# Review of the in vitro Assays

– the Tier 1 Endocrine Disruption Screening Assays

Presented by: Colleen Toole, Ph.D. Director of Project Management for CeeTox, Inc.

# CeeTox Experience

 In Vitro Laboratory (Kalamazoo, MI)  Lots of experience with the in vitro assays  > 90 in vitro studies following EDSP guidelines  ~ 30 of the Tier 1 test articles3 

# ER Binding Performance Standards

-12 -11 -10 -9 -8 -7 -6 0 25 50 75 100 125

Log Concentration (M)Mean Specific Binding (%) Estradiol

-9 -8 -7 -6 -5 -4 -3 0 25 50 75 100 125

Log Concentration (M)Mean Specific Binding (%) 19
- Norethindrone Parameter Unit Estradiol Nor e t h y nodrel
L o w e r limit Upper limit L o w e r limit Upper limit

Lo g e(Sy x ) ( i.e

. , Lo g e(R e s i d u al St d

. D ev)
-- NA 2.35 NA 2.60 Bottom plateau level % binding

- 4 1

- 5 1 Top plate a u level* % binding 94 111 90 110 (Hil l ) Slope lo g 10(M)

- 1

- 1.1

- 0.7

- 1.1

- 0.7

# Comparison of AR and ER AR E R

 Test tube based assay

 Uses 100 μ L of cytosol

 Less waste and prep time for assay

 Storage at

- 80oC up to 90 days

 Decrease runs for re

qualifying prep

 Solubility constraints

 Reporting

 Categories for binding

 Binder

 Equivocal

 Non

- binder  Test tube based assay

 Uses 300 μ L of cytosol and discards 200 μ L

 2Xs the amount of test tubes increases prep time

 No Negative Control

 Storage

– discard after 6 months

 Decrease runs for re
qualifying prep

 Solubility constraints

 Reporting

 Categories for binding

 Interactive

 Non

- interactive5 

# Determination of False Positives by Determining the K i

 

## K i 

## is the equilibrium dissociation constant (the concentration of the competing ligand/compound that will bind to half the binding sites at equilibrium)

 

## Performed when non
- sigmoidal binding curves or partial curves are observed

.

Susan Laws, Toxicological Sciences 20066 

# Determination of False Positives by Determining the K i

 

## Increasing concentrations of 3 

## H

- E2 are incubated with ERs in the presence of test material (~0, 0.5, 1 and 2 X IC50 )

 

## Lineweaver

- Burke plots are constructed

 

## The slopes obtained from the double reciprocal plots are replotted vs. concentration of the test material. The negative intercept of the slope replot is the K i0 200 400 600 800 1000 1200 1400

0 5 10 15 20 25 2 nM 1 nM 0.5 nM 0 nM

0 10 20 30 40 50 60 70

- 0.5 0 0.5 1 1.5 2 2.5 

# 17 β

- Estradiol

– True Binding Event

All the trendlines for 17 β

Estradiol intersect at the same point. Results in a linear slope replot0 20 40 60 80 100 120 140 160

0 1 2 3 4 5 6 1.2 mM 0.6 mM 0 mM

- 10 - 5 0 5 10 15 20 25 30

- 0.3

- 0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 

# Test Material A

– False Positive

T he trendlines for Test Material A do not intersect at the same point. Results in a non

- linear slope

# ERTA Control Data

- Challenges

Name logPC 50 

### logPC 10 

### logEC 50 

### Hill slope Test range 17β

- Estradiol (E2)

- 11.4 ~ - 10.1 <
- 11 - 11.3 ~
- 10.1 0.7 ~ 1.5 10 - 14 ~ 10 - 8 M 17α
- Estradiol

- 9.6 ~

- 8.1

- 10.7 ~
- 9.3
- 9.6 ~

- 8.4 0.9 ~ 2.0 10 - 12 ~ 10 - 6 M Corticosterone

– – – – 10 - 10 ~ 10 - 4 M 17α
- MT

- 6.0 ~

- 5.1 - 8.0 ~

- 6.2

– – 10 - 11 ~ 10 - 5 M 

## 17 α − MT E2

-16 -15 -14 -13 -12 -11 -10 -9 -8 -7 0 25 50 75 100 125 150 175 200

Log Concentration (M)% of Maximal induction Control

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 0 25 50 75 100

Log Concentration (M)% of Maximal Induction Control

# ERTA Stability of 1 nM E2 (PC)

# Steroidogenesis, Challenges

• 22RHC can be used to increase basal production of Estradiol and Testosterone, however cytotoxicity and solubility issues were identified at concentrations between 20

- 40 μ M as outlined in guideline.

• Delicate balance between Estradiol and Testosterone 0 20 40 60 80 100 120

0.4 1 4 10 20 40Viability

Concentration 22HC ( μ M) 22RHC Viability Assessment: MTT in H295R

# Laboratory Proficiency

• GLP studies

• laboratory staff proficient, documented and in training records

• DESTs were available after studies were completed

• Does not state in guidelines to include proficiency data in the report

# In Vitro Assays: Remaining Challenges

• Technical Support for individual questions (contact at EPA)

• Guidance for:

• Alternative vehicles

• Outlier analysis (edge effect, other)

• Revised performance criteria (more labs and data)

- 95% confidence limits (curves)

• Miniaturization of the assays

- higher throughput