Editorial Note on Part 113
Editorial Note: Nomenclature changes to part 113 appear at 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 ) L 0 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]