Use of Complete Freund’s Adjuvant (CFA) and Other Adjuvants in Research Animals

Policy

Use of CFA is permitted if the investigator provides scientific justification for its use, in particular that antibody production cannot be achieved through use of less irritating adjuvants.

Alternative adjuvants may include aluminum compounds (e.g., Alum), squalene-in-water emulsions (MF59 and AS03), monophosphoryl lipid A (MPL), Ribi adjuvants, combined with alum (AS04); adjuvants in pre-clinical development (e.g., Montanides), polymeric microparticles, saponins (e.g., Quil A QS-21, ISCOMS, ISCOMATRIX), immunostimulatory nucleic acids (e.g. CpG oligodeoxynucleotides, poly IC:LC); other toll-like receptor-agonists (e.g., flagellin, imidazoquinolines, small molecules), cationic liposome formulations (CAF) combined with immunestimulators such as trehalose dibehenate (TDB) virus-like particles, nanoparticles, 19-21 and oligonucleotide complexes; other procedures or emulsions such as subcutaneously-implanted chambers, TiterMax, EMULSIGENS, Syntex Adjuvant Formulation (SAF), and Specol.

Regardless of the adjuvant used, precautions must be used, including aseptic preparation of the injection site and sterility of the inocula to be used in animals.

With regards to alternative adjuvants, subcutaneous (SC) or intraperitoneal (IP) administration is preferred in rodents and other routes, such as intradermal administration, are discouraged and must be scientifically justified by the investigator because of local lesions that can result. Injection volume should not be more than 0.25 ml per SC site for rabbits, and 0.1 ml SC or 0.2 ml IP for mice and rats.

Approval to perform CFA injections may be granted by the IACUC if investigators are able to demonstrate that the use of preferred alternatives is unsatisfactory and there is compelling scientific justification to deviate from the recommendations made above. Records of adjuvant administration including injection volume, location, and administration date must be maintained.

With regards to the use of CFA, SC administration is the preferred route in rodents. A concentration of <0.1 mg/ml is generally recommended to decrease potential side effects. Intradermal injections and use of the foot pad as an injection site requires justification. IP injection of CFA in mice and rats, which has been documented to cause severe peritonitis, is not acceptable. Table 1 lists maximum volume per site and route of administration.

CFA should be used in only the first injection; subsequent injections requiring an adjuvant must use Freund’s incomplete or another appropriate adjuvant with a minimum of two weeks between subsequent inoculations.

Volume, route, and site of adjuvant injection should adhere to the Guidelines for Use of Adjuvants in Research published by the Animal Research Advisory Committee of the National Institutes of Health (see table below from this publication).

Table 1: Maximum Volume of Complete Freund’s Adjuvant-Antigen Emulsion (CFA-AE) per site and route of administration

Species	SC(mL)	Intradermal(mL)	Footpad (mL)
Mouse	0.1	0.05**	0.05**
Rat	0.1	0.05**	0.1**
Rabbit	0.25	0.05**	*
Non-human Primates**	Freund’s Adjuvant is not generally recommended for use in non-human primates, as it may interfere with TB testing results and cause excessive inflammation. Nevertheless, it is recognized that some models may require the use of CFA. If used, the maximum volumes should not exceed those used in rabbits and should be scientifically justified.

*not recommended
** only when justified