U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

1 

Analysis of Eight Oil Spill Dispersants Using In Vitro Tests for 

Endocrine and Other Biological Activity 

June 30, 2010 

U.S. Environmental Protection Agency 

Office of Research and Development 

Executive Summary

The U.S. Environmental Protection Agency’s Office of Research and Development was 

asked to evaluate the cytotoxicity and potential for interaction with the androgen and estrogen 

receptors (AR, ER) of eight oil spill dispersants being used, or could be considered for use, in the 

Gulf of Mexico. These are Corexit 9500 (the current product being used), DISPERSIT SPC 

1000, JD 2000, Nokomis 3-F4, Nokomis 3-AA, SAF-RON GOLD, Sea Brat #4, and ZI-400. To 

address this request, ORD staff and outside collaborators carried out a number of separate studies 

that were run using in vitro (cell-based) assays. A total of 8 cytotoxicity assays, 3 AR agonist 

assays, 1 AR antagonist assay and 4 ER agonist assays were run on the 8 dispersants, plus 

reference compounds. Tests were run across a wide range of dispersant concentrations (0.001 to 

10,000 parts per million, or ppm). Two dispersants showed a weak signal in one of the four ER 

assays, but integrating over all of the ER and AR results these data do not indicate that any of the 

eight dispersants display biologically significant endocrine activity via the androgen or estrogen 

signaling pathways. All of the dispersants showed cytotoxicity in at least one cell type at 

concentrations between 10 and 1000 ppm. Both JD 2000 and SAF-RON GOLD tend to be less 

cytotoxic than the other dispersants. Likewise, DISPERSIT SPC 1000 tends to be more cytotoxic 

than the other dispersants in the cell-based assays. 

This document has been reviewed in accordance with U.S. Environmental Protection Agency 

policy and approved for publication. Mention of trade names or commercial products does not 

constitute endorsement or recommendation for use.
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

2 

Contents 

Executive Summary........................................................................................................................ 1

Contributors .................................................................................................................................... 3

Introduction / Background .............................................................................................................. 4

Project Goals................................................................................................................................... 6

Study Summary:.............................................................................................................................. 6

Chemicals........................................................................................................................................ 7

Results............................................................................................................................................. 9

Androgen Receptor Agonist Activity ......................................................................................... 9

AR Agonist Assay 1 - Multiplexed reporter transcription unit (RTU) assay ......................... 9

AR Agonist Assay 2 - AR beta-lactamase Assay ................................................................. 10

AR Agonist Assay 3 - MDA-kb2 Androgen-responsive transcriptional activation assay.... 11

AR Agonist Assay 4 - CV-1 transient transcription assay.................................................... 14

Androgen Receptor Antagonist Activity .................................................................................. 16

AR Antagonist Assay 1 - MDA-kb2 Androgen-responsive transcriptional activation assay in 

antagonist mode .................................................................................................................... 16

Estrogen Receptor Agonist Activity ......................................................................................... 20

ER Agonist Assay 1 - Multiplexed reporter transcription unit (RTU) trans assay .............. 20

ER Agonist Assay 2 - Multiplexed reporter transcription unit (RTU) cis assay .................. 24

ER Agonist Assay 3 – ER-alpha beta-lactamase Assay ....................................................... 24

ER Agonist Assay 4 - T47D-KBluc estrogen-responsive transcriptional activation assay.. 25

Cytotoxicity............................................................................................................................... 29

Cytotoxicity Assay 1 –HepG2 Cells..................................................................................... 29

Cytotoxicity Assay 2 –AR bla Cells..................................................................................... 29

Cytotoxicity Assay 3 –ER bla Cells ..................................................................................... 30

Cytotoxicity Results (Assays 1-3) ........................................................................................ 30

Cytotoxicity Assays 4 THRU 9: measurements in T47D-KBluc, MDA-kb2, and CV-1 cells 

(MTT and CPE assessments) (5 independent assessments). ................................................ 30

Cytotoxicity Results (Assays 4-9) ........................................................................................ 31

Cytotoxicity Summary.......................................................................................................... 37

Other Molecular Targets........................................................................................................... 40

Conclusions................................................................................................................................... 44

Supplementary Information .......................................................................................................... 45

QA Summary ................................................................................................................................ 45

References..................................................................................................................................... 46
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

3 

Contributors

U.S. EPA Office of Research and Development

National Center for Computational Toxicology 

David Dix, Keith Houck, Richard Judson, Robert Kavlock, Thomas Knudsen, Stephen Little, 

Matthew Martin, David Reif, Daniel Rotroff 

National Exposure Research Laboratory 

Lora Johnson, Mike Hiatt, Tammy Jones-Lepp, John Zimmerman, Andy Grange, Brian 

Schumacher, Ed Heithmar 

National Health and Environmental Effects Research Laboratory 

Vickie S. Wilson, Phillip Hartig, L. Earl Gray Jr., Christy Lambright, Mary Cardon, Nicola 

Wrench, Johnathan Furr, Nathan Jeffay, Barbara Collins, Carol Mitchell 

National Institutes of Health

NIH Chemical Genomics Center, Menghang Xia, Srilatha Sakamuru, Ruili Huang, Paul Shinn, 

Christopher P. Austin 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

4 

Introduction / Background

The Deepwater Horizon oil spill has led to the use of large amounts of dispersant as part 

of the integrated approach dealing with the oil spill. Given this fact, questions have arisen about 

the toxicity of the chemicals used as dispersants themselves. EPA’s Office of Research and 

Development (ORD) was asked to carry out rapid studies to provide information on the potential 

for toxicity of eight commercially available dispersants. Because some of the dispersants 

reportedly include nonylphenol ethoxylates (NPEs) that can degrade to isomers of nonylphenol 

(NP), some of which are proven estrogenic compounds, the potential endocrine effects of the 

dispersants are of particular concern. For example, NPEs and NPs have been demonstrated to be 

endocrine disruptors in fish [1]. In response to the request ORD has undertaken a series of shortterm in vitro studies to determine if any of the dispersants displayed estrogenic, androgenic or 

other endocrine activity. 

ORD developed a strategy to address the questions of endocrine activity and relative 

toxicity as rapidly as possible. ORD scientists initiated several complementary studies of eight 

oil spill dispersants being used or considered for use in the Gulf. The issue was to provide some 

targeted information on the dispersants as quickly as possible. In vitro assays are well suited for 

that purpose. This work complements a study of whole animal toxicity in small fish and brine 

shrimp also being carried out by ORD. The results of that study are being simultaneously 

released with this report. 

One set of studies used a set of mammalian in vitro reporter gene assays in estrogenresponsive and androgen-responsive cells [2, 3] run in-house at ORD laboratories in RTP, NC. 

Additional studies were conducted by two external labs (NIH Chemical Genomics Center 

[NCGC] and Attagene Inc.) to run mammalian in vitro reporter gene assays to measure androgen 

and estrogen-response activity. A panel of 74 assays against non-endocrine molecular targets 

was also included in the Attagene assays. The NCGC and Attagene assays are part of the EPA 

ToxCast program [4, 5]. All assays evaluated the eight dispersants Corexit® 9500, JD 2000TM, 

DISPERSIT SPC 1000TM, Sea Brat #4, Nokomis 3-AA, Nokomis 3-F4, ZI-400 and SAF-RON 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

5 

GOLD. The performance of the assays was characterized by simultaneously running positive and 

negative control chemicals. Quantitative cytotoxicity measurements were carried out on each of 

the cell types used. All data analyses and interpretation were carried out by ORD staff. 

 

It is important to note that positive results in vitro only demonstrate that a chemical is a 

potential endocrine disruptor and that follow-up tests will likely be needed in order to refine or 

confirm the endocrine activity. For example, effects seen in vitro may not be expressed in vivo, 

so additional studies would need to be conducted to verify the in vitro results and determine if 

the potential activity was displayed in whole animals and the dosage levels required to affect 

organisms. 

. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

6 

Project Goals

1. Determine if any of the eight dispersants displayed estrogenic, androgenic or 

antiandrogenic activity in vitro using a variety of well characterized in vitro cell-based 

assays that utilize different approaches for detecting endocrine driven gene expression 

changes 

2. Determine the dispersant concentration that induced cytotoxicity in multiple cell lines 

and derive an aggregate measure of cytotoxicity that could be use to rank order the 

chemicals and to compare with in vivo toxicity data obtained in aquatic test species. 

Study Summary:

One part of the project was carried out by ORD researchers in partnership with the NIH 

Chemical Genomics Center [NCGC] and Attagene Inc. Two high throughput assay sets were run 

on the dispersants, a collection of reference chemicals for ER and AR activity, plus nonylphenol 

compounds. In addition to assays for AR and ER, this phase of the project produced data on a 

battery of other transcription factor assays which are part of multiplexed panels including AR 

and ER assays. Cytotoxicity was evaluated in three cell lines over a range of concentrations. 

 The other phase of the study was carried out in-house by ORD researchers using 

multiple assays [2, 3] to measure interaction between the eight dispersants plus reference 

chemicals and ER or AR. In particular, this work evaluated the eight dispersants for estrogen 

agonist activity in an estrogen-responsive transcriptional activation assays (ER-TA), for 

androgen agonist activity in two androgen-responsive transcriptional activation assays (AR-TA), 

MDA-kb2 and CV-1 assays and for androgen antagonist activity in the MDA-kb2 assay in 

competition with 1 nM Dihydrotestosterone (DHT). Cytotoxicity was evaluated in each assay at 

every concentration by both a biochemical assay which assessed metabolic perturbation and by a 

visual assessment of cytopathic effect on cell viability and morphology. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

7 

Chemicals  

All assays evaluated eight commercially available oil spill dispersants that were obtained 

directly from the respective manufacturers. EPA chose these eight dispersants from the 

dispersants listed on the National Contingency Plan Product Schedule based on three criteria: 1) 

lower toxicity of the dispersant or of the dispersant when mixed with oil; 2) availability of 

sufficient quantities to respond to the Gulf spill; and 3) immediate availability of samples for 

testing. These included Corexit® 9500 (Nalco Inc., Sugarland TX), JD 2000TM (GlobeMark 

Resources Ltd., Atlanta, GA), DISPERSIT SPC 1000TM (U.S. Polychemical Corp., Chestnut 

Ridge, NY), Sea Brat #4 (Alabaster Corp., Pasadena, TX), Nokomis 3-AA (Mar-Len Supply, 

Inc., Hayward, CA), Nokomis 3-F4 (Mar-Len Supply, Inc., Hayward, CA), ZI-400 (Z.I. 

Chemicals, Los Angeles, CA) and SAF-RON GOLD (Sustainable Environmental Technologies, 

Inc., Mesa, AZ). All are liquid solutions. Further information on the dispersants, including the 

limited publicly available information on the composition of dispersants is given in Appendix 

A.1. The oil spill dispersants were tested in vitro at concentrations ranging from 0.01 to 1000 

ppm in water (vol:vol). 

The assays run by NCGC and Attagene included reference compounds recommended for 

validating ER /AR assays by ICCVAM (Interagency Coordination Committee on the Validation 

of Alternative Methods)[6] and the U.S. EPA[7]. A preliminary set of reference compounds was 

obtained from stocks at EPA facilities in RTP NC. Subsequently, additional samples were 

obtained from Sigma-Aldrich (St. Louis MO). Included in the reference chemicals are both 

straight chain and branched NP isomers and corresponding example NPEs. The reference 

chemicals are 17-Trenbolone (10161-33-8), 17-Estradiol (50-28-2), Atrazine (1912-24-9), 

Bisphenol A (80-05-7), Butylbenzyl phthalate (85-68-7), Dibutyl phthalate (84-74-2), Flutamide 

(13311-84-7), Linuron (330-55-2), 4–Nonylphenol (linear) (104-40-5), p,p' –DDE (72-55-9), 

p,p'- Methoxychlor (72-43-5), Procymidone (32809-16-8), Vinclozolin (50471-44-8), 2,4,5-T 

(93-76-5), Bicalutamide (90357-06-5), Cyproterone acetate (427-51-0), Genistein (446-72-0), 4-

(tert-octyl), Phenol (140-66-9), 4-Hydroxytamoxifen (68392-35-8), 5-androstan-17-ol-3-one 

(521-18-6) and 4-Nonylphenol, (branched) (84852-15-3). The two nonylphenol ethoxylates are 

Tergitol NP-9 (127087-87-0) and Igepal CO-210 (68412-54-4). Reference chemicals (powder 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

8 

form) were solubilized in DMSO to a final concentration of 20 mM. Further information, 

including lot and batch are given in Appendix A.2. 

In the in-house ORD assays, a 17-Estradiol (E2; 50-28-2) dose response was included 

on every plate in the ER-TA assay as a positive control. 4-Nonylphenol (branched) (84852-15-3; 

Fluka) and 17-Trenbolone (Osaka Hayashi Pure Chemical Industries Ltd., CAS no. 80657-17-

6, purity 99.9%) were also tested in the estrogen mediated assays. A dihydrotestosterone (DHT; 

Sigma Chemical; CAS 55206-14-9) dose response was included as a positive control on every 

plate in the AR-TA assays. The potent androgen, 17-Trenbolone, was also tested in the 

androgen agonist assays. Dosing solutions of dispersants and reference compounds were 

prepared on-site under observation of a Quality Assurance manager. The assays used in the 

NHEERL assays have been demonstrated [2, 8] to give appropriate responses to known 

estrogenic or androgenic compounds. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

9 

Results

 More detailed assay protocols and statistical analysis methods can be found in the 

Appendices, as well as a Quality Assurance (QA) Statement. 

Androgen Receptor Agonist Activity

AR Agonist Assay 1 ­ Multiplexed reporter transcription unit (RTU) assay  

Method Summary: This assay is part of a multiplexed reporter gene panel run by Attagene Inc. 

(RTP, NC), under contract to the U.S. EPA (Contract Number EP-W-07-049). This assay 

consists of 48 human transcription factor DNA binding sites transfected into the HepG2 human 

liver hepatoma cell line as previously described[9]. This trans assay employs a mammalian onehybrid assay consisting of an additional 25 RTU library reporting the activity of nuclear receptor 

(NR) superfamily members. The human ligand-binding domain of each nuclear receptor was 

expressed as a chimera with the yeast GAL4 DNA-binding domain that activated in trans a 

5XUAS-TATA promoter, which regulated the transcription of a reporter sequence unique to 

each NR RTU. To ensure the specificity of detection, each individual trans-RTU system 

including both receptor and reporter gene was separately transfected into suspended cells 

followed by pooling and plating of the transfected cells prior to screening. The trans assay 

evaluates changes in activities of exogenous, chimeric NR-Gal4 proteins. This particular assay 

evaluated transcription for the Androgen receptor, and uses the code ATG_AR_TRANS. 

Additional detail of the method is provided in the Appendix B.1. Concentration-response 

titration points for each compound were fitted as described in Appendix C. For this analysis, 

there were either 4 replicates in 16 concentrations, except for SAF-RON GOLD which was only 

tested in 2 replicates and 8 concentrations. 

Results: No activity was seen for any of the dispersants 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

10 

AR Agonist Assay 2 ­ AR beta­lactamase Assay  

Method Summary: This assay was run at the NIH Chemical Genomics Center (NCGC; 

Rockville, MD) in collaboration with EPA as part of the Tox21 collaboration[10]. A betalactamase reporter-gene cell-based assay [GeneBLAzer® AR-UAS-bla-GripTiteTM assay 

developed by Invitrogen] was used to measure AR ligand signaling. AR-UAS-bla-GripTiteTM 

HEK 293 cells (AR bla cells) were used with assay medium containing 10% dialyzed FBS, 0.1 

mM NEAA and 1 mM sodium pyruvate. The assay was performed in clear bottom black Greiner 

1536-well plates. R1881, a synthetic androgen agonist, was used as a positive control in the 

screen. Library compounds were measured for their ability to either stimulate or inhibit the 

reporter gene activity. Compounds were screened in a titration series in 1536-well format. The 

fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured using an 

EnVision plate reader. Data was normalized relative to R1881 control (40 nM, 100%, for agonist 

mode and 10 nM, 0%, for antagonist mode), and DMSO only wells (basal, 0% for agonist mode 

and -100% for antagonist mode). Additional detail of the method is provided in the Appendix 

B.2. Concentration-response titration points for each compound were fitted as described in 

Appendix C. For this analysis, there were 8-10 replicates in 24 concentrations. 

Results: The only dispersant that showed any activity in any of the AR assays was JD 2000, 

which was active in both the NCGC ER and AR agonist and antagonist assays in all runs with 

AC50 values ranging from 100-270 ppm (AR) and 82-120 ppm (ER). There was no apparent 

cytotoxicity in any of the cell line for JD 2000 (see results below). The EMax values for JD 2000 

in all of these assays were significantly greater than the values for positive control chemicals, 

and in the antagonist assays, this dispersant looked like a “super-activator” rather than an 

antagonist. All of this data taken together indicates strongly that some non-specific activation is 

occurring that is independent of ER or AR. We have found previously that compounds identified 

as promiscuous “super-activators” in multiple beta-lactamase reporter gene assays with a narrow 

potency range (a <3-fold difference in potency is within the experimental variations of these 

assays) are mostly auto fluorescent (R Huang, unpublished data). Thus, the activity observed for 

JD 2000 is likely an artifact of the beta-lactamase assay format. Preliminary results from three 

additional beta-lactamase assays for non-steroid receptor targets all showed the JD 2000 “super-
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

11 

activation”. Considering the totality of the data, we conclude that JD 2000 does not exhibit ER or 

AR transactivation activity. To further confirm that this JD 2000 activity is non-specific and not 

due to ER or AR activation, we are running several follow-up assays with NCGC: known 

antiestrogens and antiandrogens are being used to show that JD 2000 activity is not suppressed; 

and we will complete our analysis of results for the the three non-steroid receptor beta-lactamase 

assays are being run with JD 2000 to show that this non-specific activity occurs independent of 

ER and AR. 

AR Agonist Assay 3 ­ MDA­kb2 Androgen­responsive transcriptional activation assay  

Method Summary: This assay, run in-house by NHEERL researchers, utilized MDA-kb2 

cells[2]. These cells contain endogenous human androgen receptor capable of inducing 

transcription of an androgen responsive gene (AR-TA). This assay employs a luciferase gene 

driven by the androgen responsive MMTV promoter which has been stably integrated into the 

cells. When androgen mimicking compounds (i.e. compounds that act as androgen agonists) are 

present, these cells produce luciferase in a concentration proportionate to the efficacy of the 

androgen mimic. Nine concentrations of each dispersant were tested for agonist activity. Each 

concentration was evaluated in a total of eight replicates (two independent evaluations with four 

replicates per assay). The first dilution of each sample was a 1:100 dilution (i.e. 0.01 dilution or 

10,000 ppm) of the dispersant in cell culture medium followed by eight additional 10-fold serial 

dilutions. Additional detail of the method is provided in the Appendix F. 

Results: The ability of the eight dispersants to stimulate luciferase expression in this cell line was 

compared to DHT. The DHT positive control dose induced luciferase expression in MDA Kb2 

cells in a precise and reproducible manner within and among the plates (Figure 1. DHT and 17 

-Trenbolone data in MDA Kb2 cells). None of the eight dispersants displayed any potential 

androgenicity (i.e. did not simulate luciferase induction) at any concentration in the MDA Kb2 

cell line (Figure 2 dispersant results in MDA Kb2 cells). In fact, all the dispersants displayed 

significantly reduced luciferase levels due to cytotoxicity at high dispersant concentrations. The 

synthetic androgen 17-Trenbolone acted as a full androgen agonist at relatively low 

concentrations. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

12 

MDA Kb2 cell DHT

DATA FROM FIVE PLATES

10 -12 10 -11 10 -10 10 -9 0

2

4

6

8

10

12 Plate 1

Plate 2

Plate 3

Plate 4

Plate 5

LogEC50

Plate 1

-9.843

Plate 2

-9.776

Plate 3

-9.871

Plate 4

-9.553

Plate 5

-9.923

Concentration (M)

Fold Induction

17  Trenbolone

MDA Kb2 CELLS

10-14

10-1310-1210-1110-10

10-910-810-710-6

0

2

4

6

8

10

12 LogEC50 -9.945

Concentration (M)

Fold Induction

CV1 DHT

DATA FROM 5 PLATES

-12 -11 -10 -9

0

10

20

30 PLATE 1

PLATE 2

PLATE 3

PLATE 4

PLATE 5

LogEC50

PLATE 1

-9.667

PLATE 2

-9.558

PLATE 3

-9.726

PLATE 4

-9.663

PLATE 5

-9.678

Concentration (M)

Fold Induction

Hydroxyflutamide a known antiandrogen used

as a positive control for inhibition of androgen action

10-10 10-9 10-8 10-7 10-6 10-5 10-4 0

2

4

6

8

Concentration (M)

Fold Induction

LogEC50 -6.439

Figure 1: Included are, nonlinear regression plots of the effects of the reference androgen 

dihydrotestosterone (DHT) in two androgen sensitive cell lines (MDA Kb2 upper left and CV1 

upper right), stimulatory effects of the synthetic androgen found in some aquatic systems in 

MDA Kb2 cells (lower left) and antagonism of the 1 nM DHT by the antiandrogenic drug 

hydroxyflutamide in MDA Kb2 cells. Data are expressed as fold over the media plus the ethanol 

control value. The X axis is in log scale. Values are means plus or minus standard errors of the 

mean.
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

13 

JD 2000

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

Dispersit

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

SEA BRAT

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

NOKOMIS 

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10

0

05

1015

PPM

Fold Induction

NOKOMIS 3-F4

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

COREXIT 9500

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

ZI 400

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.00000 100.00000 1000.00000 10000.00000

05

1015

PPM

Fold Induction

Saf-Ron G

0.00000 0.00010 0.00100 0.01000 0.10000 1.00000 10.0

05

1015

PPM

Fold Induction

Assessment of Potential Androgenicity in MDA Kb2 cells. The dispersants d

induce luciferase expression in an androgenic manner

Figure 2: Assessment of the potential androgenic activity of the eight dispersants in MDA Kb2 cells. Data are e

the media plus ethanol vehicle control. Values are means plus or minus standard errors of the mean. Dispersants luciferase induction over the control fold value (control fold =1). 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

14 

AR Agonist Assay 4 ­ CV­1 transient transcription assay  

Method Summary: This assay run in-house by NHEERL is similar to the MDA-kb2 in that it also 

assesses the ability of a compound to mimic an androgen. This assay, however, uses CV-1 cells 

which do not express either endogenous androgen or estrogen receptors. In contrast to the MDAkb2 assay, both the androgen receptor and the androgen responsive MMTV promoter- luciferase 

reporter constructs are introduced into the CV-1 cells for each assay via transient transfection. 

Nine concentrations of each dispersant were tested for agonist activity in both AR-TA assays. 

Each concentration was tested in quadruplicate. The first dilution of each sample was a 1:100 

dilution (i.e. 0.01 dilution or 10,000 ppm) of the dispersant in cell culture medium followed by 

eight additional 10-fold serial dilutions. Method details are provided in the Appendix F. 

Results: Similar to the results of the MDA-kb assays, DHT induced precise and reproducible 

effects on luciferase expression within and among the plates (Figure 1) and none of the eight 

dispersants displayed any potential androgenicity (i.e. did not simulate luciferase induction) at 

any concentration in the CV-1 assay (Figure 3). In fact, all the dispersants significantly reduced 

luciferase level due to cytotoxicity at high concentrations. 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

15 

JD 2000

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

DISPERSIT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

SEA BRAT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

NOKO

0.0000 0.0001 0.0010 0.0100 0

02468

10

Fold Induction

NOKOMIS 3-F4

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

COREXIT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

ZI 400

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

02468

10

PPM

Fold Induction

SAF-R

0.0000 0.0001 0.0010 0.0100 0

02468

10

Fold Induction

Assessment of Potential Androgenic activity in CV-1 cells. Th

dispersants did not induce luciferase expression in an androgenic 

Figure 3: Assessment of the potential androgenic activity of the eight dispersants in CV-1 cells. Data are expresmedia plus ethanol vehicle control. Values are means plus or minus standard errors of the mean. Dispersants did luciferase induction over the control fold value (control fold =1). 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

16 

Androgen Receptor Antagonist Activity

AR Antagonist Assay 1 ­ MDA­kb2 Androgen­responsive transcriptional activation

assay in antagonist mode  

Method Summary: The eight dispersants were also evaluated for antagonist activity in the MDAkb2 cell line, run in-house by NHEERL researchers, by testing each dispersant in the presence of 

a near maximally stimulating concentration dihydrotestosterone (1 nM DHT). In the presence of 

an anti-androgen, the luciferase activity induced by DHT would be reduced proportionally to the 

concentration of the anti-androgen. A DHT concentration-response curve was included on each 

96-well plate with the dispersants. The well-characterized antiandrogen hydroxyflutamide (CAS 

80657-17-6) was run as a positive control (Figure 1). Six concentrations of each dispersant 

ranging from 0.0001 ppm to 10 ppm were tested for antagonist activity. Higher concentrations 

were not evaluated due to cytotoxicity seen in both MTT and CPE assays (discussed later in this 

document). Each concentration was tested in quadruplicate. Additional detail of the method is 

provided in the Appendix F. 

Results: None of the eight dispersants displayed any potential antiandrogenicity (i.e. did not 

inhibit DHT-induced luciferase induction) at concentrations below 10 ppm (1E-5 dilution). At 10 

ppm several of the dispersants reduced DHT induced luciferase activity, but the effects were 

significant (by ANOVA followed by a post hoc Dunnett’s test) for the dispersants SPC 1000 and 

Nokomis 3-AA (Figure 4). As shown in Figure 5a, these two dispersants were the most toxic 

of the dispersants to MDA Kb2 cells so it is extremely unlikely that these effects represent 

competitive inhibition of DHT binding to the ligand binding domain of the androgen receptor. 

In contrast, hydroxyflutamide, used as a positive control, completely inhibited androgen-induced 

luciferase induction at concentrations about 1000 fold higher than of the concentration of DHT 

used in this assay. 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

17 

JD 2000

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

DISPERSIT

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

*

DISPERSANT CONC IN PPM

Fold Induction

SEA BRAT

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

NOKOMIS 3AA

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

*

DISPERSANT CONC IN PPM

Fold Induction

NOKOMIS 3-F4

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

COREXIT 9500

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

ZI 400

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

Saf-Ron Gold

0.00000.00010.00100.01000.10001.0000 10.0000

02468

10

DISPERSANT CONC IN PPM

Fold Induction

Assessment of potential antiandrogenicity of Dispersants in MDAKb2 cells. The dispersant did not compete with the 1 nM DHT in theassay and lower luciferase expression in an antiandrogenic manner

* indicates p < 0.01 by Dunnett's test following ANOVA

Figure 4: Assessment of the potential antiandrogenic activity of the eight dispersants in MDA Kb2 cells. Data aover the media plus ethanol vehicle control. Values are means plus or minus standard errors of the mean. DISPENokomis 3-AA were the only dispersants that significantly reduced DHT-stimulated luciferase induction (DHT can effect which we interpreted to result from cytotoxicity at 10 ppm, the highest concentration used in this assay. 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

18 

DISPERSANTS

MDA Kb2 CELL MTT ASSAY

SPC 1000

NOKOMIS 3 AA NOKOMIS 3F4 SEA BRAT JD 2000 COREXIT 9500ZI 400 SAF-RON GOLD

012345

LOG 10 EC50

DISPERSANTS

T47D CELL MTT ASSAY

SPC 1000 SEA BRAT

NOKOMIS 3 AA COREXIT 9500 NOKOMIS 3F4ZI 400 SAF-RON GOLD JD 2000

012345

LOG 10 EC50

DISPERSANTS

CV1 CELL MTT ASSAY

SPC1000 COREXIT

NOKOMIS 3AA SEA BRATZI 400 NOKOMIS 3F4 SAF-RON GOLD JD2000

012345 SPC1000

COREXITNOKOMIS 3AASEA BRATZI 400NOKOMIS 3F4SAF-RON GOLD

LOG 10 EC50 JD2000

DISPERSANT MTT DISPERSANT CPE

Rank Rank

Dispersit 1 Dispersit 1

Nokomis 3 AA 2 Corexit 2

Seabrat 3 JD 2000 3

Corexit 4 Nokomis 3 aa 4

Nokomis 3-F4 5 Nokomis 3-F4 5

ZI 400 6 Seabrat 6

JD 2000 7 ZI 400 7

Saf-Ron Gold 8 Saf-Ron Gold 8

Figure 5: Summary of the Cytotoxic effects of the eight dispersants in the MTT and visual cytopathic examinati(CPE) in three cell lines. The histograms display the EC50 values for a reduction in MTT levels (determined by nonlinear regression on Prism 5.0) for the MDA Kb2 (upper right panel), CV1 (upper right panel) and T47D Kbl
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

19 

(lower left panel) cells. In the table in the lower right panel, the overall potency of the dispersants in the MTT an

assays is shown, with a ranking of 1 being the most potent in inducing cytotoxicity and 8 being the least cytotoxidispersant. DISPERSIT SPC 1000 was ranked as the most cytotoxic by both methods and SAF-RON GOLD is t

toxic of the eight dispersants. (The appendices contain additional details on this and the EC50 values are compara multiple range test). 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

20 

Estrogen Receptor Agonist Activity

ER Agonist Assay 1 ­ Multiplexed reporter transcription unit (RTU) trans assay

Method Summary: This assay is part of a multiplexed reporter gene panel run by Attagene Inc. 

(RTP, NC), under contract to the U.S. EPA (Contract Number EP-W-07-049). This assay 

consists of 48 human transcription factor DNA binding sites transfected into the HepG2 human 

liver hepatoma cell line as previously described[9]. This trans assay employs a mammalian onehybrid assay consisting of an additional 25 RTU library reporting the activity of nuclear receptor 

(NR) superfamily members. The human ligand-binding domain of each nuclear receptor was 

expressed as a chimera with the yeast GAL4 DNA-binding domain that activated in trans a 

5XUAS-TATA promoter, which regulated the transcription of a reporter sequence unique to 

each NR RTU. To ensure the specificity of detection, each individual trans-RTU system 

including both receptor and reporter gene was separately transfected into suspended cells 

followed by pooling and plating of the transfected cells prior to screening. The trans assay 

evaluates changes in activities of exogenous, chimeric NR-Gal4 proteins. This particular assay 

evaluated transcription for the Estrogen receptor alpha, and uses the code ATG_ERa_TRANS. 

This assay was run in twice in separate weeks, and in each case, run in duplicate. For this 

analysis, there were either 4 replicates in i6 concentrations, except for SAF-RON GOLD which 

was only tested in 2 replicates and 8 concentrations. Additional detail of the method is provided 

in the Appendix B.1. Concentration-response titration points for each compound were fitted as 

described in Appendix C.

Results: We observed statistically significant ER activity in two of the dispersants in the 

Attagene trans-ER assay (Nokomis 3-F4 and ZI-400), detailed in Table 1. Figure 6 (bottom 

panels) shows the concentration-response curves for the two active dispersants, which have 

EMax (maximum efficacy) values of between 3 and 4. This is in contrast to 17-Estradiol (top 

left panel, blue curve), which has an EMax value of 20. The top right panel of the figure shows 

the corresponding reference curve for the cis-ERE assay, showing that 17-Estradiol only elicits 

a response about half of that seen in the trans assay. To help interpret these results, we 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

21 

simultaneously analyzed their performance on a set of 19 reference chemicals recommended by 

ICCVAM[6] and EPA OPPT[7]. This analysis (detailed in Appendix E) shows that these assays 

perform well for both positive and negative predictive value. The trans-ER assay correctly 

matched ICCVAM expectation for 15 of 17 reference chemicals, with one false positive and one 

false negative. A comparison of the cis and trans assays shows that the reference chemicals in 

the cis assay consistently produce EMax values about half of that seen in the trans assay. This 

would explain the absence of observable activity for these dispersants in the cis assay, because 

we do not consider curves with EMax values below 2. The other curves in the bottom panels of 

Figure 6 show data for NP and NPE compounds, described below. 

Chemical AC50 

(ppm) 

EMax R2

 p-value 

Nokomis 3-F4 16 3.9 0.65 0.00017 

ZI-400 25 3.4 0.68 0.0041 

Table 1: Summary results for the Attagene trans-ER assay for the positive dispersants. EMax: 

maximal fold change. AC50: concentration at which 50 of maximal activity is seen. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

22 

Figure 6: Concentration-response curves for the E2, NP and NPE compounds, and the two 

dispersants showing activity in Attagene trans-ER assay. Top: E2 and the 4 NP / NPE 

compounds in the Attagene trans-ER assay (left) and the Attagene cis-ERE assay (right). 

Bottom: ZI-400 and Nokomis 3-F4 in the Attagene trans-ER assay. For the nonylphenol 

compounds, only two replicates were run. 

Nonylphenol-related activity: It is known that some of the dispersants contain NPEs. Our initial 

hypothesis was that any estrogenic activity detected for the complex mixtures could be due to the 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

23 

NPEs or to NP itself generated by in situ degradation of the NPE, or residual contamination from 

synthesis of the NPE. Consequently, we tested two nonylphenols (one linear and one branched, 

technical grade) and two commercial NPEs in the Attagene assays. Table 2 shows the results of 

this analysis, and Figure 6 shows the corresponding dose-response curves for the Attagene ER 

assays. From these data, one can see that these cell-based assays show ER activity for both the 

NPs and the NPEs. The branched, technical grade NP is the most potent, but the second most 

potent is the NPE Igepal CO-210. These data indicates that the presence of an NP or NPE in a 

mixture could give rise to ER activity such as was seen for the dispersants Nokomis 3-F4 and ZI400. Public information (given in Appendix A) indicates that ZI-400 does in fact contain an 

NPE. 

Chemical Assay AC50 

(M) 

R2 EMax p-value 

4-Nonylphenol (linear) trans -ER 11 0.77 8.3 0.29 

104−40−5 cis-ERE 4.3 0.55 2.7 0.096 

4-Nonylphenol (branched) trans -ER 0.68 0.91 12 0.0049 

84852−15−3 cis-ERE 0.61 0.092 5.4 4.9E-5 

Tergitol NP-9 trans -ER 5.7 0.86 4.8 0.18 

127087−87−0 cis-ERE 5.6 0.96 2.1 0.042 

Igepal CO−210 trans -ER 2.5 0.89 8.5 0.19 

68412−54−4 cis-ERE 14 0.96 6.5 2.1E-11 

Table 2: Results of ER assays on NPs and NPEs. 

To summarize this section, estrogen receptor (ER) activity was observed in two of the 

dispersants in the Attagene trans-ER assay (ZI-400 and Nokomis 3-F4), although at relatively 

high concentrations and with low efficacy (EMax). We have also shown that NPs and NPEs are 

also active in the trans-ER assay. Therefore, the activity in ZI-400 and Nokomis 3-F4 is 

suggestive of the presence of an NP or NPE as part of the mixture. We know that this is the case 

with ZI-400. The ER effect seen for these dispersants is weak, which is also suggestive of there 

being only a relatively small amount of NPE or some other estrogenic substance in the total 

mixture. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

24 

ER Agonist Assay 2 ­ Multiplexed reporter transcription unit (RTU) cis assay

Method Summary: This assay is part of a multiplexed reporter gene panel run by Attagene Inc. 

(RTP, NC), under contract to the U.S. EPA (Contract Number EP-W-07-049). This assay 

consists of 48 human transcription factor DNA binding sites transfected into the HepG2 human 

liver hepatoma cell line as previously described[9]. A major difference between the cis and trans

system is that in cis activities of endogenous transcription factors are measured. This particular 

assay evaluated transcription for the Estrogen receptor element (ERE), and uses the code 

ATG_ERE_CIS. For this analysis, there were either 4 replicates in i6 concentrations, except for 

SAF-RON GOLD which was only tested in 2 replicates and 8 concentrations. Additional detail 

of the method is provided in the Appendix B.1. Concentration-response titration points for each 

compound were fitted as described in Appendix C.

Results: No statistically significant activity was seen for any of the dispersants 

ER Agonist Assay 3 – ER­alpha beta­lactamase Assay

Method Summary: This assay was run at the NIH Chemical Genomics Center (NCGC; 

Rockville, MD) in collaboration with EPA as part of the Tox21 collaboration[10]. A betalactamase reporter-gene cell-based assay [ER-UAS-bla GripTiteTM cell-Based Assay from 

Invitrogen] was used to measure ER signaling pathway both in agonist and antagonist modes. 

ER-UAS-bla-GripTiteTM HEK 293 cells (ER bla cells) were used with assay medium 

containing 2% charcoal/dextran treated FBS, 0.1 mM NEAA and 1 mM sodium pyruvate. Cells 

were cultured in this assay medium overnight in the flasks before the assay. The assay was 

performed in clear bottom black Greiner 1536-well plates. 17β-estradiol was used as a positive 

control in the screen. Library compounds were measured for their ability to either stimulate or 

inhibit the reporter gene activity. Compounds were screened in a titration series in 1536-well 

format. The fluorescence intensity (405 nm excitation, 460/530 nm emission) was measured 

using an EnVision plate reader. Data was normalized relative to 17β-estradiol control (20 nM, 

100%, for agonist mode and 0.5nM, 0%, for antagonist mode), and DMSO only wells (basal, 0% 

for agonist mode and -100% for antagonist mode). Concentration-response titration points for 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

25 

each compound were fitted to the Hill equation yielding concentrations of half-maximal 

stimulation (EC50), half-maximal inhibition (IC50) and maximal response (efficacy) values. For 

this analysis, there were 8-10 replicates in 24 concentrations. Additional detail of the method is 

provided in the Appendix B.3. Concentration-response titration points for each compound were 

fitted as described in Appendix C.

Results: No biologically relevant results were seen for any of the dispersants. See the description 

above under the corresponding AR assay for JD 2000. 

ER Agonist Assay 4 ­ T47D­KBluc estrogen­responsive transcriptional activation

assay

Method Summary: T47D-KBluc, is an estrogen receptor-mediated transcriptional activation 

assay (ER-TA) that detects the ability of chemicals to mimic estrogen[8]. This assay was run inhouse by NHEERL researchers. The cells contain endogenous human estrogen receptors alpha 

and beta and are stably integrated with an engineered luciferase reporter gene controlled by 

triplet estrogen response elements. When the cells are exposed to hormone mimics, the 

mimicking chemical binds the estrogen receptor and activates production of the luciferase 

reporter gene. The luciferase product is measured in a light emitting reaction. Additional detail 

of the method is provided in the Appendix F. 

Results: The ability of the eight dispersants to stimulate luciferase expression in this cell line was 

compared to 17β-Estradiol (CAS 50-28-2: a concentration-response curve to E2 was included on 

each 96 well plate with the dispersants) and to 4-Nonylphenol (branched) (CAS 84852-15-3) 

(Figure 7 a,b). 17α-Trenbolone (CAS 80657-17-6) was run as a negative control herein (Figure 

7d) and as a positive control in the assessment of androgenicity. None of the eight dispersants 

displayed any potential estrogenicity (i.e. did not simulate luciferase induction) at any 

concentration in the current investigation (Figure 8). In fact, all the dispersants significantly 

reduced luciferase levels at high concentrations due to cytotoxicity.
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

26 

4 NONYLPHENOL

10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4

0

10203040

LogEC50 -6.657

Concentration (M)

Fold Induction

 E2 luciferase induction

from nine plates

10-13 10-12 10-11 10-10

0

10203040

Fold Induction

10-13 10-12 10-11 10-10

0

20406080

100 LogEC50 Plate 1 -11.53

Plate 2 -11.44Plate 3 -11.43Plate 4 -11.2

Plate 5 -11.46

Plate 6 -11.49

Plate 7 -11.67Plate 8 -11.55Plate 9 -11.5

Concentration (M)

PERCENT

OF MAX

17 alpha Trenbolone

1e-009 1e-008 3e-008 1e-007 3e-007 1e-006 3e-006 1e-005 3e-005 1e-004 3e-004

012345

Concentration (M)

Fold Induction

Figure 7: Estradiol 17β-induced (E2) luciferase expression in the nine plates used in the current study with T47D expressed as fold over media plus ethanol vehicle control (upper left) and percent of the maximal E2 stimulation 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

27 

effects of the xenoestrogen 4-Nonlyphenol (Branched) are shown in the upper right and the lack of estrogenicity 

androgen 17-Trenbolone are shown in the lower right panels. Values are means plus or minus standard errors o

JD 2000

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

PPM

Fold Induction

Dispersit

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

PPM

Fold Induction

SEA BRAT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

PPM

Fold Induction

NOKO

0.0000 0.0001 0.0010 0.0100 0.10

012345

P

Fold Induction

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

NOKOMIS 3-F4

PPM

Fold Induction

COREXIT 9500

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

PPM

Fold Induction

ZI 400

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

012345

PPM

Fold Induction

Saf-

0.0000 0.0001 0.0010 0.0100 0

012345

Fold Induction

Dispersants do not display any indication of estrogenic activity in T47D Kb
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

28 

Figure 8: Assessment of the potential estrogenic activity of the eight dispersants in T47D Kbluc cells. Data are the media plus ethanol vehicle control. Values are means plus or minus standard errors of the mean. Dispersantluciferase induction over the control fold value (control fold =1). 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

29 

Cytotoxicity

Cytotoxicity Assay 1 –HepG2 Cells

Method Summary: Dispersants were tested for cytotoxicity against HepG2 cells in the MTT (3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay (15) following 24 

h chemical exposure to 16 concentrations with an upper concentration of 1000 ppm. All 

concentrations were run in triplicate. This assay was run by Attagene Inc. LC50 values were 

determined by fitting curves as described in Appendix C. Results of cytotoxicity assessment are 

shown below. For this analysis, there were either 4 replicates in 16 concentrations, except for 

SAF-RON GOLD which was only tested in 2 replicates and 8 concentrations. 

Cytotoxicity Assay 2 –AR bla Cells

Method Summary: Cell viability after compound treatment was measured in these AR bla cells 

using a luciferase-coupled ATP quantitation assay (CellTiter-Glo viability assay, Promega). This 

assay was run by the NIH Chemical Genomics Center. The change of intracellular ATP content 

indicates the number of metabolically competent cells after compound treatment. The cells were 

dispensed at 2,000 cells/5 L/well for AR bla cells in 1,536-well white/solid bottom assay plates 

using an FRD. The cells were incubated for 5 hrs at 37C, followed by the addition of 

compounds using the pin tool. The final concentration range for reference compounds was 11 

pM to 92 M, and 0.000144 ppm to 1209.8 ppm for dispersants. The assay plates were incubated 

for 16 hrs at 37C, followed by the addition of 5 L/well of CellTiter-Glo reagent. After 30 min 

incubation at room temperature, the luminescence intensity of the plates was measured using a 

ViewLux plate reader (PerkinElmer). Data was normalized relative to DMSO only wells (0%), 

and tetra-n-octylammonium bromide (92 M, -100%). LC50 values were determined by fitting 

curves as described in Appendix C. Results of cytotoxicity assessment are shown below. For 

this analysis, there were 8-10 replicates in 24 concentrations. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

30 

Cytotoxicity Assay 3 –ER bla Cells

Method Summary: Cell viability after compound treatment was measured in these ER bla cells 

using a luciferase-coupled ATP quantitation assay (CellTiter-Glo viability assay, Promega). This 

assay was run by the NIH Chemical Genomics Center. The change of intracellular ATP content 

indicates the number of metabolically competent cells after compound treatment. The cells were 

dispensed at 5,000 cells/5 L/well for ER bla cells in 1,536-well white/solid bottom assay 

plates using an FRD. The cells were incubated a 5 h at 37C, followed by the addition of 

compounds using the pin tool. The final concentration range for reference compounds was 11 

pM to 92 M, and 0.000144 ppm to 1209.8 ppm for dispersants. The assay plates were incubated 

for 18 hrs at 37C, followed by the addition of 5 L/well of CellTiter-Glo reagent. After 30 min 

incubation at room temperature, the luminescence intensity of the plates was measured using a 

ViewLux plate reader (PerkinElmer). Data was normalized relative to DMSO only wells (0%), 

and tetra-n-octylammonium bromide (92 uM, -100%). LC50 values were determined by fitting 

curves as described in Appendix C. Results of cytotoxicity assessment are shown below. For 

this analysis, there were 8-10 replicates in 24 concentrations. 

Cytotoxicity Results (Assays 1­3)  

Results of Cytotoxicity Assays 1-3 are summarized below the description of Assays 4-6. 

Cytotoxicity Assays 4 THRU 9: measurements in T47D­KBluc, MDA­kb2, and CV­1

cells (MTT and CPE assessments) (5 independent assessments).

Methods summary: The ability of the dispersants to produce a general toxic effect on each of the 

cell lines used in the in the NHHERL in-house assays was assessed by both observational and 

biochemical methods. First, each well of cells in every assay was evaluated by visual 

microscopic examination utilizing a five point cytopathic effect (CPE) criteria scale ranging from 

0 (no visual toxicity) to 4 (total cell death). CPE assessment criteria were as follows: 0 = no 

observed effect; 1= subtle changes suggesting effect; 2 = definite effects or death in a at least 

25% of cells; 3 = 50 to 75% of cells effected; 4 = 100% of cells effected/cell death. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

31 

Second, an assessment of the metabolic perturbation of cell health was quantitated by 

monitoring the ability of cells to metabolize 3-[4,5-Dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT)[11]. In this biochemical assay, healthy cells are capable of 

converting a yellow MTT solution into a blue dye. The healthier the cell the more blue dye 

produced. This biochemical assay is an indicator that the cells are metabolically active and is a 

measurement of general cell health. The MTT assay is a quantitative evaluation of 

mitochondrial function of the cells whereas the first method was a qualitative microscopic 

cytopathological evaluation (CPE) of cell viability and morphology 

Cytotoxicity Results (Assays 4­9)

All eight dispersants disrupted cell function and caused cell death in all three cell lines in 

the two highest concentrations (0.01 and 0.001, or 10,000 and 1,000 ppm, respectively). 

Furthermore, none of the dispersants produced any sign of cytotoxicity at concentrations below 1 

ppm (Figures 9-12). 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

32 

Dispersit

1 10 100

02468

1012

CV1MDA KB2T47DKBLUC

Concentration ppm

CELL TOXICITY SCORE

COREXIT 9500

1 10 100

02468

1012

CV1MDA KB2T47DKBLUC

Concentration ppm

CELL TOXICITY SCORE

JD 2000

1 10 100

02468

1012

Concentration ppm

CV1

MDA KB2

T47DKBLUC

CELL TOXICITY SCORE

NOKO

1 

02468

1012

CV1MDA KT47DK

Concent

CELL TOXICITY SCORE

NOKOMIS 3-F4

1 10 100

02468

1012

CV1MDA KB2T47DKBLUC

Concentration ppm

CELL TOXICITY SCORE

SEA BRAT

1 10 100

02468

1012

CV1MDA KB2T47DKBLUC

Concentration ppm

CELL TOXICITY SCORE

ZI 400

1 10 100

02468

1012

CV1MDA KB2T47DKBLUC

Concentration ppm

CELL TOXICITY SCORE

Cytopathological evaluation of dispersant cytotoxicity(eight plates/dispersant, four replicate wells/plate/conc) inMDAKb2, CV 1 and T47D Kbluc cells. Figures are rankedfrom top left to bottom right in cytotoxic potency at 1, 10and 100 ppm. At higher concentrations CPE scores were3-4 for all dispersants and no CPE was observed atconcentrations below 1 ppm. The maximum total CPEscore is 12, 4 per assay

Saf-Ro

1 

02468

1012

CV1MDA KT47DK

Concent

CELL TOXICITY SCORE

cytopathic effect: defined

no CPE none observedCPE +1 subtle changes suggesting effe

CPE+2 definite changesin more than 

CPE+3 50‐75 % of cells effected

CPE+4 100% cells effected/dead

Figure 9: 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

33 

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

JD 2000

PPM

PERCENT

OF CONTROL

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

DISPERSIT

PPM

PERCENT

OF CONTROL

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

SEA BRAT

PPM

PERCENT

OF CONTROL

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.000

1

0

255075

100125

NOKOMIS 3 

PPM

PERCENT

OF CONTROL

NOKOMIS 3-F4

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

PPM

PERCENT

OF CONTROL

COREXIT 9500

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

PPM

PERCENT

OF CONTROL

ZI 400

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100

PPM

PERCENT

OF CONTROL

Saf-Ron Go

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.000

1

0

50

100150

PPM

PERCENT

OF CONTROL

MDA Kb2 CELL MTT ASSAY OF CELL FUNCTION

Figure 10: Toxic effects of the eight dispersants on MMT levels in MDA Kb2 cells. A reduction in MTT levels 

cytotoxcity, seen with all dispersants at the two higher concentrations (1000 and 10,000 ppm) and at 100 ppm wi

dispersants. Data are expressed as percent of control (media). 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

34 

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

JD 2000

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

DISPERSIT

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

SEA BRAT

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0

50

100

PERCENT

OF CONTROL

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

NOKOMIS 3-F4

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

COREXIT 9500

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0.0010

0.0100

0.1000

1.0000

10.0000

100.0000

1000.0000

10000.0000

0

50

100

ZI 400

PPM

PERCENT

OF CONTROL

0.0000

0.0001

0

50

100

PERCENT

OF CONTROL

CYTOTOXICITY OF DISPERSANTS TO CV 1 CELLS IN THE MTT ASSAY

Figure 11: Toxic effects of the eight dispersants on MMT levels in CV-1 cells. A reduction in MTT levels is an cytotoxcity, seen with all dispersants at the two higher concentrations (1000 and 10,000 ppm) except JD 2000 (n

any concentration) and SAF-RON GOLD (toxicity was seen only at the highest concentration of 10,000 ppm). Dand Corexit also induced cytotoxicity at 100. Data are expressed as percent of control (media). 
U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

35 

Figure 12: Toxic effects of the eight dispersants on T47D Kbluc cells. A reduction in MTT levels is an indicato

with all dispersants at the two higher concentrations (1000 and 10,000 ppm) and at 100 ppm with several of the d

expressed as percent of control (media).

JD 2000

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125

PPM

PERCENT

OF CONTROL

DISPERSIT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125

PPM

PERCENT

OF CONTROL

SEA BRAT

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125

PPM

PERCENT

OF CONTROL

NOKOMIS 3AA

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000

0

1

0

255075

100125

PPM

PERCENT

OF CONTROL

NOKOMIS 3-F4

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125

PPM

PERCENT

OF CONTROL

COREXIT 9500

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125

PPM

PERCENT

OF CONTROL

ZI 400

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000.0000 10000.0000

0

255075

100125150

PPM

PERCENT

OF CONTROL

SAF-RON GOLD

0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 100.0000 1000

0

1

0

255075

100125

PPM

PERCENT

OF CONTROL

Cytotoxic effects of dispersants on T47D Kbluc cells in the MTT Assay
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

36 

The lowest observed effective concentration (LOEC) for dispersant-induced reductions in MTT, 

estrogen, androgen and antiandrogen assays are reported in Table 3. In the table, the noted changes 

in the two androgen and the estrogen agonist assays do not result from hormone-like increases in 

luciferase activity but rather represent significant reductions in luciferase expression that likely result 

from the cytotoxic effects of the dispersants. Statistical significance was determined using analysis 

of variance followed by t-tests (LSMEANS) using PROC GLM on SAS 9.1 (p< 0.01 was used as the 

critical value to determine statistical significance). 

MDA Kb2 cells CV1 cells T47D Kbluc cells 

MTT 

Cytotoxicity 

Androgen 

Antagonist 

Assay*** 

Androgen 

Agonist 

Assay 

MTT 

Cytotoxicity 

Androgen 

Agonist 

Assay 

MTT 

Cytotoxicity 

Estrogen 

Agonist Assay 

Dispersant LOEC* 

(ppm) 

LOEC 

(ppm) 

LOEC 

(ppm) 

LOEC 

(ppm) 

LOEC 

(ppm) 

LOEC 

(ppm) 

LOEC 

(ppm) 

JD 2000 100 >10 1,000 10,000** 1,000 1,000** 10,000** 

DISPERSIT 

SPC 1000 

100 10 100 

10 100 10 

100 

Sea Brat #4 100 >10 100 1,000** 1,000 10,000 1,000 

Nokomis 3-

AA 100 10 10 1,000 1,000 100 1,000 

Nokomis 3-

F4 

100 >10 10 

1,000 1,000 1,000 

1,000 

Corexit 9500 

1,000 >10 1,000 100 1,000 100 1,000 

ZI-400 1,000 >10 100 1,000 1,000 1,000 1,000 

SAF-RON 

GOLD 10,000 >10 1,000 10,000 1,000 1,000 10,000 

*LOEC (ppm) represents the lowest concentration at which the dispersant consistently reduced the MTT value. Statistical significance 

was using p<0.01 as determined using LSMEANS option of PROC GLM available on SAS 9.1. 

** LOEC concentration was equivocal (nonmonotonic response) 

*** Antagonist assay for antiandrogens was not run the three highest concentrations (10,000, 1000 and 100 ppm) to 

avoid most the confounding effects of cell death. The highest concentration was 10 ppm, Dispersants that did not 

reduce luciferase expression in this assay at any concentration were scored as >10 ppm. 

Table 3: Summary table of the Lowest Observed Effect Concentration (LOEC) of the eight 

dispersants in the MTT cytotoxicity in three cell lines, the estrogen agonist assay in T47D Kbluc 

cells, the agonist assays in CV-1 and MDA K2 cells and the antagonist assay in MDA Kb2 cells. 

Since none of the dispersants displayed any effect interpreted as result of the dispersant displaying 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

37 

endocrine activity we interpret all the results as indications of disruption of cell function and cell 

death. Since the androgen antagonist assay for antiandrogens did not include the three dispersant 

highest concentrations (10,000, 1000 and 100 ppm) to avoid most the confounding effects of overt 

toxicity (seen in the MTT assay with MDA Kb2 cells), the highest concentration in this assay was 10 

ppm, Dispersants that did not reduce luciferase expression in this assay at any concentration were 

scored as >10 ppm. In spite of this precaution, the two most cytotoxic dispersants still reduced 

luciferase expression in this assay, an effect we attribute to less overt cell toxicity. 

 The EC50 values for the dispersant dose response curves were determined using nonlinear 

regression procedures with GraphPad Prism 5.0 software (Figure 5 a,b,c). Ranking the eight 

dispersants in order of highest to lowest potency in the MTT assays and the CPE assessment in three 

cells lines indicates that there are some consistent differences among dispersants in their ability to 

disrupt the function and viability of these cell lines (Figure 5 d). Dispersant SPC 1000 appears to 

be more toxic in both MTT (below) and CPE assessments. 

Cytotoxicity Summary  

For comparison to all of the in vitro cytotoxicity assays, we also include LC50 values from 

whole animal, aquatic species lethality assays for the mysid, Americamysis bahia, in a 48-hr static 

acute toxicity test and an inland silverside, Menidia beryllina, 96-hr static acute toxicity test[12]. All 

LC50 values are plotted in Figure 13 and the numerical values are listed in Appendix D. One can 

see that the cell-based LC50 values overall vary by about two orders of magnitude, and that the 

values for any given chemical span about one order of magnitude. The rank order of cytotoxicity 

varied between the various cell types, a not unexpected finding [13]. There is overlap in the range of 

cytotoxicity for all of the dispersants. 

In order to assess, statistically, differential cytotoxicity across the eight dispersants we 

performed an ANOVA to determine pairwise if any two dispersants were more cytotoxic than the 

other. We performed this statistical test with and without multiple test correction (Bonferroni). For 

any dispersant and assay combination that did not achieve an LC50, a default value of 3000 ppm was 

used; three-fold higher than the highest concentration tested in the relevant assays. LC50 values 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

38 

greater than 3000 ppm were also set to this default value to prevent large extrapolated LC50 values 

from biasing the results. All six cell-based quantitative cytotoxicity assays were used for this 

analysis. The resulting p-values, raw and corrected, are provided in Table 4. Both JD 2000 and SAFRON GOLD tend to be less cytotoxic than the other dispersants. Likewise, DISPERSIT SPC 1000 

tends to be more cytotoxic than the other dispersants in the cell-based assays. 

The aquatic species LC50 values are almost always lower than the cell-based LC50 values. 

As with the cell-based assays, JD 2000 is the least toxic in the whole animal assay. 

Figure 13: Toxicity data for the dispersants, combining data from cell-based assays in this report 

with data on aquatic species from a concurrent EPA report [12]. Each horizontal band shows the data 

for one dispersant. Results are presented from all 6 quantitative cytotoxicity assays. Cell-based LC50 

values (concentration at which 50% lethality or effect is observed) are indicated by circles and 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

39 

squares. Aquatic species LC50 values are indicated by triangles. Note that all dispersants were tested 

in all assays, and missing data points indicate that no toxicity was seen in that assay at the highest 

concentration tested. 95% confidence intervals are shown for all assays. JD 2000 Dispersit SPC 1000 Sea Brat 4 Nokomis 3-AA Nokomis 3-F4 

 Corexit 9500  ZI-400  SAF-RON GOLD 

JD 2000 0.1456 0.308 0.1876 0.2464 0.224 0.364 1 

Dispersit SPC 

1000 0.0052 0.84 1 0.644 1 0.126 0.056 

Sea Brat 4 0.011 0.03 1 1 1 1 0.1456 

Nokomis 3-AA 0.0067 0.082 0.11 1 1 0.42 0.0756 

Nokomis 3-F4 0.0088 0.023 0.5 0.12 1 1 0.1036 

Corexit 9500 0.008 0.086 0.34 0.42 0.65 1 0.0952 

ZI-400 0.013 0.0045 0.68 0.015 0.19 0.12 0.1652 

SAF-RON 

GOLD 0.92 0.002 0.0052 0.0027 0.0037 0.0034 0.0059 

Bonferroni Corrected P-Value 

   Raw P-Value   

Table 4: Statistical comparison of LC50 cytotoxicity values from cell-based assays across the eight 

dispersants. All dispersants combinations with a p-value less <0.05 are shaded pink. All values 

below the diagonal are raw p-values derived from the ANOVA, while all values above the diagonal 

swere adjusted for multiple testing. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

40 

Other Molecular Targets

In addition to ER and AR, we also analyzed the chemical collection (dispersants plus 

reference chemicals) using a multiplexed reporter gene assay battery that evaluates activity 

against a panel of transcription factors including nuclear receptors[5, 9]. These assays were run 

by Attagene Inc. These data also provide a measure of quality control related to the specificity of 

any endocrine-related activity caused by the dispersants. The description of the assay and a 

complete list of targets is given in Appendix B.2. All of these assays were carried out twice, one 

week apart, and in each week, duplicate runs were performed. Figure 14 summarize all of the 

results for the dispersants. This plot helps illustrate several key points about the data. 

First, as the concentration of a chemical approaches the cytotoxic level, generalized cell 

stress occurs, accompanied by broad misregulation of transcription. When this threshold is 

reached, many assays in this system simultaneously activate, but this activity is assumed to be 

non-specific. One sentinel of this cell stress behavior is NRF2, which is an indicator of 

generalized oxidative stress. Therefore, if we see many assays become active at about the same 

concentration, especially if NRF2 is among them, we can discount any target specificity above 

that concentration. We see this behavior for Corexit 9500 (~50 ppm), JD 2000 (~500 ppm), 

Nokomis 3-AA (~75 ppm), Nokomis 3-F4 (~75 ppm), Sea Brat #4 (~90 ppm) and ZI-400 (~50 

ppm). 

The ER activity for Nokomis 3-F4 occurs at a concentration well below where this nonspecific behavior is indicated. For ZI-400, the confidence intervals for ER and NRF2 overlap, 

indicating a possibility that the ER result is non-specific. 

The lowest activity that is generally seen is for PXR (Pregnane-X-receptor), which is a 

xenosensor. This behavior is entirely expected, is common across many classes of organic 

chemicals, and is not in itself an indicator of toxicity. PXR has been reported to be a xenosensor 

that acts to protect against endocrine active chemicals[14]. PPAR (peroxisome proliferator 

activating receptor[15-19]) activity is observed for a number of the dispersants, at higher 

concentrations than is seen for the PXR assays. There is an extensive literature on PPAR activity 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

41 

associated with disease in rodents, although the human relevance is unclear [15-18, 20-23]. 

However, only for Corexit 9500 and Nokomis 3-AA (and potentially for SAF-RON GOLD) is 

the PPAR signal well below the level of non-specific activity. Vitamin D receptor (VDR) 

activity is seen for Sea Brat #4 and Nokomis 3-AA below but near the concentration of nonspecific behavior. 

The activity of JD 2000 cannot necessarily be dismissed as being all non-specific, despite 

it occurring at the same concentration as NRF2 activity. This is because there are only two target 

families being activated – PXR and PPAR. A similar observation can be made about 

DISPERSIT SPC 1000. At the concentration of NRF2 activity, we only see activation of two 

PXR assays and one for SREBP (SREBF1 sterol regulatory element binding transcription factor 

1) which is involved in fatty acid synthesis regulation. 

The largest effect (in terms of EMax) of any dispersant and assays is for ZI-400 and AhR 

(Aryl hydrocarbon receptor), with EMax >30. The AhR is well-known for its role in mediating 

the adaptive metabolism of xenobiotics, and also in the toxicity that follows exposure to 2,3,7,8-

tetrachlorodibenzo-p-dioxin (dioxin). This indicates the potential for the presence of a dioxinlike compound, which would be cause for concern. In the ToxCast Phase I data set[4, 5] of 309 

chemicals, we saw only three with AhR efficacy higher than is seen with ZI-400. It is not clear 

though that this effect is specific, given that it occurs in the same concentration range as activity 

in a number of other targets, and above the NRF2 AC50. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

42 

Figure 14: Summary plot of all Attagene cis and trans assays for dispersants with AC50 values 

below cytotoxicity levels. Each horizontal band displays data for a single dispersant. The x-value 

is the AC50. Points are staggered in the y-direction to make overlapping points visible. Where 

there were multiple assays for a given gene target (e.g. PPAR, PPAR, PPAR) were given a 

single symbol. For illustration, 95% confidence intervals are shown on assays for NRF2. The 

vertical red lines indicate the LC50 for cytotoxicity in the HepG2 cells. Dispersants are ordered 

by decreasing cytotoxicity LC50 values. 

The major conclusions of this section are that several of the dispersants display PXR and 

PPAR activity at concentrations below where cell stress and cytotoxicity occur. These are 

expected responses in hepatocytes to xenobiotics. The ordering of dispersants by lowest 

concentration at which bioactivity occurs is consistent with the ordering based on cytotoxicity. 

One observation of more general interest is that we are able to detect specific target-based 

bioactivity in complex mixtures such as these. This is observation is relevant to the challenges of 

real world chemical toxicity testing, wherein humans and other organisms are often exposed to 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

43 

complex mixtures rather than the pure single compounds that are the subject of typical toxicity 

testing. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

44 

Conclusions

The primary conclusions are as follows: 

For six of the eight dispersants tested we found no evidence that they would be capable of 

interacting with estrogen or androgen receptor function from testing in multiple in vitro systems. 

For the other two dispersants, there was a weak ER signal in one assay. However, integrating 

over all of the ER and AR results, these data do not indicate that any of the eight dispersants will 

display biologically significant endocrine activity via the androgen or estrogen signaling 

pathways. As mentioned previously, NPEs (and their breakdown product NPs) can be endocrine 

disruptors in fish[1], so the risk of using NPE-containing dispersants should be carefully weighed 

against the expected benefits. One limitation of the present study is that there are other routes by 

which chemicals can cause endocrine disruption, as well as other types of toxicity that have not 

been tested for here. Most importantly though, there were no indications of estrogenic activity 

for Corexit 9500, the dispersant currently being used in the Gulf of Mexico. 

All of the dispersants showed cytotoxicity in at least one cell type at concentrations 

between 10 and 1000 ppm. Both JD 2000 and SAF-RON GOLD tend to be less cytotoxic than 

the other dispersants. Likewise, DISPERSIT SPC 1000 tends to be more cytotoxic than the other 

dispersants in the cell-based assays. The aquatic species LC50 values tend to be lower than the 

cell-based LC50 values. As with the cell-based assays, JD 2000 is the least toxic in the whole 

animal assay. 
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

45 

Supplementary Information

Supplemental information, including a QA Statement, is included in the referenced Appendices. 

QA Summary

All research described in this report was conducted under a comprehensive and rigorous program 

of quality assurance (QA), as documented in the QA supplemental file. The overall goal of the 

QA program was to ensure research data were of known and acceptable quality. QA staff 

surveillance of critical research activities was an important feature of the overall QA approach 

and ensured quick and effective resolution of any problems. The conclusion of the QA review 

process is that results presented in this report accurately reflect the raw data obtained during the 

course of the research and are scientifically valid and defensible.
 

U.S. EPA/ORD Dispersant In Vitro Testing June 30, 2010 

46 

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