CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 15-13

TO: Biologics Licensees, Permittees, and Applicants 

Directors, Center for Veterinary Biologics

Veterinary Services Management Team

FROM: Byron Rippke

Director

SUBJECT: Option to Remove Back-titration Hamsters from In Vivo Potency 

Tests for Leptospira Serogroups Canicola and Icterohaemorrhagiae 

I. PURPOSE

The purpose of this notice is to inform interested parties that the Center for 

Veterinary Biologics (CVB) will allow an exemption from the titration 

requirement in vaccination-challenge potency assays for Leptospira Serogroups 

Canicola and Icterohaemorrhagiae. Removal of the back-titration hamsters could 

reduce animal use by 50% when potency testing these two fractions.

II. BACKGROUND

The codified potency tests for Leptospira Canicola Bacterins and Leptospira 

Icterohaemorrhagiae Bacterins are hamster vaccination-challenge assays 

described in title 9, Code of Federal Regulations (9 CFR), parts 113.102 and 

113.103 respectively. These tests have been targeted by the CVB as part of our 

ongoing commitment to refine, reduce, and replace animal testing.

Currently, a valid test requires ≥80% (8/10) of challenge control hamsters to 

succumb to disease and an LD50 between 10 and 10,000. The minimum LD50

requirement has always been met if at least 80% of challenge controls 

succumbed to disease; the back-titration hamsters are not required to confirm a 

minimum challenge dose is administered. The maximum LD50 constraint has 

been based on 1) reports that subpotent bacterins may occasionally test potent 

when ≥10,000 LD50 is administered and 2) historical concerns that a potent serial 

may be deemed subpotent when challenged with a higher concentration of 

Leptospira than normally seen in the field.

Tests were conducted at the CVB to evaluate the risk associated with the removal 

of the maximum LD50 limit. The CVB could not identify an increased risk of 

releasing a subpotent vaccine. A small risk was identified that some potent 

bacterins may be deemed unsatisfactory when the challenge LD50 exceeds 

10,000. The risk will vary by product.

Animal and Plant

Health Inspection

Service

Veterinary Services

Center for Veterinary

Biologics

1920 Dayton Avenue

PO Box 844

Ames, IA 50010

(515) 337-6100

An Equal Opportunity Provider and Employer
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 15-13

Page 2

III. ACTION

Firms may request an exemption from the titration requirement for the vaccinationchallenge potency assays for Leptospira Serogroups Canicola and Icterohaemorrhagiae 

when using the CVB supplied strains. The CVB will accept the following alternative in 

the Outline of Production: 

Modified 9 CFR Vaccination-Challenge Leptospira Assay

This modified approach allows a one-stage vaccination-challenge assay without backtitration hamsters followed by an option to test by the 9 CFR potency assay including

back-titration hamsters. Details are as follows: 

1) For the initial test, 10 vaccinated hamsters and 10 challenge control hamsters will 

be tested according to the 9 CFR specifications, except the LD50 of the challenge 

will not be monitored.

2) Results for the initial test shall be evaluated as follows:

a. At least 8 of 10 challenge control hamsters must succumb to disease for a 

valid test. If <8 challenge control hamsters succumb to disease, the test is 

declared a “NO TEST” and the vaccination-challenge assay without backtitration hamsters is repeated without prejudice.

b. If ≥8 of 10 vaccinates survive in a valid test, the serial is declared “SAT” 

and no additional testing is necessary. 

c. If ≤2 of 10 vaccinates survive in a valid test, the serial is declared 

“UNSAT” and no additional testing will be conducted.

d. If 3-7 of 10 vaccinates survive in a valid test, the serial may be declared 

“NO TEST” and tested by the complete codified 9 CFR method. Any 

serials not tested by the 9 CFR method will be declared UNSAT.

3) Special instructions concerning testing by the complete codified 9 CFR test, 

which includes back-titration hamsters, are as follows:

a. All tests in which the challenge exceeds 10,000 LD50 will be considered 

invalid.

b. The results from the complete codified 9 CFR test will be evaluated 

independently from the initial test. The number of survivors in the 

complete codified 9 CFR retest will not be combined or averaged with the 

number of survivors from the initial test to determine the potency of a 

serial. 

The potency assays outlined in 9 CFR 113.102 and 113.103 remain acceptable if a firm 

prefers not to request an exemption from the titration requirement. If a firm chooses to 

test by the codified methods, all tests in which the challenge exceeds 10,000 LD50 will be 

considered invalid in accordance with 9 CFR 113.102 and 113.103. 

An Equal Opportunity Provider and Employer
CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 15-13

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IV. IMPLEMENTATION

According to 9 CFR 113.4, licensees and permittees must request an exemption to the 

codified hamster potency tests if they are interested in using an alternate approach. The 

Outline of Production must specify the titration exemption for each fraction. This policy 

is effective immediately. 

An Equal Opportunity Provider and Employer