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

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

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

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

(i) International antitoxin unit.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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