IQF Framework for QSAR

1. Speciation and Metabolism Molecular Initiating Events Measurable System Effects Adverse Outcomes Parent Chemical IQF Framework for QSAR Systems Biology Chemistry/ Biochemistry QSAR 1. Identify Plausible Molecular Initiating Events 2. Design Database for Abiotic Binding Affinity/Rates 3. Explore Linkages in Pathways to Downstream Effects 4. Develop QSARs to Predict Initiating Event from Structure

2. Estrogen Receptor (ER) Toxicity Pathway Chemical interacts with Receptor at Molecular Level, Initiates a Series of events, and leads to Adverse Outcome In vitroAssays QSAR In vivo Assays Inert/Antimicrobial Chemical INDIVIDUAL POPULATION CELLULAR Response TISSUE/ORGAN MOLECULAR Initiating Event Skewed Sex Ratios; Yr Class Sex reversal; Altered behavior; Repro. Altered proteins, hormones; Ova-testis Altered Protein Expression ER Binding Toxicological Understanding Risk Assessment Relevance

3. INDIVIDUAL Response POPULATION Response TISSUE/ORGAN Response CELLULAR Respopse MOLECULAR Initiating Event Skewed Sex Ratios, Altered Repro. Chg 2ndry Sex Char Altered Repro. Altered proteins (VTG), Ova-testis Altered Protein Expression ER Binding In vitro Assay Endpts Trout cyto rtER Binding MaleTrout Liver Slice Vtg Induction ER Toxicity Pathway Male Medaka Chg in 2ndary sex characteristic; behavior? Papillary Processes Male Medaka Liver Vtg Induction In vivo Assay Endpts Sex Reversal Genetic Males to Phenotypic Females Female Medaka Liver Vtg decr. Female Medaka Fecundity Hatch Male Medaka Gonad Complete conversion to ovary

4. No Yes ChemicalUniverse Yes No No • Alkylbenzsulfonic Acids • Sulfonic Acid Dyes • p-Alkyl Fluorobenzenes • Bis–anilines • Alkoxy Anilines • Imidazolidines • Isothiazolines • Alkyl Benzthiols • Pyrrolidiones Non binder(RBA<0.00001) Contains at least one possible H-bonding site Yes Log Kow <1.3 • Oxazoles • Benzamide • Furans • Sorbitans • Triazines Possible Low Affinity Binder No Yes Type “A” Contains Phenol Fragment Belongs to known Active class II Yes • DDT-Like • Tamoxifen-Like • Multi-Cyclohydrocarbons • Alkyl Chlorobenzenes Yes Special Rule Classes IV No No Type “B” Contains identif. Type B Fragment Belongs to known Inactive class No III Yes Yes III • Alkyl Phenols • Alkoxy Phenols • Parabens • Salicylates Potency Rules / Equations No No Belong to untested class Non binder(RBA<0.00001) Yes Belongs to known Active class • Fully-hindered Alkyl Phenols Needs testing Belong to untested class with possible Type A low affinity Yes • Mixed Phenols • Alkyl Anilines • Phthalates • Branched Phenones • Cyclo Phenones • p-subst Cyclohexanols • p-subst Cyclohexanones • ring subst (o-CH3, • tri-CH3) Benzoates No Needs testing • N-alkyl Phenones • Non ring subst Benzoates Belongs to known Inactive class Yes Non binder(RBA<0.00001) • ring subst p-CH3 Benzoates • Thiophosphate Esters • Mixed Organics • Cyclic Alcohols (not • p-subst hexanols) • Cyclic Pentanones/Others • Br, I halobenzenes No Needs testing Belongs to class with possible Type B low affinity under investigation Potency Rules / Equations Contains Cycle Belongs to known Inactive class Contains 2 OH, or OH and =0, at Spec. Dist Possible High Affinity, “A-B”; “A-C”; or “A-B-C” type binder Yes V No Contains some attenuating feature steric?; other? Yes No High Binding Affinity “A-B”;“A-C” or “A-B-C” type Assess strength of attenuation steric?; other? Some Complete Non binder Ex: Progesterone Corticossterone (RBA<0.00001) No Low Affinity Binder “A-B”,“A-C” or “A-B-C” type

5. No Yes ChemicalUniverse Yes No No • Alkylbenzsulfonic Acids • Sulfonic Acid Dyes • p-Alkyl Fluorobenzenes • Bis–anilines • Alkoxy Anilines • Imidazolidines • Isothiazolines • Alkyl Benzthiols • Pyrrolidiones Non binder(RBA<0.00001) Contains at least one possible H-bonding site INDIVIDUAL Response POPULATION Response Yes Skewed Sex Ratios, Altered Repro. TISSUE/ORGAN Response CELLULAR Respopse Chg 2ndry Sex Char Altered Repro. MOLECULAR Initiating Event Altered proteins (VTG), Ova-testis Log Kow <1.3 Altered Protein Expression ER Binding • Oxazoles • Benzamide • Furans • Sorbitans • Triazines Possible Low Affinity Binder No Speciation and Metabolism Molecular Initiating Events Measurable System Effects Yes Adverse Outcomes Parent Chemical Type “A” Contains Phenol Fragment Belongs to known Active class II Yes • DDT-Like • Tamoxifen-Like • Multi-Cyclohydrocarbons • Alkyl Chlorobenzenes Yes Special Rule Classes IV No No No Type “B” Contains identif. Type B Fragment Belongs to known Inactive class III Yes Yes III • Alkyl Phenols • Alkoxy Phenols • Parabens • Salicylates Potency Rules / Equations No No Systems Biology Chemistry/ Biochemistry QSAR Belong to untested class Non binder(RBA<0.00001) Yes Belongs to known Active class • Fully-hindered Alkyl Phenols Needs testing Belong to untested class with possible Type A low affinity Yes • Mixed Phenols • Alkyl Anilines • Phthalates • Branched Phenones • Cyclo Phenones • p-subst Cyclohexanols • p-subst Cyclohexanones • ring subst (o-CH3, • tri-CH3) Benzoates No Needs testing • N-alkyl Phenones • Non ring subst Benzoates Belongs to known Inactive class Yes Non binder(RBA<0.00001) • ring subst p-CH3 Benzoates • Thiophosphate Esters • Mixed Organics • Cyclic Alcohols (not • p-subst hexanols) • Cyclic Pentanones/Others • Br, I halobenzenes No Needs testing Belongs to class with possible Type B low affinity under investigation Potency Rules / Equations

6. Speciation and Metabolism Speciation and Metabolism Molecular Initiating Events Molecular Initiating Events Measurable System Effects Adverse Outcomes Parent Chemical Parent Chemical Circulating LH, FSH LDL R HDL R LH R FSH R Cholesterol Outer mitochondrial membrane Activin StAR VTG Inhibin Fecundity Population Fecundity Population Fecundity Population Fecundity Population Inner mitochondrial membrane P450scc pregnenolone 3bHSD 17α-hydroxyprogesterone progesterone Systems Biology Chemistry/ Biochemistry QSAR P450c17 20βHSD androstenedione 17βHSD 17α,20β-P (MIS) testosterone P450arom P45011β (steroidogenic cells) 11βHSD 11-ketotestosterone estradiol VTG VTG VTG Circulating Sex Steroids / Steroid Hormone Binding Globlulin AR Estradiol + Vtg ER ER ( -) AR (+) Aromatase (CYP 19) Fenarimol 17α-trenbolone 17β-trenbolone Fadrozole Prochloraz CYP17

7. Risk Assessment Applications • Prioritization • -Systematic testing with a “well-defined” endpoint • -QSAR Decision Support System for ER molecular initiating event • - diverse chemical structures • Same approach can be taken for other molecular initiating events • AR – diverse structures – extent may be same of less than ER • systematic testing needed to develop model for defined chemical list • - assays available; may need to be optimized for anticipated • low affinity chemicals (EPA problem); EU application? • aromatase – diversity of structures causing enz inhib? • systematic testing needed to develop model for defined chemical list • - assays available; need optimization?? • other steroidogenic enzymes – (same approach as above) • Higher Tier Assessment • HPG Systems Models assist data interpretation?

8. Developmental Endpoints HPT Systems Model Identify molecular initiating events NIS TPO ( in vitro assays optimized for QSAR approach) CYP induction – T4 kinetics (gluc)