DOI: 10.5061/dryad.qz612jmt7
title: An advancement in developmental and reproductive toxicity (DART) risk assessment: evaluation of a bioactivity and exposure-based NAM toolbox

description: Traditional chemical safety assessment involves identifying the lowest
 level of a chemical that impacts endpoints measured in standardized animal
 studies together with appropriate safety factors to establish human
 exposure limits. In vitro assays have shown promise in providing points of
 departure that can be protective of human health when combined with
 exposure predictions into a bioactivity:exposure ratio (BER). Using a
 combination of broad screening tools and DART-targeted assays, we
 previously demonstrated high biological coverage of this NAM toolbox
 against a list of DART-relevant genes and pathways. To fully transition to
 an animal-free paradigm, it is crucial to establish confidence that these
 in vitro assays sufficiently represent the DART toxicity mechanisms,
 ensuring a level of protection that is safe for non-pregnant adults,
 pregnant women, and fetal populations. In this proof-of-concept study, we
 have extended the toolbox to include additional in vitro and in silico
 tools and have performed an evaluation using 37 benchmark compounds across
 49 exposure scenarios. According to existing regulatory opinions, 18 of
 these scenarios would be considered high-risk chemical exposures from a
 DART perspective. Our DART NAM toolbox approach identified 17 out of these
 18 high-risk scenarios. We further investigated the impact of gross
 physiological changes in pregnancy and the fetus on internal exposures by
 evaluating human clinical data where available for the 37 compounds. In
 most instances, the variability resulting from pregnancy or gestational
 changes falls within the range of toxicokinetic variability observed in
 the general population. This work demonstrates that protective safety
 decisions can be made for DART without generating new animal test data.
description: # An advancement in developmental and reproductive toxicity (DART) risk
 assessment: evaluation of a bioactivity and exposure-based NAM toolbox
 **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](https://doi.org/10.5061/dryad.fbg79cnx1) and [https://doi.org/10.5061/dryad.v6wwpzh57](#0){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](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15274)*. In addition a subset of samples from Biostudies accession numbers *[E-MTAB-15258](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15258)* and *[E-MTAB-15266](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15266)* and ENA project *[PRJEB75746](https://www.ebi.ac.uk/ena/browser/view/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 &lt;*assay_name*&gt;-httr-support-file*. contains the raw cell stress panel data for assay . 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 plate) | | WELL\_ID | Well ID | | DROP\_TYPE | Drop type. If blank then no drop condition is applied | | DROP\_CODE | Drop code. If blank then no drop condition is applied | | 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) | **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: | **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. 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 &lt;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: | **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. **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. ![](ReproTracker_equations.png){width="460"} | **Column name** | **Description** | | ------------------------------- | ------------------------------------------------------------------------------------------------------- | | STUDY\_DESC\_NAME | Study/project name/description | | LINEAGE | Lineage | | EXPERIMENTAL\_CODE | Experiment batch number | | EXPERIMENTAL\_CODE\_START\_DATE | Start of differentiation | | SAMPLE\_DATE | Sample date | | EXPERIMENT\_TYPE | Differentiation type | | TEST\_SUBSTANCE | Test substance name | | TEST\_SUBSTANCE\_CODE | Test substance code | | CONCENTRATION | Test concentration (uM) | | EXPOSURE\_TIME | Day of assay measurement | | EXPOSURE\_TIME\_UNIT | Exposure time unit | | WELL\_ID\_EXPOSURE | Well ID of exposure plate | | PLATE\_ID\_EXPOSURE | Plate ID of exposure plates | | BIOLOGICAL\_REPLICATE\_NUMBER | Biological replicate number | | TECHNICAL\_REPLICATE\_NUMBER | Technical replication description | | RNA\_NUMBER | RNA number | | BIOMARKER | Biomarker measured | | BIOMARKER\_DYE | Dyes used for biomarker detection | | tech\_group | Unique identifier for technical replicate group | | PLATE\_ID\_CDNA | Plate ID of CDNA plates | | WELL\_ID\_CDNA | Well ID of CDNA plates | | QPCR\_RUN\_NAME | Qpcr run name | | WELL\_ID\_QPCR | WELL\_ID\_QPCR | | VALUE\_TYPE | Value description | | VALUE | Value | | DROP\_CODE | Drop code. If NA, then no drop condition is applied | | failed | Boolean flag for when raw Ct in more than 2/3 of replicates is NA or &gt;40 | | isna | Boolean flag for when raw Ct of individual replicate is NA or &gt;40 | | mean\_CT | Mean raw Ct across non NA values of the technical replicate group | | sd | Sd of raw Ct across non NA values of the technical replicate group | | sd\_above\_0.35 | Boolean flag is sd &gt;0.35 | | sd\_above\_0.5 | Boolean flag is sd &gt;0.5 | | remove | Boolean flag to remove samples from dose-response modelling if sd &gt; 0.5, failed = TRUE, or is.na = TRUE | | amp\_value | Amplification efficiency relating to biomarker | | out\_of\_bound\_amp\_eff | Boolean if amp\_value is outside 90-110 % range | \**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: | Column name | Description | | ----------------------------- | --------------------------- | | EXPERIMENT\_TYPE | Differentiation type | | LINEAGE | Lineage | | TEST\_SUBSTANCE | Test substance name | | TEST\_SUBSTANCE\_CODE | Test substance code | | CONCENTRATION | Test concentration | | CONCENTRATION\_UNIT | Test concentration unit | | EXPOSURE\_TIME | Day of assay measurement | | EXPOSURE\_TIME\_UNIT | Exposure time unit | | EXPERIMENT | Experiment batch number | | WELL\_ID\_EXPOSURE | Well ID of exposure plate | | TECHNICAL\_REPLICATE\_NUMBER | Technical replicate number | | BIOLOGICAL\_REPLICATE\_NUMBER | Biological replicate number | | VALUE | RFU value | | VALUE\_TYPE | Value description | | ADDITIONAL\_NOTES | Plate format | | STUDY\_DESC\_NAME | Study/project/description | **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: | Column name | Description | | ------------------------------------------------------- | ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | Compound # | Test substance assigned number | | Compound name | Test substance name | | CAS | Test substance CAS number | | \ LOEC (M) | Calculated lowest observed effect concentration (LOEC) for the given readout in molar units. ND= not determined as the potency of the tested compound in the read-out assay was too high to determine E2 variations in H295R. NA = No LOEC calculated. | | Highest non-cytotoxic concentration tested in H295R (M) | Test substance highest concentration tested in the H295R assay in molar units.  | | E2/T Level | 'UP' indicating increase or 'DOWN' indicating decrease direction of change. '\**' =* the compound is showing activity but only at one concentration, at the highest concentration tested it exceeds the threshold of activity. ND = not determined\*\*\*.\* NA = no response measured | | OECD evaluation (yes/no) | Determination of positive result based on OECD criteria. YES = 2 successive concentrations exceeding the threshold of activity. | | Highest concentration tested in CALUX\* (M) | Test substance highest concentration tested in the CALUX pre-screen in molar units | | EXPOSURE\_TIME\_UNIT | Exposure time unit | *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: | Column name | Description | | --------------- | ------------------------------------------------------------------------------------------- | | Test Article ID | Test substance name | | HTC (uM) | Highest tested concentration in micromolar | | dTP (uM) | Developmental toxicity potential point of departure in micromolar. NA = no value calculated | | viability (uM) | Cell viability/cytotoxicity point of departure in micromolar. NA = no value calculated | | Source | Source of PoD; Unilever generated data or from Zunderlin et al (2020)\* | \**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](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15274)*. In addition a subset of samples from Biostudies accession numbers *[E-MTAB-15258](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15258)* and *[E-MTAB-15266](https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-15266)* and ENA project *[PRJEB75746](https://www.ebi.ac.uk/ena/browser/view/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: | **Column name** | **Description** | | :---------------------- | :--------------------------------- | | SAMPLE\_ID | Sample ID number | | CELL\_TYPE | Cell type | | TEST\_SUBSTANCE | Test substance | | CONCENTRATION | Concentration of test substance | | CONCENTRATION\_UNITS | Concentration units | | EXPOSURE\_TYPE | Exposure type | | EXPOSURE\_TIME | Exposure time | | EXPOSURE\_UNITS | Timepoint units | | TREATMENT\_VESSEL\_TYPE | Vessel type (e.g., 384 well plate) | | TREATMENT\_VESSEL\_ID | Vessel ID (i.e., of plate) | | TREATMENT\_WELL\_ID | Well ID | | FASTQ\_FILENAME | Name of fastq file | | SEQUENCING\_PLATE\_ID | Sequencing plate ID | | SEQUENCING\_WELL\_ID | Sequencing well ID |