Title: Dilution of Preservative Studies
Release Date: 20 Aug 2019 Author: Document Number: CVB-WI-0182.02

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Veterinary Services
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Service
Veterinary Services

Center for Veterinary
Biologics

1920 Dayton Avenue
PO Box 844
Ames, IA 50010

(15) 337-6100

Document Number: CVB-WI-0182 Revision: 02

Previous Number: PEL Reviewer Manual Ch. 4.1.5

Section/Area: CVB-WI-PEL-REV
Release Date: 20 Aug 2019
Notes:

Printed by: CRRYAN on 23 Aug 2019, 11:27:43 am.
For Official Use Only


Title: Dilution of Preservative Studies
Release Date: 20 Aug 2019 Author: Document Number: CVB-WI-0182.02

Dilution of Preservative Studies

Historically, the information regarding media volumes used for sterility testing in Section V.A.
of the Outline of Production was collected on inspection and provided to the laboratory. Firms
would group like products and conduct the studies on representative serials or mock ups.

Currently, studies patterned after SAM 903 are submitted during the licensure process. Firms
tend to submit data using serials of the specific product. The amount of media used for the test
and the date (with ML designation) that data to support the volume was accepted are now noted
in the filed Outline of Production.

While firms may be allowed to add up to a certain level of antibiotic within a specified range,
serials may not represent that maximum amount. Issues of the dilution of preservative not
yielding adequate media amounts have been detected pre and post licensure. Therefore, the
dilution of preservative studies should be conducted with serials at the maximum antibiotic level
allowed in the range specified in the Outline of Production. This may require spiking the
prelicensing serials or adjusting the maximum antibiotic concentration level cited in the Outline
of Production.

During the licensure process, the antibiotic concentration of the prelicensing serials should be
compared to the maximum amount allowed in the outline and the concentration of the serial(s)
used for the dilution of preservative study.

During the confirmatory testing of the prelicensing serials and check tests, an 11" vessel
screening test is being conducted. The screening test is described in Veterinary Services
Memorandum 800.120 entitled “Dilution of Preservative Screening for Sterility Testing of
Veterinary Biologics”.

This is basically an extra tube or plate of media that receives the product and the indicator
organism. If the 11" vessel shows no growth, the CVB Laboratory conducts a dilution of
preservative test in accordance with SAM 903 and 9 CFR 113.25. Therefore, a prelicensing
serial or other serial picked for testing may be reported as unsatisfactory for dilution of
preservative and a no test for sterility testing conducted in accordance with 9 CFR 113.26/27.

For live bacterial products, it indicates products with antibiotics added, dilutions 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. An
exemption may be requested. However, in an effort to evaluate this situation, the CVB laboratory
has begun inoculating an extra media vessel for all live bacterial products utilizing the purity test
in accordance with 9 CFR 113.27(b). Ifa lack of indicator organism growth in the extra tube is
seen in check or confirmatory testing, this policy will be re-evaluated. Exemptions provided may
be revoked if interference is detected.

CVB-WI-0182.02 Page | of 2

Printed by: CRRYAN on 23 Aug 2019, 11:27:43 am.
For Official Use Only


Title: Dilution Studies
Release Date: 20 Aug 2019 Author: Document Number: CVB-WI-0182.02

In the case of live E. coli products (and potentially others), the E. coli will outgrow the indicator
organisms and interfere with their growth almost completely. The use of larger amounts of media
does not circumvent this situation. In this case one might view the E. coli itself as an indicator
organism for validating the media volume. Exemptions may be requested.

CVB-WI-0182.02 Page 2 of 2

Printed by: CRRYAN on 23 Aug 2019, 11:27:43 am.
For Official Use Only