| Study Protocol |
An a priori protocol/study plan with defined mechanistic hypotheses is required, including reference to a thorough Standard Operating Procedure (SOP) that details sampling protocols and internal quality control. Health outcomes being investigated in the study plan should consider biologically plausible mechanisms. The protocol should include all details regarding study design and implementation. Note any deviations. While all details of the protocol will not be included in a journal article, the documents should be available on request and submitted for any studies being considered for pesticide registration and policy-making.
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| Validity and Reliability of Sampling |
Exposure assessed via the same methods and within the same timeframe across all groups. Samples collected under controlled conditions; contamination assessed (e.g., via blanks analysis). Contamination from storage materials, the matrix (e.g., external contamination on toenail samples), and other sources fully considered and controlled via equipment selection, cleaning, etc. All glassware, reagents, organic solvents, and water should be checked for possible interfering contaminants. For some environmental samples, the available methods may not be well standardized, such as wipe sampling for dust exposure. In these cases, the researcher must conduct research to fill any knowledge gaps (e.g., best wipe material), and conduct method validation to optimize the methods [22].
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With regard to time frame, while not all samples will necessarily be taken at the same time (e.g., studies with rolling enrollment), the researcher must evaluate whether pesticide concentrations may have changed over time (e.g., due to environmental degradation, changing pesticide use) and assess how any changes may impact results.
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For biological samples that are time-varying (within a person) due to chemical metabolism or other factors, particularly within a single day or across weeks, the researchers must assess and either standardize sample collection across participants or adjust/stratify results to account for differences.
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In instances where timing affects results, stratification or other methods for evaluating inconsistencies across measures may be utilized [3].
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For wipe sampling, consult relevant guidance (e.g., Battelle 2007 [22]).
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| Exposure Window |
The exposure window of the chosen biomarker/metric reflects the time period during which the exposure could have effects relevant to the outcome of interest. It considers induction and latency and is based on biological and clinical pathways.
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Not applicable to case-control studies. For a developmental endpoint, the metric should capture the exposure that occurred during the period of fetal development associated with that effect (e.g., for congenital heart defects, exposure occurs during cardiac development). Similarly, for cancer, the exposure must precede the diagnosis by a sufficient latency period (typically, at least 5 years for hematologic neoplasms and 10 years for epithelial neoplasms).
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| Time Integration |
The biomarker or personal/environmental sampling data correspond(s) to the most relevant exposure metric (e.g., average, cumulative, peak), based on the studied outcome and known biological variability.
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| Specificity |
Metric is specific to the exposure of interest. For biological samples, the biomarker must be the parent compound of interest or a toxicologically relevant metabolite that represents an internal dose that is well correlated with external exposure. The toxicokinetics of the parent compound in the body are understood and have been considered in choosing the metric and designing the sampling protocol.
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For example, dialkylphosphates, the urinary metabolites of organophosphates (OPs), are not specific to individual OPs, so they are not informative regarding specific OPs [23].
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For personal monitoring and environmental exposures, several exposure metrics may be needed to provide sufficient information. Confirmatory tests can be conducted to ensure positive identification of the exposure of interest [18].
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| Sensitivity |
Metric is sensitive (i.e., measurable down to a low limit of detection that is low enough to detect chemicals in a sufficient percentage of the samples to inform the causal or research question). There must also be high confidence in the ability of the instruments used to provide the needed level of sensitivity.
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| Validity and Reliability of Analytical Methods |
The analytical test methods have been validated (e.g., by assessment of repeatability within a laboratory and reproducibility of the method at multiple laboratory sites). In the case of a novel test, sufficient information must be provided regarding the within-laboratory validation, and any uncertainties should be discussed. Reliability should be assessed via intraclass correlation coefficient (ICC) or similar assessment.
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| Number of Samples and Replicates |
A sufficient number of replicates per sample is required to ensure data validation and assessment of data variability. For personal monitoring and environmental samples, ensure there are a sufficient number of total samples for the specific detection limit and/or to achieve sufficient power for the necessary statistical analysis (see example under comments).
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As noted in US EPA guidance for regulatory risk assessment, if, for example, a frequency of detection limit of five percent is used, “then at least 20 samples of a medium would be needed (i.e., one detect in 20 samples equals a five percent frequency of detection)” [24].
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If using ProUCL for environmental assessment, a minimum of 4–5 samples are required.
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| Consideration of Matrix Effects |
Adjust exposure estimate, as needed, based on the matrix and any matrix effects during sample processing. Both adjusted and unadjusted concentrations and results should be reported. Matrix effects must be investigated in the early phase of studies and used to inform the methods chosen and/or to put sample treatment remedies in place. Investigations can include comparing the response of an analyte in a standard solution and in a post-extraction spiked sample (matrix-matched standard). Preventative measures such as correcting for analyte losses during pre-treatment, may also be performed [25].
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| Sample Storage and Stability |
Verify the stability of the substance of concern in the samples, given the matrix, storage conditions, and duration of storage. Samples may have some known losses, but differences between low and high exposures can be assessed.
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For many biological samples, storage at a range of −80 to −130 degrees Celsius is preferred [26]; deviation of this general standard must be justified. Samples that have undergone thawing and refreezing must be evaluated against spiked samples subjected to the same conditions [2]. Fluctuations in storage conditions should be avoided.
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| Reporting Requirements |
The study protocol should be made publicly available. Details regarding the consideration and fulfillment of the above criteria (e.g., all sample collection, handling, processing, and storage; evidence of sample stability; analytical methods; method sensitivity, specificity, validity, and reliability; and Quality Assurance/Quality Control [QA/QC] procedures) should be reported. Limits of detection and limits of quantification should be stated for the target analyte, and the proportion of samples at or below these values should be recorded. Any deviations from the SOP/protocol and justifications for such deviations should be reported. While some of these details may need to be omitted for peer-reviewed publication, the information should be provided in supplemental material or made available upon request.
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To determine concentrations most representative of potential exposures, results above and below the limit of quantification should be considered together. Use one-half the limit of quantification as a proxy concentration if there is reason to believe the chemical is present below the limit of quantification, or use the limit of quantification value itself if there is reason to believe the concentration is closer to this value. Only use a value of zero if there is specific information indicating that the chemical is not likely to be present in a sample [24].
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