[Federal Register Volume 60, Number 52 (Friday, March 17, 1995)]
[Rules and Regulations]
[Pages 14357-14363]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 95-6647]



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DEPARTMENT OF AGRICULTURE
9 CFR Part 113

[Docket No. 92-132-2]


Viruses, Serums, Toxins, and Analogous Products; Revision of 
Standard Requirements

AGENCY: Animal and Plant Health Inspection Service, USDA.

ACTION: Final rule.

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SUMMARY: This rule amends the Standard Requirements concerning Dog 
Safety Testing; Canine Distemper Vaccine, Killed Virus; Canine 
Hepatitis Vaccine, Killed Virus; Canine Adenovirus Type 2 Vaccine, 
Killed Virus; Mink Enteritis Vaccine, Killed Virus; Canine Hepatitis 
Vaccine, Live Virus; Canine Adenovirus Type 2 Vaccine, Live Virus; and 
Canine Distemper Vaccine, Live Virus. The amendments are necessary 
because new test methods and procedures have been developed that can 
replace current test requirements and increase the validity of test 
results. The effect of the amendments is to provide new test methods 
and procedures and to relax some of the restrictions currently in 
effect. Also, the Standard Requirement for Canine Distemper Vaccine 
(Ferret Virulent) is removed because this vaccine is no longer 
manufactured.

EFFECTIVE DATE: April 17, 1995.

FOR FURTHER INFORMATION CONTACT:
Dr. David A. Espeseth, Deputy Director, Animal and Plant Health 
Inspection Service, Biotechnology, Biologics, and Environmental 
Protection, Veterinary Biologics, 4700 River Road Unit 148, Riverdale, 
MD 20737-1228, (301) 734-8245.

SUPPLEMENTARY INFORMATION:

Background

    The regulations in 9 CFR part 113 ``Standard Requirements'', 
(referred to below as the regulations) consist of test methods, 
procedures, and criteria established by the Animal and Plant Health 
Inspection Service (APHIS) for the evaluation of veterinary biological 
products based upon their purity, safety, potency, and efficacy. The 
Agency periodically reviews the regulations and amends test methods and 
procedures as required to ensure that they are consistent with current 
scientific knowledge. On July 23, 1993, we published in the Federal 
Register (see 58 FR 39467-39473, Docket No. 92-132-1) a proposed rule 
to update the regulations based upon current scientific knowledge.
    We solicited comments concerning our proposal for a 60-day comment 
period ending September 21, 1993. We received four comments by that 
date. One commenter fully supported the proposal as written. Three 
commenters suggested changes to certain sections related to the 
Standard Requirements. These comments are discussed below.
    Two commenters suggested changes to Sec. 113.204. Both commenters 
indicated that the portion of the regulations dealing with the time(s) 
of feces collection for virus detection required clarification, and 
suggested that feces collection at some point from day 4 to 8 would be 
appropriate.
    APHIS believes that the above comments have merit. APHIS agrees 
that feces collection early or late in the collection period, or more 
than once, is unnecessary. Therefore, APHIS has revised the regulations 
in Sec. 113.204(b)(2) to specify that feces are [[Page 14358]] to be 
collected once from day 4 through day 8.
    One of the two commenters stated that the term ``virus isolation'' 
should be defined and that methods other than that specified, ``virus 
isolation and/or fluorescent antibody examination,'' should be allowed 
for the detection of virus in feces. APHIS agrees that not only ``virus 
isolation'' but the whole phrase ``virus isolation and/or fluorescent 
antibody examination'' needs clarification. Therefore, we have revised 
the regulations in Sec. 113.204(b)(2) to specify that cell culture with 
fluorescent antibody examination is the acceptable method of virus 
detection. APHIS does not agree, however, with the suggestion that 
other methods of virus detection should be specified in the regulations 
presently. We believe that cell culture with fluorescent antibody 
examination is the most sensitive and specific method of virus 
detection. Should APHIS become aware of another method that is superior 
to that indicated, it would consider rulemaking to specify that method 
in the regulations.
    One of the two commenters also stated that unvaccinated control 
mink in immunogenicity studies should not be considered susceptible to 
challenge if the animals exhibit clinical signs but do not shed virus. 
The second commenter stated that the determination of virus shedding in 
animals used for immunogenicity studies should not be required if four 
or five of the five unvaccinated control mink exhibit clinical signs. 
APHIS does not agree with either commenter. We believe that the absence 
of appropriate clinical signs in vaccinated mink challenged with 
virulent mink enteritis virus together with clinical signs in 
unvaccinated control mink after challenge is sufficient evidence of the 
effectiveness of the challenge. We also believe that an effective 
vaccine against mink enteritis should prevent virus shedding. No change 
in the regulations is made in response to these comments.
    As a final comment on Sec. 113.204, one commenter criticized what 
the commenter thought was a Standard Assay Method (SAM) developed by 
the National Veterinary Services Laboratories for challenging mink with 
mink enteritis virus. No such SAM has been prepared. What has been 
prepared is more appropriately termed a ``bench protocol.'' Since the 
protocol was not addressed in the proposed rulemaking, no change to the 
regulations is made based on this comment.
    Comments on the three other Standard Requirements included in the 
proposed rule (Secs. 113.40, 113.201, and 113.306) were received from 
another commenter. Two of the comments related to the route of canine 
distemper virus challenge and the requirements for satisfactory vaccine 
performance in a repeat immunogenicity study. The commenters requested 
that Sec. 113.306 concerning live virus vaccines be changed to specify 
an intranasal rather than the traditional intracerebral route of 
challenge. No amendments to the route of challenge or repeat 
immunogenicity requirements were proposed in Sec. 113.306. An 
intracerebral challenge has been used successfully for many years with 
the live virus vaccine. It was the proposed amendments to Sec. 113.201 
that changed the route of challenge from intranasal to intracerebral 
for killed virus vaccines to be consistent with that specified for live 
virus vaccines in Sec. 113.306. Since the commenters focussed only on 
Sec. 113.306 and that section is not being amended as to route of 
challenge, no change to the regulations is made in response to these 
commenters.
    The same commenter also claimed that the proposal would result in 
the overuse of Master Seed and suggested that material obtained after 
five passages of Master Seed be used instead. APHIS disagrees with this 
comment. In requiring that Master Seed be used, the proposed change is 
consistent with other regulations in part 113. We believe that testing 
the Master Seed is necessary for a satisfactory determination of its 
use. No change to the regulations is made in response to this 
commenter.
    Based on the rationale set forth in the proposed rule and in this 
document, we are adopting the provisions of the proposed rule as a 
final rule, with the changes discussed in this document.

Executive Order 12866 and Regulatory Flexibility Act

    This proposed rule has been reviewed under Executive Order 12866. 
The rule has been determined to be not significant for the purposes of 
Executive Order 12866 and, therefore, has not been reviewed by the 
Office of Management and Budget.
    This final rule revises the current Standard Requirements for 
certain vaccines. Sections 113.201 and 113.202 are amended to revise 
the potency test performed on each serial of product so that fewer dogs 
will be used and serology will be used instead of virus challenge. Both 
of these changes will decrease the costs of production to the 
manufacturer. In Sec. 113.204, the potency test in mink is changed to 
require that virus shedding be examined. This change should result in 
only a minimal increase in cost (less than $100 per test) to the 
manufacturer. Other changes to the Standard Requirements generally 
update the Standard Requirements to reflect current scientific 
knowledge. We do not expect any increase in cost, except as noted 
above, to the 200 biologics manufacturers affected by this rule. In 
most cases, we expect the changes will actually decrease the costs for 
the manufacturers.
    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 category 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.)

Paperwork Reduction Act

    This rule contains no new information collection or recordkeeping 
requirements under the Paperwork Reduction Act of 1980 (44 U.S.C. 3501 
et seq.).

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 in conflict 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.

List of Subjects in 9 CFR Part 113

    Animal biologics, Exports, Imports, 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 113.40 is revised to read as follows:


Sec. 113.40  Dog safety tests.

    The safety tests provided in this section shall be conducted when 
prescribed in a Standard Requirement or in the filed Outline of 
Production for a biological product recommended for use in dogs. 
Serials which are not found to be satisfactory when tested pursuant to 
[[Page 14359]] the procedures in this section may not be released for 
shipment.
    (a) The dog safety test provided in this paragraph shall be used 
when the Master Seed Virus is tested for safety.
    (1) The test animals shall be determined to be susceptible to the 
virus under test by a method acceptable to the Animal and Plant Health 
Inspection Service.
    (2) Each of at least 10 susceptible dogs shall be administered a 
sample of the Master Seed Virus equivalent to the amount of virus to be 
used in one dog dose of the vaccine, by the method recommended on the 
label, and the dog shall be observed each day for 14 days.
    (3) If unfavorable reactions attributable to the virus occur in any 
of the dogs during the observation period, the Master Seed Virus is 
unsatisfactory. If unfavorable reactions occur which are not 
attributable to the Master Seed Virus, the test shall be declared 
inconclusive and may be repeated: Provided: That, if the test is not 
repeated, the Master Seed Virus shall be considered unsatisfactory.
    (b) The dog safety test provided in this paragraph shall be used 
when a serial of vaccine is tested for safety before release.
    (1) Each of two healthy dogs shall be administered 10 dog doses by 
the method recommended on the label and the dogs shall be observed each 
day for 14 days.
    (2) If unfavorable reactions attributable to the biological product 
occur during the observation period, the serial is unsatisfactory. If 
unfavorable reactions occur which are not attributable to the 
biological product, the test shall be declared inconclusive and may be 
repeated: Provided, That, if the test is not repeated, the serial shall 
be considered unsatisfactory.
    3. Section 113.201 is revised to read as follows:


Sec. 113.201  Canine Distemper Vaccine, Killed Virus.

    Canine Distemper Vaccine, Killed Virus, shall be prepared from 
virus-bearing cell culture fluids. Only Master Seed Virus which has 
been established as pure, safe, and immunogenic shall be used for 
vaccine production. All serials of vaccine shall be prepared from the 
first through the fifth passage from the Master Seed Virus.
    (a) The Master Seed Virus shall meet the applicable general 
requirements prescribed in Sec. 113.200.
    (b) The immunogenicity of vaccine prepared from the Master Seed 
Virus in accordance with the Outline of Production shall be 
established. Vaccine used for this test shall be at the highest passage 
from the Master Seed and prepared at the minimum preinactivation titer 
specified in the Outline of Production.
    (1) Twenty-five canine distemper susceptible dogs (20 vaccinates 
and 5 controls) shall be used as test animals. Blood samples drawn from 
each dog shall be individually tested for neutralizing antibody against 
canine distemper to determine susceptibility. A constant virus-varying 
serum neutralization test in cell culture using 50 to 300 TCID<INF>50 
of virus shall be used. Dogs shall be considered susceptible if there 
is no neutralization at a 1:2 final serum dilution.
    (i) The 20 dogs used as vaccinates shall be injected with one dose 
of vaccine by the method recommended on the label. If a second dose is 
recommended, the second dose shall be administered at the time 
specified on the label.
    (ii) At least 14 days after the last inoculation, the vaccinates 
and controls shall each be challenged intracerebrally with canine 
distemper virus furnished or approved by the Animal and Plant Health 
Inspection Service and observed each day for 21 days.
    (iii) If at least four of the five controls do not die and the 
survivor, if any, does not show clinical signs of canine distemper, the 
test is inconclusive and may be repeated.
    (iv) If at least 19 of the 20 vaccinated do not survive without 
showing clinical signs of canine distemper during the observation 
period, the Master Seed Virus is unsatisfactory.
    (c) Test requirements for release. Each serial shall meet the 
applicable general requirements prescribed in Sec. 113.200 and the 
special requirements for safety and potency provided in this section.
    (1) Safety test. The vaccinates used in the potency test in 
paragraph (c)(2) of this section shall be observed each day during the 
postvaccination observation period. If unfavorable reactions occur 
which are attributable to the vaccine, the serial is unsatisfactory. If 
unfavorable reactions occur which are not attributable to the vaccine, 
the test is inconclusive and may be repeated: Provided, That, if the 
test is not repeated, the serial is unsatisfactory.
    (2) Potency test--serum neutralization test. Bulk or final 
container samples of completed product shall be tested for potency 
using five susceptible dogs (four vaccinates and one control) as the 
test animals. Blood samples drawn from each dog shall be individually 
tested for neutralizing antibody against canine distemper virus to 
determine susceptibility.
    (i) A constant virus-varying serum neutralization test in tissue 
culture using 50 to 300 TCID<INF>50 of virus shall be used. Dogs shall 
be considered susceptible if there is no neutralization at a 1:2 final 
serum dilution.
    (ii) Vaccination. Each of the four vaccinates shall be injected as 
recommended on the label. If two doses are recommended, the second dose 
shall be administered at the time specified on the label. The dogs 
shall be observed each day for at least 14 days after the last 
inoculation.
    (iii) Serology. At the end of the post vaccination observation 
period, a second blood sample shall be obtained from each of the five 
dogs and the serums shall be individually tested for neutralizing 
antibody against canine distemper virus in the same manner used to 
determine susceptibility.
    (iv) Interpretation of the serum neutralization test. If the 
control has not remained seronegative at 1:2, the test is inconclusive 
and may be repeated. If at least three of the four vaccinates in a 
valid test have not developed titers based upon a final serum dilution 
of at least 1:50 and the remaining vaccinate has not developed a titer 
of at least 1:25, the serial is unsatisfactory except as provided in 
paragraphs (c)(2) (v) and (vi) of this section.
    (v) Virus challenge test. If the results of a valid serum 
neutralization test are unsatisfactory, the vaccinates and the control 
may be challenged intracerebrally with a virulent canine distemper 
virus furnished or approved by the Animal and Plant Health Inspection 
Service and each animal observed each day for an additional 21 days.
    (vi) Interpretation of the virus challenge test. For a serial to be 
satisfactory, all vaccinates must remain free from clinical signs of 
canine distemper while the control must die of canine distemper. If the 
control does not die of canine distemper, the test is inconclusive and 
may be repeated except, that if any of the vaccinates show signs or 
dies of canine distemper, the serial is unsatisfactory.
    4. Section 113.202 is revised to read as follows:


Sec. 113.202  Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, 
Killed Virus.

    Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed 
Virus, shall be prepared from virus-bearing cell culture fluids. Only 
Master Seed Virus which has been established as pure, safe, and 
immunogenic shall be used for vaccine production. All serials of 
vaccine shall be prepared from the [[Page 14360]] first through the 
fifth passage from the Master Seed Virus.
    (a) The Master Seed Virus shall meet the applicable requirements 
prescribed in Sec. 113.200.
    (b) Each lot of Master Seed Virus used for vaccine production shall 
be tested for immunogenicity by one or both of the following methods. 
Vaccine used for these tests shall be at the highest passage from the 
Master Seed and prepared at the minimum preinactivation titer specified 
in the Outline of Production.
    (1) Immunogenicity for canine hepatitis. Twenty-five canine 
hepatitis susceptible dogs shall be used as test animals (20 vaccinates 
and 5 controls). Blood samples shall be drawn from these animals and 
individual serum samples tested. The dogs shall be considered 
susceptible if the results are negative at a 1:2 final serum dilution 
in a varying serum-constant virus neutralization test using 50 to 300 
TCID<INF>50 of canine adenovirus.
    (i) The 20 dogs to be used as vaccinates shall be injected with one 
dose of vaccine and the remaining five dogs held as controls. If a 
second dose is recommended, the second dose shall be administered at 
the time specified on the label.
    (ii) Not less than 14 days after the last inoculation, each 
vaccinate and control shall be challenged intravenously with virulent 
infectious canine hepatitis virus furnished or approved by the Animal 
and Plant Health Inspection Service and observed each day for 14 days.
    (iii) If at least four of the five controls do not show severe 
clinical signs of infectious canine hepatitis, the test is inconclusive 
and may be repeated.
    (iv) If at least 19 of the 20 vaccinates do not survive without 
showing clinical signs of infectious canine hepatitis during the 
observation period, the Master Seed Virus is unsatisfactory.
    (2) Immunogenicity for canine adenovirus type 2. Thirty canine 
adenovirus type 2 susceptible dogs shall be used as test animals (20 
vaccinates and 10 controls). Blood samples shall be drawn from these 
animals and individual serum samples tested. The dogs shall be 
considered susceptible if the results are negative at a 1:2 final serum 
dilution in a varying serum-constant virus neutralization test using 50 
to 300 TCID<INF>50 of canine adenovirus.
    (i) The 20 dogs to be used as vaccinates shall be injected with one 
dose of vaccine and the remaining 10 dogs held as controls. If a second 
dose is recommended, the second dose shall be administered at the time 
specified on the label.
    (ii) Not less than 14 days after the last inoculation, the 
vaccinates and the controls shall be challenged by exposure to a 
nebulized aerosol of virulent canine adenovirus type 2 furnished or 
approved by the Animal and Plant Health Inspection Service and observed 
each day for 14 days postchallenge. The rectal temperature of each 
animal shall be taken and the presence of respiratory or other clinical 
signs of canine adenovirus type 2 noted and recorded each day.
    (iii) If at least 6 of 10 controls do not show clinical signs of 
canine adenovirus type 2 infection other than fever, the test is 
inconclusive and may be repeated.
    (iv) If a significant difference in clinical signs in a valid test 
cannot be demonstrated between vaccinates and controls using a scoring 
system approved by the Animal and Plant Health Inspection Service, the 
Master Seed Virus is unsatisfactory.
    (c) Test requirements for release. Each serial shall meet the 
applicable general requirements prescribed in Sec. 113.200, the special 
requirements for safety provided in this section, and the applicable 
potency tests provided in this section.
    (1) Safety test. The vaccinates used in the potency test in 
paragraph (c)(2) and/or (c)(3) of this section shall be observed each 
day during the postvaccination observation period. If unfavorable 
reactions occur which are attributable to the vaccine, the serial is 
unsatisfactory. If unfavorable reactions occur which are not 
attributable to the vaccine, the test is inconclusive and may be 
repeated: Provided, That, if not repeated, the serial is 
unsatisfactory.
    (2) Potency test for canine hepatitis--serum neutralization test. 
Bulk or final container samples of completed product shall be tested 
for potency using at least five susceptible dogs (four vaccinates and 
one control) as the test animals. Blood samples drawn from each dog 
shall be individually tested for neutralizing antibody against canine 
adenovirus to determine susceptibility.
    (i) A constant virus-varying serum neutralization test in tissue 
culture using 50 to 300 TCID<INF>50 of virus shall be used. Dogs shall 
be considered susceptible if there is no neutralization at a 1:2 final 
serum dilution.
    (ii) Vaccination. Each of the vaccinates shall be injected as 
recommended on the label. If two doses are recommended, the second dose 
shall be administered at the time specified on the label. The dogs 
shall be observed each day for at least 14 days after the last 
inoculation.
    (iii) Serology. At the end of the postvaccination observation 
period, a second blood sample shall be obtained from each of the dogs 
and the serums shall be individually tested for neutralizing antibody 
against canine adenovirus in the same manner used to determine 
susceptibility.
    (iv) Interpretation of the serum neutralization test. If the 
control(s) has not remained seronegative at 1:2, the test is 
inconclusive and may be repeated. If at least 75 percent of the 
vaccinates in a valid test have not developed titers based upon final 
serum dilution of at least 1:10 and the remaining vaccinate(s) has not 
developed a titer of at least 1:2, the serial is unsatisfactory except 
as provided in paragraphs (c)(2) (v) and (vi) of this section.
    (v) Virus challenge test. If the results of a valid serum 
neutralization test are unsatisfactory, the vaccinates and the 
control(s) may be challenged intravenously with a virulent canine 
hepatitis virus furnished or approved by the Animal and Plant Health 
Inspection Service and each animal observed each day for an additional 
14 days.
    (vi) Interpretation of the virus challenge test. For a serial to be 
satisfactory, all vaccinates must remain free of clinical signs of 
canine hepatitis while the control(s) must show severe clinical signs 
of canine hepatitis. If the control(s) does not show severe clinical 
signs of canine hepatitis, the test is inconclusive and may be 
repeated: Provided, That, if any of the vaccinates show signs or die of 
canine hepatitis, the serial is unsatisfactory.
    (3) Potency test for canine adenovirus type 2. Bulk or final 
container samples of completed product shall be tested for potency 
using eight susceptible dogs (five vaccinates and three controls) as 
the test animals. Blood samples drawn from each dog shall be 
individually tested for neutralizing antibody against canine adenovirus 
to determine susceptibility.
    (i) A constant virus-varying serum neutralization test in tissue 
culture using 50 to 300 TCID<INF>50 of virus shall be used. Dogs shall 
be considered susceptible if there is no neutralization at a 1:2 final 
serum dilution.
    (ii) Vaccination. Each of the five vaccinates shall be injected as 
recommended on the label. If two doses are recommended, the second dose 
shall be administered at the time specified on the label. The dogs 
shall be observed each day for at least 14 days after the last 
inoculation.
    (iii) Not less than 14 days after the last inoculation, the 
vaccinates and the controls shall be challenged by exposure to a 
nebulized aerosol of virulent canine adenovirus type 2 furnished or 
[[Page 14361]] approved by the Animal and Plant Health Inspection 
Service and observed each day for 14 days postchallenge. The rectal 
temperature of each animal shall be taken and the presence of 
respiratory or other clinical signs of canine adenovirus type 2 noted 
and recorded each day.
    (iv) If at least two of three controls do not show clinical signs 
of canine adenovirus type 2 other than fever, the test is inconclusive 
and may be repeated.
    (v) If a significant difference in clinical signs cannot be 
demonstrated between vaccinates and controls using a scoring system 
approved by the Animal and Plant Health Inspection Service and 
prescribed in the Outline of Production, the serial is unsatisfactory.
    5. Section 113.204 is amended by revising paragraphs (b)(2) and 
(b)(3) to read as follows:


Sec. 113.204  Mink Enteritis Vaccine, Killed Virus.

* * * * *
    (b) * * *
    (2) Challenge. At least 2 weeks after the last inoculation, the 
five vaccinates and the five controls shall be challenged with virulent 
mink enteritis virus and observed each day for 12 days. Fecal material 
shall be collected on one day between days 4-8 (inclusive) 
postchallenge from each test animal that remains free of enteric signs 
and tested for the presence of mink enteritis virus by cell culture 
with fluorescent antibody examination.
    (3) Interpretation. A serial is satisfactory if at least 80 percent 
of the vaccinates remain free of enteric signs and do not shed virus in 
the feces, while at least 80 percent of the controls develop clinical 
signs of mink enteritis or shed virus in the feces. If at least 80 
percent of the vaccinates remain free of enteric signs and do not shed 
virus in the feces, while less than 80 percent of the controls develop 
clinical signs of mink enteritis or shed virus in the feces, the test 
is considered inconclusive and may be repeated: Provided, That, if at 
least 80 percent of the vaccinates do not remain well and free of 
detectable virus in the feces, the serial is unsatisfactory.
    6. Section 113.305 is revised to read as follows:


Sec. 113.305  Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall 
be prepared from virus-bearing cell culture fluids. Only Master Seed 
Virus which has been established as pure, safe, and immunogenic shall 
be used in preparing the production seed virus for vaccine production. 
All serials shall be prepared from the first through the fifth passage 
from the Master Seed Virus.
    (a) The Master Seed Virus shall meet the applicable requirements 
prescribed in Sec. 113.300 except that the dog safety test prescribed 
in Sec. 113.40(a) shall be conducted by the intravenous route.
    (b) Each lot of Master Seed Virus used for vaccine production shall 
be tested for immunogenicity by one or both of the following methods:
    (1) Immunogenicity for canine hepatitis. Twenty-five canine 
hepatitis susceptible dogs shall be used as test animals (20 vaccinates 
and 5 controls). Blood samples shall be drawn from these animals and 
individual serum samples tested. The dogs shall be considered 
susceptible if the results are negative at a 1:2 final serum dilution 
in a varying serum-constant virus neutralization test using 50 to 300 
TCID<INF>50 of canine adenovirus.
    (i) A geometric mean titer of the dried vaccine produced from the 
highest passage of the Master Seed Virus shall be established before 
the immunogenicity test is conducted. The 20 dogs to be used as 
vaccinates shall be injected with a predetermined quantity of vaccine 
virus and the remaining five dogs held as uninjected controls. To 
confirm the dosage calculations, five replicate virus titrations shall 
be conducted on a sample of the vaccine virus dilution used.
    (ii) Not less than 14 days postinjection, the vaccinates and the 
controls shall each be challenged intravenously with virulent 
infectious canine hepatitis virus furnished or approved by the Animal 
and Plant Health Inspection Service and observed each day for 14 days.
    (A) If at least four of the five controls do not show severe 
clinical signs of canine hepatitis, the test is inconclusive and may be 
repeated.
    (B) If at least 19 of the 20 vaccinates do not survive without 
showing clinical signs of infectious canine hepatitis during the 
observation period, the Master Seed Virus is unsatisfactory.
    (iii) The Master Seed Virus shall be retested for immunogenicity 
for canine hepatitis in 3 years unless use of the lot previously tested 
is discontinued. Ten susceptible dogs (8 vaccinates and 2 controls) 
shall be used in the retest. Susceptibility shall be determined in the 
manner provided in paragraph (b)(1) of this section.
    (A) Each vaccinate shall be injected with a predetermined quantity 
of vaccine virus as provided in paragraph (b)(1)(i) of this section.
    (B) At least 14 days postvaccination, a second serum sample shall 
be drawn from each dog and tested for neutralizing antibody to canine 
adenovirus in the same manner used to determine susceptibility.
    (C) If the two controls have not remained seronegative at 1:2, the 
test is inconclusive and may be repeated.
    (D) if at least six of the eight vaccinates in a valid test do not 
develop titers of at least 1:10 based upon final serum dilution, the 
Master Seed Virus is unsatisfactory except as provided in paragraph 
(b)(1)(iii)(E) of this section.
    (E) if the results of a valid serum neutralization test are 
unsatisfactory, the vaccinates and the controls may be challenged as 
provided in paragraph (b)(1)(ii) of this section. A Master Seed is 
satisfactory if all vaccinates remain free of clinical signs of canine 
hepatitis, while both controls develop severe clinical signs of canine 
heptatis. If both controls do not show severe clinical signs of canine 
hepatitis, the test is inconclusive and may be repeated: Provided, 
That, if any of the vaccinates show such signs, the Master Seed Virus 
is unsatisfactory.
    (2) Immunogenicity for canine adenovirus Type 2. Thirty canine 
adenovirus type 2 susceptible dogs shall be used as test animals (20 
vaccinates and 10 controls). Blood samples shall be drawn from these 
animals and individual serum samples tested. The dogs shall be 
considered susceptible if the results are negative at a 1:2 final serum 
dilution in a varying serum-constant virus neutralization test using 50 
to 300 TCID<INF>50 of canine adenovirus.
    (i) A geometric mean titer of the dried vaccine produced from the 
highest passage of the Master Seed Virus shall be established before 
the immunogenicity test is conducted. The 20 dogs to be used as 
vaccinates shall be injected with a predetermined quantity of vaccine 
virus and the remaining 10 dogs held as uninjected controls. To confirm 
the dosage calculations, five replicate virus titrations shall be 
conducted on a sample of the vaccine virus dilution used.
    (ii) Not less than 14 days postinjection, the vaccinates and the 
controls shall be challenged by exposure to a nebulized aerosol of 
virulent canine adenovirus type 2 furnished or approved by the Animal 
and Plant Health Inspection Service and observed each day for 14 days 
postchallenge. The rectal temperature of each animal shall be taken and 
the presence of respiratory or other clinical signs of canine 
adenovirus type 2 noted and recorded each day. [[Page 14362]] 
    (A) If at least 6 of 10 controls do not show clinical signs of 
canine adenovirus type 2 infection other than fever, the test is 
inconclusive and may be repeated.
    (B) if a significant difference in clinical signs in a valid test 
cannot be demonstrated between vaccinates and controls using a scoring 
system approved by the Animal and Plant Health Inspection Service, the 
Master Seed Virus is unsatisfactory.
    (iii) the Master Seed Virus shall be retested for immunogenicity in 
3 years unless use of the lot previously tested is discontinued. Either 
10 vaccinates and 6 controls or 5 vaccinates and 3 controls shall be 
used in the retest.
    (A) If less than 4 of 6 or 2 of 3 of the controls show clinical 
signs of canine adenovirus type 2 other than fever, the test is 
inconclusive and may be repeated.
    (B) a significant difference in clinical signs shall be 
demonstrated between vaccinates and controls in a valid test as 
prescribed in paragraph (b)(2)(ii)(B) of this section.
    (iv) an Outline of Production change shall be made before 
authorization for use of a new lot of Master Seed Virus shall be 
granted by the Animal and Plant Health Inspection Service.
    (c) Test requirements for release. Each serial and subserial shall 
meet the requirements prescribed in Sec. 113.300 and in this paragraph. 
Final container samples of completed product shall be tested. Any 
serial or subserial found unsatisfactory by a prescribed test shall not 
be released.
    (1) Virus titer requirements. Final container samples of completed 
product shall be tested for virus titer using the titration method used 
in paragraph (b)(1)(i) and/or (b)(2)(i) of this section. To be eligible 
for release, each serial and each subserial shall have a virus titer 
sufficiently greater than the titer of vaccine virus used in the 
immunogenicity test(s) prescribed in paragraph (b) of this section to 
assure that when tested at any time within the expiration period, each 
serial and subserial shall have a virus titer of 10<SUP>0.7 greater 
than that used in such immunogenicity test(s) but not less than 
10.<SUP>2.5 TCID<INF>50 dose. If both immunogenicity tests in paragraph 
(b) of this section are conducted and a different amount of virus is 
used in each test, the virus titer requirements shall be based on the 
higher of the two amounts.
    7. Section 113.306 is revised to read as follows:


Sec. 113.306  Canine Distemper Vaccine.

    Canine Distemper Vaccine shall be prepared from virus-bearing cell 
culture fluids or embryonated chicken eggs. Only Master Seed Virus 
which has been established as pure, safe, and immunogenic shall be used 
for preparing the production seed virus for vaccine production. All 
serials of vaccine shall be prepared from the first through the fifth 
passage from the Master Seed Virus.
    (a) Master Seed Virus. The Master Seed Virus shall meet the 
applicable requirements prescribed in Sec. 113.300 and the requirements 
prescribed in this section.
    (1) To detect ferret virulent canine distemper virus, each of five 
canine distemper susceptible ferrets shall be injected with a sample of 
the Master Seed Virus equivalent to the amount of virus to be used in 
one dog dose and observed each day for 21 days. If undesirable 
reactions are observed during the observation period, the lot of Master 
Seed is unsatisfactory.
    (2) Master Seed Virus propagated in tissues or cells of avian 
origin shall be tested for pathogens by the chicken embryo test 
prescribed in Sec. 113.37. If found unsatisfactory, the Master Seed 
Virus shall not be used.
    (b) Each lot of Master Seed Virus used for vaccine production shall 
be tested for immunogenicity. The selected virus dose from the lot of 
Master Seed Virus shall be established as follows:
    (1) Twenty-five canine distemper susceptible dogs shall be used as 
test animals (20 vaccinates and 5 controls). Blood samples shall be 
drawn from these animals and individual serum samples tested. The dogs 
shall be considered susceptible if the results are negative at a 1:2 
final serum dilution in a varying serum-constant virus neutralization 
test using 50 to 300 TCID<INF>50 of canine distemper virus.
    (2) A geometric mean titer of the dried vaccine produced from the 
highest passage of the Master Seed Virus shall be established before 
the immunogenicity test is conducted. The 20 dogs used as vaccinates 
shall be injected with a predetermined quantity of vaccine virus and 
the remaining five dogs held as uninjected controls. To confirm the 
dosage calculations, five replicate virus titrations shall be conducted 
on a sample of the vaccine virus dilution used.
    (3) At least 14 days post-injection, the vaccinates and the 
controls shall each be challenged intracerebrally with virulent canine 
distemper virus furnished or approved by the Animal and Plant Health 
Inspection Service and observed each day for 21 days.
    (i) If at least four of the five controls do not die and the 
survivor, if any does not show clinical signs of canine distemper the 
test is inconclusive and may be repeated.
    (ii) If at least 19 of the 20 vaccinates do not survive without 
showing clinical signs of canine distemper during the observation 
period, the Master Seed Virus is unsatisfactory.
    (4) The Master Seed Virus shall be retested for immunogenicity in 3 
years unless use of the lot previously tested is discontinued. Ten 
susceptible dogs (8 vaccinates and 2 controls) shall be used in the 
retest. Susceptibility shall be determined in the manner provided in 
paragraph (b)(1) of this section.
    (i) Each vaccinate shall be injected with a predetermined quantity 
of vaccine virus as provided in paragraph (b)(2) of this section.
    (ii) At least 14 days postvaccination, a second serum sample shall 
be drawn from each dog and tested for neutralizing antibody to canine 
distemper virus in the same manner used to determine susceptibility.
    (iii) If the two controls have not remained seronegative at 1:2, 
the test is inconclusive and may be repeated.
    (iv) If at least 6 of the 8 vaccinates in a valid test do not 
develop titers of at least 1:50 based upon final serum dilution, the 
Master Seed Virus is unsatisfactory, except as provided in paragraph 
(b)(4)(v) of this section.
    (v) If the results of a valid serum neutralization test are 
unsatisfactory, the vaccinates and the controls may be challenged as 
provided in paragraph (b)(3) of this section. A Master Seed is 
satisfactory if all vaccinates remain free of clinical signs of canine 
distemper, while the two controls die with clinical signs of canine 
distemper. If the two controls do not die with clinical signs of canine 
distemper, the test is inconclusive and may be repeated: Provided, 
That, if any of the vaccinates show such signs, the Master Seed Virus 
is unsatisfactory.
    (5) An Outline of Production change shall be made before 
authorization for use of a new lot of Master Seed Virus shall be 
granted by the Animal and Plant Health Inspection Service.
    (c) Test requirements for release. Except for 
Sec. 113.300(a)(3)(ii), each serial and subserial shall meet the 
requirements prescribed in Sec. 113.300 and in this paragraph. Final 
container samples of completed product shall be tested. Any serial or 
subserial found unsatisfactory by a prescribed test shall not be 
released.
    (1) The test for pathogens prescribed in Sec. 113.37 shall be 
conducted on each serial or one subserial of avian origin vaccine.
    (2) Virus titer requirements. Final container samples of completed 
product [[Page 14363]] shall be tested for virus titer using the 
titration method used in paragraph (b)(2) of this section. To be 
eligible for release, each serial and subserial shall have a virus 
titer sufficiently greater than the titer of vaccine virus used in the 
immunogenicity test prescribed in paragraph (b) of this section to 
assure that when tested at any time within the expiration period, each 
serial and subserial shall have a virus titer of 10<SUP>0.7 greater 
than that used in such immunogenicity test but not less than 10<SUP>2.5 
TCID<INF>50 per dose.


Sec. 113.307  [Removed]

    8. Section 113.307 is removed.

    Done in Washington, DC, this 13th day of March 1995.
Terry L. Medley,
Acting Administrator, Animal and Plant Health Inspection Service.
[FR Doc. 95-6647 Filed 3-16-95; 8:45 am]
BILLING CODE 3410-34-M