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Cited by 5Effects and risks of endocrine disrupting chemicalsSexual Differentiation and DisordersNeuroendocrine regulation and behavior

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

Open access
Zaccaroni, Marco, Massolo, Alessandro, Beani, Laura, Seta, Daniele Della, Farabollini, Francesca, Giannelli, Giulietta, Fusani, Leonida, Dessì-Fulgheri, Francesco·2020·Toxicological Research
DOI 10.1007/s43188-019-00035-zPMC PMC7494694
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Abstract

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 (EE 2), 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 EE 2 during critical perinatal periods can affect male social play, we exposed 72 male Sprague-Dawley rats to EE 2 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 EE 2 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 EE 2 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.

AI snapshot

Written from the paper’s full text.

TL;DR

Developmental exposure of male Sprague–Dawley rats to low or very low doses of ethinylestradiol (EE2) altered juvenile social play, with effects depending on whether exposure occurred during gestation or lactation. Both the environmentally relevant 4 ng/kg/day dose and the clinical 400 ng/kg/day dose were effective.

Key findings

  • Total social activity was significantly affected by timing of exposure (F1,64 = 42.84, p < 0.001) and by treatment × timing interaction (F2,64 = 11.44, p < 0.001): gestational EE400 increased social activity versus oil (post hoc Sidak p < 0.01), while lactational EE4 and EE400 decreased it versus oil (both p < 0.05).
  • PCA identified three components explaining 78.37% of variance: aggressive-like play (PC1, 54.45%), defensive-like play (PC2, 12.66%), and allogrooming (PC3, 11.25%). Aggressive-like play was increased by gestational exposure and decreased by lactational exposure (timing F1,64 = 8.154, p < 0.01; interaction F2,64 = 4.754, p < 0.01).
  • Defensive-like play was decreased by treatment, with lactational exposure decreasing it regardless of dosage (timing F1,64 = 18.658, p < 0.001; interaction F2,64 = 3.373, p < 0.05).
  • Gestational EE2 increased anogenital distance and anogenital index at birth in a dose-dependent manner (AGD F2,146 = 6.68, p < 0.01; AGI F2,146 = 11,654, p < 0.001), while body weight at PND1 was not affected (F2,146 = 1.45, p > 0.2).

Methods

  • 72 male Sprague–Dawley rats were used, one male per litter, with dams treated orally by pipette with vehicle, 4 ng/kg/day EE2, or 400 ng/kg/day EE2 either during gestation (GD 5–20) or lactation (PND 1–21) using a cross-fostering design.
  • Social play was video-recorded between PND 40 and 45 in groups of four same-treatment, non-sibling males in a neutral arena for 15 min after 1 min familiarization; an observer blind to treatment scored behaviors from an ethogram.
  • Data were analyzed with PCA followed by GLM on component scores and total social activity, including treatment, timing, and cage as factors, with Sidak post hoc tests.

Limitations

  • The paper states that for lactational exposure, an effect of EE2 on dam maternal behavior cannot be ruled out, and maternal behavior was not measured; in contrast, gestational-exposed subjects were cared for by untreated foster dams.
  • The paper states caution is needed in translating results to humans because of differences between humans and rats in EE2 biotransformation, which could result in more serious effects in rats.
  • The paper states the mechanism underlying the different effects of gestational versus lactational exposure is unknown; evident from the text, this study examined only male rats, so sex-specific conclusions are limited.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1007/s43188-019-00035-z

Cites

80 of 80 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

36 of 77 cited sources checked · 20 supported · 0 flagged · accuracy not yet measured

  • Premisecitation
    § Introduction

    Estrogen can exert an organizational effect on CNS and behavior in higher vertebrates during early stages of development.

    “It is well known that estrogen can exert an organizational effect on CNS and behavior in higher vertebrates during the early stages of development see [8–10].”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [8] McEwen B (2002) Estrogen actions throughout the brain. Rec Progr Horm Res 57:357–384 source
    • cites [9] McCarthy MM (2008) Estradiol and the developing brain. Physiol Rev 88:91–134 source
    • cites [10] McCarthy MM, Arnold AP (2011) Reframing sexual differentiation of the brain. Nat Neurosci 14:677 source
  • Premisecitation
    § Introduction

    Juvenile social play contributes to the development of adult social and emotional skills in humans and non-human animals.

    “Juvenile social play contributes to the development of adult social and emotional skills in humans and non human animals [1–6]”
    Quote in the paperSubstantial claim3 of 6 sources checked
    • cites [1] Bekoff M (1974) Social play and play-soliciting by infant canids. Am Zool 14:323–340 source
    • cites [2] Van den Berg CL, Hol T, Van Ree JM, Spruijt BM, Everts H, Koolhaas JM (1999) Play is indispensable for an adequate development of coping with social challenges in the rat. Dev Psychobiol 34:129–138 source
    • cites [3] Pellis SM, Pellis VC, Bell HC (2010) The function of play in the development of the social brain. Am J Play 2:278–296
    • cites [4] Veenema AH, Bredewold R, De Vries GJ (2013) Sex-specific modulation of juvenile social play by vasopressin. Psychoneuroendocrinology 38:2554–2561 source

      The Introduction (page not provided) says juvenile social play is thought to contribute to social and emotional skill development in humans and animals. It does not specify that these are adult skills, and it presents the contribution as a hypothesis rather than an established fact. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [5] Paul MJ, Terranova JI, Probst CK, Murray EK, Ismail NI, De Vries GJ (2014) Sexually dimorphic role for vasopressin in the development of social play. Front Behav Neurosci 8:58. https://doi.org/10.3389/fnbeh.2014.00058 source
    • cites [6] Vanderschuren LJ, Trezza V (2014) What the laboratory rat has taught us about social play behavior: role in behavioral development and neural mechanisms. Curr Top Behav Neurosci 16:189–212 source

      The Concluding Remarks state that social play is important for social and emotional development, and section 3.1 describes long-lasting social impairments after isolation during the peak play period. The chapter focuses on laboratory rats; our copy does not establish the claim for humans or non-human animals generally. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Introduction

    Estrogens or estrogenic EDCs are involved in the development of play behavior in rats.

    “However, recent research showed that estrogens or estrogenic EDCs are also involved in the development of play behavior in rats [15–19].”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [15] Dessi-Fulgheri F, Porrini S, Farabollini F (2002) Effects of perinatal exposure to bisphenol A on play behavior of female and male juvenile rats. Environ Health Perspect 110:403–407 source
    • cites [16] Porrini S, Belloni V, Della Seta D, Farabollini F, Giannelli G, Dessì-Fulgheri F (2005) Early exposure to a low dose of bisphenol A affects socio-sexual behavior of juvenile female rats. Brain Res Bull 65:261–266 source
    • cites [17] Olesen KM, Jessen HM, Auger CJ, Auger AP (2005) Dopaminergic activation of estrogen receptors in neonatal brain alters progestin receptor expression and juvenile social play behavior. Endocrinology 146:3705–3712 source
    • cites [18] Ferguson SA, Law CD, Kissling GE (2014) Developmental treatment with ethinyl estradiol, but not bisphenol A, causes alterations in sexually dimorphic behaviors in male and female Sprague Dawley rats. Toxicol Sci 140:374–392 source
    • cites [19] Zaccaroni M, Massolo A, Della Seta D, Farabollini F, Giannelli G, Fusani L, Dessì-Fulgheri F (2018) Developmental exposure to low levels of ethinylestradiol affects play behavior in juvenile female rats. Neurotox Res 33:876–886 source
  • Premisecitation
    § Introduction

    Perinatal exposure to estrogen can modify behavioral developmental trajectories.

    “Perinatal exposure to estrogen can modify behavioral developmental trajectories.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] McCarthy MM (2008) Estradiol and the developing brain. Physiol Rev 88:91–134 source
  • Findingown data
    § Discussion

    Lactational exposure was as effective or more effective than gestational exposure.

    “In general, lactational exposure was as effective or more effective than gestational exposure, which is in contrast with studies reviewed by Delclos et al. [36] reporting a limited transfer of EE2 to newborns via milk in humans and rats.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Delclos KB, Weis CC, Bucci TJ, Olson G, Mellick P, Sadovova N, Latendresse JR, Thorn B, Newbold RR (2009) Overlapping but distinct effects of genistein and ethinyl estradiol (EE2) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies. Reprod Toxicol 27:117–132 source
  • Classified as probably not a substantial claim. 27
  • May combine two claims. 16

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    EE2 has been detected in some river systems in the USA and Europe due to its widespread pharmaceutical use and relatively long half-life.

    “Due to its widespread pharmaceutical use and relatively long half-life, EE2 has been detected in some river systems in the USA and Europe [23–25], and is a matter of concern for public and wildlife health [26].”
    Quote in the paperNot a substantial claim1 of 4 sources checked
    • cites [23] Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: a national reconnaissance. Environ Sci Technol 36:1202–1211 source
    • cites [24] Nash JP, Kime DE, Van der Ven LT, Wester PW, Brion F, Maack G, Stahlschmidt-Allner P, Tyler CR (2004) Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ Health Perspect 112:1725–1733 source

      The paper’s introductory passage (labeled Abstract in our copy; page unavailable) reports EE2 in European surface waters and U.S. streams, identifies it as a contraceptive-pill component, and notes its longer half-life. The Conclusions (page unavailable) also describe its persistence in aquatic environments. The paper does not establish that pharmaceutical use is widespread or explicitly attribute those detections to widespread use and the longer half-life. (LLM, conf=0.81)

      Experimental — accuracy not yet measured
    • cites [25] Johnson AC, Williams RJ (2004) A model to estimate influent and effluent concentrations of estradiol, estrone, and ethinylestradiol at sewage treatment works. Environ Sci Technol 38:3649–3658 source
    • cites [26] Wise A, O’Brien K, Woodruff T (2010) Are oral contraceptives a significant contributor to the estrogenicity of drinking water? Environ Sci Technol 45:51–60 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Introduction

    Timms et al. estimated that each year in the USA and Europe, almost 2 million women who use oral contraceptives become pregnant accidentally, primarily because of missed pills.

    “Timms et al. [20] estimated that each year in the USA and Europe, almost 2 million women who use oral contraceptives become pregnant accidentally, primarily because of missed pills.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [20] Timms BG, Howdeshell KL, Barton L, Bradley S, Richter CA, vom Saal FS (2005) Estrogenic chemicals in plastic and oral contraceptives disrupt development of the fetal mouse prostate and urethra. PNAS 102:7014–7019 source

      The introductory passage states the same figure, location, and reason: almost 2 million oral-contraceptive users in the United States and Europe become pregnant accidentally each year, primarily because of missed pills. But it introduces this as an existing estimate and cites reference 8, rather than presenting it as an estimate Timms et al. made themselves. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    The disrupting effects of EE2 at environmentally relevant levels on fish reproduction and behavior are well known.

    “The disrupting effects of EE2 at environmentally relevant levels on fish reproduction and behavior are well known [24, 27–30].”
    Quote in the paperNot a substantial claim2 of 5 sources checked
    • cites [24] Nash JP, Kime DE, Van der Ven LT, Wester PW, Brion F, Maack G, Stahlschmidt-Allner P, Tyler CR (2004) Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ Health Perspect 112:1725–1733 source
    • cites [27] Parrott JL, Blunt BR (2005) Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and demasculinizes males. Environ Toxicol 20:131–141 source
    • cites [28] Saaristo M, Craft JA, Lehtonen KK, Lindström K (2010) Exposure to 17α-ethinyl estradiol impairs courtship and aggressive behaviour of male sand gobies (Pomatoschistus minutus). Chemosphere 79:541–546 source
    • cites [29] Reyhanian N, Volkova K, Hallgren S, Bollner T, Olsson P-E, Olsén H, Hällström IP (2011) 17α-Ethinyl estradiol affects anxiety and shoaling behavior in adult male zebra fish (Danio rerio). Aquat Toxicol 105:41–48 source
    • cites [30] Sumpter JP, Jobling S (2013) The occurrence, causes, and consequences of estrogens in the aquatic environment. Environ Toxicol Chem 32:249–251 source

      In “The Practical Impacts of the Research,” the paper says EE2 affects fish at sub-ng/L concentrations and can prevent reproduction at a few ng/L. The passages from our copy do not report an effect of EE2 on fish behavior, so that part of the claim is unsupported. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    In mammals, pharmacological levels of EE2 exert important effects on reproductive physiology and behavior.

    “In mammals, pharmacological levels of EE2 exert important effects on reproductive physiology and behavior [18, 31–34].”
    Quote in the paperNot a substantial claim2 of 5 sources checked
    • cites [18] Ferguson SA, Law CD, Kissling GE (2014) Developmental treatment with ethinyl estradiol, but not bisphenol A, causes alterations in sexually dimorphic behaviors in male and female Sprague Dawley rats. Toxicol Sci 140:374–392 source

      The Discussion reports that developmental EE2 treatment altered reproductive measures and behavior in male and female Sprague Dawley rats, and Materials and Methods specifies the administered doses. The study supports the claim for rats under this treatment regimen, but does not establish the effect across mammals generally or explicitly characterize the doses as pharmacological. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [31] Arabo A, Lefebvre M, Fermanel M, Caston J (2005) Administration of 17α-ethinylestradiol during pregnancy elicits modifications of maternal behavior and emotional alteration of the offspring in the rat. Dev Brain Res 156:93–103 source
    • cites [32] Dugard M, Tremblay-Leveau H, Mellier D, Caston J (2001) Prenatal exposure to ethinylestradiol elicits behavioral abnormalities in the rat. Dev Brain Res 129:189–199 source
    • cites [33] Ferguson SA, Law CD Jr, Abshire JS (2011) Developmental treatment with bisphenol A or ethinyl estradiol causes few alterations on early preweaning measures. Toxicol Sci 124:149–160 source

      The results on dam body weight and litter outcomes (pages not provided) show that EE2-treated rats had lower gestational body weights and, at the higher dose, lower pup birth weights. But the Righting reflex and Slant board behavior results report no significant differences from controls attributable to EE2. The paper therefore supports effects on some pregnancy-related measures in rats, not the broader claim of important effects on both reproductive physiology and behavior in mammals. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [34] Mandrup KR, Jacobsen PR, Isling LK, Axelstad M, Dreisig K, Hadrup N, Vinggaard AM, Hass U, Boberg J (2013) Effects of perinatal ethinyl estradiol exposure in male and female Wistar rats. Reprod Toxicol 42:180–191 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    Administration of EE2 during development can affect a variety of reproductive anatomical and physiological endpoints in mice and rats.

    “administration of EE2 during development can affect a variety of reproductive anatomical and physiological endpoints in mice and rats [20, 36–46]”
    Quote in the paperNot a substantial claim8 of 10 sources checked
    • cites [20] Timms BG, Howdeshell KL, Barton L, Bradley S, Richter CA, vom Saal FS (2005) Estrogenic chemicals in plastic and oral contraceptives disrupt development of the fetal mouse prostate and urethra. PNAS 102:7014–7019 source
    • cites [36] Delclos KB, Weis CC, Bucci TJ, Olson G, Mellick P, Sadovova N, Latendresse JR, Thorn B, Newbold RR (2009) Overlapping but distinct effects of genistein and ethinyl estradiol (EE2) in female Sprague-Dawley rats in multigenerational reproductive and chronic toxicity studies. Reprod Toxicol 27:117–132 source
    • cites [37] Delclos KB, Camacho L, Lewis SM, Vanlandingham MM, Latendresse JR, Olson GR, Davis KJ, Patton RE, da Costa GG, Woodling KA (2014) Toxicity evaluation of bisphenol A administered by gavage to Sprague Dawley rats from gestation day 6 through postnatal day 90. Toxicol Sci 139:174–197 source

      The Results report that developmental EE2 exposure affected multiple reproductive endpoints in Sprague Dawley rats, including vaginal opening, estrous cycling, ovarian pathology, and testicular descent. This source does not establish the claim for mice, so it supports the rat portion of the sentence but not its full species scope. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [38] Derouiche L, Keller M, Martini M, Duittoz AH, Pillon D (2015) Developmental exposure to ethinylestradiol affects reproductive physiology, the GnRH neuroendocrine network and behaviors in female mouse. Front Neurosci 9:463 source
    • cites [39] Fusani L, Della Seta D, Dessì-Fulgheri F, Farabollini F (2007) Altered reproductive success in rat pairs after environmental-like exposure to xenoestrogen. Proc R Soc B 274:1631–1636 source

      The Discussion reports that developmental EE exposure altered reproductive physiology in rat pairs and cites previous rat studies reporting genital anomalies and disrupted oestrous cycles. This source does not address mice, so it supports the rat portion of the broader claim but not the mouse portion. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [40] Howdeshell KL, Furr J, Lambright CR, Wilson VS, Ryan BC, Gray LE Jr (2007) Gestational and lactational exposure to ethinyl estradiol, but not bisphenol A, decreases and rogen-dependent reproductive organ weights and epididymal sperm abundance in the male long evans hooded rat. Toxicol Sci 102:371–382 source

      The Discussion reports that developmental EE exposure affected reproductive organ development and endocrine parameters in male Long Evans rats; it also reports decreased reproductive organ weights and sperm production. This supports the rat portion of the claim, but the cited paper does not establish these effects in mice. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [41] Latendresse JR, Bucci TJ, Olson G, Mellick P, Weis CC, Thorn B, Newbold RR, Delclos KB (2009) Genistein and ethinyl estradiol dietary exposure in multigenerational and chronic studies induce similar proliferative lesions in mammary gland of male Sprague-Dawley rats. Reprod Toxicol 28:342–353 source
    • cites [42] Meyer N, Santamaria CG, Müller JE, Schumacher A, Rodriguez HA, Zenclussen AC (2019) Exposure to 17α-ethinyl estradiol during early pregnancy affects fetal growth and survival in mice. Environ Pollut 251:493–501 source
    • cites [43] Shiorta M, Kawashima J, Nakamura T, Ogawa Y, Kamiie J, Yasuno K, Shirota K, Yoshida M (2012) Delayed effects of single neonatal subcutaneous exposure of low-dose 17α-ethynylestradiol on reproductive function in female rats. J Toxicol Sci 37:681–690 source

      The Discussion reports that neonatal EE exposure affected the estrous cycle in female rats, and the section on ovulation and histopathology reports ovulatory and ovarian changes. These passages support reproductive physiological and anatomical effects in rats, but this paper does not establish the claim for mice. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [44] Shirota M, Kawashima J, Nakamura T, Kamiie J, Shirota K, Yoshida M (2015) Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats. J Toxicol Sci 40:727–738 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation
    § Discussion

    One should be cautious in translating the present results to humans because of differences between man and rat in the biotransformation of EE2, which could result in more serious effects in rats.

    “However, one should be cautious in translating the present results to humans because of differences between man and rat in the biotransformation of EE2 [66, 67], which could result in more serious effects in rats.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [66] Bořek-Dohalská L, Valášková P, Černá V, Stiborová M (2014) Role of rat cytochromes P450 in the oxidation of 17α-ethinylestradiol. Environ Toxicol Pharmacol 38:852–860 source
    • cites [67] Stiborová M, Dračínská H, Bořek-Dohalská L, Klusoňová Z, Holecová J, Martínková M, Schmeiser HH, Arlt VM (2018) Exposure to endocrine disruptors 17alpha-ethinylestradiol and estradiol influences cytochrome P450 1A1-mediated genotoxicity of benzo[a]pyrene and expression of this enzyme in rats. Toxicology 400–401:48–56 source

      The Introduction (page unavailable) identifies different CYP enzymes involved in EE2 metabolism in human and rat livers, supporting caution about translating rat findings to humans. It does not say that these differences could cause more serious effects in rats. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § Discussion

    EE2 has a low affinity for α-fetoprotein and human sex hormone-binding globulin.

    “In addition, EE2 has a low affinity for α-fetoprotein (AFP) [64] and human sex hormone-binding globulin (SHBG) [65].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [64] Hong H, Branham WS, Dial SL, Moland CL, Fang H, Shen J, Perkins R, Sheehan D, Tong W (2012) Rat α-Fetoprotein binding affinities of a large set of structurally diverse chemicals elucidated the relationships between structures and binding affinities. Chem Res Toxicol 25:2553–2566 source
    • cites [65] Hong H, Branham W, Dial S, Moland C, Fang H, Shen J, Perkins R, Sheehan D, Tong W (2015) Affinities of 125 structurally diverse chemicals and comparison with their binding to and rogen receptor, estrogen receptor, and α-fetoprotein. Toxicol Sci 143:333–348 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation
    § Materials and methods

    Treatment administered orally with a pipette appears to be much less stressful than gavage.

    “This procedure appears to be much less stressful than gavage [56, 57].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [56] Vandenberg LN, Welshons WV, VomSaal FS, Toutain P-L, Myers JP (2014) Should oral gavage be aband oned in toxicity testing of endocrine disruptors? Environ Health 13:46 source

      The Background section on stress reports that gavage causes pronounced stress responses. The section on other administration options lists feeding animals from a pipette, but does not assess its stress effects or compare them with gavage. The specific claim that pipette dosing is much less stressful is therefore not established by this paper. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [57] Gioiosa L, Palanza P, Parmigiani S, vom Saal FS (2015) Risk evaluation of endocrine-disrupting chemicals: effects of developmental exposure to low doses of bisphenol A on behavior and physiology in mice (Mus musculus). Dose-Response 13:1559325815610760 source

      The Introduction’s “Procedure, Behavioral Testing, and Results” section says pregnant mice drank treatment from a modified syringe without being handled or stressed. It does not compare their stress with that of gavaged mice or establish that the procedure is “much less stressful” than gavage; gavage appears only in a cited reference’s title. Page numbers are unavailable in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation
    § Discussion

    Early effects of steroid hormones on mammalian brain and behavior take place in a perinatal time window, in rodents from late gestation to initial post-natal life.

    “It is clearly shown by a large number of studies that early effects of steroid hormones on mammalian brain and behavior take place in a perinatal time window, in rodents from late gestation to initial post-natal life [9].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] McCarthy MM (2008) Estradiol and the developing brain. Physiol Rev 88:91–134 source

      Section IV describes a perinatal sensitive period in which gonadal steroids permanently alter brain architecture and later reproductive behavior. The aromatization section discusses late-gestation fetuses and early postnatal pups. But Section IV expressly limits this organizational principle to reproductively relevant outcomes; the claim states it more broadly as applying to early steroid effects on mammalian brain and behavior generally. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Alterations of maternal care can affect offspring.

    “alterations of maternal care can affect offspring [74, 75].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [74] Meaney MJ (2001) Maternal care, gene expression, and the transmission of individual differences in stress reactivity across generations. Annu Rev Neurosci 24:1161–1192 source
    • cites [75] Jensen Peña C, Champagne FA (2013) Implications of temporal variation in maternal care for the prediction of neurobiological and behavioral outcomes in offspring. Behav Neurosci 127:33 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    In male mice, gestational administration of Di(2-ethylhexyl) phthalate, an estrogenic chemical, increases fetal testosterone, AGD and AGI at very low doses but not at higher ones.

    “a study by Do et al. [62] showed that in male mice gestational administration of Di(2-ethylhexyl) phthalate, an estrogenic chemical, increases fetal testosterone, AGD and AGI at very low doses but not at higher ones.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [62] Do RP, Stahlhut RW, Ponzi D, vom Saal FS, Taylor JA (2012) Non-monotonic dose effects of in utero exposure to di (2-ethylhexyl) phthalate (DEHP) on testicular and serum testosterone and anogenital distance in male mouse fetuses. Reprod Toxicol 34:614–621 source

      The Discussion (page unavailable) reports elevated fetal serum testosterone at low DEHP doses and increased testicular testosterone and AGD in a subset of male fetuses; the highest dose did not differ from controls for the latter measures. Results §3.9 (page unavailable) reports a non-linear effect on weight-adjusted AGD, which may correspond to the claim’s AGI, but does not call it AGI. The copy does not characterize DEHP as an estrogenic chemical, and the AGD and testicular-testosterone findings are specifically for 1MF males, not all male fetuses. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    A diet rich in the phytoestrogens genistein and diadzein during gestation had no effect on male rat AGD at birth.

    “a diet rich in the phytoestrogens genistein and diadzein during gestation had no effect on male rat AGD at birth [77].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [77] Casanova M, You L, Gaido KW, Archibeque-Engle S, Janszen DB, Heck HDA (1999) Developmental effects of dietary phytoestrogens in Sprague-Dawley rats and interactions of genistein and daidzein with rat estrogen receptors alpha and beta in vitro. Toxicol Sci 51:236–244 source

  • May combine two claims.

    Findingown data
    § Results

    PC1 explained 54.45% of variance and included most elements of aggressive play, and was labeled aggressive-like play.

    “Principal component 1 (PC1) explained the largest percentage of variance (54.45%) and included most elements of aggressive play, and was therefore labeled as “aggressive-like play”.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    Cage had no significant effect on allogrooming.

    “Cage had not significant effect (F2,64 = 0.271, p = 0.76).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    Cage had no significant effect on aggressive-like play.

    “Cage had not significant effect (F2,64 = 2.688, p = 0.07).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    Body weight at PND1 was not affected by treatment.

    “Body weight at PND1 was not affected by treatment (F2,146 = 1.45; p > 0.2).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    No cage effect was detected on total social activity.

    “No cage effect (F2,64 = 2.41, p = 0.09) was detected.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    PC2 included most element of defensive play and was labeled defensive-like play, with variance explained 12.66%.

    “PC2 included most element of defensive play and was labeled as “defensive-like play” (variance explained = 12.66%).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    Cage had no significant effect on defensive-like play.

    “Cage had no significant effect (F2,64 = 0.520, p=0.59).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    Our data show that the effect of gestational exposure is reversed after lactational exposure, a difference that has not been reported by other researches.

    “However, our data show that this effect is reversed after lactational exposure, a difference that has not been reported by other researches.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given
    § Discussion

    Total social behavior, aggressive-like play, and defensive-like play were differently affected by gestational or lactational exposure to EE2.

    “Total social behavior, aggressive-like play, and defensive-like play were differently affected by gestational or lactational exposure to EE2.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    The development of play is influenced by perinatal testosterone through its 5α-reduced products.

    “The development of play is influenced by perinatal testosterone through its 5α-reduced products [14].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Meaney MJ, Stewart J, Poulin P, McEwen BS (1983) Sexual differentiation of social play in rat pups is mediated by the neonatal androgen-receptor system. Neuroendocrinology 37:85–90 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    Johnson and Williams suggested that 40% of ingested EE2 is found free (deconjugated) in the environment.

    “Johnson and Williams [25] suggested that 40% of the ingested EE2 is found free (deconjugated) in the environment.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [25] Johnson AC, Williams RJ (2004) A model to estimate influent and effluent concentrations of estradiol, estrone, and ethinylestradiol at sewage treatment works. Environ Sci Technol 38:3649–3658 source

  • May combine two claims.

    Premisecitation
    § Introduction

    The mammalian brain is essentially feminine in the absence of early exposure to gonadal steroid and is susceptible to the organizational action of sex hormones or their mimics.

    “In fact, the mammalian brain is essentially feminine in the absence of early exposure to gonadal steroid [11] and is susceptible to the organizational action of sex hormones or of their mimics.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Gorski RA (2002) Hypothalamic imprinting by gonadal steroid hormones. Adv Exp Med Biol 511:57–70 source

  • May combine two claims.

    Premisecitation
    § Introduction

    Rat juvenile social play is sensitive to chemical factors such as prenatal and neonatal hormones and is a useful behavioral marker of neurodevelopment.

    “Rat juvenile social play is sensitive to chemical factors such as prenatal and neonatal hormones and is a useful behavioral marker of neurodevelopment as severe deficits in this behavior are associated with neurodevelopmental disorders [7].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Blake BE, McCoy KA (2015) Hormonal programming of rat social play behavior: standardized techniques will aid synthesis and translation to human health. Neurosci Biobehav Rev 55:184–197 source

  • May combine two claims.

    Interpretationown data
    § Discussion

    Developmental exposure of male rats to low or very low doses of EE2 significantly alters social play by modifying the frequencies of its aggressive and defensive components, and this effect may vary with timing of exposure.

    “Our findings showed that developmental exposure of male rats to low or very low doses of the synthetic estrogen EE2 significantly alters social play by modifying the frequencies of its aggressive and defensive components and that this effect may vary in relation of the timing of exposure.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning
    § Materials and methods

    Each experimental subject came from a different dam.

    “Thus, only one male per litter for a total of 72 males was used for the study, i.e. each experimental subject came from a different dam.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning
    § Discussion

    As a consequence, EE2 is able to reach target areas in the brain and to affect physiology and behavior during critical time windows.

    “As a consequence, EE2 is able to reach target areas in the brain and to affect physiology and behavior during critical time windows.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § Introduction

    Isolation is a strong stressor per se and represents an important confounding factor.

    “Isolation is a strong stressor per se [53] and, although it may enhance the emergence of effects on play [7], it represents an important confounding factor.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [53] Blanchard RJ, McKittrick CR, Blanchard DC (2001) Animal models of social stress: effects on behavior and brain neurochemical systems. Physiol Behav 73:261–271 source
    • cites [7] Blake BE, McCoy KA (2015) Hormonal programming of rat social play behavior: standardized techniques will aid synthesis and translation to human health. Neurosci Biobehav Rev 55:184–197 source

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationcitation
    § Discussion

    If play is essential for the refinement of social skills, then a modification of play may have consequences on adult social behavior.

    “If play is essential for the refinement of social skills [2, 3], then a modification of play may have consequences on adult social behavior.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2] Van den Berg CL, Hol T, Van Ree JM, Spruijt BM, Everts H, Koolhaas JM (1999) Play is indispensable for an adequate development of coping with social challenges in the rat. Dev Psychobiol 34:129–138 source
    • cites [3] Pellis SM, Pellis VC, Bell HC (2010) The function of play in the development of the social brain. Am J Play 2:278–296

      Not checked in the free run.

  • May combine two claims.

    Premisecitation
    § Introduction

    Previous studies in Sprague-Dawley rats showed significant effects of a developmental administration (GD 5-PND 32) of very low (4 ng/kg/day) or low clinical EE2 doses (400 ng/kg/day) on learning and memory, sexual behavior, pain perception, anxiety and social play.

    “previous studies in Sprague–Dawley (SD) rats showed significant effects of a developmental administration (GD 5–PND 32) of very low (4 ng/kg/day) or low clinical EE2 doses (400 ng/kg/day) on learning and memory [47], sexual behavior [48, 49], pain perception [50], anxiety [51] and social play [19].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [47] Corrieri L, Della Seta D, Canoine V, Fusani L (2007) Developmental exposure to xenoestrogen enhances spatial learning in male rats. Horm Behav 51:620–625 source
    • cites [48] Della Seta D, Farabollini F, Dessì-Fulgheri F, Fusani L (2008) Environmental-like exposure to low levels of estrogen affects sexual behavior and physiology of female rats. Endocrinology 149:5592–5598 source
    • cites [49] Della Seta D, Minder I, Belloni V, Aloisi AM, Dessì-Fulgheri F, Farabollini F (2006) Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats. Horm Behav 50:301–307 source
    • cites [50] Ceccarelli I, Fiorenzani P, Della Seta D, Aloisi AM (2015) Perinatal 17α-ethinylestradiol exposure affects formalin-induced responses in middle-aged male (but not female) rats. Horm Behav 73:116–124 source

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § Materials and methods

    Video recordings were analyzed by an observer blind to treatment.

    “All sessions were recorded with a video camera, and video recordings were analyzed with ‘The Observer Video Pro 4.0’ software (Noldus Information Technology, The Netherlands) by an observer blind to treatment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    Pellis and Pellis described in Long Evans hooded male and female rats a peak of play fighting around 41-45 days of age in same-sex pairs.

    “Pellis and Pellis [55] described in Long Evans hooded male and female rats a peak of play fighting around 41–45 days of age in same-sex pairs.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] Pellis SM, Pellis VC (1990) Differential rates of attack, defense, and counterattack during the developmental decrease in play fighting by male and female rats. Dev Psychobiol 23:215–231 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Introduction

    A lack of effects with 500 ng/kg/day was observed in Mandrup et al.

    “but see a lack of effects with 500 ng/kg/day in Mandrup et al. [34].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Mandrup KR, Jacobsen PR, Isling LK, Axelstad M, Dreisig K, Hadrup N, Vinggaard AM, Hass U, Boberg J (2013) Effects of perinatal ethinyl estradiol exposure in male and female Wistar rats. Reprod Toxicol 42:180–191 source

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given
    § Materials and methods

    Testing of different experimental groups was balanced across time.

    “Testing of different experimental groups was balanced across time.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Effects of subtle variations in the concentration of hormones on development have been reported previously, as illustrated by differences in adult behavior depending on intrauterine position that affects the prenatal hormonal milieu.

    “Effects of subtle variations in the concentration of hormones on development have been reported previously, as illustrated by the differences in adult behavior depending on the intrauterine position that affects the prenatal hormonal milieu [68, 69].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [68] Ryan BC, Vandenbergh JG (2002) Intrauterine position effects. Neurosci Biobehav Rev 26:665–678 source
    • cites [69] Vom Saal F (2016) Triennial reproduction symposium: environmental programming of reproduction during fetal life: effects of intrauterine position and the endocrine disrupting chemical bisphenol A. J Anim Sci 94:2722–2736 source

  • Classified as probably not a substantial claim.

    Study design and conductnone given
    § Concluding remarks

    The experimental protocol was designed to mimic an environmental or clinical early exposure to EE2.

    “With an experimental protocol designed to mimic an environmental or clinical early exposure to EE2”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Discussion

    Low doses of bisphenol A administered through pregnancy to lactation can exert a mild but significant effect on maternal care of rat dams.

    “Our group showed that low doses of the weak estrogen bisphenol A administered through pregnancy to lactation can exert a mild but significant effect on maternal care of rat dams [76].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [76] Della Seta D, Minder I, Dessì-Fulgheri F, Farabollini F (2005) Bisphenol-A exposure during pregnancy and lactation affects maternal behavior in rats. Brain Res Bull 65:255–260 source

  • May combine two claims.

    Premisecitation
    § Discussion

    In a study by Corrieri et al., SD male rats received through their mothers very low doses of EE2 (4 ng/kg/day) or oil from GD 5 on, and a similar slight, though not significant, increase of AGD at birth was observed.

    “In a study by Corrieri et al. [47], SD male rats received through their mothers very low doses of EE2 (4 ng/kg/day) or oil from GD 5 on, and a similar slight, though not significant, increase of AGD at birth was observed.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Corrieri L, Della Seta D, Canoine V, Fusani L (2007) Developmental exposure to xenoestrogen enhances spatial learning in male rats. Horm Behav 51:620–625 source

  • May combine two claims.

    Interpretationnone given
    § Discussion

    To our knowledge, this is the first time that this effect is shown, suggesting a difference between a prenatal and a postnatal critical window of sex steroid action.

    “To our knowledge it is the first time that this effect is shown, suggesting a difference between a prenatal and a postnatal critical window of sex steroid action.”
    Quote in the paperSubstantial claim
40 of 40

  • The paper relies on this without support.

    EE2 is a powerful mimic of natural estrogen and is the active component of most contraceptive pills.

    “The synthetic estrogen ethinylestradiol (EE2) is a powerful mimic of natural estrogen and is the active component of most contraceptive pills.”

  • The paper relies on this without support.

    Unintentional exposure of the developing human fetus can occur if oral contraception is continued during the early months of undetected pregnancy.

    “Unintentional exposure of the developing human fetus can occur if oral contraception is continued during the early months of undetected pregnancy.”

  • The paper relies on this without support.

    In humans, the assumption of EE2 is mainly through fish food intake.

    “In humans, the assumption of EE2 is mainly through fish food intake.”

  • The paper relies on this without support.

    Previous work relying on castration/hormone replacement approaches often showed on–off effects of the hormones with consequent masking of those traits that typically respond in a dose-dependent manner.

    “Previous work relying on castration/hormone replacement approaches often showed on–off effects of the hormones with consequent masking of those traits that typically respond in a dose-dependent manner.”
4 of 4

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Related

Authors
Zaccaroni, M., Massolo, A., Beani, L., Seta, D. D., Farabollini, F., Giannelli, G., Fusani, L., & Dessì-Fulgheri, F.
Affiliations
  • University of Florence (IT)
  • University of Pisa (IT)
  • University of Siena (IT)
  • University of Vienna (AT)
Corresponding author
Zaccaroni, Marco
ORCID
Venue
Toxicological Research
Publisher
Springer Science and Business Media LLC
Published
2020-02-07
Volume
36
Issue
4
Pages
301-310
ISSN
  • 1976-8257 (ISSN-L)
  • 2234-2753
DOI
10.1007/s43188-019-00035-z
PMC
PMC7494694
OpenAlex
W3005685944
Semantic Scholar
63a53aadcb098fc7097fba4e5b4d5f8456937413
Cited by
5
References
82
Citations by year
2025: 1 · 2023: 1 · 2022: 2 · 2021: 1
FWCI
0.35
Type
journal article
Language
en
Open access
Licence
Locations
  • Toxicological Research (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
  • CINECA IRIS Institutial research information system (University of Pisa) (submittedVersion)
  • Florence Research (University of Florence) (submittedVersion)
Topics
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Effects and risks of endocrine disrupting chemicals
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
  • Social Sciences › Psychology › Social Psychology › Neuroendocrine regulation and behavior
Keywords
ethinyl estradiol, endocrine disruptors, social play, prenatal exposure, lactational exposure, developmental neurotoxicity, social behavior, dose-response relationship

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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