[Title 21 CFR D]
[Code of Federal Regulations (annual edition) - April 1, 1996 Edition]
[Title 21 - FOOD AND DRUGS]
[Chapter I - FOOD AND DRUG ADMINISTRATION,]
[Subchapter F - BIOLOGICS]
[Part 620 - ADDITIONAL STANDARDS FOR BACTERIAL PRODUCTS]
[Subpart D - Cholera Vaccine]
[From the U.S. Government Publishing Office]
21
FOOD AND DRUGS
7
1996-04-01
1996-04-01
false
Cholera Vaccine
D
Subpart D
FOOD AND DRUGS
FOOD AND DRUG ADMINISTRATION,
BIOLOGICS
ADDITIONAL STANDARDS FOR BACTERIAL PRODUCTS
Subpart D--Cholera Vaccine
Sec. 620.30 Cholera Vaccine.
The proper name of this product shall be Cholera Vaccine, which
shall consist of an aqueous preparation of equal parts of Ogawa and
Inaba serotypes of killed Vibrio cholerae bacteria.
[41 FR 18295, May 3, 1976]
Sec. 620.31 Production.
(a) Strains of bacteria. (1) A strain of Ogawa and a strain of Inaba
serotypes of V. cholerae shall be used in the manufacture of the
vaccine. Each serotype strain shall have been shown in controlled field
studies to yield a vaccine no less potent than vaccines prepared from
Ogawa strain 41 and Inaba strain 35A3 obtained from the Center for
Biologics Evaluation and Research.
(2) Antigenic integrity of the strains shall be verified by (i) the
agglutination of living bacteria of each serotype by cholera O Group I
antiserum; (ii) the agglutination of the Ogawa strain in monospecific
Ogawa antiserum and of the Inaba strain in monospecific Inaba antiserum;
and (iii) the absence of spontaneous agglutination of living bacteria of
either strain in 0.85 percent sodium chloride solution during incubation
for at least 5 hours at 37 deg. C.
(b) Propagation of bacteria. The culture medium for the propagation
strains shall not contain ingredients known to be capable of producing
allergenic effects in human subjects. The harvested bacteria shall be
free of extraneous bacteria, fungi, and yeasts as demonstrated by
microscopic examination and cultural methods. Bacteria of the two
serotypes shall be grown separately.
(c) Bacterial content. (1) The number of bacteria in each separate
bacterial harvest shall be determined by use of the U.S. Opacity
Standard not later than 2 hours after harvest and before treatment with
a preservative or other agent capable of altering opacity of the
bacterial suspension.
(2) The vaccine shall contain equal numbers of bacteria of the Ogawa
and Inaba serotypes, and the total number shall not exceed 8 x
109 bacteria per milliliter.
(d) Nitrogen content. The total nitrogen content of the vaccine
shall not exceed 0.3 milligram per milliliter for bacteria grown on
solid medium or 1.0 milligram per milliliter if grown in liquid medium.
In no instance shall the vaccine contain more than 0.07 milligram per
milliliter of nitrogen precipitable by the addition of an equal volume
of 10 percent trichloracetic acid.
(e) Preservative. The vaccine shall contain a preservative.
[41 FR 18295, May 3, 1976, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
Sec. 620.32 U.S. Standard preparations.
The following U.S. Standard preparations shall be obtained from the
Center for Biologics Evaluation and Research, Food and Drug
Administration, for use as prescribed in this subpart:
(a) Vaccine standard. The U.S. Standard Cholera Vaccine, Ogawa
serotype, and U.S. Standard Cholera Vaccine, Inaba serotype, shall be
reconstituted as directed for determining the potency of Cholera
Vaccine.
(b) Opacity standard. The U.S. Opacity Standard for use in
estimating the bacterial content of the vaccine and of the challenge
culture.
(c) Seed culture. Seed cultures of V. cholerae, Inaba serotype,
strain 35A3 and Ogawa serotype, strain 41, for preparation of vaccine
challenge cultures for use in the vaccine potency test.
[41 FR 18295, May 3, 1976, as amended at 49 FR 23834, June 8, 1984; 55
FR 11013, Mar. 26, 1990]
Sec. 620.33 Potency tests.
Each lot of vaccine shall be subjected to two potency tests. One
test shall determine the potency of the vaccine in comparison with the
U.S. Standard
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Cholera Vaccine, Ogawa serotype, and the other test shall determine the
potency of the vaccine in comparison with the U.S. Standard Cholera
Vaccine, Inaba serotype. At least four dilutions of each vaccine shall
be tested. Each test shall be performed as follows:
(a) Mice. Healthy mice shall be used, all from a single strain and
of the same sex, or an equal number of each sex in each group, with
individual weights between 10 and 14 grams. A group of at least 16 mice
shall be used for each dilution of each vaccine. In addition, there
shall be at least 4 groups consisting of no less than 10 mice each for
each potency test as a control for virulence titration of the challenge
suspension.
(b) Injections of vaccine. Serial dilutions, no greater than
fivefold, of the vaccine to be tested and of the appropriate serotype
standard vaccine shall be made in 0.85 percent sodium chloride solution.
The median effective dose (ED50), which is the dose of vaccine that
is expected to protect 50 percent of the animals that received the
vaccine, shall be bracketed by the dilutions used. Each mouse in each
dilution group shall receive intraperitoneally 0.5 milliliter of the
appropriate vaccine dilution. At least 87.5 percent of the mice in each
dilution group shall survive, and all surviving mice shall appear
healthy at the time of challenge.
(c) The challenge. The challenge shall be administered 12 to 16 days
after injection of the vaccine.
(1) The strains of V. cholerae for challenge shall be Ogawa 41 and
Inaba 35A3, except that V. cholerae, Inaba serotype, strain V86 may be
used instead of Inaba serotype, strain 35A3, for preparation of vaccine
challenge culture: Provided, That the source of the challenge culture
shall be identified and verified by the manufacturer as equal to that
distributed by the World Health Organization. For each test, the
challenge culture shall be taken from a batch of cultures maintained by
a method such as freeze-drying that retains constancy of virulence.
(2) The challenge and virulence titration doses shall be prepared as
follows: The bacteria for each challenge shall be harvested from a 6- to
18-hour culture grown at 36 deg.plus-minus1 deg. C, on a suitable
agar medium adjusted to pH 7.4. The harvested bacteria shall be
uniformly suspended in a diluent consisting of M/15 phosphate buffered
saline adjusted to pH 7.4 and shall contain 0.1 to 0.2 percent gelatin.
The suspension shall be free from agar particles and clumps of bacteria.
The suspension shall be adjusted to an opacity of 10 units, and diluted
in tenfold increments using the same diluent. The suspensions for the
challenge and virulence titrations shall be suspended in a 5 to 10
percent sterile gastric mucin preparation adjusted to pH 7.4. The
challenge suspension shall be prepared from whichever bacterial dilution
provides the required median lethal dose (LD50) for a 0.5
milliliter challenge dose. The LD50 is the dose of the challenge
suspension that is expected to kill 50 percent of the animals that
received the challenge. The virulence titration suspensions shall
consist of the challenge suspension and at least three dilutions of the
challenge suspension calculated to bracket the LD50 value.
(3) At least 16 surviving mice, randomly selected from each dilution
group that received vaccine, shall be inoculated intraperitoneally with
a 0.5-milliliter dose of the challenge suspension. Mice in each of the
four groups of control mice used for the virulence titration of the
challenge suspension shall be inoculated intraperitoneally with a 0.5-
milliliter dose of the challenge suspension and its respective
dilutions. The challenge dose control mice shall be inoculated last. The
interval between removal of the bacteria from the culture medium and the
inoculation of the last mouse shall not exceed 2\1/2\ hours.
(d) Recording the results. The mice shall be observed daily for 2
days following challenge. A daily record shall be maintained of the
number of mice that die. A record of the number of mice that survive
shall be made at the end of the observation period.
(e) Validity of the test. The test is valid provided: (1) The
ED50 value of the vaccine under test and the standard vaccine is
between the largest and smallest doses inoculated into the mice;
[[Page 80]]
(2) The homogeneity of the dose response lines for both the vaccine
under test and the standard vaccine is acceptable;
(3) The log-dose response lines for the vaccine under test and the
standard vaccine are shown to be parallel by an appropriate statistical
method;
(4) The results of all dilutions shall be used to calculate the
ED50 value of both the standard and test vaccine by a parallel line
bioassay method or a method statistically equivalent;
(5) The challenge dose contains between 100 and 10,000 LD50
doses; and
(6) The LD50 value of the challenge suspension contains no more
than 10,000 colony-forming units determined by plate count.
(f) Repeat tests. Repeat tests need be performed only on the
serotype which failed to meet the potency requirements prescribed in
paragraph (h) of this section. The results of each test on each serotype
meeting the criteria in paragraph (e) of this section shall be combined
by means of a geometric mean. The determination that the vaccine meets
the potency requirements shall be made from the results of not more than
three valid tests on each serotype.
(g) Estimate of the potency. The ED50 value of each vaccine
shall be calculated. The protective unit value of each serotype per
milliliter of the vaccine under test shall be calculated in terms of the
unit value of the corresponding standard vaccine.
(h) Potency requirements. The vaccine shall have a potency of not
less than 8 units per serotype per milliliter. This requirement shall be
met only if the potency for a single test is not less than 4.4 units per
serotype per milliliter, or for two tests not less than 5.3 units, or
for three tests not less than 5.7 units.
[41 FR 18295, May 3, 1976, as amended at 41 FR 46587, Oct. 22, 1976]
Sec. 620.34 Mouse toxicity test.
The final vaccine shall be demonstrated to be free from toxicity by
the following test: A group of no less than 10 and no more than 40 mice,
each mouse weighing 14 to 16 grams, shall have free access to food and
water at least 2 hours before injection and throughout the test period.
The group weight of the mice shall be determined immediately before
injection. Each mouse shall be injected intraperitoneally with a test
dose of 0.5 milliliter of undiluted vaccine. The group weight of the
mice shall be determined again at the end of 72 hours. The 72-hour
average weight per mouse shall be no less than the average weight per
mouse immediately preceding the injection. No more than 5 percent of the
total number of mice used may die during the test period; however,
neither death nor significant toxic signs attributable to the vaccine
shall result.
[41 FR 18295, May 3, 1976]
Sec. 620.35 General requirements.
(a) Freezing prohibition. Cholera Vaccine shall not be frozen at any
time.
(b) Dose. These standards are based on a total immunizing dose of
two injections of 0.5 milliliter and 1.0 milliliter, respectively, given
at intervals specified in the manufacturer's labeling.
(c) Date of manufacture. The date of manufacture shall be the date
of initiation of the last valid potency test for the Ogawa serotype or
the Inaba serotype, whichever date is earlier.
(d) Labeling. In addition to the applicable labeling provisions of
this chapter, the package label shall bear the following: (1) A
statement that the vaccine contains 8 units of each serotype antigen per
milliliter.
(2) The statement, ``DO NOT FREEZE''.
(3) The statement, ``SHAKE WELL''.
(e) Samples; protocols; official release. For each lot of vaccine,
the following material shall be submitted to the Director, Center for
Biologics Evaluation and Research, Food and Drug Administration, 8800
Rockville Pike, Bethesda, MD 20892.
(1) A sample consisting of no less than 40 milliliters of the
product. The sample may be in the final container or from the vaccine
bulk lot.
(2) A protocol which consists of a summary of the history of
manufacture of each lot including all results of each test for which
test results are requested by the Director, Center for Biologics
Evaluation and Research,
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Food and Drug Administration. The raw data and results from each potency
test performed shall be included.
(3) The product shall not be issued by the manufacturer until
written notification of official release of the lot is received from the
Director, Center for Biologics Evaluation and Research.
[41 FR 18295, May 3, 1976, as amended at 42 FR 27582, May 31, 1977; 49
FR 23834, June 8, 1984; 51 FR 15610, Apr. 25, 1986; 55 FR 11013, Mar.
26, 1990]