Caffeine attenuates estradiol-stimulated uterine growth in an OECD 440-compliant bioassay using immature rats

Caffeine is among the world's most widely consumed psychoactive substances, yet its potential to interact with the endocrine system, particularly as an estrogen mimic, remains ambiguous and inadequately characterized using validated in vivo models. Our objective is to definitively assess the intrinsic estrogenic activity of caffeine and its potential interaction with a reference estrogen using the standardized OECD 440 uterotrophic bioassay.

Immature female Sprague-Dawley rats (n = 10/group) were administered vehicle, caffeine (10, 20, or 40 mg/kg/day), 17β-estradiol (positive control), or estradiol combined with caffeine for three consecutive days. Uterine wet and blotted weights were measured 24 h post-final dose, and the uterotrophic response was evaluated.

As expected, estradiol treatment provoked a robust uterotrophic response, increasing uterine wet weight over 4-fold and blotted weight over 3.7-fold compared to controls (P < 0.001). In contrast, caffeine alone at all tested doses failed to produce any significant increase in uterine weight, indicating a lack of intrinsic estrogenic activity. Notably, co-administration of caffeine with estradiol significantly attenuated the estradiol-induced response, reducing wet weight by approximately 33% and blotted weight by 25% (P < 0.05).

These findings demonstrate that caffeine is not an estrogen agonist in this validated in vivo model. Co-administration with estradiol significantly reduced the uterotrophic response, suggesting a potential interaction that warrants further mechanistic investigation.