Sieli et al. The linear statistical model was a two by seven factorial (two treatments, seven time points). If main effects were statistically significant at p < 0.05, mean differences were determined using Fisher’s least significant difference. All data are presented as mean ± SE. Pharmacokinetic analysis. Unconjugated and total serum concentration–time profiles after oral and diet BPA-d6 exposure were ana- lyzed with a noncompartmental analysis (NCA) using WinNonlin (WinNonlin® Professional, version 5.3; Pharsight Corporation, Cary, NC, USA). We calculated area under the curve (AUC) up to the last quantifiable serum con- centration [i.e., AUC(0–24hr)], using the linear trapezoidal rule. For additional information on NCA analysis, see Supplemental Material, p. 5 (http://dx.doi.org/10.1289/ehp.1003385). The sparse data option of WinNonlin was used, allowing computation of the different SEs associated with estimated parameters (see Supplemental Material, Tables 3–5). We analyzed unconjugated BPA-d6 serum concentrations after oral exposure with a compartmental analysis using a mono- compartmental model without lag time. Pooled data were fitted using the following equation (the so-called Bateman equation): C^ t h = K01 # dose V F # ^K01 - K10h 6exp^- K10 # th - exp^- K01 # th@, [1] where C(t) is the pooled BPA-d6 serum con- centration at time t, F is the unknown bio- availability of BPA-d6, dose is the BPA-d6 dose, V is the volume of distribution (milli- liters per kilogram), K01 (per hour) is the first-order rate constant of absorption and K10 c Cumulative ingested BPA-d6 Cumulative ingested BPA-d6 determined by deconvolution analysis Light c 16 ) g k / g12 m ( 6 d - P A 8 B e v i t la 4 u m u C a 0 0 a b 4 c Dark 8 12 16 20 24 Time after BPA-d6 administration (hr) 1900 2300 0300 0700 1100 1500 1900 Dark/light cycle Figure 1. Cumulative diet exposure to BPA‑ d6 versus time and night/day cycle estimated by amount of food consumed and by deconvolution. Deconvolution analysis was performed by using the oral bolus administration as a reference to evaluate the in vivo BPA‑ d6 input rate (BW) over 11 hr (i.e, during the night). Time points with different superscripts are significantly different from each other based on log10 scale analysis (p < 0.01). 1262 (per hour) is the first-order rate constant of elimination. V/F, K10, and K01 were estimated. Iterative reweighting was used during minimi- zation process, that is, the data were weighted by the inverse of the observed value (1/Yobs) [see Supplemental Material, Figure 1 (http:// dx.doi.org/10.1289/ehp.1003385)]. The good- ness of fit of the model was assessed by using least-squares criteria and visual inspection of residuals. Weighted residuals are presented in Supplemental Material, Figure 2. For addi- tional information on compartmental analysis, see Supplemental Material, p. 6. The relative bioavailability between the two conditions of administration was calcu- lated from the following equation: F % = AUC^0 - 24 hrhdiet AUC^0 - 24 hrhoral bolus # Doseoral bolus # 100. Dosediet [2] AUC(0–24hr)oral bolus and AUC(0–24hr)diet (the AUC of unconjugated serum BPA) was estimated by noncompartmental analysis, and Doseoral bolus and Dosediet were the actual nomi- nal dose and dose ingested through the diet, respectively, with the diet dose estimated by food consumption. We also used Equation 2 to estimate the extent of BPA-d6 absorption replacing unconjugated BPA-d6 AUCs by the corresponding total BPA-d6 AUCs. We used deconvolution to evaluate in vivo drug release and delivery when data from a known drug input were available (i.e., the kinetics after the oral bolus BPA-d6 admin- istration). The BPA-d6 input rate evaluated when BPA-d6 was administered in the diet corresponded to the in vivo BPA-d6 release from food followed by a BPA-d6 delivery to the general systemic circulation. In this exper- iment, deconvolution was used to evaluate the in vivo BPA-d6 input rate (milligrams per hour) into blood of mice fed during the night. Data from 0 to 11 hours (i.e., only data col- lected during the nocturnal period) were con- sidered. The reference BPA input was obtained from the bolus oral administration for which the input into the digestive tract was instanta- neous (drenching). The deconvolution analy- sis was performed using the mean of pooled unconjugated BPA-d6 serum concentrations after diet exposure over time. More details and results of these analyses are included in Supplemental Material, pp. 5–8, 11–18, and 21; Tables 3–7; and Figures 3 and 4 (http:// dx.doi.org/10.1289/ehp.1003385). Results Measurement of external cumulative expo‑ sure rate to BPA‑ d6 in diet‑ exposed mice. By knowing the weight of BPA-d6–supplemented diet provided at the outset of the experiment, the amounts remaining at each time point of serum collection, and the weight of the mice in each cage at the time of serum col- lection, we could calculate the cumulative exposure of BPA-d6 (milligrams per kilogram) in the diet-exposed group (Figure 1). Mice were placed on the BPA-d6 supplemented diet at 1900 hours (i.e., at the end of their normal light cycle), and within the first 6 hr (i.e., by 0100 hours) the animals had eaten about 50% of the food consumed over 24 hr (Figure 1). By this time point, mice had con- sumed 6.5 ± 0.4 mg/kg BPA-d6 (mean ± SE). After 11 hr, consumption had increased to 8.6 ± 1.3 mg/kg and at 24 hr had reached 13.0 ± 1.1 mg/kg. After 7 days of consum- ing the BPA-d6–supplemented diet, the total ingested dose was 78.1 ± 0.7 mg/kg rather than the 140 mg/kg expected dose. No differences in BW were observed among the mice on the two treatment regimens, and overall body mass did not change significantly over the 7-day experimental period. In addi- tion, none of the mice showed outward signs of ill health. These data thus suggest that BPA-d6 in the feed was well tolerated. Measurement of internal exposure to unconjugated BPA‑ d6 in serum. For mice receiving the oral bolus (20 mg/kg BW), maximum concentration (Cmax) of unconju- gated BPA-d6 (21.0 ± 3.9 ng/mL, mean ± SE) occurred within 1 hr (i.e., by 2000 hours) of administration of BPA-d6 (Table 1 and Figure 2A) and declined slowly thereafter, reaching barely detectable concentrations after 24 hours (i.e., at 1900 hours the day after bolus administration). In the diet-exposed group, the estimated dose was 13 mg/kg BW over the first 24 hr, and peak BPA-d6 concentra- tions (18.8 ± 4.4 ng/mL) were not observed until 6 hr (0100 hours) after the initiation of the BPA-d6–supplemented diet (Table 1 and Figure 2A); unconjugated serum BPA-d6 concentrations declined significantly by 11 hr. However, because Cmax occurred after consuming only 6.5 mg/kg BW of BPA-d6 (Figures 1 and 2), we derived a scaled Cmax value for dietary exposure consistent with the oral bolus dose of 20 mg/kg BW [i.e., (20 mg/kg ÷ 6.5 mg/kg) × 18.8 ng/mL] result- ing in an estimated peak concentration of 57.9 ng/mL for diet exposure. Unconjugated BPA-d6 serum concentrations collected at 1900 hours from mice on BPA-d6–containing diet for 7 days were higher than unconjugated BPA-d6 serum concentrations in mice on this diet for 24 hr (13.2 ± 5.2 ng/mL versus 6.9 ± 3.3 ng/mL, p < 0.05), revealing that sig- nificant buildup of biologically active BPA-d6 had occurred during the 7-day exposure period. To compare the oral bolus and diet groups at the same external dose, we scaled the dose of the diet-exposed group to 20 mg/kg BW, and the AUC(0–24hr) was modestly greater (but not statistically significant) in the diet ovlume 119 | number 9 | September 2011 • Environmental Health Perspectives