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2nd McKim Workshop, Baltimore 2012. Identification of non-genotoxic carcinogens based on mechanisms. Jan van Benthem National Institute for Public Health and the Environment Laboratory for Health Protection Research Bilthoven, The Netherlands.

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2nd McKim Workshop, Baltimore 2012


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    1. 2nd McKim Workshop, Baltimore 2012 Identification of non-genotoxic carcinogens based on mechanisms Jan van Benthem National Institute for Public Health and the Environment Laboratory for Health Protection Research Bilthoven, The Netherlands

    2. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research - no further testing not genotoxic In vitro genotoxicity tests consider whether in vivo test is required check bioavailability check available data consider proper in vivo (follow up) test consider integration into other toxicity tests + - no further testing not genotoxic In vivo genotoxicity tests + Considered genotoxic in somatic cells Check for information on a genotoxic hazard to germ cells Carcinogenicity test

    3. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Detection of non-genotoxic carcinogens • Carcinogenicity test • (Sub)-chronic repeat dose toxicity tests • 90-day toxicity test • 6-month toxicity test • 12-month toxicity test • Negative in genotoxicity tests

    4. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research REACH Registration, Evaluation, Authorisation and Restriction of CHemicals Registratie, Evaluatie en Autorisatie van CHemische stoffen Registrement, Evaluation et Autorisation des produits CHimiques Since 1981 difference is made between new and existing chemicals (marketed before September 1981). About 99% of the total number of compounds marketed is part of this new policy In 1981, 100106 (existing) compounds were registered (30000 >> 1 tpa) Since 1993, 141 existing compounds have been tested Of about 80% of these compounds toxicological data are lacking

    5. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research REACH • New: • In future it is the responsibility of the industry and down stream users to obtain toxicological knowledge bout chemicals. Every compound is notified only once (industrial collaboration) • Major aims: • To ensure a high level of protection of human health and the environment • Safety assessment for new and existing chemicals • Classification and labelling • Risk Assessment • In less time, money and experimental animals • Promotion of tests which do not use experimental animals. • Optimal use of in vitro tests • Stimulation of development of new in vitro tests • Minimalization of test strategies

    6. No studies required In vitro geno-toxicity Acute toxicity Local toxicity In vitro geno-toxicity Repro-ductive toxicity Acute toxicity Local toxicity 28 day toxicity 90 day toxicity In vitro geno-toxicity Repro-ductive toxicity In vivo geno-toxicity Develop-mental toxicity Acute toxicity Local toxicity 28 day toxicity 90 day toxicity 2 genera-tion study In vitro geno-toxicity Repro-ductive toxicity In vivo geno-toxicity Develop-mental toxicity Acute toxicity Local toxicity 28 day toxicity 90 day toxicity 2 genera-tion study Carcino-genicity National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research REACH data requirements at different tonnage levels < 1 tpa 1 - 10 tpa 10 - 100 tpa 100 – 1000 tpa > 1000 tpa

    7. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research The Cosmetics Directive and its Seventh Amendment • September 11, 2004: • Testing ban on finished cosmetic products in the EU. • Marketing ban on cosmetic products and ingredients tested on animals outside of the EU where validated alternative tests exist. • March 11, 2009 • Testing bancosmetic ingredients or formulations in the EU. • Marketing bancosmetic products and ingredients tested on animals with the exception of repeated-dose toxicity, reproductive toxicity and toxicokinetics. • March 11, 2013 • Marketing bancosmetic products or ingredients tested on animals, irrespective of the availability of alternative non-animal tests. • The 7th Amendment requires cosmetics manufacturers and distributors to provide certain product information for the safety of the end user.

    8. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Risk associated with non-genotoxic carcinogens Mechanisms of non-genotoxic carcinogens and importance of a weight of evidence approach Lya G. Hernandez, Harry van Steeg, Mirjam Luijten, Jan van Benthem Mutation Research 682, 94–109 (2009) lya.hernandez@rivm.nl

    9. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Number non-carcinogens in IARC 1, 2A and 2B IARC group total carc non-carc percentage 1 77 64 13 16.9 2A 57 55 2 3.5 2B 237 207 30 12.7 total 371 326 45 13.8

    10. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Margin of Exposure • Margin of Exposure (MoE) = Margin of Safety (MoS) • The ratio: Most relevant exposure from animal studies • (Estimated) human exposure • Potential hazard: • Carcinogens MoE < 10,000 • Non-genotoxic carcinogens MoE < 100 for consumers • Non-genotoxic carcinogens MoE < 50 for workers • Cosmetics in Europe: MoS < 100

    11. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Approaches Rodent dose response carcinogenicity study data of the Gold database using LTD10 calculated from TD50 (Sub)-chronic dose response toxicity studies from literature using NO(A)EL RfD using EPA/IRIS data Human exposure using data from the open literature vs

    12. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Reference dose A dose (with uncertainty factors) of a daily exposure to human population that is likely to be without appreciable risk of deleterious effects during a lifetime Potential hazard: Average human daily exposure > RfD

    13. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Risk Assessment MoE from LTD10 38% (7/18) with MOE < 100 MoE from NOAEL 18% (2/11) with MOE < 50 RfD 30% (3/10) RfD > average human daily exposure Non-genotoxic carcinogens with potential human hazard: 27% (12/45) among non-genotoxic carcinogens 3.2% (12/371) among carcinogens (1, 2A, 2B)

    14. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research MoE cut off’s of EPA With an acceptable life time cancer risk of 1 in 104 MoE > 100,000 low risk for carcinogenicity 10,000 < MoE < 100,000 moderate risk for carcinogenicity MoE < 10,000 high risk for carcinogenicity For the present data: 1/45 (2%) in the low risk group 2/45 (4%) in the moderate risk group 22/45 (49%) in the high risk group No data were available for 20 remaining compounds

    15. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Comparison EU vs EPA Non-genotoxic carcinogens with potential human hazard: - Europe, MoE < 100 - 12/45 (27%) - 12/371 (3.2% of all carcinogens) - U.S.A., MoE < 100,000 - 24/45 (49%) (moderate to high risk) - 24/371 (6.5% of all carcinogens)

    16. Putative methods to detect non-genotoxic carcinogens National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research • Structure-activity relationships (SAR) and quantitative (Q)SAR • Read across • Replicative DNA synthesis • In vitro cell transformation assay • Toxicogenomics

    17. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Dissecting modes of action of non-genotoxic carcinogens in primary mouse hepatocytes Mirjam M Schaap, Edwin P Zwart, Paul FK Wackers, Ilse Hijskens, Bob van de Water, Timo M. Breit, Harry van Steeg, Martijs J. Jonker, and Mirjam Luijten. Arch. Toxicol. submitted For more information: mirjam.schaap@rivm.nl

    18. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Primary hepatocytes combined with toxicogenomics Goal Identification mechanism of action of non-genotoxic carcinogens Advantages • relevant cell type: liver major organ for toxicity • metabolic competent cells • requires only small number of animals

    19. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Experimental design • Primary mouse hepatocytes • Two-step perfusion (based on Seglen, 1976) • Hepatocytes cultured in sandwich configuration • Compounds tested • 16 non-genotoxic carcinogens • Dose ~ 90% cell viability (based on MTT) • Microarray analysis • Cells exposed for 24 hours • Affymetrix HT MG-430 PM Array plate

    20. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Non-genotoxic carcinogens

    21. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Data analyses General • Principal component analysis • Pathway analysis per individual substance Supervised clustering Selection discriminative genes  specific for mechanism? Unsupervised clustering Clustering of substances based on gene expression signature

    22. Differentially expressed genes: from 40 to >5,600 (FDR<0.05) National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Gene expression analysis - general

    23. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Results of the gene expression analysis NGTXC mode of action DEG response PCA TCE halogenated hydrocarbon 40 3 A CT halogenated hydrocarbon 56 3 A TCPOBOP constitutive androstane receptor agonist 65 3 A PB constitutive androstane receptor agonist 238 3 A TCDD arylhydrocarbon receptor agonist 567 1 A ARO arylhydrocarbon receptor agonist 762 1 B CA skin tumor promotor 281 3 A OA skin tumor promotor 282 3 A WY peroxisome proliferator 1611 1 B CF peroxisome proliferator 308 1 A CSA immunosuppressant 124 1 A FK506 immunosuppressant 2404 2 B HCE ligand-independent estrogen receptor 2088 2 B HCH ligand-independent estrogen receptor 115 3 A LAC metalloid 5693 2 C SAR metalloid 4130 2 C

    24. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Data analyses General • Principal component analysis • Pathway analysis per individual substance Supervised clustering Selection discriminative genes  specific for mechanism? Unsupervised clustering Clustering of substances based on gene expression signature

    25. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Supervised clustering Method to select genes discriminative for MoA Lac Sar CT TCE OA CA FK506 CsA Pb TCPOBOP HCH HCE TCDD Aroclor Clofibrate WY -2 -1 0 1 2

    26. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Supervised clustering - specificity Distribution random pairs (5000 permutations) • Apply supervised clustering on random pairs to confirm specificity of previous results • Level of distinction is expressed as C-value; calculated for each individual gene • Maximum C-value obtained for random pairs ~ 40 Maximal C-value

    27. Distribution random pairs (5000 permutations) Maximal C-value National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Maximal C-values ‘correct’ pairs

    28. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Unsupervised clustering Which substances have a similar gene expression profile and cluster together?

    29. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Unsupervised clustering – preliminary results

    30. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Data analyses General • Principal component analysis • Pathway analysis per individual substance Supervised clustering Selection discriminative genes  specific for mechanism? Unsupervised clustering Clustering of substances based on gene expression signature

    31. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Conclusions • Primary hepatocytes combined with toxicogenomics useful to identify mechanisms of action of some non-genotoxic carcinogens: • Peroxisome proliferators • Metalloids • Skin tumor promotors • AhR ligands • Useful tool for risk assessment of chemicals

    32. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Finding maximal transcriptome differences between reprotoxic and non-reprotoxic phthalate responses in rat testis Xiaolian Yuan, Martijs J. Jonker, Jillian de Wilde, Aart Verhoef, Floyd R.A. Wittink, Jan van Benthem, Jos G. Bessems, Betty C. Hakkert, Raoul V. Kuiper, Harry van Steeg, Timo M. Breit, and Mirjam Luijten. J. Appl. Toxicol 31, 421 – 430 (2011) For more information: mirjam.luijten@rivm.nl

    33. Category approach – hazard identification National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Category:Phthalates • mainly used as plasticizers to increase their flexibility, transparency, durability and longevity. • widely applied in children’s toys • some phthalates reprotoxic, others not Can we separate reprotoxic and non-reprotoxic phthalates by gene expression profiling? Experiment: • Oral exposure of rats to phthalates, reprotoxic and non-reprotoxic • Sacrifice after 24h of exposure: gene expression profiling and histopathology of the testis 33

    34. Gene expression profiling Acclimatization Treatment 1 -21 0 Time (days) National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Study design 7-week-old male HsdCpb:WU rats; n = 5 Animals killed 24 h after treatment

    35. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Experimental design

    36. No clear histopathological effects 24 hours after exposure to phthalates National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Histopathology Positive control (2-ME) Untreated control DHP treatment

    37. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Data analysis: 3 strategies • Identification of differentially expressed genes between treatment with individual compounds and the untreated control • Identification of differentially expressed genes between compound classes: • reprotoxic phthalates (RT) • non reprotoxic phthalates (NRT) • untreated control (NC) • 3. Identification of differentially expressed individual probes between compound classes

    38. Differentially expressed genes (FDR<0,1) range from 107 (DEHP) to 1,273 (DPeP) 1. Differentially expressed genes-individual compounds (I) RT NRT National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research

    39. Little overlap between different RT or different NRT phthalates Only 2 genes (LCMT2 and an unknown gene) are affected by all RT phthalates, these are however also affected by at least one NRT phthalate 1. Differentially expressed genes-individual compounds (II) National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research

    40. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research 2. Differentially expressed genes-compound classes (I) • Differentially expressed genes (FDR <0,05): • RT vs NRT phthalates: 469 genes • RT phthalates vs NC: 454 genes • NRT phthalates vsNC: 764 genes • No genes were differentially expressed in all 3 comparisons • 70 (51 + 19) genes not only differentially expressed in RT vs NRT phthalates, but also between either class of phthalates and NC

    41. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research 2. Differentially expressed genes-compound classes (II) Discrimination between RT and NRT phthalates based on 70 selected genes

    42. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research 3. Differentially expressed probes-compound classes (I) • Differentially expressed probes (FDR <0,05): • RT vs NRT phthalates: 1897 probes • RT phthalates vsNC: 4159 probes • NRT phthalates vsNC: 6189 probes • No probes were differentially expressed in all 3 comparisons • 269 (143 + 126) probes were not only differentially expressed in RT vsNRT phthalates but had also altered expression between either class of phthalates and NC

    43. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research 3. Differentially expressed probes-compound classes (II) Discrimination between RT and NRT phthalates based on 269 selected probes

    44. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Discussion • Although 24 hours after exposure to phthalates no histopathological effects were observed, gene expression signatures were clearly changed • Comparison of the different phthalate classes rather than individual compounds results in the identification of 70 genes or 269 probes that can discriminate between RT and NRT phthalates • We, therefore, offer a proof-of-principle for the possibility to implement toxicogenomics in hazard assessment • A combination of toxicogenomics and a category approach would allow prioritizing chemicals for toxicity testing and will as a result: • Be much faster than conventional toxicity testing • Reduce number of laboratory animals used for testing • Lead to a reduction of costs

    45. Phtalates: RIVM: Mirjam Luijten, Jan van Benthem, Harry van Steeg, Jeroen Pennings, Jillian de Wilde, Joke Robinson, Raoul Kuiper, Conny van Oostrom, Jos Bessems, Cees de Heer, ZhiChao Dang, Betty Hakkert MicroArray Department, UvA, Amsterdam: Xiaolian Yuan, Martijs Jonker, Timo Breit National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Acknowledgements Hepatocytes: RIVM: Mirjam Schaap, Mirjam Luijten, Edwin Zwart, Harry van Steeg LACDR, University Leiden: Ilse Huijskens, Bob van de Water Merck: Jan Polman, Willem Schoonen MicroArray Department, University Amsterdam: Paul Wackers, Martijs Jonker, Floyd Wittink, Timo Breit Risk assessment: Lya Hernandez, Wout Slob, Wim Mennes, André Muller, Jan van Benthem Fundings: Dutch Technology Foundation, STW 06935 NGI Netherlands Toxicogenomics Centre NTC 050-06-510 21 October 2010 | Health Canada 45

    46. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research

    47. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Number non carcinogens in IARC IARC group total carc non-carc percentage 1 77 64 13 16.9 2A 57 55 2 3.5 2B 237 207 30 12.7 3 507 471 36 7.1 total 878 797 81 9.2

    48. Phthalates - Toxicity based on side chain length National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Reprotoxic (C4-C6) Non-Reprotoxic

    49. National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research Results of the gene expression analysis NGTXC mode of action DEG response PCA TCE halogenated hydrocarbon 40 3 A TCPOBOP constitutive androstane receptor agonist 65 3 A TCDD arylhydrocarbon receptor agonist 567 1 A ARO arylhydrocarbon receptor agonist 762 1 B CA skin tumor promotor 281 3 A CT halogenated hydrocarbon 56 3 A CF peroxisome proliferator 308 1 A CSA immunosuppressant 124 1 A HCE ligand-independent estrogen receptor 2088 2 B LAC metalloid 5693 2 C OA skin tumor promotor 282 3 A PB constitutive androstane receptor agonist 238 3 A SAR metalloid 4130 2 C FK506 immunosuppressant 2404 2 B WY peroxisome proliferator 1611 1 B HCH ligand-independent estrogen receptor 115 3 A

    50. Risk estimate: many uncertainties and critical data gaps mode of action interspecies extrapolation dose-response analysis Ethics: high number of test animals involved reduction & refinement Time-consuming and costly gene expression profiles representative of toxicity prioritization new chemicals Potential relevance -omics to risk assessment National Institute for Public Health and the Environment (RIVM) Laboratory for Health Protection Research 50