2.7.2. Blood Samples

For genotoxicity evaluation, blood was obtained from a tail cut for micronucleus (MN) analysis and individual cells (comet assay) via alkaline electrophoresis.

#### *2.8. Determination of Aluminum Concentration in Breast Tissues*

Samples were previously digested in a TITAN MPS microwave oven [38]. Briefly, 0.4 ± 0.02 g of breast tissue was weighed and placed in 15 × 2.5 cm Teflon digestion tubes containing 7 mL of concentrated nitric acid (HNO3). The digestion conditions were 200 ◦C, 35 × 10<sup>5</sup> Pa, and 1600 W for 47 min [39]. The resultant acidic residue was made to a total of 100 mL using deionized water for the subsequent quantification of aluminum concentration.

The determination of aluminum concentration in the standard, certified reference material, and rat breast tissue was performed using atomic absorption spectroscopy with an AAnalyst 400 automated equipment in a graphite furnace absorption atomic spectroscopy (GFAAS) or in the electrothermal absorption atomic spectroscopy (ETAAS) mode, under the operating conditions recommended by the manufacturer [40,41]. The analytical method was previously optimized and its expanded uncertainty for quantifying aluminum by FAAS (flame absorption atomic spectroscopy) and ETAAS was subsequently validated and estimated uncertainty [42–44].

#### *2.9. Evaluation of Genomic Instability*

### 2.9.1. Histopathological Evaluation

Morphological instability was assessed by preparing histological cuts from rat mammary glands and mounting them on fixed slides using a microtome. The preparations were stained with hematoxylin and eosin and observed under a LEICA DME optical microscope under 40× and 100× magnification [45].
