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

2. Toolbox
General Scheme

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

4. Comparison and visualization functionalities in Toolbox

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

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

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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

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 Explain to see detailed info. 23

24. 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

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.
The target chemical has 9 generated metabolites falling into 8 categories 25

26. 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

27. 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

28. 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

29. 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

30. 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

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

32. 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

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

34. 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

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

36. 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

37. 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

38. 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

39. 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

40. 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)

41. 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)

42. 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)

43. 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)

44. 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)

45. 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)

46. 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)

47. 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)

48. 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)

49. 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

50. 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

51. 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

52. 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

53. 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)

54. 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)

55. 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)

56. 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

57. 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

58. Stage 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)

59. Stage 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)

60. Stage 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)

61. 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)

62. 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)

63. Stage 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)

64. Stage 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

65. The selected rows with categories are labeled with “s”
Stage 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

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

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

68. Stage 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

69. Stage 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

70. Select two referential boundaries and combined them by logically “AND”
Stage 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

71. Save the profile by clicking on “Save as” button
Stage 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

72. Give the name of the file and click “Save”
Stage 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

73. Stage 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

74. 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

75. 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

76. 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

77. * 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

78. 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

79. 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

80. 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

81. 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

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

83. Distribution of 581 chemicals from US HPV Challenge Program inventory by Benigni/Bossa profiler83

84. 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