[Code of Federal Regulations]
[Title 9, Volume 1]
[Revised as of January 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 9CFR113.406]
[Page 764-765]
TITLE 9--ANIMALS AND ANIMAL PRODUCTS
CHAPTER I--ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF
AGRICULTURE
PART 113_STANDARD REQUIREMENTS--Table of Contents
Sec. 113.406 Tuberculin, Intradermic.
Tuberculin, Intradermic, is a filtrate produced from cultures of Pn,
C, and Dt strains of Mycobacterium tuberculosis (supplied by Animal and
Plant Health Inspection Service) which has been inactivated and is non-
toxic. Each serial shall be tested for purity, safety, potency, and
special chemical tests in accordance with the conditions prescribed for
each test. A serial found unsatisfactory by any prescribed test shall
not be released.
(a) Purity test. Each serial shall be tested for purity as provided
in this paragraph.
(1) Final container samples of completed product shall be tested for
viable bacteria and fungi as prescribed in Sec. 113.26.
(2) A 20 ml sample shall be centrifuged and the sediment examined
microscopically for the presence of acidfast (Ziehl-Nielsen stain) or
other microorganisms (Gram stain). A serial which contains
microorganisms is unsatisfactory for release.
(b) Safety test. Final container samples of completed product from
each serial shall be tested for safety. Two mature guinea pigs shall be
injected subcutaneously with 1 ml and observed for 10 days. If
unfavorable reactions attributable to the product occur during the
observation period, the serial is unsatisfactory. If unfavorable
reactions occur which are not attributable to the product, the test
shall be declared inconclusive and repeated: Provided, That if the test
is not repeated, the serial shall be declared unsatisfactory.
(c) Potency test. Bulk or final container samples of completed
product from each serial shall be subjected to a comparison test using a
Reference Tuberculin supplied by Animal and Plant Health Inspection
Service. Test animals shall be 10 sensitized white female guinea pigs
from one source which weigh 500-700 grams at the beginning of the test
and which have not been used in a previous test. The comparison test
shall be conducted in accordance with
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the procedures prescribed in paragraphs (c)(1), (2), (3), (4), (5), (6),
(7), and (8) of this section.
(1) The guinea pigs shall be sensitized with a sterile heat-killed
suspension of equal amounts of strains Pn, C, and Dt of Mycobacterium
tuberculosis. The heat-killed sensitizing agent shall be injected in a
volume of 0.5 ml per guinea pig. The guinea pigs shall be considered
sensitized for testing not less than 30 days nor more than 120 days
post-injection.
(2) The guinea pigs shall be prepared for sensitivity testing at
least 4 hours prior to the injection of tuberculin. The entire abdominal
and flank areas shall be clipped, a depilatory agent applied for 5-10
minutes, the area rinsed with warm water, and dried.
(3) Dilutions of 1:100, 1:200, and 1:400 shall be prepared with the
Reference Tuberculin and the unknown tuberculin. Three test sites on
each side of and equidistant from the abdominal midline shall be chosen
on each guinea pig. Using a tuberculin syringe and needle, 0.05 ml of
each dilution shall be injected intradermally at one of the test sites
which has been randomly selected for the dilution.
(4) The sensitivity of the tuberculins shall be determined 24 hours
after injected by measuring the area of erythema. Measurements in
millimeters shall be made anterior of the greatest diameter and
perpendicular to the first measurement. The square millimeter shall be
calculated by multiplying the two measurements.
(5) The total area of response for each tuberculin tested shall be
determined by adding the areas of erythema for each dilution of each of
the test animals in a group. The sums of the areas of erythema for all
three dilutions of each tuberculin shall be added to give the total area
of tuberculin response.
(6) The total tuberculin response area of the serial being tested
shall be expressed as a percentage of the total tuberculin response area
of the Reference Tuberculin. (The total response area of the serial
divided by the total response area of the Reference Tuberculin times
100.)
(7) If the total tuberculin response area of the serial being tested
does not fall between 75 percent and 125 percent of the total tuberculin
response area of the Reference Tuberculin, the serial is unsatisfactory.
(8) Two unsensitized guinea pigs are given 0.05 ml intradermal
injections of 1:4 and 1:10 dilutions of both the serial being tested and
the Reference Tuberculin as a control for nonspecific positive
reactions. If positive reactions are observed with the Reference
Tuberculin, the test is considered a ``No Test'' and repeated. If
positive reactions are observed with the serial being tested only, the
serial is unsatisfactory.
(d) Special chemical tests and requirements. Final container samples
of completed product from each serial shall be tested as follows:
(1) Hydrogen ion concentration. The hydrogen ion concentration shall
be determined with a pH meter which has been standardized with a pH 7.0
buffer just prior to use. The pH of the product shall be 7.0 0.3.
(2) Total nitrogen determination. The nitrogen content shall be
determined by the Kjeldahl method on duplicate 15 ml samples consisting
of 5 ml from each of three vials. The total nitrogen content of the
product shall be 0.18 percent 0.06 percent.
(3) Trichloroacetic acid precipitable nitrogen. The determination of
precipitable nitrogen by a final concentration of 4 percent
trichloroacetic acid shall be made by the Kjeldahl method on duplicate
15 ml samples, consisting of 5 ml from each of three vials. The
trichloroacetic acid precipitable nitrogen content shall be 0.047
percent 0.01 percent.
(4) Phenol determination. The phenol content shall be determined by
direct titration with a standardized bromide-bromate solution. (A
correction factor of 0.04 should be subtracted from the final value in
the determination of phenol in tuberculin.) The phenol content shall be
0.54 percent 0.04 percent.
(5) Clarity. The product shall be optically clear and free from any
extraneous particles.
[39 FR 16857, May 10, 1974. Redesignated at 39 FR 25463, July 11, 1974.
Redesignated at 55 FR 35561, Aug. 31, 1990, as amended at 56 FR 66784,
Dec. 26, 1991
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