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e-CFR data is current as of August 5, 2020

Title 9Chapter ISubchapter EPart 113 → Subject Group


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


Inactivated Bacterial Products

§113.100   General requirements for inactivated bacterial products.

Unless otherwise prescribed in an applicable Standard Requirement or in the filed Outline of Production, an inactivated bacterial product shall meet the applicable requirements in this section.

(a) Purity tests. (1) Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(2) Each lot of Master Seed Bacteria shall be tested for the presence of extraneous viable bacteria and fungi in accordance with the test provided in §113.27(d).

(b) Safety tests. Bulk or final container samples of completed product from each serial shall be tested for safety in young adult mice in accordance with the test provided in §113.33(b) unless:

(1) The product contains material which is inherently lethal for mice. In such instances, the guinea pig safety test provided in §113.38 shall be conducted in place of the mouse safety test.

(2) The product is recommended for poultry. In such instances, the product shall be safety tested in poultry as defined in the specific Standard Requirement or Outline of Production for the product.

(3) The product is recommended for fish, other aquatic species, or reptiles. In such instances, the product shall be safety tested in fish, other aquatic species, or reptiles as required by specific Standard Requirement or Outline of Production for the product.

(c) Identity test. Methods of identification of Master Seed Bacteria to the genus and species level by laboratory tests shall be sufficient to distinguish the bacteria from other similar bacteria according to criteria described in the most recent edition of “Bergey's Manual of Systematic Bacteriology” or the American Society for Microbiology “Manual of Clinical Microbiology”. If Master Seed Bacteria are referred to by serotype, serovar, subtype, pilus type, strain or other taxonomic subdivision below the species level, adequate testing must be used to identify the bacteria to that level. Tests which may be used to identify Master Seed Bacteria include, but are not limited to:

(1) Cultural characteristics,

(2) Staining reaction,

(3) Biochemical reactivity,

(4) Fluorescent antibody tests,

(5) Serologic tests,

(6) Toxin typing,

(7) Somatic or flagellar antigen characterization, and

(8) Restriction endonuclease analysis.

(d) Ingredient requirements. Ingredients used for the growth and preparation of Master Seed Bacteria and of final product shall meet the requirements provided in §113.50. Ingredients of animal origin shall meet the applicable requirements provided in §113.53.

(e) Only serials tested for viricidal activity in accordance with the test provided in §113.35 and found satisfactory by such test shall be packaged as diluent for desiccated fractions in combination packages.

(f) If formaldehyde is used as the inactivating agent, and the serial has not been found satisfactory by the viricidal activity test, bulk or final container samples of completed product from each serial must be tested for residual free formaldehyde content using the ferric chloride test.1 Firms currently using tests for residual free formaldehyde content other than the ferric chloride test have until July 14, 2004 to update their Outline of Production to be in compliance with this requirement.

1The procedures for performing the ferric chloride test for residual free formaldehyde may be obtained from USDA, APHIS, Center for Veterinary Biologics-Laboratory, 1800 Dayton Road, P.O. Box 844, Ames, IA 50010.

(1) The residual free formaldehyde content of biological products containing clostridial antigens must not exceed 1.85 grams per liter (g/L).

(2) The residual free formaldehyde content of bacterins, bacterin-toxoids, and toxoids, other than those containing clostridial antigens, must not exceed 0.74 grams per liter (g/L).

[39 FR 16862, May 10, 1974. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 60 FR 14355, Mar. 17, 1995; 68 FR 35283, June 13, 2003; 79 FR 31021, May 30, 2014]

§113.101   Leptospira Pomona Bacterin.

Leptospira Pomona Bacterin shall be produced from a culture of Leptospira pomona which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira pomona fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/800th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.

(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.

(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.

(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira pomona organisms, using a dose of 10-10,000 hamster LD50 as determined by titration.

(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die of leptospirosis, the test is valid and the results shall be evaluated according to the following table:

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total dead hamsters for satisfactory serialCumulative total dead hamsters for unsatisfactory serial
110102 or less5 or more.
210205 or less6 or more.

(5) If three or four vaccinates die in the first stage, the second stage shall be conducted in a manner identical to the first stage.

(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.

[39 FR 16862, May 10, 1974, as amended at 40 FR 20067, May 8, 1975; 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.102   Leptospira Icterohaemorrhagiae Bacterin.

Leptospira Icterohaemorrhagiae Bacterin shall be produced from a culture of Leptospira icterohaemorrhagiae which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira icterohaemorrhagiae fraction shall meet the applicable requirements in §113.100 and be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/80th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.

(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.

(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.

(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira icterohaemorrhagiae organisms, using a dose of 10-10,000 hamster LD50 as determined by titration.

(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die from leptospirosis, the test is valid and the results shall be evaluated according to the following table:

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total dead hamsters for satisfactory serialCumulative total dead hamsters for unsatisfactory serial
110102 or less5 or more.
210205 or less6 or more.

(5) If three or four vaccinates die in the first stage, the second stage shall be used. The second stage shall be conducted in a manner identical to the first stage.

(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.

[39 FR 16862, May 10, 1974, as amended at 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.103   Leptospira Canicola Bacterin.

Leptospira Canicola Bacterin shall be produced from a culture of Leptospira canicola which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira canicola fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Serials found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/80th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.

(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.

(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.

(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira canicola organisms, using a dose of 10-10,000 hamster LD50 as determined by titration.

(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die from leptospirosis, test is valid and the results shall be evaluated according to the following table:

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total dead hamsters for satisfactory serialCumulative total dead hamsters for unsatisfactory serial
110102 or less5 or more.
210205 or less6 or more.

(5) If three or four vaccinates die in the first stage, the second stage shall be used. The second stage shall be conducted in a manner identical to the first stage.

(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.

[39 FR 16862, May 10, 1974, as amended at 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.104   Leptospira Grippotyphosa Bacterin.

Leptospira Grippotyphosa Bacterin shall be produced from a culture of Leptospira grippotyphosa which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira grippotyphosa fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product shall be diluted with physiological saline so that each 0.25 ml contains not more than 1/800th of the dose recommended on the label and shall be tested for potency, using the two-stage test provided in this paragraph.

(1) Vaccinates. Inject each of at least 10 but not more than 12 young adult hamsters, each weighing 50 to 90 grams, with 0.25 ml of the diluted bacterin either subcutaneously or intramuscularly, in accordance with the label recommendations for use.

(2) Controls. Retain at least 10 but not more than 12 additional hamsters from the same group as unvaccinated controls.

(3) Challenge. From 14 to 18 days postvaccination, challenge each of 10 vaccinates and each of 10 controls intraperitoneally with a suspension of virulent Leptospira grippotyphosa organisms, using a dose of 10-10,000 hamster LD50 as determined by titration.

(4) Post-challenge period. Observe the vaccinates and controls for 14 days post-challenge and record all deaths. If eight or more controls die of leptospirosis, the test is valid and the results shall be evaluated according to the following table:

Cumulative Totals

StageNumber of vaccinatesDead hamsters for acceptanceDead hamsters for rejection
1102 or less5 or more.
2205 or less6 or more.

(5) If three or four vaccinates die in the first stage, the second stage shall be conducted in a manner identical to the first stage.

(6) If the second stage is used, each serial shall be evaluated according to the second part of the table. On the basis of cumulative results, each serial shall either pass or fail.

[40 FR 17003, Apr. 16, 1975, as amended at 40 FR 23989, June 4, 1975; 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.105   Leptospira Hardjo Bacterin.

Leptospira Hardjo Bacterin shall be produced from a culture of Leptospira hardjo which has been inactivated and is nontoxic. Each serial of biological product containing Leptospira hardjo fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the test written into the filed Outline of Production.

[40 FR 17003, Apr. 16, 1975, as amended at 40 FR 20067, May 8, 1975. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.106   Clostridium Chauvoei Bacterin.

Clostridium Chauvoei Bacterin shall be produced from a culture of Clostridium chauvoei which has been inactivated and is nontoxic. Each serial of biological product containing Clostridium chauvoei fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Serials found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

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

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

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

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

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total number of deaths for a satisfactory testCumulative total number of deaths for an unsatisfactory test
1881 or less3 or more.
28164 or less5 or more.

The second stage shall be required only when exactly two animals die in the first stage. The second stage shall be conducted in a manner identical to the first stage.

[39 FR 16862, May 10, 1974, as amended at 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990 and amended at 56 FR 66784, 66785, Dec. 26, 1991]

§113.107   Clostridium Haemolyticum Bacterin.

Clostridium Haemolyticum Bacterin shall be produced from a culture of Clostridium haemolyticum which has been inactivated and is nontoxic. Each serial of biological product containing Clostridium haemolyticum fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

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

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

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

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

StageNumber of vaccinatesCumulative number of vaccinatesCumulative total number of deaths for a satisfactory testCumulative total number of deaths for an unsatisfactory test
1881 or less3 or more.
28164 or less5 or more.

The second stage shall be required only when exactly two animals die in the first stage. The second stage shall be conducted in a manner identical to the first stage.

[39 FR 16862, May 10, 1974, as amended at 40 FR 20067, May 8, 1975; 45 FR 40100, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66785, Dec. 26, 1991]

§113.108   Clostridium Novyi Bacterin-Toxoid.

Clostridium Novyi Bacterin-Toxoid shall be produced from a culture of Clostridium novyi which has been inactivated and is nontoxic. Each serial of biological product containing Clostridium novyi fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

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

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

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

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

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

(vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solutions 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 121 °C for 25 minutes; and storing at 4 °C until used.

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

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

(i) At least seven rabbits are required to make an acceptable serum pool.

(ii) Equal quantities of serum from each rabbit shall be combined and tested as a single pooled serum.

(iii) If less than seven rabbits are available, the test is invalid and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

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

(i) Make a dilution of Standard Antitoxin to contain 0.1 International Unit of antitoxin per ml.

(ii) Make a dilution of Standard Toxin in which 0.1 Lo dose is contained in a volume of 1 ml or less. Make a second dilution of Standard Toxin in which 0.1 L + dose is contained in a volume of 1 ml or less.

(iii) Combine 0.1 International Unit of Standard Antitoxin with 0.1 Lo dose of diluted Standard Toxin and combine 0.1 International Unit of Standard Antitoxin with 0.1 L + dose of diluted Standard Toxin. Each mixture is adjusted to a final volume of 2.0 ml with diluent.

(iv) Combine 0.1 Lo dose of diluted Standard Toxin with a 0.2 ml volume of undiluted serum. The mixture is adjusted to a final volume of 2.0 ml with diluent.

(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 72 hours post injection and record all deaths.

(5) Test Interpretation shall be as follows:

(i) If any mice inoculated with the mixture of 0.1 International Unit of Standard Antitoxin and 0.1 Lo doses of Standard Toxin die, the results of the serum neutralization 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 0.1 International Unit of Standard Antitoxin and 0.1 L + doses of Standard Toxin die, the results of the serum neutralization 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 0.2 ml undiluted serum with 0.1 Lo dose of Standard Toxin die, the serum is considered to contain less than 0.50 International Units per ml.

(iv) If the single pooled serum from seven or more rabbits contains less than 0.5 International Unit per ml, the serial is unsatisfactory.

[39 FR 16862, May 10, 1974, as amended at 45 FR 40101, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990; 56 FR 37825, Aug. 9, 1991, as amended at 56 FR 66784, 66785, Dec. 26, 1991]

§113.109   Clostridium Sordellii Bacterin-Toxoid.

Clostridium Sordellii Bacterin-Toxoid shall be produced from a culture of Clostridium sordellii which has been inactivated and is nontoxic. Each serial of biological product containing Clostridium sordellii fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

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

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

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

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

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

(vi) Diluent. The solution used to make proper dilutions prescribed in this test. Such solutions 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 121 °C for 25 minutes; and storing at 4 °C until used.

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

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

(i) At least seven rabbits are required to make an acceptable serum pool.

(ii) Equal quantities of serum from each rabbit shall be combined and tested as a single pooled serum.

(iii) If less than seven rabbits are available, the test is a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

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

(i) Make a dilution of Standard Antitoxin to contain 1.0 international unit of antitoxin per ml.

(ii) Make a dilution of Standard Toxin in which 1.0 Lo dose is contained in a volume of 1 ml or less. Make a second dilution of Standard Toxin in which 1.0 L + dose is contained in a volume of 1 ml or less.

(iii) Combine 1.0 International Unit Standard Antitoxin with 1.0 Lo dose of diluted Standard Toxin and combine 1.0 International Unit of Standard Antitoxin with 1.0 L + dose of diluted Standard Toxin. Each mixture is adjusted to a final volume of 2.0 ml with diluent.

(iv) Combine 1.0 Lo dose of diluted Standard Toxin with a 1.0 ml volume of undiluted serum. This mixture is adjusted to a final volume of 2.0 ml with diluent.

(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 72 hours post injection and record all deaths.

(5) Test Interpretation shall be as follows:

(i) If any mice inoculated with the mixture of 1.0 International Unit of Standard Antitoxin and 1.0 Lo doses of Standard Toxin die, the results of the serum neutralization 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 1.0 International Unit of Standard Antitoxin and 1.0 L + doses of Standard Toxin die, the results of the serum neutralization 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 1.0 ml undiluted serum with 1.0 Lo dose of Standard Toxin die, the serum is considered to contain less than 1.0 International Units per ml.

(iv) If the single pooled serum from seven or more rabbits contains less than 1.0 International Unit per ml, the serial is unsatisfactory.

[39 FR 16862, May 10, 1974, as amended at 42 FR 61247, Dec. 2, 1977; 45 FR 40101, June 13, 1980. Redesignated at 55 FR 35562, Aug. 31, 1990; 56 FR 37826, Aug. 9, 1991; 56 FR 66784, 66785, Dec. 26, 1991; 79 FR 55969, Sept. 18, 2014]

§113.110   Clostridium Botulinum Type C Bacterin-Toxoid.

Clostridium Botulinum Type C Bacterin-Toxoid shall be produced from a culture of Clostridium botulinum Type C which has been inactivated and is nontoxic. Each serial of biological product containing Clostridium botulinum Type C fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b).

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency, using susceptible mink as test animals. At least five vaccinates and three unvaccinated controls of the same source and approximately the same age shall be used.

(1) Each of the vaccinates shall be injected subcutaneously with the dose recommended on the label for mink. Twenty-one to twenty-eight days post-injection, the vaccinates and the controls shall be challenged intraperitoneally with botulinum Type C toxin which has been titrated in mice to provide for a 104.0 mouse MLD dose. The titration technique shall include inoculation of the mice intraperitoneally.

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

[39 FR 16862, May 10, 1974, as amended at 40 FR 759, Jan. 3, 1975. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.111   Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

Clostridium Perfringens Type C Toxoid and Clostridium Perfringens Type C Bacterin-Toxoid shall be produced from a culture of Clostridium perfringens Type C which has been inactivated and is nontoxic. Each serial shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b).

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

(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) L0 dose. 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 solutions shall be made by dissolving 1 gram of peptone and 0.25 grams 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) Each of at least eight rabbits of a strain acceptable to APHIS, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the largest recommended dose for any species indicated on the product label. A second equivalent dose shall be given not less than 20 days nor more than 23 days after the first does.

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

(i) At least seven rabbits are required to make an acceptable serum pool.

(ii) Equal quantities of serum from each rabbit shall be combined and tested as a single pooled serum.

(iii) If less than seven rabbits are available, the test is a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(4) The antitoxin content of the rabbit serums 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) 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.

(iv) Combine 1 ml of undiluted serum with 10 L0 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.

(5) Test Interpretation shall be as follows:

(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 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 serum with 10 L0 doses of Standard Toxin die, the serum is considered to contain less than 10 International Units per ml. and the serial is unsatisfactory

[39 FR 16862, May 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; 40 FR 41088, Sept. 5, 1975. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66785, Dec. 26, 1991; 62 FR 31330, June 9, 1997; 79 FR 55969, Sept. 18, 2014]

§113.112   Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

Clostridium Perfringens Type D Toxoid and Clostridium Perfringens Type D Bacterin-Toxoid shall be produced from a culture of Clostridium perfringens Type D which has been inactivated and is nontoxic. Each serial shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. Any serial found unsatisfactory by a prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b).

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

(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) L0 dose. 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 solutions 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) Each of at least eight rabbits of a strain acceptable to APHIS, each weighing 4-8 pounds, shall be injected subcutaneously with not more than half of the largest recommended dose for any species indicated on the product label. A second equivalent dose shall be given not less than 20 days nor more than 23 days after the first dose.

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

(i) At least seven rabbits are required to make an acceptable serum pool.

(ii) Equal quantities of serum from each rabbit shall be combined and tested as a single pooled serum.

(iii) If less than seven rabbits are available, the test is a No Test and shall be repeated: Provided, That, if the test is not repeated, the serial shall be declared unsatisfactory.

(4) The antitoxin content of the rabbit serums 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 Lo doses per ml and make a second dilution of Standard Toxin to contain 10 L+ doses per ml.

(iii) Combine 1 International Unit of Standard Antitoxin with 10 Lo doses of diluted Standard Toxin and Combine 1 International Unit of Standard Antitoxin with 10 L+ doses of diluted Standard Toxin.

(iv) Dilute 1 ml of serum with 1 ml of diluent (1:2) and combine 1 ml of this solution with 10 Lo 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.

(5) Test Interpretation shall be as follows:

(i) If any mice inoculated with the mixture of 1 International Unit of Standard Antitoxin and 10 Lo 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 area 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 serum with 10 Lo doses of Standard Toxin die, the serum is considered to contain less than 2 International Units per ml, and the serial is unsatisfactory.

[39 FR 16865, May 10, 1974; 39 FR 20783, June 14, 1974. Redesignated at 39 FR 25463, July 11, 1974, and amended at 40 FR 759, Jan. 3, 1975; 40 FR 41088, Sept. 5, 1975. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66785, Dec. 26, 1991; 62 FR 31331, June 9, 1997; 79 FR 55969, Sept. 18, 2014]

§113.113   Autogenous biologics.

Autogenous biologics shall be prepared from cultures of microorganisms which have been inactivated and are nontoxic. Such products shall be prepared only for use by or under the direction of a veterinarian under a veterinarian-client-patient relationship, Provided, That, such products may be prepared for use under the direction of a person of appropriate expertise in specialized situations such as aquaculture, if approved by the Administrator.

Each serial of an autogenous biologic shall meet the requirements in this section, and if found unsatisfactory by any prescribed test shall not be used.

(a) Seed requirements. The microorganisms used as seed to prepare autogenous biologics shall be microorganisms which are isolated from sick or dead animals in the herd of origin and which there is reason to believe are the causative agent(s) of the current disease affecting such animals.

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

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

(i) Name, address, and phone number of the owner of the herd of origin.

(ii) Attending veterinarian's name, address, and phone number.

(iii) Animal species and number in herd of origin.

(iv) Identification of microorganism(s), at least to genus.

(v) Diagnosis or clinical signs of the disease observed.

(vi) Name and address of the person who isolated the microorganism(s) and the date of isolation.

(vii) Number of doses of autogenous biologic requested and vaccination schedule.

(viii) Each adjacent herd owner's name, address, and phone number.

(ix) Number of animals and species in each adjacent herd.

(x) The attending veterinarian's or approved specialist's assessment of the involvement of the adjacent herd(s) with the disease observed.

The applicant shall give notice to the State Veterinarian or other appropriate State Official in writing when an autogenous biologic is to be used in adjacent herds.

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

(i) Names and addresses of practitioners in the area in place of the name, address, and phone number of the adjacent herd owner.

(ii) The geographic designations of the area involved.

(iii) A summary of the epidemiology of the disease situation that links the designated geographic areas with the herd of origin.

In addition, an applicant for authorization under this paragraph (a)(3) shall provide written approval from the State Veterinarian or other appropriate State Official in the State in which the autogenous biologic is to be used in nonadjacent herds.

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

(i) The attending veterinarian's or approved specialist's current assessment of the continued involvement of a herd with the originally isolated microorganism(s), including a summary of the diagnostic work that has been done to support this assessment.

(ii) Evidence of satisfactory protection from the previous use of the autogenous biologic produced from the microorganisms involved.

(iii) Any other information the Administrator may require in order to determine the need to use the microorganism to make additional serials.

(b) Restrictions. Unless otherwise authorized by the Administrator, each serial of an autogenous biologic shall be subject to the following restrictions:

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

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

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

(i) When the number of final containers in a serial or subserial is 50 or less, two final container samples from each serial and subserial shall be tested as prescribed in §113.26(b): Provided, That, 1 ml aliquots from each sample may be inoculated into five corresponding individual test vessels of each of the test media required.

(ii) Serials which are satisfactory after the third day of observation of purity test cultures and of safety test animals may be released for shipment to the customer and the tests continued throughout the required period; and

(iii) Serials released on the basis of satisfactory results of third day observations shall be immediately recalled if evidence of contamination occurs in test cultures or if any of the test animals used to demonstrate product safety, sicken, or die during the observation period.

(iv) Test summaries must be submitted to the Administrator (in c/o the Director, Center for Veterinary Biologics, Inspection and Compliance, 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010) on a quarterly basis by the 21st day of January, April, July, and October or more often as required by the Administrator.

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

(i) Purity test. Final container samples of completed product from each serial and subserial shall be tested for viable bacteria and fungi as provided in §113.26. When the number of final containers in a serial or subserial is 50 or less, two final container samples from each serial and subserial shall be tested as prescribed in §113.26(b): Provided, That, 1 ml aliquots from each sample may be inoculated into five corresponding individual test vessels of each of the test media required.

(ii) Safety test. Bulk of final container samples of completed product from each serial shall be tested for safety as provided in §113.33 (b) or §113.38.

(iii) Identification. All microorganisms used for the production of autogenous biologics shall be identified as follows: Bacteria, fungi, and mycoplasma shall be identified at least to genus and species. Viruses shall be identified at least to family. After 15 months from the date of isolation, or 12 months from the harvest date of the first serial of autogenous product produced from a microorganism, whichever comes first, characterization and identification shall be completed to strain and/or serotype before such microorganism may be used for production.

(iv) Antigenicity, or immunogenicity, and potency. Persons seeking authorization to prepare additional serials of autogenous biologics from microorganisms that are older than 24 months from the date of isolation, shall be required to conduct the following additional tests:

(A) Completed product shall be tested for antigenicity or immunogenicity in the species for which the product is recommended or in another animal species whose immunological response has been shown in the scientific literature to correlate with the response of the species for which the product is recommended. Such tests shall be conducted in accordance with a protocol developed by the licensee and approved by the Administrator and the results submitted to the Director, Center for Veterinary Biologics, Policy, Evaluation, and Licensing, 1920 Dayton Avenue, P.O. Box 844, Ames, IA 50010 for review. Microorganisms not shown to be antigenic (that is, not shown to induce a significant serological response) or immunogenic by such approved tests shall not be used for the preparation of such product.

(B) Bulk or final container samples of completed product from each serial of such autogenous biologics containing fractions for which standard requirement potency test procedures have been established shall be tested for potency in accordance with applicable standard requirement potency tests provided in 9 CFR part 113. If the culture of microorganisms used to produce such fractions is shown to be of a different strain or serotype than the reagent or challenge microorganisms used in the standard requirement potency test, reagents or challenges of the same strain or serotype as the microorganism used for production may be used.

(C) If no standard requirement potency test procedures have been established for a fraction(s) in the autogenous biologic, such fraction(s) of each serial of product shall be tested for potency using a developmental potency test described in the filed outline of production or shall at least be standardized to contain an antigenic mass for such fraction(s) that has been shown to be antigenic or immunogenic in accordance with paragraph (c)(2)(iv)(A) of this section.

[57 FR 38756, Aug. 27, 1992, as amended at 59 FR 67616, Dec. 30, 1994; 64 FR 43044, Aug. 9, 1999; 67 FR 15714, Apr. 3, 2002; 75 FR 20773, Apr. 21, 2010]

§113.114   Tetanus Toxoid.

Tetanus Toxoid shall be produced from a culture of Clostridium tetani which has been inactivated and is nontoxic. The toxoid may be either absorbed, precipitated, or purified and concentrated. Each serial of biological product containing tetanus toxoid fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial or subserial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b).

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency. A group of 10 guinea pigs consisting of an equal number of males and females weighing 450 to 550 grams shall each be injected subcutaneously with 0.4 of the largest dose recommended on the product labels.

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

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

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

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

[39 FR 16862, May 10, 1974, as amended at 46 FR 23224, Apr. 24, 1981; 50 FR 24905, June 14, 1985. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 37827, Aug. 9, 1991; 56 FR 66785, Dec. 26, 1991]

§113.115   Staphylococcus Aureus Bacterin-Toxoid.

Staphylococcus Aureus Bacterin-Toxoid shall be prepared from toxoided broth cultures of selected toxogenic strains of Staphylococcus aureus which has been inactivated and is nontoxic. Each serial of biological product containing Staphylococcus Aureus Bacterin-Toxoid shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product shall be tested for safety as provided in §113.33(b). Also, the rabbits used in the potency test provided in paragraph (c) of this section shall constitute an additional safety test. If unfavorable reactions attributable to the product occur in any of the rabbits during the observation period, the serial is unsatisfactory.

(c) Potency test. Rabbits, each weighing 2000-3000 grams, shall be used as test animals. Either a five rabbit individual serum test or an eight rabbit pooled serum test shall be conducted. At the start of the test, individual serums from the five rabbits or pooled serums from the eight rabbits shall contain less than 0.2 alpha antitoxin units per ml.

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

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

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

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

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

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

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

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

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

[39 FR 16862, May 10, 1974. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.116   Pasteurella Multocida Bacterin, Avian Isolate, Type 4.

Pasteurella Multocida Bacterin, Avian Isolate, Type 4 shall be prepared from cultures of Pasteurella multocida, avian isolate, Type 4 (Little and Lyons classification), which have been inactivated, and are nontoxic. Each serial of biological product containing Pasteurella Multocida Bacterin, Avian Isolate, Type 4, shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency, as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in 9 CFR 113.26.

(b) Safety test. Observation of the vaccinated turkeys during the prechallenge period of the potency test provided in paragraph (c) of this section shall constitute the safety test. If unfavorable reactions that are attributable to the product occur, the serial is unsatisfactory. If unfavorable reactions that are not attributable to the product occur in one turkey, test results shall be determined by observing the remaining 20 turkeys. The test is a No Test and may be repeated if unfavorable reactions that are not attributable to the product occur in two or more turkeys, but the serial is unsatisfactory if the test is not repeated.

(c) Potency test. Bulk or final container samples of completed product shall be tested for potency of the Type 4 strain, using the two-stage test provided in this paragraph. Turkeys at least 6 weeks old obtained from the same source and hatch shall be properly identified and used as provided in this paragraph.

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

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

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

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

Stage Number of vaccinates Cumulative number of vaccinates Cummulative total number of dead vaccinates for___
Satisfactory serial Unsatisfactory serial
120206 or less9 or more.
2204015 or less16 or more.

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

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

[47 FR 5795, Feb. 4, 1982; 47 FR 6817, Feb. 17, 1982, as amended at 52 FR 9117, Mar. 23, 1987. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.117   Pasteurella Multocida Bacterin, Avian Isolate, Type 1.

Pasteurella Multocida Bacterin, Avian Isolate, Type 1, shall be prepared from cultures of Pasteurella multocida, avian isolate, Type 1 (Little and Lyons classification), which have been inactivated and are nontoxic. Each serial of biological product containing Pasteurella Multocida Bacterin, Avian Isolate, Type 1, shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Observation of the vaccinated chickens during the prechallenged period of the potency test provided in paragraph (c) of this section shall constitute the safety test. If unfavorable reactions that are attributable to the product occur, the serial is unsatisfactory. If unfavorable reactions that are not attributable to the product occur in one chicken, test results shall be determined by observing the remaining 20 chickens. The test is a No Test and may be repeated if unfavorable reactions that are not attributable to the product occur in two or more chickens, but the serial is unsatisfactory if the test is not repeated.

(c) Potency test. Bulk or final container samples of completed product shall be tested for potency of the Type 1 strain, using the two-stage test provided in this paragraph. Chickens, at least 12 weeks of age, obtained from the same source and hatch, shall be properly identified and used as provided in this paragraph.

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

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

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

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

Stage Number of vaccinates Cumulative number of vaccinates Cummulative total number of dead vaccinates for___
Satisfactory serial Unsatisfactory serial
120206 or less9 or more.
2204015 or less16 or more.

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

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

[39 FR 16866, May 10, 1974; 39 FR 20368, June 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; 40 FR 23989, June 4, 1975; 47 FR 5195, Feb. 4, 1982; 52 FR 9118, Mar. 23, 1987. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.118   Pasteurella Multocida Bacterin, Avian Isolate, Type 3.

Pasteurella Multocida Bacterin, Avian Isolate, Type 3, shall be prepared from culture of Pasteurella multocida, avian isolate, Type 3 (Little and Lyons classification), which have been inactivated and are nontoxic. Each serial of biological product containing Pasteurella Multocida Bacterin, Avian Isolate, Type 3, shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency, as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Observation of the vaccinated turkeys during the prechallenge period of the potency test provided in paragraph (c) of this section shall constitute the safety test. If unfavorable reactions that are attributable to the product occur, the serial is unsatisfactory. If unfavorable reactions that are not attributable to the product occur in one turkey, test results shall be determined by observing the remaining 20 turkeys. The test is a No Test and may be repeated if unfavorable reactions that are not attributable to the product occur in two or more turkeys, but the serial is unsatisfactory if the test is not repeated.

(c) Potency test. Bulk or final container samples of completed product shall be tested for potency of the Type 3 strain, using the two-stage test provided in this paragraph. Turkeys, at least 6 weeks of age, obtained from the same source and hatch, shall be properly identified and used as provided in this paragraph.

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

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

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

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

StageNumber of vaccinatesCumulative number of vaccinatesCummulative total number of dead vaccinates for___
Satisfactory serialUnsatisfactory serial
120206 or less9 or more.
2204015 or less16 or more.

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

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

[39 FR 16862, May 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; 47 FR 5196, Feb. 4, 1982; 52 FR 9118, Mar. 23, 1987. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.119   Erysipelothrix Rhusiopathiae Bacterin.

Erysipelothrix Rhusiopathiae Bacterin shall be produced from a culture of Erysipelothrix rhusiopathiae which has been inactivated and is nontoxic. Each serial of biological product containing Erysipelothrix rhusiopathiae shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.38.

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the mouse protection test provided in this paragraph. A mouse dose shall be 110 of the least dose recommended on the label for swine. Such swine dose shall not be less than 1 ml.

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

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

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

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

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

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

eCFR graphic ec14no91.016.gif

View or download PDF

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

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

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

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

(i) If the RP obtained in the original test is one-third or less than the average RP obtained in the retests, the initial RP may be considered a result of test system error and the serial is satisfactory for potency.

(ii) If the RP value obtained in the original test is more than one-third the average RP obtained in the retests, a new average shall be determined using the RP values obtained in all tests. If the new average is less than 0.6, the serial is unsatisfactory.

[39 FR 16862, May 10, 1974, as amended at 40 FR 759, Jan. 3, 1975; 40 FR 20067, May 8, 1975; 40 FR 51414, Nov. 5, 1975; 44 FR 71408, Dec. 11, 1979; 50 FR 23795, June 6, 1985; 51 FR 23731, July 1, 1986. Redesignated at 55 FR 35562, Aug. 31, 1990; 56 FR 66558, Dec. 24, 1991; 56 FR 66784, 66785, Dec. 26, 1991]

§113.120   Salmonella Typhimurium Bacterin.

Salmonella Typhimurium Bacterin shall be prepared from a culture of Salmonella typhimurium which has been inactivated and is nontoxic. Each serial of biological product containing Salmonella typhimurium fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b).

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the mouse test provided in this paragraph. A mouse dose shall be 120 of the least dose recommended on the label for other animals which shall not be less than 2 ml.

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

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

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

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

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

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

eCFR graphic ec14no91.017.gif

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

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

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

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

(i) If the RP obtained in the original test is one-third or less than the average RP obtained in the retests, the initial RP may be considered a result of test system error and the serial is satisfactory.

(ii) If the RP value obtained in the original test is more than one-third the average RP obtained in the retests, a new average shall be determined using the RP values obtained in all tests. If the new average is less than the minimum required in paragraph (c)(7) of this section, the serial is unsatisfactory.

[40 FR 17003, Apr. 16, 1975, as amended at 42 FR 59487, Nov. 18, 1977; 48 FR 31008, July 6, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66785, Dec. 26, 1991]

§113.121   Pasteurella Multocida Bacterin.

Pasteurella Multocida Bacterin shall be prepared from a culture of Pasteurella multocida strains other than avian which have been inactivated and are nontoxic. Each serial of biological product containing Pasteurella multocida fraction shall meet the applicable requirements in §113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product from each serial and each subserial shall be tested for viable bacteria and fungi as provided in §113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in §113.33(b). The subcutaneous route is to be used.

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the mouse test provided in this paragraph. A mouse dose shall be 120 of the least dose recommended on the label for other animals which shall not be less than 2 ml.

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

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

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

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

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

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

eCFR graphic ec14no91.018.gif

View or download PDF

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

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

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

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

(i) If the RP obtained in the original test is one-third or less than the average RP obtained in the retests, the initial RP may be considered a result of test system error and the serial is satisfactory.

(ii) If the RP value obtained in the original test is more than one-third the average RP obtained in the retests, a new average shall be determined using the RP values obtained in all tests. If the new average is less than the minimum required in paragraph (c)(7) of this section, the serial is unsatisfactory.

[40 FR 17004, Apr. 16, 1975, as amended at 42 FR 59487, Nov. 18, 1977; 48 FR 31008, July 6, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66784, 66785, Dec. 26, 1991]

§113.122   Salmonella Choleraesuis Bacterin.

Salmonella Choleraesuis Bacterin shall be prepared from a culture of Salmonella choleraesuis which has been inactivated and is nontoxic. Each serial of biological product containing Salmonella choleraesuis fraction shall meet the applicable requirements in 9 CFR 113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in 9 CFR 113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in 9 CFR 113.33(b).

The subcutaneous route shall be used when the product is in combination with Pasteurella Multocida Bacterin.

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the mouse test provided in this paragraph. A mouse dose shall be 120 of the least dose recommended on the label for other animals which shall not be less than 2 ml.

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

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

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

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

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

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

eCFR graphic ec14no91.019.gif

View or download PDF

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

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

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

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

(i) If the RP obtained in the original test is one-third or less than the average RP obtained in the retests, the initial RP may be considered a result of test system error and the serial is satisfactory.

(ii) If the RP value obtained in the original test is more than one-third the average RP obtained in the retests, a new average shall be determined using the RP values obtained in all tests. If the new average is less than the minimum required in paragraph (c)(7) of this section, the serial is unsatisfactory.

[43 FR 25077, June 9, 1978, as amended at 48 FR 31008, July 6, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

§113.123   Salmonella Dublin Bacterin.

Salmonella Dublin Bacterin shall be prepared from a culture of Salmonella dublin which has been inactivated and is nontoxic. Each serial of biological product containing Salmonella dublin fraction shall meet the applicable requirements in 9 CFR 113.100 and shall be tested for purity, safety, and potency as prescribed in this section. A serial found unsatisfactory by any prescribed test shall not be released.

(a) Purity test. Final container samples of completed product shall be tested for viable bacteria and fungi as provided in 9 CFR 113.26.

(b) Safety test. Bulk or final container samples of completed product from each serial shall be tested for safety as provided in 9 CFR 113.33(b).

(c) Potency test. Bulk or final container samples of completed product from each serial shall be tested for potency using the mouse test provided in this paragraph. A mouse dose shall be 120 of the least dose recommended on the label for other animals which shall not be less than 2 ml.

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

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

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

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

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

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

eCFR graphic ec14no91.020.gif

View or download PDF

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

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

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

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

(i) If the RP obtained in the original test is one-third or less than the average RP obtained in the retests, the initial RP may be considered a result of test system error and the serial is satisfactory.

(ii) If the RP value obtained in the original test is more than one-third the average RP obtained in the retests, a new average shall be determined using the RP values obtained in all tests. If the new average is less than the minimum required in paragraph (c)(7) of this section, the serial is unsatisfactory.

[43 FR 25077, June 9, 1978, as amended at 48 FR 31009, July 6, 1983. Redesignated at 55 FR 35562, Aug. 31, 1990, as amended at 56 FR 66785, Dec. 26, 1991]

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