Agricultural Health Study http://www.aghealth.org A large, prospective cohort study Goal: Establish a large group of people associated with pesticide application and follow them for many years to evaluate the relationship between agricultural exposures and disease

Agricultural Health Study Example of a Prospective Cohort Study Time Disease Exposed to Agricultural Risk Factor Defin Popula (Certif Pestici Applica ed tion ied de tors) No disease Non- Randomized Unexposed to Agricultural Risk Factor Disease No disease Direction of Inquiry

Topics Covered Study Design Pesticide Exposure Assessment Major Exposures and Health Effects Pesticides & Human Carcinogenicity

Design Pesticide Certification Program (North Carolina and Iowa) Main Cohort Study (Cohort Identification/Questionnaire Administration) Main Cohort Study Morbidity and Mortality Follow-Up ---------------------------- Goal: 75,000 study subjects (Enrolled 89,658) Nested Case-Control Studies a. Cancer b. Non-Cancer Pesticide Exposure Assessment

Collaborating Institutions Executive Committee National Cancer Institute National Institute of Environmental Health Sciences Environmental Protection Agency National Institute for Occupational Safety and Health Field Stations University of Iowa Battelle/Centers for Public Health Research & Evaluation Coordinating Center (Westat, Inc.) Operations Committee

Key Organizations Affiliated with Iowa Field Station Center for Health Effects of Environmental Contamination College of Public Health Department of Epidemiology (AHS Iowa Field Station) Department of Occupational and Environmental Health State Health Registry of Iowa Iowa Department of Agriculture and Land Stewardship Iowa State University Extension Service

Study Overview Phase I 1993-97 Phase II 1998-03 Phase III 2004-10 Pesticide Applicators Recruited at Training (84%) Spouses of Applicators (75%) CATI follow-up interview (~70%) Buccal Cell Collection (~40%) Diet Questionnaire (~40%) CATI follow-up interview (~50%) Buccal Cell Collection (N=~1000 cancer subjects) Cancer and Mortality Follow-up Phase II 1998-03 Phase III 2004-10

Enrollment in the Agricultural Health Study by County (an additional 392 enrollees have an out-of-state address) 762 474 401 393 316 421 573 625 628 575 1,098 1,431 908 598 501 759 734 477 652 869 881 662 1,212 671 902 562 613 452 796 879 930 781 1,037 1,044 652 830 433 599 546 627 482 911 627 733 640 920 616 574 661 931 416 393 695 631 810 748 727 756 401 302 532 362 801 501 618 523 635 631 1,113 704 531 234 368 422 495 688 849 403 484 443 341 262 124 162 114 185 250 679 325 603 514 194 319 174 218 171 196 268 458

Sources for Information Questionnaires (www.aghealth.org) Environmental monitoring, literature, PHED Disease registries Biomarkers

Cohort Characteristics at Enrollment PA (Farmers) CA Spouse of PA Average age: 47.3 yrs 38.5 yrs 47.2 yrs Race: White 95% 99% 95% Nonwhite 3% 1% 2% Gender: Men 97% 96% 1% Women 3% 4% 99% Education: 12+ years 86% 94% 82% Current smoker: 15% 25% 10% Personally applied pesticides: 11+ yrs 65% 33% 18% 10+ days/yr 53% 66% 11% 60+ days/yr 4% 21% 1% (From: Alavanja MCR et al. Scand J Work Environ Health 31 Suppl 1:39-45, 2005)

Major Exposures and Health Effects under Study PESTICIDES Animals Engine exhaust Solvents Organic and inorganic dust Health Effects Cancer Respiratory Health Reproductive Health Neurologic Disease Work Place Injuries

Exposure Assessment Procedures Source: Dosemeci M et al Exposure Assessment Procedures Source: Dosemeci M et al. Annals of Occupational Hygiene 46(2):245-260, 2002 Questionnaire Data Identification of Exposure Scenarios Intensity Info. Mixing status Application type Repair equipment PPE use Work habit Personal hygiene Data from Literature Assigning Exposure Scores for Each Scenario AHS Surveys PHED Time Info. -Duration of use - Frequency of use - Calendar time Merging Subject & Chemical-specific Assessment

Exposure Metrics In the AHS Lifetime Exposure-Days (specific pesticide) = Yrs Application (specific pesticide) * Days of application/Yr (specific pesticide) Intensity-Weighted Lifetime Exposure-Days (specific pesticide) = Life time Exposure Days (to specific pesticide) * Intensity of exposure Lifetime Exposure-Days (all pesticides) = Yrs Application * Days of application/Yr High Pesticide Exposure Events = acute events involving unusually high exposures because of spills, immersions, etc.

Number of Pesticides Per Applicator, AHS 1999 - 2003 Number of Applicators Number of Pesticides Reported Used

Source of Vital Status for Cohort through December 31, 2007 (reported February 17, 2010)

Cancer Incidence Linkage through 2006 Malignant Cancer Incidence after Enrollment by Type of Applicator and State

Commercial Applicators Update of Cancer Standardized Incidence Ratios (SIR) through 2006, North Carolina and Iowa Combined (Koutros S et al. An update of cancer incidence in the Agricultural Health Study. Journal of Occupational and Environmental Medicine (in press) ) Cancer Private Applicators Commercial Applicators Spouses SIR (N) 95% CI All sites 0.85 (4316) 0.83-0.88 0.93 (219) 0.81-1.07 0.82 (1896) 0.79-0.86 Lung 0.48 (436) 0.43-0.53 0.75 (26) 0.49-1.09 0.42 (133) 0.35-0.50 Colon 0.87 (339) 0.78-0.97 0.98 (17) 0.57-1.57 0.83 (144) 0.70-0.98 Prostate 1.19 (1719) 1.14-1.25 1.28 (73) 1.00-1.61 1.05 (7) 0.42-2.15 Female breast 0.95 (33) 0.65-1.33 1.00 (770) 0.93-1.08 Multiple myeloma 1.20 (71) 0.93-1.51 1 0.94 (21) 0.58-1.44

Number of Deaths by Cause through 2007 Cause of Death Appl Spouse Total Cancer 1,624 665 2,289 Heart Disease 1,376 292 1,668 Injuries 496 66 562 Other circulatory 376 147 523 Respiratory 346 92 438 Other 662 277 939 All 4,880 1,539 6,419 % of total 8.5 4.8 7.2

Update of Cancer Standardized Mortality Ratios (SMR) through 2007, North Carolina and Iowa Combined (Waggoner JK et al. Mortality in the Agricultural Health Study: 1994-2007. American Journal of Epidemiology (in press)) Cause of Death Applicators Spouses SMR (N) 95% CI Transportation Motor vehicle - nontraffic 2.80 (25) 1.81-4.14 (0) Other injury -machine 4.15 (62) 3.18-5.31 (3) Intentional self-harm 0.57 (106) 0.46-0.69 0.32 (9) 0.15-0.61 All cancers 0.61 (1624) 0.58-0.64 0.65 (665) 0.60-0.70 Heart diseases 0.54 (1376) 0.51-0.56 0.47 (292) 0.42-0.53 Respiratory 0.38 (346) 0.34-0.43 0.30 (92) 0.24-0.37

Publications (n=137) as of September 2010 at www.aghealth.org Methods (n=11) Exposure Assessment (n=32) General (n=21) High Pesticide Exposure Events (n=6) Environmental Measures (n=5) Health Outcomes (n=87) Mortality (n=5) Cancer (n=41) Neurological (n=9) Respiratory (n=11) Reproductive (n=9) Other (n=12) Diet (n=1) Injury (n=6) Iowa Field Station 69 IRB-approved new projects into 18th year of AHS

Cancer Causal Inference in AHS: What are we looking for? Biological Initial Replication Evidence in Findings later in time Humans Iowa North Carolina License Type Dose- Response Dose- Response YES

Pesticides Evaluated for Human Carcinogenicity 18 commonly used pesticides associated with one or more of 8 cancer types Bladder, colon, leukemia, lung, skin melanoma, multiple myeloma, prostate, rectum 9 other commonly used pesticides possibly associated with one or more of 5 cancer types Colon, leukemia, multiple myeloma, NHL, rectum Prostate and multiple myeloma are furthest along for biologic evidence in humans

Prostate Cancer Risk by Exposure Status with and without a First Degree Family History (follow-up studies scheduled) Pesticide PC risk, no family history of PC PC risk, family history of PC Statistical interaction, PC history & Pesticide Exposure Odds Ratio 95% C.I. Fonofos 0.92 0.71-1.19 1.80 1.14-2.84 2.04 1.21-3.44 Coumaphos 0.86 0.57-1.28 2.17 1.24-3.82 2.58 1.29-5.28 Alavanja MCR, et al. American Journal of Epidemiology. 2003: 157 (9): 800-814. Similar results found for permethrin, phorate, chlorpyrifos.

Biologic Evidence Prostate Cancer Nested Case-Control Study (Gene x Environment Interaction) 8q24 Hormonal Pathway DNA repair Xenobiotic metabolism Lipid metabolism Telomere length DNA methylation Others

Observation from AHS (follow-up evaluations scheduled) Multiple Myeloma in excess in AHS SIR=1.34 (0.97-1.81) - Alavanja MCR, et al. Scand J Work Environ Health 2005:31 suppl 1:39-45); SIR= 1.20 (0.93-1.51) - most recent incidence data through 2006 Monoclonal gammopathy of undetermined significance (MGUS) – 2-fold significant excess in AHS (Landgren O. et al., Blood (2009), 113(25);6386-6391). Several widely used pesticides are associated with elevated MGUS levels (Landgren O. et al., Blood (2009), 113(25);6386-6391). Odds ratio for multiple myeloma = 5.7 (2.1-7.2) for heaviest users of permethrin (Rusiecki J, et al., Environ. Health Perspectives (2009), 117;581-586).

Biologic Evidence AHS Biomarker Study Markers of Potential Interest Telomere length DNA methylation MGUS Monoclonal B-cell lymphocytosis (MBL) Mitochondrial DNA copy number Immune suppression among pesticides associated lymphomas and leukemias Cytokines including interleukin IL10 and tumor necrosis factor (TNF) Chromosome aberrations and pesticides associated with lymphomas and leukemias Urinary metabolite pesticide concentrations

Epidemiology Research: Potential Contributions Characteristics No extrapolation of results from lab animal to human No extrapolation from high exposures in animal testing to lower exposure in human experience Prospective study to minimize information bias Control for confounders to obtain valid risk estimates Comprehensive exposure assessment to minimize misclassification Generate biological results to assess biological plausibility and modes of action and identify susceptible sub-populations Results generalizable for the chemical evaluated in adults (within the range of exposures of the study)

Translational Research Contrary to widely held misperceptions, AHS research has demonstrated following attributes: Pesticide applicators have accurate knowledge of pesticides they apply Questionnaire-based exposure assessments significantly correlated with field measurements of pesticides in urine and on skin Although each applicator uses a number of different pesticides, the use of these pesticides is generally not highly correlated and rarely cause confounding (an alternative explanation) Because of these attributes, US EPA recently announce in Federal Register that epidemiological data from the AHS will be used in its risk assessment of pesticides This marks significant departure from EPA practice, which has focused on short-term bioassays and animal testing in risk assessment

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