1418.65 Penicillin - streptomycin - neomycin in oil; penicillin-dihydrostreptomycin-neomycin in oil; penicillin-streptomycin-neomycin oint ment; penicillin-dihydrostrepto mycin-neomycin ointment. 141a.66 Capsules procaine penicillin in oil. 141a.67 Procaine penicillin and benzathine penicillin G in streptomycin sulfate solution; procaine penicillin and benzathine penicillin G in dihydrostreptomycin sulfate solution veterinary (procaine penicillin and benzathine penicillin G in crystalline dihydrostreptomycin sulfate solution veterinary). 141a.68 Benzathine penicillin G and streptomycin; benzathine penicillin G and dihydrostreptomycin veterinary. 141a.69 Crystalline penicillin G oral suspension, crystalline penicillin G sodium oral suspension, potassium penicillin G oral suspension. Tablets benzathine penicillin G and penicillin V. Capsules penicillin V. polymyxin in oll; procaine penicillin - dihydrostreptomycin-polymyxin in oil; procaine penicillinstreptomycin-polymyxin ointment; procaine penicillin-dihydrostreptomycin - polymyxin ointment. 141a.87 Benzathine penicillin V oral suspension; benzathine penicillin ▼ for oral suspension. 1418.88 Benzathine penicillin G in streptomycin sulfate solution veterinary; benzathine penicillin G in dihydrostreptomycin sulfate solution veterinary; potency, sterility, toxicity, pyrogens, pH. 141a.89 Procaine penicillin-neomycin-polymyxin in oil; procaine penicillinneomycin-polymyxin ointment. 141a.90 Crystalline penicillin-streptomycinpolymyxin-oxytetracycline-carbomycin powder veterinary; crystalline penicillin-dihydrostreptomy cin-polymyxin oxytetracyclinecarbomycin powder veterinary. 1418.91 Hydrabamine penicillin V (penicillin V hydrabamine salt). 141a.92 Hydrabamine penicillin V oral suspension. 141a.97 Capsules penicillin-tetracycline phosphate complex - novobiocinnystatin veterinary. 141a.98 Penicillin- streptomycin - bacitracin methylene disalicylate-neomycin ointment; penicillin-dihydrostreptomycin - bacitracin disalicylate-neomycin methylene ointment. 141a.99 Benzathine penicillin V for aqueous injection veterinary. 141a.100 Phenethicillin potassium (potassium a-phenoxyethyl penicillin). 141a.101 Phenethicillin potassium (potassium a-phenoxyethyl penicillin) tablets. 141a.102 Phenethicillin potassium (potassium a-phenoxyethyl penicillin) for oral solution. 141a.103 Methicillin sodium. 141a.104 Sodium oxacillin. 141a.105 Sodium oxacillin tablets. 141a.106 Sodium oxacillin capsules. 141a.107 Buffered methicillin sodium (buffered sodium-2, 6-dimethoxyphenyl penicillin). AUTHORITY: §§ 141a.1 to 141a.107 issued under sec. 507, 59 Stat. 463, as amended; 21 U.S.C. 357. SOURCE: §§ 141a.1 to 141a.107 appear at 27 F.R. 13001, Dec. 29, 1962. § 141a.1 Sodium penicillin, calcium penicillin, potassium pencillin; potency. (a) Cylinders (cups). Use stainless steel cylinders with an outside diameter of 8 mm. (±0.1 mm.), an inside diameter of 6 mm. (±0.1 mm.), and a length of 10 mm. (±0.1 mm.). (b) Culture media. Use ingredients that conform to the standards prescribed by the U. S. P. or N. F. (1) Make nutrient agar for the seed layer and for carrying the test organism as follows: 1.32 gm. 1,000.0 ml. dihydrogen phos Distilled water, q. 8- pH 7.0 after sterilization. In lieu of preparing the media from the individual ingredients specified in paragraph (b) (1), (2), and (3) of this section, they may be made from a dehydrated mixture which, when reconstituted with distilled water, has the same composition as such media. Minor modification of the individual ingredients specified in paragraph (b) (1), (2), and (3) of this section are permissible if the resulting media possess growth-promoting properties at least equal to the media described. the (c) Working standard. Keep working standard (obtained from the Food and Drug Administration) at room temperature in tightly stoppered vials, which in turn are kept in larger stoppered tubes containing a suitable desiccant. Weigh out carefully in an atmosphere of 50 percent relative humidity or less between 4 and 5 milligrams of the working standard and dilute with sterile 1 percent phosphate buffer (pH 6.0) to make a stock solution of any convenient concentration. Keep this solution under refrigeration and use for 2 days only. From this stock solution make appropriate working dilutions. (d) Preparation of sample. Dissolve the sample to be tested in 1.0 percent phosphate buffer, pH 6.0, to make an appropriate stock solution. (e) Preparation of plates. Add 21 ml. of agar to each Petri dish (20 x 100 millimeters). Distribute the agar evenly in the plates and allow it to harden. Use the plates the same day they are prepared. The test organism is Staphylococcus aureus (American Type Culture Collection 6538P). Maintain the test organism on agar slants and transfer to a fresh agar slant about once a week. Prepare an inoculum for the plates by transferring the culture from the agar slant into broth and incubate at 32° C.-35° C. From 16 to 24 hours thereafter add 2.0 milliliters of this broth culture to each 100 milliliters of agar which has been melted and cooled to 48° C. Mix the culture and agar thoroughly and add 4 milliliters to each of the plates containing the 21 milliliters of the uninoculated agar. Tilt the plates back and forth to spread the inoculated agar evenly over the surface. Porcelain covers glazed on the outside are used. Place four cylinders on the agar surface so that they are at approximately 90° intervals on a 2.8 cm. radius. In so placing the cylinders drop them from a height of 1⁄2 inch, using a mechanical guide or device. A suspension of the test organism may be used in place of the broth culture described above in preparing the inoculum for the seeding of plates. Prepare such suspension as follows: Wash the organisms from an agar slant which has been incubated for 24 hours at 32° C.-35° C. and stored for 24 hours at room temperature with 2.0 milliliters of sterile physiological saline onto a large agar surface such as that provided by a Roux bottle containing 300 milliliters of agar. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate 24 hours at 32° C.-35° C. and store for 24 hours at room temperature. Wash the resulting growth from the agar surface with about 50 milliliters of sterile physiological saline. Standardize this suspension by determining the dilution which will permit 20 percent light transmission through a filter at 6500 Angstrom units in a photoelectric colorimeter. (In the preparation of the suspensions of the test organism on agar slants and Roux bottles, the 24hour storage periods at room temperature may be omitted if the suspensions will permit 20-percent light transmission through a filter at 6,500 angstrom units in a photoelectric colorimeter). termine by appropriate tests the quantity of this resulting dilution to be added to each 100 milliliters of agar, which has been melted and cooled to 48° C., for the secondary layer that will give sharp, clear zones of inhibition. The suspension may be used for 1 week. De (f) Assay. Use four plates for each sample. Fill one cylinder on each plate with a 1.0 unit per milliliter dilution, and one with a 0.25 unit per milliliter dilution, of the working standard. Add the estimated dilutions of 1.0 unit per milliliter and 0.25 unit per milliliter of the sample under test to the remaining two cylinders on each plate. Carefully place the plates in racks and incubate 16 to 18 hours at 32° C.-35° C. After incubation measure the diameter of each circle of inhibition to the nearest 0.5 millimeter using a colony counter with a millimeter scale etched into the supporting glass over the light source. Other measuring devices of equal accuracy may be used. (g) Estimation of potency and error. (1) Use the accompanying chart (Chart 1) and nomograph (Chart 2) for estimating the potency and its error. To use the chart for estimating potency two values, namely, V and W, are required. For each plate calculate two values w= = (Ùμ+SH) — (uz+sz) where SH and sĩ are the diameters of the zones of inhibition in millimeters of the 1.0 unit and 0.25 unit dilutions of the standard, respectively, and uя and uz refer similarly to the corresponding dilutions of the sample under test. The value V is the sum of the v values for all plates and W is the sum of the w values for all plates. To estimate the potency locate the point on the chart corresponding to the values of V and W, and the potency can be read from the radial lines on the chart. (2) The error of the assay is estimated by using the nomograph which requires five values, namely, the potency, V, W, Rv, and Rw. Rv (the range of the v's) is the highest value of v minus the lowest value of v obtained from the individual plates. Similarly, Rw is the difference between the highest and lowest w values. After obtaining these five values, connect with a straightedge the points corresponding to v and w on the respective scales on the right side of the nomograph. Mark with a pin or sharp-pointed pencil the intersection of the straightedge and the diagonal line of the nomograph. Move the straightedge so that it connects the value of Rw on its scale and the diagonal line at the point of the pin. The value for Q is thus determined by the scale value where the straightedge crosses the line labeled "Q". T is ob tained by adding the squares of Q and Rv. On the left side of the chart connect the values of T and W with the straightedge and read the value of the ratio (error of assay-potency) where the straightedge intersects the scale of values for the ratio. This value multiplied by the potency equals the percentage error of the assay. The error of the assay calculated here estimates only how closely one assayist can check himself on any given set of dilutions of unknown and standard. It does not include any errors of weighing or errors due to variations in materials or subdivisions of a lot of penicillin. The chart for determining potency should not be used for determinations of potency lower than 50 percent or higher than 150 percent of the standard. If the potency lies outside these limits. the assay should be repeated using a higher or lower dilution. The radial lines on the chart beyond these limits permit a rough estimation of potency from as low as 5 percent to as high as 1,000 percent when low values of W are found. If the value of V or W falls outside the limits of the chart, divide both V and W by the same proper number to bring them into the range of the chart and read the potency from the radial lines as before. If 11.4 Rw is greater than W, the slope of the assay does not differ significantly from zero and the assay is invalid. (The figure 11.4 was obtained by use of Student's "t" test for determining the significance of a slope.) In certain laboratories it has been noted that with the 4 to 1 ratio, involving concentrations of 0.25 unit for the low dose, the zone of inhibition given by this dose may either be too small for accurate reading or have edges which are poorly defined. In order to permit the use of a higher concentration of penicillin for the low dose the third of the attached charts (Chart 3) may be used in assays in which the ratio of doses is 2 to 1, i. e., the high dose (SH) Is twice the low dose (sL). As in the preceding chart (Chart 1), if the potency lies outside the limits of 50 percent to 150 percent the assay should be repeated, using a lower or higher dilution. potencies beyond these limits are to be used for rough estimation purposes only. These extensions can also be used for four (or more) plate assays if both V and W are divided by the same proper number to bring them into the range of the The chart. The error of the assay using the ratio of doses 2 to 1 is estimated by using the nomograph (Chart 2) in the same manner as described for the 4 to 1 ratio of doses. However, the resultant error of the assay derived in this manner must be divided by 2 to give the correct error of the assay for the 2 to 1 ratio of doses. (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 |