The Category Approach for Predicting Mutagenicity and Carcinogenicity Laboratory of Mathematical Chemistry, University “Prof. As. Zlatarov”, Bourgas, Bulgaria

Toolbox General Scheme

Input IUCLID5 interface: XML, Web Services Transfer of data from IUCLID 5 to Toolbox

Comparison and visualization functionalities in Toolbox

Functionalities 1: Correlation between the categories of two profiling schemes Bar diagram showing the number of chemicals meeting the boundaries of two binary profiles The fist profiler has the categories: Active; Non active The second one has the categories: Binding; Non binding

Functionality 2: Correlation between two profiles by analyzing the distribution of the categories of one of the profile across the categories of the other profile The fist profile has categories: Strong, Weak, Non The second one has categories: Category1, Category2, Category3, Category4

Functionality 3: Correlation between two profiles by analyzing the distributions of their categories in case of using category combinations (working with multifunctional chemicals) When more than one category is assigned simultaneously to a chemical, then unique combinations of such categories are used

The proposed stages of the categorization approach Stage 1. Profiling databases according to endpoint specific profiles The following endpoint specific profiles were implemented Oncologic Primary Classification Mutagenicity/carcinogenicity alerts by Benigni/Bossa Micronucleus alerts by Benigni/Bossa The following databases with mutagenicity and carcinogenicity data were used: HPV Carcinogenicity containing 216 chemicals and ISSCAN containing 1129 chemicals

The proposed stages of the categorization approach Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Chemical distribution according to endpoint specific profiles is analyzed* Categories were selected highly populated by chemicals: Aromatic amines - consisting of 39 and 271 chemicals in HPV Carcinogenicity and ISSCAN, respectively Halogenated linear aliphatic types of compounds - consisting of 27 and 44 chemicals in HPV Carcinogenicity and ISSCAN, respectively The Toolbox profiles for DNA and protein binding mechanisms have been used for subcategorization of the endpoint specific categories of Aromatic amines and Halogenated linear aliphatic types of compounds The profiling for DNA and protein binding mechanisms were applied without and with using liver rat S9 metabolism *See the presentation for Assessing correlation between the categories of profiling schemes

The proposed stages of the categorization approach Stage 3. Validating the correlation between mechanistic subcategories based on DNA binding mechanisms and AMES The validation is based on comparison of the correlations for selected classes - aromatic amines and halogenated linear aliphatic types of compounds derived from: HPV Carcinogenicity and ISSCAN Stage 4. Validating the correlation between mechanistic subcategories based on DNA and protein binding mechanisms and carcinogenicity

The proposed stages of the categorization approach Stage 5. Identifying the boundaries of the combined endpoint specific and binding mechanism categories providing >75% correlation with genotoxic effects and carcinogenicity Along with AMES and carcinogenicity the correlation with other genotox effects was also studied, such as CA, MNT and CTA Stage 6. Coding boundaries of the combined categories highly correlating with the genotox and/or carcinogenicity effects Stage 7. Screening of inventories for chemicals falling in the domains of highly correlating combined categories for searching data to support the boundaries of these categories

Stage 1. Profiling databases according to endpoint specific profiles HPV Carcinogenicity database profiled according to Oncologic Primary Classifications

Stage 1. Profiling databases according to endpoint specific profiles ISSCAN database profiled according to Oncologic Primary Classifications

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Analysis of the distribution of HPV carcinogenicity database (216) according to Oncologic Primary Classification

Total number 39 chemicals Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Highly populated categories are identified Aromatic amines as one of all categories with the biggest number of chemicals. Total number 39 chemicals

Distribution of 39 Aromatic amines across Ames experimental data Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across Ames experimental data

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Sequence of steps to analyze the distribution of 39 Aromatic amines across DNA binding and Ames data

Sorted by descending order of correlation Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding and Ames data Sorted by descending order of correlation 18

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Sequence of steps to analyze the distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data

Sorted by descending order of correlation Sorted by Positive data Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Sorted by descending order of correlation Sorted by Positive data

Detailed information for generated metabolites. Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for generated metabolites. Highlight chemical to see detailed information for generated metabolites

Detailed information for metabolically generated metabolites. Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. Right click 22

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. Click Explain to see detailed info. 23

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. Click Details to see the categories of generated metabolites 24

Detailed information for metabolically generated metabolites. Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. The target chemical has 9 generated metabolites falling into 8 categories 25

Detailed information for metabolically generated metabolites. Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. Highlight metabolite then click Details to see why the metabolite falls into this category 26

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Detailed information for metabolically generated metabolites. The current metabolite has fragment highlighted in red corresponding to the category of Aromatic Amines Click on Amines to see mechanistic justification of the category 27

Click on Advance to see structural boundaries of each category Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Click on Advance to see structural boundaries of each category 28

Sorted by descending order of correlation Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across combined DNA and Protein binding categories and Carcinogenicity data Sorted by descending order of correlation

Sorted by descending order of correlation Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 39 Aromatic amines across combined DNA and Protein binding categories taking into account liver metabolism, and Carcinogenicity data Sorted by descending order of correlation

Distribution of ISSCAN Carcinogenicity database (1129) according to Oncologic Primary Classification

Total number 271 chemicals Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Highly populated categories are identified Aromatic amines is one of the categories with the highest population of chemicals. Total number 271 chemicals

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 271 Aromatic amines category across Ames experimental data

Click on Add as a target list button Highlight Aromatic amines Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Adding Aromatic amines as target list Click on Add as a target list button Highlight Aromatic amines

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Aromatic amines as a target list

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 271 Aromatic amines according to DNA binding and Ames data Sorted by descending order of correlation Categories highly correlating with Ames data

Sorted by descending order of correlation Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distributing of 271 Aromatic amines across DNA binding taking into account liver metabolism and Ames data Sorted by descending order of correlation Categories highly correlating with Ames data accounting for liver metabolism

Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distribution of 271 Aromatic amines according to combined DNA and Protein binding categories and Carcinogenicity data Sorted by descending order of correlation Categories highly correlating with Carcinogenicity data

Sorted by descending order of correlation Stage 2. Subcategorization of obtained endpoint specific categories by molecular interaction mechanisms Distributing of 271 Aromatic amines across DNA and Protein binding categories taking into account liver metabolism and Carcinogenicity data Sorted by descending order of correlation Categories highly correlating with Carcinogenicity data accounting liver metabolism

Aromatic amines (39 chemicals) Aromatic amines (271 chemicals) Stage 3. Validating the correlation between mechanistic subcategories based on DNA binding mechanisms and AMES data Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Aromatic amines (39 chemicals) Aromatic amines (271 chemicals) Stage 3. Validating the correlation between mechanistic subcategories based on DNA binding taking into account liver metabolism and AMES data Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Aromatic amines (39 chemicals) Aromatic amines (271 chemicals) Stage 4. Validating the correlation between mechanistic subcategories based on DNA and Protein binding mechanisms and Carcinogenicity data Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Aromatic amines (39 chemicals) Aromatic amines (271 chemicals) Stage 4. Validating the correlation between mechanistic subcategories based on DNA and Protein binding taking into account liver metabolism and Carcinogenicity data Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both sets of chemicals Category 1 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 2 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 3 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 4 is based on partial overlapping between two sets 47 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 5 is based on partial overlapping between two sets 48 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 6. Building profiles for categories highly correlating with the genotox and carcinogenicity effects Building profilers for screening inventories based on Oncologic classification and DNA alerts without metabolism Oncologic class 1 and DNA boundaries 1 Oncologic class 1 and DNA boundaries 2 Oncologic class 1 and DNA boundaries 3 ………………………….. Oncologic class 2 and DNA boundaries 1 Oncologic class 2 and DNA boundaries 2 Oncologic class 2 and DNA boundaries 3 …………………………………………… Oncologic class n and DNA boundaries1 Oncologic class n and DNA boundaries2 Oncologic class n and DNA boundaries3

Stage 6. Building profiles for categories highly correlating with the genotox and carcinogenicity effects Building profilers for screening inventories based on Oncologic classification and DNA alerts with metabolism Oncologic class 1 and DNA boundaries with metabolism 1 Oncologic class 1 and DNA boundaries with metabolism 2 Oncologic class 1 and DNA boundaries with metabolism 3 ………………………….. Oncologic class 2 and DNA boundaries with metabolism 1 Oncologic class 2 and DNA boundaries with metabolism 2 Oncologic class 2 and DNA boundaries with metabolism 3 …………………………………………… Oncologic class n and DNA boundaries with metabolism 1 Oncologic class n and DNA boundaries with metabolism 2 Oncologic class n and DNA boundaries with metabolism 3

Stage 6. Building profiles for categories highly correlating with the genotox and carcinogenicity effects Building profilers for screening inventories based on Mutagenicity/carcinogenicity alerts by Benigni/Bossa and DNA alerts without metabolism Benigni/Bossa class 1 and DNA boundaries 1 Benigni/Bossa class 1 and DNA boundaries 2 Benigni/Bossa class 1 and DNA boundaries 3 ………………………….. Benigni/Bossa class 2 and DNA boundaries 1 Benigni/Bossa class 2 and DNA boundaries 2 Benigni/Bossa class 2 and DNA boundaries 3 …………………………………………… Benigni/Bossa class n and DNA boundaries 1 Benigni/Bossa class n and DNA boundaries 2 Benigni/Bossa class n and DNA boundaries 3

Stage 6. Building profiles for categories highly correlating with the genotox and carcinogenicity effects Building profilers for screening inventories based on Mutagenicity/carcinogenicity alerts by Benigni/Bossa and DNA alerts with metabolism Benigni/Bossa class 1 and DNA boundaries with metabolism 1 Benigni/Bossa class 1 and DNA boundaries with metabolism 2 Benigni/Bossa class 1 and DNA boundaries with metabolism 3 ………………………….. Benigni/Bossa class 2 and DNA boundaries with metabolism 1 Benigni/Bossa class 2 and DNA boundaries with metabolism 2 Benigni/Bossa class 2 and DNA boundaries with metabolism 3 …………………………………………… Benigni/Bossa class n and DNA boundaries with metabolism 1 Benigni/Bossa class n and DNA boundaries with metabolism 2 Benigni/Bossa class n and DNA boundaries with metabolism 3

Oncologic class + Category 1 (DNA without S9) Stage 6. Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Coded boundaries Oncologic class + Category 1 (DNA without S9)

Oncologic class + Category 2 (DNA without S9) Stage 6. Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Coded boundaries Oncologic class + Category 2 (DNA without S9)

Oncologic class + Category 3 (DNA without S9) Stage 6. Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Coded boundaries Oncologic class + Category 3 (DNA without S9)

Oncologic class + Category 4 (DNA without S9) Stage 6. Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Coded boundaries Oncologic class + Category 4 (DNA without S9) 56

Oncologic class + Category 5 (DNA without S9) Stage 6. Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Coded boundaries Oncologic class + Category 5 (DNA without S9) 57

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Common categories based on analysis between two sets of aromatic amine Category 1 58 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Common categories based on analysis between two sets of aromatic amine Category 2 59 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Common categories based on analysis between two sets of aromatic amine Category 3 60 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 4 61 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 5. Identifying category boundaries in terms of endpoint specific classes and binding mechanisms providing >75% correlation with genotoxic effects and carcinogenicity Common categories identified in both set of chemicals Category 5 62 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Common categories based on analysis between two sets of aromatic amine 63 Aromatic amines (39 chemicals) Aromatic amines (271 chemicals)

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Common categories could be selected by simultaneously clicking on “Ctrl” button and on the beginning of the corresponding category row 64

The selected rows with categories are labeled with “s” Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism The selected rows with categories are labeled with “s” 65

Click on “Create scheme” button Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Click on “Create scheme” button 66

The profiler with expected categories has been performed Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism The profiler with expected categories has been performed 67

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism In order to include Aromatic amine as a part of each category, it is needed to defined new referential boundary 68

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Select Oncologic profiler and add “Aromatic Amines” as a referential category. 69

Select two referential boundaries and combined them by logically “AND” Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Select two referential boundaries and combined them by logically “AND” 70

Save the profile by clicking on “Save as” button Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Save the profile by clicking on “Save as” button 71

Give the name of the file and click “Save” Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism Give the name of the file and click “Save” 72

Stage 6. 1. Automatic generation of Profiler for screening inventories based on Oncologic and DNA alerts without metabolism The automatic generated profiler now could be used for screening. The profile has been saved 73

Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories Screening of HPVC EU inventory (4843 chemicals) by the profile: Aromatic Amines (Oncologic) and DNA binding (categories #1-5) highly correlating with AMES data

Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories Distribution of HPVC EU inventory across the profile: Aromatic Amines (Oncologic) and DNA binding (categories #1-5) highly correlating with AMES data

15 chemicals correspond to this profile Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories Distribution of HPVC EU inventory across the profile: Aromatic Amines (Oncologic) and DNA binding (categories #1-5) highly correlating with AMES data 15 chemicals correspond to this profile 76

* No information for S9 metabolism Experimental AMES data for HPVC chemicals confirming the predictive power of the identified categories Category/Total 4834 Experimental Ames data* Positive Negative No data Summary 15 10 3 2 Ar.amine (Onco) + Category 1 (DNA without S9) 4 Ar.amine (Onco) + Category 2 (DNA without S9) Ar.amine (Onco) + Category 3 (DNA without S9) 9 6 1 * No information for S9 metabolism

Oncologic class + DNA/Protein with S9 Stage 6. Profiler for screening inventories based on Aromatic Amines (Oncologic) and DNA/Protein binding accounting for metabolism (categories #1-9) Oncologic class + DNA/Protein with S9 78

Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories Screening of US HPV Challenge Program inventory (9125 chemicals) by the updated profile: Aromatic Amines (Oncologic) and DNA /Protein binding accounting for metabolism (categories #1-9) highly correlating with carcinogenicity data

Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories Distribution of US HPV Challenge Program inventory across the updated profile: Aromatic Amines (Oncologic) and DNA/Protein binding accounting for metabolism (categories #1 - 9) highly correlating with carcinogenicity data

Experimental Carcinogenicity data Stage 7. Screening of inventories for chemicals falling in highly correlating categories for searching data to support the boundaries of these categories US HPV Challenge Program (9125) chemicals were screened by the updated profile highly correlating with carcinogenicity These chemicals could be considered as potential carcinogens Inventory US HPV Challenge Program Total 9125 Experimental Carcinogenicity data ISSCAN Positive Negative Equivocal No data Profiled chemicals 581 31* 13** 3*** 534 Detailed information *31_positive.pdf **13_negative.pdf ***3_equivocal.pdf 81

Screening of 581 chemicals from US HPV Challenge Program inventory according to Mutagenicity/Carcinogenicity alerts by Benigni/Bossa profiler

Distribution of 581 chemicals from US HPV Challenge Program inventory by Benigni/Bossa profiler 83

Mutagenicity/Carcinogenicity alerts by Benigni/Bossa Distribution of 581 chemicals from US HPV Challenge Program inventory by Benigni/Bossa profiler Inventory US HPV Challenge program Total 581 Mutagenicity/Carcinogenicity alerts by Benigni/Bossa SA for genotoxic carcinogenicity SA for nongenotoxic carcinogenicity No alert for carcinogenicity Profiled chemicals 539 42 Detailed information *42_No alert.pdf *42_No alert.xls 84