Enhanced reproductive toxicity following combined exposure to ethinylestradiol and levonorgestrel: evidence of non-additive endocrine disruption in male rats

Ethinylestradiol (EE2) and levonorgestrel (LNG) are synthetic hormones widely used in contraceptives and increasingly recognized as endocrine-disrupting contaminants. Although their individual reproductive effects are documented, their combined toxicity remains poorly understood. This study evaluated the sub-chronic reproductive toxicity of EE2 and LNG, administered alone or in combination, in male Wistar rats, with emphasis on potential non-additive endocrine-disrupting interactions. Animals were orally treated with EE2 (1 µg/kg bw), LNG (5 µg/kg bw), or both for four weeks. Individual exposures caused significant alterations in testicular weight, hormone levels, oxidative stress markers, and sperm quality, whereas epididymal weight was not significantly affected. In contrast, combined exposure markedly intensified these effects, causing severe spermatogenic impairment, near-complete sperm depletion, and major histological damage in testicular tissue. This enhanced toxicity was associated with increased lipid peroxidation, reduced antioxidant defenses, and marked suppression of testosterone, FSH, and LH levels, indicating disruption of the hypothalamic–pituitary–gonadal axis. Two-way factorial analysis confirmed statistically significant EE2 x LNG interactions for testicular weight, sperm concentration, sperm motility, LH and testosterone; in each case the observed combined effect was smaller than predicted by effect addition, indicating an infra-additive rather than synergistic interaction, consistent with a floor effect at these already substantial individual effect sizes. These findings highlight the importance of assessing combined endocrine disruptor exposures, as mixture effects modify reproductive toxicity in ways not predictable from single-compound data alone.