June 27, 2014

VETERINARY SERVICES MEMORANDUM NO.800.120

TO: Veterinary Services Leadership Team

Directors, Center for Veterinary Biologics

Biologics Licensees, Permittees, and Applicants 

FROM: John R. Clifford /s/ Jack A. Shere, for

Deputy Administrator

SUBJECT: Dilution of Preservative Screening for Sterility Testing of Veterinary 

Biologics

I. PURPOSE

This memorandum provides guidance on sterility testing of veterinary biological 

products. It is meant to assist licensees, permittees, and applicants in meeting 

sterility requirements in title 9, Code of Federal Regulations (9 CFR), part 113 

sections 113.25(d), 113.26, and 113.27.

II. REPLACEMENT

This memorandum combines information that was originally provided in the 

following Center for Veterinary Biologics (CVB) Notices:

• CVB Notice 09-02 - “Dilution of Preservative Screening for Broth-based 

Sterility Tests,” dated January 22, 2009.

• CVB Notice 09-25 - “Dilution of Preservative Screening for Plate-Based 

Sterility Tests,” dated December 31, 2009. 

• CVB Notice 12-21 – “Conducting Dilution of Preservative Studies for Live 

Bacterial Vaccines,” dated October 15, 2012.

III. BACKGROUND

Sterility of veterinary biologics is determined in direct inoculation tests codified in 

9 CFR 113.26 and 113.27. Manufacturers are required, under 9 CFR 113.25(d), to 

determine the ratio of inoculum to medium, which shall result in sufficient dilution 

of such product to prevent bacteriostatic and fungistatic activity. Currently, live 

bacterial products without antibiotics or oral products may be granted an exemption 

to 9 CFR 113.25(d) upon request. 

Animal and Plant

Health Inspection

Service

Veterinary Services

1400 Independence 

Ave, SW

Washington, DC

20250

An Equal Opportunity Provider and Employer
VETERINARY SERVICES MEMORANDUM 800.120

Page 2

The amount and type of media found acceptable at each temperature and the date these 

dilution of preservative data were accepted, or the date a product exemption was given 

are recorded in section V.A. of the Outline of Production.

The following table provides references to our laboratory procedures for the above 

codified testing:

Material tested 9 CFR* Supplemental Assay 

Method (SAM)*

Killed biologics 113.26 906

Master Seed viruses

Live Viral biologics

113.27(c)

113.27(a) 908

Live poultry biologics not 

for injection 113.27(e) 909

Master Seed bacteria

Live bacterial biologics

113.27(b)

113.27(d) 928

Dilution of preservative 113.25(d) 903

*The current version of the document 

Since 2007, the CVB has been screening the media volumes indicated in the Outlines of 

Production for dilution of preservatives as serials were picked for check or confirmatory 

sterility testing. The screening test is the inoculation of a single extra tube or plate (11th

vessel) with indicator organisms and the serial. The extra vessel is observed for growth, 

with the lack of growth being suggestive of product inhibition/interference. A dilution of 

preservative test is performed in accordance with the appropriate SAM listed above. The 

data from 2007 and 2008 were combined and provided to the industry in 2009 by CVB 

Notices 09-02 and 09-25. For the broth-based tests, the data suggested that for 7% of the 

serials tested at the CVB, the dilution of preservative study may not have provided an 

adequate dilution to result in a valid test. For the plate-based test, the data suggested that 

for 35% of the serials tested at the CVB, the dilution of preservative study may not have 

provided an adequate dilution to result in a valid test.

Serials of licensed product that do not pass the full dilution of preservative testing, in 

accordance with SAM 903, are reported by the CVB as a no test for the sterility test and 

unsatisfactory for dilution of preservative. The APHIS Form 2008 is then processed as 

not to be released with a copy of the CVB Test Report provided. 

For prelicensing serials, the firm is informed of the result by correspondence from the 

assigned reviewer with the associated CVB Test Report enclosed. No license is

issued until the matter is resolved.

 An Equal Opportunity Provider and Employer
VETERINARY SERVICES MEMORANDUM 800.120

Page 3

IV. POLICY

Since insufficient media volumes to produce valid sterility tests continue to be found in 

our laboratory, the policy (as first announced in the notices) will continue as a standard 

practice. 

In some cases, past history of the product or other suggestive evidence may result in 

testing according to 9 CFR 113.25(d)/SAM 903, as well as/or instead of the eleventh 

vessel screening test.

V. IMPLEMENTATION/ APPLICABILITY

The eleventh vessel screening test and subsequent SAM 903 testing and regulatory 

actions will continue as originally implemented. Therefore, that aspect of the 

memorandum should be considered as implemented immediately. Since factors, in 

addition to the eleventh vessel, have been noted, this policy should be considered as 

implemented 30 days from publication of this memorandum.

 An Equal Opportunity Provider and Employer