The formalin test is used as a primary behavioural screen for assaying the 
antinociceptive activity of compounds in laboratory rodents. After hindpaw 
formalin injection, nociceptive behaviours are expressed in a biphasic pattern 
and correlate closely with the concentration of formalin injected. Here, the 
antinociceptive efficacy of six compounds used in the clinical treatment of 
chronic pain was compared in rats injected with either 1% or 5% formalin. 
Predictably, the anti-inflammatory drug ibuprofen (30-300 mg/kg) attenuated 
second phase (16-40 min) nociceptive behaviours in 5% formalin-injected rats; 1% 
formalin-injected rats were unaffected. The anti-epileptic drugs gabapentin 
(50-200 mg/kg) and lamotrigine (10-60 mg/kg) were antinociceptive only during 
second phase in both 1% and 5% tests. Notably, they were 2-4 times more potent 
in 1% vs 5% formalin-injected rats. The dual monoamine reuptake inhibitor 
duloxetine (3-60 mg/kg) consistently attenuated nociceptive behaviours during 
first phase and interphase in both tests. However second phase nociception was 
only attenuated in 5% formalin-injected rats giving rise to a 6 fold increase in 
potency compared with 1% formalin-injected rats. The micro-opioid receptor 
agonist morphine (1-6 mg/kg) and the combined micro-opioid receptor 
agonist/monoamine reuptake inhibitor tramadol (5-50 mg/kg) both attenuated 
nociceptive behaviours throughout the duration of both the 1% and 5% tests; no 
difference in antinociceptive potency occurred for either compound during second 
phase. Thus, for mechanistically-distinct compounds the predictive 
antinociceptive capacity of the formalin test can vary with the concentration of 
formalin injected, likely as a result of peripheral and central nociceptive 
signalling mechanisms being differentially engaged.