3.5.2. Study Design

The study involved 48 Swiss albino mice obtained from the Egyptian Organization for Biological Products and Vaccines (Vacsera, Giza, Egypt) weighing 25–30 g. The mice were housed in plastic cages at a 25 ◦C temperature under a normal light/dark cycle, with water and food provided ad libitum. The mice were left for 1 week before the experiments to adjust. They were randomly separated into 6 groups (8 mice each). The first group was considered the normal group (negative control) and received Tween 80. The tumor cells were injected in all 5 other groups (groups 2–6). Group 2 was considered the EAC control group. Groups 3 and 4 received 1 mg/kg and 2 mg/kg of ceramide **A** (**1**), respectively. Groups 5 and 6 were treated by 1 mg/kg and 2 mg/kg of ceramide **B** (**2**), respectively. Ceramide **A** (**1**) and ceramide **B** (**2**) were dissolved in Tween 80 as a vehicle. The day of tumor cell injection was considered day zero (0). Groups 3–6 were treated daily from day 7 until day 21. The in vivo study was in agreemen<sup>t</sup> with the *Guide for the Care and Use of Laboratory Animals*. The study protocol was approved by the ethical committee of the Faculty of Pharmacy at Suez Canal University (201605PHDA1).
