An Equal Opportunity Provider and Employer

CENTER FOR VETERINARY BIOLOGICS NOTICE NO. 17-06

TO: Veterinary Services Leadership Team 

Directors, Center for Veterinary Biologics 

Biologics Licensees, Permittees, and Applicants

FROM: Byron E. Rippke

Director

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

Leptospira Serogroups Pomona and Grippotyphosa

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 vaccinationchallenge potency assays for Leptospira Serogroups Pomona and Grippotyphosa. 

Removal of the back-titration hamsters could reduce animal use by 50 percent when 

potency testing these two fractions.

II. BACKGROUND

The codified potency tests for Leptospira Pomona Bacterins and Leptospira 

Grippotyphosa Bacterins are hamster vaccination-challenge assays described in title 9, 

Code of Federal Regulations (9 CFR), parts 113.101 and 113.104 respectively. The 

leptospiral codified potency tests have been targeted by the CVB as part of our ongoing 

commitment to refine, reduce, and replace animal testing. CVB Notice No. 15-13 

already allows the option to remove back-titration hamsters from in vivo potency tests 

for Leptospira Serogroups Canicola and Icterohaemorrhagiae. This Notice expands the 

option to include the remaining codified leptospiral potency assays.

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 is met 

if at least 80% of challenge controls succumb 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 LD50 limit. An increased risk of either a subpotent serial release or a potent 

bacterin being deemed unsatisfactory was not specifically identified with removal of 

the LD50 requirement. Notably, the calculated LD50 often did not correlate to 

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. 17-06

Page 2

Leptospira concentration, suggesting the LD50 requirement may not be a functional validity 

requirement. 

III. ACTION

Firms may request an exemption from the titration requirement for the vaccination-challenge 

potency assays for Leptospira Serogroups Pomona and Grippotyphosa when using the CVB 

supplied strains. The CVB will accept the following alternative in the Outline of Production: 

A 9 CFR vaccination-challenge two-stage assay without back-titration hamsters.

1. Vaccinated hamsters and challenge control hamsters will be tested according to the 

9 CFR specifications except the LD50 of the challenge will not be monitored.

2. Any serial that is deemed UNSAT cannot be retested due to concerns of an 

overchallenge.

The potency assays outlined in 9 CFR 113.101 and 113.104 remain acceptable if the 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. 

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. The Outline of Production must specify 

the backtitration exemption for each fraction. This policy is effective at the receipt of this 

notice.