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

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.

-----------------------------------------------------------------------

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 TCID50

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 TCID50 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

TCID50 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 TCID50 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 TCID50 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 TCID50 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

TCID50 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 TCID50 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 100.7 greater

than that used in such immunogenicity test(s) but not less than

10.2.5 TCID50 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 TCID50 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 100.7 greater

than that used in such immunogenicity test but not less than 102.5

TCID50 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