Table 6.

Data on the fate of BPA in humans have been published by Völkel et al., 2002, 2005^*

Criticism	Assessment of the commission
1.“Several inconsistencies are present in this report and therefore raise questions about its reliability (Völkel et al., 2002). For instance, the authors report two different values when maximal plasma concentrations (Cmax) were achieved (1.35 hours versus 4 hours).”	The authors definitely do not report two different time points for maximal plasma concentrations. In fact, they wrote, “The results from this study show that maximal plasma concentrations of d16-bisphenol A glucuronide were reached approximately 80 min after oral administration” (Völkel et al., 2002; p. 1285). The blood concentrations of d(16)-BPA-glucuronide are summarized in Figure 6 (Völkel et al., 2002; p. 1285) and show only one peak approximately 80 minutes after administration of BPA and not two peaks as claimed by Vandenberg et al. (2010a, 2010b). As clearly stated in the publication, two separate studies were performed giving identical doses. The first study was intended to provide mass balance/recovery and observe blood levels over a longer time period. Urinary and blood concentrations were determined after oral administration of BPA. In this first experiment, blood concentrations were measured 4, 8, 12, 16, 24, and 32 hours after BPA administration and the highest concentration of BPA-glucuronide was observed after the earliest examined period of 4 hours (Figure 5B in Völkel et al., 2002). In the second study blood levels of BPA-glucuronide were analyzed in shorter intervals of 0, 51, 81, 111, 141, 201, 261, 321, and 381 minutes (Figure 6 in Völkel et al., 2002) to determine initial kinetics. This resulted in a kinetic profile with a single peak after approximately 80 minutes. In fact, extrapolation of the half-life from the first study exactly predicts the levels at the sampling points in the second study and blood concentrations of BPA-glucuronide at overlapping sampling points from the two studies are essentially identical. It should be considered that the first experiment was needed to assess recovery and kinetics in blood at later time points, which cannot be studied with the design required for assessment of distribution and initial elimination from blood, because the latter requires short sampling intervals and thus blood sampling by a venous port. It should also be noted that the authors (Völkel et al., 2002) never mentioned “two different values for the time when Cmaxwas achieved” in their paper. In conclusion, this criticism of Vandenberg et al. (2010a, 2010b) is not justified.
2.“Additionally, the BPA-glucuronide levels reported in blood are higher than the total BPA concentrations measured in the same individuals.”	The blood concentrations of d(16)-BPA-glucuronide and total d(16)-BPA after glucuronidase treatment are very similar with overlapping error bars as shown in Figure 6 (Völkel et al., 2002). There is no significant difference. Vandenberg et al. may have misinterpreted Figure 6 because the peak values of d(16)-BPA-glucuronide determined by LC-MS/MS are slightly above total d(16)-BPA determined after glucuronidase treatment. However, the difference is in the range of the experimental error. In conclusion, Figure 6 of Völkel et al. (2002) clearly shows that concentrations of total and glucuronidated BPA in blood are very similar after oral administration of BPA.
3.“Finally, the authors indicate that they measured BPA metabolism in 3 women, a group of 3 men, and then in a separate group of 4 men, yet the groups of male volunteers clearly overlap, making the data compiled from combining these two groups questionable.”	The characteristics (gender, age, height, body weight) of the individuals who joined the pharmacokinetic study are given in Table 1 (Völkel et al., 2002, p. 1282), together with identification numbers. Urinary excretion was analyzed in individuals A, B, C, E, F, and G, as described in Materials and Methods. First, since a range-finding study was not carried out for all male individuals, data from four subjects had to be handled separately. Second, BPA-glucuronide concentrations in blood of these individuals were followed over relatively long periods (4, 8, 12, 16, 24, and 32 hours) after administration of BPA (Figure 5) in the first experiment. In the second experiment, the kinetics in blood were determined using shorter intervals (Figure 6). This second experiment was performed with individuals G, M, N, and O (Figure 6). Therefore, only one individual (G) took part in both studies and the determined blood concentrations are highly consistent between the two studies (see above). The commission sees no reason why this constellation should compromise the results, particularly since the study design was clearly described.
4.Vandenberg et al. (2010a, 2010b) criticized that “there was alack of acknowledgement of the likelihood of different toxicokinetics when BPA exposure is continuous compared to a single administration. Ginsberg and Rice suggested that results from the Völkel et al. study were more consistent with delayed excretion from long-term internal storage or cycling between conjugation and deconjugation (Ginsberg and Rice, 2009).”	First, it should be emphasised that the aim of the study of Völkel et al. (2002) was to analyze toxicokinetics of BPA after administration of a single dose. Therefore, this argument cannot be used to criticize the study. Second, basic pharmacokinetic calculations permit the prediction of blood concentrations after repeated doses once the half-life and clearance of a compound are known. The relevant parameters for such calculations are available from Völkel et al. (2002). It should also be kept in mind that the argument of Ginsberg and Rice (2009) is highly questionable, since it is based only on a higher blood concentration at only one time point (24 hours) in one gender where error bars clearly overlap and the differences are not statistically significant. In addition, none of the available other primate studies give any evidence for cycling of BPA. Long-term internal storage or cycling is also very unlikely, since most of the administered compound is recovered in the urine within 24 hours (Völkel et al., 2002).
5.Vandenberg et al. also criticized that “the potential for BPA to have actions at low levels (in the ng/ml range) was not considered” in the Völkel study.	Again, this is not the aim of a toxicokinetics study. The commission has the impression that Vandenberg et al. (2010a, 2010b) confuse the goals of a toxicokinetics study and risk assessment. We comment on this under “criticism 12.” The relevance of low doses of BPA has been discussed insection “Do oral low BPA doses below 5 mg/kg bw/day cause adverse health effects in laboratory animals?” of this article. It is important to note that recent pharmacokinetic modeling studies have shown that the highest reported oral exposures for BPA in the general population would result in blood concentrations much lower than concentrations claimed to cause some effects in vitro (Mielke and Gundert-Remy, 2009).
6.“Third, this toxicokinetics study was designed to assess the metabolism of BPA following oral exposure because until very recently (Stahlhut et al., 2009), it was assumed that most if not all BPA exposure in humans was occurring via the oral route. All sources of BPA have not been identified, so non-oral exposures cannot be discounted.”	This is not a valid argument against the quality of the study of Völkel et al. (2002) because this study aimed at analyzing the pharmacokinetics after oral administration of BPA. We have discussed the question of oral versus non-oral exposure in section “Specific exposure conditions” of this article. Briefly, it should be kept in mind that BPA is quantitatively excreted via the urine. Techniques to estimate overall uptake of BPA from urinary concentrations are generally accepted (and discussed in Mielke and Gundert-Remy, 2009).
7.“Finally, the possibility of differences in toxicokinetics under different physiologic paradigms was overlooked…. The differences between sexes and age groups in urinary levels of BPA found in biomonitoring studies from CDC (Calafat et al., 2005, 2008) raise the possibility that toxicokinetics of chemicals and drugs including BPA are likely to be different in fetuses and neonates compared to adults.”	Analysis of differences between neonates and adults, etc., was not the aim of the study of Völkel etal. (2002). Again, Vandenberg et al. (2010a, 2010b) mixup the goals of a toxicokinetics study and the risk assessment process. For risk assessment, safety factors are used to consider possible differences between neonates and adults. In this context, the finding that a recently published PBPK model predicted the internal exposure in newborns to be approximately 3 times higher than in adults should be considered (Mielke and Gundert-Remy, 2009). The prediction model took into account conjugation by sulfotransferases, which is already active in newborns (Milke and Gundert-Remy, 2009). Moreover, toxicokinetic data from neonatal non-human primates are available (Doerge et al., 2010b).The 2008 Calafat study simply measured urine from neonates in intensive care units and found that urine samples mostly contained conjugates, and the two groups of neonates investigated in this study had major differences in average exposures. The authors suggest that the neonates were exposed to BPA via intravenous medical devices used at one of the sites. The study of Calafat did not examine toxicokinetics of BPA.
8.“BPA kinetics were examined in six individuals administered BPA, although no information was provided about the characteristics of these subjects making it difficult to draw any conclusions from this study (Völkel et al., 2005).”	The authors give detailed characteristics of the subjects participating in the study. In Table 1 (Völkel et al., 2005, page 1749), gender, age, height, and body weight of all six individuals are stated. Further information is given in the Materials and Methods section: “All subjects enlisted in the study had to refrain from alcoholic beverages and medicinal drugs 2 days before and throughout the experiment. Subjects did not abuse alcohol and were nonsmokers. Subjects were healthy, as judged by medical examination and clinical blood chemistry.” Therefore, the statement by Vandenberg et al. (2010a, 2010b) that “no information was provided” is unsubstantiated.
9.“The authors suggested that there were no differences in kinetics between volunteers, yet a closer look shows a wide variation in BPA measurements between individuals (Völkel et al., 2005). In the three men examined, 85 % of the administered BPA dose was recovered in urine after 5 h, mostly as BPA-glucuronide. In the three women examined, 75 % of BPA was recovered as BPA-glucuronide after the same period of time.”	There is no “wide variation in BPA measurements between individuals.” The data (Völkel et al., 2002) show a 93 ± 19 nmol recovery for men and 83 ± 16 for women. Using a second technique analyzing total BPA after enzymatic cleavage ofglucuronides, recovery was 106 ±16 nmol and 92 ±16 nmol, for men and women, respectively. Therefore, the ranges overlap and there is thus no evidence for a wide variation between individuals.
10.“In two of six individuals, unconjugated BPA was detected in the urine at levels of approximately 1 ng/ml. This finding directly contradicts the conclusions reached by the study authors, who suggested that 100 % first-pass metabolism would promptly convert BPA to its conjugated metabolites.”	There is certainly no contradiction and the authors have carefully considered this aspect (Völkel et al., 2002, 2005, 2008). When deuterated d(16)-BPA was used, Völkel et al. did not detect free BPA in the urine. By contrast, when studies were performed with non-deuterated BPA, very low levels of free BPA were detected in some samples. In a further study, the authors even administered both d(16)-BPA and non-deuterated BPA (Völkel et al., 2008). Interestingly, no free d(16)-BPA but free non-deuterated BPA was detected in urine. The authors discussed contamination as a possible reason for the observation that only non-deuterated free BPA was observed. It should also be considered that 1 ng/ml of free BPA represents a very low concentration. A concentration of 1 ng/ml corresponds to a daily intake of approximately 0.025 µg/kg bw, which is 2000-fold below the tolerable daily intake (TDI) of 50 µg/kg bw/day. It should be taken into account that glucuronides of BPA in urine may release free BPA. Therefore, the presence of free BPA in urine is difficult to interpret, particularly if only concentrations close to the LOD are detected.
11.Vandenberg et al. (2010a, 2010b) criticized the fact that LODs of Völkel et al. (2002) were higher than in other studies.	It should be kept in mind that the study (Völkel et al., 2002) is a toxicokinetics study and not a biomonitoring study. In a toxicokinetic study, sensitivity should be sufficient to study the time course of a controlled dose of a chemical for a sufficient period of time. In the case of the study by Völkel et al. (2002), concentrations of d(16)-BPA in urine were above the LOD even 42 hours after administration. The LOD of 10 nM d(16)-BPA in blood is also sufficient, considering that the peak concentrations of the BPA-glucuronide in blood were approximately 800 nM and concentrations could be determined over the time frame covered by the study design. In conclusion, the commission does not see any reason why the toxicokinetics study of Völkel et al. (2002) is compromised because of the sensitivity of the analytical procedure.
12.Vandenberg et al. (2010a, 2010b) criticized the conclusion of Völkel et al. (2002) concluded from their study “that there is no risk from current human exposure levels (Völkel et al., 2002).”	No such statement can be found in the article of Völkel et al. (2002). Moreover, it was designed as a toxicokinetics study and therefore does not make any conclsuion on risk. Again, Vandenberg et al. confuse the goals of a pharmacokinetics study and of risk assessment. Toxicokinetics studies may have important implications for risk assessment; however, risk assessment is not based on toxicokinetics studies per se. Risk assessment is based on NOAELs or benchmark doses from toxicity studies in animals and on exposure assessment. Kinetics can only be used to justify acceptable margins of exposure. It should also be kept in mind that the EFSAhas never based its assessment on the outcome of any of the mentioned BPA kinetics studies but used the species differences in kinetics to conclude that the margin of exposure of 100 is conservative.

^*Aspects of these studies have been criticized (Vandenberg et al., 2010a, 2010b). Here, we assess the relevance of the criticism of Vandenberg et al. (2010a, 2010b).