## 4 Discussion  
A previous study evaluated the extract's cytotoxicity in five different cell lines: human breast adenocarcinoma (MCF-7), human non-small lung carcinoma (NCI-H460), human cervical carcinoma (HeLa), human hepatocellular carcinoma (HepG2), and porcine liver cells (PLP2) [17]. These authors reported that the extract had significant capacity to inhibit the proliferation of these tumor cell lines, while low concentrations of EE had no effect on the normal cell line. After cell studies, a correct approach is to switch to an experimental animal protocol. To the best of our knowledge, this is the first study to assess the safety of different concentrations of an elderberry colorant extract in mice. Although the FVB/n strain is normally used for genetic and transgenic experiments, wild-type FVB/n mice have also been employed in metabolic and toxicology studies [28; 29; 30; 31]. Animals supplemented with EE had considerably lower mean body weight values, particularly EE12, which was significantly inferior to the control group. _In vitro_ assays have revealed that the primary components of this extract have an anti-obesity activity [32]. In a before-and-after clinical trial, [PERSON] et al. (2008) reported that a combination of _S. nigra_ and _Asparagus officinalis_ was significantly effective in lowering the mean body weight and blood pressure while improving the participants' physical and emotional well-being and quality of life [33]. The same occurrence appeared to happen to the EE-supplemented animals, especially at the lowest dose. Given that these animals consumed identical amounts of food and that the humane endpoint analysis revealed no changes in behavior associated with toxicity, the inclusion of anthocyanins in their diet appeared to influence their body weight gain. Another study discovered that dietary supplementation with purified mulberry (_Morus australis_ Poir) anthocyanins suppressed body weight gain in high-fat-diet-fed C57 BL/6 mice by reducing the epididymal fat weight and adipocyte size [34]. A future study should explore this extract's effect on adipose tissue and how it can affect body weight gain. Food consumption was standard compared with other studies developed by our group with the same diet [27; 30]. The mice's initial reactions to the extract were not very positive, but, over time, the overall average consumption by the supplemented groups became similar to that by the control group. As this particularly occurred for the group supplemented with the highest concentration of EE, this difference may be due to the extract's flavor, which might have been too strong or overbearing. The values regarding drink intake were, however, consistent with those of the aforementioned works. The relative weight of the heart in EE12 significantly increased when compared with the other groups, probably due to residual blood after cardiac puncture. Regarding hydrophobic changes, animals from the control group exhibited a generalized distribution of these changes. According to the literature, these represent acute and reversible, sublethal cell injuries [35], and in the absence of biochemical evidence of liver damage in these animals, the changes observed in the control group were not regarded as having pathological significance. In the present study, the animals supplemented with EE at the lowest concentration showed minor and zone-restricted hydropic changes when compared with the control group. This effect may be related to the anthocyanins present in the EE, because, in chronic ethanol-induced liver injury in male Wistar rats and CCl4-induced Kunming mice [36; 37], these substances have been reported to have an hepatotoxicity effect. Our results regarding the renal function markers, particularly the urea levels in EE12, which were considerably greater than those of the control group, were in conflict with the literature, as anthocyanins are usually associated with a reduction in urea levels [38; 39].