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thermometer or any other temperature-measuring device of equal sensitivity that has been tested to determine the time necessary to reach the maximum reading.

(2) Pyrogen-free glassware. Render all glassware free from pyrogens by heating at 250° C. for not less than 30 minutes or by any other suitable method.

(3) Pyrogen-free syringes and needles. Render all syringes and needles free from pyrogens by heating at 250° C. for not less than 30 minutes or by any other suitable method.

(4) Pyrogen-free sodium chloride. Heat sodium chloride for not less than 2 hours at 200° C.

(5) Pyrogen-free sodium carbonate. Heat anhydrous sodium carbonate for not less than 4 hours at 170° C.

(b) Diluents. (1) Diluent 1 (pyrogenfree water): Prepare pyrogen-free water by collecting freshly distilled water and sterilizing it in an autoclave at 121° C. for not less than 20 minutes. Pyrogen-free water meets the requirements for the absence of pyrogens as described in §436.32(a)(3) when 10 milliliters per kilogram are administered as described in §436.32(a)(2). In testing water for the absence of pyrogens, the aliquot to be tested is made isotonic by the addition of pyrogen-free sodium chloride.

(2) Diluent 2 (pyrogen-free saline solution): Prepare an isotonic solution of sodium chloride by dissolving 9.0 grams of pyrogen-free sodium chloride (prepared as described in § 436.31(a)(4)) in pyrogen-free, distilled water (diluent 1) to make 1,000 milliliters. Sterilize in an autoclave at 121° C. for not less than 20 minutes. Pyrogen-free saline solution meets the requirements for the absence of pyrogens as described in § 436.32(a)(3) when 10 milliliters per kilogram are administered as described in § 436.32(a)(2).

(3) Diluent 3 (sterile distilled water): Prepare freshly distilled water. Sterilize in an autoclave at 121° C. for 20 minutes.

(4) Diluent 4 (sterile saline solution): Dissolve 9.0 grams of sodium chloride in distilled water to make 1,000 milliliters. Sterilize in an autoclave at 121° C. for 20 minutes.

(5) Diluent 5 (10 percent gum acacia): Dissolve 10 grams of gum acacia in ap

proximately 50 milliliters of distilled water. Allow to stand overnight at room temperature and dilute to 100 milliliters with distilled water. Filter through cotton. Store under refrigeration.

(6) Diluent 6 (0.5 percent gum acacia in distilled water). 11132

(7) Diluent 7 (1.0N hydrochloric acid). (8) Diluent 8 (0.1N hydrochloric acid). (9) Diluent 9 (0.05N sodium hydroxide).

(10) Diluent 10 (1 percent U.S.P. methylcellulose (4,000 centipoises) solution): Dissolve 1 gram of U.S.P. methylcellulose (4,000 centipoises) in 100 milliliters of distilled water. Allow to stand overnight at room temperature or until solution is complete. Store under refrigeration.

(11) Diluent 11 (0.12N sodium hydroxide).

(12) Diluent 12 (0.5 percent methylcellulose (4,000 centipoises) in distilled water). Proceed as directed in paragraph (b)(10) of this section, except use 0.5 gram of methylcellulose (4,000 centipoises).

(13) Diluent 13 (pyrogen-free sodium carbonate solution). Dissolve 25.6 grams of anhydrous pyrogen-free sodium carbonate (prepared as described in paragraph (a)(5) of this section) in 1,000 milliliters pyrogen-free, distilled water (diluent 1). Pyrogen-free, sodium carbonate solution meets the requirements for the absence of pyrogens as described in § 436.32(a)(3) when 1.0 milliliter per kilogram is administered as described in § 436.32(a)(2).

(14) Diluent 14 (0.07M sterile sodium carbonate solution). Dissolve 7.3 grams of sodium carbonate in distilled water to make 1,000 milliliters. Sterilize in an autoclave at 121° C. for 20 minutes.

(15) Diluent 15 (pyrogen-free sodium carbonate solution): Dissolve 9.9 grams of anhydrous pyrogen-free sodium carbonate (prepared as directed in paragraph (a)(5) of this section) in 1,000 milliliters of pyrogen-free, distilled water (diluent 1). Pyrogen-free sodium carbonate solution meets the requirements for the absence of pyrogens as described in § 436.32(a)(3) when 1.0 milliliter per kilogram is administered as described in § 436.32(a)(2).

(16) Diluent 16 (0.13M sterile pyrogenfree sodium carbonate solution). Dissolve 14.0 grams of anhydrous pyrogenfree sodium carbonate (prepared as described in paragraph (a)(5) of this section) in 1,000 milliliters pyrogen-free, distilled water. Sterilize in an autoclave at 121 °C for 20 minutes.

[39 FR 18944, May 30, 1974, as amended at 40 FR 51625, Nov. 6, 1975; 50 FR 48397, Nov. 25, 1985; 53 FR 13401, Apr. 25, 1988]

§ 436.32 Pyrogen test.

(a) Method 1-(1) Test animal. Use healthy, mature rabbits weighing not less than 1,800 grams each that 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.) 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. Before using an animal that has not been used for a test during the previous 2 weeks, condition it 1 to 3 days prior to pyrogen testing by conducting a sham test as directed in paragraph (a)(2) of this section, omitting the injection.

(2) Procedure. Using equipment and diluents described in §436.31, as necessary, 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 by inserting the temperature-measuring device into the rectum of the test animal to a depth of not less than 7.5 centimeters and allowing sufficient time to reach a maximum temperature, as previously determined, before taking the reading. In any one test use only those animals whose control temperatures 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 in

jection of the material is calculated. If the product is packaged for dispensing and is in a combination package with a container of diluent, dilute the product as directed in the labeling. Warm the product to be tested to approximately 37° C. Dilute the sample with sterile, pyrogen-free saline (prepared as described in §436.31(b)(2)) to the appropriate concentration specified in the individual section for each antibiotic to be tested. Inject a test dose of 1 milliliter of the diluted sample per kilogram of rabbit weight 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.

(3) Evaluation. 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.

(b) Method 2. Proceed as directed in paragraph (a) of this section, except dilute the sample with pyrogen-free water (diluent 1).

(c) Method 3. Proceed as directed in paragraph (a) of this section, except dilute the sample with pyrogen-free water (diluent 1) and inject a test dose of 2.0 milliliters of the diluted sample per kilogram of rabbit weight.

(d) Method 4. Proceed as directed in paragraph (a) of this section, except inject a test dose of 0.5 milliliter of the diluted sample per kilogram of rabbit weight.

(e) Method 5. Proceed as directed in paragraph (a) of this section, except dilute the sample with pyrogen-free water (diluent 1) and inject a test dose of 0.5 milliliter of the diluted sample per kilogram of rabbit weight.

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(f) Method 6. Proceed as directed in paragraph (a) of this section, except dilute sample with 0.05N sodium hydroxide (diluent 9).

(g) Method 7. Proceed as directed in paragraph (a) of this section, except dilute sample with sodium carbonate solution (diluent 13).

(h) Method 8. Proceed as directed in paragraph (a) of this section, except inject a test dose of 2.0 milliliters of the diluted sample per kilogram of rabbit weight.

(i) Method 9. Proceed as directed in paragraph (a) of this section, except dilute sample with pyrogen-free sodium carbonate solution (diluent 15).

(j) Method 10. Proceed as directed in paragraph (a) of this section, except dilute the sample with sodium carbonate solution (diluent 16).

[39 FR 18944, May 30, 1974, as amended at 40 FR 51625, Nov. 6, 1975; 45 FR 22921, Apr. 4, 1980; 50 FR 48397, Nov. 25, 1985; 53 FR 13401, Apr. 25, 1988]

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1 Diluent number as listed in sec. 436.31(b).
2 Milligrams of activity per milliliter.
3 Milliliters per kilogram of body weight.

4 The concentration of the test solution is expressed in units per milliliter in lieu of milligrams of activity per milliliter.

5 To prepare the test solution, proceed as directed in the individual section of the antibiotic drug regulation in this chapter for the antibiotic to be tested.

[46 FR 60568, Dec. 11, 1981, as amended at 46 FR 61071, Dec. 15, 1981; 49 FR 5096, Feb. 10, 1984]

Subpart D-Microbiological Assay Methods

§ 436.100 Laboratory equipment.

Equipment should be selected which is adequate for its intended use and should be thoroughly cleansed after each use to remove any antibiotic residues. The equipment should be kept covered when not in use. Clean glassware intended for holding and transferring the test organisms should be sterilized in a hot air oven at 200-220° C. for 2 hours. Volumetric flasks, pipettes, or accurately calibrated diluting devices should be used when diluting standard and sample solutions.

(a) Microbiological agar diffusion assay-(1) Cylinders. Use stainless steel cylinders with an outside diameter of 8 millimeters (±0.1 millimeter), an inside diameter of 6 millimeters (±0.1 millimeter), and a length of 10 millimeters (±0.1 millimeter).

(2) Plates. Plastic or glass Petri dishes may be used, having dimensions of 20 by 100 millimeters. Covers should be of suitable material.

(b) Microbiological turbidimetric assay-(1) Tubes. Tubes which give satisfactory results and have uniform length and diameter should be used. If reusable tubes are employed, care must be taken to remove not only all antibiotic residues from the previous test but also all traces of cleaning solution. (2) Colorimeter. Use a suitable photoelectric colorimeter at a wavelength of 530 millimicrons. Set the instrument at zero clear,

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absorbance

with

uninoculated broth prepared as described in the applicable method for the antibiotic being assayed.

[39 FR 18944, May 30, 1974, as amended at 41 FR 34743, Aug. 17, 1976]

§ 436.101 Solutions.

(a) Antibiotic assay solutions are prepared as follows (solution numbers 1, 2, 3, 4, and 6 correspond to those used in "Assay Methods of Antibiotics," D. C. Grove and W. A. Randall, Medical Encyclopedia, Inc., New York, N.Y. (1955), p. 222), which is incorporated by reference. Copies are available from the Medical Encyclopedia Inc., 30 East 60th St., New York, NY 11220, or available for inspection at the Office of the Federal Register, 800 North Capitol Street, NW., suite 700, Washington, DC. (1) Solution 1 (1 percent potassium phosphate buffer, pH 6.0).

Dibasic potassium phosphate: 2.0 gm.
Monobasic potassium phosphate: 8.0 gm.
Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 5.95 to 6.05 after sterilization.

(2) Solution 2 (citrate buffer solution pH 6.3).

Citric acid: 13.2 gm.

Sodium hydroxide: 7.06 gm.
Sodium citrate: 97.0 gm.

Distilled water, q.s: 1,000.0 ml.

Adjust with 10 percent citric acid solution or 10N sodium hydroxide to yield pH 6.2 to 6.4 after sterilization.

(3) Solution 3 (0.1M potassium phosphate buffer, pH 8.0).

Dibasic potassium phosphate: 16.73 gm.
Monobasic potassium phosphate: 0.523 gm.
Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 7.9 to 8.1 after sterilization.

(4) Solution 4(0.1M potassium phosphate buffer, pH 4.5).

Monobasic potassium phosphate: 13.6 gm.
Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 4.45 to 4.55 after sterilization.

(5) [Reserved]

(6) Solution 6 (10 percent potassium phosphate buffer, pH 6.0).

Dibasic potassium phosphate: 20.0 gm.

Monobasic potassium phosphate: 80.0 gm.
Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 5.95 to 6.05 after sterilization.

(7)-(9) [Reserved]

(10) Solution 10 (0.2M potassium phosphate buffer, pH 10.5).

Dibasic potassium phosphate: 35.0 gm. 10 N potassium hydroxide: 2.0 ml. Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 10.4 to 10.6 after sterilization.

(11) Solution 11 (10 percent potassium phosphate buffer, pH 2.5).

Monobasic potassium phosphate: 100.0 gm. Concentrated hydrochloric acid: 0.2 ml. (approximately).

Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 2.0 to 2.8 after sterilization.

(12) Solution 12 (10 percent potassium phosphate buffer, pH 7.0).

Monobasic potassium phosphate: 100.0 gm.
Distilled water, q.s: 1,000.0 ml.

Adjust with 18N phosphoric acid or 10N potassium hydroxide to yield a pH 6.95 to 7.05 after sterilization.

(13) Solution 13 (0.01N methanolic hydrochloric acid).

1.0N hydrochloric acid: 10.0 ml. Methyl alcohol, q.s: 1,000.0 ml.

(14) Solution 14 (2 percent sodium bicarbonate solution).

Sodium bicarbonate: 20.0 gm.
Distilled water, q.s: 1,000.0 ml.

Prepare daily.

(15) Solution 15 (80 percent isopropyl alcohol solution).

Isopropyl alcohol: 800.0 ml.

Distilled water, q.s: 1,000.0 ml.

(16) Solution 16 (0.1 M potassium phosphate buffer, pH 7.0).

Dibasic potassium phosphate: 13.6 gm.
Monobasic potassium phosphate: 4.0 gm.
Distilled water, q.s.: 1,000.0 ml.

Adjust with 18 N phosphoric acid or 10 N potassium hydroxide to yield a pH 6.8 to 7.2 after sterilization.

(17) Solution 17 (5 percent methyl alcohol in 1 percent potassium phosphate buffer, pH 6.0).

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§ 436.102 Culture media.

(a) Ingredients. Use ingredients that conform to the standards, if any, prescribed by the U.S.P. or N.F. In lieu of preparing the media from the individual ingredients specified, they may be made from dehydrated mixtures that, when reconstituted with distilled water, have the same composition as such media. Minor modifications of the individual ingredients specified in this section are permissible if the resulting media possess growth-promoting properties at least equal to the media described.

(b) Description of media. Medium numbers 1, 2, 3, 4, 5, 8, 9, 10, 11, and 13 correspond to those used in "Assay Methods of Antibiotics," D. C. Grove and W. A. Randall, Medical Encyclopedia, Inc., New York, N.Y. (1955) p. 220, which is incorporated by reference. Copies are available from Medical Encyclopedia Inc., 30 East 60th St., New York, NY, or available for inspection at the Office of the Federal Register, 800 North Capitol Street, NW., suite 700, Washington, DC. Medium numbers 18 through 21 correspond to those used in "Outline of Details for Official Microbiological Assays of Antibiotics," A. Kirshbaum and B. Arret, "Journal of Pharmaceutical Sciences," vol. 56, No. 4, April 1967, p. 512, which is incorporated by reference. Copies are available from the American Pharmaceutical Association, 2215 Constitution Ave. NW., Washington, DC 20037, or available for inspection at the Office of the Federal Register (see address in this paragraph).

(1) Medium 1.

Peptone: 6.0 gm.

Pancreatic digest of casein: 4.0 gm. Yeast extract: 3.0 gm.

Beef extract: 1.5 gm.

Dextrose: 1.0 gm.

Agar: 15.0 gm.

Distilled water, q.s: 1,000.0 ml. pH 6.5 to 6.6 after sterilization.

(2) Medium 2.

Peptone: 6.0 gm.

Yeast extract: 3.0 gm.
Beef extract: 1.5 gm.
Agar: 15.0 gm.

Distilled water, q.s: 1,000.0 ml.
pH 6.5 to 6.6 after sterilization.

(3) Medium 3. Peptone: 5.0 gm.

Yeast extract: 1.5 gm.
Beef extract: 1.5 gm.
Sodium chloride: 3.5 gm.
Dextrose: 1.0 gm.

Dipotassium phosphate: 3.68 gm.

Potassium dihydrogen phosphate: 1.32 gm. Distilled water, q.s: 1,000.0 ml. pH 6.95 to 7.05 after sterilization.

(4) Medium 4. Peptone: 6.0 gm.

Yeast extract: 3.0 gm.
Beef extract: 1.5 gm.
Dextrose: 1.0 gm.
Agar: 15.0 gm.

Distilled water, q.s: 1,000.0 ml.
pH 6.5 to 6.6 after sterilization.

(5) Medium 5. Medium 5 is the same as medium 2, except adjust the final pH to 7.8 to 8.0 after sterilization.

(6)–(7) [Reserved]

(8) Medium 8. Medium 8 is the same as medium 2, except adjust the final pH to 5.8 to 6.0 after sterilization. (9) Medium 9.

Pancreatic digest of casein: 17.0 gm.
Papaic digest of soybean: 3.0 gm.
Sodium chloride: 5.0 gm.
Dipotassium phosphate: 2.5 gm.
Dextrose: 2.5 gm.
Agar: 20.0 gm.

Distilled water, q.s: 1,000.0 ml.
pH 7.2 to 7.3 after sterilization.

(10) Medium 10. Medium 10 is the same as medium 9, except:

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