Lapas attēli
PDF
ePub

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

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).

as

(16) Diluent 16 (0.13M sterile pyrogen- jection of the material is calculated. If free sodium carbonate solution). Dis- the product is packaged for dispensing solve 14.0 grams of anhydrous pyrogen- and is in a combination package with a free sodium carbonate (prepared as de- container of diluent, dilute the product scribed in paragraph (a)(5) of this sec- as directed in the labeling. Warm the tion) in 1,000 milliliters pyrogen-free, product to be tested to approximately distilled water. Sterilize in an auto- 37° C. Dilute the sample with sterile, clave at 121 °C for 20 minutes.

pyrogen-free saline (prepared as de(39 FR 18944, May 30, 1974, as amended at 40

scribed in $ 436.31(b)(2)) to the approFR 51625, Nov. 6, 1975; 50 FR 48397, Nov. 25, priate concentration specified in the 1985; 53 FR 13401, Apr. 25, 1988]

individual section for each antibiotic

to be tested. Inject a test dose of 1 mil8 436.32 Pyrogen test.

liliter of the diluted sample per kilo(a) Method 1-(1) Test animal. Use gram of rabbit weight into an ear vein healthy, mature rabbits weighing not of each of three rabbits within 30 minless than 1,800 grams each that have utes subsequent to the control temmaintained their weight on an anti- perature reading. Record the temperabiotic-free diet for at least 1 week ture at 1, 2, and 3 hours subsequent to under the environmental conditions the injection. specified in this section. House the ani

(3) Evaluation. If no rabbit shows an mals individually in an area of uniform individual rise in temperature of 0.6° C. temperature (+3° C.) and free from dis

or more above its respective control turbances likely to excite them. Do not

temperature, and if the sum of the use animals for pyrogen tests more fre

three temperature rises does not exquently than once every 48 hours or

ceed 1.4° C., the sample meets the reprior to 2 weeks following their having

quirements for the absence of been given a test sample that was ad

pyrogens. If one or two rabbits show a judged pyrogenic. Before using an ani

temperature rise of 0.6° C. or more, or mal that has not been used for a test

if the sum of the temperature rises exduring the previous 2 weeks, condition

ceeds 1.4° C., repeat the test using five it 1 to 3 days prior to pyrogen testing

other rabbits. If not more than three of by conducting a sham test as directed

the eight rabbits show individual rises in paragraph (a)(2) of this section,

in temperature of 0.6° C. or more, and if omitting the injection.

the sum of the eight temperature rises (2) Procedure. Using equipment and

does not exceed 3.7° C., the sample diluents described in $ 436.31, as nec

meets the requirements for the absence essary, perform the test in an area

of pyrogens. where the animals are housed or under

(b) Method 2. Proceed as directed in similar environmental conditions. On

paragraph (a) of this section, except dithe day of the test: Withhold all food

lute the sample with pyrogen-free from the animals being used until after

water (diluent 1). completion of the test, except that access to water may be allowed; and de

(c) Method 3. Proceed as directed in termine the “control temperature" of

paragraph (a) of this section, except dieach animal by inserting the tempera

lute the sample with pyrogen-free ture-measuring device into the rectum

water (diluent 1) and inject a test dose of the test animal to a depth of not less

of 2.0 milliliters of the diluted sample than 7.5 centimeters and allowing suffi

per kilogram of rabbit weight. cient time to reach a maximum tem- (d) Method 4. Proceed as directed in perature, as previously determined, be- paragraph (a) of this section, except infore taking the reading. In any one test ject a test dose of 0.5 milliliter of the use only those animals whose control diluted sample per kilogram of rabbit temperatures do not deviate by more weight. than 1° C. from each other and do not (e) Method 5. Proceed as directed in use any animal with a temperature ex- paragraph (a) of this section, except diceeding 39.8° C. The control tempera- lute the sample with pyrogen-free ture recorded for each rabbit con- water (diluent 1) and inject a test dose stitutes the temperature from which of 0.5 milliliter of the diluted sample any subsequent rise following the in- per kilogram of rabbit weight.

[graphic][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed][subsumed]

(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.

(1) 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).

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.

5To 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

[blocks in formation]

$ 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 (10.1 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

[graphic]

absorbance with clear,

zero

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

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

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

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.

8 436.101 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).

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. (ap

proximately). 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).

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).

Monobasic potassium phosphate: 100.0 gm. Distilled water, q.3: 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).

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).

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.

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

Prepare daily.

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

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).

Isopropyl alcohol: 800.0 ml.
Distilled water, a.s: 1,000.0 ml.

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

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

Dibasic potassium phosphate: 13.6 gm. Monobasic potassium phosphate: 4.0 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).

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).

Dibasic potassium phosphate: 20.0 gm.

[blocks in formation]

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.

8 436.102 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.

(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:

Agar: 12.0 gm.
Polysorbate 80 (add polysorbate 80 after boil-

ing the medium to dissolve the agar): 10.0

ml. pH 7.2 to 7.3 after sterilization.

(11) Medium 11. Medium 11 is the same as medium 1, except adjust the final pH to 7.8 to 8.0 after sterilization.

(12) [Reserved] (13) Medium 13.

Peptone: 6.0 gm.
Pancreatic digest of casein: 4.0 gm.
Yeast extract: 3.0 gm.
Beef extract: 1.5 gm.

Peptone: 10.0 gm.

« iepriekšējāTurpināt »