Effects of occupational exposure to chlorpyrifos on neuropsychological function: A prospective longitudinal study

NeuroToxicology 41 (2014) 44–53

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NeuroToxicology

Effects of occupational exposure to chlorpyrifos on neuropsychological function: A prospective longitudinal study Stanley Berent a,b,c,*, Bruno Giordani a,b, James W. Albers b,c, David H. Garabrant c, Sarah S. Cohen d, Richard P. Garrison c, Rudy J. Richardson b,c a

Department of Psychiatry, University of Michigan, USA Department of Neurology, University of Michigan, USA Department of Environmental Health Sciences, University of Michigan, USA d EpidStat Institute, Ann Arbor, MI, USA b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 July 2012 Accepted 27 December 2013 Available online 18 January 2014

Background: Exposure to chlorpyrifos (CPF), an organophosphorus (OP) anticholinesterase insecticide, occurs typically in settings where multiple agents are present (e.g., agriculture) and quantitative dose measures may be absent (e.g., pesticide application). Such exposures allow few opportunities to study potential neurobehavioral effects of CPF alone. We studied the relationship between CPF exposure and behavioral function among CPF manufacturing workers, which allowed identification, measurement, and estimation of exposure and important non-exposure variables that potentially could affect study findings. Methods: A prospective longitudinal study design was used to compare neurobehavioral function over a one-year period among 53 CPF workers and 60 referent workers. Quantitative and qualitative measures were used, and potential confounders were identified and tested for possible inclusion in our statistical models. Neurobehavioral function was assessed by neuropsychological tests covering various behavioral domains that may be adversely affected by exposure to CPF in sufficient amount. Results: CPF workers had significantly greater CPF exposures during the study period than did referents at levels where physiologic effects on plasma butyrylcholinesterase (BuChE) activity were apparent and with higher 3,5,6-trichloro-2-pyridinol (TCPy/Cr) urinary excretion (p < 0.0001) and lower average BuChE activity (p < 0.01). No evidence for impaired neurobehavioral domains by either group of workers was observed at baseline, on repeat examination, or between examinations. CPF workers scored higher than referent workers on the verbal memory domain score (p = 0.03) at baseline, but there were no significant changes in verbal memory over time and no significant group-by-time interactions. Conclusions: The study provides important information about CPF exposure in the workplace by not supporting our working hypothesis that CPF exposure associated with various aspects of the manufacturing process would be accompanied by adverse neurobehavioral effects detectable by quantitative neurobehavioral testing. Some aspects making this workplace site attractive for study and also present limitations for the generalization of results to other situations that might have exposures that vary widely between and within different facilities and locations. For example, these results might not apply to occupations such as applicators with higher exposure or to workers with low educational levels. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Chlorpyrifos Neuropsychology Neurotoxicity Occupational exposure Organophosphates

1. Introduction Adverse cognitive and other neurobehavioral findings have been observed in acute and chronic, high level pesticide exposures (Kamel et al., 2005; Savage et al., 1988; Starks et al., 2012). A

* Corresponding author at: Neuropsychology Section, Department of Psychiatry, University of Michigan Health System, 2101 Commonwealth, Suite C, Ann Arbor, MI 48105, USA. Tel.: +1 734 763 9259; fax: +1 734 936 9262. E-mail address: [email protected] (S. Berent). 0161-813X/$ – see front matter ß 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2013.12.010

variety of cognitive (e.g., memory, attention, concentration, and alertness), psychomotor, and emotional (e.g., depression, anxiety, and irritability) impairments have also been reported to result from 20 or more chronic, low level organophosphorus (OP) exposures studies (Ismail et al., 2012). Ismail and colleagues, for instance, identified 17 studies in their meta-analytic study of agricultural workers and pesticide applicators (Ismail et al., 2012), and they concluded ‘‘decrements in neurobehavioral performance’’ in the pesticide exposed group in comparison to the control group. Work in these areas, however, has proven to be challenging from a scientific perspective. Such challenges are likely due to a host of

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factors, many outside a given investigators control as is often acknowledged by the authors of a given study (Blain, 2001; Clegg and van Gemert, 1999; Kamel et al., 2005; Pilkington, 2001; Starks et al., 2012; Woods et al., 1999). The settings in which exposures to OP compounds are likely to occur, for instance, frequently challenge good experimental control of important variables, such as specific exposure details and other factors that are known to affect study outcomes and other aspects of methodology. These settings often involve exposure to multiple types of compounds aside from OP compounds. Determining levels of exposure is often difficult to accomplish, and potential confounders may be difficult to identify and control. Studies that target a specific compound, e.g., chlorpyrifos (CPF), must attempt to identify and control factors with the potential for causal contribution to results, including the presence of alternative exposures. At the same time, knowing that a particular setting may be associated with measurable impairments is important from a public health viewpoint even when causality has not yet been specified completely (Farahat et al., 2003; Hogstedt et al., 1984; Rosenstock et al., 1991; Rothlein et al., 2006; Starks et al., 2012; Stephens et al., 1996). Recognizing the importance of arriving at scientifically objective answers to questions surrounding the potential toxic effects of chronic, low-level OP exposures, a committee was convened by the United Kingdom Department of Health (Woods et al., 1999). The committee reviewed existing evidence that associated chronic, low-level exposures to OP insecticides with adverse effects on neurological or neuropsychological performance. They determined that the evidence did not scientifically support a causal association, but recommended further investigation to establish whether the risk of neurological dysfunction is increased by low-level OP exposures. Other reports have made similar recommendations (Albers et al., 1999; Clegg and van Gemert, 1999). In response to these recommendations, we sought to address the question of adverse effects of chronic, low-level OP exposure on neurobehavioral function by studying individuals involved in the manufacture of these compounds. CPF manufacturing workers are a suitable population to study for a number of reasons. The most important reasons include their potential for chronic exposure, the opportunity to measure exposures reliably, their availability in a single geographic location in which standardized medical and neurobehavioral testing can be performed, the ability to control potential confounders that might influence the test performance and the availability of these workers for repeat study over time. Our working hypothesis was that workers with chronic occupational exposure to CPF develop dose-related subclinical or clinically evident adverse neurobehavioral effects demonstrable by quantitative neurobehavioral testing. 2. Methods 2.1. Study design The prospective longitudinal study design employed here has been described previously (Albers et al., 2004a, 2004b). Briefly, we evaluated two groups of workers at the Dow Chemical Company in Midland, Michigan, on two occasions, baseline and one year later. As previously reported (Albers et al., 2010), the CPF workers averaged almost a decade of exposure to chlorpyrifos, and there was no evident movement of workers from chlorpyrifos-related jobs. The referent group included workers involved in the manufacturing of Saran (a clear plastic film wrapping material) who had no current or recent occupational exposure to chlorpyrifos and no exposure between the baseline and one year evaluations. It is not possible to say that these Saran workers never worked in the CPF facility. A few may have worked there in the distant past. More importantly, we measured, directly, their

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cumulative lifetime exposure to CPF and found it to be small (Albers et al., 2004a, 2004b; Burns et al., 2006; Garabrant et al., 2009). Most importantly, none of the referents had any interim CPF exposure. No workers in either group had been exposed to known or suspected neurotoxicants other than CPF. Subjects from both groups were examined concurrently. For CPF subjects, the baseline examinations took place during a period of potential CPF exposure, followed by a second examination after approximately one year of additional exposure. Subjects from both groups were examined on any given day, and investigators were blinded as to individual group membership. The technical personnel who administered the quantitative neurobehavioral examinations also were blinded with regard to subjects’ exposure histories. Power calculations for neurobehavioral test measurements were based on 2-tailed t-tests for differences in mean responses between two groups, alpha error = 0.05, power = 0.8, based on between subject variability in change over time for domains of interest. Analyses demonstrated that we would have 80% power to detect differences in mean responses between exposed and nonexposed subjects for the major domains in the test battery. Based on an anticipated sample size of 100 subjects divided evenly between the CPF exposed and referent groups, we would have greater than 80% power to detect a mean difference of 2–3 points in general ability, 1 point in visual and verbal memory (recall test), and 2–3 points in motor ability (finger tapping), with greater power to detect larger differences. 2.2. Subject selection Methods of selecting subjects have been described previously (Albers et al., 2004a, 2004b). Briefly, eligibility for inclusion in the CPF exposed group included all workers employed in any of the Dow Chemical Company’s Midland, Michigan, buildings involved in CPF production before the study began. All Saran manufacturing workers at the Midland site also were invited to participate in the referent group. Additional inclusion criteria for all participants included being between 18 and 65 years of age and able to read, comprehend, and give their informed consent. Volunteers were excluded from participating if their histories indicated any of the following: head injury accompanied by loss of consciousness lasting more than 20 minutes, any physical or mental condition that left them unable to complete the intended protocol, or disability application or litigation against Dow Chemical Company. The CPF workers and referents worked in separate buildings that were over 1000 ft apart. They had no common ventilation and there was no opportunity for workers to mingle and/or enter each other’s work areas. The Saran workers produced Saran Wrap for food service use. There was no CPF contamination in that area. Urinary excretion of 3,5,6-trichloropyridinol (TCPy) was monitored in both study groups. The urine TCPy levels in the referents were typical of background levels in the US adult population (Hill et al., 1995). Subject selection proceeded with a group meeting in which the study was explained to all eligible participants, with opportunity to ask questions regarding the protocol and conduct of the study. Following this, participants also met individually with one member of the research team who responded to any further questions the individual might have. At that time, the participant had the opportunity to ask further questions and then read and sign the consent document if they desired to proceed as a study participant. The study protocol and informed consent document were approved by the University of Michigan Institutional Review Board for Human Subject Research and the Dow Chemical Company Human Subject Review Board. Fifty-three of the 66 eligible CPF workers (80% of the eligible population) participated in the study. Seventy-four Saran workers

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were chosen at random and asked to participate. Of these, 60 (81% of the randomly chosen population) agreed to participate. No worker in either group was excluded from participating in the study on the basis of our exclusion criteria. The second examinations were performed approximately one year after the baseline examinations and were completed on 111 of the initial 113 subjects (98% participation). The two subjects who did not participate in the second examinations were from the referent group. All participants were tested during paid work time and were paid a total of $300 in addition for submission of all 4 requested urine samples. 2.3. Exposure assessment The exposure assessment protocol has been described previously (Albers et al., 2004a, 2004b; Burns et al., 2006; Garabrant et al., 2009). In brief, we reviewed industrial hygiene records, including air level estimates that were based on personal and historical air sampling, to estimate historic cumulative CPF exposure for specific job titles. These estimates were established for the period extending from the time of initial employment to the baseline examination and for the interim period that extended from the baseline examination to after approximately one year of additional cumulative CPF exposure. CPF exposure also was assessed biologically during the year between the baseline and second examinations by urinary excretion of TCPy, a metabolite of CPF, plasma butyrylcholinesterase (BuChE) activity, and red blood cell (RBC) acetylcholinesterase activity (acetylcholinesterase [AChE]). The urine TCPy level was reported as a weighted average of four overnight collections of TCPy in mg divided by creatinine (Cr) in grams (TCPy/Cr). Weights were chosen to reflect the number of months of exposure each measurement represented. Specifics regarding weighting definitions and other aspects of our exposure assessment have been reported previously (Albers et al., 2010; Burns et al., 2006; Garabrant et al., 2009). Monthly plasma BuChE activities (m/ml) were assessed and averaged during the same interval. RBC AChE activity was measured at baseline and at the second examinations. 2.4. Questionnaires Questionnaires were administered by a trained interviewer in a standardized, semi-structured interview format at the baseline and second evaluations. Details of these questionnaires have been described previously (Albers et al., 2004a). Briefly, the baseline questionnaire included questions about demographics, medical histories, family history of neurological diseases, social history, and occupational and environmental histories that included exposure histories. Questions were developed to elicit overt symptoms relevant to cognition (e.g., memory complaints) and other potential neurological or neurobehavioral dysfunction. All positive responses resulted in additional questions about the reported symptoms. The questionnaire also contained five-level Likert scale questions (strongly agree, agree, neutral, disagree, strongly disagree) about memory, reading skills, ability to concentrate, and problem solving skills at the present time compared to 10 years previously. The second questionnaire updated information on events since the baseline interview a year earlier.

groups at baseline, and no subjects were diagnosed by a physician between baseline and one-year examinations with any new systemic illness that could potentially contribute to central nervous system (CNS) dysfunction (Albers et al., 2004a). Neurological examinations, including motor and reflex evaluations, were performed by a board-certified neurologist to identify clinically evident abnormalities and to define clinically evident impairments suggestive of encephalopathy (Albers et al., 2004a). Every subject was evaluated using a semi-structured clinical interview by a licensed neuropsychologist to clarify neurobehavioral (cognitive and emotional) symptoms elicited in the questionnaire described earlier. A number of variables can interact with and affect performance on neuropsychological measures. To ensure that subjects would be able to participate in neurobehavioral testing, we included potential exclusionary measurements in our protocol. These included, for example, an evaluation of color vision, using the Dvorine Pseudo-Isochromatic Plate Test (Dvorine, 1953), a mental status measure (Folstein et al., 1975) and the Word Memory Test (WMT; Conder et al., 1992) a computerized measure designed to detect feigning and malingering of cognitive deficits. No subject was excluded on the basis of the results from any of these additional measures. Alcohol use, or potential abuse, was explored individually with each subject, by questionnaire self-report during the neuropsychological clinical evaluation as well as during medical and neurological evaluations. Workers were asked to estimate amount of alcohol used (e.g., number of alcoholic drinks consumed per day; a drink defined as one bottle of beer, one four-ounce glass of wine, or 1.5 ounces of grain alcohol). Also, workers were asked for information pertaining to a history of adverse alcohol-related behaviors (e.g., ever being told they had a problem with alcohol or ever being arrested for driving under the influence of alcohol [DUI]). 2.6. Quantitative neurobehavioral (neuropsychological) testing A wide range of behaviors have been reported to be adversely affected by exposure to OP compounds, including attention, visual memory, visual-motor speed, problem solving, motor steadiness, and dexterity. Specific tests for this evaluation were chosen a priori and organized in seven functional areas (domains) encompassing these and related, important domains of neurobehavioral function (Berent and Trask, 2000). The majority of these tests were computer-based, e.g., CANTAB (Fray et al., 1996) and CogniSyst (Green and Allen, 1999). In choosing our tests, as stated earlier, we placed importance on neurobehavioral functions reported in various studies to be at risk for injury by exposure to CPF and even more importance on the tests we used to measure those functions. That is, we chose tests with validity, reliability, and other formal psychometric factors established previously in independent studies. As mentioned earlier, we chose to use technician administered computerized tests, predominantly. We felt that the computerized approach, which has been used and found to be effective in many studies of exposure to potential neurotoxicants (Anger, 2003), reflected established psychometric factors and would also reduce bias and provide a well standardized approach to our measurements, providing measurements that would be trusted scientifically and instructive to others working in this area. The tests used are listed in Table 1.

2.5. Clinical evaluations 2.7. Statistical analyses A general medical examination was performed by an occupational medicine specialist who also was board-certified in internal medicine to detect medical conditions that potentially produce nervous system abnormalities. The overall frequency of medical problems did not differ significantly between the CPF and referent

Data to be analyzed were entered into a computer database, and data were double-entered or hand checked against the original records for accuracy. All analyses were performed in SAS, version 9.3 (SAS Institute, Cary, NC). Analyses included comparison of

S. Berent et al. / NeuroToxicology 41 (2014) 44–53 Table 1 Neuropsychological summary domains and the primary test variables underlying each. Domain 1: general ability Wide Range Achievement Test-Third Edition (WRAT-3) (Reading Subtest) (Jastak and Wilkinson, 1993) Domain 2: attention/information processing Reaction time (RTI) (Fray et al., 1996), including simple movement, simple reaction, choice movement, and choice reaction times Rapid Visual Information Processing (RVIP) Signal Detection A Prime Score (Fray et al., 1996) Matching to Sample Visual Search Change Score in milliseconds (MTS) (Fray et al., 1996) Domain 3: memory-visual Delayed Matching to Sample (DMS) Percent Correct (Fray et al., 1996) Domain 4: memory-verbal CogniSyst Story Recall Test (CSRT) (Green and Allen, 1999), including immediate and delayed recall scores Domain 5: problem solving Stockings of Cambridge (SOC) Moves (Fray et al., 1996) Domain 6: psychomotor Motor Performance Series (MPS) (Matthews and Klove, 1964), including aiming, line error, long pin time, short pin time, tapping, and motor steadiness Domain 7: personality/mood Brief Symptom Inventory (BSI) (Derogatis and Spencer, 1982), including depression and global index

clinical examination results relevant to the neuropsychological evaluation for the CPF group and the referent group at baseline and second examinations (t-test for difference in means, Fisher exact test or Chi square test for associations). A two-tailed p-value of 0.05 was considered significant. We chose not to adjust for multiple comparisons because the standard approaches to such adjustments (e.g., Bonferroni methods) yield confidence intervals that are overly conservative, and it is far more important to evaluate the meaning of such associations and whether they make sense in the context of the other findings in the paper and in the broader literature (Savitz and Olshan, 1995). The individual, quantitative neurobehavioral test variables described in Table 1 were assessed for normality, and those that were appreciably skewed were transformed using log or inverse transformations before modeling. The primary scores for these variables were used to create the seven summary domains, which were used for the principal study analyses. For each of the seven domains, a summary domain score was created by the following methods. The scores for each primary test variable were transformed, if necessary, so that higher values indicated better performance. A T-score was calculated for each subject as the difference between the published mean score for each test minus the individual’s score divided by the published standard deviation (CeNeS Cognition, 1998) The T-score for all variables in each domain were then averaged to create a summary domain score for each subject. This procedure was done for the 1999 test results and again for the 2000 test results. Test variables were modeled using a mixed linear model with repeated measures, using the ‘‘Mixed’’ procedure in SAS. The neurobehavioral test variables were measured on two occasions (in year 1 and year 2) for each subject. The variable ‘‘group’’ (CPF or referent) was the between-subjects main effect, while the variable ‘‘time’’ (year 1 or year 2) was the within-subjects main effect. We were primarily interested in the main effect for group and the interaction term between group and time in order to determine whether CPF exposure was associated with differences between the two participant groups or changes in neurobehavioral test results over time. Using domains to aggregate data across tests allows the reader to evaluate whether groups of tests might show an association with exposure that was not clear in any individual test. In cases where the summary domain score was statistically

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significant (p < 0.05), the primary test variables constituting that domain score were considered formally. Each of the seven neurobehavioral domain scores was first modeled as a function of group (CPF or referent), time (baseline or one-year examination), group-by-time interaction, and covariates. The covariates used in all models were age, sex, education, sleepiness (Stanford Sleepiness Scale; Reeves et al., 1995) a measure of fatigue at the time of the evaluation), North American Adult Reading Test (NART-R; Blair and Spreen, 1989) estimate of baseline general ability, Personal Reaction Inventory (PRI; Crowne and Marlow, 1960) measure of motivation to perform, history of alcohol use (drinks per day), and job, family, financial, health, and social stresses. If the parameter estimate for the group-by-time effect in an adjusted model (with group, time, group-by-time, and all potential confounders) differed by less than one standard error from the parameter estimate for the simple model without covariates, we concluded that no appreciable amount of confounding was present and the simple model was appropriate to use. The simple model contained only group, time, and group-bytime. We also assessed three-way interaction terms (group-bytime-by-specified covariate) involving each covariate for every outcome variable, keeping the main effect of the covariate, group, time, and group-by-time in the model. If the p-value for the threeway interaction was <0.05, the model containing the three-way interaction was preferred over the simple model. Otherwise, the simple model was selected as the best choice for modeling the neurobehavioral outcome variables. Regression analyses for exposure main effects in models of domain scores for neurobehavioral test variables as a function of Historic Cumulative Exposure (measured from beginning of employment at the company) and Interim Cumulative Exposure (measured between the baseline and the second examinations) also were carried out for CPF workers. These models were adjusted for the confounders listed above. 3. Results 3.1. Demographic and other subject-related details As seen in Table 2, CPF subjects were comparable to referents at study baseline in terms of age, sex, body mass index (BMI), and anxiety level. The subjects were early middle age, mostly male, and mostly Caucasian. The two groups were comparable at baseline in terms of general aptitude, as measured by the NART-R index, and in terms of general mental status, as determined by clinical interview and formal testing. The overall frequency of medical problems as reported on questionnaires did not differ significantly between the CPF and referent groups at baseline. Between the baseline and second examinations, no subjects were diagnosed by a physician with a new systemic illness that could potentially contribute to CNS dysfunction. CPF workers were significantly more likely to be married. Near, but non-significant differences, were evident for education and history of self-reported arrest for DUI. Other measures of alcohol use were assessed. Both groups of participants reported an average of approximately one alcoholic drink per day, and there were no differences across the groups in terms of whether they reported having been told they had a drinking problem or whether they were identified in a medical examination as having alcoholism. 3.2. Exposure assessment Duration of work in CPF exposed areas was found to be significantly longer for CPF subjects than for referent subjects (9.72 [SD: 6.12] vs. 0.10 years [SD: 0.37]; p < 0.0001). Also, the two groups of workers differed significantly in measures of historic

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Table 2 Subject demographics and other characteristics by group, chlorpyrifos or referent, at baseline.a Variable

Chlorpyrifos mean (SD)

Referent group mean (SD)

Probability (p-value)b

Age (years) Male Caucasian Education (years) Married General ability(NART) Abnormal mental status index, (MMSE score < 24, number of subjects) Mental status symptoms from clinical examination (number of subjects complaining)

41.2 (7.5) 77.4% 92% 14.3 (2.1) 90.4% 106 (9) 0 4

41.3 (8.4) 73.3% 88% 13.6 (1.5) 67.8% 104 (9) 0 6

0.92 0.62 0.88 0.06 0.01 0.21 NA 0.75

BSI (T-score) Anxiety Depression

47.9 (11.80) 43.40 (14.18)

47.75 (13.80) 43.0 (14.71)

0.93 (NS) 0.88 (NS)

Number of alcoholic drinks/day Ever told have an alcohol problem? History of DUI

0.80 (0.9) n=6 n=3

1.30 (1.4) n=8 n = 10

0.19 (NS) 0.99 (NS) 0.08 (NS)

(NS) (NS) (NS) (NS) (Sig.) (NS) (NS)

a Abbreviations used in Table 2: NART, National Adult Reading Test; MMSE, Mini-Mental Status Examination; DUI, driving under the influence; BSI, Brief Psychiatric Inventory; NS, not statistically significant; NA, not applicable. b p-Values determined by t-tests for difference in means, Fisher’s Exact test, or Chi Square test.

cumulative CPF exposure (64.16 [SD: 59.97] vs. 0.69 [SD: 2.51] mg/ m3 days; p < 0.0001) and interim CPF exposure (6.13 [SD: 3.70] vs. 0.00 [SD: 0.00] mg/m3 days; p < 0.0001), and none of the referent subjects had any identifiable exposure to CPF in their jobs during the study period. Biological assessments revealed significant differences in exposure between the CPF group and the referent group for urine TCPy/Cr (192.13 [SD: 311.78] vs. 6.19 [SD: 2.18] mg/g-Cr; p < 0.0001), average BuChE activity (7154.72 [SD: 1728.65] vs. 8183.32 [SD: 1630.90] m/ml; p < 0.01), and ratio of the lowest BuChE activity during the study year to pre-exposure or baseline activity (0.75 vs. 0.88; p = 0.0008), all consistent with elevated CPF exposure among the CPF workers. The daily excretion of TCPy at this study site suggested an estimated daily CPF dose of about 576– 627 mg/day, an amount approximately 60% (range 0–500%) of that received by a typical worker exposed during a working day to the American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value (TLV) of 0.1 mg/m3 in air (Albers et al., 2004a; American Conference of Governmental Industrial Hygienists, 2013). AChE levels were similar for both groups at the baseline examination (6923.19 [SD: 828.33] vs. 6966.77 [SD: 766.38] m/ml; p = 0.77) and the second examination (7148.55 [SD: 750.62] vs. 7252.74 [SD: 810.92]; p = 0.48), indicating no inhibition of AChE associated with work in the CPF exposed areas in comparison to working in the referent building. The CPF exposures in our study can be compared to levels reported by the National Health and Nutrition Examination Survey (NHANES) (Centers for Disease Control [CDC], 2005) of the United States population during the same historic period. Both NHANES and our study measured urinary excretion of TCPy, a principal metabolite of CPF but not other organophosphate compounds. In NHANES, the median urinary TCPy excretion was 1.9 mg/g (micrograms/gram creatinine) and the 95th percentile was 10.3 mg/g creatinine. In our study, the CPF workers had a median TCPy excretion of 38–59 mg/g creatinine during the four urine collection periods, and the 95th percentile ranged from 346 to 7408 mg/g creatinine (Garabrant et al., 2009). We found significant inhibition of serum cholinesterase at TCPy levels exceeding 110 mg/g creatinine. 3.3. Neuropsychological outcome variables The only significant group effect was for the Memory-Verbal domain, with the CPF group performing better than the referent group on this domain (p = 0.03; Table 3). Subjects performed

significantly better in 2000 than in 1999 (time effect) on four domains: Attention/Information Processing, Problem Solving, Psychomotor, and Personality/Mood (p < 0.01 for all four domains). None of the parameter estimates group-by-time interaction was significantly different from the zero value of baseline for any of the summary domain variables (both with and without covariates in the models), allowing the simple model to be applied containing only group, time, and group-by-time. Significant three-way interactions were identified in the summary domain scores for Memory-Verbal, (family stress), and Problem Solving (age). A near-significant finding was found for MemoryVerbal and education. Regression analyses of CPF workers’ neurobehavioral performance as a function of historic and interim CPF exposures produced only two significant or near significant p-values on the verbal memory domain (p = 0.07) and the general ability level domain (p = 0.03), both as a function of Interim Cumulative Exposure and both in the direction of improved performance. The remaining parameter estimates were non-significant for summary domain scores, with p-values ranging from 0.40 to 0.98 for Historic Cumulative Exposure and 0.51–0.93 on Interim Cumulative Exposure. Interim CPF exposure, however, showed significant relationships in the underlying primary scores for story delayed recall (p = 0.05) and WRAT-3 Reading (p = 0.04), both consistent with the positive verbal memory findings reported in Table 3. Analyses for the primary test variables underlying the summary domain scores are presented in Table 4. The results of these

Table 3 Longitudinal analysis. Parameter estimates with associated probability statements (p-values) for each of the seven summary domain scores modeled as a function of group, time, group-by-time interaction, and significant group-by-time 3-way interactions for all subjects (n = 113). Domain

Group (p-value)

Time (p-value)

Group-by-time (p-value)

General ability level Attention/information processing Memory-visual Memory-verbal Problem solving Psychomotor Personality/mood

1.77 (0.10) 0.33 (0.97)

0.89 (0.07) 3.68 (<0.01)

0.45 (0.53) 0.74 (0.52)

0.72 2.20 2.31 1.54 0.61

2.18 0.51 4.49 3.78 2.07

0.73 1.48 1.34 1.27 0.71

(0.81) (0.03) (0.18) (0.41) (0.88)

(0.10) (0.70) (<0.01) (<0.01) (0.03)

(0.74) (0.21) (0.43) (0.20) (0.64)

Notes: Domain scores presented in this table have been adjusted so that higher scores represent better performance. Data presented in this table are without inclusion of covariates, as the adjusted models did not vary from the simple models.

S. Berent et al. / NeuroToxicology 41 (2014) 44–53

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Table 4 Longitudinal analysis. Parameter estimates with associated probability statements (p-values) for neurobehavioral test variables ordered by respective domains for group, time, and group-by-time interactions, (n = 113).a Domain/test Domain 1: general ability WRAT-3 reading Domain 2: attention/information processing Simple movement time Simple reaction time Choice movement time Choice reaction time RVIP a prime MTS change time Domain 3: memory-visual DMS % correctb Domain 4: memory-verbal Story immediate recall Story delayed recall Domain 5: problem solving SOC moves Domain 6: psychomotor MPS aiming time MPS line error time MPS long pin time MPS short pin time MPS tapping MPS steadiness errors Domain 7: personality/mood BSI depression BSI global index

Group (p-value) 2.71 (0.09) 31.5 (0.61) 0.01 (0.89) 27.63 (0.32) 5.33 (0.75) 0.01 (0.40) 174.52 (0.92)

Time (p-value) 1.34 (0.07) 6.89 0.01 10.29 3.56 0.01 963.86

(0.01) (0.14) (0.01) (0.06) (<0.01) (<0.01)

Group-by-time (p-value) 0.68 (0.53) 44.98 (0.04) 0.02 (0.54) 23.07 (0.18) 5.92 (0.39) 0 (0.60) 397.89 (0.21)

29,637.77 (0.68)

53,055.43 (0.12)

30,869.03 (0.53)

3.25 (0.03) 3.25 (0.13)

0.33 (0.36) 1.11 (0.14)

2.21 (0.19) 0.50 (0.79)

0.49 (0.16)

0.93 (<0.01)

0.27 (0.46)

0.07 0.18 0.07 0.02 4.85 1.55

0.18 0.51 2.45 0.07 0.92 2.32

0.07 0.22 1.00 0.03 2.26 1.66

(0.44) (0.61) (0.54) (0.84) (0.41) (0.56)

0.40 (0.92) 0.61 (0.88)

(<0.01) (<0.01) (<0.01) (0.02) (0.90) (<0.01)

1.50 (0.14) 2.07 (0.03)

(0.09) (0.18) (0.32) (0.51) (0.51) (0.34)

1.23 (0.67) 0.71 (0.64)

Note: Data presented in this table are without inclusion of covariates, as the adjusted models did not vary from the simple models. a Abbreviations used in Table 4: WRAT-3, Wide Range Achievement Test, 3rd edition; RVIP, Rapid Visual Information Processing; MTS, Matching to Sample; DMS, Delayed Matching to Sample; SOC, Stockings of Cambridge; MPS, Motor Performance Series; BSI, Brief Psychiatric Inventory. b Indicates normalized score.

analyses (both with and without covariates) indicated that the significant difference between groups on the parameter estimate for the Memory-Verbal domain reflected better performance by the CPF group than the referent group on the initial learning portion of the Story Recall Test (p = 0.03). The significant main effects for time in the analyses of summary domain scores were largely reflected in a number of the primary variables. These variables, underlying the summary domains, showed improvements in performance over time. The findings are summarized in Table 4. Although not of clear relevance since the summary domain score was not significant, the only primary variable listed in Table 4 that showed a significant group-by-time interaction was the movement speed component of the reaction time task, a test in which the CPF group evidenced greater improvement in performance speed over time than did the referent group (p = 0.04). Mean scores and variances for individual test results are presented in Table 5. 4. Discussion Our earlier publications focused on the effects of CPF exposure on findings from neurological examination of CNS and peripheral nervous system function (Albers et al., 2004a, 2004b). In the present report, we focused on quantitative examination of neuropsychological function. Our working hypothesis that workers with chronic occupational exposure to CPF develop doserelated sub-clinical or clinically evident adverse neurobehavioral effects demonstrable by quantitative neuropsychological evaluation was not supported by our findings. We found no significant group-by-time effect on any domain score. In contrast, we found that the CPF group had significantly better performance on Memory-Verbal tests at baseline. Both groups improved over time in six of seven domains, with the results being significant on four domains (Attention/Information Processing, Problem Solving,

Psychomotor, and Personality/Mood). It is possible that this improvement reflected a practice effect over time. At baseline, the CPF group, as compared to the referent group, was found to have a significantly higher percentage of married individuals and a near-significant higher education level and lower self-reported DUI history than did referent Saran workers. Threeway interactions, while judged not formally explanatory to results in the present study, called attention to the possibility that age, education, and stress could moderate outcome and should be included in future studies on this topic. The findings from demographic group comparisons and three-way interactions could have been influenced by self-selection (e.g., health-related contributions to job placement) or some other factor, e.g., cognitive reserve (Bleecker et al., 2002) possibly influencing employment longevity. It is possible that subjects with low education levels and therefore less cognitive reserve could have a greater response to exposure, i.e., educational level could modify the relationship between exposure and response. Insofar as our subjects had relatively high educational backgrounds, we had limited ability to observe any such interaction. There are many potential differences across study sites, especially across international settings, where cultural influences may be unique to a given setting. The current study site represented a setting where workers came from relatively high socio-economic groups, higher than might be found elsewhere. The workers we studied were well educated and sophisticated about the settings in which they worked and lived, as well as the availability and use of personal protective equipment. The present study site is involved in chemical manufacturing. Other sites, such as those involved in application of the same compound we studied, may involve different personnel, equipment, and safety regulations. These or other factors could result in different exposures and safety more generally. Such differences potentially limit generalization of study results and must be considered before applying our findings to other settings. These results may not apply to

S. Berent et al. / NeuroToxicology 41 (2014) 44–53

50

Table 5 Descriptive statistics (means and standard deviations) for neurobehavioral test variables ordered by respective domains for chlorpyrifos and referent subjects at baseline, one year and change over 1 year from baseline.a Domain/test

Group

N

Baseline

N

Mean (SD) Domain 1: general ability WRAT-3 reading

CPF Referent Domain 2: attention/information processing Simple movement time CPF Referent Simple reaction time CPF Referent Choice movement time CPF Referent Choice reaction time CPF Referent RVIP a prime CPF Referent MTS change time CPF Referent Domain 3: memory-visual DMS % correct CPF Referent Domain 4: memory-verbal STORY immediate recall CPF Referent STORY delayed recall CPF Referent Domain 5: problem solving SOC moves CPF Referent Domain 6: psychomotor MPS aiming time CPF Referent MPS line error time CPF Referent MPS long pin time CPF Referent MPS short pin time CPF Referent MPS tapping CPF Referent MPS steadiness errors CPF Referent Domain 7: personality/mood BSI depression CPF Referent BSI global index CPF Referent

53 60

107.62 (7.53) 104.92 (8.64)

53 58

One year follow-up

Change

Mean (SD)

Mean (SD)

108.28 (6.24) 106.50 (8.59)

0.66 (4.67) 1.26 (6.60)

53 60 53 60 53 60 53 60 53 60 53 60

472.57 (122.51) 440.32 (113.48) 341.34 (49.59) 337.92 (52.69) 468.51 (104.43) 440.88 (103.05) 351.00 (38.10) 345.67 (47.96) 0.93 (0.04) 0.919 (0.04) 3214.38 (1659.97) 3388.90 (1955.25)

53 58 53 58 53 58 53 58 53 58 53 58

418.92 (87.72) 430.18 (114.86) 330.23 (41.36) 330.73 (47.74) 435.15 (85.99) 428.35 (91.89) 341.52 (39.90) 341.37 (48.52) 0.94 (0.04) 0.934 (0.05) 2648.41 (1453.91) 2429.42 (1009.06)

53.65 (119.53) 3.89 (118.29) 11.11 (53.79) 4.86 (52.59) 33.36 (97.33) 8.55 (83.58) 9.48 (34.40) 3.17 (38.60) 0.01 (0.04) 0.014 (0.04) 565.97 (1462.63) 969.63 (1834.02)

53 60

84.78 (13.55) 83.56 (13.07)

53 58

86.67 (9.43) 86.44 (10.67)

1.89 (14.76) 2.99 (13.57)

53 60 53 60

59.41 55.75 48.49 43.42

53 58 53 58

60.90 55.41 50.31 44.96

1.49 0.56 1.82 1.29

53 60

(12.00) (12.76) (12.96) (13.61)

8.87 (1.51) 8.35 (1.76)

53 58

(12.02) (10.76) (14.59) (12.93)

9.51 (1.51) 9.31 (2)

(8.89) (9.47) (10) (10.12)

0.64 (1.82) 0.90 (2.07)

53 60 53 60 53 60 53 60 53 60 53 60

9.69 10.42 1.62 1.80 43.96 43.89 53.25 56.14 207.45 201.58 7.74 6.18

(2.32) (2.79) (1) (1.05) (4.84) (5.51) (13.54) (19.80) (29.62) (23.38) (9.73) (10.21)

53 58 53 58 53 58 53 58 53 58 53 58

8.67 8.68 1.32 1.30 42.52 41.41 50.09 52.04 206.87 203.14 3.76 3.91

(1.91) (2.22) (0.83) (0.74) (4.26) (7.22) (10.15) (23.00) (26.71) (22.2) (4.98) (5.44)

1.03 1.77 0.30 0.50 1.45 2.42 3.16 4.22 0.59 0.98 3.99 2.41

(1.97) (2.55) (0.87) (0.9) (4.49) (6.03) (12.52) (20.91) (20.77) (16.21) (7.89) (10.27)

53 60 53 60

43.40 43.00 50.09 50.70

(14.18) (14.71) (7.38) (11.59)

53 58 53 58

40.66 41.47 48.74 48.78

(13.64) (15.51) (8.57) (11.02)

2.74 1.47 1.36 2.14

(14.39) (15.95) (7.29) (8.67)

a Abbreviations used in Table 5: WRAT-3, Wide Range Achievement Test, 3rd edition; RVIP, Rapid Visual Information Processing; MTS, Matching to Sample; DMS, Delayed Matching to Sample; SOC, Stockings of Cambridge; MPS, Motor Performance Series; BSI, Brief Psychiatric Inventory.

occupations such as applicators with higher exposure or to workers with low educational levels. Any of the factors being discussed can influence or even explain a worker’s performance in a study of toxic exposure. Our study took place in a setting where cultural influences were relatively standard, where the basic needs of study participants were met, where safety and hygienic practices were taught and adhered to, and where such factors could be identified and measured. The findings cannot be generalized to all places where such studies are initiated, but they can teach much regarding the nature of exposure consequences to the current setting. Regardless of the setting, it is important to identify potential confounders and to learn from resulting interactions the nature of factors that potentially or actually modify study findings. While a study design, such as ours, might employ a formal level of statistical significance that was determined a priori, qualitative considerations of findings to enhance understanding from the study’s quantitative findings can and should be done as well (Berent and Albers, 2005; Woodworth and Schlosberg, 1964). In the present study, for example, we observed three-way interactions associated with our major domains of Memory-Verbal and

Problem Solving. These modifiers included family stress, education level, and age. Two of the interaction terms were formally significant and one was near-significant. Beyond findings of formal statistical significance, these interactions revealed variables that will likely be important to hypothesis generation (Berent and Albers, 2005) in preparing for future studies and might contribute to increasing our knowledge of their influence on other aspects of the exposures under study. Baseline comparisons were made against a history of cumulative CPF exposure that was significantly greater in the CPF group than among referents (64.16 vs. 0.69 mg/m3 days; p < 0.0001, or from another perspective, an average of nearly 10 years exposure for workers in the CPF group vs. 0.1 years for referent workers; p < 0.0001). These observations are not inconsequential, as one would expect to see an effect at the time of the initial evaluation from these prior exposures if long-term CPF exposure produced neurobehavioral impairments. The better performance by CPF workers at the initial evaluation could be seen as evidence against neurotoxicity; however, only if there was no selection bias. To the best of our knowledge, initial work assignments to CPF or referent groups were comparable, i.e., based on similar credentials.

S. Berent et al. / NeuroToxicology 41 (2014) 44–53

We cannot verify completely that some differential educational demands were not considered for employment in CPF related jobs. Similarly, it is possible that over time some CPF workers moved into assignments with less CPF exposure. A self-selection based on the presence of an adverse effect associated with CPF exposure (e.g., headache, nausea, irritability) and resulting in change in job assignment away from CPF exposure cannot be excluded, though previous observations of the workforce at the same site as the present study have found no evidence of retention bias of CPF workers compared to other company employees (Burns et al., 1998). Although our study design did not allow us to determine specifically if CPF workers had moved to other jobs prior to our study, there was no evidence to suggest that this had occurred, and the long exposure durations averaging more than 9 years and involving 80% of the eligible CPF workers argues against the possibility. In addition to the difference in historical cumulative CPF exposures between groups, the CPF group had significantly higher interim CPF exposures than did the referent group (6.13 vs. 0.00 mg/m3 days, p  0.0001). However, no between group differences were observed in terms of change in performance over time on any of the summary neurobehavioral domains, with and without consideration of potential confounders. The CPF workers were found to have a positive relationship between interim exposure and general ability (and a similar and near-significant finding for verbal memory). General ability (e.g., WRAT-3 Reading) is a domain expected to be resistant to many factors that have the potential to adversely affect neurologic function. The positive relationship between general ability and interim CPF exposure could have been related to a non-identified variable or to some as yet to be determined cause and effect relationship. In contrast to the expectation of decreased performance in association with increasing exposure to CPF, the CPF group was found by us at baseline to perform better than the referent group on the Memory-Verbal domain (see Tables 2 and 3). Starks and colleagues (2012) reported finding no consistent evidence for an association of OP use and adverse neurobehavioral test performance in their study of 701 older pesticide applicators. They did report finding a positive association between verbal learning and memory and lifetime days of use of five OP pesticides, including chlorpyrifos. Such findings, as reported by Starks et al. (2012) and in the present study, could lead to speculation that the higher scores by CPF exposed groups reflect a stimulatory or beneficial effect on neurobehavioral function at these levels of exposure. The lack of inhibition of RBC AChE in the present study suggests that direct inhibition of brain AChE under these conditions would seem unlikely. This conclusion is supported by animal studies, including studies with primates that show RBC AChE inhibition at doses lower than brain AChE (Chen et al., 1999). It may be worth noting that work reported by Moreno et al. (2008) has shown that CPF might affect dopamine and serotonin independent of AChE. Since these neurotransmitters are known to be involved in cognition and other behavioral functions, the finding could theoretically suggest an alternative mechanism for CPF-induced behavioral effects. From a clinical perspective, no subject in the present work evidenced clinical encephalopathy (Albers et al., 2004a), and instances of emotional impairment were few, mild, and explainable on the basis of non-exposure considerations, e.g., family or other life stresses as determined in clinical interview. We predominantly used computer-based tests for obtaining our neurobehavioral measurements. Some concerns have been raised about maintaining adequate subject motivation during computerized testing (Bauer et al., 2012; Letz, 2003). With this and other aspects of computerized testing in mind we included a trained test technician in the examination rather than evaluate the subject

51

unmonitored. We also used a blinded and standardized approach to test presentations for all subjects. The present study included the opportunity to compare subjects who had measurable occupational exposure to CPF to other subjects of comparable demographic characteristics but who had no such exposure. Sample size was limited to those workers available at the study site (see Section 2.2, Subject Selection). A power calculation during the planning of our research design indicated adequate power to detect differences related to our study hypotheses. Our studies were accomplished in an environment that was conducive to appropriate experimental control and sufficiently stable (e.g., in terms of work and personal environments) to allow application of our one-year follow-up model, with all but two participants in the referent group returning for reevaluation. Further, the participants in this study were drawn from the same overall population and relatively homogeneous demographically. Participation of CPF workers and of referent subjects was excellent over the entire course of study, both at baseline and one-year examinations. Other strengths were the longitudinal design, statistical modeling that included identified confounders, comprehensive outcome variables, clinical relevance of outcome variables, adequacy of exposure history, and biological measures of CPF (Garabrant et al., 2009). In terms of possible weakness in our study, the specific neurobehavioral tests we used in the present study could have been limited in terms of the functional areas we observed or in some insensitivity of our tests to potential impairments. We attempted to measure those functional areas most commonly reported to reflect toxicity-induced neurobehavioral and neurological impairments (Berent et al., 2008; Berent and Trask, 2000). In terms of sensitivity, the observed positive changes in neurobehavioral performance between the two examination periods could have reflected effects of practice and, therefore, some degree of sensitivity to normally expected changes in scores with repeated test administrations. An argument could be made that we did not follow subjects long enough to identify a change, when in fact a slow progression of neurobehavioral impairments might be present. Although possible, such a conclusion seems unlikely. Changes in performance over time were overwhelmingly in the direction of improved scores. While the majority of these improved scores may have reflected a practice effect in both groups, the sole significant group-by-time interaction reflected improved performance over time in the CPF exposed group. This is contrary to what might be expected recognizing that CPF workers had already been exposed for an average of nearly ten years at the time they entered the study. The historical cumulative and interim cumulative CPF exposure and their relationship to neurobehavioral findings also appear consistent with the lack of exposure-related changes over the one-year study period. The areas of neurobehavioral function evaluated in prior research, such as the ‘‘Phytoner Study’’ (Baldi et al., 2001) study of insecticide applicators (Farahat et al., 2003) and the more recent studies (Rothlein et al., 2006; Abdel Rasoul et al., 2008), were included in the present work, albeit with different tests. Our approach emphasized the functional areas to measure and not specific tests. It is possible that differences in tests between studies could have contributed to some of the variations in findings, although other factors also provide potential explanations for such discrepancies (e.g., differences in settings, subject populations, occupation, exposure type or time, differences in protective equipment and safety regulation and instruction, or other factors). For example, the Phytoner and Abdel Rasoul studies included pesticide applicators or farm workers who may have had ineffective protective equipment, possibly leading to higher exposure than might otherwise be the case.

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S. Berent et al. / NeuroToxicology 41 (2014) 44–53

The results of our study are relevant to those concerned with the regulation or manufacture of CPF, especially workers as well as others who may be at risk for exposure to this compound. Our findings did not support the hypothesis that workers with chronic, low level occupational exposure to CPF in manufacturing develop dose-related sub-clinical or clinically evident adverse neurobehavioral effects under the conditions of the present study. These conditions included, but were not limited to, the opportunity to study workers at a site actively involved in the manufacturing and production of CPF, the potential for chronic exposure to CPF, the opportunity to measure workers’ exposures accurately and reliably, limitations on the different chemical compounds found in the facility, presence of a suitable comparison group, and other aspects as presented and discussed in various places in this paper. The authors recognize that some aspects making this site attractive for these studies also present limitations for the generalization of results to other sites and places that might have practices that vary widely between and within different facilities and locations in a given country (including the United States, as studied here) and around the world. Conflict of interest statement In addition to funding from governmental and private industrial sources for research and related activities, some of the authors have at times been retained as consultants or served as expert witnesses in litigation for firms, agencies, or companies, including Dow Chemical Company and Dow AgroSciences, concerned with the manufacture or use of insecticides. Support of these activities has included both personal and institutional remuneration. Activities by individual authors have included the following: James W. Albers has been the Principal Investigator on research grants to the University of Michigan from Dow Chemical Company and Dow AgroSciences and recipient of a Dow Chemical Company Foundation SPHERE (Supporting Public Health and Environmental Research Efforts) Award to the University of Michigan, and he has served as a consultant and expert witness on behalf of Dow Chemical Company and Dow AgroSciences. Stanley Berent has been the an Investigator on research funded by grants to the University of Michigan, including monetary and other support from Dow Chemical Company, Dow AgroSciences, the Dow Foundation, including a SPHERE (Supporting Public Health and Environmental Research Efforts) award through the university of Michigan. In addition, he has served as a consultant and expert witness to legal firms who were representing Dow Chemical Company and Dow AgroSciences. Sarah S. Cohen received funding as a graduate student while working on research grants and research donations to the University of Michigan from Dow Chemical Company and Dow AgroSciences. David H. Garabrant has been the Principal Investigator on research grants to the University of Michigan from Dow Chemical Company. He serves on a Human and Ecological Health Advisory Committee to Dow AgroSciences. In addition, he has served as an expert witness on behalf of Dow Chemical Company and Dow AgroSciences. Richard P. Garrison has been a Co-Investigator on research grants to the University of Michigan from Dow Chemical Company and Dow AgroSciences. He has also served as a consultant and expert witness on behalf of Dow Chemical Company and Dow AgroSciences. Bruno Giordani has been a co-investigator on a research grant to the University of Michigan from Dow Chemical Company and Dow AgroSciences. Rudy J. Richardson has been the Principal Investigator on research grants and research donations to the University of

Michigan from Dow Chemical Company and Dow AgroSciences. He has also been appointed by the University of Michigan as the Dow Professor of Toxicology, a professorship endowed by the Dow Foundation. In addition, he has served as a consultant and expert witness on behalf of Dow Chemical Company and Dow AgroSciences. Transparency documents associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neuro.2013. 12.010. Acknowledgments This study was supported financially by Dow AgroSciences, Indianapolis, Indiana, USA, with additional support from Dow Chemical Company and Dow Chemical Company Foundation. Aspects of this study also were presented in a talk at the 10th International Symposium on Neurobehavioral Methods and Effects in Environmental and Occupational Health (NEUREOH) (Berent et al., 2008). We acknowledge the additional investigators who worked at some point on this project, including Brenda Gillespie, PhD, Alison Berent-Spillson, PhD, Steven P. Levine, PhD, Jonathon Raz, PhD (deceased), and other members of the Chlorpyrifos Study team: Jennifer N. Baughman, Nathan Bradshaw, Luis Casenas, Angela K. (Freymuth) Caveney, Elizabeth Humpert, Mary Kauszler, Zhuolin Li, Shelly H. Martinez, Esther Meima, and Ann M. Schultz. Also, we acknowledge receipt of a SPHERE (Supporting Public Health and Environmental Research Efforts) Award from the Dow Chemical Company Foundation, for which the authors wish to express their gratitude, and the assistance provided to us by the many Dow Chemical Company and DowAgrosciences employees who assisted at various points in supporting this research, with specific thanks to Carol J. Burns and Joel L. Mattsson. Finally, we are indebted to the Dow Chemical Company employees who volunteered their time as subjects in this study. References Abdel Rasoul GM, Abou Salem ME, Mechael AA, Hendy OM, Rohlman DS, Ismail AA. Effects of occupational pesticide exposure on children applying pesticides. Neurotoxicology 2008;29(5):833–8. Albers JW, Berent S, Garabrant DH, Giordani B, Schweitzer SJ, Garrison RP, et al. The effects of occupational exposure to chlorpyrifos on the neurologic examination of central nervous system function: a prospective cohort study. JOEM 2004a; 46:367–78. Albers JW, Cole P, Greenberg RS, Mandel JS, Monson RR, Ross JH, et al. Analysis of chlorpyrifos exposure and human health: expert panel report. J Toxicol Environ Health B: Crit Rev 1999;2:301–24. Albers JW, Garabrant DH, Berent S, Richardson RJ. Paraoxonase status and plasma butyrylcholinesterase activity in chlorpyrifos manufacturing workers. J Exp Anal Environ Epidemiol 2010;20:79–89. Albers JW, Garabrant DH, Schweitzer SJ, Garrison RP, Richardson RJ, Berent S. The effects of occupational exposure to chlorpyrifos on the peripheral nervous system: a prospective cohort study. Occup Environ Med 2004b;61:201–11. American Conference of Governmental Industrial Hygienists. TLVs and BEIs based on the documentation of the threshold limit values for chemical substances and physical agents and biological exposure indices. Cincinnati, OH: American Conference of Governmental Industrial Hygienists; 2013. Anger WK. Neurobehavioural tests and systems to assess neurotoxic exposures in the workplace and community. Occup Environ Med 2003;60:531–8. Baldi I, Filleul L, Mohammed-Brahim B, Fabrigoule C, Dartigues JF, Schwall S, et al. Neuropsychologic effects of long-term exposure to pesticides: results from the French Phytoner study. EHP 2001;109:839–44. Bauer RM, Iverson GL, Cernnich AN, Binder LM, Ruff RM, Naugle RI. Computerized neuropsychological assessment devices: joint position paper of the American Academy of Clinical Neuropsychology and the National Academy of Neuropsychology. Clin Neuropsychol 2012;26:177–96. Berent S, Albers JW. Neurobehavioral toxicology: neurological and neuropsychological perspectives, Volume. I, Foundations and methods. London and New York: Taylor and Francis; 2005. Berent S, Albers J, Garabrant D, Giordani B, Richardson R. Occupational chlorpyrifos exposure and neurobehavioral functioning in pesticide manufacturing workers. In: 10th International Symposium on Neurobehavioral Methods and Effects in Environmental and Occupational Health (NEUREOH) [Program abstract No. We-O-19]. 2008.

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