Comment submitted by Scott Phillips, MD, University of Colorado Health Science Center

Scott D. Phillips, MD, FACP, FACMT, FAACT Department of Medicine, Division of Clinical Pharmacology & Toxicology University of Colorado Health Science Center Rocky Mountain Poison & Drug Center 777 Bannock St, Mail Code# 0180 Denver CO 80204 FIFRA Scientific Advisory Panel Office of Pesticide Programs (OPP) Regulatory Public Docket (7502P), Environmental Protection Agency, 1200 Pennsylvania Ave., NW, Washington, DC 20460–0001 Docket Identification Number: EPA–HQ–OPP–2008–0274 Re: Evaluation of the Toxicity Profile of Chlorpyrifos in Conjunction with Effects Reported in Rauh et al (2006) Dear SAP Panel on Chlorpyrifos: As a physician toxicologist, I have evaluated many patients during my career. I use the traditional medical approach in caring for patients. I meet with them, take a history of their concerns and complaints, and ask probing questions to tease out certain aspects of their case. Not only do I examine the main complaint of the person, but also gather information that gives me acceptable constructs of the overall health and health risks of each case. We then perform a physical examination and any indicated laboratory tests within an iterative process, to further refine this diagnosis and consider potential treatment that may be indicated. In this process, a consideration of picogram concentrations of substances is not on my clinical radar. In fact, I am not aware that picogram concentrations of any substance have been demonstrated to cause developmental abnormalities. When evaluating a subject, I am most concerned with the clinical findings of the case and relevant health risks, such as; tobacco smoke, alcohol and other abused substances, seat belts usage, child abuse/neglect, potable water, clean air, helmet use, cholesterol and blood pressure measures. That is, health care, not health speculation. To make such firm statements about such a vanishingly small quantity of a substance (at the exclusion of tens of thousands of others), as is the case for chlorpyrifos as reported in Rauh et al (2006) and a defined medical endpoint, defies clinical common sense. It is easy to draw wrong conclusions from epidemiological studies involving low-level exposure, because most such studies have some imperfections. Associations potentially identified in epidemiological studies are not causal because they may be due to unidentified or uncontrolled confounders, to bias or to chance. Let us now examine the case of chlorpyrifos (CPF) in the Rauh paper in more detail. Chlorpyrifos (CPF) is generally well absorbed through the gastrointestinal tract and the lungs. (Aprea 1994, Nolan 1984) Dermal absorption is significantly less than other routes because it presents a barrier to penetration. (Nolan 1984) Chlorpyrifos is an inhibitor of the acetylcholinesterase (AChE) enzyme. Inhibition of AChE is believed to be the most sensitive response in all species evaluated, as well as in humans, regardless of exposure (EPA 1999, Clegg 1999). Long-term feeding studies have shown no effects at doses less than 1 mg/kg/d for studied endpoints in mammals and birds (Barron 1995). Clinically, CPF-induced toxicity results almost entirely from inhibition of AChE by the parent substance and its bioactivation product, the chlorpyrifos oxon (Namba et al. 1971) Extrapolating from laboratory animals to humans may be done in the case of chlorpyrifos because the mechanism of action of the pesticide is the same in all species examined, and the metabolism and excretion of chlorpyrifos are similar, if not identical, in humans and common laboratory animals. Nevertheless, animal experiments have not conclusively demonstrated a developmental effect or toxicity related to CPF. In animal studies, the LOAEL for acetylcholinesterase (AChE) inhibition has been reported to be 0.5-mg/kg/day dosing. The internal dose that has resulted in measurable brain AChE inhibition is > 1 mg/kg/day. (Hoberman 1998, Maurissen 2000) These levels are dramatically greater than those reported by Rauh to cause neurotoxic effects. Why is it that definitive guideline-compliant animal studies are disparate with Rauh? I have read with great interest the paper by Rauh, et al (2006). In this study the authors evaluated an ongoing cohort for developmental toxicity (DNT) based on CPF exposure. The authors describe their exposure groups based on umbilical cord plasma levels. It is important to recognize that these groups are actually dose groups, not exposure groups. The later term “exposure groups” is used as a general term in epidemiological analysis, but is in fact, very different from the internal dose that was assayed in this study. In this paper the authors did not correlate environmental sampling (exposure) with biological samples (dose). The authors divide the exposure groups (dose groups) into high and low groups as discussed below. These groups are then compared by several common statistical methods used in observational studies. While I believe that their statistical and epidemiological methods are sound, there are marked limitations to the results of this study based on the exposure and dose characterizations. Furthermore, it should be remembered the observational studies seek to determine association, not causation. Rauh, et al (2006) stratified the exposure groups (internal dose groups) into 4 categories: undetectable, and 3 tertiles of detection. Because of differences in the developmental scores in the “high exposure” group versus the other groups, the authors dichotomized the “exposure variable” (dose groups) as above and below 6.17 picograms of CPF per gram of plasma (pg/g). The within-subject percent coefficient of variation (%CV) is a measure of reproducibility. It determines the degree of closeness of repeated measurements taken on the same subject either by the same instruments or on different occasions under the same conditions. It is clear that, the smaller the %CV, the better the reproducibility. Barr (2002) reported %CV of 20% for CPF. Rauh et al did not report this in their study. Thus the accuracy of dividing the subjects by their exposure groups with such precision (6 x 10-12 grams) lends itself to bias by potentially assigning them to the wrong group (misclassification, in either direction) based on the inherent variability of a laboratory test. At picogram/gram levels, a cutoff level of 6.17 is so precise and so close to the limits of detection of the assay that it creates the possibility of misclassification. The Rauh (2006) cohort has 50 patients in the high exposure group versus 204 in the low exposure group. If the error of the test is 20 %CV from the mean, and the accuracy is 0.05 (95% confidence interval) and the SD is 0.02 pg/g then there is a possibility that some cases were misclassified which may alter the results. As clinical toxicologists, we struggle with the interpretation of AChE levels in acute and chronic poisoning cases. The same is true for vanishingly small levels of any toxicant. What does it mean? How does it relate to the signs and symptoms that we are observing? These questions cannot be answered by one manuscript. The suggestions by Rauh (2006), that CPF cord levels at a trillionth of a gram (6.17 x 10-12) are related to an adverse outcome in medicine, is not supported by the weight of the scientific evidence. The Rauh paper suggests an association, but it is not clinically recognizable by physicians, which questions its relevance. In fact, this study suggests that a level of 6.18 pg/g is associated with developmental effects, but a level of 6.16 pg/g is not. Levels that approach the extremes of measure are difficult to interpret in their clinical context ((clinically evident (what a clinician can detect in an examination of a patient) disease)) and are beyond clinical recognition by physicians. It is important to realize that the ability to accurately measure a substance near its level of detection (LOD) does not imply that it, de novo, has any adverse effects. Based on these levels, the known mechanism of action for chlorpyrifos, and confounding socioeconomic factors within this cohort, I don’t think the reported findings have toxicological or clinical relevance. The fact that they measured CPF at single picogram quantities in the plasma doesn’t mean a causal nexus exists with a DNT issue, even if there were a statistical correlation. Perhaps a more meaningful test would have been to compare all levels to the developmental scores as a continuous variable to determine a potential dose response relationship. The authors have also reported adverse effects on development with PAH’s, ETS, postpartum maternal hardship, diazinon and propoxur metabolites, yet they do not test those exposures in this study (Choi 2006, Perera 2007, Choi 2008, Perera 2005, Perera 2006). A prior paper (Whyatt et al 2003) also found diazinon; propoxur and o-phenylphenol in breathe zone air samples in this cohort. Thus, since Rauh has not controlled for substances that they have previously reported to be related to DNT, they have introduced confounding into this manuscript. It is impossible for observational epidemiological studies to understand or measure every exposure that may confound the results. Rauh et al (2006) did not adequately control this study for confounders, based on their own prior research involving different chemicals. In this case the authors are basing their results only on CPF. The Rauh paper also does not provide an indication of maternal medications consumed during pregnancy nor lactation, factors that may also confound. It is commonly known that patients do not provide accurate substance abuse histories. This bias is not malicious, but rather to maintain the appearance of being a good parent. Inferring a causal nexus with CPF in the face of unmeasured or controlled confounders just does not make clinical sense. Toxicologically, it is the equivalent of pulling one straw out of a pile of a 20 million and saying that it is the common denominator holding the haystack together. There are many medications that may have been taken by the cohort in the Rauh study that are inhibitors of AChE. These are used as medicines to treat a variety of health conditions. These conditions include myasthenia gravis, glaucoma, bladder spasm, bowel hypomotility, Alzheimer’s, and parkinsonism syndromes. Failure to consider and control for these medications or conditions, introduces bias, which may affect the study results in a positive or negative way. If effects reported by Rauh are presumably due to inhibition of AChE, then multiple factors, not just CPF, need to be considered. A reported symptom (subjective report) alone cannot be used to determine dose, exposure concentration or a possible source of an exposure. Causation analysis must be used to determine if an illness is the direct result of some substance based on a weight of the evidence approach. Furthermore, causation should be inferred from multiple studies, by different investigators at different times, and in different places. The Hill criteria detailed below are used to make an assessment of the weight of the evidence approach to causation. This methodological approach has been adopted by several agencies and bodies, including, but not limited to: the World Health Organization (2006), United States Environmental Protection Agency (2005), Agency for Toxic Substances and Disease Registry (ATSDR) 2001, and National Academy of Sciences (1999). In order to provide an appropriate framework for the present discussion, each of the Hill Criteria is discussed. These are not to be taken individually, but rather it is their collective strength that supports causation. The more criterions that are satisfied, the greater the weight of evidence in causation analysis. This provides scientific strength to the causal nexus. 1. Strength: The magnitude of the alleged association when measured using appropriate statistical tests. 2. Consistency: Different persons in different places, circumstances and times, must also observe the observed association. 3. Specificity: A single cause produces a specific, defined effect. 4. Temporality: Exposure must occur prior to development of the outcome. 5. Biological Gradient: The “dose-response” relationship. 6. Plausibility: The association is consistent with current accepted understanding of pathological mechanisms. 7. Coherence: The association is compatible with existing theory and knowledge. 8. Experiment: Introducing an appropriate experimental regimen can alter the condition. 9. Analogy: Consideration for all other possible alternative explanations. The paper by Rauh is not consistent with existing knowledge of chlorpyrifos from animal toxicology studies. Current evidence indicates that chlorpyrifos does not adversely affect reproduction or development. No effects were seen in animals tested at dose levels up to 5 mg/kg/day (Breslin 1986), despite maternal effects noted above 3 mg/kg/d. No effects on reproduction occurred in a three-generation study with rats fed dietary doses as high as 1 mg/kg/day (EPA 1989, Thompson 1971). In another study in which rats were fed 1.0 mg/kg/day for two generations, the only effect observed was a slight increase in the number of deaths of newborn offspring (Gallo 1991). Despite the positive statistical correlates in the Rauh study, their reported results are over-stated based on mechanistic toxicology of CPF, animal studies, and the clinical perspective. The dose levels in the Rauh paper are far below those studies in animals and which have been without effects on neurodevelopment. The Columbia cohort findings have simply not been demonstrated in any of the animal toxicology studies for chlorpyrifos. The Rauh study findings are not supported using a weight of the evidence approach to determine a causal nexus, and the reported results need to be considered with caution. In summary, I am concerned that important decisions could be considered based on a study (Rauh) that is flawed from a toxicological perspective. I urge the Panel to give careful and considerate thought to the overstated results of this paper that are in contrast to the known body of literature on DNT and CPF. Sincerely, Scott D. Phillips, MD, FACP, FACMT, FAACT Associate Clinical Professor Of Medicine University of Colorado Health Sciences Center Rocky Mountain Poison and Drug Center Denver, CO References Aprea C, Sciarra G, Sartolli P, et al. Biological monitoring of exposure to organophosphorus insecticides by assay of urinary alkylphosphates: influence of protective measures during manual operations with treated plants. Int Arch Occup Environ Health 1994; 66:333-338. Barr DB, Barr JR, Maggio VL, et al. A multi-analyte method for the quanti?cation of contemporary pesticides in human serum and plasma using high-resolution mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci. 2002;778:99 –111. Barron, M. G. and K. B. Woodburn (1995). "Ecotoxicology of chlorpyrifos." Rev Environ Contam Toxicol 144: 1-93. Breslin, W. 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