Safeguarding American Agriculture

Federal Relay Service

APHIS is an agency of USDA’s Marketing and Regulatory Programs (Voice/TTY/ASCII/Spanish)

An Equal Opportunity Provider and Employer 1-800-877-8339

October 15, 2012 

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-21 

TO: Biologics Licensees, Permittees, and Applicants

 Directors, Center for Veterinary Biologics

 Veterinary Services Leadership Team

 

FROM: Richard E. Hill, Jr. /s/ Richard E. Hill, Jr.

 Director

 Center for Veterinary Biologics

SUBJECT: Conducting Dilution of Preservative Studies for Live Bacterial Vaccines 

I. PURPOSE

The purpose of this Notice is to inform interested parties regarding the Center for 

Veterinary Biologics’ (CVB) interpretation of when dilution of preservative data is 

required for live bacterial vaccines. 

II. BACKGROUND

Title 9, Code of Federal Regulations (9 CFR), Part 113.27(b) describes a direct 

inoculation sterility test to be conducted on live bacterial vaccines in order to detect 

extraneous bacterial or fungal contamination. With regard to the media volume to be 

used, 9 CFR 113.27(b) provides a default media volume of 40 mL, but indicates that 

larger volumes may be needed when 9 CFR 113.25(d) testing indicates product 

interference. The CVB uses a screening test for dilution of preservative/ product 

interference when conducting confirmatory sterility testing of products. The screening 

test was first announced in CVB Notice 09-02, Dilution of Preservative Screening for 

Broth-Based Sterility Tests. 

While many live bacterial vaccines lack antibiotics in their final form, a small number of 

them have antibiotics added which could cause product interference for the detection of 

bacterial or fungal contamination. The intent of this Notice is to ensure the dilution of 

preservative requirement in 9 CFR 113.25(d) and 9 CFR 113.27 is consistently applied 

to live bacterial products. 

III. ACTION (or POLICY)

For live bacterial products with antibiotics added, dilution of preservative studies are 

required in accordance with 9 CFR 113.25(d). For live bacterial products that lack 

antibiotics at formulation, dilution of preservative studies will not be required at this 

time. However, in an effort to evaluate this situation, the CVB laboratory has begun 

United States 

Department of 

Agriculture

Animal and Plant 

Health Inspection 

Service

Veterinary Services

Center for Veterinary 

Biologics

1920 Dayton Avenue 

PO Box 844 

Ames, IA 50010 

(515) 337- 6100
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 12-21 

Page 2 

inoculating an extra media vessel for all live bacterial products utilizing the purity test in 

accordance with 9 CFR 113.27(b). If a lack of indicator organism growth in the extra 

tube is seen in check or confirmatory testing, this result will be evaluated utilizing a 

complete dilution of preservative study in accordance with SAM 903. 

For oral products, not covered by 9 CFR 113.27(e), an exemption to dilution of 

preservative may be supplied upon request. The default amount of media would then be 

used. 

Therefore, each Outline of Production should include information on the dilution of 

preservative status with the information in Section V regarding the sterility test. The date 

that the data were accepted, the date an exemption was obtained, or the statement that the 

product lacks antibiotics, should be included. 

IV. IMPLEMENTATION/ APPLICABILITY

The above policies will be implemented 30 days from the publication date of this Notice. 

As each licensee/permittee is required to review their Outlines of Production at least 

annually for accuracy and sufficiency in accordance with 9 CFR 114.8(d), we would 

anticipate that within 18 months of publication, all products and their Outlines will be in 

compliance with the above. Manufacturers that require additional time to achieve 

compliance should contact their Reviewer with the request for additional time.