# Presentation: 031150

## Background and Rationale for “Extended One-generation” Study

- Paul Foster, Ph.D.
- CIIT Centers for Health Research
- Email: foster@ciit.org

## Overview of Presentation

- 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 Estrogen, 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

- 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?

- 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?

*Sexually indifferent fetus*

*Testis formation*

- Window of
- hormone
- susceptibility

- Critical Window of Susceptibility:Male or Female?

*Pregnancy Week*

- 6

- 7-8

- ~15

*hormones*

## Prenatal Developmental Toxicology 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.

## Prenatal Developmental Toxicity Study

**Maturation**

**Gamete Production**
**& Release**

**Fertilization**

**Zygote **
**Transport**

**Implantation**

**Embryogenesis**

**Fetal**
**Development**

**Parturition**

**Lactation &**
**Postnatal**
**Development**

**Growth &**
**Development**

**Dose**

**Assessment**

**Sex Differentiation**

## EPA Prenatal Developmental Toxicity Study

**Maturation**

**Gamete Production**
**& Release**

**Fertilization**

**Zygote **
**Transport**

**Implantation**

**Embryogenesis**

**Fetal**
**Development**

**Parturition**

**Lactation &**
**Postnatal**
**Development**

**Growth &**
**Development**

**Dose**

**Assessment**

- Extended Dose Period

## General Prenatal Rat Toxicology Protocol

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

***GESTATION (days)***

- Dose Dam

**Sexual Differentiation**

- Critical
- Gap

- Breed

**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 **

## Multigeneration Reproduction 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.

## Multigeneration Reproduction Study

**Maturation**

**Gamete Production**
**& Release**

**Fertilization**

**Zygote **
**Transport**

**Implantation**

**Embryogenesis**

**Fetal**
**Development**

**Parturition**

**Lactation &**
**Postnatal**
**Development**

**Growth &**
**Development**

**Dose**

**Assessment**

## General Multigenerational Protocol

**F****0**

- Dose Parents

- Gestation

- 0

- 1

- 2

- 3

**Sexual **
**Differentiation**

- Assign to treatment
- 20/sex/dose

- Breed F0

- Dose Dams

- 0

- 3

- 20 wks

**Wean****: Gross**
**Necropsy 3/sex/litter**

**Birth**

**Breed F****1****: 20/sex/dose**

**     ****Necropsy F****1****:**
**n= 20/sex/dose, 20 litters**

**F****1**

- Dose F1

**Cull**

**Path= 10/sex/dose**

## 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’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

- 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).

## Transgenerational Study

**Maturation**

**Gamete Production**
**& Release**

**Fertilization**

**Zygote **
**Transport**

**Implantation**

**Embryogenesis**

**Fetal**
**Development**

**Parturition**

**Lactation &**
**Postnatal**
**Development**

**Growth &**
**Development**

**Dose**

**Assessment**

**Sex Differentiation**

## General Transgenerational Protocol

- Dose Dams

- GD 6

- Birth
- GD 22

- Breed
- GD 0

- Postnatal Life (weeks)

- 0

- 20

- Dose Dams

- Dose pups (optional)

- 2

- 3

- 4

- 7

**AGD**

**Areolae**

**Wean**

**VO**

**PPS**

- Necropsy all offspring
- (n= ~ 50/sex/dose/ 8 litters)

**Sexual Differentiation**

- GD 14

## The Multigeneration Study 

- 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

- 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

- 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

- 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 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.