This study confirms that the three pain models induce pain in rodents and showed that peak pain coincided with peak mechanical hypersensitivity. However, mechanical hypersensitivity remained once pain subsided, mimicking the human experience of CFA injection. These findings further our understanding of the roles of, and relationship between, these assays in the assessment of nociception and pain.

23 An effective and pain-free killing method is required to achieve the goal of euthanasia, a "good 24 death". Overdose of sodium pentobarbital (PB) by intraperitoneal (IP) injection is a widely 25 accepted technique, but questions remain regarding pain associated with administration. As PB 26 rapidly causes sedation and loss of consciousness, most studies have relied on indirect evidence 27 of pain. The objective of this study was to assess pain associated with IP PB using an appropriate 28 vehicle control.29 Adult male and female Sprague Dawley (SD) and female Wistar rats (N = 112) were block 30 randomised by sex and strain to receive one of four treatments: 1) 800 mg/kg PB (pH 11); 2) 800 31 mg/kg PB with 4 mg/kg lidocaine (PB+lido); 3) saline or 4) vehicle controls (pH 11 or 12.5).32 Behavior (Rat Grimace Scale [RGS], writhing, back arching) was evaluated at baseline, before 33 loss of righting reflex (PB and PB+lido groups), 80s, 151s and 10 min post-injection (PI; saline 34 and vehicle control groups).35 In the vehicle control groups, the RGS scores were increased at 151s PI (SD: p = 0.0008, 95%CI 36 -0.731 to -0.202) from baseline, as was relative frequency of writhing (SD: p < 0.00001; Wistar; 37 p = 0.0004). RGS scores remained elevated 10 mins PI (SD: p = 0.0070, 95%CI -0.768 to -0.118; 38 Wistar: p = 0.0236, 95%CI -0.907 to -0.0742) but the relative frequency of writhing did not (p > 39 0.05). The RGS scores and the relative frequency of writhing remained low in the PB, PB+lido 40 and saline groups (p > 0.05). Back arching increased from baseline in the PB+lido group before 41 loss of righting reflex and in the vehicle control group (SD rats) at 151s PI (p < 0.05).42 These results show that IP PB results in signs associated with pain. The sedative effects of PB 43 limit behavioral assessment.