2.5. Chronic toxicity study

The protocol was performed based on the OECD Guidelines for Testing of Chemicals (Section 452) with slight modification including rodent species, dose levels and number of animals used. Swiss albino mice (12/sex/group, total 168, bodyweight 25−40 g) and hamsters (13/sex/group, total 182, bodyweight 80−100 g) were randomly divided into seven groups as follows: Group 1 (control) normal diet without any treatment, Group 2 daily oral gavage with BNCs (0.58 g/kg bw/day in mice or 1.16 g/kg bw/day in hamsters), Groups 3–5 daily oral gavage of CNCs at low, medium and high doses in mice (0.09, 0.27 and 0.8 g/kg bw, equivalent to 0.025, 0.075, 0.225 g/kg bw of curcumin, respectively) and CNCs at low, medium and high doses in hamsters 0.18, 0.54, 1.61 g/kg bw (equivalent to 0.05, 0.15, 0.45 g/kg bw of curcumin, respectively), for 6 months. Groups 6 and 7 of both species (n = 24 mice and 26 hamsters), termed the recovery groups, were respectively given 0.58 g/kg bw/day of BNCs or the high-dose CNCs regimen daily for 6 months. Following cessation of the treatments at 6 months, animals in groups 6 and 7 were held for a further 28 days. The dose volume in all animals used was 10 mL/kg bw. The dose levels to be used for chronic toxicity testing were based on the results from the acute toxicity testing. The high-dose level in the chronic toxicity test was approximately equivalent to the medium dose level used in the acute toxicity tests: the medium and low doses used in the chronic toxicity tests were successive approximately three-fold reductions of the high dose. During the experiment, all animals were daily checked for overall health condition, body weight, morbidity and mortality. All animals were starved for 1 day before euthanasia.