April 26, 2001 

VETERINARY SERVICES MEMORANDUM NO. 800.99 

Subject: Guidelines for Using In Vitro Relative Potency Tests to Determine the 

Antigen Content of Inactivated Bovine Rhinotracheitis Vaccine 

To: Veterinary Biologics Licensees, Permittees, and Applicants 

Directors, Center for Veterinary Biologics 

I. PURPOSE 

This memorandum provides guidelines for in vitro potency tests for serial release of bulk 

or final container samples of inactivated bovine rhinotracheitis vaccine. The guidelines 

outlined below include recommended procedures for the qualification and requalification 

of in vitro reference preparations as provided in Title 9, Code of Federal Regulations, 

Part 113, Section 113.8 (9 CFR 113.8). 

II. BACKGROUND 

APHIS has published the Standard Requirement for Bovine Rhinotracheitis Vaccine in 9 

CFR 113.216. The regulation specifies that bulk or final container samples of the 

completed product must be tested for potency using a host animal serum neutralization 

test in cell culture or a virus challenge test of host animals if the results of the serum 

neutralization test are unsatisfactory. APHIS has granted an exemption to this 

requirement under 9 CFR 113.4 for firms using validated tests that determine potency by 

comparing the antigen content of the test serial to a reference preparation (Relative 

Potency) in accordance with the requirements specified in 9 CFR 113.8. Refer to 

Veterinary Services Memorandum 800.90 for additional information concerning relative 

potency assays and requirements for references. 

III. GUIDELINES 

A. In Vitro Relative Potency Tests

An in vitro relative potency test that compares the antigen content of a test serial to that 

of a reference preparation using a parallel line immunoassay or equivalent method 

acceptable to APHIS may be used to determine the antigen content of a serial of bovine 

rhinotracheitis vaccine. 

B. Requirements for the Reference

The definition of a reference is contained in 9 CFR 101.5, and the requirements for 

references used in in vitro tests for serial release are specified in 9 CFR 113.8. 

Additional guidance is provided in Veterinary Services Memorandum 800.90. 
Veterinary Services Memorandum No. 800.99 2 

1. Qualification of Final Product Master References 

a. Master References that are non-frozen final product are qualified 

in animals directly using a minimum of 20 vaccinates and 10 controls in 

the host animal immunogenicity study. 

b. Serum samples taken from animals in the reference qualification 

study that are stored frozen may be used in future reference requalification 

studies. 

2. Qualification of Frozen Master References 

a. Master References that consist of final product or purified 

antigen that is stored frozen are qualified indirectly in host animals using a 

Qualifying Serial. Qualifying Serials must be produced in accordance with 

the current filed Outline of Production and tested for immunogenicity in a 

manner acceptable to APHIS. Additional guidance on establishing a 

Qualifying Serial is provided in Veterinary Services Memorandum No. 

800.90 

b. The Qualifying Serial must be administered to a minimum of 20 

vaccinates in the host animal immunogenicity study. Use 10 unvaccinated 

animals as controls. 

c. Serum samples taken from animals in the reference qualification 

study that are stored frozen may be used in future reference requalification 

studies. 

C. Working References

1. Master References that have been qualified in a manner acceptable to 

APHIS as specified in paragraph III.B. may be used throughout dating as the 

Working Reference in the serial release potency test. Additional guidance is 

provided in Veterinary Services Memorandum No. 800.90. 

2. Qualifying Serials produced in accordance with the current filed Outline 

of Production and tested for immunogenicity in accordance with methods 

acceptable to APHIS may be used as the Working Reference in the serial release 

potency test. Additional guidance is provided in Veterinary Services 

Memorandum 

No. 800.90. 

D. Expiration Date of the Reference

1. As specified in 9 CFR 113.8, the expiration date of the Master 
Veterinary Services Memorandum No. 800.99 3 

Reference shall be equal to the dating of the product or as supported by data 

acceptable to APHIS. 

2. The expiration date of a Working Reference stored in a manner similar 

to a serial of licensed product shall be the same as the expiration date of a serial of 

the licensed product. 

3. Expired references may not be used in serial release potency tests unless 

requalified. 

E. Requalification of the Reference

References may be requalified by one of the following methods: 

1. The host animal serological test prescribed in 9 CFR 113.216(c)(2) may 

be used to requalify an expired Master Reference for use as the Working 

Reference in the serial release potency test provided that: 

a. The Master Reference is directly administered to test animals as 

non-frozen final product. 

b. Master References that are frozen (or that are purified antigens) 

are indirectly administered to animals using a Qualifying Serial 

c. Serum samples for titer determination are collected at the same 

time interval as was used in the original Master Reference qualification 

test. 

d. The serum neutralization test used to titer serum samples 

collected during the Master Reference requalification study is the same in 

vitro method that was used to titer samples when the reference was 

qualified. 

e. The serum antibody titers of test animals used in the 

qualification and requalification studies are statistically equivalent in side 

by side testing. 

2. An expired Master Reference may be requalified by using in vitro 

methods to compare to a Reference Standard that has been prepared and validated 

under the auspices of a National Control Authority or a recognized Standards 

Organization acceptable to APHIS. Additional guidance is provided in Veterinary 

Services Memorandum No. 800.90. 

3. The dating of a Working Reference that was established by testing a 

Qualifying serial in host animals may be extended by comparison to the Master 

Reference or by using the serial release potency test prescribed in 9 CFR 
Veterinary Services Memorandum No. 800.99 4 

113.216(c)(2) to measure serological response in host animals. Additional 

guidance is provided in Veterinary Services Memorandum No. 800.90. 

F. Test Interpretation

The results of each serial tested for potency using a parallel line assay or equivalent 

method acceptable to APHIS must be interpreted as specified in 9 CFR 113.8(b). To be 

eligible for release, the relative potency of each serial must be $ 1.0. 

/s/ Chester A. Gipson for 

Alfonso Torres 

Deputy Administrator 

Veterinary Services