Environmental Estrogens Ethinyl Estradiol and Bisphenol' Are we drowning in a Sea of estrogens or, i

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Environmental Estrogens Ethinyl Estradiol and
Bisphenol. Are we drowning in a Sea of
estrogensor, is this Estro-Phobia?

• L. Earl Gray Jr., PhD Senior Reproductive
  Biologist and Toxicologist Reproductive
  Toxicology Branch US Environmental Protection
  Agency
• This presentation does not necessarily reflect
  USEPA policy, but rather represents the authors
  current view on the state of the science

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Outline

• Our research Program on Endocrine Disrupting
  Chemicals
• Estrogens in aquatic systems and effects on fish
• Bisphenol A the controversy
• The press and advocates say it is dangerous
• Most regulatory agencies say current exposure
  levels are safe
• Compare in vitro and in vivo studies on BPA
  versus the drug Ethinyl Estradiol (EE2) which
  also is an environmental estrogen
• My level of concern about BPA

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Our research on Endocrine Disrupting Chemicals
(EDCs)

• Identify the cellular and molecular mechanisms of
  abnormal reproductive development
• Developed in vitro assays for these studies and
  for screening estrogens and androgens, including
  water samples
• CAFOs (concentrated animal feedlot effluents) and
  other effluents
• Global Water Research Coalition (GWRC) assessed
  the utility of various assays to screen for the
  presence of estrogenic chemicals in water
  samples.
• Screening drinking water samples for
  estrogenicity in South Africa
• USEPA Tier 1 Screening Battery for EDCs

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Why screen for estrogens?There are Valid
Concerns about Estrogens in aquatic systems

• Ethinyl estradiol (EE2) is a common contaminant
  of aquatic systems from human pharmaceutical
  usage
• Effluents also may contain natural estrogens like
  estradiol and estrone and occasionally
  alkylphenols
• Exposures to estrogens as estrogen-equivalents
  (EEQs) of 0.3 ng/L feminize fish. 1 to 5 ng/L can
  cause infertility. (Tyler and Jobling, 2008)
• 5.7 of 139 US streams had gt5 ng/L EEQs (Kolpin
  2002)
• Less data on levels of EEQs in drinking water
• In contrast, no known adverse effects of BPA in
  aquatic systems

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Widespread disruption of male fish by estrogens
in the UK (Tyler and Jobling. 2008. Bioscience)
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Why are some people so concerned about BPA?
Concern has been elevated by statements from
advocates of banning BPA like the following

• "The science is clear and the findings are not
  just scary, they are horrific," (name removed). l
  said. "When you feed a baby out of a clear, hard
  plastic bottle, it's like giving the baby a birth
  control pill.
• However, BPA does not cause infertility at any
  dose level in animal studies

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Several governmental agencies have conducted risk
assessments on the potential effects of BPA on
human health, for example.

• UK Committee on Toxicology 2001
• European Food Standards Agency 2005
• National Toxicology Program and the NTP CERHR BPA
  Expert Panel which advised the NTP
• FDA draft 2006 the current controversy

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UK COT comments on low dose reports of effects of
BPA (2001)

• The Committee considers that it is not
  appropriate, at this time, to base human health
  risk assessment on these reports, for several
  reasons.
• Firstly, it is not known whether the reported
  effects are transient or irreversible.
• Secondly, the functional significance of the
  reported effects is unknown, ...
• Thirdly, the observations of low-dose effects of
  BPA are not robust, in that they cannot be
  independently reproduced in other competent
  laboratories.
• Fourthly, other chemicals that can induce subtle
  effects on the developing reproductive organs
  inevitably cause more gross defects at higher
  doses, whereas BPA shows no such gross
  developmental toxicity.
• The European Food Standards Agency reiterated
  these comments after reviewing the new data in
  2006

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http//cerhr.niehs.nih.gov/chemicals/bisphenol/bis
phenol.html

• NTP CERHR Expert Panel
• Review of
• Bisphenol A

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Literature reviewed
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What a review of these studies of BPA revealed

• A significant percentage of studies failed to
  meet minimum standards for experimental design
  and analysis
• Among Adequate studies, BPA had not caused
• Cancer of the prostate, breast or any other
  tissue.
• It is not mutagenic
• Reproductive malformations
• Infertility
• Or consistently (at low dosage levels)
• Alter prostate weight or the weight of any
  androgen-dependent tissue
• Alter hormone levels
• Lower sperm counts
• Alter puberty in male or female rats or male mice
• Cause Obesity
• NTP-CERHR expert panel report on the reproductive
  and developmental toxicity of bisphenol A. Birth
  Defects Res B Dev Reprod Toxicol. 2008
  Jun83(3)157-395. Chapin RE, Adams J,
  Boekelheide K, Gray LE Jr, Hayward SW, Lees PS,
  McIntyre BS, Portier KM, Schnorr TM, Selevan SG,
  Vandenbergh JG, Woskie SR.

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Human Exposures
EPAs oral reference dose (RfD) is 50 µg/kg/day
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BPA levels in water samples(Chapin et al, 2008.
Birth defects research NTP CERHR BPA Expert
Panel Final report, 2007)
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Is there a Biologically Plausible Mechanism to
explain how BPA might induce adverse effects at
low doses?
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BPA is a million-fold less potent than is ethinyl
estradiol (EE2) in inducing ER-dependent gene
expression in vitro.
BPA ER gene expression
EE2 ER gene expression
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Effects of Ethinyl Estradiol (EE2) and BPA given
orally or sc on uterine weight in immature female
rats (data from published OECD interlab studies).
Orally, BPA displays partial agonist activity at
a dose a million fold higher than the ED50 of EE2.
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Administration of BPA in utero, during lactation
and after weaning does not accelerate the age of
puberty in female rats over a broad dose range
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Standard in vitro and in vivo assays used to
detect estrogenic activity reveal the BPA is a
very weak estrogen.
Recently, it has been proposed that BPA act at
low concentrations with GPR30 and membrane ERs,
however, the role of these receptors in
reproduction, if any, is unknown.
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What our study reveals about the reproductive
effects of EE2 and BPA in male and female rats
after exposure in utero and during lactation via
the dam.
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BPA had no effect
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EE2 reduces maternal weight gain and litter sizes
at birth, BPA does not
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EE2 permanently reduces F1 male body, prostate,
seminal vesicle and testis weights and sperm
counts, BPA does not
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EE2 accelerates the age at puberty, BPA does not
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EE2 exposure during fetal and neonatal life
reduced fecundity in F1 female rat offspring.
BPA did not
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The robust, comprehensive multigenerational
studies in rats are mice are consistent. BPA
dose not produce adverse effects at low dosage
levels.
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Conclusions about low dose effects - EE2 versus
BPA

• Biological plausibility of the mechanism of
  action at low dose levels
• Ethinyl estradiol is a very potent synthetic
  estrogen
• Doses as low as 10 µg/day are effective in girls
  Doses as low as 0.2-1 µg/day are effective in
  rats
• BPA is far too weak an environmental estrogen for
  binding to the ERs alpha and beta that act a
  nuclear transcription factors to account for any
  low dose effects.
• Some other proposed mechanisms of action have no
  known role in reproductive development or
  reproductive function (GRP30 and membrane ERs).

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Conclusions about low dose effects - EE2 versus
BPA Toxicity

• When EE2 is administered at doses equivalent to
  pharmacologically effective levels in humans
  administered to the mother rat in utero and
  during lactation, EE2 causes
• Maternal and neonatal effects
• Reduced maternal weight gain
• Reduced offspring viability
• Female rat offspring display
• Accelerated age at puberty
• Reproductive tract malformations
• Infertility and abnormal estrous cyclicity
• Alterations (defeminization) of some sexual
  dimorphic behaviors
• Male rat offspring display
• Reduced sperm counts
• Histopathological alterations of prostate and
  seminal vesicles
• Permanent reductions in reproductive organ
  weights
• Bisphenol A does not produce adverse effects in
  multigenerational studies in rats or mice at low
  dosage levels

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Conclusions about low dose effects - EE2 versus
BPAExposure

• Wildlife
• EE2 exposures in aquatic systems are a problem in
  aquatic ecosystems at 1 ng/L and above
• BPA exposures in such systems has not been
  associated with any adverse effects
• Humans
• EE2 exposures from drinking water and etc are not
  yet well characterized
• Total BPA exposures from all sources are in the
  50-80 ng/kg/d (median from biomonitoring data)
  with a max of about 3.5 micrograms/kg/d. (NTP
  monograph)
• Total BPA in drinking water was below the MDL in
  2/3 samples and 2-5 ng/L in a third (0.2-0.5
  ng/kg/d for a 10 kg child drinking 1 L) (NTP
  monograph)
• Human BPA exposures are well below the EPAs RfD
  of 50 micrograms/kg/d

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Our USEPA Research Team

• Multigenerational studies
• Dr LE Gray Jr
• Johnathan Furr
• Fetal endocrine and genomic studies
• Dr Kembra Howdeshell
• In vitro mechanistic studies and assay
  development
• Dr Vickie Wilson
• Dr Dieldrich Bermudez
• Ms Christy Lambright
• Dr Phillip Hartig
• Ms Mary Cardon

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The End
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BPA and estrogen in 139 streams (Kolpin et al
2002)
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• A FACA committee An Official advisory committee
  to a governmental agency
• FACA rules require
• No conflict of interest
• Public comment
• Open meetings
• Objectivity of the participants
• The Final report represents the opinion of ALL of
  the participants, consensus or not.
• Prevent special interest groups from unduly
  influencing the government

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World Health Organization EDC Definition
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Estrogenic potential of BPA in in vitro and
short-term in vivo assays
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• Levels of Concern

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Experimental design and statistical issues
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Routes of Exposure
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BPA does not cause Obesity in rodents
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Obesity page 2
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• BPA does not consistently affect T or LH levels

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Effects are small, transient and not necessarily
adverse
One dose level, Small sample size, No dose
response, Poor design
U shaped dose response for BPA not demonstrated
Result contrary To effects in recognized Guideline
studies
Lack of reproducibility Of low dose effects
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Bisphenol A