PENICILLIN ASSAY - Chart for Determining Potency as Percent of Standard from Two-Dose Plate Method; Ratio of Doses 4:1

L.

Doss

PENICILLIN ASSAY- Chart for Determining Potency as Percent of Standard from Two-Dose Plate Method

(h) Assay by alternative methods. The potency of the sample may also be determined by the iodometric method as described in § 141a.5(d), or by the standard-curve technique, using a single dose of standard and unknown. In the case of the standard-curve technique, dilute the sample to be tested to 1.0 unit per milliliter (estimated) in 1 percent phosphate buffer, pH 6.0. Place six cylinders on the inoculated agar surface so that they are at approximatly 60° intervals on a 2.8-centimeter radius. Use three plates for each sample and fill three cylinders on each plate with the 1.0 unit per milliliter standard and three cylinders with the 1.0 unit per milliliter (estimated) sample, alternating standard and sample. Incubate the plates for 16 to 18 hours at 32° C.-35° C. and measure the diameter of each circle of inhibition. Average the zone reading of the standard, and average the zone readings of the sample on the plates used. If the sample gives a larger average zone size than the average of the standard, add the difference between them to the 1.0 unit per milliliter zone size of the standard curve. If the average sample zone size is smaller than the standard value, subtract the difference between them from the 1.0 unit per milliliter zone size of the standard curve. From the curve read the concentration corresponding to these corrected values of zone sizes. Prepare concentrations for the standard curve by diluting aliquots of the standard stock solution with 1 percent phosphate buffer, pH 6.0, to give final concentrations of 0.64, 0.80, 1.0, 1.25, and 1.56 units per milliliter. Use three plates for each concentration except the 1.0 unit per milliliter concentration. Thus there will be a total of 12 plates. The 1.0 unit concentration is the reference point and is included on each plate. On each of three plates fill three cylinders with the 1.0 unit per milliliter standard and the other three cylinders with the concentration of the standard under test. Thus there will be thirty-six 1.0 unit per milliliter determinations and nine determinations for each of the other 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 concen

tration 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 unit per 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 penicillin, potassium penicillin; sterility.

Proceed as directed in § 141.2 of this chapter, using the method described in paragraph (e) (1) or (2) of that section, except use 180 milligrams (activity) in lieu of 500 milligrams, and, if using the method in paragraph (e) (2), use medium B in lieu of medium A.

[28 F.R. 5464, June 4, 1963]

§ 141a.3 Sodium penicillin, calcium penicillin, potassium penicillin; pyrogens.

(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 conditions. 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 tem

peratures 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 one or more times, using for each test five or more previously unused mice weighing 20 grams (±0.5 gram) each; if the total deaths within 48 hours is no greater than 10 percent of the total number of animals tested, inIcluding the original test, the sample is nontoxic.

[27 F.R. 13001, Dec. 29, 1962, as amended at 28 F.R. 5295, May 29, 1963]

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