This dataset provides measured in vitro intrinsic clearance rates for 54 chemicals tested using isolated hepatocytes from humans, rats, and rainbow trout. The test chemicals were selected to provide broad coverage across the industrial and pesticidal chemical space while also prioritizing chemicals of interest to EPA’s Endocrine Disruptor Screening Program (EDSP). A data evaluation framework was developed to identify results suitable for rate reporting. Acceptable results were then used to evaluate the chemical domain of applicability of the applied methods, the influence of starting substrate concentration on measured rates of intrinsic clearance, and differences in metabolic activity among species. These findings provide data for chemicals of specific interest to the EDSP. More importantly, the results provide critical guidance on future use of in vitro biotransformation assays to support high-throughput chemical risks assessments. This dataset is associated with the following publication: Black, S., J. Nichols, K. Fay, S. Matten, and S. Lynn. Evaluation and comparison of in vitro intrinsic hepatic clearance rates measured using cryopreserved hepatocytes from humans, rats, and rainbow trout. TOXICOLOGY. Elsevier Science Ltd, New York, NY, USA, 457: 152819, (2021).

This paper describes the results of an international ring trial (round robin study) of in vitro methods used to measure chemical biotransformation in fish. Researchers at six laboratories in the U.S. and Europe evaluated six test chemicals (cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene) using two in vitro systems (liver S9 fraction and isolated hepatocytes) derived from rainbow trout livers. This information was then evaluated to quantitatively characterize assay reliability (repeatability and reproducibility) and evaluate potential user bias associated with the methods. Additional calculations were performed using established models to estimate in vivo levels of hepatic clearance and predict the effects on chemical biotransformation on chemical bioaccumulation. The Science Hub dataset includes all information presented in the paper as figures and tables including, as appropriate, individual values used to calculate reported means and standard deviations. Also included are all datasets provided as Supplementary Data. This dataset is associated with the following publication: Nichols, J., K. Fay, M.J. Bernhard, I. Bischof, J. Davis, M. Halder, J. Hu, K. Johanning, H. Laue, D. Nabb, C. Schlechtriem, H. Segner, J. Swintek, J. Weeks, and M. Embry. Reliability of In Vitro methods used to measure intrinsic clearance of hydrophobic organic chemicals by rainbow trout: Results of an international ring trial. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 164(2): 563-575, (2018).

This paper describes the results of an international ring trial (round robin study) of in vitro methods used to measure chemical biotransformation in fish. Researchers at six laboratories in the U.S. and Europe evaluated six test chemicals (cyclohexyl salicylate, fenthion, 4-n-nonylphenol, deltamethrin, methoxychlor, and pyrene) using two in vitro systems (liver S9 fraction and isolated hepatocytes) derived from rainbow trout livers. This information was then evaluated to quantitatively characterize assay reliability (repeatability and reproducibility) and evaluate potential user bias associated with the methods. Additional calculations were performed using established models to estimate in vivo levels of hepatic clearance and predict the effects on chemical biotransformation on chemical bioaccumulation. The Science Hub dataset includes all information presented in the paper as figures and tables including, as appropriate, individual values used to calculate reported means and standard deviations. Also included are all datasets provided as Supplementary Data. This dataset is associated with the following publication: Nichols, J., K. Fay, M.J. Bernhard, I. Bischof, J. Davis, M. Halder, J. Hu, K. Johanning, H. Laue, D. Nabb, C. Schlechtriem, H. Segner, J. Swintek, J. Weeks, and M. Embry. Reliability of In Vitro methods used to measure intrinsic clearance of hydrophobic organic chemicals by rainbow trout: Results of an international ring trial. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 164(2): 563-575, (2018).

The purpose of this study was to compare two in vitro systems, cryopreserved trout hepatocytes and trout liver S9 fractions, used to predict in vivo levels of biotransformation in fish. This information is needed to refine modeled estimates of bioaccumulation for hydrophobic organic chemicals that undergo biotransformation. In this effort we used trout hepatocytes to measure in vitro biotransformation of 6 polycyclic aromatic hydrocarbons (PAHs). The results were compared to metabolism rates reported previously for trout liver S9 fractions. Results obtained using both in vitro systems were then used to predict measured levels of hepatic clearance for the same test chemicals exhibited by isolated perfused livers. The results of this study suggest that both in vitro systems are well suited for performing in vitro-in vivo metabolism extrapolations with fish as a means for improving modeled bioaccumulation predictions. This dataset is associated with the following publication: Fay, K., P. Fitzsimmons, A. Hoffman, and J. Nichols. Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, FL, USA, 36(2): 463-471, (2017).

The purpose of this study was to compare two in vitro systems, cryopreserved trout hepatocytes and trout liver S9 fractions, used to predict in vivo levels of biotransformation in fish. This information is needed to refine modeled estimates of bioaccumulation for hydrophobic organic chemicals that undergo biotransformation. In this effort we used trout hepatocytes to measure in vitro biotransformation of 6 polycyclic aromatic hydrocarbons (PAHs). The results were compared to metabolism rates reported previously for trout liver S9 fractions. Results obtained using both in vitro systems were then used to predict measured levels of hepatic clearance for the same test chemicals exhibited by isolated perfused livers. The results of this study suggest that both in vitro systems are well suited for performing in vitro-in vivo metabolism extrapolations with fish as a means for improving modeled bioaccumulation predictions. This dataset is associated with the following publication: Fay, K., P. Fitzsimmons, A. Hoffman, and J. Nichols. Comparison of trout hepatocytes and liver S9 fractions as in vitro models for predicting hepatic clearance in fish. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, FL, USA, 36(2): 463-471, (2017).

Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level2_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level5_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Supplemental File LTEA_Inucyte_Images.zip is comprised of 20,493 images totaling more than 15 gigabytes. Cell morphology images were acquired for each well/plate with an Essen IncuCyte™ FLR automated phase-contrast microscope located inside a tissue culture incubator. Six 96-well culture plates were loaded into the instrument and imaged for an elapsed time (~24 minutes). The IncuCyte™ software was used for image capturing and export of images in JPEG format. This dataset is associated with the following publication: Franzosa, J., J. Bonzo, J. Jack, N.C. Baker, P. Kothiya, R. Witek, P. Hurban, S. Siferd, S. Hester, I. Shah, S. Ferguson, K. Houck, and J. Wambaugh. High-throughput toxicogenomic screening of chemicals in the environment using metabolically competent hepatic cell cultures. npj Systems Biology and Applications. Springer Nature Group, New York, NY, 7: Article 7, (2021).

Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level2_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Gene expression data from the Fluidigm qRT-PCR arrays was analyzed in R (v3.6.1; R Foundation for Statistical Computing, 2019). Prior to processing through the tcpl package, each qRT-PCR primer set was annotated as an individual assay endpoint (aeid) for analyses. For each plate, well types were designated for test compound wells (t), positive controls (c), (that is phenobarbital) and neutral controls (n, DMSO). Fold-change in the number of amplification cycles needed to pass the background threshold (Ct) for 96 transcripts to (ftp://newftp.epa.gov/COMPTOX/CCTE_Publication_Data/CCED_Publication_Data/Wambaugh/ToxCast_LTEA, file LTEA_Level5_20191119.zip) were normalized to the geometric mean of three housekeeping genes (ACTB, GAPDH, POLR2A) to generate ΔCt values (cval). Prior to calculating the response values (rval), or ΔΔCt, for each transcript (n = 96) per well, the baseline value (bval), the plate-wise median of the neutral control wells, was generated for each plate (the normalization process is described in detail in supplemental file SupFile4-DeltaCTCalculation.docx). The bval was subtracted from the cval to yield the rval or log2 Fold Change per transcript. Supplemental File LTEA_Inucyte_Images.zip is comprised of 20,493 images totaling more than 15 gigabytes. Cell morphology images were acquired for each well/plate with an Essen IncuCyte™ FLR automated phase-contrast microscope located inside a tissue culture incubator. Six 96-well culture plates were loaded into the instrument and imaged for an elapsed time (~24 minutes). The IncuCyte™ software was used for image capturing and export of images in JPEG format. This dataset is associated with the following publication: Franzosa, J., J. Bonzo, J. Jack, N.C. Baker, P. Kothiya, R. Witek, P. Hurban, S. Siferd, S. Hester, I. Shah, S. Ferguson, K. Houck, and J. Wambaugh. High-throughput toxicogenomic screening of chemicals in the environment using metabolically competent hepatic cell cultures. npj Systems Biology and Applications. Springer Nature Group, New York, NY, 7: Article 7, (2021).

We introduce a new high-throughput transcriptomics (HTTr) platform comprised of a collagen sandwich primary rat hepatocyte culture and the TempO-Seq assay for screening and prioritizing potential hepatotoxicants. We selected 14 chemicals based on their risk of drug-induced liver injury (DILI) and tested them in hepatocytes at two treatment concentrations. HTTr data was generated using the TempO-Seq whole transcriptome and S1500+ assays. The HTTr platform exhibited high reproducibility between technical replicates (r>0.9) but biological replication was greater for TempO-Seq S1500+ (r>0.85) than for the whole transcriptome (r>0.7). Reproducibility between biological replicates was dependent on the strength of transcriptional effects induced by a chemical treatment. Despite targeting a smaller number of genes, the S1500+ assay clustered chemical treatments and produced gene set enrichment analysis (GSEA) scores comparable to those of the whole transcriptome. Connectivity mapping showed a high-level of reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project with high concordance between the S1500+ gene set and whole transcriptome. Taken together, our results provide guidance on selecting the number of technical and biological replicates and support the use of TempO-Seq S1500+ assay for a high-throughput platform for screening hepatotoxicants. FASTQ files and read counts data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) (GSE152128). This dataset is associated with the following publication: Lee, F., I. Shah, Y.T. Soong, J. Xing, I.C. Ng, F. Tasnim, and H. Yu. Reproducibility and Robustness of High-Throughput S1500+ Transcriptomics on Primary Rat Hepatocytes for Chemical-Induced Hepatotoxicity Assessment. Current Research in Toxicology. Elsevier B.V., Amsterdam, NETHERLANDS, 2: 282-295, (2021).

We introduce a new high-throughput transcriptomics (HTTr) platform comprised of a collagen sandwich primary rat hepatocyte culture and the TempO-Seq assay for screening and prioritizing potential hepatotoxicants. We selected 14 chemicals based on their risk of drug-induced liver injury (DILI) and tested them in hepatocytes at two treatment concentrations. HTTr data was generated using the TempO-Seq whole transcriptome and S1500+ assays. The HTTr platform exhibited high reproducibility between technical replicates (r>0.9) but biological replication was greater for TempO-Seq S1500+ (r>0.85) than for the whole transcriptome (r>0.7). Reproducibility between biological replicates was dependent on the strength of transcriptional effects induced by a chemical treatment. Despite targeting a smaller number of genes, the S1500+ assay clustered chemical treatments and produced gene set enrichment analysis (GSEA) scores comparable to those of the whole transcriptome. Connectivity mapping showed a high-level of reproducibility between TempO-Seq data and Affymetrix GeneChip data from the Open TG-GATES project with high concordance between the S1500+ gene set and whole transcriptome. Taken together, our results provide guidance on selecting the number of technical and biological replicates and support the use of TempO-Seq S1500+ assay for a high-throughput platform for screening hepatotoxicants. FASTQ files and read counts data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) (GSE152128). This dataset is associated with the following publication: Lee, F., I. Shah, Y.T. Soong, J. Xing, I.C. Ng, F. Tasnim, and H. Yu. Reproducibility and Robustness of High-Throughput S1500+ Transcriptomics on Primary Rat Hepatocytes for Chemical-Induced Hepatotoxicity Assessment. Current Research in Toxicology. Elsevier B.V., Amsterdam, NETHERLANDS, 2: 282-295, (2021).

The files in this data release are RNA seq datafiles from a study that examined the effects of the synthetic anabolic steroid 17β hydroxyestra 4,9,11 trien-3-one, trenbolone (17βT - CAS 10161-33-8), a common contaminant of wastes from confined animal feeding operations (CAFOs). Japanese quail (Coturnix japonica) were exposed in the egg and through feed to multiple doses of 17βT and liver transcriptomes were examined to identify genes and pathways directly affected by this androgenic compound. RNA was extracted from liver of adults and embryos and analyzed (1x50 bp) on an Illumina HiSeq 2000. NCBI Biosample accessions and the raw counts that were input into the differential expression analysis are provided in this dataset. The raw sequence data are available at the NCBI Sequence Read Archive under Bioproject numbers PRJNA313918 and PRJNA313931.