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

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]

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