Animals were sacrificed in proestrous.

Histopathology: Animals were injected with BrdU an hour and a half before sacrifice.The fourth and fifth mammary gland pair was excised with the right whole mounted and the left embedded in parafin for light microscopy. The different measurements and determinations were provided (++ for methods). Blinding was not reported (NR for blinding). Overall rating = NR

Estradiol: Trunk blood was collected at sacrifice in heparinized tubes and plasma was separated and stored at -80 degrees C until assayed. Steroids were extracted from plasma using diethyl ether. Percent recovery was calculated, but not reported. Plasma E2 was determined by RIA. (+ for methods). Blinding was not reported (NR for blinding).

mRNA expression: Mammary glands were collected under RNase-free conditions and frozen for subsequent RNA isolation and RT-PCR. This is a well-established method (++ for method). The purity and specificity of the PCR product was confirmed by the SYBR Green dissociation curve set up at the end of each PCR run. Blinding was not reported (NR for blinding). Overall rating = NR.

Immunohistochemistry: Sections were processed for immunohistochemistry to assess DNA synthesis, apoptosis, or expression of ER-alpha or progesterone receptor. These are well-established methods with some description provided in the study report. Criteria for measurement was reported (++ for methods). Blinding was not reported (NR for blinding). Overall rating = NR

response to E2: one pup per litter from each treatment were ovariectomized at 25d of age and immediately planted with a pump delivering vehicle or 0.5 ug/kg/day E2. Animals were killed 10 days later. Then animals were evaluated for histopathology as noted above.

Munoz-de-Toro et al. 2005

Risk of bias

HAWC