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 (non benzoate) are active but the salicylates (non - benzoate) are inactive. Figure I - 2 . Apparent binding by Octyl benzesulfonic acid showing full displacement of 3H E2 in the cytosolic rtER assay (left panel) but no agonist activity for Vtg mRNA induction (right panel). Figure I - 3 . Testing p rocedure 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 d ecline 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 (r ight panel) ) , the decline is attributed to to cyto toxicity 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 (non benzoate) 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 = n
o ctyl
- 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 Octyl benzesulfonic acid showing full displacement of 3H E2 in the cytosolic rtER assay (left panel) but no agonist activity for Vtg mRNA induction (right panel).

Figure I - 3 . Testing p rocedure for antagonism in liver slice VTG assay s . The chemicals is tested across the concentration range twice, to compete against two E2 concentrations to distinguish antagonism from cyto toxicity. When there is a significant d ecline in Vtg induction at high concentration s 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 cyto toxicity 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 × 10 4 1.0 × 10 5 1.0 × 10 6 1.0 × 10 7 1.0 × 10 8

-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 AcidLeakage % of Total LDH

Log Concentration (M) OBS- 48h CONTROL- 48h
CTRL 10 4 10 5 10 6 10 7 10 8

-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