1. IQF Framework for 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 Speciation and Metabolism Parent Chemical Molecular Initiating Events Measurable System Effects Adverse Outcomes QSAR SystemsBiology Chemistry/Biochemistry

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

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

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

5. Molecular Initiating Events Speciation and Metabolism Measurable System Effects Adverse Outcomes Parent Chemical QSAR SystemsBiology Chemistry/Biochemistry ER Binding Altered Protein Expression Chg 2ndry Sex Char Altered Repro. Skewed Sex Ratios, Altered Repro. MOLECULAR Initiating Event CELLULAR Respopse TISSUE/ORGAN Response INDIVIDUAL Response POPULATION Response Altered proteins (VTG), Ova-testis Chemical Universe Yes Special Rule Classes Log Kow <1.3 Contai ns at least one possibl e H- bondin g site Type “A” Contains Phenol Fragment Alkyl Anilines Phthalates Branched Phenones Cyclo Phenones p-subst Cyclohexanols p-subst Cyclohexanones ring subst (o-CH3, tri-CH3) Benzoates Possible Low Affinity Binder Type “B” Contains identif. Type B Fragment N-alkyl Phenones Non ring subst Benzoates Alkyl Phenols Alkoxy Phenols Parabens Salicylates Belongs to known Active class Potency Rules / Equations Potency Rules / Equations NoNo YesYes NoNo NoNo II Needs testing NoNo YesYes YesYes Belongs to class with possible Type B low affinity under investigation Belongs to known Inactive class III Belongs to known Inactive class Fully-hindered Alkyl Phenols Belong to untested class with possible Type A low affinity ring subst p-CH3 Benzoates Thiophosphate Esters Mixed Organics Cyclic Alcohols (not p-subst hexanols) Cyclic Pentanones/Others Br, I halobenzenes NoNo NoNo YesYes Belongs to known Active class Belong to untested class Needs testing Non binder (RBA<0.00001) Mixed Phenols NoNo NoNo NoNo YesYes YesYes YesYes III Non binder (RBA<0.00001) NoNo Alkylbenzsulfonic Acids Sulfonic Acid Dyes p-Alkyl Fluorobenzenes Bis–anilines Alkoxy Anilines Imidazolidines Isothiazolines Alkyl Benzthiols Pyrrolidiones IV Oxazoles Benzami de Furans Sorbitan s Triazines YesYes YesYes NoNo Ye s DDT-Like Tamoxifen-Like Multi-Cyclohydrocarbons Alkyl Chlorobenzenes

6. Molecular Initiating Events Speciation and Metabolism Measurable System Effects Adverse Outcomes Parent Chemical QSAR SystemsBiology Chemistry/Biochemistry Speciation and Metabolism Parent Chemical Molecular Initiating Events VTG Fecundity Population ER ( -) AR (+) Aromatase (CYP 19) VTG Fecundity Population VTG Fecundity Population VTG Fecundity Population CYP17 Fenarimol Fadrozole Prochloraz 17β-trenbolone 17α-trenbolone Circulating LH, FSH LDL R HDL R 11βHS D P4501 1β Activi n Inhib in Circulating Sex Steroids / Steroid Hormone Binding Globlulin FSH R LH R Outer mitochondrial membrane Inner mitochondrial membrane StAR P450sc c pregnenolo ne 3  HS D P450c17 androstened ione testostero ne Choleste rol estradiol AR ERER 17βHS D P450aro m 11- ketotestosterone progester one 17 α - hydroxyprogesteron e 17α,20β-P (MIS) 20βHSD Estradi ol + Vtg (steroidogenic cells)

7. Risk Assessment Applications 1)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) 2)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)