Results of modulation studies.

<p>Each graph shows the effect of a mixture of 16 modulators (A) or one of 16 individual modulators (B–Q), concentration indicated on the x-axis, on the ESCREEN response evoked by a reference mixture of 14 estrogens (Mixture 3d, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t004" target="_blank">Table 4</a>). Experimental results are shown as grey circles representing each replicate (duplicate testing within the assay) and obtained in three independent (A) or one (B–Q) experiment (s). Experimental results were normalized by setting the value observed for the REF_mix alone, the concentration of which was selected to evoke a concentration of ca. 50%, to 0.5. The positive control values (black circles, eight replicates per experimental plate, 25 nM estradiol) are indicated adjacent to the y-axis. A horizontal grey strap indicates the approximate range of values observed using the positive control values on all experimental plates but centered at 0.5 to give a visual indication of the random noise present in the data. A black line indicates a LOESS spline fit to the data (A–Q) and a grey line indicates linear regression fit to the data for individual modulators (B–Q). A vertical dotted line is drawn at a mixture concentration of 1.6 µM (A) or at the concentration of each modulator present at that mixture concentration (0.1 uM, B–Q). Visual inspection of the individual modulator results (B–Q) was used to identify possible modulation, i.e. when the experimental data appears to deviate from the linear regression, or when the linear regression is not horizontal, and when the magnitude of the modulation is outside the variation expected in the assay.</p

Different effects of phthalates in ERLUX and ESCREEN.

<p>Graphs show the results of testing four phthalates (DEHP, BBP, DBP, DEP) in the ERLUX (A–D) and ESCREEN (E–H) assays. Each phthalate was tested in two independent experiments in ERLUX (triplicate testing within assay) and three (DEHP) or one (BBP, DBP, DEP) experiments in ESCREEN (duplicate testing within assay).</p

Estrogenicity of individual compounds (ESCREEN).

<p>EC₁₀, EC₂₅: concentration provoking 10% and 25% effect, respectively. Values in brackets denote the upper and lower limits of the approximate 95% confidence interval; the column “RM” indicates the mathematical regression function as defined by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone.0043606-Scholze1" target="_blank">[13]</a>; θ^∧₁, θ^∧₂, θ^∧₃, θ^∧_max estimated model parameters, given for concentrations expressed in M (rounded values); θ_min were not estimated, but set to 0 relating to the mean value of the negative vehicle controls.</p

Statistical uncertainty of predicted and observed effect concentrations for mixtures (ESCREEN).

<p>CA – Concentration Addition, IA – Independent Action, CI – Confidence Interval; All predictions statistically significant to the observed ECs are shown in bold; *Effect mixture concentration for effect levels higher than the lowest estimated compound maximal model asymptote are extrapolated either (i) by assuming no contribution of this compound to the overall mixture effect (toxic unit equals zero), or (ii) by setting the compounds’ toxic unit to a fixed level equalling the value at the mixture concentration producing an effect of 0.7*θ_max (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t003" target="_blank">Table 3</a>). The right side of the interval corresponds to (i) and the left side to (ii), defining the range of possible CA predictions.</p

Test mixtures (ERLUX &amp; ESCREEN).

<p>Rounded values given for relative proportions.</p

Estrogenicity of individual compounds (ERLUX).

<p>EC₁₀, EC₂₅: concentration producing 10% and 25% effect, respectively. Values in brackets denote the upper and lower limits of the approximate 95% confidence interval; the column “RM” indicates the mathematical regression function as defined by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone.0043606-Scholze1" target="_blank">[13]</a>; θ^∧₁, θ^∧₂, θ^∧₃, θ^∧_max estimated model parameters, given for concentrations expressed in M (rounded values); θ_min were not estimated, but set to 0 relating to the mean value of the negative vehicle controls.</p

Distribution of toxic units.

<p>Each graph shows the distribution of toxic units as predicted by CA at the EC10 level for mixtures of estrogenic compounds tested in the ERLUX and ESCREEN. A) 17 component mixture at fixed mixture ratio proportional to the individual EC10’s (ERLUX, Mixture1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t004" target="_blank">Table 4</a>)). B) 16 component mixture at fixed mixture ratio proportional to the individual EC25’s (ESCREEN, Mixture3a (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t004" target="_blank">Table 4</a>)). C) 14 component mixture at fixed ratio proportional to approximate human tissue concentrations (ERLUX, Mixture 2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t004" target="_blank">Table 4</a>)). D) 13 component mixture at fixed ratio proportional to approximate human tissue concentrations (ESCREEN, Mixture 4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t004" target="_blank">Table 4</a>)).</p

Observations of estrogenicity or toxicity for chemicals screened as potential modulators.

a<p>results from modulator screening studies were classified as ‘<i>clear negative</i>’ (reduction in effect of REFmix at multiple concentrations showing an approximately sigmoid dose-response), ‘<i>possible negative</i>’ (reduction in effect of REFmix at a single concentration or multiple concentrations with a apparent linear concentration-response relationship) and ‘<i>none</i>’ (no indication of a negative or positive effect).</p>b<p>‘<i>Active’</i> indicates a positive signal in ESCREEN (estrogenicity), clearly distinguishable from assay variability and noise and usually supported by a dose-response (i.e. not reliant on data from only a single concentration).</p>c<p>‘<i>Possible toxicity’</i> indicates a decrease in value for treated wells below that of vehicle controls, this signal is small so the assignment of toxicity is not certain; ‘<i>Toxic’</i> indicates a reduction in signal evoked by increasing concentrations above those at which a chemical showed activity (estrogenicity).</p

Concentration-response curves from single chemical testing and inter-assay correlation.

<p>Graphs show the results of testing single estrogenic chemicals in the ERLUX (left) or ESCREEN (right) assays. Each single chemical was tested in three or more independent experiments. Results are shown as the best fit regression model, for which details are provided <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043606#pone-0043606-t003" target="_blank">3</a>. Middle graph shows the correlation between EC10 values obtained in the ERLUX (y-axis) and ESCREEN (x-axis) assays.</p