"All dams of treated groups were observed daily for any signs of general toxicity, bleeding, and/or mortality. Body weight was recorded weekly throughout gestation.

Assessment of reproductive teratogenicity of lufenuron. On the 20th gestational day, the dams were euthanized using sodium pentobarbital and subjected to cesarean section operations. The ovaries of both uterine horns were removed, and the corpora lutea were counted. Gravid uteri were carefully segregated, weighed, and examined. The total number of implantation sites, as well as live, resorbed, or dead fetuses, was recorded according to a previously described method.

Examination of fetal skeleton for abnormalities. Fetuses were fixed in 100% ethanol, then eviscerated and stained using Alcian blue–Alizarin red double staining method according to Young et al. The stained fetal skeleton was stored in pure glycerol to be examined for any anomalies according to Aliverti et al. using a dissecting binocular microscope (Leica, Germany) and photographed using a digital camera (Leica, Germany).

Histopathologic examination. Tissue biopsies from the liver and kidney of both mothers and fetuses from all three groups were immediately taken and fixed using 10% neutral-buffered formalin for 24 h. Preparation of paraffin sections and staining in hematoxylin and eosin was performed according to the method previously described by Bancroft et al. All sections were examined using a BX53M light microscope (Olympus, Japan) and photographed using a DP74 camera (Olympus, Japan).

Assessment of DNA fragmentation by comet assay (SCGE). Liver tissues were frozen in liquid nitrogen and gently homogenized into a powder. The extent of DNA strand breaks in the liver tissues of mothers and fetuses from the three groups was assessed using the alkaline comet assay, as previously described by Tice et al.117. Comets were analyzed using an Axio fluorescence microscope (Carl Zeiss, Germany) with an excitation filter at 524 nm and a barrier filter at 605 nm. A Komet 5.0 analysis system developed by Kinetic Imaging, Ltd. (Liverpool, United Kingdom) connected to a charge-coupled device (CCD) camera was used to measure tail length, percentage of migrated DNA, and tail moment.

Cell cycle analysis by flow cytometry through PI staining. Liver tissues acquired from the mothers and fetuses from all groups were weighed, rinsed in phosphate-buffered saline, and minced using a pair of scissors to produce approximately 1-mm fragments. Afterward, the tissues were digested using collagenase (1 ml/0.25 g tissue). Cell cycle analysis through PI staining was performed according to the method previously described by Allen and Davies. The stained cells were analyzed using an Attune flow cytometer (Applied Biosystem, USA).

Estimation of oxidative stress markers. The appropriate kits for determining malondialdehyde (MDA), glutathione peroxidase (GPX), and super oxide dismutase (SOD) were purchased from (Biodiagnostic, Egypt). Hepatic activities for MDA, GPX, and SOD were measured using a UV-2100 spectrophotometer (Qualitest, USA) according to Ohkawa et al., Paglia and Valentine, and Nishikimi et al., respectively."

P0 Female Albino Rats