2.4. Acute toxicity study

Female animals (total 29 mice and 23 hamsters) were randomly assigned to non-treated (normal control group) or intervention groups. Acute toxicity testing was carried out following OECD Guideline 423 for Testing of Chemicals with slight modification, including dose level, rodent species and number of animals used. The acute oral toxicity of CNCs was classified based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) 2003 in which the most severe toxicity is classified in category 1 (LD₅₀ ≤ 0.005 g/kg bw) and relatively low toxicity in category 5 (LD₅₀ > 2−5 g/kg bw). Agents that have very low toxicity (LD₅₀ > 5 g/kg bw) [43] are placed into the unclassified hazards category as indicated by the ∞ symbol based on OECD guidelines [44]. Most previous articles have reported LD₅₀ of curcumin to be approximately 2 g/kg bw (in GHS category 5 of OECD guidelines for oral toxicity studies) in rats and mice [45,46]. Therefore, in this study, we used the higher dose levels from OECD Guideline 423 to determine the actual dose for translation from animal to human. For rodent species, mice and hamsters were selected as described above. CNCs were given on a single occasion at low, medium and high doses, which were 0.1, 1.1 or 11 g/kg bw (equivalent to 0.03, 0.3 or 3 g/kg bw of curcumin, respectively) for mice, and 0.2, 2.1 and 21.4 g/kg bw (equivalent to 0.06, 0.6, 6 g/kg bw of curcumin, respectively) for hamsters. Mice and hamsters in the blank nanocomplexes (BNCs) group received a single oral dose of 7.9 g/kg bw BNCs and 15.4 g/kg bw BNCs, respectively. Administration of the single dose was performed by oral gavage in a volume of 10 mL/kg bw. The BNCs or CNCs powder to be administered was diluted with distilled water at a ratio of 10:1 w/v. The diluted samples were pushed through gavage tubes within 45 min. Animals in the negative control group received no intervention. Clinical signs of toxidromes (depression, rising fur, tremors, excitability, twitching, salivation, morbidity) and mortality were observed and recorded twice daily for 14 days post-treatment.