Estrogenicity of individual compounds and mixture.

<p>EC10: concentration associated with 10% proliferation rate. Values in brackets denote the upper and lower limits of the approximate 95% confidence interval based on bootstrap replicates; the column “RM” indicates the mathematical regression function, used for describing the concentration response relationships (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088808#pone.0088808-Scholze1" target="_blank">[<i>17</i>]</a> for more details). estimated model parameters, if marked by *, then held fixed, i.e. not estimated.</p

Predicted and observed estrogenic activity of a mixture of 21 components combined according to their individual EC₁₀.

<p>Observed mixture effects (black circles), controls (gray circles) and regression curve (solid black line, with 95% confidence belt as dotted black lines) are from four independent experiments, each tested on three micro-titer plates. Effect variation is expressed by box and whisker diagrams; the boxes show 1.5 interquartile ranges around the median. Predicted effects were calculated using the DA concept (solid red line), dotted red lines show the corresponding approximate 95% confidence belt. The green lines and the green shaded area are the lower and upper estimates of predictions that are based on toxic unit extrapolations (see Material & Methods for details)</p

Concentration-response curves for the 21 tested estrogenic chemicals with regression lines derived from the best fitting models for E-Screen <i>in vitro</i> data.

<p>All agents were tested in at least four independent experiments, run on up to three micro-titer plates, with each plate containing eight increasing concentrations of the test chemical in duplicates (data not shown).</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

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

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

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

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

Test mixtures (ERLUX &amp; ESCREEN).

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

Predicted and observed estrogenic activity of the mixture of 21 components shown in Figure 4.

<p>The predicted effects were calculated using the model of DA (solid red line) according to the GCA approach by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088808#pone.0088808-Howard1" target="_blank">[14]</a>. Observed mixture effects (black circles) and controls (gray circles) are from four independent experiments, each tested on three micro-titer plates. Effect variation is expressed by box and whisker diagrams; the boxes show 1.5 interquartile ranges around the median.</p