[Title 21 CFR A]
[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 650 - ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR DERMAL TESTS]
[Subpart A - Diphtheria Toxin for Schick Test]
[From the U.S. Government Publishing Office]




  21
  FOOD AND DRUGS
  7
  1996-04-01
  1996-04-01
  false
  
  A
  Subpart A
  
    FOOD AND DRUGS
    FOOD AND DRUG ADMINISTRATION,
    BIOLOGICS
    ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR DERMAL TESTS
  


               Subpart A--Diphtheria Toxin for Schick Test



Sec. 650.1   Diphtheria Toxin for Schick Test.

    The proper name of this product shall be Diphtheria Toxin for Schick 
Test, which shall be a preparation of a diphtheria toxin obtained from 
the growth of Corynebacterium diphtheriae.



Sec. 650.2   U.S. Standard preparation.

    The U.S. Standard Diphtheria Toxin for Schick Test shall be used to 
determine the Schick test dose of the product. The Schick test dose of 
the standard is that amount of the standard that, when mixed with 0.001 
unit of the U.S. Standard Diphtheria Antitoxin and injected 
intradermally in a guinea pig, will induce an erythematous reaction of 
10 mm. in diameter.



Sec. 650.3   Manufacture of Diphtheria Toxin for Schick Test.

    (a) Propagation of bacteria. The culture medium for propagation of 
the Corynebacterium diphtheriae for preparation of the parent toxin 
shall not contain ingredients known to be capable of producing 
allergenic effects in human subjects.
    (b) The parent toxin. Diphtheria Toxin for Schick Test shall be 
prepared from a parent toxin which has been demonstrated to be stable 
and which contains no less than 400 minimum lethal doses per milliliter 
or 400,000 minimum reaction doses per milliliter. A minimum lethal dose 
is the smallest amount of toxin that will kill a guinea pig weighing 
approximately 250 gm. on the fourth day after its subcutaneous 
injection. A minimum reaction dose is that amount of toxin which when 
injected intradermally into a guinea pig induces an erythematous 
reaction 10 mm. in diameter.



Sec. 650.4   Potency test.

    The dermal reactivity of each lot of the product shall be determined 
from the results of simultaneous guinea pig intradermal potency tests of 
the product under test and of the standard. The test shall be performed 
as follows:
    (a) Guinea pigs. At least four healthy female guinea pigs shall be 
used, all of the same strain and each of a size that will permit a 
random distribution of eight intradermal injections. The hair shall be 
removed from the back and both sides of each guinea pig without 
producing abrasions of the skin. The denuded skin of each animal shall 
be sectioned into four equal areas at right angles to the vertebral 
column to provide two injection sites in each of the four areas, one on 
each side of the vertebra. The test is not valid if the guinea pigs do 
not show a graded response to the graded dilutions of the Schick test 
dose of the standard toxin.
    (b) Preparation of the test doses. Four dilutions, two of the 
product under test and two of the U.S. Standard Diphtheria Toxin for 
Schick Test, shall be prepared in sterile buffered saline pH 7.4 
containing 0.2 percent gelatin. The low and high dilutions of the 
standard shall be those amounts of a Schick test dose of the standard 
which in a dose of 0.1 ml. are capable of eliciting graded erythematous 
dermal reactions between 10 mm. and 20 mm. in diameter. The low and high 
dilutions of the Schick test dose of the toxin under test shall be the 
same as those of the standard toxin and estimated to have the same 
dermal reactivity.
    (c) Inoculation. The low and high dilutions of the product (chart 
designation PL and PH) and the low and high dilutions of the 
standard (chart designations SL and SH) shall be injected 
intradermally in a volume of 0.1 ml. into each of the four guinea pigs 
according to either the following scheme, or in another scheme, provided 
it will permit comparable randomization of injection sites:

----------------------------------------------------------------------------------------------------------------
                                                               Guinea Pig Number                                
                              ----------------------------------------------------------------------------------
             Area                        1                    2                    3                   4        
                              ----------------------------------------------------------------------------------
                                  Left      Right       Left      Right     Left      Right     Left      Right 
----------------------------------------------------------------------------------------------------------------
A............................  SL         SL         SH         SH        PL        PL        PH        PH      
B............................  SH         SH         SL         SL        PH        PH        PL        PL      

[[Page 147]]

                                                                                                                
C............................  PL         PL         PH         PH        SL        SL        SH        SH      
D............................  PH         PH         PL         PL        SH        SH        SL        SL      
----------------------------------------------------------------------------------------------------------------

    (d) Calculation of test results. Between 40 and 66 hours following 
injection, a diameter of the reaction for each injection site shall be 
calculated by averaging two diameters of the reaction measured at right 
angles to each other. The average reaction for each dilution for each 
animal shall be determined, then the average diameters of the reactions 
of all of the guinea pigs for each dilution shall be calculated. The 
ratios of the reactions are determined by dividing the average diameter 
of the low dilution of the product under test by the average diameter of 
the low dilution of the standard and by dividing the average diameter of 
the high dilution of the product by the average diameter of the high 
dilution of the standard.
    (e) Potency requirement. The potency of the product under test is 
satisfactory if each calculated ratio of the reactions of the product 
under test and of the standard is 1.0. The potency of the lot under test 
is considered to be equal to that of the standard if the ratios are not 
lower than 0.77 or higher than 1.30, provided that in a single test the 
ratios are substantially the same.



Sec. 650.5   Stability test.

    A sample of each lot of the product shall be held at 37 deg. C for 
not less than 24 hours and then tested for potency as prescribed in 
Sec. 650.4. The stability of the product is satisfactory if test results 
of the sample meet the potency requirement prescribed in Sec. 650.4(e).



Sec. 650.6   Samples; protocols; official release.

    For each lot of the product, the following material shall be 
submitted to the Director, Center for Biologics Evaluation and Research:
    (a) A protocol which consists of a summary of the history of 
manufacture of each lot including all results of all tests for which 
test results are requested by the Director, Center for Biologics 
Evaluation and Research.
    (b) A sample of no less than 20 milliliters of the product.
    (c) 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.

[38 FR 32097, Nov. 20, 1973, as amended at 42 FR 27584, May 31, 1977; 49 
FR 23834, June 8, 1984; 55 FR 11013, Mar. 26, 1990]