2. Issues Related to Uncertainty in Risk Assessment High to low dose extrapolation Species to species extrapolation Mechanism of carcinogenesis Interindividual differences

3. Chemicals that are carcinogenic in animals are expected to be carcinogenic in humans Humans are assumed to be as sensitive as the most sensitive animal The dose-response is assumed to be linear Major Default Assumptions in Cancer Risk Assessment

4. Emerging Issues in Biologically-based Risk Assessment Incorporation of PBPK models Use of molecular dosimetry as a surrogate of exposure Mode of action information –Role of cell proliferation –Mutagenicity Life stage differences in susceptibility

5. Potential of Molecular Dosimetry in Risk Assessment High to low dose extrapolation –Saturation of metabolic activation –Saturation of detoxication –Saturation of DNA repair Route to route differences Species to species differences

6. Chemical Exposure (air, water, food, etc.) Internal Exposure Metabolic Activation Macromolecular BindingDetoxication DNARNAProtein Biologically Effective Dose X Efficiency of Mispairing X Cell Proliferation Biomarkers of Exposure Mutation/Initiation ProgressionCancer Biomarkers of Effect

7. c a b Increasing External Exposure Increasing Adduct Concentration Sublinear Supralinear

12. Role of Increased Cell Proliferation in Carcinogenesis Decreases time available for DNA repair Converts repairable DNA damage into nonrepairable mutations Necessary for chromosomal aberrations, insertions, deletions and gene amplification Clonally expands existing cell populations

13. 8-oxo-G, FapyGua Lipid Peroxidation Lipid Peroxide MDA, 4-HNE AP Sites DNA Base Modification DNA Base adduct ROS Sugar Damage DNA Base adduct M 1 G, edG, edA Oxidative Stress Induced-DNA Damage Glycosylase hOGG1 Glycosylase MPG Base Propenal

14. Non-smoker Lymphocytes

15. Pentachlorophenol  Used as a Pesticide and Wood Preservatives  Introduction to Humans: Air, Food and Drinking water  Mutagen, Rodent Carcinogen OH Cl Cl Cl Cl Cl OH Cl Cl OH Cl Cl O Cl Cl O Cl Cl O Cl Cl OH Cl Cl O2O2 O2-O2- H2O2H2O2 OH

16. Induced Oxidative Stress Calf Thymus DNA Exposed to TCHQ

17. Aldehydic DNA lesions (ADL) in HeLa cells exposed to H 2 O 2 (0.06-20 mM) for 15 min

18. Efficiency of Low Doses of H 2 O 2

19. DNA Alkylation N7Alkyl Guanine O6 Akyl Guanine O4 Alkyl Thymine O2 Alkyl Thymine MMS85%0.3%-- MNU DMN 70%7%0.1%0.4% ENU DEN 14%7%2%7%

21. O 4 -EtdThd O 6 -EtdGuo

23. 0 20406080100 0 20 40 60 80 100 0 20 40 60 80 100 Dose (ppm DEN) ET (pM)/dT (  M) 05101520 0 5 10 15 0 5 10 15 O 2 -ETO 4 -ET Molecular Dosimetry of DEN

24. Vinyl Chloride Vinyl chloride is a known human and animal carcinogen that induces hepatic angiosarcomas Carcinogenic response is associated with high exposure (>50 ppm) To date, 197 VC workers have developed hepatic angiosarcomas. All of them started work prior to lowering the occupational exposure 1 ppm Vinyl chloride is present in many Superfund sites and some public drinking water in ppb amounts

25. Exposure-Response for Vinyl Chloride Metabolism and Carcinogenicity

26. Formation of [ 13 C 2 ]-DNA Adducts by Vinyl Chloride

27. Miscoding Properties of Vinyl Chloride DNA Adducts 7-(2-Oxoethyl)guanine(7OEG)None N 2,3-Ethenoguanine (εG) G → A 3,N 4 -Etheno-2’-deoxycytidine (εdC) C → T C → A C → G 1,N 6 -Etheno-2’-deoxyadenosine (εdA) A → T A → C A → G

28. Mutations in VC-induced Neoplasms in Humans and Rats Marion et al., found G:C→A:T mutations in codon 13 of the c-Ki-ras-2 gene in 5/6 human hepatic angiosarcomas. Hollstein et al., found A:T →T:A mutations in codon 249 and 255 of the p53 gene in 2/4 human hepatic angiosarcomas. Bolvin-Angele did not find ras gene mutations in hepatic angiosarcomas induced by VC or vinyl fluoride.

29. Sample Spectrum of 7-OEG m/z 265  152 AST m/z 267  152 13 C 2 -OEG m/z 270  157 IST AB A. Adult rat liver(1100 ppm [ 13 C 2 ]-VC, 5days) B. Weanling rat liver (1100 ppm [ 13 C 2 ]- VC, 5days)

30. Relative Amounts of Endogenous and Exogenous DNA Adducts in Liver DNA From Rats Exposed to [ 13 C 2 ]-VC (1100 ppm, 6 hr/day, 5 days) [ 12 C 2 ]- 7OEG/ 10 5 Gua [ 13 C 2 ]- 7OEG/ 10 5 Gua [ 12 C 2 ]- N 2,3-εG/ 10 8 Gua [ 13 C 2 ]- N 2,3-εG/ 10 8 Gua [ 12 C 2 ]- 1N 6 - εdA/ 10 8 dA [ 13 C 2 ]- 1N 6 - εdA/ 10 8 dA Adult Rats at End of Exposure 0.2± 0.110.4± 2.34.1 ± 2.818.9 ± 4.94.9 ± 0.65.1 ± 0.6 2 Weeks Post Exposure 0.1 ± 0.030.4± 0.33.7 ± 3.114.2 ± 4.28.6 ± 0.9ND 4 Weeks Post Exposure 0.2 ± 0.040.1± 0.063.1 ± 1.016.9 ± 1.66.2 ± 1.3ND 8 Weeks Post Exposure 0.2 ± 0.07ND3.7 ± 1.513.2 ± 2.54.1 ± 0.5ND

31. T 1/2 and Repair Pathways For VC-Induced DNA Adducts AdductT 1/2 Repair Pathways 7OEG4 DaysChemical depurination N 2,3-εG150 DaysUnknown 1,N 6 - εdA~1 Day MPG/Aag AlkB 3,N 4 - εdC~1 DayDNA glycosylases

32. Vinyl Chloride Cancer Risk Estimates 1.6-3.7Rat PBPK/LMS1995 Clewell et al 2000 1996 1989 1994 Year 1.4Epi Chen & Blancato 4.4Rat (f) 1.0-2.3Mouse 0.3-2.8Epi PBPK/LMSEPA 0.6RatPBPK/LMSReitz et al 0.7-1.4Rat PBPK/LMS 84RatLMSEPA Inhalation Risk (per  g/m 3 x 10 -6 ) DataModelAuthor(s)

33. Use of Mechanistic Evidence in Vinyl Chloride Risk Assessment PBPK Modeling –Conversion of animal exposures to human equivalent concentrations –Route-to-route extrapolation DNA Adducts –Selection of low dose extrapolation model –Inclusion of 2-fold protection factor for young –Increased confidence in risk assessment

34. Uncertainties in Vinyl Chloride Risk Assessments Relationship between low exposure and cancer has large uncertainty. High quality human exposure data are not available for individuals with angiosarcoma. There has not been any utilization of new data on endogenous DNA adducts.

35. Formaldehyde is produced worldwide more than 20 million tons/year and used in a wide spectrum of applications. Therefore, formaldehyde exposures from environmental and occupational sources are quite common. Formaldehyde is a known animal and human carcinogen, causing nasal cancer. 1.rats: 15ppm formaldehyde induced 50% incidence of nasal carcinomas after 2 year-exposure (10ppm formaldehyde caused 22% incidence). 2.humans: “sufficient epidemiological evidence that formaldehyde causes nasopharyngeal cancer in humans” according to IARC Limited evidence to support formaldehyde inducing leukemia. 1.“strong but not sufficient evidence for a causal association between leukemia and occupational exposure to formaldehyde” based on IARC ( in 2006) 2.no mechanism of induction of leukemia in human has been identified Introduction FEMA trailers used after Hurricane Katrina 15ppm 12-month formaldehyde induced nasal tumor

36. Squamous Cell Carcinoma in a Rat Exposed to 15 ppm Formaldehyde

37. Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde

38. Formaldehyde is a ubiquitous environment pollutant, but it is also an essential metabolite in all living cells. Therefore, both endogenous and exogenous formaldehyde is present. Formaldehyde is very reactive with DNA and proteins, leading to diverse protein adducts and DNA damage. glutathione S-hydroxymethyl- glutathione ADH3 S-formylglutathione hydrolase formate CO 2 +H 2 O glutathione endogenous sources exogenous sources ALDH1A1 ALDH2 one carbon pool adduct formation Fate and metabolism of formaldehyde Adapted for IARC monograph 88

39. Experimental Design Rats were exposed to 10 ppm [ 13 CD 2 ]-formaldehyde for 6 hrs/day for 1 or 5 days and sacrificed within 2 hr. Nasal mucosa, lung, liver, spleen, thymus and bone marrow were collected for DNA adduct analysis. DNA was reduced with NaCNBH 3, hydrolyzed to nucleosides and adducts were separated by HPLC and fraction collection. ~40 µg DNA was used for nasal tissue and 200 µg for all others. Thus, 5- fold more DNA was analyzed from distal sites. Ultrasensitive UPLC-MS/MS methods were developed for N 2 - methyl-dG (detection limit 240 amol) and N 6 -CH 3 -dA (detection limit 50 amol) monoadducts. Endogenous and [ 13 CD 2 ]-adducts were measured. Lu, et al., Toxicol. Sci. 116, 441-451, 2010.

40. 1 day-exposed nasal epithelium (A), 5 day-exposed nasal epithelium (B), bone marrow (C) and spleen (D). LC-ESI-MS/MS SRM chromatograms of N 2 -Me-dG in typical tissues

41. Exposure periodTissues N 2 -HOCH 2 -dG (adducts/10 7 dG) N 6 -HOCH 2 -dA (adducts/10 7 dA) exogenousendogenousexogenousendogenous 1 day Nose 1.28±0.49 * 2.63±0.73 n.d. 3.95±0.26 Lungn.d. + 2.39±0.16 ‡ n.d. 2.62±0.24 Livern.d. 2.66±0.53 n.d. 2.62±0.46 # Spleenn.d. 2.35±0.31 n.d. 1.85±0.19 Bone Marrown.d. 1.05±0.14 n.d. 2.95±1.32 Thymusn.d. 2.19±0.36 n.d. 2.98±1.11 5 day Nose 2.43±0.782.84±1.13 n.d. 3.61±0.95 Lungn.d. 2.61±0.35 n.d. 2.47±0.55 Livern.d. 3.24±0.42 n.d. 2.87±0.65 Spleenn.d. 2.35±0.59 n.d. 2.23±0.89 Bone Marrown.d. 1.17±0.35 n.d. 2.99±0.08 Thymusn.d.1.99±0.30n.d.2.48±0.11 Formaldehyde-induced monoadducts in tissues of rats exposed to 10 ppm [ 13 CD 2 ]-formaldehyde for 1 day or 5 days # Endogenous N 6 -HOCH 2 -dA was present in control rat liver at 1.96±1.86 adducts/10 7 dA From Cheng et al., Chem. Res. Toxicol. 21, 746-751,2008.

42. Endogenous 282.2 → 166.1 m/z Exogenous 285.2 → 169.1 m/z Internal Standard 297.2 → 176.1 m/z 4.9 adducts/ 10 7 dG 9.0 adducts/ 10 7 dG 20 fmol Exposure (ppm) Exogenous adducts/10 7 dG Endogenous adducts/10 7 dG n 0.7±0.2 0.039±0.0193.62±1.33 3* 2.0±0.1 0.19±0.086.09±3.03 4** 5.8±0.5 1.04±0.245.51±1.06 4 9.1±2.2 2.03±0.433.41±0.46 5 15.2±2.1 11.15±3.014.24±0.92 5 *4-6 rats combined ** 2 rats combined Dosimetry of N 2 -hydroxymethyl-dG Adducts 15 ppm Rat NE

43. Non-Linear Dose Response for Formaldehyde DNA Monoadducts

44. Non-Human Primate Study 13 CD 2 O Exposure for 2 days (6 hours/day) Cynomolgus Macaque Tissues (to date) –Nasal Maxilloturbinate –Femoral Bone Marrow Exposure Levels –2 ppm (n=4) –6 ppm (n=4)

45. Nasal Maxilloturbinate N 2 -hydroxymethyl-dG Adducts Endogenous 282.2 → 166.1 m/z Exogenous 285.2 → 169.1 m/z Internal Standard 297.2 → 176.1 m/z Endogenous 282.2 → 166.1 m/z Exogenous 285.2 → 169.1 m/z Internal Standard 297.2 → 176.1 m/z 1.9 ppm 13 CD 2 O6.1 ppm 13 CD 2 O

46. Adduct Numbers in Primate Nasal Maxilloturinbates Exposure concentrati on Exogenous adducts/10 7 dG Endogenous adducts/10 7 dG 1.9 ppm0.25 ± 0.042.49 ± 0.39 6.1 ppm0.41 ± 0.052.05 ± 0.53 n = 3 or 4

47. Species Comparisons 1 day 2 day

48. Primate Femoral Bone Marrow Endogenous and Exogenous Adducts 1.9 ppm 13 CD 2 O6.1 ppm 13 CD 2 O 312 µg DNA 7E6 6E4 2E6 Endogenous 282.2 → 166.1 m/z Exogenous 285.2 → 169.1 m/z Internal Standard 297.2 → 176.1 m/z 178 µg DNA No Exogenous Adducts Detected with 5-10 fold >DNA 2E7 4E4 3E6 Endogenous 282.2 → 166.1 m/z Exogenous 285.2 → 169.1 m/z Internal Standard 297.2 → 176.1 m/z Note: We used ~20- 30 ug for nasal tissue

49. Adduct Numbers in Primate Bone Marrow Exposure concentrati on Exogenous adducts/10 7 dG Endogenous adducts/10 7 dG 1.9 ppmnd17.48 ± 2.61 6.1 ppmnd12.45 ± 3.63 n = 4

50. Conclusions Both cytotoxicity and genotoxicity are key events for the induction of nasal carcinoma. The sustained increase in cell proliferation that results from formaldehyde cytotoxicity “fixes” both endogenous and exogenous DNA adducts into heritable mutations. If a rat was placed in a FEMA trailer for 6 hours, only 91/100,000 formaldehyde adducts would come from the exposure. The rest are endogenous. A 6 hr exposure of a rat to the USEPA proposed safe level of formaldehyde (0.07 ppt) would induce 83/100,000,000 adducts. The lack of exogenous formaldehyde adduct formation in bone marrow and other distant sites does not support the biologic plausibility of leukemia.

51. Application to Risk Assessment Because no [ 13 CD 2 ]-N2-MedG adducts were detectable in primate bone marrow, we can state that they must be below the LOD. Therefore, the LOD represents a worst case upper bound for the amount of DNA analyzed. We have assumed that the relationship between airborne formaldehyde concentration and exogenous dG adducts is linear through zero. We calculated steady state concentrations based on the adduct half life and a 24/7 exposure. Risk estimates were calculated for all data sets.

53. Risk Assessment Model is Conservative Attributes all background risk to dG adducts. Only utilizes endogenous dG adducts, even though endogenous dA adducts are also present. Risk model is linear. Used lower 95% confidence bounds of measured endogenous adducts to generate upper 95% bounds on slopes. Made conservative assumptions on kinetics and dG adduct half-life data. Used same scaling methods used by the USEPA.

54. MicroRNA Study Acquired nasal maxilloturbinate samples (stored in RNAlater) from cynomolgus macaques from the Moeller et. al. study Isolated small RNA molecules Generated Agilent miRNA Microarray using – 2 controls – 3 2ppm formaldehyde tissue samples – 3 6ppm formaldehyde tissue samples Statistical analysis revealed 3 unique miRNAs significantly differentially expressed in monkeys exposed to 2 ppm formaldehyde (Fold Change >= +/- 1.5, ANOVA p < 0.05, FDR q < 0.10) Statistical analysis revealed 13 unique miRNAs significantly differentially expressed in monkeys exposed to 6 ppm formaldehyde

55. Significance of Findings All 3 of the significantly differentially expressed miRNAs in the 2 ppm group were also significant in the 6 ppm group, where fold change magnitudes were larger (dose-response) Many of the significant miRNAs have known associations to cancer (based on literature searches): 4 of the 13 significant miRNAs were measured as significantly differentially expressed after 1 ppm formaldehyde exposure using human lung cancer cells in the Rager et al. 2011 study published in EHP.

56. Fold Change in Cancer-related miR Significant in exposed group compared to controls * Down-regulated by formaldehyde in Rager et al. 2011

57. 1,3-Butadiene An important industrial chemical Classified by IARC, the NTP and EPA as a “Known Human Carcinogen” Epidemiologic data: SBR process is associated with increase leukemia in workers; monomer workers have increased lymphoma, but not leukemia Significant species differences in carcinogenicity: mice are much more sensitive than rats, the sites of tumors also differ. Formation of diepoxybutane is much greater in mice than rats. The diepoxide is 100-200 times more mutagenic than the monoepoxide or diolepoxide. Numerous DNA adducts are formed, with N7-guanine adducts being most prevalent.

58. Copyright ©2009 American Association for Cancer Research Goggin, M. et al. Cancer Res 2009;69:2479-2486 Figure 1. Metabolic activation of BD to reactive electrophiles and the formation of DNA adducts

59. Copyright ©2009 American Association for Cancer Research Goggin, M. et al. Cancer Res 2009;69:2479-2486 Figure 2. Dose-dependent formation of bis-N7G-BD in liver DNA of female B6C3F1 mice and female F344 rats exposed to BD by inhalation

60. Supralinear Exposure Response for Bis-N7G-BD in Female Mice

61. BD-induced N-7 Guanine Adducts in Female and Male Mice (Adducts per 10 8 Guanine) Exposure (ppm) N7-HBGBis-N7G-BDN7-THBG MaleFemaleMaleFemaleMaleFemale 0.5 ND N/A0.9 ± 0.69.5 ±1.58.3 ± 1.5 1.0 N/ANDN/A1.4 ± 1.1N/A17.1 ± 1.6 1.5 ND N/A2.2 ± 1.1 35.3 ± 6.7 31.6 ±3.6 6.25 N/A8.3 ± 4.9N/A 16.4 ± 4.62 N/A70.1 ± 8.7 62.5 N/A29 ± 11N/A 41 N/A732 ± 150 200 122 ± 20138 ± 12N/A1023914 ± 6823635 ± 353 625 659 ± 85728 ± 824N/A 202 ± 46 6205 ± 56103 ± 1140

62. Butadiene Hemoglobin Adducts

63. Human: pyr-Val-Leu*-Ser- Pro-Ala-Asp-Lys/-Thr-Asn-Val-Lys V - L - S - P - A - D - K / -T - N - V - K Mouse: pyr-Val-Leu*-Ser-Gly-Glu-Asp-Lys/-Ser-Asn-Ile-Lys V - L - S - G - E - D - K /- S - N - I - K Rat: pyr-Val-Leu*-Ser-Ala-Asp-Asp-Lys/-Thr-Asn-Ile-Lys V - L - S - A - D - D - K / -T - N - I - K * d 3 in Internal standards / hydrolyzed by trypsin pyr-Val containing α-N-Terminal Peptides (1-11)

64. Globin  Add IST (1-11) peptide  Trypsin hydrolysis (1-7 N-terminal peptide)  Centricon-3 filtration  Applying on IA column  Filtration (2 µm)  LC-ESI-MS/MS Protocol for pyr-Val-Peptide Analysis

65. Ion Chromatograms of HB-Val and Pry-Val in Control and Exposed Rodents

66. Dose Response Curves for Female Rodents After 10 Days Exposure

67. Efficiency of THBVal formation and Hprt Mutant Induction in Female B6C3F1 Mice Exposed to BD -13 -9

68. Hprt Mutation Induction and pyr-Val Adduct Formation in Female Mice Exposed to BD for 10 Days Georgieva et al. Tox. Sci. 2010

69. Lack of Gender Differences in Pyr-Val in Mice Exposed to Butadiene

70. Lack of Gender Differences in Pyr-Val in Rats Exposed to Butadiene

71. BD-induced Globin Adducts in Female Mice and Rats B6C3F 1 MiceF344 Rats Dose (ppm) HB-ValPyr-ValTHB-ValHB-ValPyr-ValTHB-Val 0.5 18.8 ± 6.423.2 ± 2.5187.4 ± 43.00.9 ± 0.10.8 ± 0.139.2 ± 6.3 1 21.5 ± 4.066.8 ± 5.9555 ± 3071.6 ± 0.51.4 ± 0.266.1 ± 9.6 1.5 26.5 ± 9.282.3 ± 6.5663 ± 1082.5 ± 0.42.0 ± 0.5116 ± 6 6.25 71.4 ± 4.5272 ± 171074 ± 2598.1 ± 0.76.7 ± 0.7257 ± 19 62.5 430 ± 701632 ± 2831185 ± 12660.0 ± 3.046.5 ± 8.3518 ± 53 200 N/A 148 ± 19123 ± 8643 ± 75 625 N/A 410 ± 15124 ± 11773 ± 74 N/A: Not available

72. Copyright ©2009 American Association for Cancer Research Goggin, M. et al. Cancer Res 2009;69:2479-2486 Figure 4. Gender differences in the formation of racemic bis-N7G-BD in B6C3F1 mice and F344 rats exposed to 625 ppm BD, and mice exposed to 200 ppm BD by inhalation for 2 wk

73. Black bars = mice; Gray bars = rats Gender and species differences in BD-induced Hprt MFs in mice and rats exposed for 2 weeks to 1250 ppm BD

74. DNA Damage Response to Butadiene Diepoxide in DT-40 Cells

75. Effect of Butadiene Exposure on Hemoglobin Adducts, Urinary Metabolites, and Indicators of Genotoxicity in Humans Albertini et al., Res. Rep. Health Eff. Inst., 1-141 (2003).

76. Czech BD Gender Study A second molecular epidemiology study was conducted in the same plant to investigate gender differences in biomarkers using 26 female controls, 23 female BD-exposed workers, 25 male controls and 30 male BD-exposed workers (Albertini RJ, et al., Chem Biol Interact. 166:63-77, 2007.) The design was similar to the 2003 study, but BD exposures were lower (Exposed Males - 0.81 mg/m 3 [0.37 ppm], Exposed Females – 0.397 mg/m 3 [0.18 ppm]). Biomarkers of exposure and effect were measured including: urinary metabolites, hemoglobin adducts, chromosomal aberrations and HPRT mutations, as well as metabolic genotypes.

77. THBVal in Male and Female Czech Butadiene Plant Workers

78. PyrVal in Male and Female Czech Butadiene Plant Workers

80. Preliminary Conclusions The presence of the pyr-Val protein adduct was demonstrated in humans. Among 95 subjects from the Czech Republic, 47 had detectable and 33 had quantifiable amounts of pyr-Val ranging from 0.08 to 0.6 pmol/g globin using the NanoUPLC-MS/MS system. The measured values for pyr-Val are much lower compared to the corresponding THB-Val in all samples, which could be an indication of the predominant hydrolytic metabolic pathway in humans. These preliminary data on occupationally-exposed BD workers do not provide support for females forming more epoxide metabolites than males at comparable exposures. Together these data suggest that humans can form pyr-Val after occupational exposure to BD, but it is also clear that pyr-Val is formed from other unknown sources. Preliminary inter-species comparisons of low exposures to BD between rodents and humans suggest that the extent of pyr-Val formation in humans is ~10-fold lower than in rats and ~100-fold lower than in mice.

81. Estimation of Molecular Dose Calculation internal dose of individual epoxides using globin adducts A : globin adduct level (mol/g) K val : The in vitro rate constant for the formation of an adduct the formation of an adduct (Fred et al 2004, Recio et al 1992, Ostermann-Golkar 1998)

82. Calculation for the EB-dose Equivalent in Humans, Rats and Mice Dose-equivalent = EB × 1 + DEB × 32 + EBdiol × 0.21

84. CONCLUSIONS The collective data from nearly 20 years of research by many investigators has provided an excellent understanding of the mode of action for butadiene carcinogenesis. It is clear that mice are much more sensitive than rats and humans as a result of more efficient metabolism of BD to BRIs. Potentially important gender differences have been demonstrated in rats and mice, where increases in DNA cross- links and mutagenesis were shown in female rats and mice, compared to males. This is most likely due to differences in DNA repair, as metabolism was not different. Using the EB-equivalents concept, we found that mice produce ~44 and 174 times greater numbers of EB dose equivalents than rats and humans, respectively. In three blinded molecular epidemiology studies, no increases in HPRT or chromosomal mutations have been demonstrated under current occupational exposures.

85. 2005 EPA Guidelines for Carcinogen Risk Assessment Linear extrapolation should be used when there are Mode Of Action data to indicate that the dose-response curve is expected to have a linear component below the POD. –Agents that are DNA-reactive and have direct mutagenic activity. The EPA Guidelines also suggest using a Framework Analysis approach to support or not support a proposed Mode Of Action.

86. IPCS/EPA Framework for Evaluating Mechanistic D ata Introduction Postulated mode of action Key events Dose-response relationship Temporal association Strength, consistency and specificity of association with key events Biological plausibility and coherence Other modes of action Assessment of mode of action Uncertainties, inconsistencies and data gaps

87. MOA Key Events Genotoxicity DNA Adducts Mutations in reporter genes Mutations in cancer genes Cancer

88. Genotoxicity A chemical is defined as genotoxic if the weight of evidence is positive in a battery of genetic toxicology assays. This is not a quantitative data set. Such data represents Hazard Identification, not Risk Assessment.

89. MOA Key Events Genotoxic DNA Adducts Mutations in reporter genes Mutations in cancer genes Cancer

90. Molecular Dosimetry of DNA Adducts DNA adducts are expected to be linear at low doses. An exception to this is when identical adducts are formed endogenously. Many forms of endogenous DNA adducts have been identified and measured. These include direct oxidative adducts, exocyclic adducts, AP sites and deamination products.

91. Linear DNA Adducts at Low Doses MMS DBP PO DMN

92. MOA Key Events Genotoxic DNA Adducts Mutations in reporter genes Mutations in cancer genes Cancer

93. Mutations Do Not Go Through Zero In contrast to most DNA adducts, mutations do not go through zero. Rather, they reach a spontaneous level that reflects the summation of endogenous DNA damage and repair that occurs in cells. The point in the dose response curve where the number of mutations significantly increase above the spontaneous level represents the point at which the exogenous DNA damage starts driving the biology that results in additional mutations.

94. Inflection point Dose Typical Mutation Dose Response

95. Historical Control Data for HPRT and TK Mutations in vitro Penman and Crespi, Environ Mol Mut 10:35-60, 1987

97. Abramsson-Zetterberg, 2003Zeiger, et al., 2007 Relationships Between DNA Adducts and Micronucleus Induction in Mice with Carcinogenic Doses of Acrylamide Twaddle et al, 2004 Tareke et al, 2006 Young, et al, 2007

99. DNA Repair Can Modulate Where Increased Mutations Occur If DNA repair is impaired or absent, the inflection point for mutations occurs at lower doses. This results from increased numbers of DNA adducts relative to a cell with normal DNA repair.

100. 6 6 Exposure-response for Mutagenesis in Drosophila Exposed By Inhalation To Propylene Oxide Nivard et al, Mut. Res. 529: 95-107, 2003. 12,000 ppm hr PO

101. Comparison of Acute and 28-Day Treatments 350 mg/kg 12.5 mg/kg/day

102. MOA Key Events Genotoxic DNA Adducts Mutations in reporter genes Mutations in cancer genes Cancer

103. Gaps in Knowledge Most mutation assays are done at high doses to establish that a compound is or is not genotoxic. There is a real need to generate dose response data at low exposures to establish NOAELs for mutation in CA, MN and surrogate genes such as hprt. These data will further establish the exposures where the background number of mutations become significantly increased.

104. Conclusions As our knowledge of carcinogenesis has expanded, concepts of “one molecule → cancer “ have little to no scientific support. Mutations in genes controlling cell proliferation and cell death appear to play major roles in the induction of cancer. While these genes are difficult to monitor in noncancer tissues, surrogate mutations can be used to examine dose response in cells, animals and humans.

105. Conclusions (cont.) Such mutations do not have linear relationships with exposure. Rather, they reach a spontaneous incidence that is driven by endogenous biological processes. The exposures where mutagenesis becomes significantly increased over background represents a scientifically based Point of Departure for setting acceptable exposures. This could be accomplished by using a Margin of Exposure approach to protect susceptible individuals.

106. ED 0.01 Carcinogenicity Study A carcinogenicity study has been conducted on Dibenzo[a,l]pyrene using 42,000 rainbow trout. This model is 50 times more sensitive than rodent bioassays due to the low background incidence of neoplasia. The EPA linear risk model over estimated the actual observed liver cancer incidence by three orders of magnitude. Bailey et al, Chem. Res. Tox., 2009

107. Biologically-Based Risk Assessment Refine estimates of dose to relevant targets through use of biomarkers of exposure and PBPK modeling Improve hazard characterization through a better understanding of the mode(s) of action for endpoints of concern Strengthen inferences regarding the shape of dose/response curves outside the range of traditional observations Identify/investigate opportunities for research in human populations, such as susceptibility factors

108. The Exposome Chris Wild proposed that we should be considering the “Exposome” for cancer etiology. Wild, C: CEBP 14: 1847-1850, 2005 –Under this view, the assessment of exposures should not be restricted to chemicals entering the body from air, water, food, smoking, etc., but should also include internally generated toxicants produced by the gut flora, inflammation, oxidative stress, lipid peroxidation, infections, and other natural biological processes. In other words, we must focus upon the ‘internal chemical environment’ arising from all exposures to bioactive chemicals inside the body More recently, Martyn Smith et. al. made similar statements. Smith, M: Chemico Biological Interactions 192: 155-159, 2011 –The question arises as to how to find the causes of the majority of de novo AMLs that remain unexplained. We propose that we should attempt to characterize the 'exposome' of human leukemia by using unbiased laboratory-based methods to find the unknown 'environmental' factors that contribute to leukemia etiology.

109. Systematic Characterization of Comprehensive Exposure-Dose-Response Continuum and the Evolution of Protective to Predictive Dose-Response Estimates