Developmental exposure to low levels of ethinylestradiol affects social play in juvenile male rats


Juvenile social play contributes to the development of adult social and emotional skills in humans and non-human animals and is therefore a useful endpoint to study the effects of endocrine disrupters on behavior in animal models. Ethinylestradiol (EE2), a widely produced, powerful synthetic estrogen is widespread in the environment mainly because it is a component of the contraceptive pill. To understand whether clinical or environmental exposure to EE2 during critical perinatal periods can affect male social play, we exposed 72 male Sprague–Dawley rats to EE2 or vehicle either during gestation (from gestation day (GD) 5 through 20) or during lactation (from postnatal day (PND) 1 through 21). Two doses of EE2 were used to treat the dams: a lower dose in the range of possible environmental exposure (4 ng/kg/day) and a higher dose similar to that received during contraceptive treatment (400 ng/kg/day). Social play was observed between PND 40 and 45. A principal component analysis (PCA) of frequencies of behavioral items observed during play sessions allowed to allocate behaviors to the two main components that we named aggressive-like play and defensive-like play. Aggressive-like play was increased by gestational and decreased by lactational exposure. Defensive-like play was decreased by treatment. For both types of play the lower dose (4 ng/kg/day) was as effective as the higher one. Total social activity was increased by gestational and decreased by lactational exposure. These findings provide further evidence that exposure to low and to very low doses of EE2 during critical periods of development can affect essential aspects of social behavior, and that the timing of exposure is critical to understand its developmental action.

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Anogenital distance


Anogenital index


Central nervous system


Endocrine disrupter chemicals

EE2 :



Estrogen receptor


Gestation day


General linear model


Principal component analysis


Principal components


Postnatal day


Sex hormone-binding globulin




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This study was supported by a grant ‘Rientro dei Cervelli’ of the Italian Ministry of University and Research (MUR) to L.F. and by a grant to F. D-F, Research Project COFIN of MIUR, Italy.

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Correspondence to Marco Zaccaroni.

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Zaccaroni, M., Massolo, A., Beani, L. et al. Developmental exposure to low levels of ethinylestradiol affects social play in juvenile male rats. Toxicol Res. 36, 301–310 (2020).

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  • Endocrine disrupters
  • Ethinylestradiol
  • Xenoestrogens
  • Social play
  • Play fighting
  • Anogenital distance
  • Developmental windows
  • Cross-fostering
  • Rat