Critical evaluation of key evidence on the human health hazards of exposure to bisphenol A

Figures & data

Table 1.  Cutting edge topics of the current controversy on BPA.

Are the studies used for regulatory purposes flawed?

Do oral low doses below 5 mg/kg bw/day cause adverse health effects in laboratory animals?

How can differences between industry sponsored and publicly sponsored studies be explained?

Swallow or inject? What is the relevance of the oral route versus implantation of pumps or intravenous injection?

Can rodents be used to extrapolate to the human situation?

To which degree are embryos, babies, or children more susceptible?

How critical is exposure by intravenous infusion in intensive care units?

How critical is tissue deconjugation of BPA-glucuronide and BPA-sulfate?

How can biomonitoring support risk evaluation?

What are the mechanisms of action of BPA? Does the multitude of mechanisms other than estrogen receptor activation make the substance more dangerous?

Why are recent governmental responses inconsistent?

Table 2.  Criticisms of the studies of Tyl et al. (2008a, 2008b, 2008c) and responses.

Criticism	Response
1. Myers et al. questioned the use of CD-1 Swiss mice because of their “aberrant insensitivity” to estrogens.	Recently, Ryan et al. (2010a) used a wide range of doses from 0.05 to 50 μg/kg bw/day for the positive control estrogen, EE2. They observed a comparable sensitivity for CD-1 mice, Long-Evans, and Sprague-Dawley rats for several adverse effects. Therefore, the use of CD-1 mice by Tyl et al. (2008a, 2008b, 2008c) is justified. Tyl et al. purchased their CD-1 mice from Charles River to avoid supplier effects. It should be considered that also vom Saal and some of his colleagues used CD-1 mice and reported low dose effects (Timms et al., 2005). Low-dose effects of BPA were also reported in Long-Evans (Akingbemi et al., 2004) and in SD rats.
2. Myers et al. considered the prostate weights reported in the study of Tyl et al. (2008a, 2008b, 2008c) “abnormally high” and suggested “that the dissection procedures for the prostate in the Tyl laboratory included other nonprostatic tissues in the weight measurements, rendering them unusable….” This criticism was also expressed by Gies et al. (2009).	Tyl (2009a, 2009b) reported that paraffin blocks and slides of the analyzed prostates are still available and indicate no evidence of extraneous tissue/fat or excessive inflammation (all recently audited by the US FDA). In a response letter (Tyl, 2009a, 2009b), the author documented that considering the age of the mice, prostate weights in their study were within the range of published data. Differences from vom Saal’s study may be explained by his post wean caging regimen. Vom Saal’s CF-1 mice were housed by sex. Pups were weaned at 23 days of age and male littermates were housed three per cage (Nagel et al., 1997). Prostate weight was determined when the mice were 6 months old. For this purpose, one male per litter was randomly selected and individually housed for 1 month before determination of prostate weights (Nagel et al., 1997). This procedure is problematic because the dominant cage male develops large androgen-dependent accessory organs, whereas subservient cage males have smaller prostates. It is questionable whether 1 month of single housing may compensate for months of group housing and its influence on sexual development of male mice. In the study of vom Saal (Nagel et al., 1997), no controls were presented with respect to the influence of group housing and compensation by single housing and the authors did not report whether they determined prostate weight only from the dominant males or if all animals were included. Since the authors wrote that “one male per litter was randomly selected…” (Nagel et al., 1997), they likely included both, dominant and subservient males. This may explain the high variability of the prostate weights in studies from vom Saal’s lab compared to much lower variability in Tyl´s studies (Tables 1 and 2 in Tyl, 2009a, 2009b). It should also be taken into account that the number of animals exposed to BPA in vom Saal’s study (n = 7) is very low also, considering that an additional confounder (dominant versus subservient mice) may be present. Much higher numbers of animals were used in Tyl’s studies (28 per group). After careful evaluation of Tyl’s studies, the criticism of Myers et al. (2009) and Gies et al. (2009) concerning prostate weights is unsubstantiated. However, several aspects may be critical in vom Saal’s studies, such as the influence of dominant versus subservient males, no available histology, and use of a now obsolete in-house strain preventing other researches to reproduce the initial positive studies. Another drawback is the statistical analysis.Concerning the criticism that crude mistakes were made by Tyl’s laboratory in determining prostate weights, it should also be considered that this laboratory has carried out a large number of reprotox studies (often sponored by the US Government) and has successfully joined interlaboratory validation studies, where the Tyl laboratory achieved some of the most precise prostate weight data and robust treatment-related effects (EPA review).
3. Myers et al. criticized the high incidence and severity of prostatitis in the animals from Tyl’s study, which may compromise their results.	The paraffin slides of the prostates of the mice from Tyl’s study are still available and have been reanalyzed by an independent pathologist. The results have been published (Tyl, 2009a, 2009b). The incidence of inflammation in CD-1 mice of Tyl’s study matches the low incidences of prostatitis seen in many mouse strains without treatment. Therefore, it is extremely unlikely that the results of Tyl et al. (2008a, 2008b, 2008c) have been compromised by prostatitis. It should be kept in mind that vom Saal’s laboratory did not include any histopathological examinations (Nagel et al., 1997). Therefore, a possible confounding influence of prostatitis could not be accounted for in their study.
4. Myers et al. criticized that the diet used in Tyl’s study contained phytoestrogens, which they claim would interfere with BPA activity.	Recently, Tyl et al. have published the genistein, daidzein, and glycitein contents of the standard diet (Purina Certified Ground Rodent Chow No. 5002) used in their study. Vom Saal and colleagues have not reported the phytoestrogen content of their diet. The majority of “normal rodent diets have similar levels of phytoestrogens.” Although it is not possible to compare diets between Tyl’s and vom Saal’s laboratories, it is extremely unlikely that the diet in Tyl’s study compromised an estrogenic response, because in studies with estradiol in which mice were fed the same standard diet as those in the BPA studies, estradiol (0.5 ppm) clearly accelerated acquisition of puberty (Tyl, 2008a).
5. Myers et al. criticized that “Tyl et al. (2008a) did not examine any neurobehavioral end points,” which Myers et al. interpreted as a “glaring omission of Tyl.”	The multigeneration study performed by Tyl et al. (2008a) represents a standard test performed according to a specific guideline that is not designed to investigate neurobehavioral endpoints. Therefore, there is certainly no “glaring omission of Tyl,” because behavior simply is not an endpoint in this study type. However, there are other studies that have focussed on neurobehavioral endpoints: Ema et al. (2001) observed no effects in their two-generation, low-dose (0.2–200 μg BPA/kg bw/day) study. Similarly, Ryan et al. (2010a) obtained negative results in a detailed study on reproductive development, function, and behavior in female rats exposed perinatally to a wide range of BPA doses (see also Sharpe, 2010). Stump et al. (2010) published the most recent study conducted according to GLP. The results show that BPA, at levels of exposure 4000-fold higher than the maximum human exposure in the general population, does not cause any discernible adverse effects in female rats. By contrast, EE2, used as a positive control in this study, caused major adverse health effects at doses in the range of those applied in early contraceptives (Ryan et al., 2010a; highlight report: Sharpe, 2010).
6. Myers et al. criticized Tyl et al. (2008a, 2008b, 2008c) for using too many animals: “…all of the studies by Tyl et al. were significantly overpowered and this is in direct violation of federal guidelines for conducting animal research, a fact about which U.S. FDA regulators seem unaware.”	The consideration of animal protection aspects should certainly be an important point in all animal testing. But even if we assume that more animals than required were used, according to statistical power analysis, this would not lead to different or incorrect conclusions from a study. However, the numbers of animals used in Tyl’s studies are appropriate—28 mice/sex/group/generation. This is in line with the OECD and US EPA toxicity testing guidelines, which require at least 20 pregnant females per group.

Table 3.  Examples of guideline-compliant BPA studies.

Study	Design	Result	Reference
Three-generation reproductive toxicity study in rats	• Six BPA dose groups (0.001–500 mg/kg/day; administration in diet);• OPPTS guideline 870.03800	• No effects in the low- dose range (0.001–5 mg/kg/day)• Reduced body and organ weights at ^350 mg/kg/day• Effects on renal and hepatic histopathology: 500 mg/kg/day• Reproductive and developmental toxicity: 500 mg/kg/day• At doses <500 mg/kg/day: no effects on prostate weights, acquisition of puberty• Systemic NOAEL: 5 mg/kg/day• Reproductive NOAEL: 50 mg/kg/day	Tyl et al., 2002
Two-generation rat study	• Daily gavage doses of 0.0.2–200 μg/kg/day• US EPA GLPs and OPPTS TG with added endocrine-sensitive and neurobehavioral end points	• No effect at any dose (consider that this is a low-dose study with 0.2 mg/kg/day as the highest dose)	Ema et al., 2001
One- and two-generation reproductive toxicity studies in mice	• Two vehicle control groups, six BPA dose groups (0.003–600 mg/kg/day; administration in diet)• Dietary estradiol as positive control (0.2 μg/kg/day to 8 mg/kg/day)• OECD GLPs	• No effects in the low-dose range (0.003–5 mg/kg/day)• Reduced body weights, increased renal and liver weights and further effects at 600 mg/kg/day. However, no adverse effects on adult reproductive structures or functions at 600 mg/kg/day.• Effects on liver histopathology at 50 mg/kg/day• Systemic NOAEL: 5 mg/kg/day• Reproductive NOAEL: 50 mg/kg/day	Tyl et al., 2008a, 2008b, 2008c
Developmental neurotoxicity study in rats	• Dietary administration of 0, 0.01, 0.1, 5, 50, and 150 mg/kg/day (mean target doses) from gestation day 0 through lactation day 21. Evaluation of F1 offspring.• OECD test guideline 426	• No treatment-related neurobehavioral effects• No evidence of neuropathology and no effects on brain morphometry• NOAEL for systemic toxicity derived from maternal and offspring body weight reductions: 5.85 and 13.1 mg/kg/day (calculated) during gestation and lactation, respectively	Stump et al., 2010

Table 4.  Key conclusions from the toxicokinetics study of Völkel et al. (2002) in human subjects using deuterated BPA [d(16)-BPA].

• 5 mg of d(16)-BPA (60–80 µg/kg/bw) were orally administered to human volunteers; blood and urine sampling was performed at predetermined time points.

• Maximum blood concentrations were measured approximately 80 minutes after administration.

• The half-life of d(16)-BPA was less than 6 hours.

• The administered doses were completely recovered in urine as d(16)-BPA-glucuronide.

Note. Similar data were obtained by Doerge et al. (2010b), Kurebayashi et al. (2002), and Tominaga et al. (2006) in non-human primates.

Table 5.  Kinetic parameters of bisphenol A in blood at comparable single oral doses in primates and rodents.

Species	Dose (µg/kg bw)	Cmax (nmol/L)	tmax (h)	t1/2 (h)	AUC (nmol × h × L^−1)	Reference
Rhesus monkeys	100	590 (BPA conjugates) 0.84 (free BPA)	0.5 (total BPA)	3.5 (total BPA) 0.39 (free BPA)	920 (BPA total) 1.5 (free BPA)	Doerge et al., 2010b
Cynomolgus monkeys	100	456 (total BPA)	1.0 (total BPB)	9.6 (total BPA)	1162 (BPA-glucuronide)	Kurebayashi et al., 2002
Human subjects	60–80	820 (BPA conjugates) <10 (free BPA)	1.35 (total BPA)	5.3 (terminal) 1.5 (initial)	2792 (BPA-glucuronide)*	Völkel et al., 2002
Sprague-Dawley rats, female	100	73 (total BPA) 0.39 (free BPA)	2 (both free and total)	4.6 (total BPA) 3.0 (free BPA)	680 (total BPA) 2.6 (free BPA)	Doerge et al., 2010a

*Calculated based on a clearance of 7.8 L/h and an oral bioavailability of 99%.

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 Cmax was 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 a lack 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 in section “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 et al. (2002). Again, Vandenberg et al. (2010a, 2010b) mix up 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 of glucuronides, 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 EFSA has 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).

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