[Federal Register Volume 62, Number 75 (Friday, April 18, 1997)]
[Rules and Regulations]
[Pages 19033-19039]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 97-10100]


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DEPARTMENT OF AGRICULTURE

Animal and Plant Health Inspection Service

9 CFR Parts 101 and 113

[Docket No. 94-051-3]
RIN 0579-AA66


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

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

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SUMMARY: We are amending the regulations to provide for the use of in 
vitro potency tests when conducting immunoassays to determine the 
relative antigen content (potency) of a serial of inactivated 
veterinary biological product once immunogenicity is established using 
host animal tests. Such tests would be conducted using unexpired 
immunogenic reference preparations and parallel line assays, or other 
methods which demonstrate linearity, specificity, and reproducibility 
at least equivalent to the parallel line assay. Firms currently using 
immunoassays which do not meet the standard in this amendment will have 
2 years from the effective date of this final rule to update their 
filed Outlines of Production. This amendment also changes the title of 
the section and adds definitions of ``Master reference,'' ``Working 
reference,'' ``Qualifying serial,'' and ``Immunogenicity'' to the 
regulations.
    The effect of this action is to standardize requirements for in 
vitro immunoassay potency tests for inactivated products which cannot 
be evaluated on the basis of virus titer or bacterial counts.

EFFECTIVE DATE: May 19, 1997.

FOR FURTHER INFORMATION CONTACT: Dr. David A. Espeseth, Director, 
Center for Veterinary Biologics, Licensing and Policy Development, VS, 
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. Tests to 
establish efficacy include immunogenicity tests in host animals using 
product which is manufactured according to specified requirements which 
include specifications for antigen content and/or animal potency. If a 
product has been tested for immunogenicity in animals and shown to 
elicit the desired immune response, it should follow that subsequent 
serials (batches) of the product manufactured to the same 
specifications should also have the same effect. Based on this premise, 
once immunogenicity is established in relation to a specific minimum 
antigen content, it should no longer be necessary to test every 
subsequent product serial for potency in animals if an evaluation of 
the relative antigen content can be made 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 a 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 
for determining the potency of serials and/or subserials of veterinary 
biological products after required animal tests are completed. Prior to 
this amendment, the in vitro test procedures prescribed in Sec. 113.8 
were only applicable to products containing live microorganisms. With 
these amendments Sec. 113.8 will be applicable to both live and 
inactivated products.
    On May 17, 1995, we published in the Federal Register (60 FR 26381-
26384, Docket No. 94-051-1) a proposal to amend the regulations 
regarding the use of in vitro potency tests in place of animal tests 
for immunogenicity. The proposed rule provided for the use of a 
parallel line assay, or other valid method, and an unexpired reference 
preparation in an in vitro immunoassay for relative antigen content to 
determine the potency of a serial of inactivated product. In proposing 
the parallel line assay or equivalent valid method and the use of an 
unexpired reference as a standard for in vitro immunoassay potency 
tests for serial release, APHIS did not intend to preclude the 
validation of existing in vitro immunoassays or the adoption of 
technological advances in antigen quantitation.
    We solicited comments concerning our proposal for 90 days ending 
August 15, 1995. We extended the comment period an additional 30 days 
ending September 14, 1995 (60 FR 36743-36744, Docket No. 94-051-2, July 
18,

[[Page 19034]]

1995). We also announced that we would be having a public hearing on 
August 1, 1995, in Ames, IA, to have further discussion related to in 
vitro testing by interested persons. We received comments from four 
licensed manufacturers, and a national trade association representing 
U.S. manufacturers of animal health products. Three comments from 
biologics producers were received at the public hearing on August 1, 
1995, in Ames, IA. While generally supportive of in vitro immunoassay 
tests for determining the relative antigen content and thereby the 
potency of products, most commenters suggested changes in one or more 
sections as proposed. Others suggested that the comment period be 
extended and the proposal be submitted to negotiated rulemaking. We 
carefully considered all of the comments we received. They are 
discussed below.
Analysis of Comments and APHIS' Response
    Four commenters requested that the comment period be further 
extended for 10 months beyond September 14, 1995, and that negotiated 
rulemaking be initiated. In response to this comment, APHIS notes that 
the history of this rulemaking began with a proposed rule published on 
May 17, 1995 (60 FR 26381-26384, Docket No. 94-051-1). The comment 
period of 90 days was extended to 120 days until September 14, 1995, in 
response to a request for an extension from a national trade 
association (See 60 FR 36743, July 18, 1995, Docket No. 94-051-2). In 
addition, a public hearing was held on In Vitro Potency Testing on 
August 1, 1995, in Ames, IA to obtain further comment on this topic. 
Contrary to the commenters' request, the comment period cannot be 
further extended for negotiated rulemaking because the initiation of 
negotiated rulemaking necessitates the withdrawal of the current 
proposal and the proposal of another rule after the conclusion of the 
negotiated rulemaking. APHIS believes that the publication of a final 
rule with appropriate consideration of responses and comments would be 
a more efficient way of handling this matter and would allay concerns 
and clarify issues raised by the commenters. Therefore, the request for 
further extension of the comment period and initiation of negotiated 
rulemaking is not granted.
    Two commenters expressed concern that by specifying that in vitro 
immunoassays used to determine relative antigen content be parallel 
line assays, APHIS would be imposing a requirement which would not 
allow the industry to take advantage of technological advances that are 
occurring in the area of antigen quantitation. APHIS proposed the 
parallel line assay as a standard for immunoassay tests for relative 
antigen content. Assay formats which are equivalent to or exceed the 
parallel line assay standard could have been used as provided for in 9 
CFR 113.4. In response to these comments, however, APHIS has amended 
Secs. 101.5(q) and 113.8(a) in the final rule to provide specifically 
for the use of other valid methods for determining relative antigen 
content which demonstrate linearity, specificity, and reproducibility 
at least equivalent to the parallel line assay.
    Five commenters recommended amending the rule to allow laboratory 
animal tests and antibody titers that have been correlated to host 
animal protection to be used to requalify or extend the dating of 
reference preparations. One of the commenters pointed out that the 
proposed standard requirement for Escherichia coli (E. coli) bacterins 
(59 FR 51390-51392, October 11, 1994) which also uses a parallel line 
immunoassay to test for potency, includes such a provision. In 
addition, the commenter interpreted the E. coli standard requirement to 
imply that in vitro assays may be used in place of reference 
requalification in host animals. In response to the commenter, APHIS 
agrees that the proposed E. coli standard requirement allows antibody 
titers and laboratory animal studies, previously correlated to 
protection, to be used to requalify reference preparations. These same 
provisions were available under the proposal to amend Secs. 101.5 and 
113.8 (See proposed terminology in Sec. 101.5(o) which provides for 
direct or indirect correlation of potency to host animal 
immunogenicity). However, by specifying in proposed Sec. 101.5(q)(1) 
that Qualifying Serials used to requalify or extend the dating of a 
Master Reference shall be ``tested for immunogenicity in host 
animals,'' APHIS may have inadvertently implied that laboratory animal 
tests could not be used for reference requalification. This was not the 
intent of the proposed regulation. In response to the commenter, the 
final rule has been amended in Sec. 101.5(q)(1) to clarify the 
definition of Qualifying Serial to provide for the use of procedures 
acceptable to APHIS which will include antibody titers and laboratory 
animal testing along with host animal immunogenicity for reference 
requalification.
    In response to the comment regarding the use of in vitro assays to 
requalify or extend the dating of a reference in place of performing 
studies in animals, in vitro tests may not be substituted for animal 
tests for reference requalification. The proposed E. coli standard 
requirement stated that an in vitro procedure may be used to monitor 
the potency of the Master Reference for indication of decline, but 
specified that the reference must be requalified when a decline in 
potency is detected. As proposed in the proposed E. coli standard 
requirement, the immunogenicity of Qualifying Serials used in reference 
requalification studies may be based on host animal studies (challenge 
or antibody titer) or laboratory animal studies as provided in 
protocols acceptable to APHIS. Therefore, to clarify these points and 
to eliminate the apparent inconsistency between the two proposed rules, 
APHIS is amending Sec. 113.8(d)(2) pertaining to in vitro testing to 
include a monitoring provision and to clarify that: (1) The monitoring 
procedure can only be used to monitor the unexpired reference to detect 
when a decline in potency has occurred between requalification 
intervals, and (2) to specify that, if such monitoring procedures 
indicate the potency of the reference is declining, the reference must 
be requalified either by testing a Qualifying Serial in host animals or 
by providing other evidence of reference immunogenicity, e.g., antibody 
titers or laboratory animal test data previously correlated to host 
animal protection, or a new reference must be prepared and qualified. 
In vitro monitoring, however, would not be a substitute for reference 
requalification at the end of product dating.
    One commenter suggested amending Sec. 113.8(c)(5) to include a 
provision to allow a firm to declare a potency test with valid lines a 
``no test'' if the firm does not have confidence in the test result. 
APHIS does not agree that it would be appropriate to declare such a 
test a ``no test''. The regulation, as proposed, allows a firm to 
retest a serial two times when the initial test shows that potency is 
less than the required minimum potency. The commenter's suggestion, 
however, would make potency testing subjective and allow a firm to 
disregard valid results that are not consistent with a desired outcome. 
Conceivably, a serial with unsatisfactory test results could be 
retested indefinitely. In response to this comment, APHIS has clarified 
provisions for the retesting of such serials and permitted up to three 
retests to be performed. Provisions have also been added to permit the 
potency test to be repeated under certain specified conditions.
    Two commenters requested that firms be allowed more than two years 
to

[[Page 19035]]

convert currently approved in vitro immunoassays that are described in 
filed Outlines of Production that are not parallel line assays, to 
parallel line assays or to another method which demonstrates linearity, 
specificity, and reproducibility at least equivalent to the parallel 
line assay. They believed that the two-year timetable will have a 
negative impact on new product development, and therefore result in 
fewer new products on the market. In response, APHIS realizes that some 
firms may require more than two years to convert to parallel line 
assays or other valid methods. However, two years from the effective 
date of the final rule should be adequate time for most firms to 
validate their immunoassays and requalify references for existing 
products, considering that a single reference requalification procedure 
may be applicable to several different products. Also, those firms 
experiencing difficulty in meeting the time period may be granted 
additional time, if justified, by requesting an extension as provided 
in the regulations. Therefore, no change to the regulations is made in 
response to these comments.
    One commenter requested that the definition of ``Master Reference'' 
in Sec. 101.5(o) be amended to include options and directions for 
stabilizing and storing reference preparations. The commenter believed 
that this will result in more options for treating the references. 
APHIS does not agree that the rule needs to be amended. The definition 
of a ``Master Reference'' does not limit the options available to firms 
when it comes to stabilizing, storing, lyophilizing, or freezing Master 
References provided that such procedures are described in the filed 
Outline of Production. Specifying such procedures in the definition, 
however, would limit the industry to the procedures defined. Since the 
proposed definition does not limit the available options, no change to 
the regulations is made in response to this comment.
    Another commenter requested clarification of proposed Sec. 101.5(p) 
of the regulations. The commenter inquired if a purified antigen 
preparation could serve as the Working Reference. As proposed in 
Sec. 101.5(p) of the regulations, the Working Reference may be the 
Master Reference, and since the Master Reference may be a purified 
preparation of the protective immunogen (antigen), it follows that a 
purified antigen can serve as the Working Reference. Therefore, no 
change to the regulations is made in response to this comment.
    One commenter recommended amending proposed Sec. 101.5(q)(1) of the 
rule to require Qualifying Serials for reference requalification to be 
produced at the minimum antigen level specified in the Outline of 
Production instead of specifying that the geometric mean relative 
potency not exceed 1.0 when compared to the Master Reference. The 
commenter reasoned that, by specifying that the amount of antigen in 
the Qualifying Serial not exceed the amount of antigen contained in the 
Master Reference, the antigen level contained in the Master Reference 
is a more appropriate benchmark (measure of protection) than is the 
antigen content specified in the Outline of Production. The commenter 
believed that the amount of antigen specified in the Outline of 
Production should establish the antigen requirement for the Qualifying 
Serial. APHIS does not agree with the commenter's recommendation. In 
measuring relative potency, the antigen level used to demonstrate host 
animal protection becomes the benchmark by which other serials are 
measured and is the level of antigen to be contained in a Qualifying 
Serial that is used to determine if the Master Reference is still 
protective and therefore eligible for continued use in the potency 
assay. The commenter's recommendation of using a regular production 
serial and devising a calculation procedure to show antigen equivalency 
is an indirect method that was considered by APHIS and determined to be 
inappropriate and less meaningful than the provision in the APHIS 
proposal. Therefore, no change to the regulations is made in response 
to this comment.
    Two commenters expressed confusion regarding proposed 
Secs. 113.8(a)(4) (i) and (ii) of the rule. The commenters noted that 
although Sec. 113.8(a)(4) refers to in vitro methods for determining 
the potency of inactivated products, the cited examples, i.e., 
determining log10 virus titer and determining the live bacterial 
count only apply to live products. APHIS agrees that the wording of 
proposed Sec. 113.8(a)(4) is contradictory and has amended the final 
rule, eliminating the contradictory sections, by incorporating the 
provisions of Sec. 113.8(a)(4) into Sec. 113.8(a)(3) as follows:

    (3) Establishing a satisfactory potency test for the product in 
accordance with the following provisions:
    (i) Potency of live products may be determined by log10 
virus titer or determining the live bacterial count based on the 
protective dose used in the Master Seed immunogenicity test plus an 
adequate overage for adverse conditions and test error; and
    (ii) Potency for inactivated products may be determined using 
tests for relative antigen content by comparing the antigen content 
of the test serial to a reference preparation using a parallel line 
immunoassay or equivalent method which measures linearity, 
specificity, and reproducibility in a manner acceptable to APHIS.

    One commenter requested that the phrase ``an appropriate 
difference'' referred to in proposed Sec. 113.8(b)(5) be further 
defined. Proposed Sec. 113.8(b)(5) pertains to in vitro potency tests 
for live vaccines in which potency is measured in terms other than 
log10 virus titer or live bacterial counts, e.g., Marek's Disease 
vaccines in which potency is measured in terms of plaque forming units 
(PFU). Generally, an appropriate difference pertains to how a serial is 
determined to have satisfactory potency when the initial potency test 
determines that the serial contains less than the number of PFU's 
specified in the Outline of Production (OP) or standard requirement and 
the manufacturer elects to retest the serial to rule out test system 
error as the cause of the unsatisfactory test result. In accordance 
with Sec. 113.8(b)(5), the manufacturer must specify in the OP the 
difference between the average PFU count obtained in the retest and the 
PFU count obtained in the initial test so that the initial test may be 
considered a result of test system error. The commenter did not suggest 
what this appropriate difference in PFU or organism count should be. 
APHIS has noted that the appropriate difference between test results 
may be different for each product and this is the reason the proposed 
rule specified that this value should be placed in the product Standard 
Requirement or filed OP. From data submitted to APHIS, however, it is 
also noted that an acceptable guideline for determining such 
appropriate difference would be if the difference between the average 
PFU count obtained in the retest and the count obtained in the initial 
test exceeds 20 per cent. However, because no specific value was 
proposed by the commenter, and there is a need to address specific 
product differences, no change to the regulation is made in response to 
this comment.
    One commenter proposed that tests for relative antigen content 
which cannot be termed satisfactory or unsatisfactory should be called 
``no tests'' and be eligible for unlimited retesting without prejudice. 
In response, APHIS points out that Sec. 113.8(c)(1) of the regulation 
classifies a test that results in no valid lines as a ``no test''. 
Typically, this designation is used when a deficiency in the test 
system renders an invalid test result which is

[[Page 19036]]

unsuitable for reaching a conclusion regarding the potency of a serial; 
such serials may be retested. An equivocal test as that test is used in 
Sec. 113.8(c)(2), is a test that results in valid lines which are not 
parallel. Therefore, the test is considered inconclusive and the serial 
cannot be termed satisfactory or unsatisfactory. In order to clarify 
the proper handling and disposition of serials of product with 
equivocal test results, APHIS has amended Secs. 113.8 (c) (4) and (5) 
regarding the retest of serials with equivocal test results due to a 
lack of parallelism by specifying (1) the number of times such serials 
may be retested and, (2) the disposition of the serial based on the 
results of the retest.
    Four comments were received related to proposed Sec. 113.8(d)(2). 
The commenters requested that: (1) Stabilized Master References be 
allowed to serve as Working References; (2) Master References be 
allowed an initial dating period at least twice as long as that allowed 
for a regular serial of product; and (3) Frozen references be allowed 
an initial expiration dating of 5 years, provided that they are 
monitored by in vitro methods. In response to these comments regarding 
item (1), APHIS notes that proposed Sec. 101.5(o) of the regulation 
specifies that the Master Reference may be used as the Working 
Reference. Regarding item (2), proposed Sec. 113.8(d)(2) specifies that 
the dating of the reference shall be equal to the dating of the product 
or as supported by data acceptable to APHIS. Stability can be 
demonstrated by repeat testing of the reference over time or by 
demonstrating that the reference has maintained immunogenicity after 
being stored for a period of time equal to or greater than the dating 
period requested. Regarding item (3), allowing longer dating for 
references based on special treatments or storage conditions may be 
justified if such treatments or storage conditions are better able to 
maintain the stability of the reference. Section 113.8(d)(2) provides 
for determining the stability of the reference on the basis of 
confirming the immunogenicity in a manner acceptable to APHIS. This 
would include data from a stabilized monitored reference demonstrating 
stability in a manner acceptable to APHIS. Therefore, a reference may 
be allowed to have an initial dating longer than that for a regular 
production serial, provided that the request for the longer initial 
dating is supported by appropriate preliminary data and provides for 
monitoring stability to determine when the potency of the reference 
starts to decline and for taking appropriate steps to requalify or 
replace such a reference.
    In response to the commenters, APHIS has amended the regulations to 
allow frozen references an initial dating period of 5 years, provided 
that the request for such initial dating is supported by preliminary 
data and a frozen storage protocol, including monitoring procedures, 
acceptable to APHIS. As amended, Sec. 113.8(d)(2) reads as follows:

    (d)(2) * * * 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, except that 
frozen references may have an initial dating of up to 5 years, 
Provided, That the request for dating of frozen references beyond 
the dating of the product is supported by preliminary data 
acceptable to APHIS and includes provisions for monitoring the 
stability of the reference to determine when the potency starts to 
decline and for taking the appropriate steps to requalify a 
reference with declining potency either by testing a Qualifying 
Serial in host animals or by providing other evidence of 
immunogenicity, e.g., antibody titers or laboratory animal test data 
previously correlated to host animal protection in a manner 
acceptable to APHIS. Prior to the expiration date, such reference 
may be granted an extension of dating, Provided, That its 
immunogenicity has been confirmed using a Qualifying Serial of 
product in a manner acceptable to APHIS. * * *

    APHIS received two comments on proposed Sec. 101.5(q)(2) inquiring 
into the rationale for requiring the qualifying serial used to extend 
the dating of a Master Reference to be prepared within 6 months of 
initiating a requalification test. The commenters believed that the 6 
month restriction limited their options relating to production 
schedules and antigen manufacture. APHIS proposed the 6 month 
restriction as a means of assuring that qualifying serials used to 
extend the dating of a reference would be representative of the firm's 
current production method. APHIS agrees with the commenters regarding 
the potential restrictive aspects of the 6 month requirement and has 
amended Sec. 101.5(q)(2) in response to the comment to be more 
consistent with our intent as follows:

    (2) Qualifying serials used to requalify or extend the dating 
period of a Master Reference shall be determined to be immunogenic 
in accordance with methods deemed appropriate by APHIS as provided 
in paragraph (a)(1) of this section, and, in addition, shall be 
within their permitted dating period and have been prepared in 
accordance with the production method described in the currently 
filed Outline of Production.

    APHIS received one comment requesting clarification of proposed 
Sec. 113.8(d)(1) concerning confirmation of the protective dose 
established for live products in the Master Seed immunogenicity test 
after three years. In response to this comment, confirming the accuracy 
of the protective dose for live products three years after completion 
of a satisfactory immunogenicity test is specified in the Standard 
Requirements for live viral vaccines, and in the filed Outline of 
Production for products where standards have not been codified. 
Including a reference to this requirement for live viral vaccines in 
Sec. 113.8(d)(1) corrects an omission and provides notification of the 
requirement to those unfamiliar with this provision of the regulations. 
As specified in the codified requirements for individual live viral 
vaccines, only one retest is required. No change to the regulations is 
made in response to this comment.
    We received two comments regarding the definition of a ``Qualifying 
Serial'' in Sec. 101.5(q)(1). The commenter expressed concern that 
limiting a qualifying serial to a relative potency, when compared to 
the Master Reference, of not greater than 1.0 is too restrictive. The 
commenters suggested that the normal tolerance limits of 15 
per cent for parallel line immunoassays could cause a Qualifying Serial 
set at 1.0 to be as low as 0.85, which means that it may not pass a 
requalification test in animals. APHIS does not agree that requiring 
the Qualifying Serial to have a mean relative potency of not greater 
than 1.0 is too restrictive. As the commenter is probably aware, test 
assay variation is to be expected. Usually, a manufacturer will 
optimize the test system to determine how much variation is normal, and 
adjust the antigen levels so that the risk of failing a requalification 
test in animals is minimized. The alternative would require APHIS to 
include tolerance limits in the regulations. APHIS does not agree that 
such tolerance limits are necessary. The individual manufacturers can 
optimize antigen levels based on their individual experiences with test 
assay variation to assure that a Qualifying Serial with a mean relative 
potency of not greater than 1.0 will pass the requalification test in 
animals. No change to the regulations is made in response to this 
comment.
    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 the changes discussed in this document.

[[Page 19037]]

Executive Order 12866 and Regulatory Flexibility Act

    This 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 amendment allows 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 or demonstrates linearity, specificity, and reproducibility 
equivalent to the parallel line assay using an unexpired reference 
preparation. This amendment affects all licensed manufacturers of 
veterinary biologicals utilizing in vitro relative potency immunoassays 
for determining the potency of animal biological products. There are 
currently approximately 118 veterinary biologics establishments that 
may be affected by this rule. According to the Small Business 
Administration regulations, most of them would be classified as small 
entities. The majority of these establishments currently utilize in 
vitro relative potency tests to release serials of veterinary 
biological products. Since potency testing is already required under 
Sec. 113.5 of the regulations and since this rule does not require the 
use of in vitro relative potency tests, any additional cost imposed by 
the validity requirements specified in this rule should be minimal. 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 products. This rule does not restrict 
the firm's discretion to choose the most appropriate test for its 
product. The rule only prescribes validity requirements for in vitro 
immunoassays for relative potency. The overall effect of this amendment 
will 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 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 12988

    This final rule has been reviewed under Executive Order 12988, 
Civil Justice Reform. It is not intended to have retroactive effect. 
This rule would not preempt any State or local laws, regulations, or 
policies, unless they present an irreconcilable conflict with this 
rule. There are no administrative procedures which must be exhausted 
prior to a judicial challenge to the provisions of this rule.

Paperwork Reduction Act

    In accordance with the Paperwork Reduction Act of 1995 (44 U.S.C. 
3501 et seq.), the information collection or recordkeeping requirements 
included in this 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.

Regulatory Reform

    This action is part of the President's Regulatory Reform 
Initiative, which, among other things, directs agencies to remove 
obsolete and unnecessary regulations and to find less burdensome ways 
to achieve regulatory goals.

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 are amended as follows:

PART 101--DEFINITIONS

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

    Authority: 21 U.S.C. 151-159; 7 CFR 2.22, 2.80, and 371.2(d).

    2. Section 101.5 is 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 
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. The preparation of a Master Reference as 
described in a filed Outline of Production may be:
    (1) A completed serial of vaccine or bacterin prepared in 
accordance with a filed Outline of Production;
    (2) A purified preparation of a 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 Animal and Plant Health Inspection Service 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 accordance with methods deemed appropriate 
by the 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 five or more replicates; or other valid assay methods for 
determining relative antigen content which demonstrate linearity, 
specificity, and reproducibility at least equivalent to the parallel 
line assay and are acceptable to the Animal and Plant Health Inspection 
Service.
    (2) Qualifying serials used to requalify or extend the dating 
period of a Master Reference shall be determined to be immunogenic in 
accordance with methods deemed appropriate by the Animal and Plant 
Health Inspection Service as provided in paragraph (a)(1) of this 
section, and, in addition, shall be within their permitted dating 
period and have been prepared in accordance with the production method 
described in the currently filed Outline of Production.
    (r) Immunogenicity. The ability of a biological product to elicit 
an immune response in animals as determined by test methods or 
procedures acceptable

[[Page 19038]]

to the Animal and Plant Health Inspection Service.

PART 113--STANDARD REQUIREMENTS

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

    Authority: 21 U.S.C. 151-159; 7 CFR 2.22, 2.80, and 371.2(d).

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


Sec. 113.8  In vitro 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 satisfactory potency for the product in accordance 
with the following provisions:
    (i) Potency for live products may be determined by log10 virus 
titer or determining the live bacterial count based on the protective 
dose used in the Master Seed immunogenicity test plus an adequate 
overage for adverse conditions and test error; and
    (ii) Potency for inactivated products may be determined using tests 
for relative antigen content by comparing the antigen content of the 
test serial to a reference preparation using a parallel line 
immunoassay or equivalent method which measures linearity, specificity, 
and reproducibility in a manner acceptable to APHIS.
    (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:
* * * * *
    (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) and (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 an unexpired reference by a parallel line immunoassay 
or other procedure acceptable to APHIS.\1\ Firms currently using 
immunoassays which do not satisfy this requirement 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 unless 
granted an extension by the Administrator based on a showing by the 
firm seeking the extension that they have made a good faith effort with 
due diligence to achieve compliance. 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 method for evaluating relative antigen content, 
Supplemental Assay Method 318, and relative potency calculation 
software are available from the United States Department of 
Agriculture, Animal and Plant Health Inspection Service, Veterinary 
Services, National Veterinary Services Laboratories, Center for 
Veterinary Biologics--Laboratory, 1800 Dayton Road, P. O. Box 844, 
Ames, Iowa 50010.
---------------------------------------------------------------------------

    (1) A test that results in no valid lines is considered a ``no 
test'' and may be repeated.
    (2) An initial test (test 1) 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 (test 1) shows that potency equals or 
exceeds the required minimum potency, the serial is satisfactory 
without additional testing.
    (4) If the initial test (test 1) is an equivocal test due to lack 
of parallelism, the serial may be retested up to three times (tests 2, 
3, and 4) with disposition to be as specified in paragraphs (c)(4)(i) 
and (ii) of this section; Provided, That, if the serial is not retested 
or the other provisions of this section are not satisfied, the serial 
shall be deemed unsatisfactory.
    (i) If: The first retest (test 2) following an initial equivocal 
test; the second retest (test 3) following two consecutive equivocal 
tests (tests 1 and 2); or the third retest (test 4) following three 
consecutive equivocal tests (tests 1, 2, and 3) shows that the potency 
equals or exceeds the required minimum potency, the serial is 
satisfactory.
    (ii) If the first retest (test 2) following an initial equivocal 
test shows that potency is less than the required minimum potency, 
disposition of the serial will be based on the outcome of retests 2 and 
3 (tests 3 and 4) as follows: if either retest (test 3 or 4) shows that 
potency is less than the required minimum potency, the serial is 
unsatisfactory. If either retest 2 or retest 3 (tests 3 or 4) is an 
equivocal test, or in the event that each retest (tests 2, 3, and 4) 
following an initial equivocal test is also an equivocal test, the 
accumulated test results shall be considered indicative of a lack of 
potency and release of the serial withheld. In which case, the licensee 
may submit data confirming the continued validity of the test system to 
APHIS for review and approval. If the data are acceptable to APHIS, the 
potency test may be repeated by the firm, subject to the provisions 
specified in paragraphs (i) and (ii) and confirmatory testing by APHIS.
    (5) If the initial test (test 1) shows that potency is less than 
the required minimum potency, the serial may be retested a minimum of 
two times (tests 2 and 3) but not more than three times (tests 2, 3, 
and 4) with disposition as specified in paragraphs (c)(5) (i) and (ii) 
of this section; Provided, That, if the

[[Page 19039]]

serial is not retested or the other provisions of this section are not 
satisfied, the serial shall be deemed unsatisfactory.
    (i) If two consecutive retests (tests 2 and 3) show that potency of 
the serial equals or exceeds the required minimum potency, the serial 
is satisfactory. If one of the two retests (test 2 or 3) shows that the 
potency is less than the required minimum potency, the serial is 
unsatisfactory.
    (ii) If one of the retests (tests 2 or 3) shows that the potency 
equals or exceeds the required minimum potency and the other retest 
(test 2 or 3) is an equivocal test, a third retest (test 4) may be 
performed. If the third retest (test 4) shows that the potency of the 
serial equals or exceeds the required minimum potency, the serial is 
deemed satisfactory. If both retests (tests 2 and 3) or if the third 
retest (test 4) is an equivocal test, the accumulated test results 
shall be considered indicative of a lack of potency and release of the 
serial withheld, in which case the licensee may submit data confirming 
the continued validity of the test system to APHIS for review and 
approval. If the data are acceptable to APHIS, the potency test may be 
repeated by the firm, subject to the provisions specified in paragraphs 
(c)(4) (i) and (ii) and (c)(5) (i) and (ii) of this section, and 
confirmatory testing by APHIS.
    (d) Repeat immunogenicity tests. (1) The accuracy of the protective 
dose established for live products in the Master Seed immunogenicity 
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 or equivalent methods 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, except that frozen 
references may have an initial dating of up to 5 years, Provided, That 
the request for dating of the frozen references beyond the dating of 
the product is supported by preliminary data acceptable to APHIS and 
includes provisions for monitoring the stability of the reference to 
determine when the potency starts to decline and for taking the 
appropriate steps to requalify a reference with declining potency 
either by testing a Qualifying Serial in host animals or by providing 
other evidence of immunogenicity, e.g., antibody titers or laboratory 
animal test data previously correlated to host animal protection in a 
manner acceptable to APHIS. Prior to the expiration date, such 
reference may be granted an extension of dating, Provided, That its 
immunogenicity has been confirmed using a Qualifying Serial of product 
in a manner acceptable to APHIS. The dating period of the Master 
Reference and Working Reference may be extended 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, except that frozen references 
may have an initial dating period of 5 years, 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.
* * * * *
    Done in Washington, DC, this 15th day of April 1997.
Donald W. Luchsinger,
Acting Administrator, Animal and Plant Health Inspection Service.
[FR Doc. 97-10100 Filed 4-17-97; 8:45 am]
BILLING CODE 3410-34-P