DOI: 10.5061/dryad.v6wwpzh57
title: Advancing systemic toxicity risk assessment: Evaluation of a NAM-based toolbox approach

description: For many years, a method that allowed systemic toxicity safety assessments
 to be conducted without generating new animal test data, seemed out of
 reach. However, several different research groups and regulatory
 authorities are beginning to use a variety of in silico, in chemico, and
 in vitro techniques to inform safety decisions. To manage this transition
 to animal-free safety assessments responsibly, it is important to ensure
 that the level of protection offered by a safety assessment based on new
 approach methodologies (NAMs), is at least as high as that provided by a
 safety assessment based on traditional animal studies. To this end, we
 have developed an evaluation strategy to assess both the level of
 protection and the utility offered by a NAM-based systemic safety
 “toolbox.” The toolbox comprises physiologically based kinetic models to
 predict internal exposures, and bioactivity NAMs designed to give broad
 coverage across many different toxicity modes of action. The output of the
 toolbox is the calculation of a bioactivity:exposure ratio (analogous to a
 margin of internal exposure), which can be used to inform decision-making.
 In this work, we have expanded upon an initial pilot study of 10 chemicals
 with an additional 38 chemicals and 70 consumer exposure scenarios. We
 found that, for the majority of these (&gt;90%), the NAM-based
 workflow is protective of human health, enabling us to make animal-free
 safety decisions for systemic toxicity and preventing unnecessary animal
 use. We have also identified critical areas for improvement to further
 increase our confidence in the robustness of the approach.
description: # **Readme for the supplementary data/results package for Cable et el.
 2025.** **Contents** The NGRA toolbox described in the manuscript is
 composed of four new approach methodology (NAM) platforms. These are:
 1.      PBK modelling for estimation of internal exposures 2.      In
 vitro pharmacological profiling (IPP) to assess activity of chemicals
 against receptors of pharmacological interest 3.      A cellular stress
 panel (CSP) to assess potential of chemical to induce chemical stress
 4.      A high-throughput transcriptomics (HTTr) platform to assess
 biological activity towards other endpoints Data for each NAM are shared
 within this package. The contents of the package are as follows: **Cell
 stress panel data/Data** – all data generated within the CSP platform. The
 data are all comma separated value (.csv) file types. File
 __&lt;*assay_name*&gt;-httr-support-file*. contains the raw cell stress panel data for assay &lt;*assay_name*&gt;. The data are stored in a flat format, with each row corresponding to unique measurement sample, with the following columns: | **Column name** | **Description** | | :------------------- | :--------------------------------------------------------------- | | SAMPLE\_ID | Sample ID number | | FILE\_NAME | File name | | ASSAY\_TYPE | Assay type | | MEASUREMENT\_TYPE | Type of measurement | | CELL\_TYPE | Cell type | | CELL\_BATCH\_ID | Cell Batch ID | | SUPPLIER\_ID | Supplier ID | | MEASUREMENT\_DATE | Measurement date | | TEST\_SUBSTANCE | Test substance | | CONTROL\_FLAG | Control Flag (1 indicates positive control, -1 negative control) | | CONCENTRATION | Concentration of test substance | | CONCENTRATION\_UNITS | Concentration units | | VALUE | Value of measurement in MEASUREMENT\_TYPE | | VALUE\_UNITS | Units of measurement | | EXPOSURE\_TYPE | Exposure type | | EXPOSURE\_TIME | Exposure time | | TIMEPOINT\_UNITS | Timepoint units | | VESSEL\_ID | Vessel ID (i.e., of plate) | | VESSEL\_TYPE | Vessel type (e.g., 384 well place) | | WELL\_ID | Well ID | | DROP\_TYPE | Drop type | | DROP\_CODE | Drop code | | CYPR\_ID | Cyprotex (CRO which generated the data) internal ID | | CYP\_ID | Cyprotex study ID (appears in filename) | | ROUND\_ID | Study round ID (appears in filename) | ** ** **HTTr data/Data** – all raw data generated using high throughput transcriptomics. Contains the counts table (Phase3_round1_publication_counts.csv, Phase3_round2_publication_counts.csv), meta data (Phase3_round1_publication_meta.csv, Phase2_round1_publication_meta.csv), manifest (190620 Human Whole Transcriptome 2.0 Manifest.xlsx, 20SEPT2021+Human+Whole+Transcriptome+2.1+Manifest.xlsx) and attenuation manifest (190620 Human Whole Transcriptome 2.0 Attenuation Manifest.xlsx, 100739 Human Whole Transcriptome 2.1 Attenuation Factors.xlsx) generated within the HTTr platform. The metadata tables indicate which manifest version was used to generate the counts (see PROBE_MANIFEST_VERSION column). The counts table has following format: each row corresponds to a probe (e.g., CHAC1_1279, as indicated by the 1st column) and each column to a sample ID (e.g., S_50-50ratio_HG2_P1). Numerical entries are probe counts per condition. The meta data files gives details of each condition and contains the following columns: | **Column name** | **Description** | | :----------------------------- | :--------------------------------------------------------------- | | SAMPLE\_ID | Sample ID number | | CELL\_TYPE | Cell type | | CELL\_BATCH\_ID | Cell batch ID | | TEST\_SUBSTANCE | Test substance | | CONTROL\_FLAG | Control flag (1 indicates positive control, -1 negative control) | | CONCENTRATION | Concentration | | CONCENTRATION\_UNITS | Concentration units | | EXPOSURE\_TYPE | Exposure type | | EXPOSURE\_TIME | Exposure time | | TIMEPOINT\_UNITS | Timepoint units | | VESSEL\_ID | Vessel ID | | VESSEL\_TYPE | Vessel type | | WELL\_ID | Well ID | | FASTQ\_FILENAME | FastQ filename | | SUPPLIER | Supplier | | MEASUREMENT\_DATE | Measurement date | | NUM\_UNMAPPED\_READS | Number unmapped reads | | NUM\_MAPPED\_READS | Number mapped reads | | PERCENT\_MAPPED\_READS | Percent mapped reads | | SEQUENCING\_PLATE\_ID | Sequencing plate ID | | SEQUENCING\_WELL\_ID | Sequencing well ID | | ATTENUATION\_MANIFEST\_VERSION | Attenuation manifest version | | PROBE\_MANIFEST\_VERSION | Probe manifest version | | LYSIS\_BUFFER | Lysis buffer | | FLOW\_CELL\_CHANNEL | Flow cell channel |   The attenuation manifest is a table that contains two columns: the probe ID and the corresponding attenuation factor.  The manifests provided contains the following columns: | **Column name** | **Description** | | :------------------------ | :---------------------------------------- | | Probe Name | Probe ID | | Gene Symbol | Gene symbol | | Entrez ID | Entrez ID | | ENSEMBL Gene ID | ENSEMBL Gene ID | | Probe Sequence | Sequence of the probe | | Probe Genomic Coordinates | Genomic coordinate of the probe | | Transcripts Covered | Transcripts that are covered by the probe |   **IPP data** – data and results (screening and concentration-response) for the IPP platform. The file ‘IPP results Supp Table Paper.xlsx’ contains two sheets, ‘%response screening’ and ‘IC50 dose response Bayesian mod’. The first sheet contains a table with the following columns: | **Column name** | **Description** | | :--------------- | :---------------------------- | | Assay name | Name of the assay | | Ligand/control | Ligand used in the experiment | | Response/Readout | Type of readout |   These are followed by additional columns for each test substance and screening concentration (as indicated by the column header). Values are the percentage of the response, with those exceeding 50% highlighted in bold. The second sheet (‘IC50 dose response Bayesian mod’) contains the IC50 values that were calculated from the follow dose response experiments. The table follows the same format as in the first sheet, except where the entries are the 95% confidence interval for the IC50 value.   The file ‘IPP Supplementary Data dose response Paper’ contains the dose response data, which each sheet corresponding to a single chemical (as indicated by the sheet name). The data in each sheet is organised as a table, with the following columns: | **Column name** | **Description** | | :-------------- | :-------------------------------------------- | | Compound | Compound that was tested | | Target | Pharmacological target | | Replicate | Replicate number | | Dose (uM) | Dose in micromolar | | Response % | Response of target to compound in percentage. |   If no dose response experiment was conducted for a particular chemical, then the sheet will appear blank.   **PBK data** – contains text files which were generated automatically using the software Gastroplus. Each text file contains all the input and output details for each PBK run (see main text for details), which the name of text indicating the chemical, exposure scenario and PBK parameterisation level.   **Contact** For additional queries pertaining to the data/results package, please contact the primary author of the main manuscript (S Cable).