Slide 1 of 38 Office of Pesticide Programs Drs. Vicki Dellarco & Anna Lowit Health Effects Division Office of Pesticide Programs Mode of Action/ Human Relevance Analysis For Incorporating Mechanistic Data in Human Health Risk May 2006

Slide 2 of 38 Uses of Mechanistic Data in Risk Assessment Identify Key biological (precursor) events leading to adverse toxicities (Mode of Action) Inform Human relevance of animal findings Dose response extrapolation Life stage susceptibilities Understand Common pathways of toxicity (cumulative risk assessment) Promote Consistent harmonized approach to risk assessment for all health endpoints

Slide 3 of 38 “Mechanism of action” (more detailed understanding at biochemical & molecular level) versus “Mode of action” (identification of key & obligatory steps) Exposure Toxicity Key event

Slide 4 of 38 Mode of Action Framework EPA’s Guidelines for Carcinogen Risk Assessment 1996 Proposed Revisions  put forth the notion of understanding mode of action versus mechanism of action 1999 Interim Guidance  introduced mode of action framework 2005 Final Guidance  minor rewording of MOA framework

Slide 5 of 38 Mode of Action Framework Postulated mode of action Identify sequence of key events on the path to cancer Experimental support Concordance of dose-response for key events with that for tumors Temporal relationships for key events & tumors Biological plausibility & Coherence Strength, consistency & specificity Other modes of action Identify uncertainties Conclusion

Slide 6 of 38 Human Relevance Framework Risk Sciences Institute-ILSI Comparability or concordance analysis of the key events & relevant biology between the laboratory species & humans  Tumor Responses: Meek et al., 2003, Critical Reviews in Toxicology Vol 33/Issue 6,  Reproductive, Developmental, Neurtoxocity Responses: Seed et al., 2005 Critical Reviews in Toxicology Vol 35/Issue 8-9, extended human relevance analysis to include mutagenic carcinogens & noncancer end points WHO/IPCS Human Relevance Framework (in preparation)

Slide 7 of 38 Human Relevance Framework Based on three analyses: Is the Weight of Evidence sufficient to establish a MOA in animals (MOA Framework)? Are the key events in the animal MOA plausible in humans? Taking into account kinetic/dynamic factors, is the animal MOA plausible in humans?

Slide 8 of 38 Relevant or Unknown Human Relevance Not Relevant in Humans 2.) Is the Animal MoA Plausible in Humans? Yes No Human Relevance Framework Human Relevance Framework Not Sufficient 1.) Is the Weight of Evidence Sufficient to Establish a MoA in Animals? 3.) Taking Into Account Kinetic and Dynamic Factors, Is the Animal MoA Plausible in Humans? Yes No, Continue with the Dose Response & Exposure Assessment

Slide 9 of 38 Assessing an Animal Mode of Action General Points: Applicable to all chemicals, to all endpoints, and to all modes of action Evaluation of MOA for tumors or (other adverse effects) in different organs MOA in different organs may or may not be the same Site concordance between animals & humans

Slide 10 of 38 Assessing an Animal Mode of Action General Points: When a substance operates via a novel MOA, the analysis is focused on the chemical & entails a detailed evaluation via the MOA Framework When a substance produces an adverse effect consistent with an already established & peer reviewed MOA through which other chemicals have been shown to operate, the analysis is focused on the established MOA & a determination of whether the substance operates via the same key events established for the pathway

Slide 11 of 38 Assessing an Animal Mode of Action for Human Relevance General Points: Concordance Analysis of key events is for the MOA & is not necessarily a chemical specific evaluation. Chemical specific & generic information relevant to the toxicity process can be valuable

Slide 12 of 38 “EPA’s Cancer Assessment Review Committee (CARC) classified atrazine as “not likely to be carcinogenic to humans”. Vinclozolin--“Since the androgen receptor is widely conserved across species lines, antiandrogenic effects would be expected in humans.” MOA: Inform Human Relevance

Slide 13 of 38 Alachlor - “... a margin of exposure (MOE) approach (indicative of a non-linear dose response) should be used for the risk assessment.” Cacodylic Acid – “... nonlinear default approach (i.e., derivation of a reference dose or margin of exposure) is regarded as the more appropriate dose response extrapolation approach...” Chloroform – “... a nonlinear approach is more appropriate for low-dose extrapolation.” MOA: Inform Dose Response Extrapolation

Slide 14 of 38 Case Study Cacodylic Acid (Dimethylarsinic acid)

Slide 15 of 38 DMA V Mode of Action Science Issue Paper: “Mode of Carcinogenic Action for Cacodylic Acid (Dimethylarsinic Acid, DMA V ) and Recommendations for Dose Response Extrapolation” (July 26, 2005) ic_acid/ Revised issue paper will be publicly available this spring. EPA’s Science Advisory Board (SAB) reviewed the special issue paper in September, 2005 Draft SAB report December 27, 2005

TMAs III TMAs V Methylation Reduction Metabolism of Arsenic Alternate steps of oxidative methylation & reduction Pesticide Chemical

Slide 17 of 38 DMA V : MODE OF ACTION ANALYSIS Extensive experimental cellular and laboratory animal data Weight of Evidence

Slide 18 of 38 DMA V : Available Cancer Data No epidemiology data Standard rodent bioassay Bladder carcinogen in rats  via feed -100 ppm (9.4 mg/kg bw per day)  via drinking water- 50 & 200 ppm  females more sensitive than males Not carcinogenic in mice  Up to 500 ppm in B6C3F (Gurr et al., 1989)  121 ppm in C57 XC3H/Anf or AKR (NCI 1969)

Slide 19 of 38 Mode of Action Measurable Key Events in Target Tissue DMA III Metabolite Hyperplasia Urothelial Toxicity Regenerative Proliferation Urinary Bladder Tumors Sustained Urinary bladder from a female F344 rat treated with 100 ppm DMA V BrdU Labeling

Slide 20 of 38 Compensatory regeneration in rat bladder at weeks 8 & 10 following ingestion of DMA V

Slide 21 of 38 Dose Response Concordance Temporal Dose (mg/kg bw/day) Metabolism DMA V  DMA III Urothelial Toxicity Regenerative Proliferation Urothelial Hyperplasia Transitional Cell Carcinoma 0.2 (2 ppm) + (wk ± 0.01 uM ) + (wk 10-6/10, grade 3 or 4) (10 ppm) + (wk ± 0.02 uM) + (wk 3-2/7, grade 3) (wk- 10; 8/10, grade 3 or 4) slight (wk X inc) -- 4 (40 ppm) + (wk ± 0.09 uM) + (wk 3-7/7, grade 3) (wk 10-5/10, grade 3 or 4) + (wk X inc ) + (wk 10- 4/10) (100 ppm) + (wk ± 0.15 uM) + (6 hrs-6/7, grade 3) (24 hrs-4/7, grade 3 or 4) (wk 2 6/10, grade 5)(wk 10-0/10, grade 4 or 5) + (wk X inc) (wk 2-3.9X inc) (wk X inc) + (wk 8-7/10) (wk 10-9/10) + (papilloma first obs at wk 107; carcinoma first obs at wk 87) Association of Key Precursor Events & Bladder Tumors in F344 Rats

Slide 22 of 38 Cacodylic Acid: Key Events Temporal Relationship DMA III  DMA V Urothelial Cytotoxicity Regenerative Proliferation Hyperplasia Tumors 6 hours 1 Week 8-10 weeks 104 weeks Urinary bladder from a female F344 treated with 100 ppm DMA V BrdU labeling Urinary bladder tumors

Slide 23 of 38 Cacodylic Acid: Key Events Cytotoxicity/Regenerative Proliferation Strength, Consistency & Specificity Consistency of association found in repeated experiments within a lab & among different labs Inhibition of DMA V  DMA III reduced cytotoxicity Cessation of exposure to DMA V results in recovery of tissue (i.e., hyperplasia) Biological Plausibility & Coherence Regenerative proliferation associated with persistent toxicity appears to be a risk factor for bladder cancer in humans

Slide 24 of 38 Characterization of Cacodylic Acid’s Genotoxicity Neither DMA V or DMA III are direct acting point/gene mutagens Both are clastogenic but DMA III is the more potent In vitro data only DNA damage appears to result from an indirect mechanism (ROS/oxidative damage)  DMA III  DMA V

Slide 25 of 38 Chromosomal Aberrations For the oxidative DNA damage to be relevant to the carcinogenic process (i.e., clonally expanded), stable chromosomal mutations must be formed Formation of chromosomal mutations requires DNA replication because chromosomal alterations are produced by errors of replication on a damaged DNA template. frequency of chromosomal mutations will be a function of the regenerative proliferative response. All these events--genetic errors, cytotoxicity, stimulation in cell proliferaiton -- must occur to result in bladder tumors.

Slide 26 of 38 Other Modes of Action or Key Events No other MOA with sufficient scientific support Direct DNA reactivity Formation of solids Changes in urinary chemistry & physiology

Slide 27 of 38 Mode of Action Conclusions Sequence of key events leading to bladder tumors measurable & supported by robust data Biologically plausible Uncertainties do not discount scientific support cellular target for cytotoxicity not understood unknown cytotoxic metabolites found in urine (after drinking water exposure)

Slide 28 of 38 Urothelial Cytotoxicity ? Human Relevance of DMA V ’s Mode of Action Metabolism to DMA III Regenerative Proliferation Hyperplasia & Bladder tumors

Slide 29 of 38 Concordance Analysis of Key Events in Rats & Humans: Qualitative & Quantitative Plausibility Key EventRatsHumans Presence of DMA III in urine Yes Yes (based on As i ) Persistent cytototoxicity YesPossible Persistent regenerative prolif/hyperplasia YesPossible Bladder Tumors YesPossible

Slide 30 of 38 DMA V Mode of Action (MOA) The SAB concurred with EPA’s conclusions 1.Rat data developed for DMA V most appropriate data for quantifying cancer risk 2.MOA for the development of bladder tumors in rats established 3.The rat MOA is expected to be plausible in humans 4.The MOA supports nonlinear extrapolation of cancer risk to DMA V

Slide 31 of 38 Dose response extrapolation approach Dose response extrapolation should be based on considerations of MOA which supports nonlinearity Must be sufficient DMA III to produce cell killing & sufficient cell killing to lead to regenerative proliferation Cytotoxicity & enhanced proliferation need to be sustained  Frequency of chromosomal mutations dependent on enhanced proliferation & on generation of ROS (DMA III  DMA V ) Point of Departure based on cell proliferation should be protective of DMA’s carcinogenic & promoting effects

Slide 32 of 38 Dose Response Considerations Cancer Guidelines describe a two- step dose-response process which separates Modeling the observable range of data Extrapolation to lower doses Nonlinear extrapolation Preferred approaches  PBPK Model--internal dosimetry at the target tissue –e.g. DMA III  BBDR Model—predict biological effect –e.g., two stage clonal growth Interim approach  Identify a point of departure (POD) based on benchmark dose modeling  Apply uncertainty and safety factors POD Key event Dose Response

Slide 33 of 38 MOA informs low-dose extrapolation? MOA Established? BBDR model? 1. Fit data in observable range 2. Linear extrapolation from POD Use model RfD/RfC or MOE Yes Yes, nonlinear No Yes, linear (including mutagenic MOA) Quantitative Dose-response Assessment

Slide 34 of Mean Response dose Hill Model with 0.95 Confidence Level BMD 10 BMDL 10 Benchmark Dose Modeling: Regenerative Proliferation

Slide 35 of 38 Biological Event Duration Feeding Duration Drinking water 10%1%10%1% BMD (mg/kg/day) BMDL (mg/kg/day) BMD (mg/kg/day) BMDL (mg/kg/day) BMD (mg/kg/day) BMDL (mg/kg/day) BMD (mg/kg/day) BMDL (mg/kg/day) Tumor104 weeks weeks Hyperplasia 10 weeks weeks weeks BrdU labeling (proliferation) 10 weeks Not determined. Available data not suitable for modeling. Cytotoxicity 3 weeks No reliable dose-response data available 10 weeks Cacodylic Acid: Summary of benchmark dose estimates and lower 95% confidence limits for cytotoxicity, BrdU labeling index, hyperplasia and tumor data. (Doses in mg/kg/day)

Slide 36 of 38 Estimate target tissue dose using various dose metrics (e.g., DMA V, DMA III or TMAO concentration in urine or bladder tissue) associated with bladder tumor development using DMA V PBPK model. Current status: Agency developing mouse model first then scale to rats and humans Use PBPK model to estimate the environmental exposure to DMA V required to achieve the same target tissue dose to bladder. PBPK Model Application to DMA Risk Assessment DMA V oral exposure metabolism elimination Q: What human exposure to DMA V is required to produce the same target tissue dose of DMA to bladder that results in tumors in rats exposed to DMA V ?

Slide 37 of 38 Summary Human Relevance Framework Identify key events Assist in dose response assessment Assist in rodent to human extrapolation Promote harmonization of risk assessment for all endpoints