[Code of Federal Regulations]
[Title 21, Volume 7]
[Revised as of April 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR610.12]
[Page 68-73]
TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN
SERVICES (CONTINUED)
PART 610_GENERAL BIOLOGICAL PRODUCTS STANDARDS--Table of Contents
Subpart B_General Provisions
Sec. 610.12 Sterility.
Except as provided in paragraphs (f) and (g) of this section, the
sterility of each lot of each product shall be demonstrated by the
performance of the tests prescribed in paragraphs (a) and (b) of this
section for both bulk and final container material.
(a) The test. Bulk material shall be tested separately from final
container material and material from each final
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container shall be tested in individual test vessels as follows:
(1) Using Fluid Thioglycollate Medium--(i) Bulk and final container
material. The volume of product, as required by paragraph (d) of this
section (hereinafter referred to also as the ``inoculum''), from samples
of both bulk and final container material, shall be inoculated into test
vessels of Fluid Thioglycollate Medium. The inoculum and medium shall be
mixed thoroughly and incubated at a temperature of 30 to 35 [deg]C for a
test period of no less than 14 days and examined visually for evidence
of growth on the third, fourth, or fifth day, and on the seventh or
eighth 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 milliliter shall be transferred on the third, fourth, or fifth day
of incubation, from each of the test vessels and inoculated into
additional vessels of the medium. The material in the additional vessels
shall be incubated at a temperature of 30 to 35 [deg]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.
(ii) Final container material containing a mercurial preservative.
In addition to the test prescribed in paragraph (a)(1)(i) of this
section, 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 [deg]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 paragraph
(a)(1)(i) of this section, except that the medium shall be Soybean-
Casein Digest Medium and the incubation shall be at a temperature of 20
to 25 [deg]C.
(b) Repeat tests. If growth appears in any of the test media during
testing of either bulk or final container material, the test may be
repeated to rule out faulty test procedures as follows:
(1) Repeat bulk test. Only one repeat bulk test may be conducted.
The volume of inoculum to be used for the repeat bulk test shall be as
prescribed in paragraph (d)(1) of this section. The repeat test shall be
performed using the procedure prescribed in paragraph (a)(1)(i) of this
section.
(2) First repeat final container test. The number of test samples
and the volumes of product used for the first repeat test shall be as
prescribed in paragraph (d)(2) of this section. For products that do not
contain a mercurial preservative, the repeat test shall be performed,
using both Fluid Thioglycollate Medium and Soybean-Casein Digest Medium,
following the procedures prescribed in paragraphs (a)(1)(i) and (a)(2),
respectively, of this section. If the product contains a mercurial
preservative, the repeat test shall be performed using Fluid
Thioglycollate Medium and the procedures prescribed in paragraphs (a)(1)
(i) and (ii) of this section.
(3) Second repeat final container test. If growth appears in any of
the first repeat final container tests, all tests of the first repeat
final container test shall be repeated, provided there was no evidence
of growth in any test of the bulk material. The test samples used for
the second repeat final container test shall be twice the number used
for the first repeat final container 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
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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 used for each test medium or each
incubation temperature of a test medium 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 milliliter or less, the entire contents shall be
tested. If the amount of material in the final container is more than
1.0 milliliter, the volume tested shall be the largest single dose
recommended by the manufacturer or 1.0 milliliter, whichever is larger,
but no more than 10 milliliters of material or the entire contents from
a single final container need be tested. If more than 2 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 for each test medium or each incubation
temperature condition, 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.)
(e) Culture medium--(1) Formulae. (i) The formula for Fluid
Thioglycollate Medium is as follows:
Fluid Thioglycollate Medium
1-cystine................................ 0.5 gm.
Sodium chloride.......................... 2.5 gm.
Dextrose (C6H12O6[middot]H2)O)........... 5.5 gm.
Granular agar (less than 15% moisture by 0.75 gm.
weight).
Yeast extract (water-soluble)............ 5.0 gm.
Pancreatic digest of casein.............. 15.0 gm.
Purified water........................... 1,000.0 ml.
Sodium thioglycollate (or thioglycolic 0.5 gm.
acid--0.3 ml).
Resazurin (0.10% solution, 1.0 ml.
freshly prepared).
pH after sterilization 7.10.2.
(ii) The formula for Soybean-Casein Digest Medium is as follows:
Soybean-Casein Digest Medium
Pancreatic Digest of Casein.............. 17.0 gm.
Papaic Digest of Soybean Meal............ 3.0 gm.
Sodium Chloride.......................... 5.0 gm.
Dibasic Potassium Phosphate.............. 2.5 gm.
Dextrose (C6H12O6[middot]H2O)............ 2.5 gm.
Purified water........................... 1,000.0 ml.
pH after sterilization 7.30.2.
(2) Culture media requirements--(i) Definition of a lot of culture
medium and test requirements. A lot of culture medium is that quantity
of uniform material identified as having been thoroughly mixed in a
single vessel, dispensed into a group of vessels of the same composition
and design, sterilized in a single autoclave run, and identified in a
manner to distinguish one lot from another. Each lot of culture medium
shall be tested for its growth-promoting qualities unless it meets the
exception for dehydrated culture medium described in this subpart. The
growth-promoting quality test shall be performed on the smallest sized
vessel used in an autoclave run. When using a single batch of dehydrated
culture medium, a manufacturer need not perform growth-promoting tests
on each lot of prepared liquid medium, provided that a validation
program exists for autoclaves used to sterilize the culture medium, and
the manufacturer has received approval for this practice from the
Director, Center for Biologics Evaluation and Research or the Director,
Center for Drug Evaluation and Research.
(ii) Test organisms, strains, characteristics, identity, and
verification. Two or more strains of microorganisms that are exacting in
their nutritive and aerobic/anaerobic requirements shall be used to test
the growth-promoting qualities of each lot of test medium. When using
Fluid Thioglycollate medium, both an aerobic and an anaerobic test
microorganism shall be chosen. When using Soybean Casein Digest Medium,
the yeast, Candida albicans, shall be one of the two test microorganisms
chosen. Manufacturers shall choose the strains of microorganisms from
the chart in this paragraph.
[[Page 71]]
------------------------------------------------------------------------
Incubation
Medium Test microorganisms temperature
------------------------------------------------------------------------
Spore-formers
Fluid Thioglycollate........... 1. Bacillus subtilis 30 to 35
(ATCC No. 6633). [deg]C.
2. Clostridium Do.
sporogenes (ATCC No.
11437).
Non-spore-formers
3. Candida albicans Do.
(ATCC No. 10231).
4. Micrococcus luteus Do.
(ATCC No. 9341).
5. Bacteroides Do.
vulgatus (ATCC No.
8482).
Spore-formers
Soybean-Casein Digest.......... 1. Bacillus subtilis 20 to 25
(ATCC No. 6633). [deg]C.
Non-spore-formers
2. Candida albicans Do.
(ATCC No. 10231).
3. Micrococcus luteus Do.
(ATCC No. 9341).
------------------------------------------------------------------------
ATCC strains of microorganisms described in this section are
available from the American Type Culture Collection, 10801 University
Blvd., Manassas, VA 20110. Periodic tests shall be performed to verify
the integrity of the test organisms in accordance with Sec. 610.18 (a)
and (b). The results of these periodic tests shall be recorded and
retained in accordance with Sec. 600.12(b) of this chapter.
(iii) Storage and maintenance of cultures of test organisms.
Cultures of the test organisms used to determine the growth-promoting
qualities of the medium shall be stored in a manner that will prevent
cross contamination or loss of identity, at a temperature and by a
method that will retain the initial characteristics of the organisms and
ensure freedom from contamination and deterioration. If the test
organisms are stored in the freeze-dried state, or frozen, they shall be
reconstituted or thawed, whichever is applicable, and plated
periodically to verify the colony count of the suspension. If the test
suspensions are stored in a state other than freeze-dried or frozen,
they shall be plated, and a colony count shall be performed at the time
of each growth-promoting quality test to assure that not more than 100
organisms are used per test vessel. The results of tests for
verification of the colony count shall be recorded and retained in
accordance with Sec. 600.12(b) of this chapter.
(iv) Storage and condition of media. A medium shall not be used if
the extent of evaporation affects its fluidity, nor shall it be reused
in a sterility test of the product. Fluid Thioglycollate Medium shall be
stored in the dark at room temperature if the vessels are unsealed.
Sealed vessels shall be stored at the manufacturer's specified storage
temperature. Fluid Thioglycollate Medium shall not be used if more than
the upper one-third of the medium has acquired a pink color. The medium
may be restored once by heating on a steam bath or in free-flowing steam
until the pink color disappears. The design of the test vessel for Fluid
Thioglycollate Medium shall provide favorable aerobic and anaerobic
conditions for growth of the microorganisms throughout the test period.
Soybean-Casein Digest Medium shall be stored in the dark at 20 to 25
[deg]C. Unsealed vessels of either medium may be stored for more than 10
days at the proper temperature, provided they are tested monthly for
growth-promotion and found to be satisfactory. Sealed vessels of either
medium may be stored at the proper temperature for a period of time not
to exceed 1 year, provided they are tested for growth-promotion every 3
months and found to be satisfactory. The results of such testing shall
be recorded and retained in accordance with Sec. 600.12(b) of this
chapter.
(v) Criteria for a satisfactory growth-promoting quality test. (a)
One hundred or fewer organisms of each strain tested shall be used. The
test is satisfactory if evidence of growth appears within 7 days in all
vessels inoculated. If a lot of medium fails to support the growth of
any test organism, or if the test results show that more than 100
organisms of a strain were used or are necessary to promote growth in
the lot of medium being tested, or if the growth is not a pure culture
of the test
[[Page 72]]
organism, a second test may be performed. If it fails the second test,
the lot of medium shall be rejected.
(b) Inoculated Fluid Thioglycollate Medium shall be incubated at 30
to 35 [deg]C for 7 days. If the test medium is to be used in determining
the sterility of a product containing a mercurial preservative, a second
test shall be performed in accordance with paragraph (e)(2)(v)(a) of
this section, except that the test shall be incubated at 20 to 25 [deg]C
for 7 days. Inoculated Soybean-Casein Digest Medium shall be incubated
at 20 to 25 [deg]C for 7 days. The sterility of each lot of medium shall
be confirmed by the incubation of uninoculated control test vessels for
7 days at the temperature(s) for that particular medium. The lot of
medium is satisfactory if no growth is observed in the control test
vessels within the incubation period. The tests for growth-promoting
qualities of culture media may be performed simultaneously with
sterility testing of biological products, provided the sterility test is
considered invalid if the test medium shows no growth response.
(vi) Volume of culture medium. The volume of each culture medium
shall be determined for each bulk and final container sterility test
required for each product. The ratio of the volume of inoculum to the
volume of culture medium shall result in a dilution of the product that
is not bacteriostatic or fungistatic, except for products to be tested
by membrane filtration. The volume of inhibitors or neutralizers of
preservatives added should be considered in determining the proper ratio
of inoculum/medium. 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 viable in the product being tested, and
incubated at the appropriate temperature for no less than 7 days.
(f) Membrane filtration. Bulk and final container material or
products containing oil products in water-insoluble ointments may be
tested for sterility using the membrane filtration procedure set forth
in the United States Pharmacopeia (23d Revision, 1995), section entitled
``Test Procedures Using Membrane Filtration,'' pp. 1689 to 1690, which
is incorporated by reference in accordance with 5 U.S.C. 552(a) and 1
CFR part 51. Copies are available from the United States Pharmacopeial
Convention, Inc., 12601 Twinbrook Pkwy., Rockville, MD 20852, or
available for inspection at the Center for Drug Evaluation and
Research's Division of Medical Library, 5600 Fishers Lane, rm. 11B-40,
Rockville, MD, or at the National Archives and Records Administration
(NARA). (For information on the availability of this material at NARA,
call 202-741-6030, or go to: http://www.archives.gov/federal--register/
code--of--federal--regulations/ibr--locations.html). Except that:
(1) The test samples shall conform with paragraph (d) of this
section; and
(2) 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\1/2\C in lieu of the test in Soybean-Casein
Digest Medium.
(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 as
prescribed in paragraph (a)(1)(i) of this section, one test using an
incubation temperature of 18 to 22 [deg]C, the other test using an
incubation temperature of 30 to 37 [deg]C, in lieu of performing one
test using an incubation temperature of 30 to 35 [deg]C, provided that
growth-promoting quality tests have been performed at these
temperatures.
(3) [Reserved]
(4) Test precluded or not required. (i) The tests prescribed in this
section need not be performed for Whole Blood, Cryoprecipitated AHF,
Platelets, Red Blood Cells, Plasma, Source Plasma, Smallpox Vaccine,
Reagent Red Blood Cells, Anti-Human Globulin, or Blood Grouping Reagent.
(ii) Where a manufacturer submits data which the Director, Center
for Biologics Evaluation and Research or the Director, Center for Drug
Evaluation and Research, finds adequate to
[[Page 73]]
establish that the mode of administration, the method of preparation, or
the special nature of the product precludes or does not require a
sterility test or that the sterility of the lot is not necessary to
assure the safety, purity, and potency of the product, the Director may
exempt a product from the sterility requirements of this section subject
to any conditions necessary to assure the safety, purity, and potency of
the product.
(5) Number of final containers more than 20, less than 200. If the
number of final containers in the filling is more than 20 or less than
200, the sample shall be no less than 10 percent of the containers.
(6) Number of final containers--20 or less. If the number of final
containers 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.
(7) Samples--large volume of product in final containers. For
Albumin (Human) and Plasma Protein Fraction (Human), when the volume of
product in the final container is 50 milliliters 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.
(8) Diagnostic biological products not intended for injection. For
diagnostic biological products not intended for injection, (i) only the
Fluid Thioglycollate Medium test incubated at 30 to 35 [deg]C is
required, (ii) the volume of material for the bulk test shall be no less
than 2.0 milliliters, 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.
(9) 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.
(h) Records. The records related to the testing requirements of this
section shall be prepared and maintained as required by Sec. Sec.
211.167 and 211.194 of this chapter.
[38 FR 32056, Nov. 20, 1973, as amended at 41 FR 4015, Jan. 28, 1976; 41
FR 10428, Mar. 11, 1976; 44 FR 11754, Mar. 2, 1979; 49 FR 15187, Apr.
18, 1984; 49 FR 23834, June 8, 1984; 50 FR 4133, Jan. 29, 1985; 51 FR
44906, Dec. 15, 1986; 53 FR 12764, Apr. 19, 1988; 55 FR 11013, Mar. 26,
1990; 62 FR 48175, Sept. 15, 1997; 67 FR 9587, Mar. 4, 2002; 69 FR
18803, Apr. 9, 2004; 70 FR 14985, Mar. 24, 2005]