Accepted Alternative Methods for Biologics and Vaccine Testing
Vaccines improve human and animal health and welfare by preventing the spread of infectious diseases. Testing of vaccines and other biologics to ensure efficacy and safety requires large numbers of animals, many of which experience pain and distress during testing. Therefore, identification of methods that would reduce or eliminate the need for animal testing for vaccines is a high priority for NICEATM and ICCVAM.
U.S. regulatory agencies have issued guidances on use of available alternative methods that replace, reduce, or refine (enhance animal well-being and lessen or eliminate pain and distress) animal use for vaccine testing.
Methods for Safety Testing
Exemption to Requirement for Animal Safety Testing of Veterinary Biologics
The U.S. Department of Agriculture (USDA) issued Veterinary Services Memorandum 800.116 (2013), which provides guidance under which manufacturers of vaccines, inactivated bacterial products, and antibody products may request an exemption to animal safety testing. To be considered, specific products must have a documented history of acceptable safety results and controlled manufacturing processes that ensure batch-to-batch consistency and sterility.
Relevance of the Target Animal Safety Test for Batch Safety Testing of Veterinary Vaccines
U.S. regulations require that all veterinary vaccine batches must meet defined safety criteria prior to release. If vaccine potency/efficacy testing is conducted in laboratory animal models or in vitro, separate safety tests in the targeted end-use animal are typically required. Under certain circumstances, U.S. regulations (9 CFR 113.4) provide for exemption from testing, such as when consistency of safety is demonstrated in a sufficient number of consecutive batches.
Methods for Potency Testing
USDA Guidelines for Validation of In Vitro Potency Assays
Validation is a process by which the reliability and relevance of an assay are established for its intended application. USDA Veterinary Services Memorandum 800.112, updated in 2015, provides guidance for validating veterinary biologics potency assays, and clarifies information found in Veterinary Services Memorandum 800.50 and in title 9 of the Code of Federal Regulations, sections 102.3(b)(2)(ii) and 113.8(a)(3)(ii).
Reduction and Replacement Alternatives for Potency Testing of Veterinary Leptospira Vaccines
Leptospirosis is a serious and often fatal disease caused by bacteria of the genus Leptospira. Over 500,000 people develop leptospirosis each year. The disease also affects livestock, pets, and wildlife. The current leptospirosis vaccine potency test is performed in hamsters; the test has a lethal endpoint, takes more than five weeks to complete, and requires laboratory personnel to handle live Leptospira bacteria.
The USDA developed alternative in vitro potency tests for veterinary vaccines protecting against several Leptospira interrogans serovars (strains) including Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. These tests compare the levels of protective antigen to a qualified reference standard, confirming the potency of tested vaccine production lots without using animals.
The USDA published the in vitro assay methods as Supplemental Assay Methods (SAMs) in 2009. All four SAMs were revised in 2017.
The USDA provides information on use of the in vitro tests and guidance for obtaining an exemption to the requirement for potency testing of Leptospira bacterins in hamsters in Veterinary Services Memorandum 800.102 (2013).
In October 2015, the Center for Veterinary Biologics (CVB) issued CVB Notice 15-13, which describes an exemption from the titration requirement in vaccination-challenge potency assays for Leptospira serogroups Canicola and Icterohaemorrhagiae. CVB Notice 17-06, issued in 2017, expanded this guidance to include serogroups Pomona and Grippotyphosa. Removal of back-titration hamsters can reduce animal use by 50% for vaccine potency testing.
USDA Guidance on Rabies Vaccine Potency Testing
CVB Notice 13-10 (July 2013) eliminates the upper limit LD50 for a valid challenge test as a validity requirement when conducting the rabies virus potency test. Tests in which the challenge LD50 is greater than the upper limit are now acceptable. This is expected to reduce the number of animals used for rabies vaccine testing.
Alternative Test Procedure for Tuberculin, PPD Bovis, Intradermic
Tuberculosis is an infectious disease that can cause serious illness or death but may also infect people without causing illness. The tuberculin test, a screening test for tuberculosis, is important for controlling the spread of the disease. Tuberculin test reagents are tested for potency using guinea pigs. USDA Veterinary Services Memorandum 800.114 (2012) describes an alternate testing procedure for tuberculin test reagents that uses 15 rather than 43 guinea pigs per test.
USDA Guidance on the Use of Humane Endpoints and Methods in Animal Testing of Biological Products
In May 2012, the USDA Center for Veterinary Biologics (CVB) issued guidance on the use of humane endpoints and methods in animal testing of biological products. CVB Notice No. 12-12 includes specific guidance regarding the use of humane endpoints in biological products testing, including the rabies challenge test. The guidance strongly encourages the use of anesthesia for intracerebral inoculation of mice during rabies vaccine testing, and encourages the use of analgesics in animal studies and potency testing when it can be shown this does not affect the study outcome. CVB Notice 12-12 incorporates recommendations for refinement of rabies vaccine testing made by participants at the October 2011 NICEATM-sponsored workshop on alternative methods for rabies vaccine potency testing.
Acceptance of Cell-Based Potency Assay for Botulinum Neurotoxin Products
In addition to its well-known cosmetic applications, botulinum neurotoxin is used to treat a variety of illnesses. In 2011, the U.S. Food and Drug Administration accepted a method developed by Allergan, Inc., that replaces animal use for testing the stability and potency of botulinum neurotoxin type A products. A presentation on this method was given at the November 2006 NICEATM-sponsored workshop on alternatives for botulinum toxin testing.
USDA Guidelines on Master Reference Qualification and Requalification for Vaccine Potency Assays
USDA Veterinary Services Memorandum 800.211 (2011) provides guidance on the length of time that reference standards for veterinary vaccine potency testing may be used. The new guidance allows Master References for serial release of most inactivated products to be used continually for up to 15 years if the product was licensed before January 1, 2011, and the appropriate data has been generated, submitted, and approved by the USDA. This will significantly reduce the use of host animals (including dogs, cats, and cows) for Master Reference requalification. As part of this guidance, Veterinary Services “...encourages the development of potency assays that are completely in vitro as part of the effort to reduce, refine, and replace the use of animals in testing (the “three Rs”).”
ELISA Test for Batch Potency Testing of Veterinary Erysipelas Vaccines
Erysipelas is an infectious livestock disease. Infected animals may show no symptoms, or may develop an acute or chronic disease that can be fatal. The bacterium that causes erysipelas can be spread by contaminated waste, feed, water, soil, and bedding. Biting insects and other animals also serve as vectors for erysipelas. Vaccination of livestock is key to control of the disease.
The original erysipelas vaccine potency test used 30 pigs per dose, took up to four weeks to perform, and required development of clinical disease in control animals. An alternative in vitro potency test for veterinary erysipelas vaccines is described in USDA SAM 613 (2009). This test compares the level of protective antigen to a qualified reference standard, confirming the potency of tested production lots of vaccine without using animals.
Serology Test for Batch Potency Testing of Human Tetanus Vaccines
Tetanus can be fatal to animals and humans. The bacterium that causes the disease is widespread, found in soil and in the gastrointestinal tracts of animals. Vaccination is important for prevention of tetanus, and periodic booster vaccines are needed to maintain lifelong immunity.
The traditional potency test required prior to release of each batch of tetanus vaccines, a vaccination–challenge test in guinea pigs, requires large numbers of animals and involves the development of disease in control animals. An alternative serological method refines animal use by using an ELISA test or a toxin-binding inhibition (or “ToBI”) test to measure the amount of tetanus antibodies in the blood of immunized guinea pigs. Because potency is assessed by antibody production rather than by protection against disease, this method eliminates the pain and distress experienced by the animals that develop disease in the challenge test.
The FDA provides for in vitro tests of vaccine potency in 21 CFR 610.10 .
Other Guidance on Biologics Testing
USDA Policy Concerning the Use of Humane Endpoints in Biologics Testing
USDA CVB Notice 04-09 (2004) clarified the USDA policy on the use of humane endpoints for animal challenge potency tests of biologics. The notice covers potency tests that involve administration of viable virus, bacteria, or bacterial toxin to animals in doses expected to be lethal. It states that moribund animals (animals exhibiting clinical signs consistent with the expected disease and unable to rise or move) may be humanely euthanized and considered as deaths as outlined in 9 CFR 117.4.