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
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Use of Toxicological Pathways for Hazard Assessment in OECD (Q)SAR Toolbox:
Use of Toxicological Pathways for Hazard Assessment in OECD (Q)SAR Toolbox:. LMC, Bourgas University, Bulgaria Chemical Management Center, NITE, Japan Fraunhofer Institute for Toxicology and Experimental Medicine, Germany OECD, Environment Directorate, Paris - PowerPoint PPT Presentation
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Use of Toxicological Pathways for Hazard Assessment in OECD (Q)SAR Toolbox:
McKim ConferenceSeptember 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
MolecularInitiating
Events
Chemical Speciation
and
Metabolism
MeasurableSystem Effects
Adverse Outcomes
ParentChemical
Conceptual Framework of SAR/QSARConceptual 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
ParentChemical
IQF Framework for QSARIQF Framework for QSAR
Black Box Models
Rapid but not mechanistically transparent
MolecularInitiating
Events
Speciation
and
Metabolism
MeasurableSystem Effects
Adverse Outcomes
ParentChemical
IQF Framework for QSARIQF 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
QSARQSAR
Systems Systems BiologyBiology
Chemistry/Chemistry/BiochemistryBiochemistry
QSARQSAR
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
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
Assumptions in the model:1. Chemicals always penetrate stratum corneum2. Formation of protein conjugates is a premise for ultimate effect3. Metabolism may play significant role in skin sensitization
ModelSimulator of skin metabolism ∩ QSAR models
Parent
Metabolism
Phase II
Phase II
Reactivespecies
Reactivespecies
Reactivespecies
S-PrW sensitization
S-PrW sensitization
S-Pr
S sensitization
S-PrS 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:
Molecular Initiating Events and Toxicological Pathways
General Consideration
Molecular LevelMechanism of chemical interactions
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Molecular Level
Distribution in lipid phaseProtein binding Arylcarboxylate aminolysis Michael-type addition Schiff base formation … DNA binding Quinones Hydrazines …
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Molecular Level
Distribution in lipid phaseProtein binding Arylcarboxylate aminolysis Michael-type addition Schiff base formation … DNA binding Quinones Hydrazines …
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 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 1Mechanism 2Mechanism 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 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
System 1 –System 2 –System 3 –...
Molecular Level
Chemistry/Biochemistry
Chemistry/Biochemistry
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
System biologyHepatotoxicity mechanism:• Oxidant stress• Mitochondrial damage• Apoptosis• Degradation of membrane phospholipid• Aberration of ion channel• Increase of enzyme activition of drug metabolism• Inflammatory responses• …
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
System 1 –System 2 –System 3 –...
Molecular Level
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
Cell Effects•Hepatocyte•Changes in the tubular epithelium•…
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
System biology
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Cell LevelSystem biology/Effect
System biology
Tissue, Organ and Body Observed Effects
Symptomology
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Cell LevelSystem biology/Effect
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
System biology
Tissue, Organ and Body Observed Effects
Symptomology
TissueEffect 1Effect 2Effect 3...
OrganEffect 1Effect 2Effect 3...
BodyEffect 1Effect 2Effect 3...
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Cell LevelSystem biology/Effect
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
System biology
Tissue, Organ and Body Observed Effects
Symptomology
TissueEffect 1Effect 2Effect 3...
OrganEffect 1Effect 2Effect 3...
BodyEffect 1Effect 2Effect 3...
Molecular initiating event(s) and subsequent downstream effects
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Cell LevelSystem biology/Effect
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
System biology
Tissue, Organ and Body Observed Effects
Symptomology
TissueEffect 1Effect 2Effect 3...
OrganEffect 1Effect 2Effect 3...
BodyEffect 1Effect 2Effect 3...
Molecular initiating event(s) and subsequent downstream effects
Molecular Level
Chemistry/Biochemistry
Mechanism of chemical interactions
Mechanism 1Mechanism 2Mechanism 3…
Receptor 1 –Receptor 2 – Receptor 3 – …
Cell LevelSystem biology/Effect
System 1 –System 2 –System 3 –...
Effect 1 Effect 2Effect 3...
System biology
Tissue, Organ and Body Observed Effects
Symptomology
TissueEffect 1Effect 2Effect 3...
OrganEffect 1Effect 2Effect 3...
BodyEffect 1Effect 2Effect 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
No. Structure NOEL* TargetOrgans
No. Structure NOEL* TargetOrgans
No. Structure NOEL* TargetOrgans
1 1 Erythrocyte 6 12ErythrocyteLiverKidney
11 80ErythrocyteLiverKidney
2 <5 Erythrocyte 7 40 Erythrocyte 12 1000
3 10
ErythrocyteLiverKidneyThyroid
8 30ErythrocyteLiverHeart
13 300
4 10 ErythrocyteLiver
9 <15
ErythrocyteLiverKidneyTestes
14 300
5 2 ErythrocyteLiver
10 20 ErythrocyteKidney
* mg/ kg/ day
O NH2
NH2
N
O
O
NH2N
O
O
O
HN
HN
H2N
NH2
H2N
H2N
HO NH2
HO NH2
H2N
ClS
OHO
O
H2N
S
OHO
O
O
S NH2HO
O
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
★ ★★ ★
★ ★★
★★ ★
★★ ★★
★▲
▲
▲ ▲
▲
▲
■
0 200 400 600 800 1000Dose (mg/kg/day)
HN
HNH2N
NH2H2N
NH2O NH2
NH2
NO
O
ONH2
NO
OHO NH2
NH2
Cl SO3H
NH2
SO3H
SO3H
NH2
Anemia findings○: 0, ▲: 1, ■: 2, ★: >3
RBC↓, Hb↓, HTC↓, GPT ↑ Reticulocytes↑ hemosiderin pigmentation in the spleen hematopoiesis↑ etc.
HO NH2
Comparison of the intensities of anemia for 14 aromatic amines
0
200
400
600
800
1000
-2 -1 0 1 2 3
Dos
e (m
g/kg
/day
s)
logP (CLOGP)
LOELs for anemia
RBC↓, Hb↓, HTC↓Reticulocytes↑hemosiderin pigmentation in the spleenhematopoiesis↑ etc.
Relationships between LOEL for anemia and logKow for 14 aromatic amines
Water solubleanilines (logKow<0 )have NO EFFECT
NH2
NHOHNH+
CYP450in the Liver
Glucuronide and sulfate conjugation Urine
NO
Adducts with DNA, Blood proteins: Hb, Alb
Hb Met Hb
NH2
OH
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
0
200
400
600
800
1000
-2 -1 0 1 2 3
Dos
e (m
g/kg
/day
s)
logP (CLOGP)
HO NH2
HO NH2
LOELs for anemia
RBC↓, Hb↓, HTC↓Reticulocytes↑hemosiderin pigmentation in the spleenhematopoiesis↑ 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) 61-67.
HBI = f (ΔE#)
NH+
R
NH2
R
ΔE#
Nitrenium ionE
Reaction pathway
Validation of Mechanism #1
-70
-60
-50
-40
-30
-20
-10
0
0 2 4 6 8 10
Cha
nge
in R
BC
at
200
mg
/kg/
day
Caluculated HBI
Calculated HBI (E# [eV]) vs. change in RBC in RDT test
H2N
NH2
H2N
HN
HN
Validation of Mechanism #1
in vitro, female rat liverR. Kato et al., Jpn. J. Pharmacol., 19 (1969) 53 – 62
HN NH2
+
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/dayNo Hemolysis
Findings[Johnnsen et al., Toxicol.
Lett., 30 (1986) 1-6.]
Repeated DoseToxicityTest
CH2
O
Metabolite of N-methylanilines
Validation of Mechanism #1
-70
-60
-50
-40
-30
-20
-10
0
0 5 10 15 20 25
Cha
nge
in R
BC
at
200
mg
/kg/
day
Caluculated HBI
H2N
NH2
H2N
HN
HN
Using HBI of the metabolite(Aniline)
H2N
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)
★
★
▲
■
0 200 400 600 800 1000Dose (mg/kg/day)
■
▲
▲★
HN
HNH2N
NH2H2N
NH2O NH2
NH2
NO
O
ONH2
NO
OHO NH2
HO NH2 NH2
Cl SO3H
NH2
SO3H
SO3H
NH2
Comparison of the effect on the kidney for 14 aromatic amines
0
200
400
600
800
1000
-2 -1 0 1 2 3
Dos
e (m
g/kg
/day
s)
logP (CLOGP)
LOELs for the kidney
HO NH2
HO NH2
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
NH2
NHOHNH+
CYP450in the Liver
Glucuronide and sulfate conjugation Urine
NO
Adducts with DNA, Blood proteins: Hb, Alb
Hb Met Hb
NH2
OH
Mechanism underlying the toxic effects exerted by anilines on kidney
Mechanism 2
Mechanism underlying the toxic effects exerted by anilines
NH2
OH
O
O
NH2
OH
NH2
OH
O
O ×
NH2
OH
NH2
OH
>
binding with the SH proteins
RDT Effect on the kidney Effect initiating mechanism
Basophilictubule,proximal(100, 500)
Necrosis, Tubularepithelium,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 BuildingBased on the link between chemical and toxicological
mechanisms
Category #3. If logKow > 1 and as parent or metabolite has alerting groups interacting with: