Use of Toxicological Pathways for Hazard Assessment in OECD (Q)SAR Toolbox: McKim Conference September 2008, Duluth, USA LMC, Bourgas University, Bulgaria Chemical Management Center, NITE, Japan Fraunhofer Institute for Toxicology and Experimental Medicine, Germany OECD, Environment Directorate, Paris International QSAR Foundation, USA

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

Molecular Initiating Events Chemical Speciation and Metabolism Measurable System Effects Adverse Outcomes Parent Chemical Conceptual Framework of SAR/QSAR Rather than developing statistical models of complex endpoints, key molecular initiating events become the “well-defined” endpoints for QSAR. Gil Veith; International QSAR Foundation

Adverse Outcomes Parent Chemical IQF Framework for QSAR Black Box Models Rapid but not mechanistically transparent

Molecular Initiating Events Speciation and Metabolism Measurable System Effects Adverse Outcomes Parent Chemical 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 QSAR SystemsBiology Chemistry/Biochemistry QSAR

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

Categorization and QSAR  The categories concept is part of the historical description of QSARs  QSARs are quantitative models of key mechanistic processes which result in the measured activity

Each QSAR estimate is a result of two predictions:  Qualitative prediction of predominant interaction mechanisms and hazard identification (defined by category)  Quantitative prediction of the intensity (potency) of the specific mechanisms of interaction (predicted by QSAR) Wrong definition for the mechanism of underlying reaction could result in using of a wrong QSAR for the potency estimate Categorization and QSAR

Example  Phenols are polar narcotics, uncouplers or electrophilic chemicals.  QSAR models for predicting acute effects for each mechanism have comparable uncertainty  The potency of the electrophilic mechanism can be orders of magnitude greater than polar narcotics  Wrong categorization of chemicals could cause significant errors in defining the potency

The logic for selecting a specific model (category) for a specific chemical is the cornerstone of regulatory acceptance Categorization and QSAR Basic Assumption for Regulatory Acceptance OECD QSAR AD-Hoc group meeting, Madrid, April 2007

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

Modelled human health endpoints in Toolbox  Sensitization Lung Skin  Genotoxicity AMES bacterial mutagenicity Chromosomal aberration  Irritation/corrosion Eye Skin

Commonality between the modelled endpoints The effects could be characterized by:  Single toxicological pathway  Strong dependency on initiating molecular events (e.g. on molecular structure)  Small impact of subsequent biological processes (“short” toxicological pathways)

Molecular Initiating Events Speciation and Metabolism Measurable System Effects Adverse Outcomes Parent Chemical QSAR Framework for modeled endpoint QSAR Biological processes Initiating chemical/Biochemical Interactions QSAR

Molecular Initiating Events Speciation and Metabolism Adverse Outcomes Parent Chemical QSAR QSAR QSAR Framework for modeled endpoint Initiating chemical/Biochemical Interactions

Epidermis Dermis Hypodermis Vein Protein conjugates Penetration Protein conjugates Metabolism Lymph Mechanism of skin sensitization Subject of modeling Assumptions in the model: 1.Chemicals always penetrate stratum corneum 2.Formation of protein conjugates is a premise for ultimate effect 3.Metabolism may play significant role in skin sensitization

Model Simulator of skin metabolism ∩ QSAR models Parent Metabolism Phase II Reactive species Reactive species Reactive species S-Pr W sensitization S-Pr W sensitization S-Pr S sensitization S-Pr S sensitization No sensitization QSAR

Conclusion: The categorization of substances according to chemical mechanisms governing the initiating reaction with protein or DNA is good enough for predicting human health effects resulting from single and “short” toxicological pathways

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

General characterization by the following grouping schemes: Substance information Predefined Mechanistic: Acute Toxicity MOA (OASIS) Protein binding (OASIS) DNA binding (OASIS) Electron reach fragments (Superfragments) BioBite Cramer Classification Tree (ToxTree) Veerhar/Hermens reactivity rules (ToxTree) Lipinski rules (MultiCase) Chemical input ProfilingCategory Definition Filling data gap ReportEndpoints Toolbox logical sequence of components usage

Molecular Initiating Events and Toxicological Pathways General Consideration

Molecular Level Mechanism of chemical interactions

Mechanism 1 Mechanism 2 Mechanism 3 … Molecular Level

Distribution in lipid phase Protein binding Arylcarboxylate aminolysis Michael-type addition Schiff base formation … DNA binding Quinones Hydrazines … Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Molecular Level

Distribution in lipid phase Protein binding Arylcarboxylate aminolysis Michael-type addition Schiff base formation … DNA binding Quinones Hydrazines … Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Molecular Level

Mechanism of chemical interactions Initiating event/Receptor Activation of AP-1 、 NF-kB 、 EpRE in hepatocyte →Activation of JNK/AP-1 pathway Activation of estrogen Signals → Proliferation of bile duct cell and hepatocyte injury Activation of MAPK Signals - Apoptosis … Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Molecular Level

Mechanism of chemical interactions Initiating event/Receptor Activation of AP-1 、 NF-kB 、 EpRE in hepatocyte →Activation of JNK/AP-1 pathway Activation of estrogen Signals → Proliferation of bile duct cell and hepatocyte injury Activation of MAPK Signals - Apoptosis … Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Cell Level System biology/Effect Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Cell Level System biology/Effect Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … System 1 – System 2 – System 3 –... Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Cell Level System biology/Effect System biology Hepatotoxicity mechanism: Oxidant stress Mitochondrial damage Apoptosis Degradation of membrane phospholipid Aberration of ion channel Increase of enzyme activition of drug metabolism Inflammatory responses … Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … System 1 – System 2 – System 3 –... Molecular Level

Mechanism of chemical interactions Cell Level System biology/Effect Cell Effects Hepatocyte Changes in the tubular epithelium … Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Cell Level System biology/Effect System biology Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Cell Level System biology/Effect System biology Tissue, Organ and Body Observed Effects Symptomology Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Cell Level System biology/Effect System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... System biology Tissue, Organ and Body Observed Effects Symptomology Tissue Effect 1 Effect 2 Effect 3... Organ Effect 1 Effect 2 Effect 3... Body Effect 1 Effect 2 Effect 3... Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Cell Level System biology/Effect System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... System biology Tissue, Organ and Body Observed Effects Symptomology Tissue Effect 1 Effect 2 Effect 3... Organ Effect 1 Effect 2 Effect 3... Body Effect 1 Effect 2 Effect 3... Molecular initiating event(s) and subsequent downstream effects Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Cell Level System biology/Effect System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... System biology Tissue, Organ and Body Observed Effects Symptomology Tissue Effect 1 Effect 2 Effect 3... Organ Effect 1 Effect 2 Effect 3... Body Effect 1 Effect 2 Effect 3... Molecular initiating event(s) and subsequent downstream effects Molecular Level Chemistry/Biochemistry

Mechanism of chemical interactions Mechanism 1 Mechanism 2 Mechanism 3 … Receptor 1 – Receptor 2 – Receptor 3 – … Cell Level System biology/Effect System 1 – System 2 – System 3 –... Effect 1 Effect 2 Effect 3... System biology Tissue, Organ and Body Observed Effects Symptomology Tissue Effect 1 Effect 2 Effect 3... Organ Effect 1 Effect 2 Effect 3... Body Effect 1 Effect 2 Effect 3... Molecular initiating event(s) and subsequent downstream effects Molecular Level Chemistry/Biochemistry

1.One complex endpoint (e.g., 28days RDT) could be conditioned by more than one toxicological pathway (blood toxicity, liver damage, kidney damage) Conclusion: 2.(Q)SAR models should be associated with a single toxicological pathway 3.Chemicals which interact by different toxicological pathways should be out of the model mechanistic domain

Conclusion: 4.The categorization of substances according to chemical mechanisms governing the initiating reactions with protein or DNA is not enough for predicting human health effects resulting from multiple and complex toxicological pathways 5.The link between chemical and toxicological mechanisms and respective categorization schemes needs to be identified

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

Case study: Twenty-eight day repeat dose oral toxicity test of chemicals (28d RDT) 1.Data produced by:  Safety examination of existing chemicals in NITE- Japan; under Japanese Chemical Substances Control Law;  Fraunhofer Institute for Toxicology and Experimental Medicine, Hanover, Germany 2. Categorization of chemicals for predicting 28d RDT is based on analysis of data by NITE and LMC

28-day RDT tests conducted on male rats that tested 14 aromatic amines

Categorization of Anilines 1.Based on their effects on two organs:  Blood  Kidney

Categorization of Anilines 1.Based on their effects on two organs:  Blood  Kidney

Blood Toxicity: Blood toxicity effects:  decrease in erythrocyte count (RBC)  hemoglobin level (Hb)  Hematocrit (HTC)  glutamic-pyruvic transaminase (GPT)  increase in the number of reticulocytes  hemosiderin pigmentation in the spleen  increase in hematopoiesis  etc.

Toxicity scale of Intensity Intensity scale: basis of the number of effects indicative of toxicity  strong  medium  weak  non

Toxicity scale of Intensity Example: determining LOEL of N-ethylaniline:  Test doses - 0, 5, 25, 125 mg/kg/day  Decrease in RBC only has been observed at 5 mg/kg  Decrease in RBC, Hb and HTC–at 25 and 125 mg/kg  Hence, LOEL for hemolysis is 5mg/kg/day

★★ ★★ ★★ ★ ★ ★★ ★ ★★★ ★ ▲ ▲ ▲▲ ▲ ▲ ■ Dose (mg/kg/day) Anemia findings ○: 0, ▲: 1, ■: 2, ★ : >3 RBC↓, Hb↓, HTC↓, GPT ↑ Reticulocytes↑ hemosiderin pigmentation in the spleen hematopoiesis↑ etc. Comparison of the intensities of anemia for 14 aromatic amines

NOELs for anemia RBC↓, Hb↓, HTC↓ Reticulocytes↑ hemosiderin pigmentation in the spleen hematopoiesis↑ etc. Water soluble anilines (logKow<0 ) have NO EFFECT Relationships between NOEL for anemia and logKow for 14 aromatic amines

LOELs for anemia RBC↓, Hb↓, HTC↓ Reticulocytes↑ hemosiderin pigmentation in the spleen hematopoiesis↑ etc. Relationships between LOEL for anemia and logKow for 14 aromatic amines Water soluble anilines (logKow<0 ) have NO EFFECT

CYP450 in the Liver Glucuronide and sulfate conjugation Urine Adducts with DNA, Blood proteins: Hb, Alb Hb Met Hb Mechanism 1 Mechanism 2 These mechanisms of initiating reactions will be used to develop toxicological mechanism based categories Mechanism underlying the toxic effects exerted by anilines

LOELs for anemia RBC↓, Hb↓, HTC↓ Reticulocytes↑ hemosiderin pigmentation in the spleen hematopoiesis↑ etc. Mechanism 2 Mechanism 1 Relationships between LOEL for anemia and logKow for 14 aromatic amines

Hemoglobin binding index (HBI) HBI = (mmole compound/mole Hb)/(mmole compound/kg body weight) Sabbioni, Environ. Health Perspect. 102 (1994) HBI = f (ΔE # ) ΔE # Nitrenium ion E Reaction pathway Validation of Mechanism #1

Calculated HBI (  E # [eV]) vs. change in RBC in RDT test Validation of Mechanism #1

in vitro, female rat liver R. Kato et al., Jpn. J. Pharmacol., 19 (1969) 53 – 62 + Rats 28 days >5 mg/kg/day Hemolysis (Japanese CSCL) Rats 28 days >6 mg/kg/day Hemolysis (EU Risk Assessment Report) Rats 90 days <150 mg/kg/day No Hemolysis Findings [Johnnsen et al., Toxicol. Lett., 30 (1986) 1-6.] Repeated Dose Toxicity Test Metabolite of N-methylanilines Validation of Mechanism #1

Using HBI of the metabolite(Aniline) Validation of Mechanism #1

Categorization of Anilines 1.Based on their effects on two organs:  Blood  Kidney

Effect on the kidney ○ ： Nothing ▲ ： Weak (Kidney wt↑) ■ ： Medium (Other) ★： Strong (Necrosis) ★ ★ ▲ ■ Dose (mg/kg/day) ■ ▲ ▲ ★ Comparison of the effect on the kidney for 14 aromatic amines

LOELs for the kidney Different effect of the chemical as compared with the anemia. This will be related with interaction mechanism Relationships between LOEL for the kidney and logKow for 14 aromatic amines

CYP450 in the Liver Glucuronide and sulfate conjugation Urine Adducts with DNA, Blood proteins: Hb, Alb Hb Met Hb Mechanism underlying the toxic effects exerted by anilines on kidney Mechanism 2 Mechanism underlying the toxic effects exerted by anilines

× ＞ binding with the SH proteins RDT Effect on the kidney Effect initiating mechanism Basophilic tubule, proximal (100, 500) Necrosis, Tubular epithelium, proximal (500) Kidney, wt ↑ (720)

Category building based on the link between chemical and toxicological mechanisms

Category Building Category #1. Water soluble aromatic amines – logKow ≤ 0 – no effect Based on the link between chemical and toxicological mechanisms Category #2. If 0<logKow ≤ 1; it is eliminated by mechanism #2 and as parent or metabolite has alerting groups interacting with:  Proteins (such as quinone imines; Mechanisms #2) – strong kidney toxicity and weak blood toxicity (Category #2a)  DNA or blood proteins (Mechanisms #1) – week blood toxicity (Category #2b)

Category Building Based on the link between chemical and toxicological mechanisms Category #3. If logKow > 1 and as parent or metabolite has alerting groups interacting with:  Proteins (Mechanisms #2) – week kidney toxicity (Category #3a)  DNA (Mechanisms #1) – strong blood toxicity (Category #3b)

LogKow<0 Chemical

Yes No toxic effect LogKow<0 Chemical

No Yes 0<LogKow<1 No toxic effect LogKow<0 Chemical

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert Week kidney toxicity Yes

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert Week kidney toxicity Yes No No kidney toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert Week kidney toxicity Yes No No kidney toxicity Parent or metabolites have or form alert interacting with DNA

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert Week kidney toxicity Yes No No kidney toxicity Parent or metabolites have or form alert interacting with DNA Yes Strong blood toxicity

No Yes 0<LogKow<1 Yes No toxic effect LogKow<0 Chemical Parent or metabolites have or form protein binding alert Yes Strong kidney toxicity No No/Week kidney toxicity Parent or metabolites have or form DNA binding alert Yes Week blood toxicity No No blood toxicity No Parent or metabolites have or form protein binding alert Week kidney toxicity Yes No No kidney toxicity Parent or metabolites have or form alert interacting with DNA Yes Strong blood toxicity No No blood toxicity

Demonstrated by using RDT database, Fraunhover ITEM, Hanover, Germany New Pilot Functionalities in Toolbox

Input chemical

Chemical category based on molecular initiating event

Measured RDT and Genotoxicity data are extracted from the OASIS genotox and RDT databases

OECD Toolbox Species Organ/System Effect/Tissue rat mouse immune system intestine kidney larynx liver lung lymph node mammary gland nervous system dermal drinking water feed gavage inhalation oral unspecified Route weight decreased weight increased cell proliferation changed enzyme activity degeneration inflammation metaplasia Alanine aminotransferase Alkaline phosphatase Lactate dehydrogenase Specification NOEL LOEL RDT Structure Endpoint

OECD Toolbox Filtering Reference Study Endpoint Route Bioassay (Species) *Gender *Exposure duration Organ/Tissue Effect Study information Supplemental Information Rat Mouse … Strain Study sex Effect sex Study duration, days Study duration, categories Post exposure Reliability Author(s) Title Source Date Dermal Drinking water Feed Gavage … Immune system Intestine Kidney … Weight decreased Weight increased Cell proliferation … New Filtering Functionality *Items not in SIDS tree

Target endpoint: LOEL, feed, rat Damaged organs and effects are unknown before chemical mechanism based categorization

Chemical grouping based on initiating molecular reaction

Target endpoint: LOEL, feed, rat Damaged organs and effects should be defined based on chemical mechanism based categorization

Subcategorization based on chemical interaction mechanisms and/or structure based similarity

Subcategorization based on toxicological mechanisms resulting from underlying chemical interaction mechanisms

Missing information on organs expected to be damaged as a result of initiating reaction

Missing information on effects expected on damaged organs as a result of initiating reaction

Assumed Toxicological Pathway Binding with DNA Tumour formation in liver Chemical MechanismsToxicological Mechanisms Aromatic Amines

Defining toxicological pathway and building the mechanism data base is critical for the Toolbox project Chemical MechanismsToxicological Mechanisms

Chemical categorization Grouping by Chemical Mechanisms Toxicological categorization Grouping by Toxicological Mechanisms Predicted initiating reactions for the target chemical Predicted toxicological outcome for the target chemical

Outline  Conceptual framework of QSAR  Categorization and QSAR  Predicting human health endpoints in Toolbox  Molecular initiating events and toxicological pathways  Case study with 28d RDT  Mechanism database in Toolbox

The Mechanism Database in the Toolbox Project

System biology Symptomology Chemistry/Biochemistry

Molecular LevelCell Level System biology Tissue, Organ and Body Symptomology Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Molecular Level Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Functions Pathways Processes Molecular Level Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Molecular Level Functions Pathways Processes Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemistry/Biochemistry Chemical Mechanism Database

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Tissue Organ Body Response Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemical Mechanism Database Chemistry/Biochemistry Expert observation

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Tissue Organ Body Response Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemical Mechanism Database Chemistry/Biochemistry Expert observation Toxicological Mechanism Database

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Mechanism Knowledge Database Tissue Organ Body Response Expert observation Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemistry/Biochemistry

Cell Level System biology Tissue, Organ and Body Symptomology Search / Collection of in vitro Test Information Initiating reaction (Q)SAR Categorization Mechanism Knowledge Database (Toxicological pathways) Tissue Organ Body Response Molecular Level Receptor 1 – Receptor 2 – Receptor 3 – Receptor 4 –... System 1 – System 2 – System 3 – System 4 –... Effect 1 Effect 2 Effect 3 Effect 4... Functions Pathways Processes Chemistry/Biochemistry Expert observation

Contributors:

O. Mekenyan S. Dimitrov T. Pavlov G. Chankov A. Chapkanov Laboratory of mathematical Chemistry, Bourgas, Bulgaria

Chemical Management Center, NITE, Japan Case study on RDT of aromatic amines Chemical vs. toxicological mechanisms (Project of NEDO Japan) Jun Yamada Yuki Sakuratani

Fraunhofer Institute for Toxicology and Experimental Medicine, Department Chemical Risk Assessment, Hanover, Germany Data from REPDOSE Database (Project of CEFIC LRI) Inge Mangelsdorf Sylvia Escher Annette Bitsch

Environment Directorate, OECD, Paris Bob Diderich Terry Schultz

International QSAR Foundation, USA Gilman Veith