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

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

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

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 Role of endogenous DNA damage

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

c a b Increasing External Exposure Increasing Adduct Concentration Sublinear Supralinear

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

Sources of DNA Damage Spontaneous DNA Chemistry Depurination TA GC TA TA C TA Deamination

Endogenous AP Sites in Rat Tissues and Human Liver [Nakamura and Swenberg, 1999]

Oxidative Stress Induced-DNA Damage

Aldehydic DNA lesions (ADL) in HeLa cells exposed to H 2 O 2 ( mM) for 15 min

Efficiency of Low Doses of H 2 O 2

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

Induced Oxidative Stress Calf Thymus DNA Exposed to TCHQ

Steady-state Amounts of Endogenous DNA Damage Endogenous DNA LesionsNumber per Cell Abasic sites10,000-50,000 OHEtG3,000 7-(2-Oxoethyl)guanine3,000 8-oxodG2,400 Formaldehyde1,000-4,000 Acetaldehyde1, Methylguanine1,200 AcrdG120 M 1 dG60 N 2,3-Ethenoguanine36 1N 2 -Etheno dG30 1N 6 -Etheno dA12 Total20,000-60,000

Chemical-Specfic 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%

O 4 -EtdThd O 6 -EtdGuo

Dose (ppm DEN) ET (pM)/dT (  M) O 2 -ETO 4 -ET Molecular Dosimetry of DEN

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

Exposure-Response for Vinyl Chloride Metabolism and Carcinogenicity

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

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

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.

Sample Spectrum of 7-OEG m/z 265  152 AST m/z 267  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)

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± ± ± ± ± ± Weeks Post Exposure 0.1 ± ± ± ± ± 0.9ND 4 Weeks Post Exposure 0.2 ± ± ± ± ± 1.3ND 8 Weeks Post Exposure 0.2 ± 0.07ND3.7 ± ± ± 0.5ND

None 1100 ppm VC; 4 wk 10 ppm VC; 4 wk None None known Exposure 110 ± ± ± 0.8SD rat hepatocytes 16 ± 5Human colon 17 ± 2 Human liver 10 ± ± 2.1 SD rat brain N 2,3-  G / 10 8 G Sample N 2,3-  G in Control and VC-exposed Samples

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

Vinyl Chloride Cancer Risk Estimates Rat PBPK/LMS1995 Clewell et al Year 1.4Epi Chen & Blancato 4.4Rat (f) Mouse Epi PBPK/LMSEPA 0.6RatPBPK/LMSReitz et al Rat PBPK/LMS 84RatLMSEPA Inhalation Risk (per  g/m 3 x ) DataModelAuthor(s)

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

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.

Formaldehyde is One of the Oldest Chemicals in the World One-carbon pool Methanol metabolism Amino Acid metabolism Lipid Peroxidation P450 dependent demethylation (O-, N-, S-methyl) Sources of Endogenous Formaldehyde Formaldehyde was Part of the Origin of Life

Ubiquitous Environmental Chemical Global production is >20 million tons/yr Wide use in industrial and consumer products Carcinogenic in rodent bioassays Listed as a human carcinogen  NTP 2011, IARC 2006 Mode of Action is complex  Cytotoxic/cell proliferation  Mutagenic  Site of contact vs distant sites  Endogenously formed in all cells

Carcinogenesis Bioassays CIIT/Battelle studies in rats and mice –12 month sacrifice/interim report –18 month data published in Cancer Research (Swenberg,et al 1980) –Final report and Cancer Research paper on the study (Kerns, et al. 1983) CIIT expanded the exposure range and mechanistic designs in a second bioassay published in Cancer Research (Monticello, et al, 1996) Subsequent cancer bioassays –Inhalation studies –Oral studies

Tumor Incidence and Cell Proliferation in Rats Exposed to Formaldehyde

Epidemiology of Formaldehyde and Cancer Nasopharyngeal Cancer –The NCI cohort found an increase in NPC, while other studies have been negative. Only 1 plant out of 10 had an increased incidence of NPC The same plant was in a region known for silversmithing and metal working, two known causes of NPC. The extent of formaldehyde exposure was not associated with the increase in NPC. While biologic plausibility is clearly present, the lack of consistency between studies and the lack of an exposure relationship in positive studies weakens the conclusion. Confounding cannot be eliminated.

Epidemiology of Formaldehyde and Cancer (Cont.) Myeloid Leukemia –No evidence has been provided that demonstrates that formaldehyde gets to sites distant to the portal of entry. –While several studies have shown associations, equal numbers of studies have not. –No mechanisms have been identified for the induction of leukemia by formaldehyde. –Thus, the biologic plausibility of inhaled formaldehyde causing leukemia is weak.

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

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 Capillary-MS/MS and UPLC-MS/MS methods were developed for N 2 -methyl-dG (detection limit 240 and 20 amol) and N 6 -CH 3 -dA (detection limit 50 and 5 amol) monoadducts. Endogenous and [ 13 CD 2 ]-adducts were measured.

The formation of N2-hydroxymethyl-dG originating from both endogenous and exogenous formaldehyde.

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

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 ±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± day Nose 2.43± ±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, ,2008.

Endogenous → m/z Exogenous → m/z Internal Standard → 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± ± ±1.33 3* 2.0± ± ±3.03 4** 5.8± ± ± ± ± ± ± ± ± *4-6 rats combined ** 2 rats combined Dosimetry of N 2 -hydroxymethyl-dG Adducts 15 ppm Rat NE

Non-Linear Dose Response for Formaldehyde DNA Monoadducts

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)

Nasal Maxilloturbinate N 2 -hydroxymethyl-dG Adducts Endogenous → m/z Exogenous → m/z Internal Standard → m/z Endogenous → m/z Exogenous → m/z Internal Standard → m/z 1.9 ppm 13 CD 2 O6.1 ppm 13 CD 2 O

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

Species Comparisons 1 day 2 day

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 → m/z Exogenous → m/z Internal Standard → m/z 178 µg DNA No Exogenous Adducts Detected with 5-10 fold >DNA 2E7 4E4 3E6 Endogenous → m/z Exogenous → m/z Internal Standard → m/z Note: We used ~ ug for nasal tissue

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

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.

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.

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.

Epigenetic Mode of Action Studies Adduction to histone lysines Altered expression of miRNAs – Primate maxilloturbinates, bone marrow, WBC and CD34+ cells – Rat tissues following 28 days of inhalation exposure to 2 ppm (6 hr/day, 7 days/week) DNA methylation in tissues from inhalation exposed rats and primates

Discovered in early 1990s Recognized as important biological regulators in early 2000s (Filipowicz, 2008) DNA miRNA mRNA Protein Transcription Translation Transcription 1. Decay of target mRNA 2. Translational repression 3. Cleavage of newly translated polypeptides miRNAs regulate gene expression in three ways: MicroRNAs (miRNAs) are Important Epigenetic Regulators of Gene Expression

Nonhuman Primate Project Cynomolgus macaques were exposed to 0, 2, or 6 ppm 13 CD 2 formaldehyde for 6 h/day for 2 days Time-matched control macaques received clean air under the same conditions RNA samples were collected from the nose RNA samples were hybridized to miRNA microarrays to compare genome-wide miRNA expression profiles of formaldehyde-exposed versus unexposed samples

Formaldehyde Exposure Changes the Expression Levels of 13 miRNAs in the Nonhuman Primate Nasal Epithelium Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the nasal epithelium of nonhuman primates. 3 and 13 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed upon exposure to 2 ppm and 6 ppm formaldehyde, respectively. Computationally predicted mRNA targets for miR-125b

RT-PCR of selected miRNAs with altered expression upon exposure to formaldehyde.

Systems Biology-Based Analysis of the Transcriptional Targets Predicted to be Regulated by miR-125b Revealed an Enrichment for Apoptosis Signaling Apoptosis Signaling p = Predicted targets of formaldehyde- responsive miR-125b are shown in BLUE

All Apoptosis-Related Targets of miR-125b Showed Decreased Expression in Formaldehyde-Exposed Samples Formaldehyde Exposure ↑ miR-125b Expression ↓ Predicted Targets of miR-125b ** * Significantly altered at the expression level by formaldehyde exposure in the rat nasal epithelium (Andersen et al. 2010)

Rodent Project Design Nasal Epithelium Bone Marrow White Blood Cells  Rats were exposed to 2 ppm 13 CD 2 formaldehyde for 6 h/day for 28 days  Time-matched control rats received clean air under the same conditions  RNA samples were collected from the nose, circulating white blood cells, and bone marrow  RNA samples were hybridized to the Agilent Rat miRNA Microarray to compare genome-wide miRNA expression profiles of formaldehyde- exposed versus unexposed samples Genome-wide miRNA expression profiles were assessed throughout three regions: (1) nose, (2) circulating white blood cells, and (3) bone marrow

Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the rat nasal epithelium. 59 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed after 28 days of exposure to 2 ppm formaldehyde. Formaldehyde Exposure Changes the Expression Levels of 59 miRNAs in the Rat Nasal Epithelium  miR-203 shows decreased expression levels by formaldehyde exposure within the nonhuman primate and rodent nose  Evaluated miR-203 further by predicting its transcriptional targets

Systems Biology-Based Analysis of the Transcriptional Targets Predicted to be Regulated by miR-203 Revealed an Enrichment for Carcinogenesis-Related Signaling * * * Involved in carcinogenesis Altered at the expression level by formaldehyde in the rodent nasal epithelium (Andersen et al. 2010) * * * ** * * * p < **  Member of the RAS oncogene family  Altered at the expression level in nasopharyngeal carcinoma (Zheng et al. 2007) Most significant network associated with miR-203 targets

Plot displaying the fold changes in miRNA expression induced by formaldehyde exposure in the rat leukocytes. 8 miRNAs were significantly (FC ≥ ±1.5, p<0.05, q<0.10) differentially expressed after 28 days of exposure to 2 ppm formaldehyde. Formaldehyde Exposure Changes the Expression Levels of 8 miRNAs in Rat White Blood Cells miR-31 shows formaldehyde- induced increased expression in the rat nose and circulating white blood cells

Summary of the Number of miRNAs with Altered Expression by Formaldehyde Exposure throughout Multiple Regions of the Body Nasal Epithelium Bone Marrow White Blood Cells 59 miRNAs 8 miRNAs 0 miRNAs

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 times more mutagenic than the monoepoxide or diolepoxide. Numerous DNA adducts are formed, with N7-guanine adducts being most prevalent.

Goggin, M. et al. Cancer Res 2009;69: Metabolic activation of BD to reactive electrophiles and the formation of DNA adducts

Goggin, M. et al. Cancer Res 2009;69: Dose-dependent formation of bis-N7G-BD in liver DNA of female B6C3F1 mice and female F344 rats exposed to BD by inhalation

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 ± ± ± N/ANDN/A1.4 ± 1.1N/A17.1 ± ND N/A2.2 ± ± ± N/A8.3 ± 4.9N/A 16.4 ± 4.62 N/A70.1 ± N/A29 ± 11N/A 41 N/A732 ± ± ± 12N/A ± ± ± ± 824N/A 202 ± ± ± 1140

Butadiene Hemoglobin Adducts

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

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

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

Copyright ©2009 American Association for Cancer Research Goggin, M. et al. Cancer Res 2009;69: 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

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

DNA Damage Response to Butadiene Diepoxide in DT-40 Cells

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 mg/m 3 [0.37 ppm], Exposed Females – 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.

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

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

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.

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

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.

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

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

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.

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

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.

Linear DNA Adducts at Low Doses MMS DBP PO DMN

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

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.

Inflection point Dose Typical Mutation Dose Response

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

The Formation of Endogenous and Exogenous N 2 - Ethylidene-dG in TK6 Cells Using [ 13 C 2 ]-Acetaldehyde

The Sum of Endogenous and Exogenous N 2 - Ethylidene-dG in TK6 Cells Using [ 13 C 2 ]-Acetaldehyde

Micronucleus Formation in TK6 Cells exposed to [ 13 C 2 ]-Acetaldehyde

Survival of TK6 Cells exposed to [ 13 C 2 ]-Acetaldehyde

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

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.

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

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

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

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.

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.

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.

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

The Exposome Chris Wild proposed that we should be considering the “Exposome” for cancer etiology. Wild, C: CEBP 14: , 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: , 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.

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