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. Author manuscript; available in PMC: 2018 Jun 14.
Published in final edited form as: Toxicol Sci. 2010 Mar 5;115(2):614–620. doi: 10.1093/toxsci/kfq073

Rebuttal of “Flawed Experimental Design Reveals the Need for Guidelines Requiring Appropriate Positive Controls in Endocrine Disruption Research” by (Vom Saal 2010)

Leon Earl Gray Jr *, Bryce Ryan **, Andrew K Hotchkiss *, Kevin M Crofton *
PMCID: PMC6002156  NIHMSID: NIHMS966224  PMID: 29910598

The developmental effects of the potent environment estrogen Ethinyl Estradiol (EE2) addresses important Agency research needs and EE2 is not just a “positive control”

Ethinyl estradiol is an extremely potent estrogen that is found at significant concentrations in a number of aquatic systems globally. In this regard, it is not just a positive control for the potential estrogenic effects of BPA. It has been clearly shown to be causally related to vitellogenin levels and ovotestes in male fish, altered reproductive function and population levels in field studies (Jobling et al. 1998; Jobling et al. 2006; Kidd et al. 2007). As a result of the recognition of the widespread prevalence of EE2 and natural estrogens concern has arisen about potential effects of these estrogens on human health from drinking water exposure. For this reason, the US Environmental Protection Agency, Office of Water has included EE2 on the Contaminant Candidate List 3 (CCL 3). “CCL 3 is a list of contaminants that are currently not subject to any proposed or promulgated national primary drinking water regulations, that are known or anticipated to occur in public water systems, and which may require regulation under the Safe Drinking Water Act (SDWA).” http://www.epa.gov/ogwdw000/ccl/ccl3.html.

One of the objectives of our study was to determine if male or female LE rat offspring were adversely affected by EE2 at dosage levels significantly below those that affect pregnant and lactating rats, to inform the regulatory process. While the dose levels that produced effects in these life stages did not differ, the effects in the female offspring were permanent and much more severe than the effects of EE2 in the dams.

Why the Long Evans (LE) rat and several other rat strains are excellent animal models for the study of the potential adverse effects of xenoestrogens

One reason why the LE rat is an excellent model for the study of the effects of environmental estrogens is that the effects of hormones including estrogens on behavioral sex differentiation of the female rat have been extensively studied over the last 45 years. Neonatal exposure to estrogens has been shown to alter reproductive (lordosis) and nonreproductive behaviors, the structure and function of specific brain regions, estrous cyclicity, ovulation, fertility and more recently, gene expression and protein (e.g. kisspeptin) levels in the hypothalamus. As a result, there are well validated protocols for the study of xenoestrogens like BPA for determining if specific behaviors like lordosis and saccharin preference are imprinted by neonatal treatments (McCarthy 2008) (Barraclough and Gorski 1962; Levine and Mullins 1964; Valenstein et al. 1967). In addition, in the last twenty years there are literally thousands of published studies using LE rats for behavioral observations, including scores of studies on lordosis behavior.

Why the argument that LE rats and other rat strains are “insensitive strains” is not consistent with pharmacological and toxicological data showing that humans and the LE and SD rat strains display similar sensitivities to EE2

The LE and SD rat strains also are excellent animal models for the study of the effects of EE2 and other environmental estrogens because the sensitivity of these rat strains is very similar to the sensitivity of humans to EE2. For example, a multigenerational study conducted at the National Center for Toxicological Research for the NTP (Latendresse et al. 2009) administered EE2 in the diet at 0.2, 1.1 or 5.8 µg/kg/d to SD rats. EE2 reduced body weight of the female rats at 1.1 and 5.8 µg/kg/d, altered the age at Vaginal Opening (an index of puberty), induced abnormal estrous cycles at 5.8 µg/kg/d and induced non-neoplastic uterine lesions at all dose levels including 0.2 µg/kg/d; a dose equivalent to some of the lowest dose of EE2 used in human birth control pills, pills that contain 5–10 fold more progestins than EE2.

Our critics (Vom Saal 2010) stated that we should have demonstrated the sensitivity of our strain to EE2 before conducting the developmental study. In fact, the first two experiments in the paper (Ryan et al. 2010) clearly demonstrated that the LE and SD display identical dose response increases in EE2 induced uterine weight and lordosis behavior.

In addition to their use for studies with EE2 and BPA, developmental studies (transgenerational, multigenerational or neonatal) with LE and SD rat strains have detected the estrogenic effects of orally administered estradiol (Tyl et al. 2008b) and xenoestrogens like methoxychlor (Goldman et al. 1986; Gray et al. 1985; Gray et al. 1999a; Gray et al. 1989; Gray et al. 1988), nonylphenol (Chapin et al. 1999), and genistein (Naciff et al. 2002) (Casanova et al. 1999; Latendresse et al. 2009). Furthermore, in pubertal studies, weanling female LE and/or SD female rats displayed the expected (anti)estrogen-like responses to several chemicals including EE2, tamoxifen, octylphenol and methoxychlor.

The authors of the letter (Vom Saal 2010) criticize our study by asserting that the “clinically effective dose of EE2 in oral contraceptives is <0.5 µg/kg/day. Ryan et al. (Ryan et al.) thus report no effects of EE2 in their animal model at doses sufficient to cause temporary sterility in 99.7% of women who properly use oral contraceptives” However, comparing combination birth control pills to administration of EE2 alone is like comparing apples and oranges. Oral contraceptives include not only EE2 but also five to ten fold higher doses of progestins. Even with the combination pills, some doctors recommend that women use another form of birth control in the first month since the medication may not be fully effective immediately. In contrast to women using birth control pills, the dams in our study were exposed only to EE2, the dams were treated for only about 33 days and the offspring were never exposed directly, being exposed transplacentally and through the milk.

When EE2 is used by itself as a human pharmaceutical, relatively high dose levels are required to produce the desired outcomes. For example, EE2 doses ranging from 100 – 1000 µg/day have been used to limit final height in girls and boys (Rooman et al. 2005; Schmitt et al. 1992) (Svan et al. 1991). The menopausal drugs Estinyl and Feminone contained 20 to 500 µg EE2 per pill. When EE2 was administered as a postcoital contraceptive agent, administration of 5 mg per pill was effective but 2–3 mg was only partially effective (Blye 1973; Haspels 1972).

If one uses a body weight of 50 kg for adult women as a reference, the doses of EE2 that produce adverse effects in women, when the medication does not include a progestin, range from 100 to 5000 µg/day, which is equivalent to 2 to 100 µg/kg body weight /day. In our study, we saw statistically significant adverse effects on the dams at 1.5 µg/kg/day, effects in the male and female offspring exposed indirectly to 5 µg/kg/day, with reduced fecundity and malformations of the external genitalia of females having ED50s of 2.6 and 3.9 µg/kg/day. Taken together, these results demonstrate the LE and SD rats are most appropriate animal models for the study of EE2 since the sensitivity to this chemical, and likely other estrogens, is similar to the human sensitivity to EE2.

In their critique of our study (Vom Saal 2010) the authors state that “the lowest effect dose for EE2 in the LE rat (5 µg/kg/day) was 2,500-fold higher than the maternal dose required to stimulate effects on offspring in mice (Thayer et al., 2001)”. However, reanalysis of these same data by NIEHS statisticians (National Toxicology Program 2001)(p A-52) “did not achieve the same level of significance” as reported by Thayer et al. (2001). At 5 months of age significant (p<0.05) increases in prostate weight in male offspring were noted in the NTP report at 2 and 0.2 µg/kg/d.

Even if one assumes that the effect of EE2 on the male mouse prostate weight cited by vom Saal et al (2010) can be independently reproduced and one also assumes that this is an adverse effect, then the difference between the results of Thayer et al (2001) and the constellation of adverse effects that we obtained is closer to ten fold than 2,500 fold. Since independent laboratories using robust study designs have been unable, to date, to replicate any of the reported increases in prostate weight in the mouse induced by in utero estrogens like DES, estradiol, or BPA, the importance of these observations is uncertain. In addition, the physiological and toxicological significance of a small increase in male mouse prostate weight has yet to be established. The lack of reproducibility of the original reported effect of BPA on prostate weight (Nagel et al. 1997) has been explicitly cited by several governmental agencies (Japan (Nakanishi et al. 2007), European Food Safety Authority (Scientific Panel on food additives 2006), European Union (EU 2010), Canada (Canada 2008) and the NTP CERHR (National Toxicology Program 2007)).

If estrogen- or xenoestrogen-induced increases in prostate weight of the mouse were reproduced by independent laboratories, the significance of such effects to other mammalian species would still remain uncertain since this effect has not been reported in any study with any other mammalian species including the rat. In contrast, we do know that the smaller prostates obtained with in utero exposure to antiandrogens (vinclozolin, procymidone and p,p’ DDE) in several rat strains eventually develop inflammatory lesions (Ostby et al. 1999) (Cowin et al. 2008) (Gray et al. 1999b).

In contrast to the results reported by Thayer et al (Thayer et al., 2001), the results of transgenerational studies with estrogens including EE2 in rats and mice demonstrate that SD and LE female rat offspring display malformations, infertility and histopathological lesions of the reproductive tract at dose levels similar to or below those that produce severe effects in mice, for example, for EE2 ((Kirigaya et al. 2006; Yasuda et al. 1977, 1981; Yasuda et al. 1985a; Yasuda et al. 1985b; Yasuda et al. 1987; Yasuda et al. 1986a; Yasuda et al. 1986b)), and estradiol 17 beta ((Biegel et al. 1998; Tyl et al. 2008b)). In addition, rats display similar sensitivities to sc injections of estrogens as do CD-1 mice (Padilla-Banks et al. 2001) (ED50s for sc estradiol 17β are 22 and 7.8 µg/kg/day for SD rat versus CD-1 mouse).

Our study is certainly not the only study to find that BPA did not exhibit any estrogenic activity when administered orally. Oral treatment with BPA also failed to produce any estrogen-like reproductive effects in Wistar, Alderley-Park, SD, F344 and LE rats and CF-1 and CD-1 mice at any dose level in several large robust multigenerational and transgenerational studies. These were conducted under both GLP and non-GLP conditions by scientists from government (Ema et al. 2001; Howdeshell et al. 2008; Ryan et al. 2010), academia (Yoshino et al. 2002), industry (Ashby et al. 1999; Cagen et al. 1999a, b; Tinwell et al. 2002) and contract laboratories (Tyl 2003; Tyl et al. 2008a; Tyl et al. 2002).

Why the argument that LE rats and other rat strains are “insensitive strains” is not consistent with the cellular and molecular biology of the action of estrogens on different target tissues

The "insensitive rat" argument has been used for almost a decade in some quarters to try to dismiss every well-conducted rat study that obtained negative results with BPA. It is based on a failure to recognize the basic endocrinology underlying the cellular and molecular basis for tissue-specific responses to estrogens in different strains of rats. Several traits have been shown to be estrogen sensitive in rats including induction of pituitary tumors (Wiklund and Gorski 1982) (Gorski et al. 1997; Wendell and Gorski 1997)), prolactin regulation in the pituitary (Lawson et al. 1984), thymic involution (Gould et al. 2006) (Gould et al. 2000), uterine pyometra (Gould et al. 2005), liver carcinogenesis (Pandey et al. 2005) and mammary cancer (Gould et al. 2004) to name a few. It is evident that the degree of estrogen sensitivity varies from tissue to tissue and no single rat strain is more or less sensitive than another for all traits. The complexity of the sensitivity to estrogens among rat strains arises at several levels of biological organization (Diel 2002). The genes regulated by estrogen receptors are tissue-specific and map to several different chromosomes. There are tissue-specific coactivators and corepressors that combine with ER homodimers to form the transcriptional complex that binds to the promoter (Katzenellenbogen et al. 2000; Thomas et al. 2004). In addition, there are at multiple DNA response elements expressed (Sanchez et al. 2002) that regulate the relative potency of estrogens and xenoestrogens in different tissues (Hall and Korach 2002). In no case has it been demonstrated that either the SD or LE rat strain is completely insensitive to an estrogen. Therefore, one cannot conclude that the SD or LE rat strains are insensitive to the effects of estrogens and the results of studies showing no adverse effects with BPA cannot be dismissed because of the strain of rat used by the investigator.

Consistent with this conclusion, several expert panels have addressed the “insensitive strain” argument and dismissed this as being without “scientific merit” (Canada 2008; Chapin et al. 2008; NTP 2008; Scientific Panel on food additives 2006).

How the effects of leaching of Bisphenol A (BPA) from polycarbonate cages have been grossly exaggerated

Another criticism of our study was that we used polycarbonate cages to house our animals. Vom Saal et al. (Vom Saal 2010) stated that “One potential contributor to the low sensitivity to estrogen in this experiment is the use of polycarbonate cages made from BPA”. This comment arises from a study published by Howdeshell et al. (Howdeshell et al. 2003) which reported that polycarbonate animal cages released BPA into water at room temperature. The BPA concentration was measured in water samples that had been incubated at room temperature for one week in new polycarbonate, polysulfone and polypropylene cages and from used (“visibly worn, with patches of opaque plastic and some areas of rough, pitted surface inside the cage”) and used, leaky polycarbonate cages. When tested for estrogenic activity using an MCF-7 human breast cancer cell proliferation assay, “significant estrogenic activity was detected only in water samples from the used polycarbonate cages and one of the polypropylene cages. Howdeshell et al (2003) stated that “These experimental conditions are directly relevant because it is common for aquatic laboratory animals to be housed in polycarbonate cages.” The relevance to rodents is less obvious.

Howdeshell et al (2003) reported BPA concentrations in water samples up to 310 micro g/L from used leaky polycarbonate animal cages whereas the BPA concentration was much lower in used polycarbonate cages Detectable levels of BPA were released from new polycarbonate cages (up to 0.3 micro g/L) as well as new polysulfone cages (1.5 micro g/L) and nonylphenol was detected in one polypropylene cage.

Howdeshell et al (2003) also housed 19 day old prepubertal female mice in used cages with drinking water in polycarbonate bottles for one week and then measured the uterine weight in the females. Housing prepubertal female mice in used polycarbonate cages did not significantly increase uterine weight.

Since the cages used in our study were clear, not “visibly worn, with patches of opaque plastic and some areas of rough, pitted surfaces” they would not be expected to release compounds with “significant estrogenic activity”. If we accept the methods and results of the Howdeshell et al study, then our cages would have released 0.011 µg/d (0.3 µg /L ml/7 days in 250 ml) whereas the highest value from their old leaky cage released about 8.6 µg /d (310 µg/L/7d in 195 ml). Given that the weanling rat displays an increase in uterine weight at oral BPA administration with 200 mg/kg/d in the rat (Laws et al. 2000) and an increase in uterine labeling with bromodeoxyuridine (but not weight) (Tinwell et al. 2000) in the mouse the amount of BPA “released” was about a million-fold lower than the dose that would increase uterine weight in the rat. The highest amount of BPA released from one of the old leaky cages was about is about thousand-fold below the uterotropic dose. (We used the data from the rat because oral exposure to BPA has not been shown to increase uterine weight at any dose in prepubertal mice even though they have been treated with doses as high as 300 mg/kg/d (Tinwell et al. 2000).)

Given a focus of the criticism in the vom Saal et al. letter (Vom Saal 2010) on the strains of rat and caging materials used for BPA rodent studies, it seems ironic that five out of six of the authors of the letter that have conducted studies with BPA in rats used either the SD or the LE rat strain. In addition, the caging used by several of the authors for their BPA studies in rodents included polycarbonate cages. A majority of these authors failed to specify the type of caging material used indicating that they did not consider this an important factor.

Governmental regulatory agencies have concluded that studies like ours (Ryan et al. 2010) are useful for risk assessment whereas many of the “positive” low dose studies were “inadequate” or the results have not replicated

Vom Saal et al. (Vom Saal 2010) have criticized our study as being “flawed”. However, when our data were reviewed recently by regulatory agencies, these agencies stated that the study is useful for risk assessment. In addition, several other well designed, robust studies of BPA using SD rats (Tyl et al. 2002) (Ema et al. 2001) have been described as “adequate and of high utility” and as “a well conducted thorough study”.

In contrast to these well conducted studies, when the published rodent research on BPA was reviewed by the NTP CERHR BPA Expert Panel (Chapin et al. 2008; National Toxicology Program 2007), the European Food Safety Agency and/or the NTP/NIEHS Endocrine Disruptors Low Dose Peer Review Panel (National Toxicology Program 2001) a significant percentage of the publications that reported effects of BPA at low dose levels were described as “inadequate” for methodological or statistical reasons, “not replicable”, “extremely limited” or of uncertain toxicity and relevance to human health risk assessment. Identified problems were, lack of a concurrent control group, lack of any statistical analysis (Schonfelder et al. 2002), invalid experimental design (Hunt et al. 2003) and/or statistical analysis (Zoeller et al. 2005) (vom Saal et al. 1998) (Akingbemi et al. 2004), thousand-fold errors in reporting of the administered dose of BPA (Markey et al. 2001), and intracerebellar injections of BPA (Zsarnovszky et al. 2005).

Taken together, an objective analysis of all the scientific information on BPA shows that the discrepancies in the literature among “positive” and “negative” low dose studies are not due to differences in rat strains, rodent species, caging, or likely even diet (since many investigators use the same diet we employed in our studies). In addition, the discrepancies do not arise from GLP versus non-GLP studies or the nature of the institutions where the work was conducted. Rather, the factors that need to be considered in evaluating the low dose BPA research are the quality of the research and the relevance of the results to human health. The difference between studies that are deemed “adequate” versus “inadequate” for risk assessment have included relevance of the route of exposure, sample size, validity of the experimental design and statistical analysis (accounting for litter-effects and repeated measures in the data analysis), reproducibility of the effects by other investigators, and the nature of the effect (adverse or causally linked to an adverse effect experimentally). To date, a large proportion of the “positive” low dose literature is deemed not to have met these standards of quality and, until such criteria are attained, regulatory agencies are not likely to consider the results of these studies over those of high quality.

Summary

In summary, our two publications on EE2 and BPA (Howdeshell et al. 2008; Ryan et al. 2010) demonstrate that oral administration of EE2 during gestation and lactation produces adverse effects in the treated dams and their offspring at dose levels similar to the dose levels of EE2 used pharmacologically in humans. Severe, permanent effects including malformations, infertility and defeminization of reproductive and non reproductive sexually dimorphic behaviors were seen in female LE rat offspring. None of the EE2-induced alterations displayed a nonmonotonic dose response. In Toto, EE2 altered approximately 25 different endpoints in the dams, F1 males and females in an estrogenic-like manner with the NOAEL being 0.5µg/kg/day. In contrast BPA did not display any estrogenicity at dosage levels including the highest dose of 200 µg/kg/day (the NOAEL for this study) indicating that BPA is at least 400 fold less potent than EE2 as a developmental reproductive toxicant. The claim that "The science is clear and the findings are not just scary, they are horrific. When you feed a baby out of a clear, hard plastic bottle, it's like giving the baby a birth control pill." (http://rcp.missouri.edu/articles/vomsaal.html) is inaccurate as EE2 caused infertility and many other effects in the offspring and BPA did not.

In this rebuttal we describe

  • That the developmental effects of the potent environment estrogen Ethinyl Estradiol (EE2) addresses important Agency research needs and that EE2 is not just a “positive control”.

  • Why the Long Evans (LE) rat and several other rat strains are excellent animal models for the study of the potential adverse effects of xenoestrogens.

  • Why the argument that LE rats and other rat strains are “insensitive strains” is not consistent with
    • The pharmacological and toxicological data showing that humans and these rat strains display similar sensitivities to EE2
    • The cellular and molecular biology of the action of estrogens on different target tissues

  • How the effects of leaching of Bisphenol A (BPA) from polycarbonate cages have been grossly exaggerated.

  • That governmental regulatory agencies have concluded that studies like ours (Ryan et al. 2010) are useful for risk assessment whereas many of the “positive” low dose studies were “inadequate” or the results have not been replicated.

Footnotes

In response to the letter to Toxicological Sciences by Vom Saal et al. (2010) that provided criticisms of our recent paper, we have prepared a categorical rebuttal.

DISCLAIMER: The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, ORD, U. S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.

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