Clinical Observations

Standard clinical observations were performed within 4 hours post dosing on all study days. Animals were observed for signs of cholinesterase inhibition with specific signs recorded, such as weakness, lethargy, tremors, eye-bulging, salivation, lacrimation, and diarrhea.

Body and Organ Weights

Animals were weighed on the first day of dosing and on the day of necropsy. During necropsy, the entire liver and brain were removed, and organ weights were recorded for each animal.

Clinical Pathology

Animals were terminated in random order by CO₂/O₂ (70%/30%) anesthesia one day after the final day of dosing. Blood samples were taken via cardiocentesis. Five mL of blood was collected into a tube void of anticoagulant and the serum harvested for clinical chemistry, total thyroxine (T₄), and cholinesterase measurements. The following clinical chemistry parameters were measured on an Olympus AU400e chemistry analyzer (Olympus America, Inc., Irvin, TX) using reagents obtained from Beckman Coulter (Brea, CA) or Diazyme (Poway, CA): urea nitrogen, creatinine, total protein, albumin, sorbitol dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bile acids, cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and cholinesterase. Total T₄ was measured using an MP Biomedical T4 radioimmunoassay kit with an Apex automatic gamma counter (ICN Micromedic Systems, Inc., Huntsville, AL). Toxicological study data tables are presented in Appendix B.

Sample Collection for Transcriptomics

Liver transcriptomics were performed on samples taken from three animals per dose group (randomly selected). Half the left liver lobe was processed for RNA isolation. Specifically, three pieces (3-mm cubes) were dissected and transferred to a weigh boat containing liquid nitrogen. Once flash frozen, the liver tissue for each animal was placed into a single, prechilled 2-mL cryotube and stored at or below −70°C. Frozen liver samples were shipped to the Battelle Biomedical Research Center (West Jefferson, OH) on dry ice.

RNA Isolation and cDNA Synthesis

The frozen liver tissues were submerged in 10 volumes of prechilled RNAlater^®-ICE (Life Technologies, Carlsbad, CA) and stored at −20°C ± 10°C for a minimum of 16 hours. The tissues were removed from the RNAlater^®-ICE and weighed. Each liver tissue sample, weighing between 21 and 30 mg, was added to lysis buffer and homogenized using plastic disposable pestles (Fisher Scientific, Pittsburgh, PA). Following homogenization, samples were stored at −70°C ± 10°C until RNA was isolated. Samples were thawed and centrifuged. RNA was extracted from the supernatant, subjected to DNase digestion, and isolated using the Qiagen RNeasy Mini Kit (Cat #: 74104; Qiagen, Valencia, CA). Each RNA sample was analyzed for quantity and purity by UV analysis using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). All samples were evaluated for RNA integrity using an RNA 6000 Nano Kit with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and then stored at −70°C ± 10°C until further processing.

Total RNA (100 ng), isolated from each liver sample, was used to synthesize single-stranded DNA, which was subsequently converted into a double-stranded complementary DNA (cDNA) template for transcription. An in vitro transcription (IVT) reaction, which incorporates biotinylated ribonucleotide analogs, then was used to create labeled amplified RNA (aRNA). This RNA target preparation was performed using the Affymetrix GeneChip^® 3’ IVT Express Kit (Cat #: 901228; Affymetrix Inc., Santa Clara, CA) and an Eppendorf Mastercycler^® thermal cycler (Eppendorf, Hamburg, Germany).

Labeled aRNA was fragmented and subsequently hybridized to the Affymetrix Rat Genome 230 2.0 Array (Cat #: 900505; 31,099 probe sets) using an Affymetrix GeneChip^® Hybridization Oven 645. The arrays were washed and stained using the Affymetrix GeneChip^® Hybridization Wash and Stain kit (Cat #: 900720) and a Fluidics Station 450 according to the Affymetrix-recommended protocol (FS450_0001). After washing and staining, arrays were scanned using an Affymetrix GeneChip^® Scanner 3000 7G, and the raw microarray data (.CEL files) were acquired using Affymetrix GeneChip^® Command Console^® Software. The Rat Genome 230 2.0 Array provides coverage of more than 30,000 known transcripts; although the array provides comprehensive coverage of global transcript expression, of note is that it does not cover the entirety of the rat transcriptome.

Analysis of GeneChip Data Quality

Quality control measurements were evaluated to determine if the data generated from each Affymetrix GeneChip^® array were of sufficient quality. Affymetrix-recommended guidelines for evaluating quality were used to evaluate the output files for each GeneChip^® array using the R/Bioconductor package, Simpleaffy.⁹ The following quality control parameters were evaluated for each array: average background, scale factor, percentage of genes scored as present, 3’ to 5’ ratios for the internal control genes beta-actin and glyceraldehyde-3-phosphate dehydrogenase, values for hybridization control transcripts, and values for poly (A) controls.

For samples that failed to pass quality control evaluation due to insufficient data quality, an additional round of RNA isolation and cDNA synthesis was performed and additional GeneChip^® arrays were run, which were designated with –R after each sample number.

Statistical Analysis of Body Weights, Organ Weights, and Clinical Pathology

Two approaches were employed to assess the significance of pairwise comparisons between dosed and vehicle control groups in the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Williams^10,11 and Dunnett.¹² Hormone data and clinical chemistry, which typically have skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley¹³ and Dunn.¹⁴ The Jonckheere test¹⁵ was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams or Shirley test) was more appropriate for pairwise comparisons than a test that assumes no monotonic dose response (Dunnett or Dunn test). Trend-sensitive tests were used when the Jonckheere test was significant at p ≤ 0.01.

Prior to analysis, values identified by the outlier test of Dixon and Massey¹⁶ were examined by National Institute of Environmental Health Sciences staff. Values from animals suspected of illness due to causes other than experimental exposure and values that the laboratory indicated as inadequate due to measurement problems were eliminated from the analysis.

A no-observed-effect level (NOEL) was identified as the highest dose not showing a significant (p ≤ 0.05) pairwise difference relative to the vehicle control group. A lowest-observed-effect level (LOEL) was identified as the lowest dose demonstrating a significant (p ≤ 0.05) pairwise difference relative to the vehicle control group.

Benchmark Dose Analysis of Body Weights, Organ Weights, and Clinical Pathology

Clinical pathology, body weight, and organ weight endpoints that exhibited a significant trend and pairwise test were submitted in batch for automated benchmark dose (BMD) modeling analysis. For body weight, the BMD and benchmark dose lower confidence limit (BMD_L) were reported as not determined when there were no significant results. BMD modeling and analysis was conducted using a modification of Benchmark Dose Modeling Software (BMDS) version 2.7.0. Data sets were executed using the Python BMDS interface (https://pypi.python.org/pypi/bmds; version 0.11), which allows for batch processing of multiple data sets. Data for all endpoints submitted were continuous. A default benchmark response (BMR) of 1 standard deviation (relative to control) was used for all data sets. The following BMDS 2.7.0 models were used to model the means of the data sets:

• Linear
• Polynomial 2°, 3°, 4°, 5°
• Power
• Hill
• Exponential M2, M3, M4, M5

Multiple versions of the polynomial model were executed, from a polynomial of degree 2 to a polynomial of degree equal to the number of dose groups minus 1 (e.g., if a data set had five dose groups, a 2°, 3°, and 4° polynomial model would be executed). Models were initialized using BMDS 2.7.0 model defaults, including restricting the power parameter of the power model and n-parameter of the Hill model to >1 and the beta parameters of the polynomial model to positive or negative, depending on the mean response direction of the data set. For all models, either a constant or nonconstant variance model was selected as outlined in U.S. Environmental Protection Agency (EPA) BMD technical guidance¹⁷ and was implemented in the BMDS 2.7.0 software.

After model execution, BMDs were selected using the model recommendation procedures generally described¹⁷ and the automated decision logic described in Wignall et al.¹⁸ and summarized in Appendix D, Table D-1. Models were placed into one of four possible bins, depending on the results and the bin recommendation logic:

1. Failure: model did not successfully complete
2. Nonviable model (NVM): model successfully completed but with serious issues
3. Not reportable (NR): model is identified and meets all acceptability criteria with the exception of the estimated BMD being below the lower limit of extrapolation (<1/3 the lowest nonzero dose tested); BMD reported as <1/3 the lowest nonzero dose tested and BMD_L is not reportable
4. Viable model: candidate for recommended model without warning

If only one model was in the viable model bin, it was selected as the best-fitting model. If the viable bin had more than one model, consistent with EPA guidance,¹⁷ either the model with the lowest Akaike information criterion (AIC) or lowest BMD_L was selected. If the range of BMD_L values was sufficiently close (less than threefold different), the AIC value was used; otherwise, the BMD_L value was used. If no model was recommended, no BMD was presented in the results. Details on the analysis criteria and decision tree are provided in Appendix D, Table D-1 and Figure D-1, respectively. To avoid effects of model extrapolation, BMD values derived from viable models that were threefold lower than the lowest nonzero dose tested were reported as <1/3 the lowest nonzero dose tested and corresponding BMD_L values were not reported.

Benchmark Dose Analysis of Transcriptomics Data

The BMD analysis of the transcriptomic data was performed in accordance with NTP best practices for genomic dose-response modeling as reviewed by an independent panel of experts in October 2017. These recommendations are described in the 2018 publication, National Toxicology Program Approach to Genomic Dose Response Modeling.¹⁹

Probe set intensities from raw microarray data (.CEL files from Affymetrix Rat Genome 230 2.0 Arrays) were normalized by applying the Robust Multi-array Average (RMA) algorithm from the genomics analysis tool, GeneSpring GX 12.6 (Agilent Technology, Foster City, CA). The microarray studies of multiple organophosphate phosphates (data to be reported elsewhere) were performed at the same time such that .CEL files from those related studies were normalized together with the data sets collected in this study. Principal component analysis (PCA) of the primary RMA-normalized data indicated a batch effect; due to randomization of the samples in the processing and detailed metadata capture, the source of the batch effect could be identified as the hybridization date. To correct the batch effect, the primary normalized data were loaded into Partek Genomic Suite version 6.16.0812 (St. Louis, MO) and annotated with chemical exposure/dose group and hybridization date annotations. The ANOVA-based remove batch effect function in Partek Genomic Suite then was used to remove quantitative impacts from the hybridization date batch effect. Quality control of the batch-corrected, normalized data was performed by visual inspection, using a PCA plot and normalized intensity histograms (Appendix C).

Dose-response analyses of RMA-normalized, batch-corrected probe set intensities from the EHDP study samples were performed using BMDExpress 2.20.0148 beta²⁰ (https://github.com/auerbachs/BMDExpress-2/releases), an updated version of BMDExpress 1.41 that uses an updated modeling approach. First, control genes (AFFX-) were removed from each data set. A trend test (the Williams trend test,^10,11 p ≤ 0.05) and fold change filter (1.5-fold change up or down relative to the vehicle control group for probe sets) was applied to the data set to remove probe sets demonstrating no response to chemical exposure from subsequent analysis. These filter criteria were empirically determined, with the goal of balancing false discovery with reproducibility. The criteria are consistent with the MicroArray Quality Control recommendations to combine the nominal p value threshold with a fold change filter to maximize replicability of transcriptomic findings across labs. The following dose-response models were fit to the probe sets that passed the trend test and fold change filter:

• Hill
• Power
• Linear
• Polynomial 2°
• Exponential M2, M3, M4, M5

All gene expression data analyzed in BMDExpress were log2 transformed, and thus nearly all probe sets exhibit constant variance across the doses. For this reason and for efficiency purposes, each model was run assuming constant variance. Lacking any broadly applicable guidance regarding the level of change in gene expression considered to be biologically significant, a BMR of 1 standard deviation (relative to the fit at control) was used in this study. This approach enables standardization of the BMR between apical endpoints and transcriptomic endpoints and provides a standard for use across multiple chemicals tested in this rapid screening paradigm. The expression direction (upregulated or downregulated) for each probe set was determined by a trend test intrinsic to the model executables (provided by EPA) contained in BMDExpress.

To identify the best-fit model for each fitted probe set, the AIC values for each fitted model were compared and the model with the lowest AIC selected. The best model for each probe set was used to calculate the BMD, BMD_L, and BMD upper confidence limit (BMD_U). The specific parameter settings, selected from the BMDExpress software when performing probe set-level BMD analysis, were as follows: maximum iterations – 250, confidence level – 0.95, BMR factor – 1 (the multiplier of the SD that defined the BMD), restrict power – no restriction, and constant variance – selected. The specific model selection setting in the BMDExpress software when performing probe set-level BMD analysis was as follows: best poly model test – lowest AIC, flag Hill model with ‘k’ parameters – <1/3 the lowest nonzero dose tested, and best model selection with flagged Hill model – include flagged Hill model. The inclusion of the flagged models is a deviation from EPA guidance. The justification for this deviation relates to subsequent use of the data in which the probe set BMD values are grouped into gene sets from which a median BMD is derived. If the probe sets were removed from the analysis or forced to another model, the probe set might not be counted in the gene set analysis and could lead to loss of “active” gene sets. Importantly, most of the probe sets that produce flagged Hill models show highly potent responses and should therefore be counted in the analysis.

To perform Gene Ontology (GO; annotation accession date: 03/09/18) gene set analysis, only GO terms with ≥10 and ≤250 annotated genes measured on the gene expression platform were considered. Before sorting genes into the GO terms, the best-fit model for each probe set was subjected to a filtering process to remove those probe sets (1) with a BMD >highest dose tested, (2) that mapped to more than one gene, (3) that had a global goodness-of-fit p value ≤0.1, and (4) with a BMD_U/BMD_L ratio >40. GO terms that were at least 5% populated and contained three genes that passed the above criteria were considered “active” (i.e., responsive to chemical exposure). For this report, GO terms populated with identical sets of differentially expressed genes were filtered to limit redundancy in reporting based on the following selection criteria: (1) highest percentage populated and (2) most specific/highest GO level. Redundant GO terms failing to differentiate on the basis of these criteria were retained and reported. A complete list of “active” GO terms can be found in Appendix F. To avoid effects of model extrapolation, GO terms exhibiting BMD values below the lower limit of extrapolation (<1/3 the lowest nonzero dose tested) were reported as <1/3 the lowest nonzero dose tested and corresponding BMD_L and BMD_U values were not reported.

To perform Individual Gene Analysis, a Defined Category Analysis in BMDExpress was performed that mapped probe sets to genes using a probe-to-gene annotation file. In short, the best-fit model for each probe set was subjected to a filtering process to remove those probe sets (1) with a BMD >highest dose tested, (2) that mapped to more than one gene, (3) that had a global goodness-of-fit p value ≤0.1, and (4) with a BMD_U/BMD_L ratio >40. For genes that had more than one probe set represented on the microarray and passed the above filtering, a median BMD was used to estimate the BMD, BMD_L, and BMD_U values. To ensure only genes with a robust response were assessed for potency, genes with probe sets that had a median fold change <|2| were removed prior to reporting. A complete list of genes and their corresponding metrics can be found in Appendix F. To avoid effects of model extrapolation, genes exhibiting BMD values below the lower limit of extrapolation (<1/3 the lowest nonzero dose tested) were reported as <1/3 the lowest nonzero dose tested and corresponding BMD_L and BMD_U values were not reported.

A summary of the BMDExpress gene expression analysis pipeline used in this study is shown in Figure D-2.

Data Accessibility

Primary and analyzed data used in this study are available to the public at https://doi.org/10.22427/NIEHS-DATA-NIEHS-02.²¹