*3.6. Toxicity*

The toxicity of each treatment was assessed by histopathological analysis of the kidney, liver, and spleen. No abnormalities were observed in the macro and microscopic analyses of the organs obtained from negative control animals, as well as from not infected rats treated with higher doses of LIMOX (hLIMOX-Control). In contrast, large mononucleartype inflammatory infiltrates were present in the livers of infected and untreated animals (positive control) (Figure 4). Minimal evidence of inflammation in this organ was also discovered in infected rats treated with phytotherapeutical regimens (Table 5). Correspondingly, this finding was accompanied by significantly increased AST levels (compared with the negative control), however, heightened levels of this transaminase were also observed in animals treated with BNZ (Figures 5 and 6).

**Figure 4.** Organ toxicity assessment. Histological images taken using a 40× objective on Hematoxylin and Eosin stained tissue sections. Negative control: untreated, uninfected animals; hLIMOX-Control: not infected animals treated with higher doses of an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (170 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); Positive control: untreated, infected animals; LIMOX: infected animals treated with a mixture of an essential oil fraction of *Lippia alba* carvone chemotype (enriched in limonene) (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX plus benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). Figures are representative of six independent experiments.


**Table 5.** Macroscopic and microscopic findings in organs of Wistar rats after receiving treatments.

RW: Relative weight; SEM: standard error of the mean; ̇: mean; Negative: untreated, uninfected animals; hLIMOX-Control: not infected animals treated with higher doses of an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (170 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); Positive: untreated, infected animals; LIMOX: infected animals treated with an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX and benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). Data are representative of six independent experiments and values are expressed in mean ± SEM. \* Size defined as ratio between maximum diameter and maximum length.

> Additionally, splenomegaly was evident in 16.6% of the animals in the LIMOXBNZtreated experimental group and in 50% of the animals belonging to the LIMOX-treated and positive control groups (Figure 4). In contrast, a significant reduction in the relative weight of this organ was evident in animals subjected to BNZ treatment (Figure 4 and Table 5). In addition, therapies which included that same compound (i.e., BNZ or LIMOXBNZ) induced the presence of hemosiderophages in the spleens of some of the treated animals (16.6%) (Figure 3). In a similar manner, kidney toxicity was apparent in 16.6% of the rats treated with BNZ, presenting as a pale brown color in the macroscopic analysis (Figure 3), but without altering the histopathological or biochemical parameters in the organ. With respect to BUN, a statistically significant elevation in this marker was observed in the LIMOX group compared to the reference treatment (BNZ) (*p* = 0.0048), but with no difference in creatinine values (Figure 5).

**Figure 5.** Toxicity of the tested therapies in the liver and spleen. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen. \* *p* ≤ 0.005 compared to the Benznidazole group. Negative control: untreated, uninfected animals; hLIMOX-Control: not infected animals treated with higher doses of an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (170 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); Positive control: untreated, infected animals; LIMOX: infected animals treated with a mixture of an essential oil fraction of *Lippia alba* carvone chemotype (enriched in limonene) (68.9 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX plus benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). \* *p* ≤ 0.05. Data are representative of six independent experiments and values are expressed in mean ± SEM.

With respect to hemogram parameters, thrombocytosis was associated with the LIMOX and LIMOXBNZ therapies, and was statistically significant in relation to the other treatments (*p* = 0.018 and *p* = 0.017, respectively). Regarding the leukocyte count, all infected groups presented a tendency (not statistically significant) towards higher counts than uninfected animals. In addition, the presence of atypical lymphocytes could be observed in the rats treated with BNZ, with a significant difference in relation to the other groups. The remaining hematological parameters did not exhibit significant alterations (Figure 6). An evaluation of the phytotherapies' toxicity shows that high doses of LIMOX (2.5 times the therapeutic dose) applied to not infected animals (hLIMOX-Control) caused minor signs of toxicity, particularly a change in the color of the kidney (exhibiting a pale

brown shade in the macroscopic analysis), in 16.6% of the treated rats. Likewise, a statistically significant peripheral blood neutrophilic leukocytosis was also observed in this same group of animals.

**Figure 6.** Hematological findings in Wistar rats infected with *Trypanosoma cruzi*. Hcto: Hematocrit; HGB: Hemoglobin; Negative: untreated, uninfected animals; hLIMOX-Control: not infected animals, treated with higher doses of an essential oil fraction of *Lippia alba* carvone chemotype enriched in limonene (170 mg/kg/day) and with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); Positive: untreated, infected animals; LIMOX: infected animals treated with an essential oil fraction of *Lippia alba* carvone chemotype (enriched in limonene) (68.9 mg/kg/day) with added caryophyllene oxide (Sigma-Aldrich) (70 mg/kg/day); LIMOXBNZ: infected animals treated with LIMOX plus benznidazole (7.9 mg/kg/day); BNZ: infected animals treated with benznidazole (100 mg/kg/day). \* *p* < 0.05 compared to the negative control group; \*\* *p* < 0.05 compared to the BNZ group; \*\*\* *p* < 0.005 compared to the negative control group \*\*\*\* *p* < 0.0001 compared to the negative control group. Data are representative of six independent experiments and values are expressed in mean ± SEM.
