Accepted Alternative Methods for Biologics and Vaccine Testing

USDA has updated Veterinary Services Memorandum 800.116 (2017, originally issued 2013). This memorandum 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. Controlled manufacturing processes that ensure batch-to-batch consistency and sterility are also required.

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.

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. It also 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).

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 traditional 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. USDA developed alternative in vitro potency tests for veterinary vaccines protecting against several Leptospira interrogans serovars (strains). These strains include 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.

USDA published the in vitro assay methods as Supplemental Assay Methods (SAMs) in 2009. All four SAMs were revised in 2017.

• USDA SAM 624: ELISA test for batch potency testing of Leptospira interrogans serovar Pomona
• USDA SAM 625: ELISA test for batch potency testing of Leptospira interrogans serovar Canicola
• USDA SAM 626: ELISA test for batch potency testing of Leptospira kirschneri serovar Grippotyphosa
• USDA SAM 627: ELISA test for batch potency testing of Leptospira interrogans serovar Icterohaemorrhagiae

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 USDA 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.

A separate group of hamsters is also required to propagate and maintain virulent strains for the codified test and developmental needs associated with regulated vaccines. Over 2,500 hamsters per year per facility are estimated to be used for propagation of virulent Leptospira. As a result, in 2018 the CVB developed a cryopreservation protocol for the commonly used Leptospiral strains and provides cryopreserved virulent Leptospira upon request.

A 2022 review paper coauthored by NICEATM scientists (Rogers et al. 2022) examined progress toward replacing animal use for vaccine batch potency testing with available in vitro methods.

CVB Notice 13-10 (issued by USDA in July 2013) eliminated 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.

Tuberculosis is an infectious disease that can cause serious illness or death. It 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.

In May 2012, the USDA Center for Veterinary Biologics (CVB) issued CVB Notice No. 12-12, which provides 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 during the injection of virus into the brains of mice during rabies vaccine testing. The use of analgesics in animal studies and potency testing is also encouraged when it is shown not to 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.

In addition to its well-known cosmetic applications, botulinum neurotoxin is used to treat a variety of illnesses. In 2011, FDA 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 Veterinary Services Memorandum 800.211 (2011, updated 2023) provides guidance on the length of time that reference standards for veterinary vaccine potency testing may be used. The guidance allows Master References for serial release of most inactivated products to be used continually for up to 15 years from the date of initial qualification (or requalification) via host animal immunogenicity study if there are no obvious changes in the behavior of the Master Reference in the serial release assay. This will significantly reduce the use of host animals (including dogs, cats, and cows) for Master Reference requalification.

The 2023 update also includes guidance on reference qualification and requalification, as well as information from Veterinary Services Memorandum 800.90, Guidelines for Veterinary Biological Relative Potency Assay and Reference Preparations Based on ELISA Antigen Quantification, which was removed from the USDA's Center for Veterinary Biologics website in 2016.

Erysipelas is an infectious livestock disease. Infected animals may show no symptoms or may develop an acute or chronic disease that can be fatal. 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 Supplemental Assay Method 613 (2009, updated 2016). This test compares the level of protective antigen to a qualified reference standard. Through this comparison, confirmation of the potency of tested production lots of vaccine can be performed without using animals.

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 the release of each batch of tetanus vaccines, a vaccination-challenge test in guinea pigs, uses 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 test. These tests 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.

FDA provides for in vitro tests of vaccine potency in 21 CFR 610.10.

USDA Center for Veterinary Biologics 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.