¦— j» United States Prevention, Pesticides EPA 740-C-09-006
Environmental Protection and Toxic Substances October 2009
%#Crri A9ency
Endocrine Disruptor
Screening Program Test
Guidelines
OPPTS 890.1300:
Estrogen Receptor
Transcriptional Activation
(Human Cell Line (HeLa-
9903))
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NOTICE
This guideline is one of a series of test guidelines established by the Office of
Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection
Agency for use in testing pesticides and chemical substances to develop data for submission to
the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, etseq.), and section
408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
The OPPTS test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test,
and is also used by EPA, the public, and the companies that are subject to data submission
requirements under TSCA, FIFRA and/or the FFDCA. As a guidance document, these
guidelines are not binding on either EPA or any outside parties, and the EPA may depart from
the guidelines where circumstances warrant and without prior notice. The procedures contained
in this guideline are strongly recommended for generating the data that are the subject of the
guideline, but EPA recognizes that departures may be appropriate in specific situations. You
may propose alternatives to the recommendations described in these guidelines, and the
Agency will assess them for appropriateness on a case-by-case basis.
For additional information about OPPTS harmonized test guidelines and to access the
guidelines electronically, please go to http://www.epa.gov/oppts and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-0150
through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human Cell
Line (HeLa-9903))
(a) Scope.
(1) Applicability. This guideline is intended to meet testing requirements of
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C.
136, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21
U.S.C. 346a).
(2) Background. The Endocrine Disruptor Screening Program (EDSP)
reflects a two-tiered approach to implement the statutory testing
requirements of FFDCA section 408(p) (21 U.S.C. 346a). In general, EPA
intends to use the data collected under the EDSP, along with other
information, to determine if a pesticide chemical, or other substances, may
pose a risk to human health or the environment due to disruption of the
endocrine system.
This test guideline is intended to be used in conjunction with other
guidelines in the OPPTS 890 series that make up the full screening
battery under the EDSP to identify substances that have the potential to
interact with the estrogen, androgen, or thyroid hormone (Tier 1
"screening"). The determination will be made on a weight-of-evidence
basis taking into account data from the Tier 1 assays and other
scientifically relevant information available. The fact that a substance may
interact with a hormone system, however, does not mean that when the
substance is used, it will cause adverse effects in humans or ecological
systems.
Chemicals that go through Tier 1 screening and are found to have the
potential to interact with the estrogen, androgen, or thyroid hormone
systems will proceed to the next stage of the EDSP where EPA will
determine which, if any, of the Tier 2 tests are necessary based on the
available data. Tier 2 testing is designed to identify any adverse
endocrine-related effects caused by the substance, and establish a
quantitative relationship between the dose and that endocrine effect.
(3) Source. OPPTS developed this guideline through a process of
harmonization with the testing guidance and requirements published by
the Organization for Economic Cooperation and Development (OECD)
(Ref. 16)
(b) Purpose. In vitro transcriptional activation (TA) assays are based upon the
production of a reporter gene product induced by a chemical, following binding of
the chemical to a specific receptor and subsequent downstream transcriptional
activation. TA assays using activation of reporter genes are screening assays
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that have long been used to evaluate the specific gene expression regulated by
specific nuclear receptors, such as the estrogen receptors (ERs) (Refs. 3, 4, 5 &
6). They have been proposed for the detection of estrogenic transactivation
regulated by the ER (Refs. 7, 8, & 9). The nuclear ERs exist as at least two
subtypes, termed a and (3, encoded by distinct genes and with different tissue
distribution, relative ligand binding affinities and biological functions. Nuclear
ERa mediates the classic estrogenic response, therefore models currently being
developed to measure ER activation mainly relate to ERa. The aim of this TA
assay is to evaluate the ability of a chemical to function as an ERa ligand and
activate an agonist response, for screening and prioritization purposes but can
also provide mechanistic information that can be used in a weight of evidence
approach. The definitions and abbreviations used in this Test Guidelines are
described in Appendix 1.
Initial Considerations and Limitations. Estrogen agonists act as ligands for
ERs, and may activate the transcription of estrogen responsive genes. This
interaction may have the potential to trigger adverse health effects by disrupting
estrogen-regulated systems. This Test Guideline describes an assay that
evaluates TA mediated by the hERa. This process is considered to be one of the
key mechanisms of possible endocrine disruption related health hazards,
although there are also other important endocrine disruption mechanisms.
These include:
~ Actions mediated via other nuclear receptors linked to the endocrine
system and interactions with steroidogenic enzymes
~ Metabolic activation or deactivation of hormones
~ Distribution of hormones to target tissues
~ Clearance of hormones from the body
This Test Guideline exclusively addresses TA of an estrogen-regulated reporter
gene by agonist binding to the hERa, and therefore it should not be directly
extrapolated to the complex in vivo situation of estrogen regulation of cellular
processes. Furthermore, this Test Guideline does not address antagonist
interaction with the hERa and subsequent effect on transcription.
This test method is specifically designed to detect hERa-mediated TA by
measuring chemiluminescence as the endpoint. However, non-receptor-
mediated luminescence signals have been reported at phytoestrogen
concentrations higher than 1 |jM due to the over-activation of the luciferase
reporter gene (Refs. 10 & 11). While the dose response curve indicates that true
activation of the ER system occurs at lower concentrations, luciferase expression
obtained at high concentrations of phytoestrogens or similar compounds
suspected of producing phytoestrogen-like over-activation of the luciferase
reporter gene needs to be examined carefully in stably transfected ER TA assay
systems (Appendix 2).
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(d) Principle of the Test. The TA assay using a reporter gene technique is an in
vitro tool that provides mechanistic data. The assay is used to signal binding of
the estrogen receptor with a ligand. Following ligand binding, the receptor-ligand
complex translocates to the nucleus where it binds specific DNA response
elements and transactivates a firefly luciferase reporter gene, resulting in
increased cellular expression of luciferase enzyme. Luciferin is a substrate that
is transformed by the luciferase enzyme to a bioluminescence product that can
be quantitatively measured with a luminometer. Luciferase activity can be
evaluated quickly and inexpensively with a number of commercially available test
kits.
The test system provided in this guideline utilizes the hERa-HeLa-9903 cell line,
which is derived from a human cervical tumor, with two stably inserted
constructs:
~ The hERa expression construct (encoding the full-length human receptor).
~ A firefly luciferase reporter construct bearing five tandem repeats of a
vitellogenin Estrogen-Responsive Element (ERE) driven by a mouse
metallothionein (MT) promoter TATA element.
The mouse MT TATA gene construct has been shown to have the best
performance, and so is commonly used. Consequently this hERa-HeLa-9903 cell
line can measure the ability of a test chemical to induce hERa-mediated
transactivation of luciferase gene expression.
Data interpretation for this assay is based upon whether or not the maximum
response level induced by a test chemical equals or exceeds an agonist
response equal to 10% of that induced by a maximally inducing (1 nM)
concentration of the positive control (PC) 17(3 estradiol (E2) (i.e., the PC10). Data
analysis and interpretation are discussed in greater detail in section (f)(1) through
(f)(3).
(e) Procedure.
(1) Cell Line. Use the stably transfected hERa-HeLa-9903 cell line for the
assay. The cell line can be obtained from the Japanese Collection of
Research Bioresources (JCRB) Cell Bank1.
Use only cells characterized as mycoplasma-free in testing. RT PCR (Real
Time Polymerase Chain Reaction) is the method of choice for a sensitive
detection of mycoplasm infection (Refs. 12,13 & 14).
1 JCRB Cell Bank : National Institute of Biomedical Innovation, 7-6-8 Asagi Saito, Ibaraki-shi,
Osaka 567-0085, Japan Fax: +81-72-641-9812
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Stability of the Cell Line. To monitor the stability of the cell line, use E2,
17a-estradiol, 17a-methyltestosterone, and corticosterone as the
reference chemicals, and include a complete concentration response
curve in the test concentration range provided in Table 1 at least once
each time the assay is performed. Comparative results to those provided
in Table 1 are recommended.
Cell Culture and Plating Conditions. Maintain cells in Eagle's Minimum
Essential Medium (EMEM) without phenol red, supplemented with 60
mg/L of antibiotic Kanamycine and 10% dextran-coated-charcoal-treated
fetal bovine serum (DCC-FBS), in a CO2 incubator (5% CO2) at 37±1 °C.
Upon reaching 75-90% confluency, cells can be subcultured at 10 ml_ of
0.4 x 105 - 1 x 105 cells/mL for 100 mm cell culture dish. Suspend cells
with 10% FBS-EMEM (which is the same as EMEM with DCC-FBS) and
then plate into wells of a microplate at a density of 1 * 104 cells/100
|jL/well. Next, pre-incubate the cells in a 5% CO2 incubator at 37°±1 °C for
3 hours before the chemical exposure. Use plastic-ware free of estrogenic
activity.
To maintain the integrity of the response, grow the cells for more than one
passage from the frozen stock in the conditioned media and do not culture
them for more than 40 passages. For the hERa-HeLa-9903 cell line, this
will be less than three months.
The DCC-FBS can be prepared as described in Appendix 3, or obtained
from commercial sources.
Acceptability Criteria.
(i) Positive and Negative Reference Chemicals. Prior to and during
the study, verify the responsiveness of the test system using the
appropriate concentrations of a strong estrogen: E2, a weak
estrogen (17a-estradiol), a very weak agonist (17a-
methyltestosterone) and a negative compound (corticosterone).
Acceptable range values derived from the validation study are
given in Table 1 (Ref. 2). Include these 4 concurrent reference
chemicals with each experiment. It is recommended that the
results fall within the given limits. If this is not the case, it is
suggested that the reason for the failure to meet the acceptability
criteria be determined {e.g., cell handling, and serum and
antibiotics for quality and concentration) and the assay repeated.
Obtaining values within the recommended range will help ensure
minimum variability of EC50, PC50 and PC10 values. Consistent use
of materials for cell culturing is also essential. The four concurrent
reference chemicals, which are included in each experiment
(conducted under the same conditions including the materials,
passage level of cells and technicians), ensure the sensitivity of the
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assay when the PC10S of the three positive reference chemicals,
and the PC50S and EC50S (when they can be calculated (see Table
1)) fall within the recommended ranges.
Table 1. Acceptable Range Values of the 4 Reference Chemicals for the STTA Assay
(means ± 2 standard deviations).
Name
logPC50
logPCio
logEC50
Hill slope
Test range
17p-Estradiol (E2)
CAS No: 50-28-2
-11.4 ~ -10.1
<-11
-11.3 ~ -10.1
0.7-1.5
10 14 - 10 s M
17a-Estradiol
CAS No: 57-91-0
-9.6 ~-8.1
-10.7 ~-9.3
-9.6 ~-8.4
0.9-2.0
10"12~10"6M
Corticosterone
CAS No: 50-22-6
-
-
-
-
10_10~ 104M
17a-Methyltestosterone
CAS No: 58-18-4
-6.0 ~-5.1
-8.0 ~-6.2
-
-
10~11 ~ 10"5 M
(ii) Positive and Vehicle Controls. Test the positive control (PC) (1
nM of E2) at least in triplicate in each plate. Test the vehicle that is
used to dissolve a test chemical as a vehicle control (VC) at least in
triplicate in each plate. If the PC uses a different vehicle than the
test chemical, include another vehicle control at least in triplicate on
the same plate with the PC in addition to this original vehicle
control.
(iii) Fold-induction. The target mean luciferase activity of the PC (1
nM E2) is at least 4-fold that of the mean vehicle control on each
plate. This criterion is established based on the reliability of the
endpoint values from the validation study (historically between four-
and 30-fold).
With respect to the quality control of the assay, the target fold-
induction corresponding to the PC10 value of the concurrent PC (1
nM E2) is to be greater than 1+2SD (standard deviations) of the
fold-induction value (=1) of the concurrent VC. For prioritization
purposes, the PC10 value can be useful to simplify the data analysis
required compared to a statistical analysis. Although a statistical
analysis provides information on significance, such an analysis is
not a quantitative parameter with respect to concentration-based
potential, and so is less useful for prioritization purposes.
(5) Chemicals to Demonstrate Laboratory Proficiency. Prior to testing
unknown chemicals in the STTA assay, confirm the responsiveness of the
test system by each laboratory, at least once for each newly prepared
batch of cell stocks taken from the frozen stock by independent testing of
the 10 proficiency chemicals listed in Table 2. Perform this at least in
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duplicate, on different days, and compare the results to Table 2. Please
justify any deviations.
Table 2. List of Proficiency Chemica
S.
Compound
CAS No.
Class2
Test
concentration
range
Note
Diethylstilbestrol (DES)
56-53-1
Positive
0
-&>
1
O
co
17a-Ethynyl estradiol (EE)
57-63-6
Positive
0
-&>
1
o
co
Hexestrol
84-16-2
Positive
10"13- 10"7M
Genistein
446-72-0
Positive
LO
b
I
CM
b
Cytotoxic
at (0.01)4, 0.1 and 1 mM
Estrone
53-16-7
Positive
10""- 10° M
Butyl paraben
94-26-8
Positive
b
I
b
Cytotoxic at (0.1)4 and 1 mM
1,3,5-
T ris(4hydroxyphenyl)benzene1
15797-
52-1
Positive
10 12 - 10 5 M
Cytotoxic at 100 |j.M. PCmax
approx 15% of PC
Binds to hERa and has ER
antagonist activity
Dibutyl phthalate (DBP)
84-74-2
Negative3
^r
b
I
b
Cytotoxic at 1 mM
Atrazine
1912-24-
9
Negative
^r
b
I
b
Cytotoxic4 at 1 mM
Corticosterone
50-22-6
Negative
^r
b
I
o
b
If not cytotoxic at 1 mM, then
that is to be the highest
tested concentration
Compound selected to challenge solubility and cytotoxicity.
2See Table 5 for definitions of positive and negative.
3Negative for ERa mediated transcriptional activation but may not be negative for non-ERp mediated
transcriptional activation. Thus a positive result in this assay with DBP would indicate that the system is
detecting other than pure ERa mediated activity and is therefore unacceptable.
Cytotoxicity is close to 80%.
(6) Vehicle. Use dimethyl sulfoxide (DMSO), or appropriate solvent, at the
same concentration used for the different positive and negative controls
and the test chemicals as the concurrent vehicle control. Dissolve each
test substance in a solvent that solubilizes that test substance and is
miscible with the cell medium. Water, ethanol (95% to 100% purity) and
DMSO are suitable vehicles. If DMSO is used, do not exceed 0.1% (v/v).
For any vehicle, demonstrate that the maximum volume used is not
cytotoxic and does not interfere with assay performance.
(7) Preparation of Test Chemicals. Generally, dissolve the test chemicals
in DMSO or other suitable solvent, and serially dilute with the same
solvent at a common ratio of 1:10 in order to prepare solutions for dilution
with media.
(8) Solubility and Cytotoxicity: Considerations for Range Finding.
Conduct a preliminary test to determine the appropriate concentration
range of chemical to be tested, and to ascertain whether the test chemical
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may have any solubility and cytotoxicity problems. Initially, chemicals are
tested up to the maximum concentration of 1 |jl/ml, 1 mg/ml, or 1 mM,
whichever is the lowest. Based on the extent of cytotoxicity or lack of
solubility observed in the preliminary test, perform the first definite run for
the test chemical at log serial dilutions starting at the maximum acceptable
concentration {e.g., 1 mM, 100 |jM, 10 |jM, etc.). Note the presence of
cloudiness, precipitate or cytotoxicity. Adjust concentrations in the
second, and if necessary third run as appropriate to better characterize
the concentration-response curve and to avoid concentrations which are
found to be insoluble or to induce excessive cytotoxicity.
For ER agonists, the presence of increasing levels of cytotoxicity can
significantly alter or eliminate the typical sigmoidal response and are a
consideration when interpreting the data. Use cytotoxicity testing methods
that can provide information regarding 80% cell viability, utilizing an
appropriate assay based upon laboratory experience.
Should the results of the cytotoxicity test show that the concentration of
the test substance has reduced the cell number by 20% or more, this
concentration is regarded as cytotoxic, and concentrations at or above the
cytotoxic concentration should be excluded from the evaluation.
Chemical Exposure and Assay Plate Organization. The procedure for
chemical dilutions (Steps-1 and 2) and exposure to cells (Step-3) can be
conducted as follows:
Step 1: Dilute each test chemical by serial dilution in DMSO, or
appropriate solvent, and add to the wells of a microtitre plate to
achieve final serial concentrations as determined by the
preliminary range finding test (typically in a series of, for example
1 mM, 100 |jM, 10 |jM, 1 |jM, 100 nM, 10 nM, 1 nM, 100 pM, and
10 pM (10~3-10~11 M)) for triplicate testing.
Step 2: Chemical dilution: First dilute 1.5 |jL of the test chemical in the
solvent to a concentration of 500 |jL of media.
Step 3: Chemical exposure of the cells: Add 50 |jL of dilution with media
(prepared in Step-2) to an assay well containing 104 cells/100
|jL/well.
The recommended final volume of media required for each well is 150 |jL.
Test samples and reference chemicals can be assigned as shown in
Table 3.
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Table 3. Example of Plate Concentration Assignment of the Reference Chemicals in
the Assay Plate.
Row
17a-Methyltestosterone
Corticosterone
17a-Estradiol
E2
1
2
3
4
5
6
7
8
9
10
11
12
A
cone 1 (10 |jM)
->
->
100 |jM
->
->
1 |jM
->
->
10 nM
->
->
B
cone 2 (1 |jM)
-
-
10 |jM
-
-
100
nM
-
-
1 nM
-
-
C
cone 3 (100 nM)
-
-
1 |jM
-
-
10
nM
-
-
100 pM
-
-
D
cone 4 (10 nM)
->
->
100 nM
->
->
1 nM
->
->
10 pM
->
->
E
cone 5 (1 nM)
-
-
10 nM
-
-
100
pM
-
-
1 pM
-
-
F
cone 6 (100 pM)
-
-
1 nM
-
-
10
pM
-
-
0.1 pM
-
-
G
cone 7 (10 pM)
->
->
100 pM
->
->
1 pM
->
->
0.01 pM
->
->
H
VC
->
->
->
->
->
PC
->
->
->
->
->
Plate controls = VC: Vehicle control (DMSO); PC: Positive control (1 nM E2)
Test the reference chemicals (E2, 17a-Estradiol, 17a-methyl testosterone
and corticosterone) in every run (Table 3). Include 1) PC wells treated with
1 nM of E2 that can produce maximum induction of E2, and 2) VC wells
treated with DMSO (or appropriate solvent) alone, in each test assay plate
(Table 4). If cells from different sources {e.g., different passage number,
different lot, etc.,) are used in the same experiment, the test the reference
chemicals with each cell source.
Table 4. Example of Plate Concentration Assignment of Test and Plate Control
Chemicals in the Assay Plate.
Row
Test Chemical 1
Test Chemical 2
Test Chemical 3
Test Chemical 4
1
2
3
4
5
6
7
8
9
10
11
12
A
cone 1 (10 |jM)
->
->
1 mM
->
->
1 |jM
->
->
10 nM
->
->
B
cone 2 (1 |jM)
->
->
100 |jM
->
->
100 nM
->
->
1 nM
->
->
C
cone 3 (100 nM)
->
->
10 uM
->
->
10 nM
->
->
100 pM
->
->
D
cone 4 (10 nM)
->
->
1 uM
->
->
1 nM
->
->
10 pM
->
->
E
cone 5 (1 nM)
->
->
100 nM
->
->
100 pM
->
->
1 pM
->
->
F
cone 6 (100 pM)
->
->
10 nM
->
->
10 pM
->
->
0.1 pM
->
->
G
cone 7 (10 pM)
->
->
1 nM
->
->
1 pM
->
->
0.01 pM
->
->
H
VC
->
->
->
->
->
PC
->
->
->
->
->
Confirm the lack of edge effects, as appropriate, and if edge effects are
suspected, alter the plate layout to avoid such effects. For example, a
plate layout excluding the edge wells can be employed.
After adding the chemicals, incubate the assay plates in a 5% CO2
incubator at 37±1°C for 20-24 hours to induce the reporter gene products.
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Special considerations will need to be applied to those compounds that
are highly volatile. In such cases, nearby control wells may generate false
positives, and it is important they be considered in light of expected and
historical control values. In the few cases where volatility may be of
concern, the use of "plate sealers" may help to effectively isolate individual
wells during testing, and is therefore recommended in such cases.
Conduct repeat definitive tests for the same chemical on different days, to
ensure independence.
(10) Luciferase assay. A commercial luciferase assay reagent [e.g., Steady-
Glo® Luciferase Assay System (Promega, E2510, or equivalents)] or a
standard luciferase assay system {e.g., Promega, E1500, or equivalents)
can be used for the assay, as long as the results match the acceptability
criteria as defined in this assay. Select the assay reagents based on the
sensitivity of the luminometer to be used. When using the standard
luciferase assay system, use the Cell Culture Lysis Reagent {e.g.,
Promega, E1531, or equivalents) before adding the substrate. Follow the
manufacturer's instructions when using the luciferase reagent.
Analysis of Data. To obtain the relative transcriptional activity to PC (1 nM of
E2), the luminescence signals from the same plate can be analyzed according to
the following steps (other equivalent mathematical processes are also
acceptable):
Step 1: Calculate mean value for the VC.
Step 2: Subtract the mean value of the VC from each well value to normalize
the data.
Step 3: Calculate the mean for the normalized PC.
Step 4: Divide the normalized value of each well in the plate by the mean value
of the normalized PC (PC=100%). The final value of each well is the
relative transcriptional activity for that well compared to the PC
response.
Step 5: Calculate the mean value of the relative transcriptional activity for each
concentration group of the test chemical. There are two dimensions to
the response: the averaged transcriptional activity (response) and the
concentration at which the response occurs (see following section).
(1) Considerations for Induction of EC50, PC50 and PC10. The full
concentration response curve is required for the calculation of the EC50,
but this may not always be achievable or practical due to limitations of the
test concentration range (for example due to cytotoxicity or solubility
problems). However, as the EC50 and maximum induction level
(corresponding to the top value of the Hill-equation) are informative
parameters, report these parameters where possible. For the calculation
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of EC50 and maximum induction level, use appropriate statistical software
{e.g., Graphpad Prism statistical software).
If the Hill's logistic equation is applicable to the concentration response
data, calculate the EC50 by the following equation (Ref 15):
Y=Bottom + (Top-Bottom) / (1+10 exp ((log EC50 -X) x Hillslope))
Where:
X is the logarithm of concentration; and,
Y is the response and Y starts at the Bottom and goes to the Top in a sigmoid curve.
Bottom is fixed at zero in the Hill's logistic equation.
For each test chemical, provide the following:
~ The RPCMax which is the maximum level of response induced by a test
chemical, expressed as a percentage of the response induced by 1 nM E2
on the same plate, as well as the PCMax (concentration associated with the
RPCMax)-
~ For positive chemicals, the concentrations that induce the PC10 and, if
appropriate, the PC50.
The PCX value can be calculated by interpolating between 2 points on the
X-Y coordinate, one immediately above and one immediately below a PCX
value. Where the data points lying immediately above and below the PCX
value have the coordinates (a,b) and (c,d) respectively, then the PCX value
may be calculated using the following equation:
log[PCx] = log[c]+(x-d)/(d-b)
Descriptions of PC values are provided in Figure 1 below.
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Figure 1. Example of How to Derive PC-values. The PC (Positive control; 1 nM of E2)
is included on each assay plate.
fold induction of PC (1 nM E2)-4-
fold induction corresponding to PC10 „
mean (=1) + 2SD of fold induction
¦2y4
tS >
L
f\
mean (= 1) of fold induction <
PL-iu ^ IL /
¦¦¦¦¦¦¦¦¦¦¦¦¦¦ ¦ v ^ ,
of vc < ^:^v.dv ::
°f VC ^ oil 17 I 11 lh.
10 pM 100 pM lnMllOnM 10C
f -
PC10 PC50
100%
50%
10%
0%
100 nM 1 ||M 10 MM
Vehicle Positive
control control
(2) Performance Standards.
To be acceptable, the following are considered:
• The mean luciferase activity of the positive controls (1 nM E2) is at least
4-fold that of the mean vehicle control on each plate.
• The fold induction corresponding to the PC10 value of the concurrent PC
(1 nM E2) is greater than 1+2SD of the fold induction value (=1) of the
VC (vehicle control).
• The results of 4 reference chemicals are within the acceptable range
(Table 1).
• Be reproducible.
(3) Data Interpretation Criteria. Base the results on two (or three)
independent runs. If two runs give comparable and therefore reproducible
results, it is not necessary to conduct a third run. Data interpretation
criteria are shown in Table 5. Positive results will be characterized by
both the magnitude of the effect and the concentration at which the effect
occurs. Expressing results as a concentration at which a 50% (PC50) or
10% (PC10) of positive control values are reached accomplishes both of
these goals. However, a test chemical is determined to be positive, if the
maximum response induced by the test chemical (RPCMax) is equal to or
exceeds 10% of the response of the positive control in at least two of two
or two of three runs, while a test chemical is considered negative if the
RPCMax fails to achieve at least 10% of the response of the positive control
in two of two or two of three runs.
Table 5. Positive and N
egative Decision Criteria.
Positive
If the RPCMax is obtained that is equal to or exceeds 10% of the response of
the positive control in at least two of two or two of three runs.
Negative
If the RPCMax fails to achieve at least 10% of the response of the positive
control in two of two or two of three runs.
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The EPA intends to provide a calculation spreadsheet with the posting of
this guideline on the Agency's Web site (Ref. 17) that may be utilized to
determine PC10, PC50 and PCiviax-
Obtaining PC10 or PC50 values at least twice is sufficient, unless, the
resulting base-line for data in the same concentration range shows
variability with an unacceptably high coefficient of variation (CV; %). In
such a case, the data may not be considered reliable and it is
recommended that the source of the high variability be identified. The
target CV of the raw data triplicates (i.e. luminescence intensity data) of
the data points that are used for the calculation of PC10 is less than 20%.
Meeting the acceptability criteria indicates the assay system is operating
properly, but it does not ensure that any particular run will produce
accurate data. Duplicating the results of the first run is the best insurance
that accurate data were produced (see above).
Where more information is required in addition to the screening and
prioritization purposes of this TG for positive test compounds, particularly
for PC 10-PC49 chemicals, as well as chemicals suspected to over stimulate
luciferase, it can be confirmed that the observed luciferase-activity is
solely an ERa-specific response, using an ERa antagonist (see Appendix
3).
(g) Test Report. Include the following information in the test report:
~ Test substance:
• Identification information (e.g., molecular weight, lot, supplier,
expiration date) and CAS Number, if known
• Physical nature and purity
• Physicochemical properties relevant to the conduct of the study
• Stability of the test substance
~ Solvent/Vehicle:
• Characterization (nature, supplier and lot)
• Justification for choice of solvent/vehicle
• Solubility and stability of the test substance in solvent/vehicle, if known
~ Cells:
• Type and source of cells
• Number of cell passages
• Methods for maintenance of cell cultures
~ Test conditions:
• Report cytotoxicity data (and justifications for the method of choice)
and solubility limitations, as well as:
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- Composition of media, CO2 concentration
- Concentration of test chemical
- Volume of vehicle and test substance added
- Incubation temperature and humidity
- Duration of treatment
- Cell density during treatment
- Positive and negative reference chemicals
- Duration of treatment period
- Luciferase assay reagents (Product name, supplier and lot)
- Acceptability and data interpretation criteria.
~ Reliability check:
• Fold inductions for each assay plate
• Actual logECso, logPCso, logPCio and Hill slope values for concurrent
reference chemicals
~ Results:
• Raw and normalized data of luminescent signals
• Concentration-response relationship, where possible
• RPCMax, Piviax, PC50 and/or PC10 values, as appropriate
• EC50 values, if appropriate
• Statistical analyses, if any, together with a measure of error {e.g.,
SEM, SD, CV or 95% CI) and a description of how these values
were obtained.
~ Discussion of the results.
~ Conclusion.
(h) References.
1. CERI (2006). Draft validation report of TA assay using HeLa-hER-9903 to detect
estrogenic activity. Available at:
http://www.oecd.Org/document/62/0.3343.en 2649 34377 2348606 1 1 1 1,00.
html.
2. Takeyoshi, M., Yamasaki, K., Sawaki, M., Nakai, M., Noda, S. and Takatsuki, M.
(2002). The efficacy of endocrine disruptor screening tests in detecting anti-
estrogenic effects downstream of receptor-ligand interactions. Toxicol. Lett, 126,
91-98.
3. Jefferson, W.N., Padilla-Banks, E., Clark, G. and Newbold R. (2002). Assessing
estrogenic activity of phytochemicals using transcriptional activation and immature
mouse uterotropic responses. J. Chromat. B., Ill, 179-189.
4. Sonneveld, E., Riteco, J.A., Jansen, H.J., Pieterse, B., Brouwer, A., Schoonen,
W.G. and van der Burg, B. (2006). Comparison of in vitro and in vivo screening
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models for androgenic and estrogenic activities. Toxicol. Sci., 89, 173-187.
5. Escande, A., Pillon, A., Servant, N., Cravedi, J.P., Larrea, F., Muhn, P., Nicolas,
J.C., Cavailles, V. and Balaguer, P. (2006). Evaluation of ligand selectivity using
reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem.
Pharmacol., 71, 1459-1469.
6. Gray, L.E. Jr. (1998). Tiered screening and testing strategy for xenoestrogens and
antiandrogens. Toxicol. Lett., 102-103, 677-680.
7. EDSTAC (1998). Endocrine Disruptor Screening and Testing Advisory Committee
(EDSTAC) Final Report. Available at:
http://www.epa.gov/scipolv/oscpendo/pubs/edspoverview/finalrpt.htm.
8. ICCVAM (2003). ICCVAM Evaluation of In Vitro Test Methods for Detecting
Potential Endocrine Disruptors: Estrogen Receptor and Androgen Receptor
Binding and Transcriptional Activation Assays. Available at:
http://iccvam.niehs.nih.qov/methods/endocrine.htm#fineval.
9. Escande, A., Pillon, A., Servant, N., Cravedi, J.P., Larrea, F., Muhn, P., Nicolas,
J.C., Cavailles, V. and Balaguer, P. (2006). Evaluation of ligand selectivity using
reporter cell lines stably expressing estrogen receptor alpha or beta. Biochem.
Pharmacol., 71, 1459-1469.
10. Kuiper, G.G., Lemmen, J.G., Carlsson, B., Corton, J.C., Safe, S.H., van der Saag,
P.T., van der Burg, B. and Gustafsson, J.A. (1998). Interaction of estrogenic
chemicals and phytoestrogens with estrogen receptor beta. Endocrinol., 139,
4252-4263.
11. Spaepen M, Angulo AF, Marynen P, Cassiman J J. (1992). Detection of bacterial
and mycoplasma contamination in cell cultures by polymerase chain reaction.
FEMS Microbiol Lett. 78(1), 89-94.
12. Kobayashi H, Yamamoto K, Eguchi M, Kubo M, Nakagami S, Wakisaka S, Kaizuka
M, Ishii H (1995). Rapid detection of mycoplasma contamination in cell cultures by
enzymatic detection of polymerase chain reaction (PCR) products. J Vet Med Sci.
57(4), 769-71.
13. Dussurget 0, Roulland-Dussoix D (1994). Rapid, Sensitive PCR-based Detection
of Mycoplasmas in Simulated Samples of Animal Sera. Appl Environ Microbiol.
60(3), 953-9.
14. De Lean A, Munson, P.J. Rodbard D (1978). Simultaneous Analysis of Families of
Sigmoidal Curves: Application to Bioassay, Radioligand Assay, and Physiological
Dose-response Curves. Am J Physiol. 235, E97-EI02.
15. OECD (2005). Guidance Document on The Validation and International
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Acceptance of New or Updated Test Methods for Hazard Assessment. OECD
Series on Testing and Assessment. No. 34. Paris, France.
EPA. OPPTS Harmonized Test Guidelines. Available on-line at:
http://www.epa.gov/oppts (select "Test Methods & Guidelines" on the left side
navigation menu). You may also access the guidelines in
httpV/www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-
2009-0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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Appendix 1
Definitions and Abbreviations
Agonist: A substance that binds to a specific receptor and triggers a response in the
cell. It mimics the action of an endogenous liqand that binds to the same receptor.
Antagonist: A type of receptor liqand or chemical that does not provoke a biological
response itself upon binding to a receptor, but blocks or dampens agonist-mediated
responses.
Anti-estrogenic activity, the capability of a chemical to suppress the action of 17(3-
estradiol mediated through estrogen receptors.
CV: Coefficient of variation
Cytotoxicity: the harmful effects to cell structure or function ultimately causing cell
death and can be a result of a reduction in the number of cells present in the well at the
end of the exposure period or a reduction of the capacity for a measure of cellular
function when compared to the concurrent vehicle control.
DCC-FBS: Dextran-coated charcoal treated fetal bovine serum.
DMSO: Dimethyl sulfoxide
E2: 17(3-estradiol
EC50 value, the concentration of agonist that provokes a response halfway between the
baseline (Bottom) and maximum response (Top).
EE: 17a-ethynyl estradiol
ER; Estrogen receptor
ERE: Estrogen Response Element
Estrogenic activity, the capability of a chemical to mimic 17(3-estradiol in its ability to
bind to and activate estrogen receptors. hERa mediated specific estrogenic activity can
be detected in this Test Guideline.
FBS: Fetal bovine serum
hERa: Human estrogen receptor alpha
MT: Metallothionein
OHT: 4-Hydroxytamoxifen
PC: Positive control
PC™: the concentration of a test chemical at which the response in an agonist assay is
10% of the response induced by positive control (E2 at 1nM) in each plate
PC50: the concentration of a test chemical at which the response in an agonist assay is
50% of the response induced by positive control (E2 at 1nM) in each plate
PCMax- the concentration of a test chemical inducing the RPCMax
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RPCMax: maximum level of response induced by a test chemical, expressed as a
percentage of the response induced by 1 nM E2 on the same plate
RT PCR: Real Time polymerase chain reaction
SD: Standard deviation
STTA: Stably Transfected Transcriptional Activation Assay.
TA: Transcriptional activation
Validation, a process based on scientifically sound principles by which the reliability
and relevance of a particular test, approach, method, or process are established for a
specific purpose. Reliability is defined as the extent of reproducibility of results from a
test within and among laboratories over time, when performed using the same
standardized protocol. The relevance of a test method describes the relationship
between the test and the effect in the target species and whether the test method is
meaningful and useful for a defined purpose, with the limitations identified. In brief, it is
the extent to which the test method correctly measures or predicts the (biological) effect
of interest, as appropriate (16).
VC: The vehicle that is used to dissolve test and control chemicals is tested solely as
vehicle without dissolved chemical.
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Appendix 2
False Positives: Assessment of Non-receptor Mediated Luminescence Signals
1. False Positives
False positives might be generated by non-ER-mediated activation of the luciferase
gene, or direct activation of the gene product or unrelated fluorescence. Such effects
are indicated by an incomplete or unusual dose-response curve. If such effects are
suspected, examine the effect of an ER antagonist {e.g., 4-hydroxytamoxifen (OHT) at
non-toxic concentration) on the response. The pure antagonist ICI 128780 may not be
suitable for this purpose as a sufficient concentration of ICI 128780 may decrease the
vehicle control value, and this will affect the data analysis.
To ensure validity of this approach, the following needs to be tested in the same plate:
• Agonistic activity of the unknown chemical with / without 10 |jM of OHT
• Vehicle Control (VC)(in triplicate)
• OHT (in triplicate)
• 1 nM of E2 (in triplicate) as agonist Positive Control (PC)
• 1 nM of E2 + OHT (in triplicate)
3. Data Interpretation Criteria
Note: Treat all wells with the same concentration of the vehicle.
• If the agonistic activity of the unknown chemical is NOT affected by the treatment
with ER antagonist, it is classified as "Negative".
• If the agonistic activity of the unknown chemical is completely inhibited, apply the
decision criteria.
• If the agonistic activity at the lowest concentration is equal to, or is exceeding,
PC10 response the unknown chemical is inhibited equal to or exceeding PC10
response. The difference in the responses between the non-treated and treated
wells with the ER antagonist is calculated and considered as the true response to
be used for the calculation of the appropriate parameters to enable a classification
decision to be made.
4. Data Analysis
• Check the performance standard.
• Check the CV between wells treated under the same conditions.
• Calculate the mean of the VC.
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• Subtract the mean of VC from each well value not treated with OHT.
• Calculate the mean of OHT.
• Subtract the mean of the VC from each well value treated with OHT.
• Calculate the mean of the PC.
• Calculate the relative transcriptional activity of all other wells relative to the PC.
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Appendix 3
Preparation of Serum treated with Dextran Coated Charcoal (DCC)
The treatment of serum with dextran-coated charcoal (DCC) is a general method for
removal of estrogenic compounds from serum that is added to cell medium, in order to
exclude the biased response associated with residual estrogens in serum. 500 ml_ of
fetal bovine serum (FBS) can be treated by this procedure.
Components
The following materials and equipment will be needed:
Materials
Activated charcoal
Dextran
Magnesium chloride hexahydrate (MgCl2-6H20)
Sucrose
1 M HEPES buffer solution (pH 7.4)
Ultrapure water produced from a filter system
Equipment
Autoclaved glass container (size should be adjusted as appropriate)
General Laboratory Centrifuge (that can set temperature at 4°C.)
Procedure
The following procedure is adjusted for the use of 50 ml_ centrifuge tubes:
[Day-1] Prepare dextran- coated charcoal suspension with 1 litre of ultrapure water
containing 1.5 mM of MgCh, 0.25 M sucrose, 2.5 g of charcoal, 0.25 g dextran and 5
mM of HEPES and stir it at 4°C, overnight.
[Day-2] Dispense the suspension in 50 ml_ centrifuge tubes and centrifuge at 10000
rpm at 4°C for 10 minutes. Remove the supernatant and store half of the charcoal
sediment at 4°C for the use on Day-3. Suspend the other half of the charcoal with
FBS that has been gently thawed to avoid precipitation, and heat-inactivated at 56°C
for 30 minutes, then transfer into an autoclaved glass container such as an
Erlenmeyer flask. Stir this suspension gently at 4°C, overnight.
[Day-3] Dispense the suspension with FBS into centrifuge tubes for centrifugation at
10000 rpm at 4°C for 10 minutes. Collect FBS and transfer into the new charcoal
sediment prepared and stored on Day-2. Suspend the charcoal sediment and stir
this suspension gently in an autoclaved glass container at 4°C, overnight.
[Day-4] Dispense the suspension for centrifugation at 10000 rpm at 4°C for 10
minutes and sterilize the supernatant by filtration through 0.2 |jm sterile filter. This
DCC treated FBS should be stored at -20°C and can be used for up a year.
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