The formalin test: an evaluation
of the method
Arne Tjolsen, Odd-Geir Berge, Steinar
Hunskaar, Jan Henrik Rosland and Kjell Hole
Pain, 51 (1992) pp 5-17
Formalin – the substance
An aqueous solution of 37% (weight/weight)
Ex: a 10% formalin solution contains approx. 3.7% formaldehyde.
Response: biphasic (early and late phase); consisting of
licking and biting of the injected paw.
Duration: tonic; approx. 1h is suited for a nociceptive test
Most studies have used rodents
Other species used: primates, cats, rabbits,
crocodiles and domestic fowls.
The time course may be different in different
Site of injection
Subcutaneous injection into the plantar surface
of the hindpaw
More nociceptive-specific than forepaw licking
Other site of injection: possible to affect the
intensity and pattern of the response
Volume and concentration
Volume: 20-25 μl in mice and 30-100 μl in rats
Concentration: from 1 to 5%
Effect: - long-lasting edema
- macroscopic changes (ranging from depilation
and scarring to ulceration) at the injection site
High concentration: freezing
Low concentration: sub-maximal response (without a
ceiling effect), and does not induce ulceration and long
The two phases
Early phase: an acute pain which is due to direct
chemical stimulation of nociceptors (C-fiber activation). It
starts immediately after injection and lasts for 5 min.
Late phase: a tonic pain which is dependent on
peripheral inflammation and functional changes in
central processing. It starts approx. 15-20 min. after
injection and lasts for 20-40 min.
The two phases are separated by a quiescent period in
which there is no apparent pain behavior.
Experimental results have indicated:
Substance P and bradykinin participate in early
Histamine, serotonin, prostaglandins and
bradykinin are involved in late phase.
The ambient temperature: critical in late phase
Ex: After 1% of formalin injection (0.4% formaldehyde), the animals
show weaker response in a room temperature of 20-21℃ than at 23-
24℃ or 26-28℃ in late phase.
Explanation: inflammatory reactions develop more slowly at lower
No significant difference in the magnitude of
response in early phase.
Reproducibility and robustness
Exposure to stress strongly influences the
behavior after formalin injection.
Animals that are well handled and adapted to
the environment exhibit the typical nociceptive
behavior more consistently.
Enviromental stress factors: sounds, odours,
bright light, elevated atmospheric pressure or
activity by humans.
1st method: weighted scores
0 – no elevation of the injected paw
1 – reduction in the weight put on the injected paw
2 – total elevation of the injected paw
3 – licking, biting and shaking of the injected paw
2nd method: one single parameter
only scoring the time spent licking and biting the injected
paw, or the total time the paw is kept elevated from the
Physiology and pharmacology
Weak analgesics such as acetylsalicylic acid and
paracetamol have evident effects in formalin test.
NSAID indomethacin has been shown to reduce the
response in late phase of the formalin test.
Tolerance to the antinociceptive effect of morphine is
less easily produced in the formalin test than in the reflex
Ex: In patients experiencing chronic pain, the degree of tolerance to
morphine is limited and not situation specific.
Therefore, formalin test has been argued to be a more
useful model of clinical pain.