day and on the last day of the test period. Results of each examination shall be recorded. If the inoculum renders the medium turbid so that the absence of growth cannot be determined reliably by visual examination, portions of this turbid medium in amounts of no less than 1.0 ml. shall be transferred on the third, fourth, or fifth day of incubation, from each of the test vessels and inoculated into additional vessels of medium. The material in the additional vessels shall be incubated at a temperature of 30° to 32° C. for no less than 14 days. Notwithstanding such transfer of material, examination of the original vessels shall be continued as prescribed above. The additional test vessels shall be examined visually for evidence of growth on the third, fourth, or fifth day of incubation and on the seventh or eighth day and on the last day of the incubation period. If growth appears, repeat tests may be performed as prescribed in paragraph (b) of this section and interpreted as specified in paragraph (c) of this section.

(li) Final container material containing a mercurial preservative. In addition to the test prescribed in subparagraph (1) (i) of this paragraph, final container material containing a mercurial preservative shall be tested using Fluid Thioglycollate Medium following the procedures prescribed in such subparagraph, except that the incubation shall be at a temperature of 20° to 25° C.

(2) Using Soybean-Casein Digest Medium. Except for products containing a mercurial preservative, a test shall be made on final container material, following the procedures prescribed in subparagraph (1)(1) of this paragraph, except that the medium shall be Soybean-Casein Digest Medium and the incubation shall be at a temperature of 20° to 25° C.

(b) Repeat tests-(1) Repeat bulk test. If growth appears in the test of the bulk material, the test may be repeated to rule out faulty test procedures by testing at least the same volume of material.

(2) First repeat final container test. If growth appears in any test (Fluid Thioglycollate Medium or SoybeanCasein Digest Medium) of final container material, the test may be repeated to rule out faulty test procedures by testing material from a sample of at

(3) Second repeat final container test. If growth appears in any first repeat final container test (Fluid Thioglycollate Medium or Soybean-Casein Digest Medium), that test may be repeated provided there was no evidence of growth in any test of the bulk material and material from a sample of twice the number of final containers used in the first test is tested by the same method used in the first test.

(c) Interpretation of test results. The results of all tests performed on a lot shall be considered in determining whether or not the lot meets the requirements for sterility, except that tests may be excluded when demonstrated by adequate controls to be invalid. The lot meets the test requirements if no growth appears in the tests prescribed in paragraph (a) of this section. If repeat tests are performed, the lot meets the test requirements if no growth appears in the tests prescribed in paragraph (b) (2) or (3) of this section, whichever is applicable.

(d) Test samples and volumes-(1) Bulk. Each sample for the bulk sterility test shall be representative of the bulk material and the volume tested shall be no less than 10 ml. (Note exceptions in paragraph (g) of this section.)

(2) Final containers. The sample for the final container and first repeat final container test shall be no less than 20 final containers from each filling of each lot, selected to represent all stages of filling from the bulk vessel. If the amount of material in the final container is 1.0 ml. or less, the entire contents shall be tested. If the amount of material in the final container is more than 1.0 ml., the volume tested shall be the largest single dose recommended by the manufacturer or 1.0 ml., whichever is larger, but no more than 10 ml. of material or the entire contents from a single final container need be tested. If more than two filling machines, each with either single or multiple filling stations, are used for filling one lot, no less than 10 filled containers shall be tested from each filling machine, but no more than 100 containers of each lot need be tested. The items tested shall be representative of each filling assembly and shall be selected to represent all stages of the filling operation. (Note exceptions in paragraph (g) of this section.)

(2) Culture media requirements—(i) Growth promoting qualities. Each lot of dehydrated medium bearing the manufacturer's identifying number, or each lot of medium prepared from basic ingredients, shall be tested for its growthpromoting qualities using not more than 100 organisms of two or more strains of microorganisms that are exacting in their nutritive and aerobic-anaerobic requirements.

(ii) Conditions of medium and design of test vessels. A medium shall not be used if the extent of evaporation affects its fluidity, nor shall it be reused in a sterility test. Fluid Thioglycollate Medium shall not be used if more than the upper one-third has acquired a pink color. The medium may be restored once by heating on a steam bath or in freeflowing steam until the pink color disappears. The design of the test vessel for Fluid Thioglycollate Medium shall be such as is shown to provide favorable aerobic and anaerobic growth of microorganisms throughout the test period.

(iii) Ratio of the inoculum to culture medium. The ratio of the inoculum to the volume of the culture medium resulting in a dilution of the product that is not bacteriostatic or fungistatic shall be determined for each product, except for those tested by membrane filtration. Vessels of the product-medium mixture(s)

and control vessels of the medium shall be inoculated with dilutions of cultures of bacteria or fungi which are sensitive to the product being tested, and incubated at the appropriate temperature for no less than 7 days. Inhibitors or neutralizers of preservatives may be considered in determining the proper ratio.

1

(f) Membrane filtration. Bulk and final container material of products containing oil or products in water insoluble ointments shall be tested for sterility using the membrane filtration procedure set forth in The United States Pharmacopeia (18th Revision, 1970), section entitled "Membrane Filtration," pages 853854, except that (1) the test samples shall conform with paragraph (d) of this section, (2) the temperature of incubation for the test using Fluid Thioglycollate Medium shall be 30° to 32° C. and (3) in addition, for products containing a mercurial preservative, the product shall be tested in a second test using Fluid Thioglycollate Medium incubated at 20° to 25° C. in lieu of the test in Soybean-Casein Digest Medium. Such Membrane Filtration section is hereby incorporated by reference and deemed published herein. The United States Pharmacopeia is available at most medical and public libraries and copies of the pertinent section will be provided to any manufacturer affected by the provisions of this subchapter upon request to the Director, Bureau of Biologics or the appropriate Information Center Offices listed in 45 CFR Part 5. In addition, an official historic file of the material incorporated by reference is maintained in the office of the Director, Bureau of Biologics.

(g) Exceptions. Bulk and final container material shall be tested for sterility as described above in this section, except as follows:

(1) Different sterility tests prescribed. When different sterility tests are prescribed for a product in this subchapter.

(2) Alternate incubation temperatures. Two tests may be performed, in all respects as prescribed in paragraph (a) (1) (i) of this section, one test using an incubation temperature of 18° to 22° C., the other test using an incubation temperature of 35° to 37° C., in lieu of performing one test using an incubation temperature of 30° to 32° C.

1 1 Coples may be obtained from: United States Pharmacopeial Convention, Inc., 12601 Twinbrook Parkway, Rockville, MD 20852.

(3) Different tests equal or superior. A different test (such as membrane filltration as set forth in paragraph (f) of this section) may be performed provided that prior to the performance of such test a manufacturer submits data which the Commissioner of Food and Drugs, finds adequate to establish that the different test is equal or superior to the tests described in paragraphs (a) and (b) of this section in detecting contamination and makes the finding a matter of official record.

(4) Test precluded or not required. The tests prescribed in this section need not be performed for Whole Blood (Human), Cryoprecipitated Antihemophilic Factor (Human), Platelet Concentrate (Human), Leukocyte Typing Serum, Red Blood Cells (Human), Single Donor Plasma (Human), Source Plasma (Human), Smallpox Vaccine and other similar products concerning which the Commissioner of Food and Drugs, finds that the mode of administration, the method of preparation or the special nature of the product precludes or does not require a sterility test.

(5) Viscid or turbid products. Alternative Thioglycollate Medium may be used in place of Fluid Thioglycollate Medium for the testing of products that are viscid or turbid or otherwise do not lend themselves to culturing in Fluid Thioglycollate Medium, provided it has been freshly prepared or has been heated on a steam bath or in free-flowing steam and cooled just prior to use and is used in a suitable vessel that will maintain aerobic and anerobic conditions throughout the incubation period. The formula for the Alternative Thioglycollate Medium follows:

ALTERNATIVE THIOGLYCOLLATE MEDIUM

in a filling is 20 or less, the sample shall be two final containers, or the sample need be no more than one final container, provided (i) the bulk material met the sterility test requirements and (ii) after filling, it is demonstrated by testing a simulated sample that all surfaces to which the product was exposed were free of contaminating microorganisms. The simulated sample shall be prepared by rinsing the filling equipment with sterile 1.0 percent peptone solution, pH 7.1±0.1, which shall be discharged into a final container by the same method used for filling the final containers with the product.

(8) Samples—large volume of product in final containers. For Normal Serum Albumin (Human), Normal Human Plasma, Antihemophilic Plasma (Human), Plasma Protein. Fraction (Human) and Fibrinogen (Human) when the volume of product in the finai container is 50 ml. or more, the final containers selected as the test sample may contain less than the full volume of product in the final containers of the filling from which the sample is taken: Provided, That the containers and closures of the sample are identical with those used for the filling to which the test applies and the sample represents all stages of that filling.

(9) Diagnostic products not intended for injection. For diagnostic products not intended for injection, (i) only the Thioglycollate Medium test incubated at 30° to 32° C. is required, (ii) the volume of material for the bulk test shall be no less than 2.0 ml., and (iii) the sample for the final container test shall be no less than three final containers if the total number filled is 100 or less, and, if greater, one additional container for each additional 50 containers or fraction thereof, but the sample need be no more than 10 containers.

(10) Immune globulin preparations. For immune globulin preparations, the test samples from the bulk material and from each final container need be no more than 2.0 ml.

[38 FR 32056, Nov. 20, 1973, as amended at 40 FR 4304, Jan. 29, 1975] § 610.13

Purity.

Products shall be free from extraneous material except for unavoidable bacteriophage. In addition, products shall be tested as provided in paragraphs (a) and (b) of this section.

(a) Test for residual moisture. Each lot of dried product shall be tested for residual moisture and other volatile substances.

(1) Procedure. The test for dried products shall consist of measuring the maximum loss of weight in a weighed sample equilibrated over anhydrous P,O. at a pressure of not more than one mm of mercury, and at a temperature of 20° to 30° C. for as long as it has been established is sufficient to result in a constant weight.

(2) Test results; standard to be met. The residual moisture and other volatile substances shall not exceed 1 percent except that for BCG Vaccine they shall not exceed 1.5 percent, for Measles Virus Vaccine, Live, Attenuated; MeaslesSmallpox Vaccine, Live; Rubella Virus Vaccine, Live; and Antihemophilic Factor (Human), they shall not exceed 2 percent; for Modified Plasma (Bovine); Thrombin; Fibrinogen; Streptokinase; Streptokinase-Streptodornase; and AntiInfluenza Virus Serum for the Hemagglutination Inhibition Test, they shall not exceed 3 percent; and for Hepatitis Associated Antibody (Anti-Australia Antigen) for the Reversed Passive Hemagglutination Test, they shall not exceed 4.5 percent.

(b) Test for pyrogenic substances. Each lot of any product intended for use by injection shall be tested for pyrogenic substances by intravenous injection into rabbits as provided in subparagraphs (1) and (2) of this paragraph: Provided, That notwithstanding any other provision of this subchapter, the test for pyrogenic substances is not required for the following products: Products containing formed blood elements; Cryoprecipitated Antihemophilic Factor (Human); Single Donor Plasma (Human); Source Plasma (Human); Normal Horse Serum; Normal Rabbit Serum; bacterial viral and rickettsial vaccines and antigens; toxolds; toxins, allergenic extracts; venoms; diagnostic substances and trivalent organic arsenicals.

(1) Test dose. The test dose for each rabbit shall be at least 3 milliliters per kilogram of body weight of the rabbit and also shall be at least equivalent proportionately, on a body weight basis, to the maximum single human dose recommended, but need not exceed 10 ml. per kilogram of body weight of the rabbit, except that: (1) Regardless of the human dose recommended, the test dose per kilogram of body weight of each rab

bit shall be, at least 1 milliliter for immune globulins derived from human blood, at least 3 milliliters for Normal Human Plasma, and at least 30 milligrams for Fibrinogen (Human); (ii) for Streptokinase, Streptokinase-Streptodornase, Aggregrated Radio-Iodinated (I) Albumin (Human), Radio-Chromater (Cr) Serum Albumin (Human), Radio-Iodinated (1) Serum Albumin (Human) and Radio-Iodinated (I) Serum Albumin (Human), the test dose shall be at least equivalent proportionately on a body weight basis to the maximum single human dose recommended.

(2) Procedure. Products shall be tested for freedom from pyrogenic substances by intraveneous injection of the test dose into three or more rabbits in overt good health and by recording for each rabbit a control temperature taken within one hour prior to injection, and three additional temperatures taken one, two, and three hours after injection. For purposes of subparagraph (3) of this paragraph, if there is no temperature increase over the control temperature (1.e. where the temperature remains unchanged or falls), the temperature rise shall be considered as zero. If there is an increase in temperature over the control temperature, the temperature rise shall be the difference between the highest of the three hourly readings and the control temperature reading.

(3) Test results; standards to be met. The results recorded for all rabbits used In all tests of a lot of a product shall be included in determining whether the standard for purity is met. The product fails to meet test requirements if onehalf or more of all rabbits show a temperature rise of 0.6° C. or more or if the average temperature rise of all rabbits is 0.5° C. or more.

(c) Different tests equal or superior A different test for residual moisture may be performed provided that prior to its performance the manufacturer submits data which the Commissioner of Food and Drugs finds adequate to establish that the different test is equal or superior to the test described in paragraph (a) of this section and makes the finding a matter of official record.

[38 FR 32056, Nov. 20, 1973, as amended at 39 FR 9660, Mar. 13, 1974] § 610.14

Identity.

The contents of a final container of each filling of each lot shall be tested

for identity after all labeling operations shall have been completed. The identity test shall be specific for each product in a manner that will adequately identify it as the product designated on final container and package labels and circulars, and distinguish it from any other product being processed in the same laboratory. Identity may be established either through the physical or chemical characteristics of the product, inspection by macroscopic or microscopic methods, specific cultural tests, or in vitro or in vivo immunological tests.

§ 610.15 Constituent materials.

(a) Ingredients, preservative, diluents, adjuvants. All ingredients used in a licensed product, and any diluent provided as an aid in the administration of the product, shall meet generally accepted standards of purity and quality. Any preservative used shall be sufficiently nontoxic so that the amount present in the recommended dose of the product will not be toxic to the recipient, and in the combination used shall not denature the specific substances in the product below the minimum acceptable potency within the dating period when stored at the recommended temperature. Products in multiple dose containers shall contain a preservative, except that a preservative need not be added to Yellow Fever Vaccine, Poliovirus Vaccine, Live, Oral, or to viral vaccines labeled for use with the jet injector, or to dried vaccines when the accompanying diluent contains a preservative. An adjuvant shall not be introduced into a product unless there is satisfactory evidence that it does not affect adversely the safety or potency of the product. In no event shall the recommended individual dose of a biological product contain more than 0.85 milligram of aluminum, determined by assay, or more than 1.14 milligrams of aluminum, determined by calculation on the basis of the amount of aluminum compound added.

(b) Extraneous protein; cell culture produced vaccines. Extraneous protein known to be capable of producing allergenic effects in human subjects shall not be added to a final virus medium of cell culture produced vaccines intended for injection. If serum is used at any stage, its calculated concentration in the final medium shall not exceed 1:1,000,000.

(c) Antibiotics. A minimum concentration of antibiotics, other than penicillin,

may be added to the production substrate of viral vaccines.

§ 610.16 Total solids in serums.

Except as otherwise provided by regulation, no liquid serum or antitoxin shall contain more than 20 percent total solids. § 610.17 Permissible combinations.

Licensed products may not be combined with other licensed products either therapeutic, prophylactic or diagnostic, except as a license is obtained for the combined product. Licensed products may not be combined with nonlicensable therapeutic, prophylactic, or diagnostic substances except as a license is obtained for such combination. § 610.18 Cultures.

(a) Storage and maintenance. Cultures used in the manufacture of products shall be stored in a secure and orderly manner, at a temperature and by a method that will retain the initial characteristics of the organisms and insure freedom from contamination and deterioration.

(b) Identity and verification. Each culture shall be clearly identified as to source strain. A complete identification of the strain shall be made for each new stock culture preparation. Primary and subsequent seed lots shall be identified by lot number and date of preparation. Periodic tests shall be performed as often as necessary to verify the integrity of the strain characteristics and freedom from extraneous organisms. Results of all periodic tests for verification of cultures and determination of freedom from extraneous organisms shall be recorded and retained.

Subpart C-Standard Preparations and
Limits of Potency