1

Appendix I: Section 7 Supplemental, Figures I-1 to I-4.

Figure I-1. Difference in activity in two similar chemical groups in the trout-based assays 

used for the ERES TrSet and the HTP assays. In the TrSet the chemicals listed from both 

of these chemical groups are Binders, whereas in the HTP assays the Parabens (nonbenzoate) are active but the salicylates (non-benzoate) are inactive. 

Figure I-2. Apparent binding by Octylbenzesulfonic acid showing full displacement of 3HE2 in the cytosolic rtER assay (left panel) but no agonist activity for Vtg mRNA induction 

(right panel). 

Figure I-3. Testing procedure for antagonism in VTG assay. The chemicals is tested across 

the concentration range twice, to compete against two E2 concentrations to distinguish 

antagonism from cytotoxicity. When there is a significant decline in Vtg induction at high 

concentrations of chemical, but it is not shifted by different E2 co-treatment concentrations 

(-9logM or -7logM; left panel) and it coincides with significant toxicity (measured by LDH 

leakage (right panel)), the decline is attributed to to cytotoxicity not antagonism.

Figure I-4. The results of a Ki assay showed the plot of slope vs ligand was not linear for 

OBS, indicating the binding was not true competitive binding. A Ki determination for 

Kaempferol showing competitive inhibition is shown for comparison.
2

Figure I-1. Difference in activity in two similar chemical groups in the trout-based assays 

used for the ERES TrSet and the HTP assays. In the TrSet the chemicals listed from both 

of these chemical groups are Binders, whereas in the HTP assays the Parabens (nonbenzoate) are active but the salicylates (non-benzoate) are inactive. 

Chemical name abbreviations: 

Parabens: MethylP = methylparaben [99-76-3]; EthylP: = ethylparaben [120-47-8]; PropylP = 

propylparaben [94-13-3]; ButylP = butylparaben [94-26-8]; BenzylP = benzyl-4-

hydroxybenzoate [94-18-8]; HeptylP = n-heptyl-4-hydroxybenzoate [1085-12-7]; OctylP = noctyl-4-hydroxybenzoate [1219-38-1]Salicylates: MethylS = methylsalicylate [119-36-8]; 

MethylAT = methylatrate [4707-47-5]; BenzyzylS = benzylsalicylate [118-58-1]; HexylS = 

hexylsalicylate [6259-76-3]; TMcyS = 3,3,5-Trimethylcyclohexyl salicylate [118-56-9].
3

Figures I-2 to I-4 show experimental data from several diagnostic assays that taken together 

indicate the apparent ER binding by octylbenensulfonic acid (OBS) is not true competitive 

binding. 

Figure I-2. Apparent binding by Octylbenzesulfonic acid showing full displacement of 3HE2 in the cytosolic rtER assay (left panel) but no agonist activity for Vtg mRNA induction 

(right panel). 

Figure I-3. Testing procedure for antagonism in liver slice VTG assays. The chemicals is 

tested across the concentration range twice, to compete against two E2 concentrations to 

distinguish antagonism from cytotoxicity. When there is a significant decline in Vtg 

induction at high concentrations of chemical, but it is not shifted by different E2 cotreatment concentrations (-9logM or -7logM; left panel) and it coincides with significant 

toxicity (measured by LDH leakage (right panel)), the decline is attributed to to 

cytotoxicity not antagonism.

4-Octylbenzenesulfonic Acid

[6149-03-7]

rtER Binding

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 0

10

20

30

40

50

60

70

80

90

100

110

120

E2

Competitor (M)

Specific Binding (%)

OBS

1.0×104

1.0×105

1.0×106

1.0×107

1.0×108

-10 -9 -8 -7 -6 -5 -4 -3 -2

rtER Gene Expression

E2

OBS

CONTROL

CTRL

Log Concentration (M)

Vtg mRNA (copy #/400 ng total RNA)

0

10

20

30

40

50

60

70

-10 -9 -8 -7 -6 -5 -4 -3 -2

rt Liver Slice Viability by LDH Leakage

4-Octylbenzenesulfonic Acid

Leakage % of Total LDH

Log Concentration (M)

OBS- 48h

CONTROL- 48h

CTRL

104

105

106

107

108

-10 -9 -8 -7 -6 -5 -4 -3 -2

rtER Gene Expression_antagonism assay

4-Octylbenzenesulfonic Acid

E2 at -7logM + OBS

E2

CTRL

E2 at -9logM + OBS

E2

CTRL

CTRL

Log Concentration (M)

Vtg mRNA (copy #/400 ng total RNA)
4

 

Figure I-4. The results of a Ki assay showed the plot of slope vs ligand was not linear for 

OBS, indicating the binding was not true competitive binding. A Ki determination for 

Kaempferol showing competitive inhibition is shown for comparison.

Concentration Ki 

(μM)

IC50/5 330.5

IC50/2 174.7

IC50 67.3

2X IC50 14.4

5X IC50 3.28

Concentration Ki (μM)

IC50/5 1.9

IC50/2 1.5

IC50 1

2X IC50 0.6

5X IC50 1.1

0 100 200 300 400 500 600 700

0

1000

2000

3000

4000

5000

Ki determination in rtER binding assay

4-Octylbenzenesulfonic Acid

OBS (μM)

Slope

0 25 50 75 100 125 150

0

2500

5000

7500

10000

12500

15000

Ki = competitive binding

Kaempferol

KMF (uM)

Slope