General commentary: 28-Day oral chronic toxicity study of arctigenin in rats

The strengths of this study include its study design, which incorporates both male and female rats considering sex as a biological variable, the dosing regimen/protocol including the sub-chronic exposure studies, as well as the detailed description of the results and comprehensive discussion in the body part of the manuscript. However, we realized some inaccuracy throughout the description of results and, importantly, in the abstract. Specifically, the authors did not include a key finding wherein the high dose (3x (36 mg/kg) and 10x (120 mg/kg) dose of ARC) arms did not cause histopathological abnormalities, but the minimal dose (1x (12 mg/kg) dose of ARC) caused abnormalities that matched what was observed in the vehicle/placebo control group, an intrinsic pathology inherent to the rat model used.The results indicated no significant differences in body weight or food intake between treatment groups, across both sexes. Although the authors reported "a significant reduction in body weight in the high dose group", the corresponding figure (Figure 1A-B) showed overlapping lines across groups with a lack of statistics. Furthermore, the placebo (vehicle) group showed a marked reduction in food intake on Day 14 (Figure 1C), which was not addressed in the Results section nor Discussion section. In the abstract, it reads "The high dosage of Arctigenin only decreased the body weight at day 4". Again, the body weight showed no difference between the three artigenin doses and the vehicle controls on day 4, as shown in Figure 1. Clear statistical analysis of these days/time-points would benefit the study and allow for a more robust interpretation of results.Additionally, urine, hematological and blood biochemistry, and electrolyte parameters did not differ significantly with ARC treatment. Histopathological abnormalities in various organs were only observed in the minimal dose group (12 mg/kg), while the 3x and 10x ARC dose groups were histologically normal. Importantly, the AUC study showed that the 10x dose group exhibited 2 to 5 times higher blood concentration of ARC than the 3x ARC treatment group, indicating high exposure; however, both 3x and 12x ARC groups of rats exhibited an absence of pathological abnormalities. A toxicokinetic study (Figure 4 and Table 9) was not shown for the 1x ARC treatment group that exhibited pathological abnormalities.The authors acknowledged and adequately discussed their results, i.e. the abnormalities were not presented in the high-dose ARC groups, but in the minimal dose ARC group. In their discussion, the authors suggested that the lack dose-related pathological changes might be due to an intrinsic pathology in the rats, as the vehicle/placebo group exhibits the same "pathology" as the minimal dose group. Indeed, several "representative" images of abnormalities were derived from the placebo group (Figures 2B,2C, 2D, 2F, 2G, 2H, 3A, 3B, 3G). However, these results were not accurately described in the abstract.We would like to emphasize the importance of including a stronger and more comprehensive summary of the results of the study throughout the body of the abstract. The abstract serves as the primary lens through which busy clinicians, researchers and consumers engage with a study, consequently, its clarity and precision are paramount, as it largely determines the perceived significance and credibility of the work. In its current form, the abstract, the authors conclude that the minimal adverse effect dose is 12 mg/kg in the absence of acknowledging the observation that higher doses, including 3x and 10X ARC doses, did not produce adverse effects and, in fact, were histologically normal. Therefore, the omission of these results from the abstract may suggest and influence readers to perceive that the 12 mg/kg is the lowest dose causing adverse effects. A more appropriate approach would be to conduct experiments in a model that does not have intrinsic pathologies that are commonly observed during vehicle/placebo interventions to avoid confounding factors.