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 articles
3

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 Norethynodrel

Lower limit Upper limit Lower limit Upper 

limit

Loge(Syx) (i.e., Loge(Residual Std.Dev) -- NA 2.35 NA 2.60

Bottom plateau level % binding -4 1 -5 1

Top plateau level* % binding 94 111 90 110

(Hill) Slope log10(M)-1 -1.1 -0.7 -1.1 -0.7
Comparison of AR and ER

ER AR

 Test tube based assay

 Uses 100 μL of cytosol

 Less waste and prep time 

for assay

 Storage at -80oC up to 90 

days

 Decrease runs for requalifying prep

 Solubility constraints

 Reporting 

 Categories for binding

 Binder

 Equivocal

 Non-binder

 Test tube based assay

 Uses 300 μL of cytosol and 

discards 200 μL

 2Xs the amount of test 

tubes increases prep time

 No Negative Control

 Storage – discard after 6 

months

 Decrease runs for requalifying prep

 Solubility constraints

 Reporting 

 Categories for binding

 Interactive

 Non-interactive
5

Determination of False Positives by 

Determining the Ki

 Ki 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

2006
6

Determination of False Positives by 

Determining the Ki

 Increasing concentrations of 3H-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 Ki 
0

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

17β-Estradiol –True Binding Event 

All the trendlines for 17βEstradiol intersect at the same 

point.

Results in a linear slope replot 
0

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

The trendlines for Test 

Material A do not intersect at 

the same point.

Results in a non-linear slope 


ERTA Control Data -Challenges

Name logPC50 logPC10 logEC50 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 ~ 10-4M 

17α-MT -6.0 ~ -5.1 -8.0 ~ -6.2 – – 10-11 ~ 10-5 M 

17α−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 μM as 

outlined in guideline.

• Delicate balance between Estradiol and Testosterone 

0

20

40

60

80

100

120

0.4 1 4 10 20 40

Viability

Concentration 22HC (μM)

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