Endocrine Disruption and Risk Assessment

1
Endocrine Disruption and Risk Assessment

• Tox 513
• Presented by Kara E Warner
• June 1, 2004

2
Endocrine Disruptor - Defined

• An exogenous agent which interferes with the
  synthesis, secretion, transport, binding action,
  or elimination of natural hormones in the body
  which are responsible for the maintenance of
  homeostasis, reproduction, development, or
  behavior (Kavlock et al. 1996).
• By this definition, nearly any exogenous stressor
  could be considered an endocrine disruptor.
• An exogenous chemical substance or mixture that
  alters the structure or function(s) of the
  endocrine system and causes adverse effects at
  the level of the organism, its progeny,
  populations, or subpopulations of organisms,
  based on scientific principles, data,
  weight-of-evidence, and the precautionary
  principle (EDSTAC 1998).

3
Major Endocrine Organs in Humans

• Endocrine Disruptor Research, Risk Assessment,
  and Regulatory Strategy is primarily concerned
  with
• Gonads
• Thyroid
• Pituitary
• Hypothalamus

4
Hypothalamus
PRH PRIH
MRH MRIH
TRH
SS
GnRH
GHRH
CRH
Anterior Pituitary
MSH
PRL
TSH
FSH
LH
ACTH
GH
Melanin producing cells
Mammary Gland
Gonads
Somatic cells, muscle, bone, liver
Thyroid Gland
Adrenal gland
5
Thyroid Function
Hypothalamus ? TRH (Thyrotropin releasing
hormone Pituitary ? TSH (Thyrotropin) Thyroid
Gland ? Thyroxine (T4) Triiodothyronine (T3)
active forms that bind thyroid receptor on
almost all cell types

• Major functions
• Regulation of metabolism
• Regulation of body temperature in homeotherms
• Regulation of growth and differentiation
• Post-hatch metamorphosis, smoltification, in fish
• Amphibian metamophosis
• Molting in amphibians, reptiles, and birds, etc.

6
Adrenal Function

• Glucocorticoid Cortisol
• Promote conversion of proteins to carbohydrates
• Fight or flight responses, stress responses
• Immune suppression
• Mineralocorticoid Aldosterone
• Sodium reabsorption potassium secretion into
  urine
• Regulation of blood osmotic conditions in
  conjunction with the renin-angiotensin system
  (blood pressure)
• Osmotic regulation
• Gonadocorticoid Testosterone
• May be responsible for female libido and source
  of estrogen post-menopause

7
Hypothalamus
()
CRH
(-)
Anterior Pituitary
(-)
(-)
()
ACTH
Zona reticularis
Zona glomerulosa
Zona fasiculata
Aldosterone
Reproductive Steroids
Cortisol
Adrenal Gland
8
Reproductive Hormones

• Ovaries
• Progesterone and Estrogen
• Egg production and release
• Embryo implantation and maturation
• Parturition and lactation
• Secondary sex characteristics
• Testes
• Testosterone
• Suppresses female gonadal development
• Sperm production
• Libido and secondary sex characteristics

9
Hypothalamus
()
GNRH(s)
(-)
Anterior Pituitary
()
FSH
LH
()
()
()
(-)
()
Thecal cells
Granulosa cells
()
Progesterone
()
Progesterone
Androgen
Androgen
Estrogen
OVARY
CIRCULATION
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Ovarian Steroidogenesis
LH
FSH
()
()
()
LDL, HDL receptors
LDL, HDL receptors
() LH
() LH / FSH
Cholesterol
Cholesterol
Pregnenalone
Pregnenalone
Progesterone
Progesterone
Androgen
Androgen
Aromatase (CYP19) ()FSH
Estrogen
Thecal cells (outer follcle)
Granulosa cells (inner follicle)
11
Hypothalamus
()
GNRH
(-)
Anterior Pituitary
(-)
FSH
LH
()
(-)
()
(-)
Leydig cells
Sertoli cells
Inhibin
Androgen
Estrogen (FSH)
TESTES
CIRCULATION
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Hormone Action
H R Hormone (H) Receptor (R)
Initiation of a biological response
Via plasma membrane receptors
Via nuclear receptors

• Receptor Characteristics
• Saturability
• High affinity
• Stereo- and pharmacospecificity
• Tissue specificity
• Correlation with biological response.

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Signal Transduction(Ex G-protein activating
receptors)
Signal amplification
Ligand
Receptor binding activates multiple G proteins.
Membrane receptor
G
G
AC
AC
G proteins activate multiple adenylate cyclase
molecules.
G
AC
Each adenylate cyclase molecule generates
multiple cAMP molecules. Each cAMP activates a
protein kinase A molecule. Each PKA molecule
phosphorylates multiple copies of enzyme X which
produces many molecules of product.
cAMP
Phosphorylated enzyme product
PKA
Phosphorylated enzyme
14
Nuclear Receptors
H hormone R receptor HSP heat shock
protein SRE steroid response element
DNA
SRE
Promoter
Nucleus
mRNA
Plasma binding protein
Protein
Phenotypic Response, Altered Cell Function
15
EDCs Historical Perspective

• EDCs are not new
• H. Burlington and V. Lindeman. 1950. Effect of
  DDT on testes and secondary sex characters of
  white leghorn cockerels. Proc. Soc. Exper. Biol.
  Med. 7448-51.
• Many of the chemicals now identified as EDCs have
  been in use since the WWII era or earlier.
• Substantial, but scattered evidence for potential
  endocrine disrupting effects of synthetic
  chemicals has been accumulating since the 1960s.

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EDCs Historical Perspective

• July 26-28 1991, Multidisciplinary group met at
  the Wingspread Conference Center in Racine WI, to
  assess what was known about chemically-induced
  alterations in sexual development in wildlife and
  humans.
• Issued a consensus statement that gave rise to
  the endocrine disruption paradigm.
• 1996 Our Stolen Future by Theo Colborn, Dianne
  Dumanoski, and John Peterson Myers was published.
• Focus for public attention and debate regarding
  the endocrine disruption issue.

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OSF Scientific Detective Story

• Great Lakes Herring Gulls
• Two females occupying nests
• DDT reported to disrupt sexual development of
  males
• Dr. F. vom Saal, U. Missouri.
• Studies on effects of womb position on mouse
  behavior
• Postulated that small changes in steroid exposure
  in utero manifests as significant behavioral
  differences in adult animals
• Short term, low level exposure during critical
  stage of development may have long-lasting
  implications.

http//www.ourstolenfuture.org/NewScience/reproduc
tion/wombmate.htm
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OSF Scientific Detective Story

• Thalidomide (1962)
• Mild sedative, marketed 1957-early 60s,
  treatment for morning sickness
• Severe disruption of limb development
  (phocomelia)
• Exposure in week 5-8 of pregnancy severe
  developmental disruption
• Later exposure no significant effect
• Not just a matter of dose, but more crucially
  dependent on the timing of exposure.

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OSF Scientific Detective Story

• Diethylstilbesterol
• Non-steroidal synthetic estrogen, widely
  prescribed in post-WWII years, miscarriage
  prevention.
• In utero exposure linked to development of rare
  clear cell vaginal cancer in female offspring
• Also linked to severe reproductive tract
  deformities in women
• (Inconclusively) linked to abnormal sperm,
  undescended testes, and immune system
  dysfunctions in men exposed in womb.
• Timing of exposure critical (exposure before week
  10)
• Some effects did not manifest until many years
  after birth
• Rodent studies were predictive of effects
  ultimately observed in humans

20
Sources of EDCs

• There are chemicals in the environment that can
  act as hormones or disrupt hormone function.
• Phytoestrogens
• Known and used by humans since ancient times for
  contraception etc.
• Sterilization of herbivores
• Relatively weak and present during human
  evolution
• Examples indole-3-carbinol (cruciferous
  cabbage family vegetables), genistein (in soy
  products)
• Xenobiotics
• Much more recent, no time for evolutionary
  adaptation
• Examples
• DDT identified as a weak estrogen (pesticide)
• DDE anti-androgen (pesticide breakdown product)
• Vinclozolin anti-androgen (anti-fungal agent)
• Nonylphenol xenoestrogen (PVC, polyethylene,
  breakdown project of many detergents, shampoos,
  etc. surfactants)
• Bisphenol A xenoestrogen (polycarbonate
  plastics, food beverage containers)

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Dioxin

• Low doses (near environmental) during critical
  periods of prenatal development have long-term
  adverse effect on male reproductive development.
• Up to 56 reduction in sperm counts
• Reproductive tract abnormalities
• Behavioral changes
• EPA repeated studies and found effects on females
  as well
• In adults, takes a large dose to impair
  fertility.
• Pre-natal stages 100 x more sensitive.
• Mechanism not well understood.
• Immune system effects occur.

Source OSF
22
(No Transcript)
23

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Wildlife at risk from EDCs ?

• St. Lawrence Beluga population
• Hermaphrodite washed up
• Lower reproductive success than northern
  populations.
• Florida Panthers
• Sterile individuals, sperm abnormalities, low
  sperm counts, immune impairment, thyroid
  problems.
• 13/17 males had undescended testicles
• Lake Apopka alligators
• Penis sizes ½ to 1/3 normal
• Associated with chemical spill at site (dicofol)
• Red eared slider turtles
• Prevalence of intersex very few males
• Deformed raptors in rogue valley OR
• Female mink eating fish w/ 0.3-5 ppm PCBs failed
  to reproduce
• Endocrine system highly conserved among species

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Wildlife at risk from EDCs ?

• In Our Backyard
• Willamette River, OR
• Increased prevalence of skeletal deformities in
  some species of fish in certain areas of the
  river
• Deformities may be related to infection by
  parasitic organism spread by snails
• Deformities may be related to unidentified
  compounds in river water
• Immune compromised fish?
• Bone development effects?

26
Wildlife at risk from EDCs ?

• In Our Backyard
• Frog Populations
• Increased prevalence of skeletal deformities in
  some species of frogs
• Deformities may be related to UV exposure
• Deformities may be related to infection by
  parasitic organism spread by snails
• Deformities may be related to compounds in pond
  water
• Role of EDCs?

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Wildlife at risk from EDCs ?

• In Our Backyard
• Frog Populations
• Some evidence exists for decreased larynx size,
  decreased testosterone levels in male frogs
• Increased prevalence of intersex (hermaphroditic)
  frogs in wild and lab populations
• Condition may be related to compounds in pond
  water
• Atrazine!

Hayes, et al. 2002
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Wildlife at risk from EDCs ?

• Atrazine!
• progesterone
• 17-hydroxyprogesterone
• androstenedione
• (aromatase)
• testosterone estradiol
• (5 ?-reductase)
• dihydrotestosterone

Hayes, et al. 2002
29
Several general mechanisms for EDCs

• Mimicking effects of natural hormones by binding
  their receptor sites
• Antagonizing effects of hormones by blocking
  their receptors
• Direct or indirect reaction with the hormone in
  question
• Alteration of natural patterns of hormone
  synthesis
• Altering hormone receptor levels
• Others?

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Consideration of Low Doses

• Does a threshold dose exist for receptor-mediated
  toxicity?
• Rationale is that background levels of endogenous
  hormones are already biologically active,
  therefore any additional exposure exceeds the
  threshold.
• Are the dose-response curves monotonic, or
  non-monotonic?

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Traditional Dose-Response
32
Nonmonotonic Dose-Response
vom Saal, F, BG Timms, MM Montano, P Palanza, KA
Thayer, SC Nagel, MD Dhar, VK Ganjam, S
Parmigiani and WV Welshons. 1997. Prostate
enlargement in mice due to fetal exposure to low
doses of estradiol or diethylstilbestrol and
opposite effects at high doses. Proceedings of
the National Academy of Sciences USA 942056-61.

33
Welshons, WV, KA Thayer, BM Judy, JA Taylor, EM
Curran and FS vom Saal. 2003. Large effects from
small exposures. I. Mechanisms for endocrine
disrupting chemicals with estrogenic activity.
Environmental Health Perspectives
doi10.1289/ehp.5494
34
Endocrine Disruptors Legislation

• On November 29, 1995, the Senate unanimously
  passed S. 1316, the Safe Drinking Water Act
  (SDWA) Amendments of 1995
• Estrogenic Substances Screening Program.
  Subsection 20(1) further amends the Act's
  research provisions by adding a new section (j)
  requiring EPA to develop and implement a
  screening program to determine whether certain
  substances may have an effect in humans that is
  similar to an effect produced by a naturally
  occurring estrogen or other endocrine effect. In
  implementing this screening program, EPA must
  test all active and inert ingredients used in
  pesticides and may test any other substance to
  which a widespread population may be exposed. For
  any substance found to have a potential adverse
  health effect, EPA is required to take action,
  including regulatory action, as is necessary to
  protect human health. Within 4 years of
  enactment, EPA must report to Congress the
  findings of the screening program and
  recommendations for further testing and research
  or other actions.

35
Endocrine Disruptors Legislation

• Food Quality Protection Act (FQPA) 1996
• Bill number P.L. 104-170.
• The Food Quality Protection Act (FQPA) of 1996
  was unanimously passed by Congress and signed
  into law by President Clinton. The FQPA
  dramatically changed how the US Environmental
  Protection Agency regulates pesticides through
  the Federal Food, Drug, and Cosmetic Act (FFDCA)
  and  the Federal Insecticide, Fungicide, and
  Rodenticide Act (FIFRA).
• Health-Based Safety Standard for Pesticide
  Residues in Food The new law establishes a
  strong, health-based safety standard for
  pesticide residues in all foods. It uses "a
  reasonable certainty of no harm" as the general
  safety standard.

36
Endocrine Disruptors Legislation

• Food Quality Protection Act (FQPA) 1996
• Special Provisions for Infants and Children
• Requires an explicit determination that
  tolerances are safe for children.
• Includes an additional safety factor of up to
  ten-fold, if necessary, to account for
  uncertainty in data relative to children.
• Requires consideration of children's special
  sensitivity and exposure to pesticide chemicals.
• Endocrine Disruptors Incorporates provisions for
  endocrine testing, and also provides new
  authority to require that chemical manufacturers
  provide data on their products, including data on
  potential endocrine effects.
• Pesticide Registration Renewal Requires EPA to
  periodically review pesticide registrations, with
  a goal of establishing a 15-year cycle, to ensure
  that all pesticides meet updated safety
  standards.

37
EDSTAC

• Under the Federal Advisory Committee Act of 1972
  the EPA formed the Endocrine Disruptor Screening
  and Testing Advisory Committee (EDSTAC) in 1996.

• Develop a flexible process to select and
  prioritize chemicals and pesticides for
  screening, recognizing the need to obtain and
  utilize appropriate exposure information in
  setting priorities
• 2. Develop a process for identifying new and
  existing screening tests and mechanisms for their
  validation
• 3. Agree on a set of available, validated
  screening tests for early application and
• 4. Develop a process and criteria for deciding
  when additional tests, beyond screening tests,
  are needed and how any of these additional tests
  will be validated.

38
EDSTAC Conceptual Framework

• General Considerations
• Want minimal number of screens and tests
  necessary to make sound decisions.
• Provide data that can be used for a wide variety
  of uses.
• Make best use of information generated in other
  screening and testing programs (particularly data
  on physiological changes in low dose groups)
• Adaptive to new science
• Specific Considerations
• Well defined endpoints
• Use minimal number of animals needed for
  scientifically valid results.
• Results should support further research
• Weight of evidence approach
• Reporting in format that facilitates database
  development and ease of information sharing.
• Clearly defined statistical and biological
  decision criteria.

39
EDSTAC Conceptual Framework

• SCOPE
• Relevant for human health and ecological effects
• Screening and testing will initially emphasize
  identification and characterization of effects
  that enhance, mimic, or inhibit estrogen,
  androgen, and thryroid-related processes.
• More screens to be developed as the science
  evolves.
• Should be capable of evaluating individual
  chemicals and mixtures.
• EPA has already developed screening and testing
  protocols for carcinogenicity, developmental and
  reproductive toxicity, and neurotoxicity.
• Priority setting sorting and prioritization of
  chemical substances and mixtures for evaluation
  in screening and testing batteries.
• Tier 1 Screening to detect chemical substances
  and mixtures capable of acting on endocrine
  systems.
• Tier 2 Testing to determine, characterize, and
  quantify the nature of the endocrine disrupting
  properties by prior information and/or tier 1
  screening.

40
EDSTAC Priority Setting

• Chemicals for which there is insufficient
  endocrine disruption data will be prioritized for
  Tier 1 screening (approx. 62,000 chemicals).
• Chemicals for which there is sufficient,
  functionally equivalent, data available may
  proceed directly to either Tier 2 (approx.
  500-600 compounds) or hazard assessment (approx.
  50-100 compounds).

41
EDSTAC Priority Setting

• Information that could be used for initial
  sorting
• Exposure information
• Biological sampling data
• Environmental, occupational, consumer product,
  and food-related data
• Environmental releases
• Production volume
• Fate and transport data and models
• Effects information
• Toxicological laboratory studies
• Epidemiological and field studies
• SARs, QSARs, effects models
• Results of high throughput screening
• Limited set of mixtures spanning diverse physical
  and chemical properties
• Selected by expert judgement
• Use to validate screening methods for use with
  mixtures.
• Recommended inclusion of naturally occurring
  non-steroidal estrogens (ex. Phytoestrogens,
  mycotoxins, etc.).

42
EDSTAC Tier 1 Screening

• Purpose obtain a minimum, yet sufficient, set
  of valid, reliable data to detect whether a
  chemical substance or mixture may interact with
  the endocrine system.
• Characteristics
• Inexpensive, quick and easy to perform
• Validated and standardized ASAP
• Sensitivity given priority over specificity (err
  on the side of false positive Type I errors
  rather than false negative Type II errors)
• Capture multiple endpoints reflecting multiple
  modes of endocrine action.
• Broadly predictive across species, gender, age,
  etc.
• Yield a definite or for determining how or
  whether to proceed to tier 2.

43
EDSTAC High Throughput Pre-screening

• Subset of in vitro tier 1 assays that could be
  conducted with the assistance of automated
  technology
• Primary purpose is to provide preliminary
  biological activity information for a large
  number of chemicals in a relatively short period
  of time
• Transcriptional activation assays
• Recommended for all high production volume
  chemicals (gt 10,000 lbs per year) and all
  pesticides

44
EDSTAC Tier 1 Screening

• Tier 1 screening battery intended to meet 5
  criteria
• Maximize sensitivity to minimize false negatives,
  permitting an acceptable level of false positives
• Include a range of organisms representing known
  or anticipated differences in metabolic activity
• Detect all known modes of action for the
  endpoints of concern
• Include a sufficient range of taxonomic groups
• Incorporate sufficient diversity and
  complementarity among endpoints and assays to
  draw weight of evidence conclusions

45
EDSTAC Tier 1 Screening
Recommended Tier 1 screening battery In vitro ER
binding / transcriptional activation assay AR
binding / transcriptional activation
assay Steroidogenesis assay with minced testis
46
Estrogen or xenoestrogen
1
???
Nuclear Factors
Non-genomic effects
ER
ERE-Luc
DNA Binding
Protein Phosphorylation of ER Ligand-Independent
Activation
Peptide Growth Factor
Luciferase
ER-Responsive Genes
Estrogenic Effects
47
EDSTAC Tier 1 Screening
Recommended Tier 1 screening battery In vitro ER
binding / transcriptional activation assay AR
binding / transcriptional activation
assay Steroidogenesis assay with minced testis In
vivo Rodent 3-day uterotrophic assay
(subcutaneous) Rodent 20-day pubertal female
assay with thyroid Rodent 5-7 day Hershberger
Assay Frog metamorphosis assay Fish gonadal
recrudescence assay
48

• Rodent 3-day uterotrophic assay
• Ovariectomized or immature mice or rats
• Exposure to estrogens increases uterine weight
• Vaginal and uterine histology for additional
  endpoints.
• Anti-estrogens can be assessed with
  co-administration of E2
• Well established assay
• Rodent 20-day pubertal female assay with thyroid
• Oral dosing in weanling rats initiated at 21 days
  of age
• Endpoint days to vaginal opening (VO).
• Secondary endpoints age at first estrus, and
  onset of estrous cyclicity, ovary weight, thyroid
  for histology, and serum T4 and TSH.
• Can detect estrogen agonists antagonists,
  aromatase inhibitors, altered HPG function,
  altered thyroid.
• Very well established assay.
• Hershberger Assay
• Castrated male rat T xenobiotic to detect
  androgen antagonists
• Castrated male rat xenobiotic to detect
  androgen agonists.
• Ventral prostrate and seminal vesicle weights
  measured after 4-7 days treatment by oral gavage.
• Comparison of effects on the two tissues can
  reveal effects on 5-alpha-reductase

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T3/T4
TSH
Acquisition of hypothalamic sensitivity to
thyroid hormones
TRH
Premetamorphosis
Postmetamorphosis
Prometamorphosis
Climax
Fore-limb emergence to complete tail reabsorption
Hind limbs, rapid differentiation
Frog Metamorphosis Assay
50
Fish gonadal recrudescence

• Mature fish under winter (short day length,
  cool temperatures) exhibit regressed secondary
  sex characteristics and gonad maturation.
• During reproductive season, gonadotropin
  secretion increases, gonads and gametes develop,
  secondary sex characteristics are displayed.
  Involves HPG axis and reproductive steroid
  signaling.
• Fathead minnow (Pimephales promelas)
• Exposed to test substance under increasing light
  and temperature conditions

• Primary endpoints
• Development of secondary sex char.
• Ovary and testis weights
• GSI
• Gamete maturation
• Vitellogenin induction

51
EDSTAC Tier 1 Screening
Recommended Tier 1 screening battery In vitro ER
binding / transcriptional activation assay AR
binding / transcriptional activation
assay Steroidogenesis assay with minced testis In
vivo Rodent 3-day uterotrophic assay
(subcutaneous) Rodent 20-day pubertal female
assay with thyroid Rodent 5-7 day Hershberger
Assay Frog metamorphosis assay Fish gonadal
recrudescence assay Potential alternatives Placent
al aromatase assay Modified rodent 3-day
uterotrophic assay (i.p.) Rodent 14-day intact
adult male assay with thyroid Rodent 20-day
thyroid/pubertal male assay
52
EDSTAC Tier 2 Testing

• Selection of tier 2 tests should be based on tier
  1 screening results.
• Should provide information useful for
  human/ecological hazard assessment.
• Should be both sensitive and specific
• Include parental/offspring developmental
  endpoints (two-generation studies) to adequately
  evaluate all life stages.
• Meant to encompass effects on fertility, mating,
  embryonic development, neonatal growth and
  development, and transformation for juvenile
  stage to sexual maturity.
• Include life cycle of both viviparous and
  oviparous organisms.
• Be conducted over a full range of doses
• Doses should be administered by a relevant route
  of exposure.
• Be conducted in accordance with GLP

53
EDSTAC Tier 2 Testing

• Mammalian tests
• Two generation reproductive toxicity
• An alternative, less comprehensive test
• Alternative mammalian reproductive
• One-generation test
• Non-mammalian multigeneration tests
• Avian reproduction
• Fish life cycle
• Mysid life cycle
• Amphibian development and reproduction
• Priority given to conducting tests for those
  species/taxa to which exposure is known or
  expected to occur.
• Endpoint selection may be tailored to provide
  results only for those endpoints of concern
  identified in Tier 1.

54
Two Generation Mammalian Reproductive Toxicity
Study

• Method TSCA 799.9380, OPPTS 870.3800
• (OPPTS office of prevention, pesticides, and
  toxic substances.)
• Designed to comprehensively evaluate the effects
  of a chemical on the gonadal function, estrous
  cycles, mating behavior, fertilization,
  implantation, pregnancy, parturition, lactation,
  weaning, and the offspring ability to achieve
  adulthood and successfully reproduce through two
  generations. One litter per generation.
• Generally oral exposures (food, water, or
  gavage).
• Minimum of three treatment levels plus control
• Minimum of 20 males and enough females to yield
  at least 20 pregnant females, per treatment group.

55
Avian Reproduction
Mallard
Northern Bobwhite

• Based on EPA Avian Reproduction Test Guidelines
  (OPPTS 850.2300).
• Exposure of adults prior to onset of maturation
  and egg laying, continues through egg laying
  period, offspring are exposed in early
  development by material deposited in egg yolk by
  the females.
• Endpoints eggs laid, cracked eggs, eggshell
  thickness, viable embryos, chick survival to 14
  d.
• Additional observations Circulating steroid
  concentrations, thyroid hormones, major organ
  weights, gland weights, bone development, leg and
  wing bone weights, morphometric indices, gonadal
  somatic indices, functional tests, and
  reproductive capability of offspring.

56
Fish Life Cycle Test

• Based on fish life cycle test guidelines (OPPTS
  850.1500)
• Fathead minnow recommended. Alternative species
  may be used if appropriate.
• Exposed from fertilization through development,
  maturation, reproduction, and early development
  of offspring with a test duration of up to 300
  days.
• Fish species have widely varying reproductive
  strategies including oviparity, to ovoviparity,
  to true viviparity.

8 replicates of 200 embryos

• Minimum of 5 concentrations plus controls.

hatch
4 replicates of 25 juveniles
2 replicates of 50 embryos per treatment
4 weeks
2 replicates of 25 adults
hatch
2 replicates of 25 juveniles
Sexual maturity
4 replicates of breeding pairs (1 male/2 females)
4 weeks
Final Endpoint collection
57
Mysid Life Cycle Test

• Invertebrate growth, reproduction, and
  development are under endocrine control.
• Invertebrate endocrine systems and hormones are
  not directly analogous to those of vertebrates.
• Ecdysone is a steroid hormone that regulates
  growth and molting in arthropods. Functional and
  structural similarities to estrogen.
• Morphogenetic and reproductive development of
  arthropods is controlled in part by juvenile
  hormone (JH).
• Chemicals with estrogenic properties are reported
  to have altered normal function of ecdysone
  systems.

Ecdysone
Estradiol
58
Mysid Life Cycle Test

• Based on the mysid shrimp chronic life cycle test
  (OPPTS 850.1350)
• Will be adapted to include reproductive and
  developmental endpoints particularly relevant to
  endocrine disruption.
• Considered unlikely to be relevant for
  vertebrates.
• Uncertain how representative the test will be for
  all invertebrates.
• Tentatively favored over daphnia because mysids
  are sexually dimorphic, whereas daphnia undergo
  parthenogenetic reproduction for most of its life
  cycle. (reproduction by development of
  unfertilized female gamete)

59
Amphibian Development and Reproduction

• Proposes a test that exposes larvae through
  metamorphosis and reproduction
• Represents an oviparous poikilotherm other than
  fish
• Metamorphosis, growth, and reproduction is
  relatively well understood for frogs,
  particularly Xenopus
• There was no standardized method that could be
  rapidly adapted for endocrine screening, but many
  promising methods in the research literature
  exist
• High priority for development and standardization

60
Validation and Standardization

• Purpose of VS is to provide sufficient data to
  allow informed decisions about the relative
  merits of the screening and testing battery
  component assays and alternatives considering
• Sensitivity
• Specificity
• Technical complexity
• Inter- and intra-laboratory variability
• Time
• Cost
• Validation the scientific process by which the
  reliability and relevance of an assay method are
  evaluated for the puropose of supporting a
  specific use (ICCVAM, 1996) The Interagency
  Coordinating Committee on the Validation of
  Alternative Methods
• Relevance refers to the ability of the assay to
  measure the biological effect of interest.
• Reliability is an objective measure of inter- and
  intra-laboratory reproducibility.
• Standardization definition of conditions under
  which the assay is run. Can be considered a
  critical part of the validation process.
• Food Quality Protection Act specifically calls
  for validation of the screening and testing
  program.

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Money

• It is estimated that standardization and
  validation of the proposed assays will cost 50
  million.
• EPA Fiscal year budgets for EDC work 1999 3.2
  million 2000 7.7 million
• Industry has expressed little willingness to
  contribute, although they are funding endocrine
  disruptor research of their own (Syngenta!).
• Estimated cost of tier 1 screening for a single
  chemical 200,000- 400,000
• Estimated cost of tier 2 screening for a single
  chemical up to 1-2.5 million.

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A partial list of suspected EDCs

• Persistent organohalogens
• Benzenehexachloride (T)
• 1,2-dibromoethane (R)
• Chlorform (R)
• Dioxins and furans (E)
• Octachlorostyrene (T)
• Polybrominated biphenyls (PBBs E,T)
• PCBs (E,A,T,R)
• PBDEs (T)
• Pentachlrophenol (T)

• Other
• Butylated hydroxyanisole (BHA E)
• Phthalates (E, A)
• Benzophenone (E)
• Bisphenol A (E)
• Bisphenol F (E)
• Benzo(a)pyrene (A)
• Carbendazim (R)
• Ethane Dimethane Sulphonate (R)
• PFOS (Perfluoroctane sulfonate) (T, R)
• Alkyphenols (E)
• Resorcinol (T)
• Styrene dimers, trimers (E)
• Cd (E)
• Pb (R)
• Hg (R, T)

Hormone System(s) affected Tthyroid
Eestrogen Aandrogen Rreproductive
63
A partial list of suspected EDCs

• Pesticides
• Acetochlor (T)
• Aldrin (E)
• Amitrol (T)
• Carbaryl (E)
• Chlordane (A)
• p,p-DDE (A)
• Dieldrin (E)
• Ethylene thiourea (T)
• Fenbuconazole (T)
• Fipronil (T)
• Heptaclor epoxide (T, R)
• Karate (T)
• Ketoconazole (R)
• Linuron (A)
• Mancozeb (T)
• Methomyl (T)
• Metribuzen (T)
• Nitrofen (T)

• Alachlor (T)
• Allethrin, d-trans (E)
• Atrazine (HPA)
• Chlofentezine (T)
• DDT (E)
• Dicofol (E)
• Endosulfan (E)
• Fenarimol (E)
• Fenitrothion (A)
• Fenvalarate (E)
• Heptachlor (T)
• Iprodione (A)
• Kepone (Chlrodecone, E)
• Lindane (A, E)
• Malathion (T)
• Maneb (T)
• Methoxychlor (E)
• Mirex (A)
• Nonachlor, trans (E)

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Web resources

• EPA http//www.epa.gov/endocrine/
• OECD http//www.oecd.org/EN/document/0,,EN-docum
  ent-524-14-no-24-6685-0,00.html
• NICNAS http//www.nicnas.gov.au/foreign/endocrin
  e.htm
• Our Stolen Future www.ourstolenfuture.org
• ICCVAM http//iccvam.niehs.nih.gov/methods/endoc
  rine.htm http//iccvam.niehs.nih.gov/
• EDSTAC final report http//www.epa.gov/scipoly/o
  scpendo/history/finalrpt.htm

65
?
warnerk_at_onid.orst.edu