[Federal Register Volume 60, Number 95 (Wednesday, May 17, 1995)]
[Proposed Rules]
[Pages 26381-26384]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-12152]



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DEPARTMENT OF AGRICULTURE
9 CFR Parts 101 and 113

[Docket No. 94-051-1]
RIN 0579-AA66


Viruses, Serums, Toxins, and Analogous Products; In Vitro Tests 
for Serial Release

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Proposed rule.

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SUMMARY: We are proposing to amend the regulations regarding the use of 
in vitro potency tests in place of animal tests for immunogenicity to: 
change the title of the section; prescribe requirements for in vitro 
immunoassays used to determine the relative antigen content of 
inactivated biological products; require that such immunoassays be 
parallel line assays based upon unexpired reference preparations; 
require that in vitro tests for relative antigen content be converted 
to parallel line assays within 2 years; specify procedures and 
requirements for qualifying or requalifying reference preparations for 
inactivated products; and add certain definitions to the regulations.
    The effect of the amendment would be to standardize the methods 
used to determine the relative potency of inactivated biological 
products.

DATES: Consideration will be given only to comments received on or 
before August 15, 1995.

ADDRESSES: Please send an original and three copies of your comments to 
Docket No. 94-051-1, Regulatory Analysis and Development, PPD, APHIS, 
Suite 3C03, 4700 River Road Unit 118, Riverdale, MD 20737-1238. Please 
state that your comments refer to Docket No. 94-051-1. 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, 4700 River Road Unit 148, Riverdale, MD 20737-1237, (301) 734-
8245.

SUPPLEMENTARY INFORMATION: 

Background

    The regulations pertaining to the testing of biologics provide that 
no biological product shall be released (for sale) prior to the 
completion of tests prescribed to establish the product to be pure, 
safe, potent, and efficacious (9 CFR 113.5). Efficacy refers to the 
specific ability of the product to effect the result for which it is 
offered when used as recommended by the manufacturer. Studies conducted 
to establish efficacy include immunogenicity tests in host animals 
using product manufactured according to specific requirements which 
include specifications for antigen content and/or animal potency. These 
requirements apply to every serial of product which is produced. 
Therefore, if a product has been tested for immunogenicity in animals 
and shown to have the desired effect, it follows that subsequent 
serials (batches) of the product manufactured to the same 
specifications should also have the same effect.
    Once immunogenicity is established in relation to a specific 
minimum antigen content in a product, it should no longer be necessary 
to test every subsequent serial for potency in animals if an evaluation 
can be made with reasonable certainty of the relative antigen content 
by testing the serial or subserial in an acceptable in vitro test 
system. Therefore, when properly qualified and validated, in vitro 
immunoassays that determine relative antigen content of the product can 
serve as acceptable substitutes for potency tests that otherwise would 
need to be performed in animals.
    The regulations in 9 CFR 113.8 pertain to the use of in vitro tests 
in place of animal tests for determining the potency of veterinary 
biological products. Currently, the in vitro tests prescribed in 
Sec. 113.8 which include determining the log10 virus titer and 
performing live bacterial counts are only applicable to veterinary 
biologicals which contain live microorganisms. The changes and test 
procedures prescribed in this proposal would make Sec. 113.8 applicable 
to both live and inactivated products by prescribing validity 
requirements for in vitro test systems used, in place of animal tests, 
to test for the potency of inactivated products.
    We are proposing to amend the title of Sec. 113.8 to read: ``In 
vitro potency tests for serial release.'' This change is intended to 
clarify the fact that the in vitro procedures described in Sec. 113.8 
are applicable to in vitro tests used to release serials or subserials 
of veterinary biological products after the prescribed animal 
protection studies required for licensing have been completed. In the 
case of inactivated products, the proposal specifies that in vitro 
immunoassays (test systems) which compare the relative antigen content 
(relative potency) of a test serial to a reference preparation must be 
parallel line assays using an unexpired reference preparation whose 
potency has been correlated directly or indirectly to immunogenicity in 
host animals.
    In addition, the proposal would require: confirming the accuracy of 
the protective dose established for live products 3 years after the 
initiation of the host animal immunogenicity study; and confirming the 
immunogenicity of reference preparations used in immunoassays for 
inactivated biological products prior to their expiration. The 
expiration date for a reference would be equal to the dating of the 
product or as supported by data acceptable to APHIS.
    APHIS is proposing these amendments because current requirements 
for many of the immunoassays being used to release serials or 
subserials of product do not have uniform validity criteria and do not 
include a provision to confirm periodically the immunogenicity of the 
reference used in such immunoassays. The proposed amendment would 
standardize the requirements for in vitro potency tests for relative 
antigen content and update and improve the reliability of such tests 
that are currently included in filed outlines of production. The 
proposed amendment does not specify a particular immunoassay provided 
that it is a parallel line assay using an unexpired reference 
preparation. While there is not a generally accepted ``best'' 
immunoassay, there is general agreement that an acceptable 
[[Page 26382]] immunoassay must demonstrate linearity, specificity, and 
reproducibility; and that the reference must be capable of eliciting a 
protective immune response in animals for as long as it serves as the 
reference.
    APHIS has selected the parallel line assay because it demonstrates 
linearity, specificity, and reproducibility, and also compares the 
``similarity'' of the responses elicited by the test and reference 
preparations in the immunoassay. APHIS feels that the ``similarity'' 
feature is critical to the acceptance of any in vitro immunoassay 
purporting to measure relative antigenic content. We realize that the 
proposed amendment would necessitate the revalidation of immunoassays 
currently contained in some filed outlines of production and propose to 
implement the requirements as set out below.
    Firms with filed outlines of production for licensed products with 
in vitro potency tests that are immunoassays that are not parallel line 
assays would be allowed 2 years after the effective date of the final 
rule to come into compliance with the proposed amendments. In the 
interim, immunoassays, utilizing unexpired references, contained in 
previously approved outlines of production would continue to be allowed 
for serial release of previously licensed fractions but would not be 
acceptable for fractions not previously licensed to the firm. Firms 
with filed outlines of production for licensed product with in vitro 
potency tests for relative antigen content would be required to use 
unexpired references. References that have expired or that are about to 
expire would need to be requalified or have the dating period extended 
in accordance with protocols and time schedules acceptable to APHIS.
    APHIS has determined that immunoassays that are not based on a 
parallel line assay using an unexpired reference may not provide 
reliable relative potency data in all instances. Such instances include 
the determination of relative potency based on a reference preparation 
that may have expired or the extrapolation of data based upon standard 
curves that may not be proportional between serial and reference. These 
amendments are being proposed in order to provide greater assurance 
that a serial of product provides adequate potency in all instances.
    We are also adding to the regulations in Sec. 101.5 definitions of 
the term ``immunogenicity'', and the terms ``master reference'', 
``working reference'', and ``qualifying serial'' as they apply to 
reference preparations used in in vitro immunoassays.
    Licensees, researchers, and scientists at the National Veterinary 
Services Laboratories, U.S. Department of Agriculture, have cooperated 
in the development of this proposed rule. We are therefore proposing to 
amend Secs. 101.5 and 113.8 as set forth below.

Executive Order 12866 and Regulatory Flexibility Act

    This proposed rule has been reviewed under Executive Order 12866. 
The rule has been determined to be no significant for purposes of 
Executive Order 12866, and therefore, has not been reviewed by the 
Office of Management and Budget.
    This proposed amendment, if adopted, would allow any valid in vitro 
immunoassay to be used in determining the relative antigen content of 
an inactivated veterinary biological product, provided that it 
satisfies the parallel line criteria and that it is conducted using an 
unexpired reference preparation that has been tested, directly or 
indirectly, for immunogenicity in a manner acceptable to APHIS. This 
amendment would affect all licensed manufacturers of veterinary 
biologicals utilizing in vitro relative potency immunoassays for 
determining the potency of inactivated products. This proposal, 
however, does not impose any additional economic burden since the 
testing of product for potency is already required under Sec. 113.5 of 
the regulations and outlines of production are routinely amended and 
updated. Section 113.5 specifies that no biological product shall be 
released prior to the completion of tests prescribed in a filed outline 
of production or standard requirement to establish that the product is 
pure, safe, potent, and efficacious. In the absence of a standard 
requirement prescribing a specific potency test for inactivated 
products, the firms develop a potency test suitable for their product, 
and designate such tests in the outline of production that is filed 
with APHIS. Currently, firms are using host animal tests, laboratory 
animal tests, and a variety of in vitro immunoassays as potency tests 
for inactivated products. This proposed rule does not restrict the 
firm's discretion to choose the most appropriate test for its product. 
The proposed rule would only prescribe validity requirements for in 
vitro immunoassays for relative potency. The overall effect of this 
proposed amendment would be to standardize in vitro immunoassays that 
are used to determine the potency of inactivated veterinary biological 
products.
    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 12372

    This program/activity is listed in the Catalog of Federal Domestic 
Assistance under No. 10.025 and is subject to Executive Order 12372, 
which requires intergovernmental consultation with State and local 
officials. (See 7 CFR part 3015, subpart V.)

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

    In accordance with the Paperwork Reduction Act of 1980 (44 U.S.C. 
3501  et seq.), the information collection or recordkeeping 
requirements included in this proposed rule have been approved by the 
Office of Management and Budget (OMB), and there are no new 
requirements. The assigned OMB control number is 0579-0013.

List of Subjects

9 CFR Part 101

    Animal biologics.

9 CFR Part 113

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

    Accordingly, 9 CFR parts 101 and 113 would be amended as follows:

PART 101--DEFINITIONS

    1. The authority citation for part 101 would continue to read as 
follows:

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

    2. Section 101.5 would be amended by adding new paragraphs (o), 
(p), (q), and (r) to read as follows:


Sec. 101.5  Testing terminology.

 * * * * *
    (o) Master Reference. A Master Reference is a reference whose 
potency is correlated, directly or indirectly, to host animal 
immunogenicity. The Master Reference may be used as the working 
reference in in vitro tests for relative potency. The Master Reference 
[[Page 26383]] may also be used to establish the relative potency of a 
serial of product used in requalification studies and to establish the 
relative potency of working references. A Master Reference may be:
    (1) A completed serial of vaccine or bacterin prepared in 
accordance with a field Outline of Production;
    (2) A purified preparation of the protective immunogen or antigen; 
or
    (3) A nonadjuvanted harvested culture of microorganisms.
    (p) Working Reference. A Working Reference is the reference 
preparation that is used in the in vitro test for the release of 
serials of product. Working References may be:
    (1) Master References; or
    (2) Serials of product that have been prepared and qualified, in a 
manner acceptable to APHIS, for use as reference preparations.
    (q) Qualifying Serial. (1) A serial of biological product used to 
test for immunogenicity when the Master or Working Reference is a 
purified antigen or nonadjuvanted harvest material. Qualifying serials 
shall be produced in accordance with the filed Outline of Production, 
tested for immunogenicity in host animals in accordance with protocols 
acceptable to Animal and Plant Health Inspection Service, and have a 
geometric mean relative potency, when compared to the Master Reference, 
of not greater than 1.0 as established by independent parallel line 
assays with 5 or more replicates.
    (2) Qualifying serials used to requalify or extend the dating 
period of a Master Reference in a repeat immunogenicity test shall 
satisfy all criteria prescribed above and, it addition, shall have been 
prepared within 6 months of requalifying testing, i.e., the initiation 
of the repeat immunogenicity test.
    (r) Immunogenicity. The ability of a biological product to elicit 
an immune response in animals as determined by test methods or 
procedures acceptable to the Animal and Plant Health Inspection 
Service.

PART 113--STANDARD REQUIREMENTS

    3. 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).

    4. Section 113.8 would be amended as follows:
    a. The section heading would be revised to read as set forth below.
    b. Paragraph (a) would be revised to read as set forth below.
    c. Paragraph (b) the introductory text would be revised to read as 
set forth below.
    d. Paragraph (b)(5) would be revised to read as set forth below.
    e. Paragraph (c) would be redesignated as paragraph (e) and new 
paragraphs (c) and (d) would be added to read as set forth below.
    f. In redesignated paragraph (e), in the introductory text, the 
reference to ``paragraph (b)'' would be removed and ``Paragraphs (b) 
and (c)'' would be added in its place. In paragraph (e)(4), the 
reference to ``paragraphs (c)(1),'' would be removed and ``paragraphs 
(e)(1),'' would be added in its place.


Sec. 113.8  In vitro potency tests for serial release.

    (a) Master Seed which has been established as pure, safe, and 
immunogenic shall be used for preparing seed for production as 
specified in the Standard Requirements or in the filed Outline of 
Production. The Administrator may exempt a product from a required 
animal potency test for release when an evaluation can, with reasonable 
certainty, be made by:
    (1) Subjecting the master seed to the applicable requirements 
prescribed in Secs. 113.64, 113.100, 113.200, and 113.300;
    (2) Testing the Master Seed for immunogenicity in a manner 
acceptable to the Animal and Plant Health Inspection Service (APHIS);
    (3) Establishing a satisfactory potency for live products based on 
the protective dose used in the Master Seed immunogenicity test plus an 
adequate overage allowance for adverse conditions and test error; and
    (4) For inactivated products, determining the potency of each 
serial or subserial, or both, using an accepted test system. Acceptable 
potency tests shall include:
    (i) Determining the log10 live virus titer;
    (ii) Determining the live bacterial count; or
    (iii) For inactivated products, determining the relative antigen 
content, as compared with a reference, using a parallel line 
immunoassay.
    (b) In the case of live products, each serial and subserial of 
desiccated product derived from an approved Master Seed and bulk or 
final container samples of each serial of completed liquid product 
derived from an approved Master Seed shall be evaluated by a test 
procedure acceptable to APHIS. On the basis of the results of the test, 
as compared with the required minimum potency, each serial and 
subserial shall either be released to the firm for marketing or 
withheld from the market. The evaluation of such products shall be made 
in accordance with the following criteria:
    (1) * * *
* * * * *
    (5) Exceptions. When a product is evaluated in terms other than 
log10 virus titer or organism count, an appropriate difference 
between the average potency value obtained in the retests and the 
potency value obtained in the initial test shall be established for use 
in paragraphs (b)(3) or (b)(4) of this section to evaluate such 
products and shall be specified in the Product Standard Requirement or 
filed Outline of Production.
    (c) In the case of inactivated products, bulk or final container 
samples of completed product from each serial derived from an approved 
Master Seed, shall be evaluated for relative antigen content (potency), 
as compared with a reference, by a parallel line immunoassay procedure 
acceptable to APHIS. Firms currently using immunoassays which do not 
meet the requirements of a parallel line assay shall have 2 years from 
the effective date of the final rule to update their filed Outlines of 
Production to be in compliance with this requirement. On the basis of 
the results of such test procedures, each serial that meets the 
required minimum potency shall be released to the firm for marketing; 
each serial not meeting the required minimum potency shall be withheld 
from the market. The evaluation of such products shall be made in 
accordance with the following criteria:
    (1) A test that results in no valid lines is considered a no test 
and may be repeated.
    (2) An initial test that results in valid lines that are not 
parallel is considered a valid equivocal test. Release of the serial 
may not be based on such test since the result cannot be termed 
``satisfactory'' or ``unsatisfactory''.
    (3) If the initial test shows that potency equals or exceeds the 
required minimum potency, the serial is satisfactory without additional 
testing.
    (4) If the initial test is an equivocal test due to lack of 
parallelism, the serial may be retested up to three times: Provided, 
That, if the test is not repeated, the serial shall be deemed 
unsatisfactory.
    (i) If more than 50% of all valid repeat tests show that potency 
equals or exceeds the required minimum potency, the serial is 
satisfactory.
    (ii) If greater than 50% of all valid repeat tests show either lack 
of parallelism or that potency is less than the required minimum 
potency, the serial is unsatisfactory. [[Page 26384]] 
    (5) If the initial test shows that potency is less than the 
required minimum potency, the serial may be retested. If retested, two 
additional tests, must be conducted: Provided, That, if the serial is 
not retested, the serial shall be deemed unsatisfactory.
    (i) If more than 50% of all valid tests show that potency equals or 
exceeds the required minimum potency, the serial is satisfactory.
    (ii) If more than 50% of all valid tests show either lack of 
parallelism or that potency is less than the required minimum potency, 
the serial is unsatisfactory.
    (d) Repeat immunogenicity tests.
    (1) The accuracy of the protective dose established for live 
products in the Master Seed immunogenicty test and defined as live 
virus titer or live bacterial count shall be confirmed in 3 years in a 
manner acceptable to APHIS, unless use of the lot of Master Seed 
previously tested is discontinued.
    (2) All determinations of relative antigen content using parallel 
line immunoassays shall be conducted with an unexpired reference. The 
lot of reference used to determine antigenic content shall have an 
initial dating period equal to the dating of the product or as 
supported by data acceptable to APHIS. Prior to the expiration date, 
such reference may be granted an extension of dating by confirming its 
immunogenicity using a Qualifying Serial of product. Tests to establish 
or confirm immunogenicity of references shall be conducted in a manner 
acceptable to APHIS. The dating period of the Master Reference and 
Working Reference may be extended as supported by data acceptable to 
APHIS if the minimum potency of the Master Reference is determined to 
be adequately above the minimum level needed to provide protection in 
the host animal. If a new Master Reference is established, it shall be 
allowed an initial dating period equal to the dating of the product or 
as supported by data acceptable to APHIS.
* * * * *
    Done in Washington, DC, this 11th day of May 1995.
Terry L. Medley,
Acting Administrator, Animal and Plant Health Inspection Services.
[FR Doc. 95-12152 Filed 5-16-95; 8:45 am]
BILLING CODE 3410-34-M