Figure 1. Names, acronyms, structures, and Chemical Abstracts Service (CAS) numbers of test chemicals.

Figure 2. Individual chemical in vitro (T47D-KBluc assay, panel a) and in vivo (ovariectomized rat uterotrophic, oral administration, panel b) dose response curves. Symbols and error bars represent mean ± standard error, dashed horizontal line indicates zero baseline. Data file for this figure follows on the next two pages.

Table 1. In vitro (T47D-KBluc) dose response curve parameters (slope, EC50 (M)) and relative potency factors (RPF; using EE2 as reference compound) for eight estrogenic compounds.

Chemical Slope ± SE EC50 (95% CI) RPF

EE2 1.81 ± 0.19 9.98e-13 (8.81e-13 – 1.13e-12) 1.00

E2 1.46 ± 0.09 1.68e-12 (1.52e-12 – 1.86e-12) 0.594

BPC 1.46 ± 0.11 5.71e-09 (5.03e-09 – 6.48e-09) 0.000175

BPAF 1.40 ± 0.11 2.06e-08 (1.81e-08 – 2.35e-08) 0.0000485

BPA 1.61 ± 0.18 1.27e-07 (1.08e-07 – 1.50e-07) 0.00000784

BPS 1.93 ± 0.27 6.43e-07 (5.30e-07 – 7.80e-07) 0.00000155

BBP 4.07 ± 0.87 1.31e-06 (1.12e-06 – 1.53e-06) 0.000000763

MET 2.49 ± 0.38 1.47e-06 (1.26e-06 – 1.72e-06) 0.000000679

E2 BPA BPS BPC BPAF MET BBP EE2Log molar Mean SD n Mean SD n Mean SD n Mean SD n Mean SD n Mean SD n Mean SD n Mean SD n

-15.000 3.436 3.763 6

-14.523 -1.206 3.367 6

-14.000 -5.835 5.255 6

-13.523 -9.476 9.99135 -9.790 2.923 6

-13.000 1.726 20.90038 -5.997 2.266 6

-12.523 8.335 10.27644 12.177 4.894 6

-12.000 32.711 20.70445 48.165 11.308 6

-11.523 68.899 17.84445 82.708 7.827 6

-11.000 96.374 4.23538 98.229 4.939 6

-10.523 100.074 4.31341 96.174 5.183 6

-10.000 99.581 4.15336 -0.625 10.965 10 93.756 3.308 6

-9.523 98.286 4.38128 3.800 10.389 10 -2.197 11.123 6 93.826 0.272 2

-9.000 6.846 11.004 11 0.341 11.891 13

-8.523 33.781 9.563 11 3.064 8.047 13

-8.000 -1.608 15.14118 81.935 5.053 11 32.197 8.235 13 -9.106 7.273 6

-7.523 9.214 16.72118 -8.148 16.957

17 107.990 5.225 11 68.375 10.655 13 -4.818 3.812 4 -9.246 9.372 5

-7.000 44.298 15.18318 -2.021 12.460

17 117.168 7.687 11 98.725 2.776 13 -3.484 8.526

12 -10.370 10.197 6

-6.523 84.232 10.81018 18.940 10.673

17 116.425 5.979 3 109.206 4.810 9 -1.615 6.861

12 -8.784 5.360 6

-6.000 104.243 14.92510 60.718 15.209

17 109.708 6.049 6 22.134 10.853

12 25.995 7.366 6

-5.699 32.608 5.842 4 86.187 6.538 6

-5.523 106.374 9.296 4 86.523 10.14816 66.267 9.176

12 98.950 2.034 6

-5.222 65.831 10.438 8

-5.000 106.883 2.773 8 86.456 15.50511 69.637 7.542

12

Summary data for T47D-KBluc dose-response curves in Figure 2a

Uterotrophic assay body mass and tissue mass (mean±SEM) for single chemical, oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05). Summary data for Figure 2b.

ChemicalDose

(mg/kg/d)Sample size

(# rats)Initial body weight (g)

Body weight gain (g)a

Uterus wet(mg)

Uterus blotted(mg)

Uterine fluid(mg)

E2

(+) control 8 306.3±16.4 1.7±2.3* 286.1±51.7* 158.9±11.2* 131.2±40.4*0 8 312.8±16.2 11.7±2.6 41.7±5.2 35.7±4.9 6.0±1.0

0.001 5 273.8±6.6 15.4±2.2 43.7±4.2 38.0±3.6 5.7±1.80.003 5 278.3±4.9 15.1±1.3 42.9±3.3 36.8±3.4 6.0±1.00.01 5 268.9±8.5 14.9±2.2 48.7±7.5 42.0±6.6 6.6±1.20.03 5 263.7±7.7 13.0±1.8 66.7±2.4* 59.1±2.5* 7.6±0.90.1 7 294.4±17.2 7.9±2.6 81.5±9.4* 70.0±6.9* 11.6±2.60.3 7 280.6±21.8 1.1±2.9* 131.0±10.6* 106.5±6.9* 24.5±4.3*1 5 367.1±10.4 -11.3±1.3* 241.3±34.9* 159.1±15.2* 82.2±21.5*3 5 356.5±11.2 -11.5±1.6* 234.6±18.4* 150.1±9.0* 84.5±10.2*

BBP

(+) control 4 311.0±14.5 -5.8±1.8* 135.4±8.0* 104.1±5.4* 31.3±8.4*0 4 326.4±2.7 5.4±1.0 31.6±3.8 27.1±3.2 4.4±0.9

400 4 341.7±10.8 5.5±0.7 31.7±2.6 25.9±1.9 5.9±0.9600 4 348.0±9.6 4.6±1.7 34.8±2.2 31.0±1.7 3.8±0.9800 4 347.8±9.3 3.7±1.6 32.5±1.8 26.9±2.1 5.7±0.5

BPA

(+) control 5 362.7±13.1 -7.6±2.3* 238.0±34.7* 153.3±5.8* 84.7±30.6*0 5 380.2±13.1 2.9±1.7 39.1±5.2 34.2±4.5 4.9±0.7

200 6 342.6±15.4 -0.8±1.8 43.0±6.3 37.1±5.3 5.9±1.1400 5 353.2±15.1 -2.5±2.7 58.3±1.5 50.9±0.9 7.4±1.0600 3 353.7±6.2 -5.6±2.7* 58.9±6.7 50.4±5.0 8.5±1.9800 6 370.5±22.0 -3.7±3.7 81.3±20.9* 65.6±13.8* 15.8±7.4

BPAF

(+) control 6 322.9±3.1 -29.3±3.1* 974.9±65.6* 341.3±17.9* 633.6±66.6*0 4 335.6±2.3 1.6±2.4 118.2±10.1 109.6±9.6 1.0±1.0

3.125 3 322.6±5.5 -0.6±2.2 138.6±13.3 124.1±8.4 14.5±6.26.25 3 321.1±2.4 2.3±2.4 124.1±19.8 113.0±16.2 11.1±3.612.5 3 318.0±6.1 -0.7±4.8 141.2±19.4 130.6±17.9 10.5±2.125 6 322.1±5.8 -1.9±1.9 155.0±16.4 133.6±13.6 21.3±5.150 6 318.9±5.0 -13.0±7.3 261.3±65.7* 159.7±32.5 101.6±42.1100 6 319.9±2.2 -11.4±1.7 219.1±32.5* 179.7±14.0* 39.4±19.6300 6 331.8±5.3 -27.7±5.1* 323.3±58.7* 230.4±15.2* 92.9±47.6

BPC

(+) control 6 338.2±40.4 -2.1±3.7 327.4±41.6* 163.4±11.0* 164.0±31.6*0 6 304.3±25.4 6.2±2.8 42.7±4.7 35.9±4.0 6.8±0.9

1.2 3 252.6±9.3 14.7±1.0 31.7±5.0 27.3±4.3 4.4±1.24 6 305.3±22.9 7.3±3.9 34.0±4.5 30.6±3.5 3.5±1.2

13.5 6 338.0±36.9 4.8±5.0 47.3±4.9 40.8±3.9 6.5±1.145 6 327.8±40.4 1.4±5.9 113.3±14.0* 93.1±10.0* 20.2±4.2*150 6 328.9±33.3 0.1±3.4 148.9±12.0* 122.0±5.6* 26.8±6.7*500 6 327.9±33.7 -2.7±3.4 176.1±12.1* 134.7±9.8* 41.3±7.9*

Uterotrophic assay body mass and tissue mass (mean±SEM) for single chemical, oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05). Summary data for Figure 2b.

ChemicalDose

(mg/kg/d)Sample size

(# rats)Initial body weight (g)

Body weightgain (g)a

Uterus wet(mg)

Uterus blotted(mg)

Uterine fluid(mg)

BPS

(+) control 6 338.2±9.9 -10.4±1.4* 287.3±24.9* 162.4±7.8* 124.9±22.8*0 6 342.6±15.1 0.9±1.3 31.6±2.9 25.4±3.0 6.3±0.450 6 364.5±8.2 -0.5±0.6 55.0±6.9* 44.9±5.3* 10.1±1.9100 6 359.8±16.8 -0.7±1.3 63.0±7.1* 54.8±6.0* 8.2±1.5200 6 357.1±12.8 -3.5±2.4 77.2±8.6* 64.9±7.7* 2.3±1.9400 5 369.4±18.0 -2.8±2.8 139.5±36.8* 107.2±17.5* 32.3±19.7*800 6 376.2±13.2 -4.2±1.3 156.7±13.4* 121.4±7.6* 35.3±6.8*

EE2

(+) control 3 348.7±7.2 -13.3±1.0 1026.6±50.0* 61.3±19.0* 665.3±36.8*0 3 344.7±17.5 -1.0±1.3 103.0±7.8 89.8±7.1 13.2±2.3

0.0005 3 318.6±7.3 -9.7±5.4 118.4±14.5 107.9±12.7 10.6±2.20.001 3 350.9±15.4 -2.6±2.7 115.9±10.0 107.9±9.1 8.0±1.00.0025 3 356.2±4.7 -5.4±1.5 142.3±37.0 128.7±35.6 13.6±2.30.005 3 345.7±2.5 -6.0±1.6 132.3±17.4 118.8±15.2 13.5±3.10.01 3 353.9±3.2 -12.3±6.5 244.5±64.6* 175.3±12.1* 69.2±52.60.05 2 313.5±19.2 -8.5±3.6 526.6±24.4* 206.2±4.9* 320.5±9.6*0.2 3 323.8±1.8 -13.5±0.6 874.4±120.0* 309.2±21.5* 565.2±106.2*

MET

(+) control 4 307.2±7.0 -8.1±0.3* 1107.2±236.5* 395.8±39.6* 711.4±207.7*0 3 311.9±0.1 0.2±0.7 91.5±10.5 83.4±6.2 8.1±4.4

62.5 4 319.3±5.4 0.1±1.7 202.0±12.4* 168.2±10.2* 33.8±2.7*125 4 316.8±2.5 -1.5±0.2 319.7±37.7* 229.4±22.8* 90.3±26.9*250 4 314.7±19.6 -7.4±0.8* 478.3±50.6* 316.4±44.8* 161.9±49.8*500 4 310.1±6.7 -9.7±2.0* 574.2±62.1* 307.0±9.0* 267.2±67.9*

Table 2. In vivo (ovariectomized rat uterotrophic assay, oral gavage) dose response curve parameters (slope, ED50 (mg kg-1 d-1)) and relative potency factors (RPF; using EE2 as reference compound) across three endpoints (uterus blotted weight, uterus wet weight, and uterine fluid weight) for seven estrogenic compounds.

Blotted Wet Fluid

Chem Slope ± SE

ED50

(95% CI) RPF Slope ± SE

ED50

(95% CI) RPF Slope ± SE

ED50

(95% CI) RPF

EE2 0.69± 0.14

0.0179 (0.00922 – 0.0346)

1.00 1.08± 0.17

0.0208 (0.0138 – 0.0315)

1.00 1.89± 0.56

0.0216 (0.0123 – 0.0379)

1.00

E2 1.09± 0.17

0.103 (0.0756 – 0.138)

0.173 1.17± 0.18

0.166 (0.127 – 0.216)

0.126 1.48± 0.29

0.249 (0.190 – 0.328)

0.0866

BPC 1.70± 0.34

39.8 (30.6 – 51.7)

0.000449 0.92± 0.15

95.4 (66.1 – 137.7)

0.000218 0.85± 0.25

286.8 (148.7 – 553.3)

0.0000753

BPAF 0.75± 0.19

209.1 (111.0 – 394.0)

0.0000854 0.62± 0.15

465.2 (198.0 – 1093.0)

0.0000448 0.47± 0.26

872.9 (49.7 – 15346.0)

0.0000247

BPA 1.40± 0.70

1118.0 (529.1 – 2360.0)

0.0000160 1.34 ± 0.72

1286.0 (504.8 – 3277.0)

0.0000162 1.26± 1.13

1737.0 (225.0 – 13418.0)

0.0000124

BPS 0.91± 0.14

150.0 (113.8 – 197.5)

0.000119 0.83± 0.13

259.2 (194.2 – 345.9)

0.0000804 1.05± 0.28

676.1 (412.7 – 1108.0)

0.0000319

MET 1.08± 0.22

71.4 (49.6 – 102.8)

0.000250 0.90± 0.13

126.7 (98.9 – 162.3)

0.000164 0.83± 0.20

104.4 (67.9 – 160.5)

0.000207

No values reported for BBP because it was negative in the oral uterotrophic assay

Table 3. Predicted ED50 (mg kg-1 d-1) values for individual chemicals in the ovariectomized rat uterotrophic assay across three endpoints (uterus blotted weight, uterus wet weight, and uterine fluid weight). Predictions were calculated using in vitro (T47D-KBluc) relative potency factors with EE2 as the reference compound. Predicted to observed ratios (P:O) indicate accuracy of prediction (P:O = 1.0 indicates perfect prediction).

Blotted Wet Fluid

Chem.Predicted

ED50 P:OPredicted

ED50 P:OPredicted

ED50 P:OEE2 - - - - - -

E2 0.030 0.3 0.035 0.2 0.036 0.1

BPC 102.1 2.6 119.1 1.2 123.5 0.4

BPAF 368.3 1.8 429.8 0.9 445.5 0.5

BPA 2276.2 2.0 2656.2 2.1 2753.1 1.6

BPS 11497.1 76.6 13416.5 51.8 13906.0 20.6

BBP 23387.6 - 27292.0 - 28287.8 -

MET 26284.2 368.3 30672.2 242.1 31791.3 304.5

8

Figure 3. Predicted (black dashed lines) versus observed (grey circles and black solid lines except panel b where E2 is represented by grey squares) dose response curves for uterus blotted weight in the ovariectomized rat uterotrophic assay (oral gavage). Predicted lines were generated using in vitro (T47D-KBluc) relative potency factors with 17α-ethinyl estradiol as a reference compound. Panel (a) includes additional data estimated from prior studies on the uterotrophic response to BBP (*data from Brady et al. 2000 represents uterine wet weight).

9

Figure 4. Observed data (gray circles) versus dose addition (DA) models of binary (BPAF+MET; BPS+MET) and multi-chemical (BPAF+BPC+BPS+EE2+MET) mixtures in the ovariectomized rat uterotrophic assay (oral gavage). DA models were generated using individual compound in vitro (T74D-Kbluc, black dashed lines) and in vivo (black solid lines) response data. Observed data represent mean ± 95% CI; statistical significance based on model predictions overlapping the observed 95% CI. Data file follows on the next page

10

Uterotrophic assay body mass and tissue mass (mean±SEM) for binary and multi-chemical mixtures via oral gavage exposures to ovariectomized rats (*denotes significant difference from 0 dose control, p<0.05). Summary data for Figure 4.

Chemical Dose(mg/kg/d)

Sample size(# rats)

Initial body weight (g)

Body weightgain (g)a

Uterus wet(mg)

Uterus blotted(mg)

Uterine fluid(mg)

BPAF+MET

(+) control 10 336.5±9.2 -14.8±1.0* 935.2±71.2* 333.3±28.9* 593.0±51.5*0 9 338.8±7.9 -2.6±1.5 100.4±6.8 85.1±4.7 15.2±3.2

62.5 8 342.5±4.1 -3.4±1.4 179.4±11.5* 159.3±10.0* 20.1±4.0125 8 320.1±8.8 -7.6±3.5 229.5±13.3* 193.1±13.2* 36.4±7.7*250 9 337.2±5.0 -7.4±1.1 316.1±17.8* 210.1±7.2* 102.5±15.4*375 9 326.1±8.2 -20.8±3.0* 410.7±47.8* 227.4±13.9* 176.2±42.9*500 8 336.5±8.5 -26.7±3.8* 669.3±47.0* 244.8±16.6* 415.5±53.5*

BPS+MET

(+) control 5 355.5±9.0 -6.9±1.0* 167±10.6* 127.2±5.9* 39.8±6.2*0 5 341.4±10.0 7.1±1.7 31.6±2.7 26.6±2.2 5.0±0.615 5 346.4±12.3 3.1±1.3 27.8±2.0 24.4±1.6 3.4±1.145 5 331.6±3.7 2.1±1.1 39.5±2.2 32.9±2.0 6.6±0.4135 6 366.7±7.1 -1.4±2.0* 65.8±4.2* 57.3±3.5* 8.4±0.8405 6 371.8±5.2 -0.3±1.1* 97.9±10.7* 82.5±8.1* 15.4±3.0*1000 5 349.9±4.2 -4.8±3.5* 145.4±11.2* 119.3±10.2* 26.1±2.9*

BPAF+BPC+BPS+EE2+MET

(+) control 5 376.0±10.0 -6.5±2.2* 245.2±29.7* 156.4±8.9* 88.7±27.1*0 4 356.1±9.9 3.8±0.9 37.8±1.5 33.1±2.1 4.7±0.729 5 326.3±7.0 2.2±0.8 43.6±3.6 38.2±2.9 5.4±0.958 5 362.2±13.1 -2.9±2.5 81.3±4.2* 71.5±4.0* 9.8±1.1117 5 331.9±9.3 -5.8±2.2* 92.6±4.3* 79.2±3.0* 13.3±1.8*235 4 323.1±7.6 -6.1±1.9* 114.7±19.3* 96.7±16.1* 18.0±3.4*470 5 370.7±13.1 -3.3±3.0 156.0±10.4* 122.9±9.1* 33.0±3.9*

11

Observed and predicted (Pred.) ED50 values (mg kg-1 d-1) for binary (BPAF+MET, BPS+MET) and multi-chemical (BPAF+BPC+BPS+EE2+MET) mixtures in the ovariectomized rat uterotrophic assay across three endpoints (blotted uterus weight, wet uterus weight, uterine fluid weight). Predicted values were calculated using individual chemical response data from in vivo (uterotrophic) and in vitro (T47D-KBluc) experiments and the dose addition model. Predicted to observed ratios (P:O) indicate the accuracy of the prediction (P:O=1 represents perfect prediction).

Blotted Wet Fluid

Mix.

ObservedED50

(95% CI)

In vivo In vitro ObservedED50

(95% CI)

In vivo In vitro ObservedED50

(95% CI)

In vivo In vitro

Pred. P:O Pred. P:O Pred. P:O Pred. P:O Pred. P:O Pred. P:O

BPAF+MET

79.3(54.7-115.0)

118.0 1.5 709.8 7.7 197.4(172.8-225.5)

224.9 1.1 608.2 3.6 249.4(213.0-291.9)

221.3 0.9 735.6 2.9

BPS+MET

162.6(128.5-205.7)

110.1 0.7 14108.0 86.8 191.6(151.0-243.1)

192.9 1.0 16463.0 85.9 337.6(243.8-467.5)

241.3 0.7 17064.0 50.5

BPAF+BPC+BPS+EE2+MET

95.5(72.1-126.4)

93.2 1.0 237.3 2.5 135.7(104.0-176.9)

164.1 1.2 278.0 2.0 211.5(161.0-277.9)

222.8 1.1 288.2 1.4

12

In vitro gene expression methods with data sheets following this

T47D-KBluc gene expression

T47D-KBluc cells were harvested in duplicate from maintenance media (T47DS-1 and T47DS-2; see Materials and Methods for media composition), withdrawal media (T47DD-1 and T47DD-2; see Materials and Methods for media composition), and following ~24 hr exposure to withdrawal media containing 30 pM 17β-estradiol (E2-1 and E2-2), 300 pM 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H pyrazole (PPT-1 and PPT-2; ERα agonist, CAS# 263717-53-9), or 30 nM 2,3-bis(4-hydroxyphenyl) propionitrile (DPN-1 and DPN-2; ERβ agonist, CAS# 1428-67-7). Cells were homogenized, mRNA was extracted and converted to cDNA and run on Qiagen Breast Cancer qPCR plates (CAT# 330231 PAHS-131A) in a BioRad iCycler. Table S1 below reports the CT values with ERα (ESR1) and ERβ (ESR2) genes highlighted in bold. It is apparent from the qPCR results that ERβ mRNA was minimally expressed, even following exposure to the ERβ specific agonist DPN. CV-1 cells were also homogenized and analyzed in duplicate in order to assess the capacity of the gene arrays to discriminate between cell types.

T47D-KBluc transcriptional activation

Experiments were conducted identical to those described in the Materials and Methods. Cells were exposed to a reference compound (E2), ERα specific agonist (PPT, identified above), and ERβ specific agonist (DPN, identified above) across a range of concentrations. Luciferase activity was normalized to % of control using saturating concentrations of E2 and dose response curves were graphed using GraphPad Prism. The ERα agonist (PPT) was ~1000-fold more potent than the ERβ agonist (DPN) indicating the dominance of the ERα receptor subtype in the T47D-KBluc assay response for the culture of this cell line currently maintained in our laboratory (Figure S1). DPN is an ERβ agonist, however there is some degree of cross-reactivity with ERα.

13

Expression (CT) of genes in CV-1 cells and in T47D-KBluc cells under culture conditions and exposed to three estrogen receptor agonists with varying receptor subtype specificity.

Gene symbol CV1-1 CV1-2 T47DS-1 T47DS-2

T47DD-1

T47DD-2 E2-1 E2-2 PPT-1 PPT-2 DPN-1 DPN-2

ABCB1 23.3 22.2 36.8 - - - - - - - - -ABCG2 - - 35.3 35.6 35.6 - 33.5 35.8 36.6 - 35.7 -

ADAM23 - 34.6 - - - 35.9 - 35.8 - - - -AKT1 25.9 24.8 24.1 24.0 23.8 22.7 22.8 23.5 24.1 24.3 24.6 23.7

APC - - 29.6 29.8 28.8 27.7 28.1 28.9 29.1 29.6 29.8 28.9AR 35.9 - 26.1 26.8 24.9 24.0 24.7 25.2 25.0 24.6 25.7 24.8

ATM 36.7 35.4 27.7 27.7 27.7 26.5 26.9 27.8 27.9 28.0 28.7 27.8BAD 29.1 27.9 26.1 26.2 25.5 24.4 24.9 25.7 26.4 26.1 26.9 25.8

BCL2 26.0 24.7 29.7 29.2 29.7 29.7 28.4 30.1 32.5 30.8 33.5 32.3BIRC5 30.0 29.4 29.6 30.1 30.9 30.3 28.4 30.5 31.6 31.8 31.7 31.1

BRCA1 26.7 25.6 26.4 25.8 27.7 25.9 25.5 26.2 27.0 28.0 27.8 26.9BRCA2 - - 28.3 27.5 29.6 29.0 27.5 28.7 28.8 29.6 29.5 28.7CCNA1 28.9 27.8 36.5 - - - - - - - - -CCND1 24.6 23.5 25.7 25.2 26.6 25.5 25.2 25.6 26.6 27.5 27.6 26.8CCND2 34.5 34.0 34.2 37.1 35.7 33.6 35.9 36.1 36.1 35.8 35.4 37.0CCNE1 25.9 24.6 28.1 28.1 27.9 27.3 26.3 27.1 27.9 28.4 28.6 27.8

CDH1 35.6 37.2 22.9 23.1 22.0 20.8 21.2 22.1 22.5 22.8 23.1 21.8CDH13 27.7 26.2 29.6 29.7 29.0 28.2 28.7 29.5 29.2 30.4 30.3 29.2CDK2 25.7 24.1 25.6 25.4 25.8 24.5 23.7 24.6 26.0 26.3 26.4 25.6

CDKN1A 32.3 31.4 24.8 24.9 25.7 24.4 24.8 25.4 26.4 26.0 27.1 25.7CDKN1C 27.6 26.8 33.0 32.4 31.8 30.8 30.6 32.4 31.9 31.3 31.7 30.9CDKN2A 37.9 - 27.5 27.5 26.4 25.1 25.5 26.7 27.6 27.2 28.1 27.1

CSF1 25.6 24.6 30.6 30.5 32.9 31.2 30.0 31.4 31.8 32.2 32.1 31.3CST6 36.6 38.0 35.5 33.1 30.8 29.6 28.6 30.9 32.6 32.2 32.8 32.1

CTNNB1 25.0 23.5 23.0 22.8 22.9 21.4 22.1 22.7 23.5 23.6 23.9 22.8CTSD 23.9 22.7 22.7 22.6 23.8 22.7 20.9 21.6 21.0 22.4 21.9 21.0

EGF 36.7 34.5 28.0 28.1 28.8 27.7 27.8 28.2 27.1 27.0 27.8 26.6EGFR 26.2 25.5 27.1 27.6 27.1 26.6 26.3 27.6 28.6 28.5 28.7 28.3

ERBB2 27.2 26.0 26.7 26.7 24.1 23.4 23.9 24.7 25.1 25.4 25.6 24.6ESR1 36.0 34.8 25.7 26.1 25.8 25.0 24.1 25.3 25.1 25.5 25.5 24.8ESR2 32.7 31.3 32.7 32.6 34.6 33.9 30.6 31.8 31.9 35.1 32.0 33.0

FOXA1 28.5 27.5 24.7 24.5 24.5 23.4 24.0 24.5 25.0 25.0 25.3 24.2GATA3 31.1 29.6 24.6 24.1 24.6 22.9 23.2 24.0 24.6 25.0 25.2 24.3

14

GLI1 33.4 33.6 33.8 34.1 35.5 34.1 34.8 35.3 34.7 36.5 35.1 35.4GRB7 26.5 25.9 28.0 27.3 27.3 25.7 26.1 26.9 26.9 26.6 27.4 26.3

GSTP1 - 36.7 30.0 29.9 32.5 30.7 32.0 32.9 30.5 31.5 30.9 29.8HIC1 29.5 28.8 34.0 34.5 35.3 33.9 33.7 35.0 35.0 33.0 35.5 34.8

ID1 23.3 22.3 25.1 25.1 23.5 22.4 25.7 25.8 25.7 26.3 26.1 25.3IGF1 33.6 34.1 30.9 31.3 31.7 30.9 29.9 30.5 30.0 30.8 30.8 30.1

IGF1R 30.4 29.4 24.5 25.0 25.6 24.7 24.8 25.5 25.9 26.8 26.4 25.4IGFBP3 - - 32.9 35.4 30.5 29.6 29.8 30.7 32.0 33.4 32.6 32.1

IL6 34.6 33.6 - - - 34.1 - - - 36.2 - -JUN 24.7 23.4 25.8 27.5 24.4 23.2 25.2 25.5 26.3 26.8 26.7 25.7

KRT18 23.3 22.3 21.2 22.1 19.4 18.4 18.1 18.5 18.7 19.4 19.7 18.7KRT19 - - 19.8 19.8 19.1 17.9 18.2 18.8 19.0 19.9 19.9 18.8KRT5 - 36.5 - - - - - - - - - -KRT8 32.7 33.1 22.6 22.3 21.1 19.6 19.1 19.9 20.3 21.0 21.1 19.9

MAPK1 23.9 23.0 24.2 23.6 24.5 23.0 23.6 24.3 25.0 25.0 25.8 24.6MAPK3 30.0 29.6 26.6 26.5 26.6 25.9 25.9 26.9 27.4 27.7 28.1 26.9MAPK8 26.4 25.5 26.1 25.7 26.3 25.6 25.7 26.3 26.6 26.9 27.1 26.7MGMT 26.5 25.5 25.0 24.8 25.0 24.0 24.6 25.0 25.1 25.7 25.7 24.8MKI67 28.0 26.3 24.7 24.5 25.8 24.7 23.4 24.0 25.3 27.0 26.1 25.3MLH1 27.3 26.0 24.6 24.7 24.7 23.6 23.8 24.3 24.7 25.5 25.5 24.2MMP2 27.7 26.9 - - - - - - - - - -MMP9 35.6 33.6 30.6 31.1 30.9 30.1 29.6 30.7 30.5 30.9 32.1 30.6MUC1 - - 22.6 22.7 21.8 20.6 21.3 22.1 21.9 22.2 22.5 21.4MYC 29.1 27.7 27.0 26.7 30.8 29.0 27.8 28.4 29.1 30.3 30.2 29.4

NME1 22.9 21.9 22.0 21.6 23.1 21.2 21.2 21.7 22.3 22.8 23.0 22.1NOTCH1 - - 29.6 28.8 30.3 28.7 27.9 28.6 30.2 30.4 30.6 29.6

NR3C1 25.7 24.5 28.3 27.8 27.7 26.5 26.7 28.1 28.1 27.5 28.6 27.6PGR - - 20.7 20.2 22.9 21.9 21.0 21.7 22.3 23.7 23.2 22.5

PLAU 34.5 34.2 34.8 35.1 32.2 30.4 31.4 31.9 32.6 32.5 32.0 31.1PRDM2 - 34.9 26.4 26.0 25.5 24.6 25.3 25.7 26.3 26.2 26.7 25.6

PTEN 25.1 23.9 23.5 23.1 23.4 22.5 22.6 23.8 23.9 24.3 24.6 23.7PTGS2 36.5 35.8 35.6 35.6 35.7 34.8 - 35.4 35.9 - 36.2 -

PYCARD 29.4 28.6 28.7 28.5 27.7 27.0 26.8 28.3 28.2 27.9 28.7 27.7RARB 27.9 26.8 30.7 30.7 30.2 29.3 28.8 30.7 29.9 30.6 30.7 29.8

RASSF1 27.9 27.3 28.7 28.0 28.8 27.8 27.1 27.5 28.7 28.6 28.5 28.2RB1 25.5 24.5 25.6 25.1 26.1 24.5 24.9 25.6 26.1 26.8 26.7 25.6

SERPINE1 21.5 20.8 36.2 35.1 34.9 33.6 35.7 35.2 34.4 34.0 34.7 33.9

15

SFN 29.6 28.6 25.9 25.7 26.5 24.8 23.8 24.6 25.9 26.1 26.5 25.6SFRP1 27.0 26.7 - 37.7 - - - - 38.2 - - -

SLC39A6 26.0 24.9 24.6 24.1 24.5 23.3 23.8 24.4 24.8 25.1 25.6 24.7SLIT2 32.6 31.8 - - - - - 36.1 - - - -SNAI2 25.1 24.0 - - 35.4 33.8 - - 33.4 34.9 - 34.3

SRC 27.9 26.8 27.0 26.3 27.1 26.5 26.6 27.3 27.7 27.9 28.2 27.6TFF3 30.9 29.8 21.3 20.8 24.7 23.7 22.1 22.9 23.1 25.0 23.9 23.0

TGFB1 24.3 23.0 28.9 28.4 30.2 29.6 27.5 28.8 30.3 30.6 31.1 30.2THBS1 31.3 30.8 22.5 22.5 24.1 22.9 22.3 23.1 24.3 24.0 24.6 23.5

TP53 - - 24.2 23.8 25.3 23.9 23.8 24.8 25.7 25.4 25.9 25.3TP73 31.7 30.7 30.7 30.5 30.9 30.1 29.0 30.9 30.6 30.6 30.8 30.3

TWIST1 32.3 32.1 29.6 29.7 28.5 27.0 27.6 28.6 31.9 31.4 31.2 30.6VEGFA 22.5 21.4 25.4 25.0 26.6 25.2 25.0 25.8 26.7 26.6 27.1 26.5

XBP1 22.7 21.9 19.8 19.7 21.5 20.6 20.0 20.7 20.4 21.0 20.9 20.0B2M 35.9 34.9 21.5 21.0 22.2 20.8 21.2 21.9 21.9 22.3 22.7 21.5

HPRT1 24.2 22.9 25.9 25.7 26.6 25.3 25.5 25.4 26.4 27.0 27.2 26.1RPL13A 23.2 22.3 21.4 21.1 22.3 20.9 21.7 22.5 22.8 22.9 23.4 22.5GAPDH 24.4 23.0 20.5 20.7 20.7 19.5 20.0 20.7 21.2 21.7 21.8 20.9

ACTB 18.8 17.8 19.0 18.7 18.2 16.7 17.4 17.9 18.5 19.5 19.2 18.0HGDC 37.9 - - 37.7 38.4 38.1 32.9 - - - - 36.9

RTC 22.7 22.3 22.7 23.0 22.4 22.5 21.8 23.1 22.8 22.7 22.4 23.2RTC 23.3 22.4 23.1 22.9 21.3 22.9 22.1 23.6 23.3 22.9 23.3 23.6RTC 23.6 23.5 23.6 23.5 21.8 23.3 22.6 23.9 23.6 22.9 23.6 23.8PPC 18.3 18.5 18.9 18.4 16.0 18.7 18.5 18.7 18.6 19.3 18.6 18.7PPC 18.7 19.0 18.5 18.8 18.7 19.0 19.0 19.0 18.9 19.6 19.0 19.0PPC 18.6 18.8 18.5 18.8 17.5 18.7 17.2 18.9 18.6 19.5 18.8 18.9

16

Identification of genes from Qiagen Breast Cancer array

Symbol Description Gname

ABCB1

ATP-binding cassette, sub-family B (MDR/TAP), member 1 ABC20/CD243/CLCS/GP170/MDR1/MGC163296/P-GP/PGY1

ABCG2

ATP-binding cassette, sub-family G (WHITE), member 2

ABC15/ABCP/BCRP/BCRP1/BMDP/CD338/CDw338/EST157481/MGC102821/MRX/MXR/MXR1

ADAM23

ADAM metallopeptidase domain 23 MDC3

AKT1V-akt murine thymoma viral oncogene homolog 1 AKT/MGC99656/PKB/PKB-ALPHA/PRKBA/RAC/RAC-ALPHA

APC Adenomatous polyposis coli BTPS2/DP2/DP2.5/DP3/GSAR Androgen receptor AIS/DHTR/HUMARA/HYSP1/KD/NR3C4/SBMA/SMAX1/TFM

ATM Ataxia telangiectasia mutatedAT1/ATA/ATC/ATD/ATDC/ATE/DKFZp781A0353/MGC74674/TEL1/TELO1

BADBCL2-associated agonist of cell death BBC2/BCL2L8

BCL2 B-cell CLL/lymphoma 2 Bcl-2BIRC

5Baculoviral IAP repeat containing 5 API4/EPR-1

BRCA1 Breast cancer 1, early onset BRCAI/BRCC1/BROVCA1/IRIS/PNCA4/PSCP/RNF53

BRCA2 Breast cancer 2, early onset

BRCC2/BROVCA2/FACD/FAD/FAD1/FANCB/FANCD/FANCD1/GLM3/PNCA2

CCNA1 Cyclin A1 -

CCND1 Cyclin D1 BCL1/D11S287E/PRAD1/U21B31

CCND2 Cyclin D2 KIAK0002/MGC102758

CCNE1 Cyclin E1 CCNE

CDH1Cadherin 1, type 1, E-cadherin (epithelial) Arc-1/CD324/CDHE/ECAD/LCAM/UVO

CDH13

Cadherin 13, H-cadherin (heart) CDHH/P105

CDK2 Cyclin-dependent kinase 2 p33(CDK2)

17

CDKN1A

Cyclin-dependent kinase inhibitor 1A (p21, Cip1) CAP20/CDKN1/CIP1/MDA-6/P21/SDI1/WAF1/p21CIP1

CDKN1C

Cyclin-dependent kinase inhibitor 1C (p57, Kip2) BWCR/BWS/KIP2/WBS/p57

CDKN2A

Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)

ARF/CDK4I/CDKN2/CMM2/INK4/INK4A/MLM/MTS-1/MTS1/P14/P14ARF/P16/P16-INK4A/P16INK4/P16INK4A/P19/P19ARF/TP16

CSF1Colony stimulating factor 1 (macrophage) MCSF/MGC31930

CST6 Cystatin E/M -CTNN

B1Catenin (cadherin-associated protein), beta 1, 88kDa CTNNB/DKFZp686D02253/FLJ25606/FLJ37923

CTSD Cathepsin D CLN10/CPSD/MGC2311EGF Epidermal growth factor HOMG4/URG

EGFREpidermal growth factor receptor ERBB/ERBB1/HER1/PIG61/mENA

ERBB2

V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) CD340/HER-2/HER-2/neu/HER2/MLN 19/NEU/NGL/TKR1

ESR1 Estrogen receptor 1 DKFZp686N23123/ER/ESR/ESRA/Era/NR3A1ESR2 Estrogen receptor 2 (ER beta) ER-BETA/ESR-BETA/ESRB/ESTRB/Erb/NR3A2

FOXA1 Forkhead box A1 HNF3A/MGC33105/TCF3A

GATA3 GATA binding protein 3 HDR/MGC2346/MGC5199/MGC5445

GLI1 GLI family zinc finger 1 GLI

GRB7Growth factor receptor-bound protein 7 -

GSTP1 Glutathione S-transferase pi 1 DFN7/FAEES3/GST3/GSTP/PI

HIC1 Hypermethylated in cancer 1 ZBTB29/ZNF901/hic-1

ID1

Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein ID/bHLHb24

IGF1Insulin-like growth factor 1 (somatomedin C) IGF-I/IGF1A/IGFI

IGF1R Insulin-like growth factor 1 CD221/IGFIR/IGFR/JTK13/MGC142170/MGC142172/MGC18216

18

receptorIGFB

P3Insulin-like growth factor binding protein 3 BP-53/IBP3

IL6Interleukin 6 (interferon, beta 2) BSF2/HGF/HSF/IFNB2/IL-6

JUN Jun proto-oncogene AP-1/AP1/c-JunKRT1

8 Keratin 18 CYK18/K18KRT1

9 Keratin 19 CK19/K19/K1CS/MGC15366KRT5 Keratin 5 CK5/DDD/EBS2/K5/KRT5AKRT8 Keratin 8 CARD2/CK8/CYK8/K2C8/K8/KOMAP

K1Mitogen-activated protein kinase 1

ERK/ERK2/ERT1/MAPK2/P42MAPK/PRKM1/PRKM2/p38/p40/p41/p41mapk

MAPK3

Mitogen-activated protein kinase 3

ERK1/HS44KDAP/HUMKER1A/MGC20180/P44ERK1/P44MAPK/PRKM3

MAPK8

Mitogen-activated protein kinase 8 JNK/JNK1/JNK1A2/JNK21B1/2/PRKM8/SAPK1

MGMT

O-6-methylguanine-DNA methyltransferase -

MKI67

Antigen identified by monoclonal antibody Ki-67 KIA

MLH1

MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) COCA2/FCC2/HNPCC/HNPCC2/MGC5172/hMLH1

MMP2

Matrix metallopeptidase 2 (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) CLG4/CLG4A/MMP-II/MONA/TBE-1

MMP9

Matrix metallopeptidase 9 (gelatinase B, 92kDa gelatinase, 92kDa type IV collagenase) CLG4B/GELB/MANDP2/MMP-9

MUC1Mucin 1, cell surface associated

CD227/EMA/H23AG/KL-6/MAM6/MUC-1/MUC-1/SEC/MUC-1/X/MUC1/ZD/PEM/PEMT/PUM

MYCV-myc myelocytomatosis viral oncogene homolog (avian) MRTL/bHLHe39/c-Myc

NME1Non-metastatic cells 1, protein (NM23A) expressed in AWD/GAAD/NB/NBS/NDKA/NDPK-A/NDPKA/NM23/NM23-H1

19

NOTCH1 Notch 1 TAN1/hN1

NR3C1

Nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) GCCR/GCR/GR/GRL

PGR Progesterone receptor NR3C3/PR

PLAUPlasminogen activator, urokinase ATF/UPA/URK/u-PA

PRDM2

PR domain containing 2, with ZNF domain HUMHOXY1/KMT8/MTB-ZF/RIZ/RIZ1/RIZ2

PTENPhosphatase and tensin homolog

10q23del/BZS/DEC/GLM2/MGC11227/MHAM/MMAC1/PTEN1/TEP1

PTGS2

Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) COX-2/COX2/GRIPGHS/PGG/HS/PGHS-2/PHS-2/hCox-2

PYCARD

PYD and CARD domain containing ASC/CARD5/MGC10332/TMS/TMS-1/TMS1

RARB Retinoic acid receptor, beta HAP/NR1B2/RRB2RASS

F1Ras association (RalGDS/AF-6) domain family member 1 123F2/NORE2A/RASSF1A/RDA32/REH3P21

RB1 Retinoblastoma 1 OSRC/RB/p105-Rb/pRb/pp110

SERPINE1

Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 PAI/PAI-1/PAI1/PLANH1

SFN Stratifin YWHASSFRP

1Secreted frizzled-related protein 1 FRP/FRP-1/FRP1/FrzA/SARP2

SLC39A6

Solute carrier family 39 (zinc transporter), member 6 LIV-1

SLIT2 Slit homolog 2 (Drosophila) FLJ14420/SLIL3/Slit-2SNAI

2 Snail homolog 2 (Drosophila) MGC10182/SLUG/SLUGH1/SNAIL2/WS2D

SRC

V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) ASV/SRC1/c-SRC/p60-Src

TFF3 Trefoil factor 3 (intestinal) ITF/P1B/TFITGFB

1Transforming growth factor, beta 1 CED/DPD1/LAP/TGFB/TGFbeta

20

THBS1 Thrombospondin 1 THBS/THBS-1/TSP/TSP-1/TSP1

TP53 Tumor protein p53 FLJ92943/LFS1/P53/TRP53TP73 Tumor protein p73 P73

TWIST1 Twist homolog 1 (Drosophila) ACS3/BPES2/BPES3/CRS1/SCS/TWIST/bHLHa38

VEGFA

Vascular endothelial growth factor A MGC70609/MVCD1/VEGF/VPF

XBP1 X-box binding protein 1 TREB5/XBP2B2M Beta-2-microglobulin -

HPRT1

Hypoxanthine phosphoribosyltransferase 1 HGPRT/HPRT

RPL13A Ribosomal protein L13a L13A/TSTA1

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase G3PD/GAPD/MGC88685

ACTB Actin, beta PS1TP5BP1HGD

CHuman Genomic DNA Contamination HIGX1A

RTC Reverse Transcription Control RTCRTC Reverse Transcription Control RTCRTC Reverse Transcription Control RTCPPC Positive PCR Control PPCPPC Positive PCR Control PPCPPC Positive PCR Control PPC

21

22

-14 -12 -10 -8 -6 -4-20

0

20

40

60

80

100

120

Log concentration (M)

Res

pons

e (%

of m

ax)

E2EC50 = 2.2e-12

PPTEC50 = 2.3e-11

DPNEC50 = 1.4e-8

Supplementary Figure S1. T47D-KBluc dose response curves and EC50 values for 17β-estradiol (E2), the ERα agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H pyrazole (PPT), and the ERβ agonist 2,3-bis(4-hydroxyphenyl) propionitrile (DPN).

23

Supplementary Table S4. Histopathological evaluation results of rat uteri from individual chemical uterotrophic assays using oral gavage administration. Evaluation was conducted by an American Board of Pathology certified pathologist at Experimental Pathology Laboratories, Inc. (Durham, NC). Description of evaluation parameters on following pages.

ChemicalDose

(mg/kg/d)

Sample size

(# rats)

Increased cell height

Grade 1

Increased cell height

Grade 2

Columnar differentiation

Grade 1

Columnar differentiation

Grade 2

BBP

(+) control 4 - 4 - 40 4 - - - -

400 4 - - - -600 4 - - - -800 4 - - - -

BPA

(+) control 5 - 5 - 50 5 - - - -

200 6 - - - -400 5 - - - -600 3 - - - -800 6 1 - 1 -

BPAF

(+) control 6 - 6 - 60 4 - - - -

3.125 3 - - - -6.25 3 - - - -12.5 3 - - - -25 6 - - - -50 6 4 - 4 -100 6 5 1 5 1300 5 3 2 3 2

BPC

(+) control 6 - 6 - 60 6 - - - -

1.2 3 - - - -4 6 - - - -

13.5 6 - - - -45 6 1 - 1 -150 6 6 - 6 -500 6 3 3 3 3

BPS

(+) control 6 - 6 - 60 6 - - - -50 6 - - - -100 6 - - - -200 6 4 - 4 -400 5 5 - 5 -800 6 5 1 5 1

24

Supplementary Table S4 cont. Histopathological evaluation results of rat uteri from individual chemical uterotrophic assays using oral gavage administration. Evaluation was conducted by an American Board of Pathology certified pathologist at Experimental Pathology Laboratories, Inc. (Durham, NC). Description of evaluation parameters on following pages.

ChemicalDose

(mg/kg/d)

Sample size

(# rats)

Increased cell height

Grade 1

Increased cell height

Grade 2

Columnar differentiation

Grade 1

Columnar differentiation

Grade 2

EE2

(+) control 3 - 3 - 30 3 - - - -

0.0005 3 - - - -0.001 3 - - - -0.0025 3 - - - -0.005 3 - - - -0.01 3 1 - 1 -0.05 2 2 - 2 -0.2 3 - 3 - 3

MET

(+) control 4 - 4 - 40 3 - - - -

62.5 4 - - - -125 4 2 2 2 2250 4 1 3 1 3500 5 2 3 2 3

25

Description of histopathological evaluation of uteri

Uterine sections from ovariectomized female Sprague Dawley rats were examined by

light microscopy for the presence of findings, including: 1) changes in epithelial cell height and

2) luminal epithelium change from cuboidal to columnar differentiation. Findings were graded

one to five depending on severity. The severity grading scale was minimal = 1, mild = 2,

moderate = 3, moderately severe = 4 or severe = 5. For all groups, the “baseline” for uterine

epithelial changes was a simple appearing epithelial lining consisting of low cuboidal to low

columnar epithelium. The nucleus of epithelial cells occupied more cell space than the

cytoplasm.

For all groups, changes in uterine epithelial cell height was diagnosed as greater when

increases in cytoplasm were recognized; grade 1 or minimal change represented a ratio of 1:1

(nucleus:cytoplasm), grade 2 or mild change represented a ratio of 1:2 (nucleus:cytoplasm).

Uterine epithelial change from cuboidal to columnar differentiation was diagnosed when most

epithelial cells were clearly more columnar in shape than controls.

Grade 1 (Minimal): This corresponds to a histopathologic change ranging from inconspicuous

to barely noticeable but so minor, small, or infrequent as to warrant no more than the least

assignable grade. For multifocal or diffusely-distributed lesions, this grade was used for

processes where less than approximately10% of the tissue in an average high-power field was

involved. For focal or diffuse hyperplastic/hypoplastic/ atrophic lesions, this grade was used

when the affected structure or tissue had undergone a less than approximately 10% increase or

decrease in volume.

Grade 2 (Mild): This corresponds to a histopathologic change that is a noticeable but not a

prominent feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was

26

used for processes where between approximately 10% and 25% of the tissue in an average high-

power field was involved. For focal or diffuse hyperplastic/hypoplastic/atrophic lesions, this

grade was used when the affected structure or tissue had undergone between an approximately

10% to 25% increase or decrease in volume.

27