AN EVALUATION OF THE OECD QSAR TOOLBOX

PROFILERS FOR IDENTIFYING DNA REACTIVE AND

GENOTOXIC CHEMICALSTerry SchultzProfessor Emeritus, The University of Tennessee-Knoxville & Secretariat, OECD, Paris

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Outlook• The Toolbox• 1st Exercise: Goal, Methods & Results• 2nd Exercise: Goal, Methods & Results• 3rd Exercise: Goal, Methods & Results• Conclusions

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The OECD QSAR Toolbox The seminal feature of the Toolbox is its ability to quickly evaluate all members of a category for common toxicological behaviour or consistent trends within important regulatory endpoint data.

This is done by first profiling the chemical by type, class or defined hazard identifiers and then selecting a category or subcategory.

This feature often links the chemicals in the category to a single mechanism or mode of action.

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Advantages of the Profile/Category Approach

1) it shifted emphasis to intrinsic chemical activity,

2) it allows for entire categories of chemicals to be assessed when only a few members are tested,

3) it allows for filling data gaps using read-across and trend analysis, and not just (Q)SAR models, and

4) it enables defensible hazard assessment through mechanistic comparisons without testing.

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1st McKim Workshop on Reducing Data Redundancy in Cancer Assessment

The participants agreed that chemical which are QSAR predicted to be DNA reactive (+) and are also AMES test positive (+) would be chemicals of “very high concern” for the Rodent Cancer Assay.

But you can have direct or indirect DNA-reactivity and AMES positive w/o S9 and w/ S9.

So the first exercise was to look for members of the direct DNA-reactivity and AMES positive w/o S9 category.

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?

MicroNucl ?

Protein OASIS

Generate metabolites

N

Y

Y

Receptor-Based Epigenetic

Screen

Low Carcinogenicity

Potential

Y

Chrom Ab ?

MicroNucl ?

Refine TIMES/Structural alerts

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

General Flow Diagram for Screening Large Inventories for Carcinogenicity

Focus Area of this Discussion

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?

MicroNucl ?

Protein OASIS

Generate metabolites

N

Y

Y

Receptor-Based Epigenetic

Screen

Low Carcinogenicity

Potential

Y

Chrom Ab ?

MicroNucl ?

Refine TIMES/Structural alerts

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

Chemicalinput Profiling

CategoryDefinition

Fillingdata gap Report

Endpoints

Toolbox Work Flow

OASIS databasefor AMES mutagenicity

QSAR Toolbox profiles for DNA binding by OASIS by OECD Direct

DNA Reactive & Ames Positive w/o S9

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Factors Which Effect Makeup of Chemical CategoryHow one defines the initial target chemical.For Example: Using CH4 as the initial target chemical and profiling for chemicals containing a C-atom one retrieves 8684 chemicals from the Toolbox.Whether one used DNA-binding OASIS or DNA-binding OECD.This impacts the number and definitions of the SAs which are fired.

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?

MicroNucl ?

Protein alerts?

Generate metabolites

N

Y

Y

Receptor-Based

Epigenetic Screen

Low Carcinogenit Potential

Y

2832

Chrom Ab ?

MicroNucl ?

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

Refine TIMES/Structural alerts

N

Y

Initial Database

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?

MicroNucl ?

Protein alerts?

Generate metabolites

N

Y

Y

Receptor-Based

Epigenetic Screen

Low Carcinogenit Potential

Y

2832

745

Chrom Ab ?

MicroNucl ?

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

Refine TIMES/Structural alerts

N

Y

OASIS Direct DNA Binder in Initial Database

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?

MicroNucl ?

Protein alerts?

Generate metabolites

N

Y

Y

Receptor-Based

Epigenetic Screen

Low Carcinogenit Potential

Y

2832

745

Chrom Ab ?

MicroNucl ?

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

Refine TIMES/Structural alerts

N

307

Y

OASIS Direct DNA Binders & Ames Positive w/o S9 in Initial Database

OASIS (+/+) category

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020406080

100120140

Distribution of 307 across classes

134

28 25 24 19 17 16 15 13 12 8 8 7 6

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DNA-binding OECD Eliminated:

Nitro Aromatic Compounds, Aromatic Amines, PAHs etc.Classes where it is unclear that a bacterial test would mimic the reactions well.

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2nd Exercise

Designed to look for which members of the +/+ category have been evaluated in a Rodent Cancer Assay (RCA) and to report what classes they represent.

Chemicalinput Profiling

CategoryDefinition

Fillingdata gap

Report

Endpoints

Toolbox Work Flow

CPDB Carcinogenic Potency Data Base and/or ISSCAN Carcinogenicity & mutagenicitydata base

Direct DNA Reactive &

Ames Positive w/o S9

Inventory

Direct DNA

reactive

Indirect DNA

reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?MicroNucl

?

Protein alerts?

Generate metabolites

N

Y

Y

Receptor-Based

Epigenetic Screen

Low Carcinogenit Potential

Y

Chrom Ab ?

MicroNucl ?

N

YY

N

Oxidative stress?

In vivo Mammal

Tests

Protein Reactive

Return for further screening

Refine TIMES/Structural alerts

N

307

243No data

53Positive

11Negative

Carcinogenicity Data

Y

RCA for +/+ Category

Carcinogenicity of 307 +/+ chemicals

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020406080

100120140160

17 3 5 4 2 1 3 1 4 3 3 1 1 3 1 1 1 0 1 2 0 0 1 0 0 3 0 0 0

Distribution of 53 compounds across carcinogenicity classes

CounterPositive carcinog

134

28 25 24 19 17 16 15 13 12 8 8 7 6

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3RD EXERCISE

Designed to look at which DNA-reactions are represented by the (+/+) chemicals that are also positive in the RCA.

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Major Classes

Aliphatic Halides [CX]; 17 CRA positive; 2 CRA negative (1-chlorobutane; 2-chloroethanol)Stressed Hetero-Ring Systems(e.g. C1CO1); 13 CRA positive

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Minor Classes

SN2 addition at an sp3 carbon atom sulfates and sulfonates; 4 CRA positivePhosphorates; 2 CRA positiveHydazines; 1 CRA positive

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SummaryIn this discussion we used a hypothesis testing scheme with the QSAR Toolbox; the DNA-binding profiler generate the initial hypothesis and an in vitro test (AMES testing w/o S9) tested the hypotheses.

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Summary

We maintain that chemicals that pass this hypothesis testing are “chemicals of high concern” for cancer.

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SummaryWe then asked which of these chemicals of high concern have been tested in the RCA?What are the DNA reactions covered by this suite of RCA-tested chemical?

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ConclusionThere is experimental evidence that there are opportunities to reduce the use of RCA, especially for chemicals with an aliphatic halide or stressed hetero-ring sub-structure.