[Federal Register Volume 59, Number 195 (Tuesday, October 11, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-25056]


[[Page Unknown]]

[Federal Register: October 11, 1994]


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DEPARTMENT OF AGRICULTURE
Animal and Plant Health Inspection Service

9 CFR Part 113

[Docket No. 93-039-1]

 

Viruses, Serums, Toxins and Analogous Products; Standard 
Requirement for Escherichia Coli Bacterins

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Proposed rule.

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SUMMARY: We are proposing to amend the regulations by adding a Standard 
Requirement for Escherichia coli bacterins. This amendment would 
provide a uniform procedure to demonstrate the immunogenicity of 
reference bacterins made from E. coli master seed bacteria. This 
amendment would also provide uniform procedures to requalify a 
reference bacterin for which the dating period has expired. In 
addition, the amendment would specify the potency test requirements for 
the release of serials of E. coli bacterins for immunogenicity and 
potency.

    The purpose of this regulation is to standardize the requirements 
for the production and testing of E. coli bacterins for immunogenicity 
and potency.

DATES: Consideration will be given only to comments received on or 
before December 12, 1994.

ADDRESSES: Please send an original and three copies of your comments to 
Chief, Regulatory Analysis and Development, PPD, APHIS, USDA, room 804, 
Federal Building, 6505 Belcrest Road, Hyattsville, MD 20782. Please 
state that your comments refer to Docket No. 93-039-01. Comments 
received may be inspected at USDA, room 1141, South Building, 14th 
Street and Independence Avenue SW., Washington, DC, between 8 a.m. and 
4:30 p.m., Monday through Friday, except holidays. Persons wishing to 
inspect comments are requested to call ahead (202) 690-2817 to 
facilitate entry into the comment reading room.

FOR FURTHER INFORMATION CONTACT:
Dr. David A. Espeseth, Deputy Director, Veterinary Biologics, BBEP, 
APHIS, USDA, room 838, Federal Building, 6505 Belcrest Road, 
Hyattsville, MD 20782, (301) 436-8245.

SUPPLEMENTARY INFORMATION: 

Background

    Standard Requirements are prescribed in 9 CFR part 113 for the 
preparation and testing of veterinary biological products. A Standard 
Requirement consists of test methods, procedures, and criteria which 
define the standards of purity, safety, potency, and efficacy for a 
given type of veterinary biological product. Where a Standard 
Requirement for a product does not exist in the regulations, production 
procedures and specifications for purity, safety, and potency of a 
product are provided in an Outline of Production filed with the Animal 
and Plant Health Inspection Service. Once uniform standards for a type 
of product are established, they are codified in the regulations.
    Because there is no Standard Requirement in 9 CFR part 113 for 
Escherichia coli (E. coli) bacterins, each manufacturer of these 
products has devised its own procedures, which are usually a part of a 
filed Outline of Production, to meet the requirements of the Virus-
Serum-Toxin Act that all veterinary biological products be pure, safe, 
potent, and efficacious. Therefore, even though all of the procedures 
and methods must be satisfactory to the Animal and Plant Health 
Inspection Service, there are a number of different procedures which 
are used by firms producing biological products containing E. coli as 
one of the components. At this time, we are prepared to propose adding 
a new Sec. 113.124 to the standards which would contain uniform test 
methods, procedures, and criteria to be used by licensed manufacturers 
of E. coli bacterins to provide assurance that these bacterins are 
immunogenic and potent.

Immunogencity

    The effectiveness of a veterinary biologic to ameliorate disease in 
animals is of paramount importance to the consumers of veterinary 
biological products. The test for immunogenicity ascertains the 
efficacy of a veterinary biologic. Based on experience with 
immunogenicity tests, the Animal and Plan Health Inspection Service is 
proposing uniform requirements for establishing the immunogenicity of a 
Master Reference made from E. coli master seed. This would be 
accomplished by specifying in proposed Sec. 113.124(a): (1) The minimum 
number of pregnant dams to vaccinate and the number of nonvaccinated 
controls; (2) procedures for challenge of neonates with virulent E. 
coli; and (3) the criteria for satisfactory protection against 
challenge and thereby demonstration of immunogenicity of the Master 
Reference. The immunogenicity test of master seed bacteria described in 
proposed Sec. 113.124(a) would be required of all new product license 
applications prior to licensure. The test would establish the 
immunogenicity of the Master Reference used in the relative potency 
test required for release of serials of biological product.
    As do all biologics, reference bacterins may lose potency and 
immunogenicity with the passage of time. The loss of potency and 
immunogenicity is not constant for all biologics because of differences 
between master seeds, production methods, adjuvants, diluents, 
conditions of storage and other variables used in the manufacture of 
biologics. Therefore the proposed amendment in Sec. 113.124(b) 
describes the testing that must be conducted on an E. coli Master 
Reference to determine if it is still sufficiently potent and 
immunogenic after its date of expiration to permit extending the date 
of expiration.

Potency

    Different potency tests are currently used for E. coli bacterns. 
Potency tests used to compare serials of product to a reference 
bacterin are: mouse tests, guinea pig tests, and enzyme linked 
immunosorbent assays (ELISA). As proposed, new Sec. 113.124(c) would 
require the use of a standard in vitro method for testing serials of E. 
coli bacterins. The proposed standard method is a parallel line 
immunoassay that compares, for both the unknown and the reference, the 
linear regression of the optical density (OD) versus the logarithm of 
antigen concentration determined by ELISA. The Animal and Plant Health 
Inspection Service has proposed use of this standard in vitro potency 
test for E. coli bacterins
because its use provides three advantages: (1) It ensures the use of a 
valid test method and makes potency testing more uniform; (2) it 
ensures that serials of product are potent before release; and (3) it 
reduces significantly the number of laboratory animals required for 
testing E. coli bacterins.
    If adopted, this standard test would require that manufacturing 
firms have personnel proficient in performing the ELISA procedure and 
the equipment necessary to perform the tests.

Executive Order 12866 and Regulatory Flexibility Act

    This proposed rule has been reviewed under Executive Order 12866. 
The rule has been determined to be not significant for purposes of 
Executive Order 12866, and, therefore, has not been reviewed by the 
Office of Management and Budget.
    This proposed rule, if adopted, would aid firms manufacturing E. 
coli bacterins. The proposal contains Standard Requirements for 
immunogenicity testing which would provide uniformity among firms 
instead of each firm having to meet the Animal and Plant Health 
Inspection Service requirements by its own designed methods. This would 
reduce a firm's cost of research and development needed to design a 
method to test immunogenicity. The proposed rule would prescribe in 
vitro potency testing of serials of product with minimal costs to 
manufacturing firms since the National Veterinary Services Laboratories 
of the Animal and Plant Health Inspection Service have developed and 
standardized the tests and would provide the critical reagents needed 
to perform the tests.
    The manufacturers producing E. coli bacterins would need to have 
personnel proficient in conducting ELISA tests. The necessary equipment 
and personnel, however, should already be available for other routine 
procedures in most biologic, research and diagnostic laboratories. 
Also, the ELISA in vitro potency test is less expensive than the mouse 
potency test now used by the majority of firms producing E. coli 
bacterins.
    Under these circumstances, the Administrator of the Animal and 
Plant Health Inspection Service has determined that this action would 
not have a significant economic impact on a substantial number of small 
entities.

Executive Order 12778

    This proposed rule has been reviewed under Executive Order 12778, 
Civil Justice Reform. If this proposed rule is adopted: (1) All State 
and local laws and regulations that are in conflict with this rule will 
be preempted; (2) no retroactive effect will be given to this rule; and 
(3) administrative proceedings will not be required before parties may 
file suit in court challenging this rule.

Paperwork Reduction Act

    This proposed rule contains no new information collection or 
recordkeeping requirements under the Paperwork Reduction Act of 1980 
(44 U.S.C. 3501 et seq.).

List of Subjects in 9 CFR Part 113

    Animal biologics, Export, Imports, Reporting and recordkeeping 
requirements.

    Accordingly, 9 CFR part 113 would be amended as follows:

PART 113--STANDARD REQUIREMENTS

    1. The authority citation for part 113 would continue to read as 
follows:

    Authority: 21 U.S.C. 151-159; 7 CFR 2.17, 2.51, and 371.2(d).

    2. A new Sec. 113.124 would be added to read as follows:


Sec. 113.124  Escherichia Coli Bacterin (E. coli).

    Bacterins for the prevention of colibacillosis in mammalian 
neonates born to vaccinated dams shall be prepared from bacterial 
cultures which are inactivated and nontoxic. Each lot of Master Seed 
Bacteria and serial or subserial of product containing Escherichia coli 
shall meet the applicable requirements described in Sec. 113.100 and 
the special requirements prescribed in this section. A lot of Master 
Seed Bacteria found unsatisfactory by any prescribed test shall not be 
used. A serial or subserial found unsatisfactory by any prescribed test 
shall not be released.
    (a) Each lot of Master Seed Bacteria shall be tested for 
immunogenicity in each species for which the bacterin is recommended. 
The immunogenicity of the Master Seed Bacteria shall be established as 
follows:
    (1) A Qualifying Serial of product with a minimum level of potency 
shall be produced from the highest allowable passage of the Master Seed 
Bacteria for use in the immunogenicity test. The allowable passage 
level must be specified in the filed Outline of Production. The 
relative potency of the Qualifying Serial compared to the References 
shall be established as provided in paragraph (b) of this section.
    (2) At least 30 pregnant cows or heifers (20 vaccinates and 10 
controls) or at least 13 sows or gilts (8 vaccinates and 5 controls) 
shall be used as test animals. The animals shall be randomly divided 
between vaccinates and controls. If the neonates to be protected are 
ovine or caprine, the number of test animals must be specified in a 
protocol approved by the Animal and Plant Health Inspection Service.
    (3) Pregnant dams used as vaccinates shall be injected with one 
dose of the serial of product by the method recommended on the label. 
If two doses are recommended, the second dose shall be administered at 
the time interval recommended on the label. Serum samples shall be 
collected from each dam immediately prior to each inoculation, 2 weeks 
after the last inoculation, and at parturition. Colostral samples shall 
be collected from each dam at parturition.
    (4) Challenge of the neonates.
    (i) Challenge culture(s), one for each E. coli pilus type for which 
protection is claimed, shall be provided or approved by the Animal and 
Plant Health Inspection Service. The challenge culture(s) used in the 
efficacy test(s) shall be aliquoted into individual doses, and stored 
frozen at minus 70  deg.C until needed.
    (ii) After parturition, each neonate shall be allowed to suckle 
normally for at least 4 hours. Four to 12 hours after parturition, each 
neonate shall be weighed, have a serum sample collected, and then be 
challenged.
    (iii) Each neonate shall receive orally a predetermined dose of 
challenge culture.
    (iv) Each neonate shall be examined at least twice daily for 7 days 
postchallenge for signs of colibacillosis. Neonates that die shall be 
necropsied to determine the cause of death.
    (5) Interpretation of results.
    (i) If the mortality of the neonates from nonvaccinated bovine dams 
is less than 60 percent, the test is inconclusive and may be repeated.
    (ii) If at least 80 percent of the neonates from vaccinated bovine 
dams do not survive without showing clinical signs of colibacillosis, 
the immunogenicity of the serial of product and the Master Seed 
Bacteria is unsatisfactory.
    (iii) For porcine, ovine, and caprine neonates, if a statistically 
significant greater number of neonates from vaccinated dams do not 
survive, or the survivors do not show a significant reduction in 
clinical signs of colibacillosis, or both, when compared with neonates 
from nonvaccinated control dams, the immunogenicity of the serial of 
product and the Master Seed Bacteria is unsatisfactory. The level of 
significance required is p <0.05. Clinical signs shall be evaluated by 
a method of scoring and statistical analysis acceptable to the Animal 
and Plant Health Inspection Service.
    (b) References for in vitro potency tests.
    (1) Comparison of the Qualifying Serial and Working References
    (i) If the Qualifying Serial is the Working Reference, no 
comparative potency testing between the Qualifying Serial and the 
reference is required.
    (ii) If the Qualifying Serial is not the Working Reference, then 
the potency of the Qualifying Serial relative to the Working Reference 
shall be determined using a parallel line immunoassay that is approved 
by the Animal and Plant Health Inspection Service. To be acceptable, 
the geometric mean of the relative potency values obtained for the 
Qualifying Serial in at least 5 independent parallel line immunoassays 
shall be less than or equal to 1.00.
    (iii) If the Master Reference is different from either the Working 
Reference or the Qualifying Serial, or both, the dilution of the Master 
Reference with a potency equal to that of the Working Reference shall 
be determined by using a parallel line immunoassay as specified in 
paragraph (b)(1)(ii) of this section.
    (2) Dating period for References. References shall have an initial 
dating period equal to the dating period of the product or as supported 
by data acceptable to the Animal and Plant Health Inspection Service. 
The dating period begins on the date of initiation of the 
immunogenicity test or repeat immunogenicity test. The expiration 
date(s) of References shall be stated in the Outline of Production.
    (3) Requalifying a Master Reference.
    (i) To requalify and extend the dating period of a Master 
Reference, a repeat immunogenicity test shall be conducted using a 
Qualifying Serial of product as defined in Sec. 101.5(q)(2). If the 
Master Reference or Working Reference is a serial of product, the 
Qualifying Serial becomes the new Master Reference or the new Working 
Reference.
    (ii) The protocol and the method of evaluation of the repeat 
immunogenicity test to requalify a Master Reference must be approved by 
the Animal and Plant Health Inspection Service. The results of the 
immunogenicity test specified in paragraphs (a)(2) through (a)(5) of 
this section will be a determining factor in what constitutes an 
appropriate test to requalify a Master Reference. Different methods of 
requalifying a Master Reference are:
    (A) Challenge of neonates born to vaccinated host animals.
    (B) Challenge of vaccinated animals of another species whose 
immunological response has been shown to correlate with protection of 
neonates of the species for which the product is recommended.
    (C) Serum and colostral antipilus titers of dams, or serum 
antipilus titers of neonates, or both, when antibody titers show a 
meaningful correlation to protection in the original immunogenicity 
test specified in paragraphs (a)(2) through (a)(5) of this section.
    (iii) Requalifying and extending the dating period of a Master 
Reference may also be done by monitoring the potency of the Master 
Reference by in vitro methods over time by procedures approved by the 
Animal and Plant Health Inspection Service and then conducting a repeat 
immunogenicity test as in paragraphs (b)(3)(i) and (b)(3)(ii) of this 
section when any decline in potency is detected.
    (4) An Outline of Production change must be approved by the Animal 
and Plant Health Inspection Service to provide an extension of the 
expiration date of a Master Reference.
    (c) Test requirements for release of serials.
    (1) Each serial and subserial shall meet the applicable 
requirements prescribed in Sec. 113.100 and the requirements of this 
paragraph. Any serial or subserial found unsatisfactory by a prescribed 
test shall not be released.
    (2) Potency test. Bulk or final container samples of completed 
product shall be tested for potency using the parallel line immunoassay 
and the Working Reference correlated directly to the immunogenicity 
test described in paragraph (a) or indirectly as described in paragraph 
(b)(1) of this section. The potency test(s) must be specific for each 
pilus type for which protection is claimed. The antigen capture 
antibody and the antigen indicator monoclonal antibody for the in vitro 
potency test is supplied by the Animal and Plant Health Inspection 
Service. To be satisfactory and eligible for release, each pilus 
antigen in each serial of product shall have a relative potency greater 
than or equal to 1.00 when compared to the Working Reference(s). 
Serials not satisfactory on the initial relative potency test may be 
retested in accordance with Sec. 113.8 (c)(1) through (c)(5).

    Done in Washington, DC, this 4th day of October 1994.
Terry L. Medley,
Acting Administrator, Animal and Plant Health Inspection Service.
[FR Doc. 94-25056 Filed 10-7-94; 8:45 am]
BILLING CODE 3410-34-M