An Equal Opportunity Provider and Employer

VETERINARY SERVICES MEMORANDUM NO. 800.201 

TO: Veterinary Services Leadership Team

Directors, Center for Veterinary Biologics

Biologics Licensees, Permittees, and Applicants

FROM: Jack A. Shere

Deputy Administrator

SUBJECT: General Licensing Considerations: Backpassage Studies

I. PURPOSE

This memorandum provides guidance related to title 9, Code of Federal Regulations, 

parts 102.5 and 104.5. It provides information and recommendations about the design 

and conduct of backpassage studies to support an application for a U.S. Veterinary 

Biological Product License or U.S. Veterinary Biological Product Permit for 

Distribution and Sale. 

Although this memorandum represents current policy regarding reversion to virulence 

studies, it does not confer rights for, or on, any person and does not operate to bind 

Animal and Plant Health Inspection Service (APHIS) or the public. An alternative 

approach is acceptable if such approach satisfies the requirements of the applicable 

statute, regulations, or both. 

II. CANCELLATION

This memorandum replaces Veterinary Services (VS) Memorandum No. 800.201 dated 

June 25, 2008.

III. BACKGROUND

The Center for Veterinary Biologics-Policy Evaluation and Licensing (CVB-PEL) 

requests that license and permit (for distribution and sale) applicants conduct 

backpassage studies to evaluate the stability of Master Seeds for conventional modified 

live or live recombinant vaccines to provide assurance that such vaccine microorganisms will not revert to virulence when administered to the host animal. Live 

vaccines are those that may be capable of replication in the target animal, stimulate a 

useful immune response, and generally cannot be completely characterized by chemical 

and physical tests alone. 

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence

Ave, SW

Washington, DC

20250
VETERINARY SERVICES MEMORANDUM NO. 800.201

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 An Equal Opportunity Provider and Employer

APHIS is a member of the International Cooperation on Harmonization of Technical 

Requirements for the Registration of Veterinary Medicinal Products (VICH). This policy is 

consistent with VICH requirements for licensure of live vaccines. VICH Guideline (GL) 41, 

Examination of live veterinary vaccines in target animals for absence of reversion to 

virulence, outlines the requirements to demonstrate a lack of reversion to virulence. 

One objective of VICH is to promote harmonization of technical regulatory requirements for 

veterinary medicinal products among regulatory agencies in different countries. APHIS is 

committed to seeking scientifically based harmonized technical requirements for veterinary 

biological products. Developed under the principles of the VICH, VICH GL 41 provides a 

unified standard for the European Union, Japan, and the United States to facilitate the mutual 

acceptance of clinical data by the relevant regulatory authorities for reversion to virulence 

studies. VICH members developed this guideline with consideration of the current practices 

in the European Union, Japan, and the United States, together with those of Australia and

New Zealand. The results of a study conducted as per VICH guidelines, or per the guidelines 

in VS Memorandum No. 800.201, should be acceptable to APHIS to demonstrate lack of 

reversion to virulence.

Backpassage studies consist of successively propagating vaccine Master Seed through a 

series of backpassages in vivo. Applicants administer the Master Seed microorganism to a 

group of susceptible host animals and, after an appropriate incubation time, recover the 

microorganism from these animals and administer it to a second group of susceptible host 

animals. Applicants should conduct a minimum of five such successive passages.

IV. GUIDELINES 

A. General

1. Study Protocols. Applicants should submit a detailed protocol, including the 

criteria for determining reversion, for CVB-PEL review before initiating a 

backpassage study.

2. Preliminary Data. The CVB recommends that applicants submit preliminary data 

from studies conducted to evaluate the route of administration and procedures for 

recovery and to assess the expected rate of recovery of the vaccine microorganism 

from test animals with the proposed protocol. The CVB-PEL will consider the 

backpassage requirement fulfilled when the applicant confirms preliminary data 

indicating that the applicant cannot recover the vaccine microorganism from 

vaccinates by using a group of 10 animals in a follow-up study performed as 

outlined in section IV.B of this memorandum.

3. Passage Procedures. In progressing from one backpassage to the next, applicants 

may concentrate recovered material between passages but APHIS prohibits in vitro 

propagation between passages.
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 An Equal Opportunity Provider and Employer

4. Study Animals. Applicants should conduct the backpassage studies in the most 

susceptible species of animals recommended on the label. Applicants must 

conduct the study in animals of the most susceptible age, regardless of minimum 

age recommendations on the label. These test animals should also be susceptible 

(seronegative) to the vaccine microorganism being tested. Applicants must include 

a justification for the species, age, and sex of the test animals in the protocol 

submitted for review.

5. Combining Backpassage Studies with Shed-and-Spread Studies. If the route of 

administration for backpassage studies determined from preliminary work is the 

same as the route of administration recommended on the label, applicants may 

expand backpassage studies to also collect data on shed and spread of the vaccine 

microorganism; otherwise, the CVB-PEL requires a separate shed-spread study. 

B. First Backpassage

1. Route of Administration. Administer the vaccine Master Seed to a group of host 

animals by the route most likely to lead to replication and reversion of the 

microorganism to virulence. 

2. Numbers of Animals. Use two to five animals, as needed, to ensure reisolation and 

continued backpassage (see table in VICH GL 41 on probability of reisolation). 

Use ten animals to confirm failure to recover the vaccine microorganism from a 

preliminary study (see IV.A.2.). 

3. Dosage. Administer test animals at least a typical vaccine dose (not an 

immunogenicity test dose). The applicant should formulate a typical vaccine dose 

at a titer that is above the targeted release dose of the product and includes

overage for the expected loss of titer over dating and testing variation.

4. Recovery of the Microorganism. After a period consistent with the 

pathophysiology of the progression of the disease in a naturally infected animal, 

attempt to recover the vaccine microorganism from the most appropriate tissues 

or secretions collected from treated animals.

C. Successive Backpassages

1. Passage Procedures. Administer recovered material (pooled material is 

acceptable) from animals in the preceding treatment group to animals in 

successive groups by the same route as in the first passage.

2. Number of Animals for Each Successive Passage. Based on the expected rate of 

recovery, treat two to five animals as needed to provide a high probability of 

reisolation. 
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 An Equal Opportunity Provider and Employer

3. Observations. Observe treated animals for clinical signs indicative of reversion of 

the vaccine strain to virulence. Test operators should assess clinical signs that 

indicate administration of the material caused adverse effects to the animal.

4. Number of Passages. Make at least five backpassages (four successive 

backpassages beyond the first backpassage).

5. Maintenance Period. Maintain test animals from the last backpassage group for at 

least 21 days after administration of the recovered microorganism, unless 

otherwise justified.

6. Characterization. Characterize the microorganism isolated from the last 

backpassage phenotypically and/or genotypically and compare it with the Master 

Seed to evaluate genetic stability and reversion to virulence.