins. These shall be observed parallel with the titration of one or more unknown antitoxins.

(ii) Two guinea pigs shall be used as test animals for each dilution of the unknown antitoxin. A 3.0 ml dose shall be injected subcutaneously into each animal.

(6) Controls shall be observed until they are down and are unable to rise or stand under their own power. At this time they are euthanized and the time of death is recorded in hours. For a satisfactory test, the controls must reach this point with clinical signs of tetanus within 24 hours of each other and within an overall time of 60 to 120 hours. The clinical signs to be observed are increased muscle tonus, curvature of the spine, asymmetry of the body outline when the resting animal is viewed from above, generalized spastic paralysis, particularly of the extensor muscles, inability to rise from a smooth surface when the animal is placed on its side, or any combination of these signs. If the control guinea pigs do not respond in this manner, the entire test shall be repeated.

(7) Potency of an unknown antitoxin is determined by finding the mixture which will protect the test animal the same as the Standard Toxin-Antitoxin mixture. Test animals dying sooner than the controls indicate the unit value selected in that dilution was not present, whereas those living longer indicate a greater unit value.

[39 FR 16859, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974, and amended at 40 FR 760, Jan. 3, 1975; 40 FR 41996, Sept. 10, 1975; 43 FR 1479, Jan. 10, 1978; 50 FR 24905, June 14, 1985. Redesignated at 55 FR 35561, Aug. 31, 1990; 61 FR 51776, Oct. 4, 1996; 64 FR 43045, Aug. 9, 1999]

§113.452   Erysipelothrix Rhusiopathiae Antibody.

Erysipelothrix Rhusiopathiae Antibody is a specific antibody product containing antibodies directed against one or more somatic antigens of Erysipelothrix rhusiopathiae. Each serial shall be tested as provided in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Each serial shall meet the applicable general requirements provided in §113.450.

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

(1) In the first stage, each of 40 Swiss mice, each weighing 16 to 20 grams, shall be injected subcutaneously with 0.1 ml of product (dried product shall be rehydrated according to label directions). Twenty-four hours postinjection, the injected mice and 10 additional mice designated controls shall be challenged subcutaneously with the same culture of Erysipelothrix rhusiopathiae.

(2) If less than eight of the 10 controls die from erysipelas within 7 days post-challenge, the test is invalid. All dead mice shall be examined to determine if the cause of death was Erysipelothrix rhusiopathiae infection.

(3) The mice injected with product shall be observed for 10 days postchallenge and all deaths recorded. The second stage shall be required when 7-10 of the mice injected with product die in the first stage. The second stage shall be conducted in a manner identical to the first stage.

(4) The results of the test shall be evaluated according to the following table:

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total number of deaths for a satisfactory testCumulative total number of deaths for an unsatisfactory test
140406 or less11 or more.
2408012 or less13 or more.

[39 FR 16859, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974, as amended at 40 FR 20067, May 8, 1975; 40 FR 23989, June 4, 1975. Redesignated at 55 FR 35561, Aug. 31, 1990; 61 FR 51776, Oct. 4, 1996; 64 FR 43045, Aug. 9, 1999]

§113.453   [Reserved]

§113.454   Clostridium Perfringens Type C Antitoxin.

Clostridium Perfringens Type C Antitoxin is a specific antibody product containing antibodies directed against the toxin of Clostridium perfringens Type C. Each serial shall be tested as provided in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Each serial shall meet the applicable general requirements provided in §113.450.

(b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Beta Antitoxin provided in this section. Dried products shall be rehydrated according to label directions.

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

(i) International antitoxin unit. (I.U.) That quantity of Beta Antitoxin which reacts with L0 and L+ doses of Standard Toxin according to their definitions.

(ii) L0dose. The largest quantity of toxin which can be mixed with one unit of Standard Antitoxin and not cause sickness or death in injected mice.

(iii) L+dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice.

(iv) Standard antitoxin. The Beta Antitoxin preparation which has been standardized as to antitoxin unitage on the basis of the International Clostridium perfringens Beta Antitoxin Standard and which is either supplied by or acceptable to Animal and Plant Health Inspection Service. The antitoxin unit value shall be stated on the label.

(v) Standard toxin. The Beta toxin preparation which is supplied by or is acceptable to Animal and Plant Health Inspection Service.

(vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solution shall be made by dissolving 1 gram of peptone and 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25 minutes; and storing at 4 °C. until used.

(2) The antitoxin content of the test sample shall be determined as follows:

(i) Make a dilution of Standard Antitoxin to contain 10 International Units of antitoxin per ml.

(ii) Make one dilution of Standard Toxin to contain 10 L0 doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml.

(iii) Dilute 1 ml of the test sample with 49 ml of diluent and combine 1 ml of this dilution with 1 ml of the Standard Toxin diluted to contain 10 L0 doses.

(iv) Combine 10 International Units of Standard Antitoxin with 10 L0 doses of diluted Standard Toxin and combine 10 International Units of Standard Antitoxin with 10 L+ doses of diluted Standard Toxin.

(v) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour and hold in ice water until injections of mice can be made.

(vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for each toxin-antitoxin mixture. A dose of 0.2 ml shall be injected intravenously into each mouse. Conclude the test 24 hours post-injection and record all deaths.

(3) Test Interpretation. (i) If any mice inoculated with the mixture of 10 International Units of Standard Antitoxin and 10 L0 doses of Standard Toxin die, the results of the test are a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(ii) If less than 80 percent of the mice inoculated with the mixture of 10 International Units of Standard Antitoxin and 10 L+ doses of Standard Toxin die, the results of the test are a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(iii) If any mice inoculated with the mixture of Clostridium Perfringens Type C Antitoxin diluted 1:50 and 10 L0 doses of Standard Toxin die, the antitoxin is considered to contain less than 500 International Unit per ml and the serial is unsatisfactory.

[39 FR 16859, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974. Redesignated at 55 FR 35561, Aug. 31, 1990, as amended at 56 FR 66784, Dec. 26, 1991; 61 FR 51777, Oct. 4, 1996]

§113.455   Clostridium Perfringens Type D Antitoxin.

Clostridium Perfringens Type D Antitoxin is a specific antibody product containing antibodies directed against the toxin of Clostridium perfringens Type D. Each serial shall be tested as provided in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Each serial shall meet the applicable general requirements provided in §113.450.

(b) Potency test. Bulk or final container samples of completed product from each serial shall be tested using the toxin-neutralization test for Epsilon Antitoxin provided in this section. Dried products shall be rehydrated according to label directions.

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

(i) International antitoxin unit. (I.U.) That quantity of Epsilon Antitoxin which reacts with L0 and L+ doses of Standard Toxin according to their definitions.

(ii) L0dose. The largest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and not cause sickness or death in injected mice.

(iii) L+dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice.

(iv) Standard antitoxin. The Epsilon Antitoxin preparation which has been standardized as to antitoxin unitage on the basis of the International Clostridium perfringens Epsilon Antitoxin Standard and which is either supplied by or acceptable to Animal and Plant Health Inspection Service. The antitoxin unit value shall be stated on the label.

(v) Standard toxin. The Epsilon toxin preparation which is supplied by or is acceptable to Animal and Plant Health Inspection Service.

(vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solution shall be made by dissolving 1 gram of peptone and 0.25 gram of sodium chloride in each 100 ml of distilled water; adjusting the pH to 7.2; autoclaving at 250 °F. for 25 minutes; and storing at 4 °C. until used.

(2) The antitoxin content of the test sample shall be determined as follows:

(i) Make a dilution of Standard Antitoxin to contain 1 International Unit of antitoxin per ml.

(ii) Make one dilution of Standard Toxin to contain 10 L0 doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml.

(iii) Dilute 1 ml of the test sample with 33 ml of diluent and combine 1 ml of this dilution with 1 ml of the Standard Toxin diluted to contain 10 L0 doses.

(iv) Combine 1 International Unit of Standard Antitoxin with 10 L0 doses of Standard Toxin and combine 1 International Unit of Standard Antitoxin with 10 L+ doses of Standard Toxin.

(v) Neutralize all toxin-antitoxin mixtures at room temperature for 1 hour, and hold in ice water until injections of mice can be made.

(vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for each toxin-antitoxin mixture. A dose of 0.2 ml shall be injected intravenously into each mouse. Conclude the test 24 hours post-injection and record all deaths.

(3) Test Interpretation. (i) If any mice inoculated with the mixture of 1 International Unit of Standard Antitoxin and 10 L0 doses of Standard Toxin die, the results of the test are a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(ii) If less than 80 percent of the mice inoculated with mixture of 1 International Unit of Standard Antitoxin and 10 L+ doses of Standard Toxin die, the results of the test are a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(iii) If any mice inoculated with the mixture of Clostridium Perfringens Type D Antitoxin diluted 1:34 and 10 L0 doses of Standard Toxin die, the antitoxin is considered to contain less than 34 International Units per ml and the serial is unsatisfactory.

[39 FR 16859, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974, as amended at 40 FR 760, Jan. 3, 1975. Redesignated at 55 FR 35561, Aug. 31, 1990, as amended at 56 FR 66784, Dec. 26, 1991; 61 FR 51777, Oct. 4, 1996]

§§113.456-113.498   [Reserved]

§113.499   Products for treatment of failure of passive transfer.

A product for the treatment of failure of passive transfer (FPT) shall contain a specified minimum quantity of IgG per dose and shall be recommended for use only in neonates of the same species as that of antibody origin. A product for oral administration shall not be recommended for use in animals more than 24 hours of age, while one for parenteral administration shall only be recommended for use in neonatal animals. Each serial shall meet the applicable general requirements provided in §113.450 and be tested for potency as provided in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Qualification of an IgG Reference Product. An IgG Reference Product (reference) shall be a serial of product that is manufactured according to the filed Outline of Production, properly qualified, and used to assess the potency of subsequent product serials, as described in paragraph (c) below. The reference shall be qualified as follows:

(1) At least 20 newborn, colostrum-deprived animals of the species for which the product is recommended shall be randomly selected.

(2) Blood samples shall be taken from each animal.

(3) Each animal shall be administered one dose of reference by the recommended route and shall be observed for 24 hours.

(i) Any adverse reactions shall be recorded.

(ii) The dosage of reference administered to each animal shall be in accordance with label directions. Label directions may indicate a single dosage regardless of weight, in which case the animals in the study shall be at or near the maximum weight for neonates of the species.

(4) After 24 hours, blood samples shall be taken from each animal.

(5) Pretreatment and post treatment serum IgG concentrations shall be concurrently determined for each animal using a radial immunodiffusion (RID) method acceptable to APHIS and described in the filed Outline of Production for the product.

(6) Concurrently, using the same method, five IgG measurements shall be made on an IgG Species Standard supplied or approved by APHIS. The IgG Species Standard shall be a preparation that contains IgG specific for the species in question at a concentration acceptable to APHIS.

(7) For an IgG Reference Product to be satisfactory, all animals used to qualify the reference must remain free of unfavorable product-related reactions and at least 90 percent of the paired serum samples must reflect an increase in IgG concentration (posttreatment minus pretreatment concentration) equal to or greater than the IgG concentration of the IgG Species Standard.

(b) Antibody functionality. Prior to licensure, the prospective licensee shall perform a neutralization study, or another type of study acceptable to APHIS, to demonstrate functionality of product antibody.

(c) Potency. Bulk or final container samples of completed product from each serial shall be tested for IgG content as provided in this paragraph. Samples of the test serial and of an IgG Reference Product established in accordance with paragraph (a) of this section shall be concurrently tested for IgG content by the RID method referred to in paragraph (a)(5) of this section. Five IgG measurements shall be made on each. If the IgG level per dose of the test serial does not meet or exceed that of the reference, one complete retest, involving five IgG measurements on both the reference and two samples of the test serial, may be conducted. If, upon retest, the average IgG level per dose of the two samples of the test serial does not meet or exceed that of the reference, or if a retest is not conducted, the serial is unsatisfactory.

[61 FR 51777, Oct. 4, 1996]

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