The procedures involving animals and their care were performed in Brescia according to the Union Council Directive of 22 September 2010 (2010/63/UE). The study was approved by the Ethical Committee of Animal Experimentation of the Hospital and the Italian Minister of Health (407/2018-PR). All care was taken to use the minimum number of animals.

C57BJ/6 male mice (Harlan, Udine) were housed in same-sex groups of 4 per cage on a 12:12-h light/dark cycle; animal rooms were maintained at a temperature of 23 °C. Estrogen-free diet was purchased from Dottori Piccioni S.r.L. Via Guglielmo Marconi, 29/31 Gessate (MI, Italy) (https://totofood.it/, assessed on 7 June 2021). The diet was prepared in pellets (the composition is reported in Table S1, Supplementary Materials).

The treatment started when mice were three weeks old and lasted for four months. Animals were divided randomly in nine experimental groups: control mice were fed with the base diet (estrogen-free diet) while experimental groups were fed with the base diet enriched with two different concentrations of E2, BPA, DES, or TBT (according to previous studies [74]). All the chemicals were obtained from Sigma-Aldrich, Milano, Italy, dissolved in DMSO and further diluted before their addition to the diet, for homogeneous preparations. These are the doses used: E2 (stock solution 97%, cat. number E8515; 5 or 50 µg/kg diet); BPA (stock solution 99%, cat. number 239658; 5 or 500 µg/kg diet); DES (stock solution 99%, cat. number D-4628; 0.05 or 50 µg/kg diet); and TBT (stock solution 96%, cat. number T50202; 0.5 or 500 µg/kg diet).

The normal food consumption in adult mice corresponds to 15g/100g body weight/day [75]; since mice used in this experiment had a mean body weight of 30g, it was considered an approximate consumption of 4.5 g food/day was appropriate. Accordingly, in this case mice were exposed daily to approximately 0.15–1.5 µg/g body weight of E2, 0.15–15 µg/g body weight of BPA, 0.0015–1.5 µg/g body weight of DES, and 0.015–15 µg/g body weight of TBT.

Body weights were recorded at the end of the experiment, before sacrifice (see Table 1).

Food consumption was monitored every two days as the difference between the weight of the pellets supplied and that consumed. Spilled food, if any, was collected in apposite trays underneath the food containers, measured, and taken into account.

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