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

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]

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