Background and rationale for extended one generation study
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Background and Rationale for “Extended One-generation” Study. Paul Foster, Ph.D. CIIT Centers for Health Research Email: foster@ciit.org. Overview of Presentation.

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Background and rationale for extended one generation study

Background and Rationale for “Extended One-generation” Study

Paul Foster, Ph.D.

CIIT Centers for Health Research

Email: foster@ciit.org


Overview of presentation
Overview of Presentation Study

  • Provide an outline of the basic test protocols that provide information on Endocrine Active Chemicals (EAC’s), with an emphasis on the number of animals examined.

  • Comment on the strengths and weaknesses of the current multigeneration reproduction study.

  • Provide some information on how improved design may generate enhanced information for risk assessment of EAC’s.


Edsp tiered screening and testing strategy for e strogen a ndrogen and t hyroid activity eat
EDSP Tiered Screening and Testing Strategy for StudyEstrogen, Androgen and Thyroid Activity (EAT)

  • Tier I Screening - Detect Interaction with the Endocrine System

    • Short term in vitro screens, e.g. receptor binding, transactivation

    • Short term in vivo pharmacological screens e.g Hershberger, uterotrophic assays

    • If positive go to Tier II

  • Tier II Testing - Determine and Characterize Endocrine Disrupting Effects

    • Multigeneration reproduction study

    • Data used for Risk Assessment


Purpose of tier 2 test
Purpose of Tier 2 test Study

  • Provide “definitive” information on hazard characterization of EACs (EPA).

    • Confirm or refute observations noted in Tier 1 screens/ assays.

    • Utilize in utero (most sensitive) exposure.

    • Identify activity vs. end points for which concern has been raised in humans (eg hypospadias, cryptorchidism,sperm); i.d. other endocrine-like activity.

    • Provide dose-response information to be used in risk assessment.


Why is exposure during perinatal development important
Why is exposure during perinatal development important? Study

  • Likely to produce permanent effects

    • Effects initiated in utero may not be manifest until adulthood.

  • Timing of exposure may be as important as dose level

    • Embryo/fetal sensitivity versus dam

  • Hormonal interplay for normal development can provide unique, sensitive targets for toxicity


Critical window of susceptibility male or female

Development is Study

TOTALLY

Hormone-dependent

Development

is largely hormone-

independent

Critical Window of Susceptibility:Male or Female?

Sexually indifferent fetus

Pregnancy Week

6

Testis formation

hormones

7-8

Window of

hormone

susceptibility

~15


Prenatal developmental toxicology study
Prenatal Developmental Toxicology Study Study

  • Fetuses examined grossly prior to birth.

  • No pathology conducted

  • Expected to detect agents producing major effects on skeletal and visceral structures.

  • Examination of all offspring increases the probability of detecting low incidence phenomena.


Background and rationale for extended one generation study

Prenatal Developmental Toxicity Study Study

Sexual

Maturation

Gamete Production

& Release

Growth &

Development

Lactation &

Postnatal

Development

Fertilization

Assessment

Zygote

Transport

Parturition

Sex Differentiation

Fetal

Development

Dose

Implantation

Embryogenesis


Background and rationale for extended one generation study

EPA Prenatal Developmental Toxicity Study Study

Sexual

Maturation

Gamete Production

& Release

Growth &

Development

Lactation &

Postnatal

Development

Fertilization

Assessment

Zygote

Transport

Parturition

Extended Dose Period

Fetal

Development

Dose

Implantation

Embryogenesis


General prenatal rat toxicology protocol
General Prenatal Rat Toxicology Protocol Study

GESTATION (days)

Sexual Differentiation

Critical

Gap

Dose Dam

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Necropsy dams, examine half of the

Fetuses for visceral, other half skeletal

Alterations (~3 pups/sex/litter/method)

From 20 litters; n= ~ 60/sex/ dose

Breed


Multigeneration reproduction study
Multigeneration Reproduction Study Study

  • Provides substantial information on the effects of agents on reproduction.

    • Fertility, fecundity, pregnancy, gametes etc

  • More limited information on (postnatal) development

    • Pup survival, growth, developmental landmarks etc

  • Currently considered by EPA the “definitive” Tier 2 test for EAC’s.


Background and rationale for extended one generation study

Multigeneration Reproduction Study Study

Sexual

Maturation

Gamete Production

& Release

Growth &

Development

Lactation &

Postnatal

Development

Fertilization

Assessment

Zygote

Transport

Parturition

Dose

Fetal

Development

Implantation

Embryogenesis


General multigenerational protocol
General Multigenerational Protocol Study

F0

Dose Parents

Gestation

Assign to treatment

20/sex/dose

0

1

2

3

Sexual

Differentiation

Breed F0

F1

Dose F1

Dose Dams

0

3

20 wks

Necropsy F1:

n= 20/sex/dose, 20 litters

Breed F1: 20/sex/dose

Wean: Gross

Necropsy 3/sex/litter

Birth

Cull

Path= 10/sex/dose


How well does the multigeneration study characterize eac s
How well does the multigeneration study characterize EAC’s?

  • It covers the critical developmental windows for sexual differentiation.

    • Should detect potent estrogens and antiandrogens

  • Only 1/sex/litter of F1examined at adulthood

    • Limited power to detect reproductive tract malformations (prenatal study examines ALL offspring) ie may produce false negatives.

    • If an end point in the control population is variable, statistical vs. biological significance can be problematic.


How well does the multigeneration study characterize eac s1
How well does the multigeneration study characterize EAC’s?

  • A number of developmental end points are not included, or are only triggered in the F2 generation, which stops at weaning.

    • Nipples, AGD

  • Gross necropsy at weaning is helpful, but not sufficient.

    • Unlikely to detect subtle malformations (eg epispadias)

    • Cannot detect effects on organ systems not yet developed (eg sperm production, prostate)


Alternate assay system
Alternate Assay System EAC’s?

  • EPA is considering an alternate assay systems for potential use.

  • Transgenerational study design.

    • Fewer litters, more offspring examined.

    • More end points related to EAC’s always measured (eg nipple retention, AGD, pathology).


Background and rationale for extended one generation study

Transgenerational Study EAC’s?

Sexual

Maturation

Gamete Production

& Release

Growth &

Development

Lactation &

Postnatal

Development

Fertilization

Assessment

Zygote

Transport

Parturition

Sex Differentiation

Fetal

Development

Dose

Implantation

Embryogenesis


General transgenerational protocol
General Transgenerational Protocol EAC’s?

Sexual Differentiation

Dose Dams

Breed

GD 0

GD 6

GD 14

Birth

GD 22

Dose pups (optional)

Dose Dams

Postnatal Life (weeks)

0

2

3

4

7

20

Necropsy all offspring

(n= ~ 50/sex/dose/ 8 litters)

PPS

AGD

Wean

VO

Areolae


The multigeneration study
The Multigeneration Study EAC’s?

  • The multigeneration reproduction study was originally designed to provide significant information on reproductive toxicity and, to a more limited extent, postnatal development.

  • This study is now being utilized for hazard characterization of Endocrine Active Chemicals, where postnatal development is a key indicator of adverse response.

  • How could the design of the study be improved to meet this goal?


Key questions to be addressed 1
Key Questions to be addressed -1 EAC’s?

  • Should the same degree of scientific rigor applied in prenatal developmental studies be used in postnatal evaluations?

    • Increase the number of F1 animals examined at adulthood?

      • Increase the ability to detect hazard and provide improved dose-response information?

      • Improve end points routinely examined?

      • Do the cost, effort, logistics (# of animal rooms etc) justify the potential gains?


Key questions to be addressed 2
Key Questions to be addressed - 2 EAC’s?

  • Should consideration be given to the design of a transgenerational test protocol to specifically meet the needs of hazard characterization of EAC’s ?

    • Potential use as a substitute for the costly, labor intensive, multigeneration study?

    • Potential use as an intermediate tier - acting as a bridge to older, multigen studies?

    • Potential to trigger a “definitive” study containing increased animals/ end points ?


Objectives for proposed one generation extension study
Objectives for Proposed EAC’s?“One-Generation Extension” Study

  • Determine whether some of the effects from perinatal exposure to well characterized antiandrogens (di-n-butyl phthalate, vinclozolin) that can be readily detected after puberty are missed in weanling animals of the F1 generation.

  • Determine whether some of these effects occur at an incidence that would go undetected if only 1 male per litter is retained past puberty and examined at adulthood.


Objectives for proposed one generation extension study1
Objectives for Proposed EAC’s?One-Generation Extension Study

  • Select dose levels of characterized agents that will produce clear effects and approximate a LOAEL.

  • Use numbers of litters comparable to that used in a standard multigeneration/ prenatal toxicology study.

  • Since the exposure period is limited, use a route of exposure that maximizes control of administered dose on a mg/kg/d basis.