PENICILLIN ASSAY- Chart for Determining Potency

as Percent of Standard from Two-Dose Plate Method

concentrations on the curve. Incubate the plates for 16 to 18 hours at 32° C.-35° C. and measure the diameter of each circle of inhibition. Average the readings of 1.0 unit per milliliter concentration and the readings of the concentration tested on each set of three plates, and average also all 36 readings of the 1.0 unit per milliliter concentration. The average of the 36 readings of the 1.0 unit per milliliter concentration is the correction point for the curve. Correct the average value obtained for each concentration to the figure it would be if the 1.0 unit per milliliter reading for that set of three plates were the same as the correction point. Thus, if in correcting the 0.8 uniter milliliter concentration, the average of the 36 readings of the 1.0 unit per milliliter concentration is 18 millimeters and the average of the 1.0 unit per milliliter concentration on this set of three plates is 17.8 millimeters, the correction is +0.2 millimeter. If the average reading of the 0.8 unit per milliliter concentration of these same three plates is 17.0 millimeters, the corrected value is then 17.2 millimeters. Plot these corrected values including the average of the thirty-six 1.0 unit per milliliter concentrations on two-cycle semilog paper, using the concentrations in units per milliliter as the ordinate (logarithmic scale) and the diameter of the zone of inhibition as the abscissa. Draw the standard curve through these points, either by inspection or by means of the following equations:

L= calculated zone diameter for the lowest concentration of the standard curve, H= calculated zone diameter for the highest concentration of the standard curve,

c= average zone diameter of 36 readings

of the 1.0 unit per milliliter standard, a, b, d, e-corrected average values for the 0.64, 0.80, 1.25, and 1.56 units per milliliter standard solutions, respectively.

Plot the values obtained for L and H and connect with a straight line.

(i) Potency. The potency of sodium penicillin, calcium penicillin, and potassium penicillin is satisfactory when assayed by the methods described in this section if the immediate containers are represented to contain:

(1) 200,000 units or less and contain 85 percent or more of the number of units so represented;

(2) More than 200,000 units and contain 90 percent or more of the units so represented.

§ 141a.2 Sodium penicillin, calcium pen

icillin, potassium penicillin; sterility.

(a) Culture medium. In the test for bacteria, use U. S. P. fluid thioglycolate medium I or a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such medium and has growth-promoting, buffering, and oxygen-tension-controlling properties equal to or better than those of such medium. In the preparation of the medium from either the individual ingredients or any dehydrated mixture avoid contamination with calcium. In the test for molds and yeasts use U. S. P. Sabouraud Liquid Medium (Modified).

(b) Conduct of test for bacteria. Add not more than 10 milliliters of sterile distilled water, or sterile physiological salt solution, to each immediate container in the sample to be tested. From each of not less than seven immediate containers transfer aseptically the equivalent of approximately 300 milligrams, or the entire contents if the container is packaged to contain less than 300 milligrams, to individual tubes (38 x 200 millimeter size) each containing 75-100 milliliters of thioglycolate medium and sufficient penicillinase to completely inactivate the penicillin used in the test. (Prior to use, the tubes containing the medium with added penicillinase are incubated at 32° C.-35° C. for not less than 24 hours and examined for sterility. After adding the penicillin to the tubes let them stand at room temperature for 2 hours, with frequent shaking. To one of such tubes add 1.0 milliliter of a 1:1,000 dilution of an 18-24 hour broth culture of M. pyogenes var. aureus (P. C. I.-209P and American Type Culture Collection 6538P). Incubate all tubes at 32° C.-35° C. for 5 days. The batch meets the requirements of the test for bacteria if on the first or second test the control tube and no other tube shows growth, or if the number of tubes (excluding the control tubes) that show growth in three or more consecutive tests is not more than 10 percent (to compensate for contamination that may have been induced during the test) of the total number of samples tested.

(c) Conduct of test for molds and yeasts. Add not more than 10 milliliters of sterile distilled water or sterile physiological salt solution to each immediate container in the sample to be tested. From each of not less than four immediate containers transfer aseptically the equivalent of approximately 300 milligrowth in three or more consecutive tainer is packaged to contain less than 300 milligrams, to individual tubes each containing 75-100 milliliters of U. S. P. Sabouraud Liquid Medium. Incubate all tubes at approximately 25° C. for 5 days. The batch meets the requirements of the test for molds and yeasts if on the first or second test no tube shows growth. or if the number of tubes that show growth in three or more consecutive tests is not more than 10 percent (to compensate for contamination that may have been induced during the test) of the total number of samples tested. § 141a.3 Sodium penicillin, calcium penicillin, potassium penicillin; pyro

gens.

(a) Temperature recording. Use an accurate clinical thermometer or any other temperature-recording device of equal sensitivity that has been tested to determine the time necessary to reach the maximum reading. Insert the temperature-recording device into the rectum of the test animal to a depth of not less than 7.5 centimeters and allow sufficient time to reach a maximum temperature, as previously determined, before taking the reading.

(b) Test animal. Use healthy, mature rabbits, each weighing not less than 1500 grams and which have maintained their weight on an antibiotic-free diet for at least 1 week under the environmental conditions specified in this section. House the animals individually in an area of uniform temperature (±3° C. (±5° F.)) and free from disturbances likely to excite them. Do not use animals for pyrogen tests more frequently than once every 48 hours or prior to 2 weeks following their having been given a test sample that was adjudged pyrogenic. One to 3 days before using an animal that has not been used for a test during the previous 2 weeks, condition it by conducting a sham test as directed under paragraph (c) of this section, omitting the injection.

(c) Procedure. Perform the test in an area where the animals are housed or under similar environmental condi

tions. On the day of the test, withhold all food from the animals being used until after completion of the test, except that access to water may be allowed, and determine the "control temperature" of each animal. In any one test use only those animals the control temperatures of which do not deviate by more than 1°. C. from each other, and do not use any animal with a temperature exceeding 39.8° C. The control temperature recorded for each rabbit constitutes the temperature from which any subsequent rise following the injection of the material is calculated. Render the syringes, needles, and glassware free from pyrogens by heating at 250° C. for not less than 30 minutes or by any other suitable method. Warm the product to be tested to approximately 37° C. Dilute the sample with sterile, pyrogen-free distilled water to a concentration of 2,000 units per milliliter. Inject 1 milliliter per kilogram into an ear vein of each of three rabbits within 30 minutes subsequent to the control temperature reading. Record the temperature at 1, 2, and 3 hours subsequent to the injection. If no rabbit shows an individual rise in temperature of 0.6° C. or more above its respective control temperature, and if the sum of the three temperature rises does not exceed 1.4° C., the sample meets the requirements for the absence of pyrogens. If one or two rabbits show a temperature rise of 0.6° C. or more, or if the sum of the temperature rises exceeds 1.4° C., repeat the test, using five other rabbits. If not more than three of the eight rabbits show individual rises in temperature of 0.6° C. or more, and if the sum of the eight temperature rises does not exceed 3.7° C., the sample meets the requirements for the absence of pyrogens. § 141a.4 Sodium penicillin, calcium pen

icillin, potassium penicillin; toxicity. Inject intravenously each of five mice, within the weight range of 18 to 25 grams, with 0.5 milliliter of a solution of the sample prepared by diluting with sterile distilled water to approximately 4,000 units per milliliter. The injection should be made over a period of not more than 5 seconds. If no animal dies within 48 hours, the sample is nontoxic. If one or more animals die within 48 hours, repeat the test with five unused mice weighing 20 grams (±0.5 gram) each; if all animals survive the repeat test, the sample is nontoxic.

§ 141a.5 Sodium penicillin, calcium penicillin, potassium penicillin; moisture, pH, microscopical test for crystallinity.

(a) Moisture. In an atmosphere of about 10 percent relative humidity, transfer about 100 milligrams of the finely powdered sample to a tared weighing bottle or weighing tube equipped with a capillary-tube stopper, the capillary having an inside diameter of 0.20 millimeter-0.25 millimeter. Weigh the bottle or tube and place it in a vacuum oven without removing the stopper and dry at a temperature of 60° C. and a pressure of 5 millimeters of mercury or less for 3 hours. At the end of the drying period, fill the vacuum oven with air dried by passing it through a drying agent such as sulfuric acid or silica gel. Place weighing bottle or tube in a desiccator over a desiccating agent such as phosphorous pentoxide or silica gel, allow to cool to room temperature, and reweigh.

(b) pH. Dilute the sample to be tested with carbon-dioxide-free distilled water so that the resulting solution contains 30 milligrams per milliliter. Determine the pH of this solution at 25° C. using a pH meter equipped with a glass and a calomel electrode.

(c) Microscopical test for crystallinity of sodium penicillin and potassium penicillin. Mount in mineral oil and examine by means of a polarizing microscope. Crystalline penicillin shows resolvable particles which reveal the phenomena of birefringence (interference colors) and extinction positions on revolving the microscope stage. Crystalline penicillin also reveals diagnostic refractive indices when examined by the immersion method.

(d) Heat stability-(1) Crystalline penicillin, crystalline penicillin. G. Store a weighed sample (approximately 30

Units of penicillin G per milligram=

milligrams) of crystalline penicillin in an unstoppered 50-milliliter Erlenmeyer flask for 4 days in an electric oven at 100° C.±1°. At the end of this period it does not show a loss of more than 10 percent of its original potency when determined as follows: Dilute a weighed sample (approximately 30 milligrams) with a 1-percent phosphate buffer at pH 6.0 to a concentration of approximately 1.2 milligrams per milliliter (2,000 units per milliliter). Add 2.0-milliliter aliquots to each of two 125-milliliter glass-stoppered Erlenmeyer or iodine flasks. To one add 2.0 milliliters of 1 N NaOH and allow to stand at room temperature for 15 minutes. At the end of this time add 2.0 milliliters of 1.2 N HCI and add 10 milliliters of 0.01 N I (prepared from 0.1 N I, U. S. P.). (Equal volumes of 1 N NaOH and 1.2 N HCl when mixed give pH 1.0.) After 15 minutes, titrate the excess iodine, using 0.01 N Na2S2O3. Toward the end of the titration add one drop of starch solution or about 5.0 milliliters of CCl. Continue the titration by the addition of 0.01 to 0.02-milliliter portions of 0.01 N Na2S2O3, shaking vigorously after each addition. The end point is reached when the blue color of the starch-iodine complex is discharged or when the CC1 layer becomes colorless. To the second flask add 10 milliliters of 0.01 N I, and titrate immediately with 0.01 N Na2S2O, for the blank determination. Divide the difference in titers by a factor, F, which is the number of milliliters of 0.01 N I, absorbed by 1.0 milligram of sodium penicillin G working standard, to obtain the milligrams of penicillin sodium salt. Determine the factor F by actual standardization against the sodium penicillin G working standard, using the above method.

Units of penicillin O per milligram=