Contents

The NAM toolbox described in the manuscript is composed of four new approach methodology (NAM) platforms. These are:

1.      In vitro pharmacological profiling (IPP) to assess activity of chemicals against receptors of pharmacological interest

2.      A cellular stress panel (CSP) to assess potential of chemicals to induce chemical stress

3.      The ReproTracker assay to assess the perturbation of early embryonic development in cardiomyocyte, hepatocyte-like (HLC) and neuronal cell differentiation from human induced pluripotent stem cells (hiPSCs).

4.      The devTox quickPredict assay to predict the concentration which elicts developmental toxicity using hiPSCs.

5.      The H295R steroidogenesis assay with AR/ER CALUX detection described in OECD TG 456.

6.      HTTr metadata of generated data using the TempO-Seq platform (raw data reposited in Biostudies)

Data for each NAM are shared within this package. Some data from the IPP, CSP and HTTr assays have previously been shared also at https://doi.org/10.5061/dryad.fbg79cnx1 and https://doi.org/10.5061/dryad.v6wwpzh57{style="font-size: 11pt;"}. Where assay data have been analysed internally (SERs, Unilever), raw and pre-processed data has been shared here ready for dose response modelling. Where PoDs have been generated by the assay provider, only these values have been shared in this data package. HTTr data specific to this study are given under Biostudies accession number E-MTAB-15274. In addition a subset of samples from Biostudies accession numbers E-MTAB-15258 and E-MTAB-15266 and ENA project PRJEB75746 were included in this study and are specified here.

The contents of this package are as follows:

Cell Stress Panel – all data generated within the CSP platform. The data are all comma separated value (.csv) file types. File <CYP_ID><ROUND_ID><assay_name>-httr-support-file*. contains the raw cell stress panel data for assay <assay_name>. The data are stored in a flat format, with each row corresponding to unique measurement sample, with the following columns:

IPP – data and results (screening and concentration-response) for the IPP platform.

The file ‘IPP results Master Table.xlsx’ contains two sheets, ‘%response screening’ and ‘IC50 dose response 'Bayesian PoDs’.

The first sheet contains a table with the following columns:

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.

The second sheet (‘IC50 dose response Bayesian pod’) contains the IC50 values that were calculated from the following dose response experiments for responses over 50%. The table follows the same format as in the first sheet, except the entries are the 95% confidence interval for the IC50 value. Blank values exist where dose response modelling was not performed (where response <50%). 

The file ‘Supplementary_Data_IPP_response.xlsx’ contains the dose response data, where each sheet corresponds to a single chemical (as indicated by the sheet name). The data in each sheet is organised as a table, with the following columns:

If no dose response experiment was conducted for a particular chemical, then the sheet will appear blank.

ReproTracker - raw and processed Ct values from the biomarker qPCR and raw RFU values from the AlamarBlue cytotoxicity assay. Contains combined metadata and Ct values of samples tested in the qPCR assay in file Reprotracker_norm_cts.csv and extracted RFU values from the AlamarBlue assay in file Reprotracker_AlamarBlue.csv.

Data in the combined metadata Ct values files has can be found in 'ReproTracker_round1_norm_cts.csv' for 'evaluation round 1' data and 'ReproTracker_round2_round3_norm_cts.csv' for 'evaluation round 2 and 3'. Data have been split based on differences in experimental design and availability of metadata at run time of the individual studies. Each row relates to a sample-biomarker-value-type combination. The VALUE_TYPE column and associated VALUE column contains raw (CT) and processed data (ECT, log_ECT, logEdeltaCt) with logEdeltaCT values being the final normalised values used for dose response.

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**Column names in upper case are those provided by assay provider (Toxys) and those in lower case are generated in data processing. *Only VALUE_TYPEs of Cq were provided by Toxys and are raw data, other values are processed/calculated values.

Note: The SOX1 biomarker was not used/considered for analysis based on recommendation from Toxys regarding biological relevancy

AlamarBlue files contains Relative Fluorescence Units (RFU) values used for cytotoxicity modelling. The AlamarBlue assay was not run for the round 1 study. Data is organised with the following columns:

H295R - Steroidogenesis assay with AR/ER CALUX detection LOECs (Lowest Observed Effect Concentrations) and CALUX pre-screening results.

The file ‘H295R results summary.csv' gives the LOECs of the H295R steroidogenesis assay using ER- and AR-CALUX. 'Calux results summary.csv' gives the pre-screening CALUX assay LOECs. Data is organised with the following columns:

*The H295R assay medium is diluted at least 100-fold for the CALUX® assay. The concentration stated in the  'Calux results summary' is thus the highest non-cytotoxic concentration tested in H295R ('H295R results summary') divided by 100. This concentration must be lower than the LOEC in the “pre-screening CALUX®” columns to ensure that the observed activity is specific to H295R hormones and cannot be attributed to test compound carry over.

**not determined as the potency of the tested compound in the read-out assay was too high to determine E2 variations in H295R

devTox quickPredict PoDs - cell viability and developmental toxicity potential PoD results.

The file 'devTox_quickPredict_PoDs.xlsx' contains a table of PoDs generated for both cell viability and development toxicity potential (dTP), the table's contents are described below:

*Zurlinden TJ, Saili KS, Rush N, Kothiya P, Judson RS, Houck KA, Hunter ES, Baker NC, Palmer JA, Thomas RS, Knudsen TB. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. Toxicol Sci. 2020 Apr 1;174(2):189-209. doi: 10.1093/toxsci/kfaa014. PMID: 32073639; PMCID: PMC8527599.

HTTr – HTTr data specific for this study consist of TempOSeq data and are given under Biostudies accession number E-MTAB-15274. In addition a subset of samples from Biostudies accession numbers E-MTAB-15258 and E-MTAB-15266 and ENA project PRJEB75746 were included in this study and are specified here. The metadata are given here, with each row corresponding to an single sample, with the following columns: