Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 500-234-8 | CAS number: 68891-38-3 1 - 2.5 moles ethoxylated
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Specific investigations: other studies
Administrative data
Link to relevant study record(s)
- Endpoint:
- biochemical or cellular interactions
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09-05-2022 - 09-08-2022
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- This study was based on the paper of Hatherell et. al. (Identifying and Characterizing Stress Pathways of Concern for Consumer Safety in Next-Generation Risk Assessment, Toxicol Sci. 2020;176(1):11-33).
An in vitro cell stress panel study was conducted to identify concentrations associated with induction of cellular stress pathways and therefore potentially adverse health effects. First, the solubility of SLES C12-14 linear (hereafter referred to as SLES) in matrix matched conditions was assessed. Then a cytotoxicity assessment utilising HepG2 cells measuring ATP & LDH content (Promega) was conducted to determine the maximum concentrations of SLES to use within the cell stress panel. HepG2 cells were exposed to SLES for 24h and a variety of cell stress assessments (18 assays assessing mitochondrial toxicity, cell stress, and cell health) was carried out. The BIFROST statistical model was used to analyse the concentration-response data for each biomarker and estimate a minimum effect level. Following this, a global point of departure (PoD) was calculated to represent a minimum effect level across all the cellular stress assays. - GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- other: Mitochondrial toxicity, cell stress, and general cell health
- Details on results:
- After exposure to SLES for 24 hours, all but 5 of the 36 biomarkers assessed (AhR Translocation, Membrane Permeability, DNA Content, Phospho-p53 and Heme oxygenase 1) showed a response to treatment with the PoDs calculated to be in the range of 14 µM (CDS = 1.00) to 540 µM (CDS = 0.81). From the data across all assays, a global PoD of 9.8 µM was calculated.
Several biomarkers were considered to respond to treatment with SLES under the conditions of this assay, though the majority of responses were associated with cytotoxicity in the test system (see below). - Conclusions:
- Based on the results of an in vitro cell stress panel study, several biomarkers were considered to respond to treatment with SLES, though the responses were associated with cytotoxicity in the test system. The global PoD is calculated to be 9.8 µM.
- Executive summary:
SLES was assessed in a low-tier broad-spectrum, cell stress panel in HepG2 cells consisting of 36 biomarkers representing oxidative stress, DNA damage, inflammation, endoplasmic reticulum stress, metal stress, heat shock, hypoxia as well as mitochondrial toxicity and general cellular health, measured predominantly using high content imaging. Measurements for the cell stress were analysed using Bayesian concentration-response models called BIFROST (Bayesian inference for region of signal threshold) to obtain the Point of Departure (PoD) estimate in terms of the nominal concentration. The results of this in vitro cell stress panel study indicate that several biomarkers responded to treatment with SLES, though the responses were associated with cytotoxicity in the test system. The global PoD is calculated to be 9.8 µM.
- Endpoint:
- toxicogenomics
- Remarks:
- transcriptomics assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23-03-2022 to 23-05-2022
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- other: Composition of specific tested batch
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The objective of this study was to determine the effect of SLES C12-14 Linear (hereafter referred to as SLES) on RNA expression. HepG2, MCF-7 and HepaRG cells were exposed to SLES for 24 hours. An initial cytotoxicity study was conducted to determine doses for the main cell lysate generation (both lactate dehydrogenase (LDH) and cellular adenosine triphosphate (ATP) content were assessed). Cell lysate samples were generated and analysed by transcriptomics, in addition matching cytotoxicity measurements were taken (LDH content only). Two different models were used to interpret the transcriptomics data: (1) the global point of departure (PoD) derived using the BIFROST model and (2) the minimum bench mark dose level (BMDL; the lower bound of the pathway-average Benchmark concentration) obtained using BMDexpress. The global PoD is designed to identify a gene-level ‘no observed transcriptional effect level’ (NOTEL) whereas the BMDL is intended to identify the lowest concentration that causes a perturbation of the transcriptome at the pathway level.
- GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- other: cellular responsiveness
- Details on results:
- The full data sets for the different cell lines are attached below as full reports.
- Conclusions:
- The results indicate that, in the cell lines tested, there is no change to gene expression levels at concentrations up to 1.4 µM.
- Executive summary:
A transcriptomics assay was performed with SLES to characterise perturbations in RNA transcription across the whole genome and to provide a non-targeted approach to capture biological effects potentially undetected using the other bioactivity platforms. Multiple cell models (HepG2, HepaRG, and MCF-7) were included to increase biological coverage and two different models were used to analyse the transcriptomics data and estimate a Point of Departure (PoD): the global PoD derived using the BIFROST model, and the minimum benchmark dose lower confidence limit (BMDL) (the lower bound of the pathway-average Benchmark concentration) obtained using BMDexpress2. The global PoD is designed to identify a gene-level ‘no observed transcriptional effect level’ (NOTEL) whereas the BMDL is intended to identify the lowest concentration that causes a perturbation of the transcriptome at the pathway level. This is useful additional information because isolated changes in individual genes may not represent a meaningful biological difference, whereas changes in the expression of several genes within a pathway may be more biologically relevant.
The lowest PoD observed in the HTTr assay was in the MCF-7 cell line. Of 12375 probes analysed, only 108 had a confidence score of >0.5 (indicating the target of the probe was differentially expressed at some concentration less than the maximum tested). The lowest PoD using the BIFROST method was 1.4µM (nominal concentration) due to a . Pathway analysis was also conducted. Pathway BMDLs are significant if they have >2 genes found within the pathway, have >1 gene with a significant BMDL and a 2-tailed fisher p-value <0.1. No pathways were found to be significantly perturbed by treatment of SLES in MCF-7 cells so no significant pathway BMDLs were calculated. The lowest BMDL was 43.2µM (nominal concentration) in HepG2 cells. The biological relevance of the single gene perturbation in expression in MCF-7 cells is unknown but using this as the lowest PoD is the most conservative approach in calculating BERs from HTTr data.
- Endpoint:
- mechanistic studies
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- December 2022 - February 2023
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- according to guideline
- Guideline:
- other: OECD, Guidance Document on Good In Vitro Method Practices (GIVIMP), OECD Series on Testing and Assessment, No. 286, OECD Publishing, Paris.
- Version / remarks:
- 2018
- Deviations:
- no
- Principles of method if other than guideline:
- The goal of this study was to assess the embryotoxicity potential of Alcohols, C12-14, ethoxylated, sulfates, sodium salts (hereafter referred to as SLES), by by measuring effects on differentiation of human induced pluripotent stems cells (hiPSCs) on the compounds. Differentiation of hiPSCs is used to mimic early human development and perturbations of the underlying signalling events have been shown to be predictive of embryotoxic substances (Jamalpoor et al., 2022).
The ReproTracker assay is based on differentiation of hiPSCs into cardiomyocytes, hepatocytes and neural rosettes, which arise from mesoderm, endoderm and ectoderm, respectively. The influence of SLES on this differentiation process was determined by monitoring alterations in expression patterns of tissue specific biomarkers in a dose dependent way. To better determine exposure doses the range of doses to be tested, a dose range finding (DRF) study was performed first.
Following completion of the DRF experiment, a top concentration for progression into the tri-lineage differentiation assay was selected for SLES. An initial top concentration of 203.30 μM was used in the first round of differentiation towards all lineages; however, unacceptable cytotoxicity was encountered during the liver differentiation protocol. Therefore, SLES was retested in a second round of differentiation towards this lineage. When performing the second round of differentiation towards liver, the fold-dilution for the CRC was changed from 2- to 3-fold for all tested substances.
Selected test concentrations for differentiation were 6.35 - 203.3 µM for heart and neural lineages, and 0.24 - 58.4 µM for liver.
--
Jamalpoor A et al. (2022). A novel human stem cell-based biomarker assay for in vitro assessment of developmental toxicity. Birth Defects Research 114, 1210-1228. - GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- developmental toxicity / teratogenicity
- Dose / conc.:
- 0 other: µM
- Remarks:
- Solvent only (dH2O)
- Dose / conc.:
- 6.35 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 12.71 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 25.41 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 50.83 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 101.65 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 203.3 other: µM
- Remarks:
- Heart and neural differentiation
- Dose / conc.:
- 0.24 other: µM
- Remarks:
- Liver differentiation
- Dose / conc.:
- 0.72 other: µM
- Remarks:
- Liver differentiation
- Dose / conc.:
- 2.16 other: µM
- Remarks:
- Liver differentiation
- Dose / conc.:
- 6.49 other: µM
- Remarks:
- Liver differentiation
- Dose / conc.:
- 19.47 other: µM
- Remarks:
- Liver differentiation
- Dose / conc.:
- 58.4 other: µM
- Remarks:
- Liver differentiation
- Details on results:
- Dose-range finding/Cytotoxicity Testing:
Based on the data presented in Figure 1 (see attachment), a concentration of 203.3 µM was selected as the maximum concentration in the tri-lineage differentiation assay.
Differentiation:
There were no observed morphological differences between treated and untreated differentiated cells. In all three lineages, differentiation progressed as anticipated.
Of the six biomarkers considered indicative of embryotoxic potential (BMP4 day 7, MYH6 day 14, FOXA2 day 7, AFP day 21, PAX6 days 7 and 13 and NESTIN day 13), two (HLC biomarker AFP and neural rosette biomarker PAX6) showed a dose response to treatment with SLES. Calculated BMDLs were 2.7 and 4.7 µM, respectively (see also Figures 11 and 12 in attachment). - Conclusions:
- The effect of SLES on the differentiation of hiPSCs to cardiomyocytes, hepatocytes and neural rosettes was assessed in the ReproTracker assay. Incubation with SLES did not cause any morphological changes, and the cells differentiated as anticipated. Statistical analysis of the dose response of two genes associated with potential embryotoxicity (liver AFP and neural PAX6) returned PODs of 2.7 and 4.7 µM, respectively.
- Executive summary:
SLES was tested in the Reprotracker assay, which is a human induced pluripotent stem cell (hiPSC)-based biomarker assay that assesses embryotoxicity. Pluripotent stem cells, under specified growth conditions, can be triggered to differentiate into all 3 germ layers (meso-, endo- and ectoderm) and further to the development of specific cell lineages, such as hepatocytes, cardiomyocytes and neuronal cells. Such differentiation processes are complex and tightly regulated and are susceptible to perturbations by teratogenic substances. By assessing the expression pattern of specific differentiation-related genes via qRT-PCR (OCT4 as a pluripotency marker, BMP4 as a mesoderm marker, FOXA2 as an endoderm marker, and MYH6, AFP, and PAX6/NESTIN as the cardiomyocyte-, hepatocyte-, and neural rosette-specific markers, respectively), the progress of differentiation and whether a compound interferes with embryonic development can be followed.
Incubation with SLES did not cause any morphological changes, and the cells differentiated as anticipated. Statistical analysis of the dose response of two genes associated with potential embryotoxicity (liver AFP and neural PAX6) returned PODs of 2.7 and 4.7 µM, respectively.
- Endpoint:
- biochemical or cellular interactions
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2022
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The developmental toxicity potential of SLES was assessed by the use of Stemina's devTOX quickPredict assay. The assay was conducted on human induced pluripotent stem cells (hiPSCs) and the disturbance in cellular metabolism caused by a 48-hour exposure to SLES was measured.
Upon exposure of hiPSCs to a test article, the ornithine and cysteine concentration in the media are measured. A decrease of the ornithine and cysteine ratio (o/c ratio) indicates developmental toxicity potential, and a PoD can be defined. Cell viability is tested in parallel to understand whether metabolic changes occur independently of effects on cell health. A decrease in cell viability may result from a cytotoxic, cytostatic, or antiproliferative effect of the test article, which could also negatively impact embryonic development. The developmental toxicity potential (dTP, o/c ratio) and toxicity potential (TP, cell viability) PoDs are calculated from the respective dose response curves using the hiPSC developmental toxicity threshold (dTT, 0.85) described in Palmer et al. (2013). The lowest PoD (either dTP or DT) is taken as the final assay PoD.
The assay was conducted in accordance with the following publications:
Palmer JA, Smith AM, Egnash LA, Conard KR, West PR, Burrier RE, Donley ELR and Kirchner FR (2013). Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening. Birth Defects Research (Part B) 98, 343-363.
Zurlinden TJ, Saili KS, Rush N, Kothiya P, Judson RS, Houck KA, Hunter ES, Baker NC, Palmer JA, Thomas RS and Knudsen TB (2020). Profiling the ToxCast library with a pluripotent human (H9) stem cell line-based biomarker assay for developmental toxicity. Toxicological Sciences 174, 189-209.
Palmer J (2021). Developmental Toxicity of Chemicals Tested in devTOXqP. Stemina internal documentation, updated May 2021. - GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- developmental toxicity / teratogenicity
- Details on results:
- At the exposure levels evaluated, SLES elicited a response in the o/c ratio independent of changes in cell viability. The lower point of departure was the dTP (o/c ratio) of 39.5 µM.
The positive control elicited a response in the o/c ratio of 0.13 within the defined acceptance criteria, and the negative control a o/c ratio response of 0.92, establishing that hiPSC metabolism was within assay specifications.
See also table below and figures attached in "Overall remarks, attachments". - Conclusions:
- In this in vitro study, the developmental toxicity potential of SLES was assessed by measuring its impact on human induced pluripotent stem cell (hiPSC) metabolism and cell viability at test concentrations of 3-10,000 µM. Upon exposure of hiPSCs to a test article, the ornithine and cysteine concentration in the media are measured. A decrease of the ornithine and cysteine ratio (o/c ratio) of the test article at or above that concentration indicates developmental toxicity potential. At the exposure levels evaluated, SLES elicited a response in the o/c ratio independent of changes in cell viability. The point of departure was the dTP (o/c ratio) of 39.5 µM.
- Executive summary:
The developmental toxicity potential of SLES was assessed in an in vitro assay (Stemina devTOX quick predict) using human induced pluripotent stem cells (hiPSCs). Cells were incubated with SLES at concentrations of 3 - 10,000 µM.
Upon exposure of hiPSCs to a test article, the ornithine and cysteine concentration in the media are measured. A decrease of the ornithine and cysteine ratio (o/c ratio) indicates developmental toxicity potential, and a PoD can be defined. Cell viability is tested in parallel to understand whether metabolic changes occur independently of effects on cell health. A decrease in cell viability may result from a cytotoxic, cytostatic, or antiproliferative effect of the test article, which could also negatively impact embryonic development. The developmental toxicity potential (dTP, o/c ratio) and toxicity potential (TP, cell viability) PoDs are calculated from the respective dose response curves using the hiPSC developmental toxicity threshold (dTT, 0.85).
At the exposure levels evaluated, SLES elicited a response in the o/c ratio independent of changes in cell viability. The point of departure was the dTP (o/c ratio) of 39.5 µM.
- Endpoint:
- biochemical or cellular interactions
- Remarks:
- In vitro test battery including binding assays, cellular and nuclear receptor functional and enzyme assays.
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 10, 2022 - January 3, 2023
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- other: Composition of specific tested batch
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Topic S 7 A Safety Pharmacology Studies for Human Pharmaceuticals
- Version / remarks:
- 2001, CPMP/ICH/539/00
- Deviations:
- yes
- Remarks:
- See "Principles of method if other than guideline"
- Principles of method if other than guideline:
- This study was performed to evaluate the specific binding, enzymatic activity and coactivator recruitment of SLES C12-14 Linear (hereafter referred to as SLES). An in vitro test battery was used including binding assays, cellular and nuclear receptor functional and enzyme assays.
Testing was done with a single concentration, instead of a range of concentrations as recommended in the guidance. The choice is based on the recommendations by Jenkinson et al (Journal of Pharmacological and Toxicological Methods 105:106869 (2020)), who recommend 10 µM as a suitable concentration when only a single concentration is tested. The assays were performed under non-GLP circumstances. The study was conducted at a Research Facility that generally works under GLP and general quality controls were applied. This implies that the non-GLP status has no further consequences for the outcome of the study. - GLP compliance:
- no
- Remarks:
- The study was conducted at a Research Facility that generally works under GLP and general quality controls were applied.
- Type of method:
- in vitro
- Endpoint addressed:
- toxicity to reproduction / fertility
- developmental toxicity / teratogenicity
- other: systemic toxicity
- Dose / conc.:
- 10 other: µM
- Remarks:
- The choice was based on the recommendations of Jenkinson et al (Journal of Pharmacological and Toxicological Methods 105:106869 (2020)), who recommend 10 µM as a suitable concentration when only a single concentration is tested.
- Details on results:
- Inhibition or stimulation exceeding 50% compared to the control value were considered to represent significant effects of the test compound. Such an effect was only observed for LXR alpha (h) agonist radioligand with relative binding of 54.3% compared to the control, though there was no accompanying activity in the functional agonist assay; an IC50 value of 2.0E-04 M was calculated in the follow-up LXR alpha (h) (antagonist effect) functional assay.
- Conclusions:
- SLES C12-14 Linear (hereafter referred to as SLES) was tested in a battery of assays including binding assays, cellular and nuclear receptor functional assays and enzyme assays.
The only effect with inhibition or stimulation exceeding 50% compared to the control value (considered to represent a significant effect of the test compound) was observed for LXR alpha (h) agonist radioligand with relative binding of 54.3% compared to the control. However in the more functionally-relevant IPP co-activator recruitment assay, SLES was only able to inactivate the LXR pathway at a relatively high concentration (IC50 = 0.2 mM) and was not able to activate the LXR pathway.
These results indicate that SLES does not interfere with targets with proven links to adverse reactions in humans, including targets relevant for reproduction and development. - Executive summary:
SLES was tested in an in vitro pharmacological profiling (IPP) platform containing 73 targets with known safety liabilities that were tested in binding, enzymatic, coactivator recruitment, and luciferase assays. Forty-four of the targets have been associated with in vivo adverse drug reactions by the pharmaceutical industry including: 24 G-protein-coupled receptors (GPCRs), 7 enzymes, 2 nuclear receptors, 8 ion channels, and 3 transporters. In response to the ECHA compliance check, to expand the above biological coverage, into targets implicated in DART, a further 29 targets were added to the panel, including: 5 nuclear hormone receptors, 11 basic helix-loop-helix transcription factors 11 enzymes, 1 GPCR, and 1 structural protein.
The only effect with inhibition or stimulation exceeding 50% compared to the control value (considered to represent a significant effect of the test compound) was observed for LXR alpha (h) agonist radioligand with relative binding of 54.3% compared to the control. However in the more functionally-relevant IPP co-activator recruitment assay, SLES was only able to inactivate the LXR pathway at a relatively high concentration (IC50 = 0.2 mM) and was not able to activate the LXR pathway.These results indicate that SLES does not interfere with targets with proven links to adverse reactions in humans, including targets relevant for reproduction and development.
- Endpoint:
- biochemical or cellular interactions
- Remarks:
- Panel of effect-based in vitro bioassays
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 16, 2022 - November 2, 2022
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- other: Composition of specific tested batch
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- This study was performed to evaluate the transcriptional activation ability of SLES C12-14 Linear (hereafter referred to as SLES). In vitro effect-based responses and cytotoxicity were determined using a panel of bioassays, consisting of thirteen CALUX® assays, a human thyroid peroxidase (hTPO)-inhibition assay and the H295R steroidogenesis assay. All assays except for the TPO-inhibition assay were performed in the absence and presence of a modular metabolic fraction (rat liver S9).
CALUX® bioassays comprise human bone cell lines (U2-OS), incorporating the firefly luciferase reporter gene coupled to Responsive Elements (REs) as a reporter gene for the presence of compounds activating the specific pathways linked to these REs (Sonneveld et al., 2005 (Toxicol Sci 83, 136-148); Van der Burg et al., 2013 (John Wiley & Sons, Inc. pp. 519-532)). The TPO-inhibition assay measures the effect on thyroid hormone synthesis. The in vitro H295R Steroidogenesis Assay (H295R) utilises human adenocarcinoma cell line NCI-H295R. and assesses the effect on steroidogenesis by quantifying the amount of 17β-estradiol (E2) and testosterone (T) produced by the cells and comparing this to cells exposed to vehicle control only. Results are expressed as lowest effect concentrations (LEC) in molar (M) final in well. - GLP compliance:
- no
- Type of method:
- in vitro
- Endpoint addressed:
- toxicity to reproduction / fertility
- developmental toxicity / teratogenicity
- other: systemic toxicity
- Conclusions:
- SLES was tested in a broad panel of effect-based in vitro CALUX® bioassays, both in the absence and presence of metabolic activation (S9). Receptor activity and cytotoxicity of SLES was unaffected by the presence of metabolic enzymes. The compound did not interact with the majority of nuclear hormone receptors or pathways investigated. The activation of the PXR CALUX® assay suggested that it is recognised as "foreign". The compound was found to interact with all three peroxisome proliferator activated receptors PPARα, PPARγ and PPARδ, although only PPARα agonism and PPARγ antagonism met the OECD criteria; such activity is not surprising, since PXR and PPAR receptors are involved in lipid metabolism and homeostasis and are endogenously activated by lipid-like compounds [1], which share structural similarities to SLES. For the same reason [2], SLES was found to interact with the H295R steroidogenesis pathway, which drives the lowest platform PoD in the CALUX® assay.
--
[1] Varga, T., Z. Czimmerer, and L. Nagy, PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2011. 1812(8): p. 1007-1022.
[2] Ito, T., et al., Stomach secretes estrogen in response to the blood triglyceride levels. Communications Biology, 2021. 4(1): p. 1364. - Executive summary:
SLES was tested in a broad panel of effect-based in vitro bioassays, consisting of twenty-five CALUX® assays, a hTPO-inhibition assay and the H295R steroidogenesis assay, both in the absence and presence of a metabolic module containing rat liver S9. Overall, SLES receptor activity and cytotoxicity was not affected by the presence of metabolic enzymes. The compound did not interact with the nuclear hormone receptors (ERα, AR, TRβ, GR, PR), although it did influence the hormonal axis by causing an increased estrogen production in the H295R steroidogenesis assay. The compound did not interact with LXR or RAR, or the TCF pathway. The activation of the PXR CALUX® assay suggested that it is recognised as "foreign". The compound was found to interact with all three peroxisome proliferator activated receptors PPARα, PPARγ and PPARδ, although only PPARα agonism and PPARγ antagonism met the OECD criteria. The interaction with the PXR and PPAR receptors is not surprising, since PXR and PPAR receptors are involved in lipid metabolism and homeostasis and are endogenously activated by lipid-like compounds [Ref1], which share structural similarities to SLES. For the same reason [Ref2], SLES was found to interact with the H295R steroidogenesis pathway, which drives the lowest platform PoD in the CALUX® assay.
--
[1] Varga, T., Z. Czimmerer, and L. Nagy, PPARs are a unique set of fatty acid regulated transcription factors controlling both lipid metabolism and inflammation. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2011. 1812(8): p. 1007-1022.
[2] Ito, T., et al., Stomach secretes estrogen in response to the blood triglyceride levels. Communications Biology, 2021. 4(1): p. 1364.
Referenceopen allclose all
Results from the controls:
Cytotoxicity assay:
The positive controls induced a response with both cellular ATP and LDH release for HepG2 cells.
Assay results:
Responses were observed for each biomarker in at least 1 positive control, in at least 2 of the 3 biological replicates where there is more than 1 positive control per assay.
Results SLES:
Solubility:
A solubility assessment was initially performed in DMSO. SLES had very limited solubility in DMSO (data not reported). Further solubility assessments in water demonstrated a maximum solubility of 250mM (data not shown) so a top dose of 1250 µM was chosen for the cytotoxicity screen.
Cytotoxicity:
Responses were observed with both LDH and ATP biomarkers in HepG2 cells.
The minimum PoD (median) was 280µM for HepG2. These values were multiplied by a factor of 3 to obtain estimated cytotoxic concentrations of 840µM for HepG2. Following expert review of the analysis, 850 µM for HepG2 was chosen as the maximum concentration for the main study.
Cell Stress Panel Test Results:
HepG2 cells were treated with various concentrations of SLES (0.0519-850 μM) for 24 h before assessment in the cell stress panel.
SLES showed significant activity in HepG2 cells following 24 h incubation. There was significant cell loss at the top 2 doses tested (213-850 µM) and the majority of biomarker responses also occur at these doses. Exceptions include Oxidative stress (DHE) although this is likely also due to cytotoxicity as the response decreases with dose. This is also true for the three biomarkers within the extracellular flux (Seahorse) assay (ECAR, OCR & Reserve Capacity) which represent mitochondrial toxicity, as again a decrease is seen for all 3 biomarkers and all with a high confidence score of 1.0.
The global PoD across all the measured biomarkers, based on nominal concentrations, was determined to be 9.8 µM.
Assay Specific results:
Please see figures (section 2-20) in Report figures (BIFROST) attached below ("Overall remarks, attachments").
1. AhR Translocation Assay:
Cell Count:
Mean CDS: 0.90
Expected PoD: 120 µM
PoD percentiles (µM) | ||||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 33 | 38 | 60 | 82 | 110 | 150 | 170 |
2 | 0.69 | 95 | 120 | 220 | 440 | >850 | >850 | >850 |
3 | 1 | 39 | 45 | 63 | 78 | 100 | 130 | 140 |
Nuclear Area:
Mean CDS: 0.17
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.22 | 56 | 77 | >850 | >850 | >850 | >850 | >850 |
2 | 0.09 | 260 | 400 | >850 | >850 | >850 | >850 | >850 |
3 | 0.19 | 80 | 99 | >850 | >850 | >850 | >850 | >850 |
DNA Structure:
Mean CDS: 0.83
Expected PoD: 130 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 73 | 79 | 98 | 110 | 130 | 160 | 170 |
2 | 0.5 | 100 | 120 | 310 | >850 | >850 | >850 | >850 |
3 | 1 | 46 | 49 | 60 | 68 | 81 | 100 | 110 |
AhR Translocation:
Mean CDS: 0.13
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.16 | 81 | 120 | >850 | >850 | >850 | >850 | >850 |
2 | 0.064 | 300 | 730 | >850 | >850 | >850 | >850 | >850 |
3 | 0.16 | 140 | 170 | >850 | >850 | >850 | >850 | >850 |
2. Apoptosis, Necrosis and Cell Cycle Arrest Assay:
Cell Count:
Mean CDS: 0.87
Expected PoD: 82 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 22 | 26 | 45 | 60 | 76 | 110 | 120 |
2 | 1 | 18 | 21 | 37 | 51 | 65 | 98 | 110 |
3 | 0.62 | 46 | 59 | 150 | 470 | >850 | >850 | >850 |
Nuclear Area:
Mean CDS: 0.80
Expected PoD: 330 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 170 | 190 | 230 | 280 | 360 | 490 | 540 |
2 | 1 | 160 | 170 | 210 | 260 | 350 | 490 | 530 |
3 | 0.4 | 230 | 260 | 630 | >850 | >850 | >850 | >850 |
DNA Structure:
Mean CDS: 0.90
Expected PoD: 130 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 65 | 71 | 94 | 120 | 150 | 320 | 360 |
2 | 1 | 46 | 49 | 63 | 82 | 100 | 130 | 140 |
3 | 0.7 | 74 | 92 | 200 | 310 | >850 | >850 | >850 |
Membrane permeability:
Mean CDS: 0.25
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.35 | 120 | 190 | 620 | >850 | >850 | >850 | >850 |
2 | 0.21 | 390 | 520 | >850 | >850 | >850 | >850 | >850 |
3 | 0.19 | 430 | 600 | >850 | >850 | >850 | >850 | >850 |
Caspase 3/7 intensity:
Mean CDS: 0.98
Expected PoD: 66 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 48 | 49 | 53 | 57 | 61 | 74 | 79 |
2 | 1 | 43 | 44 | 48 | 51 | 55 | 69 | 76 |
3 | 0.95 | 49 | 51 | 60 | 74 | 160 | >850 | >850 |
DNA Content:
Mean CDS: 0.11
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.13 | 310 | 460 | >850 | >850 | >850 | >850 | >850 |
2 | 0.097 | 400 | 590 | >850 | >850 | >850 | >850 | >850 |
3 | 0.11 | 520 | 650 | >850 | >850 | >850 | >850 | >850 |
3. DNA damage (phospho-p53 & pH2AX) assay:
Cell Count:
Mean CDS: 0.79
Expected PoD: 160 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.73 | 70 | 85 | 150 | 320 | >850 | >850 | >850 |
2 | 0.66 | 72 | 88 | 170 | 430 | >850 | >850 | >850 |
3 | 1 | 40 | 46 | 64 | 74 | 86 | 110 | 120 |
Nuclear Area:
Mean CDS: 0.00
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.0003 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
2 | 0.0003 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
3 | 0.002 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
DNA Structure:
Mean CDS: 0.04
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.016 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
2 | 0.013 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
3 | 0.078 | 480 | 610 | >850 | >850 | >850 | >850 | >850 |
DNA Damage (pH2AX):
Mean CDS: 0.99
Expected PoD: 93 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.99 | 59 | 62 | 75 | 93 | 140 | 260 | 480 |
2 | 0.97 | 56 | 61 | 76 | 96 | 190 | 640 | >850 |
3 | 1 | 51 | 53 | 58 | 63 | 71 | 88 | 94 |
Phospho-p53:
Mean CDS: 0.36
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.12 | 720 | 770 | >850 | >850 | >850 | >850 | >850 |
2 | 0.21 | 650 | 700 | >850 | >850 | >850 | >850 | >850 |
3 | 0.75 | 310 | 380 | 550 | 660 | >850 | >850 | >850 |
4. ER Stress Assay Panel 1:
Cell count:
Mean CDS: 1.00
Expected PoD: 100 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 57 | 63 | 83 | 97 | 110 | 140 | 150 |
2 | 1 | 55 | 60 | 79 | 94 | 110 | 140 | 150 |
3 | 1 | 65 | 72 | 100 | 120 | 140 | 180 | 200 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 85 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 54 | 56 | 66 | 75 | 89 | 110 | 120 |
2 | 1 | 59 | 61 | 72 | 82 | 96 | 120 | 130 |
3 | 1 | 64 | 67 | 82 | 97 | 110 | 140 | 140 |
DNA Structure:
Mean CDS: 1.00
Expected PoD: 89 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 48 | 50 | 62 | 80 | 100 | 130 | 140 |
2 | 1 | 54 | 58 | 75 | 95 | 120 | 150 | 160 |
3 | 1 | 46 | 50 | 70 | 98 | 120 | 150 | 160 |
Endoplasmic reticulum:
Mean CDS: 1.00
Expected PoD: 170 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 61 | 71 | 130 | 180 | 270 | 450 | 480 |
2 | 1 | 66 | 77 | 140 | 190 | 310 | 480 | 500 |
3 | 1 | 57 | 69 | 120 | 140 | 160 | 180 | 190 |
BiP:
Mean CDS: 0.66
Expected PoD: 520 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.56 | 250 | 320 | 570 | 760 | >850 | >850 | >850 |
2 | 0.71 | 230 | 330 | 510 | 640 | >850 | >850 | >850 |
3 | 0.7 | 150 | 270 | 510 | 640 | >850 | >850 | >850 |
5. ER Stress Assay Panel 2:
Cell Count:
Mean CDS: 1.00
Expected PoD: 100 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 61 | 66 | 85 | 100 | 120 | 150 | 160 |
2 | 1 | 64 | 70 | 88 | 100 | 120 | 150 | 160 |
3 | 1 | 66 | 71 | 91 | 110 | 130 | 150 | 160 |
Nuclear Area:
Mean CDS: 0.99
Expected PoD: 130 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 73 | 78 | 99 | 120 | 140 | 170 | 170 |
2 | 0.99 | 90 | 98 | 120 | 140 | 160 | 190 | 190 |
3 | 0.98 | 89 | 96 | 120 | 140 | 160 | 190 | 200 |
DNA Structure:
Mean CDS: 0.84
Expected PoD: 99 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 49 | 54 | 69 | 85 | 110 | 140 | 140 |
2 | 0.53 | 68 | 77 | 140 | 210 | >210 | >210 | >210 |
3 | 1 | 47 | 52 | 68 | 85 | 100 | 130 | 140 |
ATF4:
Mean CDS: 1.00
Expected PoD: 74 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 53 | 54 | 60 | 67 | 79 | 100 | 110 |
2 | 1 | 58 | 60 | 69 | 78 | 91 | 110 | 120 |
3 | 1 | 54 | 56 | 63 | 71 | 83 | 100 | 110 |
PERK:
Mean CDS: 0.61
Expected PoD: 150 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.61 | 71 | 83 | 130 | 180 | >210 | >210 | >210 |
2 | 0.57 | 110 | 120 | 170 | 200 | >210 | >210 | >210 |
3 | 0.64 | 88 | 100 | 150 | 180 | >210 | >210 | >210 |
CHOP:
Mean CDS: 0.89
Expected PoD: 89 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 35 | 38 | 49 | 63 | 94 | 130 | 140 |
2 | 0.66 | 59 | 73 | 130 | 180 | >210 | >210 | >210 |
3 | 1 | 42 | 46 | 60 | 76 | 110 | 140 | 150 |
6. HCS GSH, ROS, MMP & ATP Assay
Cell count:
Mean CDS: 1.00
Expected PoD: 68 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 46 | 49 | 56 | 63 | 72 | 93 | 100 |
2 | 1 | 52 | 54 | 64 | 73 | 86 | 110 | 120 |
3 | 1 | 46 | 48 | 56 | 63 | 73 | 94 | 100 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 93 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 53 | 54 | 64 | 75 | 92 | 210 | 300 |
2 | 1 | 61 | 64 | 86 | 120 | 180 | 310 | 350 |
3 | 1 | 49 | 51 | 59 | 70 | 88 | 220 | 300 |
DNA Structure:
Mean CDS: 1.00
Expected PoD: 70 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 23 | 30 | 62 | 76 | 88 | 110 | 110 |
2 | 1 | 27 | 33 | 57 | 94 | 110 | 140 | 140 |
3 | 1 | 22 | 27 | 57 | 66 | 75 | 94 | 100 |
Mitochondrial mass:
Mean CDS: 1.00
Expected PoD: 63 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 48 | 49 | 53 | 57 | 68 | 92 | 100 |
2 | 1 | 48 | 49 | 53 | 58 | 71 | 95 | 100 |
3 | 1 | 51 | 52 | 57 | 64 | 76 | 100 | 110 |
Mitochondrial membrane potential:
Mean CDS: 1.00
Expected PoD: 72 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 44 | 54 | 66 | 73 | 83 | 99 | 110 |
2 | 1 | 44 | 54 | 67 | 74 | 84 | 100 | 110 |
3 | 1 | 49 | 53 | 63 | 70 | 79 | 96 | 100 |
Oxidative stress (DHE):
Mean CDS: 1.00
Expected PoD: 51 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 11 | 14 | 28 | 63 | 77 | 96 | 100 |
2 | 1 | 14 | 16 | 30 | 67 | 110 | 140 | 150 |
3 | 1 | 13 | 15 | 27 | 44 | 100 | 140 | 150 |
GSH content:
Mean CDS: 1.00
Expected PoD: 73 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 51 | 54 | 62 | 69 | 78 | 96 | 100 |
2 | 1 | 48 | 50 | 59 | 66 | 76 | 95 | 100 |
3 | 1 | 59 | 62 | 74 | 84 | 96 | 120 | 120 |
Cellular ATP:
Mean CDS: 1.00
Expected PoD: 58 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 45 | 47 | 52 | 56 | 63 | 84 | 93 |
2 | 1 | 43 | 45 | 51 | 56 | 65 | 91 | 100 |
3 | 1 | 44 | 45 | 50 | 54 | 61 | 84 | 93 |
7. HCS MMP and ATP (TMRE)
Cell count:
Mean CDS: 0.04
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.059 | 620 | 820 | >850 | >850 | >850 | >850 | >850 |
2 | 0.022 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
3 | 0.048 | 680 | >850 | >850 | >850 | >850 | >850 | >850 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 160 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 130 | 130 | 150 | 160 | 170 | 190 | 200 |
2 | 1 | 130 | 130 | 150 | 170 | 180 | 200 | 210 |
3 | 1 | 120 | 130 | 140 | 150 | 170 | 180 | 190 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 83 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 56 | 62 | 78 | 90 | 100 | 130 | 140 |
2 | 1 | 52 | 57 | 70 | 80 | 92 | 110 | 120 |
3 | 1 | 52 | 57 | 70 | 79 | 91 | 110 | 120 |
Mitochondrial mass:
Mean CDS: 1.00
Expected PoD: 56 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 23 | 25 | 33 | 55 | 66 | 83 | 90 |
2 | 1 | 22 | 26 | 57 | 69 | 79 | 98 | 110 |
3 | 1 | 24 | 30 | 53 | 62 | 72 | 89 | 96 |
Mitochondrial membrane potential:
Mean CDS: 1.00
Expected PoD: 82 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 43 | 49 | 67 | 80 | 95 | 120 | 130 |
2 | 1 | 50 | 55 | 71 | 84 | 100 | 130 | 140 |
3 | 1 | 51 | 56 | 72 | 84 | 100 | 130 | 140 |
Cellular ATP:
Mean CDS: 1.00
Expected PoD: 62 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 40 | 42 | 47 | 51 | 58 | 90 | 100 |
2 | 1 | 49 | 51 | 58 | 64 | 72 | 96 | 110 |
3 | 1 | 49 | 52 | 58 | 64 | 72 | 94 | 100 |
8. Inflammation & pH Assay:
Cell count:
Mean CDS: 1.00
Expected PoD: 70 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 46 | 49 | 59 | 68 | 79 | 99 | 110 |
2 | 1 | 50 | 53 | 62 | 70 | 80 | 99 | 110 |
3 | 1 | 50 | 53 | 62 | 70 | 81 | 100 | 110 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 140 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 60 | 68 | 110 | 170 | 240 | 310 | 320 |
2 | 1 | 80 | 90 | 120 | 140 | 160 | 180 | 190 |
3 | 1 | 65 | 73 | 100 | 130 | 150 | 170 | 190 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 120 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 72 | 80 | 160 | 190 | 200 | 240 | 260 |
2 | 1 | 68 | 71 | 89 | 110 | 130 | 240 | 300 |
3 | 1 | 59 | 62 | 73 | 86 | 100 | 130 | 140 |
Intracellular pH:
Mean CDS: 1.00
Expected PoD: 80 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 50 | 53 | 62 | 71 | 81 | 100 | 110 |
2 | 1 | 48 | 51 | 61 | 70 | 81 | 100 | 110 |
3 | 1 | 56 | 62 | 83 | 100 | 120 | 170 | 200 |
HIF1alpha:
Mean CDS: 1.00
Expected PoD: 89 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 54 | 58 | 74 | 88 | 110 | 130 | 140 |
2 | 1 | 56 | 59 | 72 | 84 | 100 | 130 | 140 |
3 | 1 | 61 | 65 | 80 | 94 | 110 | 140 | 140 |
ICAM1:
Mean CDS: 1.00
Expected PoD: 110 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 57 | 62 | 85 | 100 | 130 | 160 | 170 |
2 | 1 | 59 | 66 | 87 | 110 | 130 | 160 | 170 |
3 | 1 | 66 | 74 | 98 | 120 | 140 | 160 | 170 |
9. Inflammatory Response ELISA (Flow Cytometry) IL8 only
Cell count:
Mean CDS: 1.00
Expected PoD: 73 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 45 | 49 | 64 | 75 | 88 | 110 | 130 |
2 | 1 | 45 | 48 | 62 | 75 | 90 | 120 | 120 |
3 | 1 | 44 | 48 | 61 | 71 | 83 | 110 | 110 |
IL-8:
Mean CDS: 1.00
Expected PoD: 62 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 27 | 34 | 53 | 68 | 88 | 130 | 150 |
2 | 1 | 28 | 34 | 52 | 65 | 82 | 120 | 130 |
3 | 1 | 23 | 27 | 42 | 54 | 68 | 100 | 110 |
10. LDH Assay:
LDH release:
Mean CDS: 1.00
Expected PoD: 76 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 58 | 61 | 70 | 80 | 96 | 120 | 130 |
2 | 1 | 54 | 56 | 61 | 66 | 74 | 91 | 99 |
3 | 1 | 57 | 60 | 69 | 78 | 93 | 120 | 120 |
11. Metal Stress Assay:
Cell count:
Mean CDS: 1.00
Expected PoD: 72 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 42 | 45 | 57 | 68 | 83 | 110 | 120 |
2 | 1 | 44 | 47 | 56 | 64 | 76 | 100 | 110 |
3 | 1 | 52 | 55 | 68 | 80 | 96 | 120 | 130 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 87 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 33 | 35 | 42 | 49 | 61 | 120 | 130 |
2 | 1 | 57 | 66 | 110 | 140 | 160 | 210 | 230 |
3 | 1 | 48 | 53 | 78 | 100 | 120 | 150 | 150 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 99 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 51 | 53 | 73 | 97 | 120 | 150 | 150 |
2 | 1 | 56 | 59 | 82 | 100 | 120 | 150 | 160 |
3 | 1 | 57 | 61 | 89 | 110 | 130 | 150 | 150 |
Metallothionein:
Mean CDS: 1.00
Expected PoD: 81 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 42 | 46 | 67 | 82 | 100 | 180 | 190 |
2 | 1 | 50 | 52 | 62 | 72 | 86 | 110 | 110 |
3 | 1 | 52 | 56 | 71 | 88 | 110 | 130 | 140 |
MTF1:
Mean CDS: 0.91
Expected PoD: 430 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.95 | 180 | 220 | 340 | 460 | 570 | >850 | >850 |
2 | 0.93 | 70 | 91 | 390 | 520 | 630 | >850 | >850 |
3 | 0.84 | 160 | 210 | 380 | 540 | 710 | >850 | >850 |
12. Mitochondrial Oxidative Stress Assay (PGC1alpha)
Cell count:
Mean CDS: 1.00
Expected PoD: 79 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 51 | 54 | 65 | 75 | 86 | 110 | 120 |
2 | 1 | 59 | 63 | 75 | 85 | 98 | 120 | 130 |
3 | 1 | 54 | 57 | 67 | 76 | 87 | 110 | 120 |
Nuclear Area:
Mean CDS: 1.00
Expected PoD: 100 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 44 | 46 | 55 | 66 | 88 | 170 | 300 |
2 | 1 | 58 | 63 | 84 | 120 | 200 | 330 | 380 |
3 | 0.99 | 55 | 59 | 83 | 110 | 150 | 250 | 410 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 81 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 57 | 60 | 71 | 84 | 100 | 120 | 130 |
2 | 1 | 55 | 58 | 71 | 85 | 100 | 130 | 150 |
3 | 1 | 50 | 52 | 60 | 72 | 88 | 110 | 120 |
Mitochondrial ROS:
Mean CDS: 0.99
Expected PoD: 490 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 230 | 260 | 370 | 470 | 550 | 660 | 680 |
2 | 1 | 260 | 300 | 410 | 500 | 580 | 680 | 710 |
3 | 0.96 | 300 | 340 | 480 | 580 | 670 | 810 | >850 |
PGC1alpha:
Mean CDS: 0.97
Expected PoD: 140 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.99 | 51 | 57 | 86 | 120 | 190 | 450 | 500 |
2 | 0.91 | 70 | 82 | 150 | 250 | 520 | >850 | >850 |
3 | 1 | 38 | 43 | 62 | 81 | 100 | 130 | 140 |
13. Osmotic & Heat Shock Stress Assay:
Cell count:
Mean CDS: 1.00
Expected PoD: 68 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 45 | 49 | 62 | 73 | 86 | 110 | 120 |
2 | 1 | 42 | 45 | 54 | 62 | 74 | 97 | 110 |
3 | 1 | 45 | 48 | 59 | 68 | 80 | 100 | 110 |
Nuclear Area:
Mean CDS: 0.94
Expected PoD: 490 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.99 | 210 | 240 | 380 | 510 | 620 | 750 | 790 |
2 | 0.99 | 240 | 300 | 440 | 540 | 630 | 740 | 780 |
3 | 0.85 | 160 | 200 | 460 | 620 | 760 | >850 | >850 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 84 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 41 | 44 | 56 | 71 | 90 | 120 | 130 |
2 | 1 | 59 | 64 | 83 | 100 | 120 | 150 | 160 |
3 | 1 | 55 | 58 | 71 | 84 | 100 | 120 | 130 |
Heat Shock Response (Hsp70):
Mean CDS: 1.00
Expected PoD: 63 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 51 | 52 | 57 | 61 | 67 | 80 | 85 |
2 | 1 | 53 | 54 | 58 | 61 | 67 | 79 | 84 |
3 | 1 | 53 | 54 | 58 | 62 | 67 | 79 | 84 |
Osmotic stress (NFAT):
Mean CDS: 1.00
Expected PoD: 140 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.99 | 69 | 76 | 100 | 120 | 150 | 310 | 450 |
2 | 1 | 100 | 110 | 140 | 170 | 200 | 260 | 290 |
3 | 1 | 75 | 83 | 110 | 130 | 150 | 200 | 230 |
14. Oxidative Stress Assay (Heme oxygenase 1 and NRF2):
Cell count:
Mean CDS: 0.96
Expected PoD: 130 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.87 | 77 | 92 | 150 | 240 | 450 | >850 | >850 |
2 | 1 | 51 | 60 | 85 | 100 | 130 | 190 | 240 |
3 | 1 | 47 | 55 | 78 | 96 | 120 | 180 | 210 |
Nuclear area:
Mean CDS: 0.83
Expected PoD: 96 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.48 | 28 | 39 | 150 | >850 | >850 | >850 | >850 |
2 | 1 | 24 | 29 | 47 | 62 | 82 | 130 | 160 |
3 | 1 | 24 | 33 | 61 | 92 | 140 | 300 | 410 |
DNA structure:
Mean CDS: 0.33
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.11 | 690 | 760 | >850 | >850 | >850 | >850 | >850 |
2 | 0.47 | 320 | 370 | 570 | >850 | >850 | >850 | >850 |
3 | 0.41 | 390 | 450 | 680 | >850 | >850 | >850 | >850 |
NRF2:
Mean CDS: 0.81
Expected PoD: 540 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.56 | 350 | 450 | 710 | 820 | >850 | >850 | >850 |
2 | 0.92 | 150 | 200 | 490 | 630 | 720 | >850 | >850 |
3 | 0.96 | 77 | 130 | 420 | 560 | 660 | 790 | >850 |
Heme oxygenase:
Mean CDS: 0.07
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.046 | 790 | >850 | >850 | >850 | >850 | >850 | >850 |
2 | 0.053 | 760 | 840 | >850 | >850 | >850 | >850 | >850 |
3 | 0.11 | 590 | 670 | >850 | >850 | >850 | >850 | >850 |
15. Oxidative Stress Assay (SRXN1 Only):
Cell count:
Mean CDS: 0.96
Expected PoD: 110 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 45 | 52 | 74 | 93 | 120 | 150 | 170 |
2 | 0.87 | 59 | 71 | 120 | 180 | 350 | >850 | >850 |
3 | 1 | 39 | 45 | 67 | 86 | 110 | 140 | 150 |
Nuclear Area:
Mean CDS: 0.01
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.0091 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
2 | 0.0023 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
3 | 0.0047 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
DNA structure:
Mean CDS: 0.03
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.061 | 670 | 800 | >850 | >850 | >850 | >850 | >850 |
2 | 0.0083 | >850 | >850 | >850 | >850 | >850 | >850 | >850 |
3 | 0.033 | 770 | >850 | >850 | >850 | >850 | >850 | >850 |
SRXN1:
Mean CDS: 0.86
Expected PoD: 140 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 73 | 77 | 87 | 93 | 100 | 130 | 140 |
2 | 0.59 | 130 | 140 | 210 | 540 | >850 | >850 | >850 |
3 | 1 | 77 | 82 | 93 | 99 | 110 | 130 | 140 |
16. Phospholipidosis and Steatosis Assay:
Cell count:
Mean CDS: 1.00
Expected PoD: 110 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 68 | 80 | 130 | 180 | 240 | 330 | 370 |
2 | 1 | 33 | 37 | 51 | 63 | 78 | 110 | 110 |
3 | 0.99 | 57 | 64 | 91 | 120 | 160 | 310 | 420 |
Nuclear Area:
Mean CDS: 0.98
Expected PoD: 230 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 130 | 150 | 190 | 210 | 240 | 370 | 430 |
2 | 0.93 | 130 | 150 | 190 | 310 | 570 | >850 | >850 |
3 | 1 | 120 | 130 | 150 | 180 | 230 | 390 | 450 |
DNA structrure:
Mean CDS: 0.36
Expected PoD: Mean CDS less than 0.5, value omitted.
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.42 | 120 | 150 | 460 | >850 | >850 | >850 | >850 |
2 | 0.31 | 69 | 85 | 730 | >850 | >850 | >850 | >850 |
3 | 0.34 | 92 | 120 | 690 | >850 | >850 | >850 | >850 |
Phospholipidosis:
Mean CDS: 1.00
Expected PoD: 250 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 190 | 200 | 230 | 270 | 330 | 430 | 470 |
2 | 0.99 | 150 | 160 | 180 | 200 | 290 | 480 | 550 |
3 | 1 | 180 | 190 | 210 | 230 | 270 | 390 | 430 |
Steatosis:
Mean CDS: 1.00
Expected PoD: 71 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 51 | 54 | 66 | 82 | 120 | 210 | 230 |
2 | 1 | 44 | 45 | 51 | 57 | 70 | 93 | 100 |
3 | 1 | 47 | 49 | 55 | 60 | 70 | 91 | 98 |
17. Seahorse Mitochondrial toxicity assay with pre-incubation:
Pre-incubated Func Mito:
Mean CDS: 1.00
Expected PoD: 20 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 13 | 14 | 17 | 19 | 23 | 28 | 29 |
2 | 1 | 12 | 13 | 16 | 18 | 21 | 26 | 28 |
3 | 1 | 14 | 15 | 19 | 22 | 25 | 29 | 31 |
Pre-incubated Func Mito Reserve Capacity:
Mean CDS: 1.00
Expected PoD: 14 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 11 | 13 | 18 | 22 | 29 | 53 | 78 |
2 | 1 | 2.8 | 6.4 | 9 | 10 | 11 | 14 | 15 |
3 | 1 | 6.8 | 8.2 | 11 | 12 | 13 | 16 | 17 |
Pre-incubated Func Mito ECAR:
Mean CDS: 1.00
Expected PoD: 17 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 11 | 12 | 14 | 17 | 19 | 25 | 28 |
2 | 1 | 12 | 13 | 15 | 17 | 19 | 24 | 25 |
3 | 1 | 12 | 12 | 15 | 17 | 19 | 24 | 26 |
18. XBP1 Assay:
Cell count:
Mean CDS: 0.93
Expected PoD: 110 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 54 | 57 | 66 | 74 | 85 | 110 | 120 |
2 | 0.8 | 74 | 84 | 160 | 220 | 510 | >850 | >850 |
3 | 1 | 58 | 61 | 69 | 77 | 88 | 110 | 120 |
Nuclear Area:
Mean CDS: 0.60
Expected PoD: 650 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 0.75 | 310 | 400 | 640 | 740 | >850 | >850 | >850 |
2 | 0.15 | 650 | 730 | >850 | >850 | >850 | >850 | >850 |
3 | 0.89 | 260 | 320 | 540 | 650 | 740 | >850 | >850 |
DNA structure:
Mean CDS: 1.00
Expected PoD: 57 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 41 | 42 | 47 | 52 | 58 | 79 | 91 |
2 | 1 | 42 | 44 | 53 | 64 | 87 | 160 | 190 |
3 | 1 | 39 | 40 | 44 | 48 | 52 | 72 | 85 |
XBP1:
Mean CDS: 0.92
Expected PoD: 140 µM
|
| PoD percentiles (µM) | ||||||
Plate | CDS | 2.5th | 5th | 25th | 50th | 75th | 95th | 97.5th |
1 | 1 | 49 | 57 | 83 | 100 | 130 | 170 | 200 |
2 | 0.77 | 45 | 63 | 150 | 280 | 750 | >850 | >850 |
3 | 1 | 68 | 81 | 110 | 140 | 170 | 230 | 260 |
Control results
Cell Lysate Generation
Chlorpromazine induced a response with both cellular ATP and LDH release in all three cell lines, while CCCP induced a dose response for cellular ATP in all three cell lines and for LDH in HepaRG and HepG2 cell lines. Chlorpromazine demonstrated a positive response in the LDH assay in all three cell lines across the plates subsequently sequenced. MRM’s for the transcriptomics controls were assessed in all the cell models. Results demonstrated that the controls were in the correct positions (appropriate wells) on all plates (data not shown).
Sequencing – TempO-Seq
Principle Component Analysis (PCA) showed that samples and controls cluster in a generally expected way (figure attached below in "Overall remarks, Attachments"), indicating proper functioning of the assay.
Samples from each cell line (MCF-7, HepG2 or HepaRG) cluster together and the bulk lysate controls, which were generated using MCF-7 cells, cluster with the MCF-7 samples.
Cytotoxicity assay
Responses were observed with both biomarkers (LDH and ATP) in HepG2 and MCF-7 cells as well as LDH release in HepaRG cells. Cellular ATP was not found to decrease in HepaRG.
The minimum PoD (median) for each cell type was 460 µM for HepaRG, 280 µM for HepG2 and 230 µM for MCF-7. Following the strategy for dose setting detailed in the method section above, these values were multiplied by a factor of 3 to obtain estimated cytotoxic concentrations of 1400 µM for HepaRG, 840 µM for HepG2 and 690 µM for MCF-7. Following expert review of the analysis, 1250 µM for HepaRG and 850 µM for HepG2 and MCF-7 were chosen as maximum concentrations for the main study.
LDH assay
Cytotoxicity was observed for all three cell lines.
BMDExpress2 - MCF-7
Normalised and rlog transformed data was inputted into BMDExpress2 for dose response modelling.
Probe Level Dose Response:
After the Williams Trend Test pre-filter (using a 1.5 fold change (FC) and p < 0.05 filter), 44 probes were seen to have a significant monotonic dose response with SLES treatment across the concentrations tested. Out of those 44 probes, 8 probes had a significant model and BMDL fit to them. Significant model fits were defined as those which had a fit p filter >0.1 and BMDLs are deemed significant if both the BMDL/BMDU ratio <40 and BMD <the highest tested concentration (see Figure 3 and 4 in HTTr BMDExpress Analysis SLES MCF-7.pdf attached).
Probe level PoDs and statistics:
PoD (μM) | |
Avg of 20 BMDLs with highest FC | 79.0 |
Avg of 25th-75th percentile of BMDLs | 97.5 |
Pathway level dose response:
Within BMDExpress2, probes which passed the Williams Trends Test are mapped to Reactome pathways. Pathway level BMDLs are calculated by taking a mean of all the probe BMDLs which are mapped to the pathways and are deemed as significant (using previously described probe significance criteria). Pathway BMDLs are considered significant if they have >2 genes found within the pathway, have >1 gene with a significant BMDL and a 2-tailed fisher p-value <0.1. No pathways were found to be significantly perturbed by SLES treatment.
No significant pathway BMDLs were calculated.
Pathway level PoDs and statistics:
PoDs and statistics could not be calcualted due to there being no significant BMDLs calculated.
BMDExpress2 - HepG2
Normalised and rlog transformed data was inputted into BMDExpress2 for dose response modelling.
Probe Level Dose Response:
After the Williams Trend Test pre-filter (using a 1.5 FC and p < 0.05 filter), 734 probes were seen to have a significant monotonic dose response with SLES treatment across the concentrations tested. Out of those 734 probes, 449 probes had a significant model and BMDL fit to them. Significant model fits were defined as those which had a fit p filter >0.1 and BMDLs are deemed significant if both the BMDL/BMDU ratio <40 and BMD <the highest tested concentration (see Figure 4 and 5 in HTTr BMDExpress Analysis SLES HepG2.pdf attached).
Probe level PoDs and statistics:
PoD (μM) | |
Avg of 20 BMDLs with highest FC | 71.4 |
Avg of 25th-75th percentile of BMDLs | 118 |
Pathway level dose response:
Within BMDExpress2, probes which passed the Williams Trends Test are mapped to Reactome pathways. Pathway level BMDLs are calculated by taking a mean of all the probe BMDLs which are mapped to the pathways and are deemed as significant (using previously described probe significance criteria). Pathway BMDLs are considered significant if they have >2 genes found within the pathway, have >1 gene with a significant BMDL and a 2-tailed fisher p-value <0.1. 149 pathways were found to be significantly perturbed by SLES treatment.
The accumulation plot of significant pathway mean BMDLs and accumulation plots of significant probes with marked probes in the pathway, can be found in the report attached below (Figure 6 and 7 in HTTr BMDExpress Analysis SLES HepG2.pdf).
Pathway level PoDs and statistics:
PoD (μM) | |
Avg of 20 lowest pathway BMDLs | 56.9 |
The lowest pathway BMDL | 43.2 |
Avg of 20 pathway BMDLs with lowest 2-tail fisher P values | 77.3 |
BMDExpress2 - HepaRG
Normalised and rlog transformed data was inputted into BMDExpress2 for dose response modelling.
Probe Level Dose Response:
After the Williams Trend Test pre-filter (using a 1.5 FC and p < 0.05 filter), 446 probes were seen to have a significant monotonic dose response with SLES treatment across the concentrations tested. Out of those 446 probes, 268 probes had a significant model and BMDL fit to them. Significant model fits were defined as those which had a fit p filter >0.1 and BMDLs are deemed significant if both the BMDL/BMDU ratio <40 and BMD <the highest tested concentration (see Figure 3 and 4 in HTTr BMDExpress Analysis SLES HepaRG.pdf attached).
Probe level PoDs and statistics:
PoD (μM) | |
Avg of 20 BMDLs with highest FC | 61.3 |
Avg of 25th-75th percentile of BMDLs | 144 |
Pathway level dose response:
Within BMDExpress2, probes which passed the Williams Trends Test are mapped to Reactome pathways. Pathway level BMDLs are calculated by taking a mean of all the probe BMDLs which are mapped to the pathways and are deemed as significant (using previously described probe significance criteria). Pathway BMDLs are considered significant if they have >2 genes found within the pathway, have >1 gene with a significant BMDL and a 2-tailed fisher p-value <0.1. 132 pathways were found to be significantly perturbed by SLES treatment.
The accumulation plot of significant pathway mean BMDLs and accumulation plots of significant probes with marked probes in the pathway, can be found in the report attached below (Figure 5 and 6 in HTTr BMDExpress Analysis SLES HepaRG.pdf).
Pathway level PoDs and statistics:
PoD(μM) | |
Avg of 20 lowest pathway BMDLs | 94.8 |
The lowest pathway BMDL | 66.2 |
Avg of 20 pathway BMDLs with lowest 2-tail fisher P values | 122 |
BIFROST- MCF-7
Summary results (see accompanying figures in HTTr_Analysis_SLES_C12-14_MCF-7.pdf attached):
Global PoD (µM): 1.4
Num. probes analysed: 12375
Num. CDS>0.5: 108
Num. CDS=1.0: 0
Lowest responding probe (50th percentile): DOCK8_21416
Second lowest responding probe (50th percentile): TMPRSS2_91261
Third lowest responding probe (50th percentile): PGBD4_16952
Probe with largest fold-increase in expression: MRPS36_4304
Probe with largest fold-decrease in expression: PGBD4_16952
BIFROST- HepG2
Summary results (see accompanying figures in HTTr_Analysis_SLES_C12-14_HepG2.pdf attached):
Global PoD (µM): 13
Num. probes analysed: 11885
Num. CDS>0.5: 172
Num. CDS=1.0: 1
Lowest responding probe (50th percentile): AIF1_16224
Second lowest responding probe (50th percentile): TSPAN10_15129
Third lowest responding probe (50th percentile): MTAP_23096
Probe with largest fold-increase in expression: CYP1A1_1698
Probe with largest fold-decrease in expression: ADAMTS13_90224
BIFROST- HepaRG
Summary results (see accompanying figures in HTTr_Analysis_SLES_C12-14_HepaRG.pdf attached):
Global PoD (µM): 2.9
Num. probes analysed: 11747
Num. CDS>0.5: 564
Num. CDS=1.0: 12
Lowest responding probe (50th percentile): PLIN4_25165
Second lowest responding probe (50th percentile): CREB3L3_29025
Third lowest responding probe (50th percentile): PDK4_28868
Probe with largest fold-increase in expression: PCK1_27655
Probe with largest fold-decrease in expression: CYP7A1_89758
Summary of PODs (in µM)
AlamarBlue PODs (cytotoxicity) | qPCR gene expression PODs (embryotoxicity) | Platform POD | |||||
Heart day 14 | Liver day 21 | Neural day 13 | Heart day 14 (MYH6 down-regulation) | Liver day 21 (AFP down-regulation) | Neural day 13 (PAX6 down-regulation) | Neural day 13 (NESTIN down-regulation) | Liver day 21 (AFP down-regulation) |
106.6 | 30.0 | No POD | No POD | 2.7 | 4.7 | No POD | 2.7 |
"No POD" indicates that there was no dose response and/or the POD could not be calculated. PODs could also not be calculated for BMP4 day 7, FOXA2 day 7 or PAX6 day 7.
Test Material | o/c Ratio dTP (µM) | Cell viability TP (µM) |
SLES | 39.5 | 264.8 |
Study 1: Binding Assays (including receptors, ion channels, transporters and other enzymes)
Binding Assays - SLES Results:
Assay Name | Test Compound | Test Concentration | % Inhibition of Control Specific Binding | ||
1st | 2nd | mean | |||
A2A(h) (agonist radioligand) | SLES | 1.0E-05 M | -17 | -6 | -11.5 |
α1A(h) (antagonist radioligand) | SLES | 1.0E-05 M | -8.9 | -11.9 | -10.4 |
α2A(h) (antagonist radioligand) | SLES | 1.0E-05 M | -11.4 | -11.1 | -11.2 |
β1(h) (agonist radioligand) | SLES | 1.0E-05 M | -10.4 | -10 | -10.2 |
β2(h) (antagonist radioligand) | SLES | 1.0E-05 M | 5 | 0.1 | 2.5 |
AT1 (h) (antagonist radioligand) | SLES | 1.0E-05 M | 8.8 | 3.7 | 6.2 |
BZD (central) (agonist radioligand) | SLES | 1.0E-05 M | -31.1 | -9.7 | -20.4 |
CB1(h) (agonist radioligand) | SLES | 1.0E-05 M | -7.2 | 1.1 | -3 |
CB2(h) (agonist radioligand) | SLES | 1.0E-05 M | 0.9 | 0.7 | 0.8 |
CCK1 (CCKA) (h) (agonist radioligand) | SLES | 1.0E-05 M | -1.9 | -15.2 | -8.6 |
D1(h) (antagonist radioligand) | SLES | 1.0E-05 M | 0.7 | 1.5 | 1.1 |
D2S(h) (agonist radioligand) | SLES | 1.0E-05 M | 2.4 | 5.4 | 3.9 |
ETA(h) (agonist radioligand) | SLES | 1.0E-05 M | 6.7 | 6 | 6.3 |
NMDA (antagonist radioligand) | SLES | 1.0E-05 M | 0.5 | 8.9 | 4.7 |
H1(h) (antagonist radioligand) | SLES | 1.0E-05 M | -2.5 | 3.8 | 0.6 |
H2(h) (antagonist radioligand) | SLES | 1.0E-05 M | -5.5 | -7.8 | -6.6 |
MAO-A (antagonist radioligand) | SLES | 1.0E-05 M | 12.7 | 16.5 | 14.6 |
M1(h) (antagonist radioligand) | SLES | 1.0E-05 M | -11 | -10.4 | -10.7 |
M2 (h) (antagonist radioligand) | SLES | 1.0E-05 M | -7.3 | -2.9 | -5.1 |
M3(h) (antagonist radioligand) | SLES | 1.0E-05 M | -18.4 | -8 | -13.2 |
N neuronal α4β2 (h) (agonist radioligand) | SLES | 1.0E-05 M | 3.7 | -5.8 | -1.1 |
δ (DOP) (h) (agonist radioligand) | SLES | 1.0E-05 M | 25.8 | 21.1 | 23.5 |
kappa (h) (KOP) (agonist radioligand) | SLES | 1.0E-05 M | 3.2 | -7 | -1.9 |
μ (MOP) (h) (agonist radioligand) | SLES | 1.0E-05 M | 12 | 14.7 | 13.4 |
LXR alpha (h) agonist radioligand | SLES | 1.0E-05 M | 52.7 | 55.9 | 54.3 |
5-HT1A(h) (agonist radioligand) | SLES | 1.0E-05 M | 20.5 | 12.7 | 16.6 |
5-HT1B (h) (antagonist radioligand) | SLES | 1.0E-05 M | -6.5 | -29 | -17.7 |
5-HT2A(h) (agonist radioligand) | SLES | 1.0E-05 M | -44.5 | -52.4 | -48.4 |
5-HT2B(h) (agonist radioligand) | SLES | 1.0E-05 M | -43.5 | -37.3 | -40.4 |
5-HT3(h) (antagonist radioligand) | SLES | 1.0E-05 M | 4.2 | 0.6 | 2.4 |
GR (h) (agonist radioligand) | SLES | 1.0E-05 M | -13.1 | -2.6 | -7.8 |
AR(h) (agonist radioligand) | SLES | 1.0E-05 M | -5.5 | -9.8 | -7.6 |
V1a(h) (agonist radioligand) | SLES | 1.0E-05 M | -4.5 | -4.7 | -4.6 |
Ca2+ channel (L, dihydropyridine site) (antagonist radioligand) | SLES | 1.0E-05 M | -10 | 9.8 | -0.1 |
Potassium Channel hERG (human)- [3H] Dofetilide | SLES | 1.0E-05 M | -10.7 | -22.2 | -16.4 |
KV channel (antagonist radioligand) | SLES | 1.0E-05 M | -46.9 | -13.9 | -30.4 |
Na+ channel (site 2) (antagonist radioligand) | SLES | 1.0E-05 M | -0.3 | 0.6 | 0.1 |
norepinephrine transporter(h) (antagonist radioligand) | SLES | 1.0E-05 M | -8.1 | 9.9 | 0.9 |
dopamine transporter(h) (antagonist radioligand) | SLES | 1.0E-05 M | -3.8 | -3 | -3.4 |
5-HT transporter (h) (antagonist radioligand) | SLES | 1.0E-05 M | -0.1 | 3.9 | 1.9 |
Please see Figure 1 in Result figures (screening) in “Overall remarks, Attachments”.
Binding Assays - Reference Compound Results:
Assay Name + Compound I.D. | IC50(M) | Ki(M) | nH |
A2A(h) (agonist radioligand) | |||
NECA | 2.5E-08 M | 2.0E-08 M | 0.9 |
α1A(h) (antagonist radioligand) | |||
WB 4101 | 5.2E-10 M | 2.6E-10 M | 1.7 |
α2A(h) (antagonist radioligand) | |||
yohimbine | 4.8E-09 M | 2.1E-09 M | 0.9 |
β1(h) (agonist radioligand) | |||
atenolol | 2.3E-07 M | 1.3E-07 M | 1.2 |
β2(h) (antagonist radioligand) | |||
ICI 118551 | 1.0E-09 M | 3.4E-10 M | 1.0 |
AT1 (h) (antagonist radioligand) | |||
saralasin | 3.2E-10 M | 1.6E-10 M | 0.8 |
BZD (central) (agonist radioligand) | |||
diazepam | 9.0E-09 M | 7.6E-09 M | 1.6 |
CB1(h) (agonist radioligand) | |||
CP 55940 | 9.1E-09 M | 2.8E-09 M | 1.6 |
CB2(h) (agonist radioligand) | |||
WIN 55212-2 | 1.6E-09 M | 1.0E-09 M | 1.5 |
CCK1 (CCKA) (h) (agonist radioligand) | |||
CCK-8s | 9.7E-11 M | 7.3E-11 M | 1.1 |
D1(h) (antagonist radioligand) | |||
SCH 23390 | 4.6E-10 M | 1.9E-10 M | 1.1 |
D2S(h) (agonist radioligand) | |||
7-OH-DPAT | 2.2E-09 M | 9.0E-10 M | 1.5 |
ETA(h) (agonist radioligand) | |||
endothelin-1 | 3.2E-11 M | 1.6E-11 M | 1.1 |
NMDA (antagonist radioligand) | |||
CGS 19755 | 4.0E-07 M | 3.3E-07 M | 0.7 |
H1(h) (antagonist radioligand) | |||
pyrilamine | 1.8E-09 M | 1.2E-09 M | 1.2 |
H2(h) (antagonist radioligand) | |||
cimetidine | 6.5E-07 M | 6.4E-07 M | 1.0 |
MAO-A (antagonist radioligand) | |||
clorgyline | 1.4E-09 M | 8.0E-10 M | 2.0 |
M1 (h) (antagonist radioligand) | |||
pirenzepine | 3.7E-08 M | 3.2E-08 M | 1.0 |
M2 (h) (antagonist radioligand) | |||
methoctramine | 5.4E-08 M | 3.8E-08 M | 1.2 |
M3 (h) (antagonist radioligand) | |||
4-DAMP | 1.5E-09 M | 1.1E-09 M | 1.3 |
N neuronal α4β2 (h) (agonist radioligand) | |||
nicotine | 7.6E-09 M | 2.5E-09 M | 1.1 |
δ (DOP) (h) (agonist radioligand) | |||
DPDPE | 3.2E-09 M | 1.8E-09 M | 1.2 |
kappa (h) (KOP) (agonist radioligand) | |||
U50488 | 8.3E-10 M | 4.5E-10 M | 1.6 |
μ (MOP) (h) (agonist radioligand) | |||
DAMGO | 8.0E-10 M | 3.3E-10 M | 1.2 |
LXR alpha (h) agonist radioligand | |||
T0901317 | 8.2E-08 M | 6.5E-08 M | 0.8 |
5-HT1A(h) (agonist radioligand) | |||
8-OH-DPAT | 7.5E-10 M | 3.7E-10 M | 1.1 |
5-HT1B (h) (antagonist radioligand) | |||
Serotonine | 1.8E-07 M | 7.9E-08 M | 0.9 |
5-HT2A(h) (agonist radioligand) | |||
(±)DOI | 1.7E-10 M | 1.3E-10 M | 0.7 |
5-HT2B(h) (agonist radioligand) | |||
(±)DOI | 3.3E-09 M | 1.6E-09 M | 0.9 |
5-HT3(h) (antagonist radioligand) | |||
MDL 72222 | 1.4E-08 M | 9.8E-09 M | 1.0 |
GR (h) (agonist radioligand) | |||
dexamethasone | 4.9E-09 M | 2.5E-09 M | 0.9 |
AR(h) (agonist radioligand) | |||
testosterone | 7.1E-09 M | 3.2E-09 M | 1.4 |
V1a(h) (agonist radioligand) | |||
[d(CH2)51,Tyr(Me)2]-AVP | 4.7E-10 M | 2.9E-10 M | 0.9 |
Ca2+ channel (L, dihydropyridine site) (antagonist radioligand) | |||
nitrendipine | 5.3E-10 M | 2.8E-10 M | 0.8 |
Potassium Channel hERG (human)- [3H] Dofetilide | |||
Terfenadine | 7.4E-08 M | 5.1E-08 M | 1.0 |
KV channel (antagonist radioligand) | |||
α-dendrotoxin | 1.3E-10 M | 1.0E-10 M | 0.9 |
Na+ channel (site 2) (antagonist radioligand) | |||
veratridine | 6.7E-06 M | 6.0E-06 M | 0.9 |
norepinephrine transporter(h) (antagonist radioligand) | |||
protriptyline | 4.8E-09 M | 3.5E-09 M | 1.4 |
dopamine transporter(h) (antagonist radioligand) | |||
BTCP | 8.4E-09 M | 4.4E-09 M | 0.8 |
5-HT transporter (h) (antagonist radioligand) | |||
imipramine | 2.9E-09 M | 1.3E-09 M | 1.2 |
Cellular and Nuclear Receptor Functional Assays:
Agonist Effect - SLES Results:
Assay Name | Test Compound | Test Concentration | % of Control Agonist Response | ||
1st | 2nd | Mean | |||
LXR alpha (h) (agonist effect) | SLES | 1.0E-05 M | 0.1 | 0 | 0.1 |
CAR (h) (agonist effect) | SLES | 1.0E-05 M | -14.2 | -13.6 | -13.9 |
PPARα (h) (agonist effect) | SLES | 1.0E-05 M | 1 | 1.7 | 1.3 |
PPARδ (h) (agonist effect) | SLES | 1.0E-05 M | 3.3 | 3.9 | 3.6 |
PPARγ (h) (agonist effect) | SLES | 1.0E-05 M | 0 | 0 | 0 |
PXR (h) (agonist effect) | SLES | 1.0E-05 M | -15.3 | -10.7 | -13 |
PR (h) (agonist effect) | SLES | 1.0E-05 M | -0.4 | -0.4 | -0.4 |
VDR (h) (agonist effect) | SLES | 1.0E-05 M | 0.1 | 0 | 0 |
Please see Figure 2 in Result figures (screening) in “Overall remarks, Attachments”.
Agonist Effect - Reference Compound Results:
Assay Name + Compound I.D. | EC50(M) | nH |
LXR alpha (h) (agonist effect) | ||
T0901317 | 5.1E-07 M | n/a |
CAR (h) (agonist effect) | ||
CITCO | 2.8E-07 M | n/a |
PPARα (h) (agonist effect) | ||
GW 7647 | 5.6E-09 M | n/a |
PPARδ (h) (agonist effect) | ||
GW 0742 | 2.3E-09 M | n/a |
PPARγ (h) (agonist effect) | ||
rosiglitazone | 3.7E-07 M | n/a |
PXR (h) (agonist effect) | ||
T0901317 | 2.1E-08 M | n/a |
PR (h) (agonist effect) | ||
progesterone | 2.5E-09 M | n/a |
VDR (h) (agonist effect) | ||
calcitriol | 2.1E-09 M | n/a |
Antagonist Effect - SLES Results:
Assay Name | Test Compound | Test Concentration | % Inhibition of Control Agonist Response | ||
1st | 2nd | Mean | |||
LXR alpha (h) (antagonist effect) | SLES | 1.0E-05 M | -1.3 | -2.1 | -1.7 |
CAR (h) (antagonist effect) | SLES | 1.0E-05 M | 11.2 | -2.3 | 4.5 |
PPARα (h) (antagonist effect) | SLES | 1.0E-05 M | -7.5 | -5.6 | -6.5 |
PPARδ (h) (antagonist effect) | SLES | 1.0E-05 M | -0.7 | -6.6 | -3.6 |
PPARγ (h) (antagonist effect) | SLES | 1.0E-05 M | -1.7 | -4 | -2.9 |
PXR (h) (antagonist effect) | SLES | 1.0E-05 M | -38.2 | -33.9 | -36.1 |
PR (h) (antagonist effect) | SLES | 1.0E-05 M | -1.1 | 0.5 | -0.3 |
VDR (h) (antagonist effect) | SLES | 1.0E-05 M | 1.3 | -3.5 | -1.1 |
Please see Figure 3 in Result figures (screening) in “Overall remarks, Attachments”.
Antagonist Effect - Reference Compound Results:
Assay Name + Compound I.D. | IC50(M) | KB(M) | nH |
CAR (h) (antagonist effect) | |||
Clotrimazole | 9.3E-06 M | 9.9E-07 M | n/a |
PPARα (h) (antagonist effect) | |||
GW 9662 | 4.4E-07 M | 1.7E-08 M | n/a |
PPARδ (h) (antagonist effect) | |||
GSK 0660 | 1.3E-05 M | 2.0E-06 M | n/a |
PPARγ (h) (antagonist effect) | |||
GW 9662 | 3.3E-08 M | 8.2E-09 M | n/a |
PXR (h) (antagonist effect) | |||
clotrimazole | 6.5E-06 M | 5.4E-07 M | n/a |
PR (h) (antagonist effect) | |||
mifepristone | 8.9E-09 M | 2.7E-10 M | n/a |
Enzyme and Assays - SLES Results:
Assay Name | Test Compound | Test Concentration | % Inhibition of Control Values | ||
1st | 2nd | Mean | |||
COX1(h) | SLES | 1.0E-05 M | 29.7 | 28.4 | 29.1 |
COX2(h) | SLES | 1.0E-05 M | -30.1 | -29 | -29.6 |
PDE3A (h) | SLES | 1.0E-05 M | -8.9 | 0.2 | -4.3 |
PDE4D2 (h) | SLES | 1.0E-05 M | 22.9 | 8.8 | 15.8 |
ACE (h) | SLES | 1.0E-05 M | -31.1 | -31.6 | -31.3 |
ACE-2 (h) | SLES | 1.0E-05 M | -37.9 | -39.3 | -38.6 |
Lck kinase (h) | SLES | 1.0E-05 M | -19 | -13 | -16 |
acetylcholinesterase (h) | SLES | 1.0E-05 M | -4.7 | -2.8 | -3.7 |
carbonic anhydrase II (h) | SLES | 1.0E-05 M | -6.9 | -1.1 | -4 |
HDAC1 (h) | SLES | 1.0E-05 M | -13.3 | 2.8 | -5.2 |
Aromatase / CYP19A1 | SLES | 1.0E-05 M | -9 | 17.7 | 4.4 |
Thyroperoxidase | SLES | 1.0E-05 M | 8.1 | 1 | 4.6 |
Please see Figure 4 in Result figures (screening) in “Overall remarks, Attachments”.
Enzyme and Assays - Reference Compound Results:
Assay Name + Compound I.D. | IC50(M) | nH |
COX1(h) | ||
Diclofenac | 1.7E-08 M | 1.7 |
COX2(h) | ||
NS398 | 2.0E-07 M | 1.6 |
PDE3A (h) | ||
milrinone | 3.6E-07 M | 0.8 |
PDE4D2 (h) | ||
Ro 20-1724 | 1.7E-07 M | 0.6 |
ACE (h) | ||
captopril | 7.3E-10 M | 1.2 |
ACE-2 (h) | ||
Ac-GG-26-NH2 | 6.1E-07 M | 1.4 |
Lck kinase (h) | ||
staurosporine | 3.6E-08 M | 2.0 |
acetylcholinesterase (h) | ||
galanthamine | 6.0E-07 M | 1.0 |
carbonic anhydrase II (h) | ||
acetazolamide | 3.1E-08 M | 1.6 |
HDAC1 (h) | ||
trichostatin A | 6.0E-09 M | 1.1 |
Aromatase / CYP19A1 | ||
Letrozole | 4.1E-10 M | 1.2 |
Thyroperoxidase | ||
Methimazole (MMI) | 1.5E-08 M | 1.3 |
Remaining results - SLES Results:
Assay Name | Test Compound | Test Concentration | % Inhibition of Control Values | ||
1st | 2nd | Mean | |||
DNMT1 Human DNA Methyltransferase Enzymatic Assay, Cerep | SLES | 1.0E-05 M | 40.0 | 23.7 | 31.8 |
Remaining results - Reference Compound Results:
Assay Name + Compound I.D. | IC50(M) | nH |
DNMT1 Human DNA Methyltransferase Enzymatic Assay, Cerep | ||
SAH | 1.6E-07 M | 0.9 |
Study 2:
Experimental (complete) Results:
Assay Name | Batch* | Spec. | Rep. | Conc. | % Inh. | IC50* | Ki | nH | R |
Compound: SLES | |||||||||
11-beta-Hydroxysteroid Dehydrogenase 1 (11-beta-HSD1) | 495136 | hum | 2 | 10 μM | 6 | ||||
HMG-CoA Reductase | 495029 | hum | 2 | 10 μM | -9 | ||||
Steroid 5α-Reductase | 495130 | rat | 2 | 10 μM | 19 | ||||
Colchicine | 495153 | rat | 2 | 10 μM | 13 |
* Batch: Represents compounds tested concurrently in the same assay(s).
hum=Human
Assay Name | Batch* | Spec. | Tissue | Rep. | Conc. | Criteria | Resp. | Ag. | Ant. | R |
Compound: SLES | ||||||||||
Estrogen Receptor alpha (ERα) Coactivator | 494901 | hum | Insect cells | 2 | 10 µM | ≥± 50% | -1% | ND | ||
Estrogen Receptor alpha (ERα) Coactivator | 494902 | hum | Insect cells | 2 | 10 µM | ≥± 50% | ND | 9% | ||
Estrogen Receptor beta (ERβ) Coactivator | 494903 | hum | Insect cells | 2 | 10 µM | ≥± 50% | -2% | ND | ||
Estrogen Receptor beta (ERβ) Coactivator | 494904 | hum | Insect cells | 2 | 10 µM | ≥± 50% | ND | 5% | ||
Retinoic Acid Receptor, RARA (RARα) | 494892 | hum | E. coli | 2 | 10 µM | ≥± 50% | 2% | ND | ||
Retinoic Acid Receptor, RARA (RARα) | 494893 | hum | E. coli | 2 | 10 µM | ≥± 50% | ND | 1% | ||
Retinoid X Receptor Alpha (RXRα) Coactivator | 494897 | hum | Insect cells | 2 | 10 µM | ≥± 50% | -1% | ND | ||
Retinoid X Receptor Alpha (RXRα) Coactivator | 494898 | hum | Insect cells | 2 | 10 µM | ≥± 50% | ND | -5% | ||
Retinoid X Receptor Beta (RXRβ) Coactivator | 494899 | hum | Insect cells | 2 | 10 µM | ≥± 50% | -1% | ND | ||
Retinoid X Receptor Beta (RXRβ) Coactivator | 494900 | hum | Insect cells | 2 | 10 µM | ≥± 50% | ND | 15% | ||
Thyroid Receptor Alpha Coactivator | 494894 | hum | Insect cells | 2 | 10 µM | ≥± 50% | 1% | ND | ||
Thyroid Receptor Beta Coactivator | 494895 | hum | Insect cells | 2 | 10 µM | ≥± 50% | 1% | ND |
* Batch: Represents compounds tested concurrently in the same assay(s). Ag.=Agonist; Ant.=Antagonist; Resp.=Response; ND=Assay Test Not Done hum=Human
Reference Compound results:
Assay Name | Reference Compound | Historical | Concurrent | |||
IC50* | Ki | nH | Batch * | IC50* | ||
11-beta-Hydroxysteroid Dehydrogenase 1 (11-beta-HSD1) | PF 915275 | 0.041 μM | 495136 | 0.035 μM | ||
HMG-CoA Reductase | Lovastatin | 0.074 μM | 495029 | 0.054 μM | ||
Steroid 5α-Reductase | Finasteride | 0.036 μM | 495130 | 0.032 μM | ||
Colchicine | Colchicine | 2.80 μM | 2.80 μM | 1.30 | 495153 | 1.62 μM |
* Batch: Represents compounds tested concurrently in the same assay(s).
Historical | Concurrent ‡ | |||
Assay Name | Reference Compound | IC50/EC50* | Batch* | IC50/EC50 |
Estrogen Receptor alpha (ERα) Coactivator - Ag. | Estriol | 3.0 nM | 494901 | 4.26 nM |
Estrogen Receptor alpha (ERα) Coactivator - Ant. | ICI182780 | 13.0 nM | 494902 | 6.60 nM |
Estrogen Receptor beta (ERβ) Coactivator - Ag. | Estriol | 3.10 nM | 494903 | 4.61 nM |
Estrogen Receptor beta (ERβ) Coactivator - Ant. | ICI182780 | 7.30 nM | 494904 | 5.15 nM |
Retinoic Acid Receptor, RARA (RARα) - Ag. | TTNPB | 0.88 nM | 494892 | 0.44 nM |
Retinoic Acid Receptor, RARA (RARα) - Ant. | Ro-415253 | 4.10 nM | 494893 | 2.32 nM |
Retinoid X Receptor Alpha (RXRα) Coactivator - Ag. | Fluorobexarotene | 3.60 nM | 494897 | 3.14 nM |
Retinoid X Receptor Alpha (RXRα) Coactivator - Ant. | UVI3003 | 0.055 μM | 494898 | 0.12 μM |
Retinoid X Receptor Beta (RXRβ) Coactivator - Ag. | Fluorobexarotene | 9.90 nM | 494899 | 8.74 nM |
Retinoid X Receptor Beta (RXRβ) Coactivator - Ant. | HX531 | 6.30 μM | 494900 | 6.60 μM |
Thyroid Receptor Alpha Coactivator - Ag. | T3 | 0.12 nM | 494894 | 0.17 nM |
Thyroid Receptor Beta Coactivator - Ag. | T3 | 0.37 nM | 494895 | 0.34 nM |
* Batch: Represents compounds tested concurrently in the same assay(s).
‡ For some assays only a single concentration of reference compound is tested.
Study 3:
ATP Concentration: 90 µM
Sample | Counts | Mean (Counts - Blanks) | Activity (% Control) | Mean | SD* |
SLES @ 10µM | 9043 | 9092 | 101 | 104 | 5 |
9604 | 107 | ||||
Control | 8441 | 8739 | 94 | 100 | 7 |
8608 | 96 | ||||
9124 | 102 | ||||
9710 | 108 | ||||
Blank | 211 | / | / | / | / |
252 | / |
* NB. Where n = 2, the value reported here is actually range / √ 2
Study 4: Binding Assays (including receptors, ion channels, transporters and other enzymes)
Binding Assays - SLES Results:
Assay Name | Test Compound | Test Concentration | % Inhibition of Control Specific Binding | ||
1st | 2nd | mean | |||
LXR alpha (h) agonist radioligand | SLES | 1.00E-08 M | -13.6 | -26.9 | -20.3 |
1.00E-07 M | -5.9 | -9.6 | -7.7 | ||
3.00E-07 M | 2.8 | 8.6 | 5.7 | ||
1.00E-06 M | -10.3 | -10.1 | -10.2 | ||
3.00E-06 M | 20 | 19.4 | 19.7 | ||
1.00E-05 M | 68.5 | 70.3 | 69.4 | ||
3.00E-05 M | 75.9 | 75.9 | 75.9 | ||
3.00E-04 M | 88.4 | 89.3 | 88.9 |
IC50(M): 4.5E-06 M
Ki(M): 3.6E-06 M
nH: 2.0
Please see Figure 1 in Result figures (dose response) in “Overall remarks, Attachments”.
Binding Assays - Reference Compound Results:
LXR alpha (h) agonist radioligand: T0901317
IC50(M): 1.3E-07 M
Ki(M): 1.0E-07 M
nH: 1.0
Cellular and Nuclear Receptor Functional Assays:
Agonist Effect - SLES Results:
Assay Name | Test Compound | Test Concentration | % of Control Agonist Response | ||
1st | 2nd | Mean | |||
LXR alpha (h) (agonist effect) | SLES | 1.00E-08 M | -0.1 | 0 | 0 |
1.00E-07 M | 0 | 0 | 0 | ||
3.00E-07 M | 0 | 0 | 0 | ||
1.00E-06 M | 0.1 | 0 | 0 | ||
3.00E-06 M | 0.1 | 0 | 0.1 | ||
1.00E-05 M | 0.1 | 0 | 0 | ||
3.00E-05 M | 0.4 | 0.1 | 0.2 | ||
3.00E-04 M | -0.4 | -0.5 | -0.4 |
EC50 value not calculable. Concentration-response curve shows less than 25% effect at the highest validated testing concentration.
Please see Figure 2 in Result figures (dose response) in “Overall remarks, Attachments”.
Agonist Effect - Reference Compound Results:
LXR alpha (h) agonist effect : T0901317
EC50(M): 6.1E-07 M
nH: n/a
Antagonist Effect - SLES Results:
Assay Name | Test Compound | Test Concentration | % of Control Agonist Response | ||
1st | 2nd | Mean | |||
LXR alpha (h) (antagonist effect) | SLES | 1.00E-08 M | 2.3 | -3.2 | -0.5 |
1.00E-07 M | 0.9 | 68.3* | 0.9 | ||
3.00E-07 M | -1.9 | 1.7 | -0.1 | ||
1.00E-06 M | 0.3 | -1.3 | -0.5 | ||
3.00E-06 M | 0.5 | -2.6 | -1 | ||
1.00E-05 M | -0.3 | -0.1 | -0.2 | ||
3.00E-05 M | 0.7 | -3.4 | -1.3 | ||
3.00E-04 M | 98.1 | 100.4 | 99.3 |
* That replicate was excluded from the calculation
IC50(M): 2.0E-04 M
KB(M): 2.7E-05 M
nH: n/a
Please see Figure 3 in Result figures (dose response) in “Overall remarks, Attachments”.
Antagonist Effect - Reference Compound Results:
LXR alpha (h) agonist effect : T0901317
EC50(M): 6.1E-07 M
nH: n/a
Table 4 - Summary of results (see also Figure 16 in "Attachments"; individual assay results are provided below)
| SLES | |||
| No S9 | + S9 | ||
Assay | LEC | PC50 | LEC | PC50 |
Cytotox CALUX® | 2.1E-4 | 2.5E-4 | 3.1E-4 | 3.6E-4 |
ERα CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-ERα CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
AR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-AR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
TRβ CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-TRβ CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
PR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-PR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
GR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-GR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
PXR CALUX® | 6.3E-5 | >1E-4 | 6.3E-5 | >1E-4 |
anti-PXR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
LXR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-LXR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
RAR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-RAR CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
PPARα CALUX® | 1.3E-5 | >1E-4 | 2.9E-5 | >1E-4 |
anti-PPARα CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
PPARγ CALUX® | 9.1E-5 | >1E-4 | 1.0E-4 | >1E-4 |
anti-PPARγ CALUX® | 1.7E-5 | >1E-4 | 2.5E-5 | >1E-4 |
PPARδ CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
anti-PPARδ CALUX® | >1E-4 | >1E-4 | >1E-4 | >1E-4 |
TCF CALUX® | >1E-3 | >1E-3 | >1E-3 | >1E-3 |
anti-TCF CALUX® | >1E-3 | >1E-3 | >1E-3 | >1E-3 |
hTPO | NA | NA | NA | NA |
H295R (E2) | 1E-5 | NA | NA | NA |
H295R + preincubation (E2) | 2E-5 | NA | 2E-5 | NA |
H295R (T) | >1E-5 | NA | NA | NA |
H295R + preincubation (T) | >2E-5 | NA | >2E-5 | NA |
Values are concentrations (M) final in well. LEC = lowest effect concentration. Italicised values are based on only one datapoint exceeding the activity threshold. Bold values are based on at least two datapoints exceeding the activity threshold. PC50 = concentration where the activity equals 50% of the maximum activity of the assay’s reference compound. NA = not applicable.
Cytotoxicity CALUX® results
The compound becomes cytotoxic at ~2E-4M (see Figure 1).
CALUX® results
(anti) ERα CALUX® assay results
The compound does not activate the ERα CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. Also on the anti- ERα CALUX® assay, the activity threshold is not reached (see Figure 2).
(anti) AR CALUX® assay results
The compound does not activate the AR CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. For the anti-AR CALUX® assay, the activity threshold is reached at the highest concentration tested. However, since this concentration is very close to cytotoxicity, this decrease in signal is most likely due to the onset of cytotoxicity rather than specific receptor antagonism (see Figure 3).
(anti) TRβ CALUX® assay results
The compound does not activate the TRβ CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. For the anti- TRβ CALUX® assay, the activity threshold is reached at the highest concentration tested. However, since this concentration is very close to cytotoxicity, this decrease in signal is most likely due to the onset of cytotoxicity rather than specific receptor antagonism (see Figure 4).
(anti) PR CALUX® assay results
The compound does not activate the PR CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. Also on the anti- PR CALUX® assay, the activity threshold is not reached (see Figure 5).
(anti) GR CALUX® assay results
The compound does not activate the GR CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. Also on the anti- GR CALUX® assay, the activity threshold is not reached (see Figure 6).
(anti) PXR CALUX® assay results. The compound activates the PXR CALUX® assay, although only one concentration point exceeds the activity threshold. No difference is observed in the presence of rat liver S9 fraction. On the anti- PXR CALUX® assay, the activity threshold is not reached (see Figure 7).
(anti) LXR CALUX® assay results. The compound does not activate the LXR CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. Also on the anti- LXR CALUX® assay, the activity threshold is not reached (see Figure 8).
(anti) RAR CALUX® assay results. The compound does not activate the RAR CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. For the anti- RAR CALUX® assay, the activity threshold is reached at the highest concentration tested. However, since this concentration is very close to cytotoxicity, this decrease in signal is most likely due to the onset of cytotoxicity rather than specific receptor antagonism (see Figure 9).
(anti) PPARα CALUX® assay results. The compound is slightly active, i.e., two data points exceed the activity threshold, in the PPARα CALUX® assay in the absence and presence of rat liver S9 fraction, at a concentration of ~1E-5 M. The compound does not show antagonistic behaviour on the anti- PPARα CALUX® assay (see Figure 10).
(anti) PPARγ CALUX® assay results. The compound shows a concentration dependent increase in signal in the PPARγ CALUX® assay which reaches the threshold of activity, albeit only at the highest tested concentration. On the anti- PPARγ CALUX® assay the compound shows a dose-dependent decrease in signal. Two datapoints exceed the threshold of activity; the compound therefore classifies as a PPARγ antagonist with a LEC of ~2E-5 M (see Figure 11).
(anti) PPARδ CALUX® assay results. Although the compound shows a slight concentration dependent increase in signal in the PPARδ CALUX® assay, the signal does not reach the threshold of activity. The compound also does not show antagonistic behaviour on the anti- PPARδ CALUX® assay (see Figure 12).
(anti) TCF CALUX® assay results. The compound does not activate the TCF CALUX® assay, neither in the absence nor presence of rat liver S9 fraction. On the anti- TCF CALUX® assay the compound shows a dose-dependent signal decrease, but this decrease coincides with the cytotoxicity data and is therefore most likely not caused by pathway inhibition (see Figure 13).
hTPO inhibition assay results
The compound shows a very strong concentration dependent increase in light signal. This is most likely not the effect of direct interaction with hTPO, but most likely an artificial enhancing effect on the luminescence reaction of the luminol-H2O2 system (see discussion section). Because of the magnitude of this effect, it is not possible to reliably assess whether the compound inhibits hTPO (see Figure 14).
H295R steroidogenesis assay results
Results for estrogen and androgen production are expressed as fold-induction above production of estrogens and androgens in vehicle control incubations (see Table 4 for LOEC values).
The compound significantly increases estrogen production by H295R cells, both as a "pure compound" and after preincubation with metabolic enzymes. Effects on androgen production are less straightforward to assess; although the "pure compound" clearly shows a concentration dependent decrease, this curve overlaps with the green cytotoxicity curve (see Figure 15).
Description of key information
The bioactivity of Alcohols, C12-14, ethoxylated, sulfates, sodium salts (SLES) was assessed in a battery of in vitro tests relevant to systemic, reproductive, and developmental toxicity. Concentration-response curves were derived for 36 cell stress markers measured in HepG2 Cells and High Throughput Transcriptomics was conducted in HepG2, HepaRG and MCF7 cells. In vitro pharmacological profiling (IPP) of SLES against 73 targets was conducted, with specific follow-up with CALUX functional assays where necessary. Specific assays relating to developmental toxicity (Reprotracker, devTOXqp) were also conducted. Lowest reported Points of Departure (PoDs) were adjusted to correct for the activity of SLES (70.23%), and ranged from 0.98 - 46.49 µM.
This data was used in a Next Generation Safety Assessment (NGSA), which is an exposure-led approach to safety assessment that employs the use of these New Approach Methodologies (NAMs). A full description of this approach can be found in Section 13.2 (Narrative Document/Next Generation Safety Assessment).
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.