[Federal Register Volume 60, Number 89 (Tuesday, May 9, 1995)]
[Rules and Regulations]
[Pages 24547-24549]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-11294]



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DEPARTMENT OF AGRICULTURE
9 CFR Part 113

[Docket No. 93-071-2]


Viruses, Serums, Toxins, and Analogous Products; Detection of 
Extraneous Agents by the Fluorescent Antibody Technique

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

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SUMMARY: We are amending the regulations concerning testing by the 
fluorescent antibody technique for extraneous agents (viruses) in cells 
of animal origin that are used in the manufacture of veterinary 
biologics. The amendment allows the use of alternative fluorochromes 
that may be conjugated to an antibody, revises the list of extraneous 
agents to be tested for, and includes extraneous agents for which 
equine cells are to be tested. In addition, the word ``agent'' is 
replaced with the word ``virus'' since this is the agent being tested 
for. The amendment is necessary to update the requirements related to 
the testing for extraneous viruses.

EFFECTIVE DATE: June 8, 1995.

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

    In accordance with the regulations contained in 9 CFR part 113, 
standard requirements are prescribed for the preparation of veterinary 
biological products. A standard requirement consists of specifications, 
procedures, and test methods which define the standards of purity, 
safety, potency, and efficacy for a given type of veterinary biological 
product. Microorganisms, animal cells, and ingredients of animal origin 
used in production are required to be tested for extraneous viruses. In 
part, this involves testing for the presence of extraneous viruses by 
the fluorescent antibody technique described in Sec. 113.47. When the 
current standard requirement was established, fluorescent antibodies 
were constructed by conjugating antibodies to one of the fluorochromes, 
fluorescein. Fluorochromes are any of a variety of chemicals used in 
cytochemistry to produce a secondary fluorescence in the specimen. In 
the intervening years, additional fluorochromes have been developed for 
use as cytochemical markers or stains.
    Standard requirements included in the regulations specify that 
cells, master seed virus, and most ingredients of animal origin used in 
the production of biological products be tested for contaminating 
bacteria, fungi, mycoplasma, cytopathogenic organisms, viruses, 
hemadsorbing agents, and extraneous agents (viruses) detectable by the 
fluorescent antibody technique. The presence of specific fluorescence 
associated with the use of certain antibodies, in comparison with the 
[[Page 24548]] appropriate controls, is an indication of the presence 
of the contaminating antigen or extraneous virus against which the 
antibody was made.
    Current Sec. 113.47 lists the types of extraneous viruses against 
which fluorescein-conjugated antibodies are to be used in testing cells 
from certain species of animals. New viruses have since been identified 
as animal pathogens. No viruses which are disease agents of horses are 
included in the current Sec. 113.47. As new knowledge has developed, 
testing for these agents has become necessary.
    On March 21, 1994, we published in the Federal Register (59 FR 
13257-13259, Docket No. 93-071-1) a proposal to amend the regulations 
by revising Sec. 113.47 to allow the use of additional fluorescent 
stains in the testing of extraneous viruses by the fluorescent antibody 
technique. The current regulations in Sec. 113.47 limit the stain used 
in the fluorescent antibody test to fluorescein (a fluorochrome). Other 
fluorochromes, when conjugated to antibodies, may be expected to 
perform as well as fluorescein in the test for extraneous viruses. The 
amendment thus allows for the use of alternative fluorochromes in such 
tests. The term ``fluorescein-conjugated antibody'' is replaced with 
``fluorochrome-conjugated antibody'' everywhere it appears in 
Secs. 113.47 and 113.52(b)(2) (i) and (ii).
    The current regulations in Sec. 113.47 lists the specific 
extraneous viruses against which antibodies are used in the testing of 
certain types of cells. We have revised the list of cell types to be 
tested for extraneous viruses to include equine cells. Those using 
other cells for the production of biologics may also be required to 
test for specific viruses before such use is approved.
    We solicited comments concerning our proposal for a 60-day comment 
period ending April 20, 1994. We received one comment by that date from 
a manufacturer of veterinary biological products. We carefully 
considered the comment which is discussed below.
    The comment was in two parts The first was that the fluorescent 
antibody (FA) test was not as sensitive for detecting bluetongue virus 
as seroconversion in sheep, and the use of the FA test would require 
firms to introduce this organism into their testing facility. The 
second was that testing of bovine cells for bovine respiratory 
syncytial virus was also a new requirement.
    In response to the comment, the Animal and Plant Health Inspection 
Service has found that the FA test is a sensitive, specific, and 
accurate test for the detection of extraneous viruses in seeds, cells 
and ingredients of animal origin. When used with the required controls, 
the FA test detects the presence of specific antigens indicative of the 
presence of specific extraneous viruses. The use of positive and 
negative controls with the FA test eliminates the incidence of false 
positives and false negatives. The FA test also has the advantage of 
relieving the firms from the need to locate and maintain seronegative 
sheep.
    APHIS acknowledges that requiring firms to test for the bluetongue 
virus will result in the introduction of the agent onto their premises. 
This should, however, pose no greater problem to the firms than 
introducing other viruses for the purpose of conducting these tests. If 
no firm's biosecurity is adequate to contain such viruses as those 
causing bovine virus diarrhea, canine parvoviral diarrhea, or 
pseudorabies, it should be able to contain bluetongue virus. Further, 
there is no threat of human disease from the use of bluetongue virus in 
association with this test as bluetongue virus is not known to affect 
humans.
    With respect to the requirement for testing bovine cells for bovine 
respiratory syncytical virus, APHIS has determined that this virus may 
be present as an extraneous agent in cells of bovine origin. One of the 
major purposes of the rule is to update the list of extraneous viruses 
for which seeds, cells, and ingredients of animal origin are tested. 
The new requirements are based on current knowledge of animal diseases. 
No changes are made to the regulations in response to the commenter.
    Therefore, based on the rationale set forth in the proposed rule 
and in this document, we are adopting the provisions of the proposal as 
a final rule with one conforming change in Sec. 113.300(c)(1).

Executive Order 12866 and Regulatory Flexibility Act

    This final rule has been reviewed under Executive Order 12866. This 
rule has been determined to be not significant for the purposes of 
Executive Order 12866, and, therefore, has not been reviewed by the 
Office of Management and Budget.
    These amendments should not have a significant economic impact on 
manufacturers since they will broaden the range of fluorochrome stains 
that may be used in conducting the fluorescent antibody test and will 
revise the list of extraneous agents for which various cell types are 
to be tested with the fluorescent antibody technique. The amendments 
will thus remove outdated requirements and provide flexibility in the 
types of antibody that may be used in tests for extraneous agents. On 
balance, therefore, there should be no net increase in testing 
requirements over the current requirements.
    Under these circumstances, the Administrator of the Animal and 
Plant Health Inspection Service has determined that this action will 
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 final rule has been reviewed under Executive Order 12778, 
Civil Justice Reform. This rule: (1) Preempts all State and local laws 
and regulations that are inconsistent with this rule; (2) has no 
retroactive effect; and (3) does not require administrative proceedings 
before parties may file suit in court challenging this rule.

Paperwork Reduction Act

    This rule contains no 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, Exports, Imports, and Reporting and recordkeeping 
requirements.

    Accordingly, 9 CFR part 113 is amended as follows:

PART 113--STANDARD REQUIREMENTS

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

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

    2. Section 113.47 is revised to read as follows:


Sec. 113.47  Detection of extraneous viruses by the fluorescent 
antibody technique.

    The test for detection of extraneous viruses by the fluorescent 
antibody technique provided in this section shall be conducted when 
prescribed in an applicable Standard Requirement or in a filed Outline 
of Production for a product.
    (a) Monolayer cultures of cells (monolayers), at least 7 days after 
the [[Page 24549]] last subculturing, shall be processed and stained 
with the appropriate antiviral fluorochrome-conjugated antibody as 
specified in paragraph (b) of this section.
    (1) Three groups of one or more monolayers shall be required for 
each specific virus prescribed in paragraph (b) of this section.
    (i) At the time of the last subculturing, one group of test 
monolayers shall be inoculated with approximately 100-300 FAID<INF>50 
of the specific virus being tested for as positive controls.
    (ii) One group of monolayers shall be the ``material under test.''
    (iii) One group of monolayers, that are of the same type of cells 
as the test monolayers and that have been tested as prescribed in 
Secs. 113.51 or 113.52 (whichever is applicable), shall be prepared as 
negative controls.
    (2) Each group of monolayers shall have a total area of at least 6 
cm\2\.
    (3) Positive control monolayers may be fixed (processed so as to 
arrest growth and assure attachment of the monolayer to the surface of 
the vessel in which they are grown) before 7 days after subculturing if 
fluorescence is enhanced by doing so, Provided, That a monolayer of the 
material under test is also fixed at the same time as the positive 
control and a monolayer of the material under test is also fixed at 
least seven days after subculturing. Monolayers that are fixed before 7 
days after subculturing shall be stained at the same time as the test 
monolayers and negative controls fixed at least 7 days after 
subculturing.
    (b) The antiviral fluorochrome-conjugated antibodies to be used 
shall depend on the type of cells required to be tested for extraneous 
viruses as specified in an applicable Standard Requirement or in a 
filed Outline of Production. Antiviral fluorochrome-conjugated 
antibodies specific for the extraneous viruses shall be applied to each 
respective type of cell in accordance with the following list. Under 
certain circumstances, additional tests may need to be conducted, as 
determined by the Administrator. When a specific antiviral 
fluorochrome-conjugated antibody is used in testing for the listed 
extraneous viruses specified in more than one cell type, it need only 
be applied to the most susceptible cell type.

(1) All cells shall be tested for:
    (i) Bovine virus diarrhea virsus;
    (ii) Reovirus; and
    (iii) Rabies virus.
(2) Bovine, caprine, and ovine cells shall, in addition, be tested for:
    (i) Bluetongue virus;
    (ii) Bovine adenoviruses;
    (iii) Bovine parvovirus; and
    (iv) Bovine respiratory syncytial virus.
(3) Canine cells shall, in addition, be tested for:
    (i) Canine coronavirus;
    (ii) Canine distemper virus; and
    (iii) Canine parvovirus.
(4) Equine cells shall, in addition, be tested for:
    (i) Equine herpesvirus; and
    (ii) Equine viral arteritis virus.
(5) Feline cells shall, in addition, be tested for:
    (i) Feline infectious peritonitis virus; and
    (ii) Feline panleukopenia virus.
(6) Porcine cells shall, in addition, be tested for:
    (i) Porcine adenovirus;
    (ii) Porcine parvovirus;
    (iii) transmissible gastroenteritis virus; and
    (iv) Porcine hemagglutinating encephalitis virus.
(7) Firms that do not have rabies virus on premises either for research 
or production purposes are exempt from having to produce positive 
rabies virus control monolayers. Fixed positive rabies virus control 
monolayers will be provided by the National Veterinary Services 
Laboratories.

    (c) After staining, each group of monolayers shall be examined for 
the presence of specific fluorescence attributable to the presence of 
extraneous viruses.
    (1) If the material under test shows any evidence of specific viral 
fluorescence, it is unsatisfactory and may not be used; Provided, That, 
if specific fluorescence attributable to the virus being tested for is 
absent in the positive control monolayers, the test is inconclusive and 
may be repeated.
    (2) If the fluorescence of the monolayers inoculated with the 
specific virus as positive controls is equivocal, or if the negative 
monolayers show equivocal fluorescence indicating possible viral 
contamination, or both, the test shall be declared inconclusive, and 
may be repeated; Provided, That, if the test is not repeated, the 
material under test shall be regarded as unsatisfactory for use in the 
production of biologics.
    3. Section 113.52, paragraph (b)(2)(i) and (ii), is revised to read 
as follows:


Sec. 113.52  Requirements for cell lines used for production of 
biologics.

* * * * *
    (b) * * *
    (2) * * *
    (i) At least two monolayers shall be stained with an antispecies 
fluorochrome-conjugated antibody unrelated to the species of origin of 
the MCS.
    (ii) At least two monolayers shall be stained with an antispecies 
fluorchrome-conjugated antibody specific to the species of origin of 
the MSC.
* * * * *


Secs. 113.51, 113.52 and 113.53  [Amended]

    4. In the following places, the word ``agents'' are removed and the 
word ``viruses'' added in its place:
    a. Section 113.51, paragraph (c)(3)(ii).
    b. Section 113.52, paragraph (f)(4)(ii).
    c. Section 113.53, paragraph (c)(6)(ii).
    5. In Sec. 113.300, paragraph (c)(1), the term ``fluorescein'' is 
removed and the term ``fluorochrome'' added in its place.

    Done in Washington, DC, this 1st day of May 1995.
B. Glen Lee,
Acting Administrator, Animal and Plant Health Inspection Service.
[FR Doc. 95-11294 Filed 5-8-95; 8:45 am]
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