Our Stolen Futurea book by Theo Colborn, Dianne Dumanoski, and John Peterson Myers
 
 

 

 

6 April 2008

Willhite, CC, GL Ball and CJ McLellan. 2008. Derivation of a bisphenol A oral reference dose (RfD) and drinking-water equivalent concentration. Journal of Toxicology and Environmental Health, Part B, 11:2, 69 – 146.


 
   
   

By Frederick vom Saal and J.P. Myers

Willhite et al. have presented a highly biased, inaccurate and incomplete review of scientific research on potential adverse effects of bisphenol A (BPA).  The paper is replete with many serious flaws.  Six glaring errors, summarized below, are sufficient to discredit its analysis and conclusions:

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1: Willhite et al. grossly misrepresent the state of the “low dose” BPA literature. For example, in their abstract they state: “Adverse health effects in mice and rats may be induced after parenteral injection or after massive oral doses.”

This statement implies that massive oral doses or parenteral injection are necessary to cause adverse health effects, ie., without them there are no adverse health effects. Contrary to this assertion, almost 200 peer-reviewed publications by independent academic and government scientists show a wide range of effects at and below the LOAEL (50 mg/kg/day) that was used to calculate the current EPA reference dose.  Of those, 38 find adverse effects beneath the current reference dose of 50 µg/kg/day after oral administration. Referring to these as “massive oral doses” is false and misleading. (References for these low-dose oral administration studies are provided below).

2: Willhite et al. ignore an important body of literature on observed levels of bioactive, unconjugated BPA in human blood, which allows comparisons to levels known to cause harm in animals.

This led Willhite et al. to conclude that humans are exposed to only very small amounts of BPA.

Willhite et al. do not acknowledge much of the literature showing that significant biologically-active (unconjugated) BPA has been reported in the blood of people in numerous studies. Willhite et al. cite only a subset of these published studies and dismiss those they do cite because they depend upon ELISA, an immunological assay that shows some cross-reactivity with other compounds. 

In fact, a series of studies which are not dependent upon ELISA documents >1 ppb levels in people from the general population. For example, Willhite et al. do not cite a study Schonfelder et al. (2002), whose analytical tools had a limit of detection (LOD) of 0.04 nM or 0.01 ng/ml.

One hundred percent of the people examined in the Schonfelder et al. study had levels of BPA in their blood above the LOD, and the median (and mean) for pregnant females was about 4 ng/ml. This is virtually identical to the value for conjugated BPA in urine reported by the CDC (Calafat et al. 2008). As pointed out in another review by CDC scientists, if BPA were, in fact, rapidly and completely metabolized, similar to other non-persistent rapidly metabolized chemicals, the ratio of metabolized BPA in urine would be at least 1000 greater than the concentration of parent BPA in blood (Barr et al. 2005).

3: Willhite et al. use a scientifically invalid justification for ignoring a large number of published studies.

Willhite et al. propose that dosage by injection or implantation of micro-osmotic pumps is inappropriate for work with fetuses or neonates, and use that as justification to ignore many experiments that used these methods.  By using this criterion, they exclude a large number of studies that used subcutaneous injections and micro-osmotic pumps, even those the serum concentrations produced in these experiments were often below the median level reported in humans today (Vandenberg et al. 2007), and even though most of these exposures caused adverse effects.

This criterion has been shown to be invalid for newborn rodents and is also highly likely to be equally invalid for human neonates (Taylor et al. 2008). While route of dose administration is important for later stages of life, nothing in the published scientific literature suggests that in fetuses or neonates (rodent or human) parenteral dosing is unacceptable. 

This is obvious for the fetus:  what matters is the serum concentration produced by dosing the mother, irrespective of the pathway of delivery.  If the serum concentrations produced by these pathways of administration in the pregnant female are within the range observed in people, then the experiments are relevant and should not be excluded.

With respect to neonates, the crucial observation is that animals at this stage of life do not have mature liver enzyme systems capable of detoxifying chemicals like BPA.  This is not new: it is based on decades of research on the ontogeny of the liver enzymes involved in conjugating (inactivating) chemicals such as BPA and drugs such as diethylstilbestrol (DES), which happens to be conjugated by the same glucuronosyltransferase as BPA. 

Just how invalid this criterion is becomes readily apparent in light of research on a synthetic estrogen closely related to BPA, DES.  There is a 30+- year track record of using subcutaneous injections of DES in neonatal experiments with mice that has proven to be remarkably and consistently predictive of health effects in people (Newbold 1995).  Willhite et al.’s proposal to ignore parenteral dosing ignores that rich and well-established scientific literature.

Thus, in addition to the large number of oral administration studies showing effects of BPA at doses below the current EPA reference dose, there are also a large number of studies that used subcutaneous injections to deliver doses to neonatal rats and mice below the reference dose, and these studies report a wide range of adverse effects. These studies were inappropriately dismissed by Willhite et al.  Using their approach one would argue that the vast and widely accepted literature on DES is invalid.

4. Willhite et al. set arbitrary standards of sample size for rejecting studies, ignoring basic statistical principles.  This allows them to eliminate many of the most sophisticated studies, even though the studies eliminated were designed using sample size criteria from the US National Institute of Health (NIH).

Sample size in an NIH-funded study is based on historical data, and power analysis is required for approval by animal care and use committees at universities. Arbitrary and excessive numbers of animals, beyond the number required to achieve statistical significance, cannot be used in an experiment, nor should they be used based on standards that have been accepted without question by generations of toxicologists.

By using an arbitrary sample size cut-off, Willhite et al. ignore the basic fact, taught in introductory statistics, that a calculation of statistical significance corrects for small sample size.  A significance level of .01 indicates the same level of confidence in a study with a sample size of 10 as one with a sample of 100.  With a small sample, either the difference must be greater or the variance smaller (or both) to achieve the same level of significance found with a large sample.

Statistical power in an experiment (the ability to detect a real effect) is based on variance (which depends on the competence of the scientist in addition to other sources of variability unrelated to treatment), and whether the endpoint measured involves changes in frequency of an event (for example, a cancer) or changes in a continuous variable.  The tradition in toxicological studies focusing on cancers or malformations (which Willhite et al. specifically use as their example on p 118) is to use group sizes as large as 20, because they are measuring changes in the frequency of an event.  Most studies of hormonally active chemicals, such as BPA, however, use continuous variables, where appropriate statistical power is easier to achieve with smaller sample sizes.

Using the large numbers that Willhite et al. require would be considered unacceptable by university animal care committees and would violate NIH guidelines for the use of animals in experiments if power analysis showed that a statistically significant result is likely to be found with a smaller sample. 

For example, Willhite et al. criticize specifically a study by Timms et al. (2005) that examined prostate volume in mice following in utero exposure.  Their sample size of 5-6 per group was based on extensive prior studies by the investigators, who used standard power analysis to calculate that this group size should be sufficient to detect an effect, if indeed it was occurring as predicted.  They found a 2-fold increase in prostate volume with very low variance, which led to P values of less than 0.005. Willhite et al. considered this study unusable even though it followed well-established guidelines from NIH and was published in the Proceedings of the National Academy of Sciences.  In so doing, with this and many other studies, they revealed a profound ignorance of basic statistical principles.

5. Willhite et al. conclude that daily human exposure to BPA is far below the reference dose of 50 µg/kg/day. This is inconsistent with what is known about the pharmacokinetics of BPA based on animal studies and on repeatedly observed levels in humans.

According to multiple studies using a variety of sensitive and specific analytical techniques, median serum levels of parent unconjugated BPA in people in developed countries are approximately 2 ng/ml (Vandenberg et al. 2007).  To achieve serum levels in animal experiments at this level requires doses of hundreds of micrograms per kilogram per day (Vandenbergh et al. 2007, Taylor et al. 2008).  If humans metabolize BPA more rapidly than rodents, as is often claimed—including by Willhite et al.—human daily intake of BPA is likely to be as much as 10 times higher than the current reference dose (Vandenberg et al. 2007).

6: Willhite et al. misrepresent the sensitivity of modern analytical chemistry.

In their abstract, Willhite et al. offer the following as a major conclusion: “Controlled ingestion trials in healthy adult volunteers with 5 mg d16-BPA were unable to detect parent BPA in plasma despite exquisitely sensitive (limit of detection = 6 nM) methods” [6 nM  = 1.4 ng/ml]. 

In fact, scientists now regularly use analytical methods for BPA that are more than 100 times more sensitive that what Willhite et al. describe as “exquisitely sensitive.”  Dozens of experiments with animals have shown that the levels reported by these truly more sensitive techniques are capable of causing adverse effects. Schonfelder et al. (cited above) used a technique 140 times more sensitive than what Willhite et al. describes as an “exquisitely sensitive” method.

By suggesting that the analytical method is ‘exquisitely sensitive,’ Willhite et al. infer that this level of exposure to BPA results in unmeasurably low serum levels.  Instead, the method used in that study was insensitive compared to modern standards.

References cited:

Barr, D. B., R. Y. Wang and L. L. Needham. 2005. Biologic monitoring of exposure to environmental chemicals throughout the life stages: requirements and issues for consideration for the National Children's Study. Environmental Health Perspectives 113(8): 1083-1091.

Calafat, A. M., X. Ye, L. Y. Wong, J. A. Reidy and L. L. Needham. 2008. Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003-2004. Environmental Health Perspectives 116(1): 39-44.

Haseman, J. K., A. J. Bailer, R. L. Kodell, R. Morris and K. Portier. 2001. Statistical issues in the analysis of low-dose endocrine disruptor data. Toxicological Sciences 61(2): 201-210.

Newbold R. 1995. Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens. Environmental Health Perspectives 103 Suppl 7:83-7.

Schonfelder, G., W. Wittfoht, H. Hopp, C. E. Talsness, M. Paul and I. Chahoud. 2002. Parent bisphenol A accumulation in human maternal-fetal-placental unit. Environmental Health Perspectives 110: A703-A707.

Taylor, J. A., W. V. Welshons and F. S. vom Saal. 2008. No effect of route of exposure (oral; subcutaneous injection) on plasma bisphenol A throughout 24 hr after administration in neonatal female mice. Reproductive Toxicology. 25(2): 169-176.

Timms, B. G., K. L. Howdeshell, L. Barton, S. Bradley, C. A. Richter and F. S. vom Saal. 2005. Estrogenic chemicals in plastic and oral contraceptives disrupt development of the mouse prostate and urethra. Proc. Natl. Acad. Sci. 102: 7014-7019.

Vandenberg, L. N., R. Hauser, M. Marcus, N. Olea and W. V. Welshons. 2007. Human exposure to bisphenol A (BPA). Reproductive Toxicology 24(2): 139-177.

vom Saal, F. S. and C. Hughes. 2005. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environmental Health Perspectives 113: 926-933.

Waldman, P. 2005. Common industrial chemicals in tiny doses raise health issue. Wall Street Journal. New York. July 25, 2005, Page 1.

Peer-reviewed studies reporting effects due to oral administration of BPA below the EPA reference dose:

Adriani, W., D. Della Seta, F. Dessi-Fulgheri, F. Farabollini and G. Laviola (2003). Altered profiles of spontaneous novelty seeking, impulsive behavior, and response to D-amphetamine in rats perinatally exposed to bisphenol A. Environ. Health Perspect. 111: 395-401.

Akingbemi, B. T., C. M. Sottas, A. I. Koulova, G. R. Klinefelter and M. P. Hardy (2004). Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells. Endocrinology 145(2): 592-603.

Al-Hiyasat, A. S. and H. Darmani (2006). In vivo effects of BISGMA-a component of dental composite-on male mouse reproduction and fertility. J Biomed Mater Res A 78(1): 66-72.

Al-Hiyasat, A. S., H. Darmani and A. M. Elbetieha (2002). Effects of bisphenol A on adult male mouse fertility. Eur. J. Oral Sci. 110: 163-167.

Aloisi, A. M., D. Della Seta, C. Rendo, I. Ceccarelli, A. Scaramuzzino and F. Farabollini (2002). Exposure to the estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats. Brain Res. 937(1-2): 1-7.

Bindhumol, V., K. C. Chitra and P. P. Mathur (2003). Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology 188(2-3): 117-124.

Ceccarelli, I., D. Della Seta, P. Fiorenzani, F. Farabollini and A. M. Aloisi (2007). Estrogenic chemicals at puberty change ERalpha in the hypothalamus of male and female rats. Neurotoxicol Teratol 29(1): 108-115.

Chitra, K. C., C. Latchoumycandane and P. P. Mathur (2003). Induction of oxidative stress by bisphenol A in the epididymal sperm of rats. Toxicology 185(1-2): 119-27.

Chitra, K. C., K. R. Rao and P. P. Mathur (2003). Effect of bisphenol A and co-administration of bisphenol A and vitamin C on epididymis of adult rats: A histological and biochemical study. Asian J Androl 5: 203-208.

Della Seta, D., I. Minder, V. Belloni, A. M. Aloisi, F. Dessi-Fulgheri and F. Farabollini (2006). Pubertal exposure to estrogenic chemicals affects behavior in juvenile and adult male rats. Horm Behav 50(2): 301-307.

Della Seta, D., I. Minder, F. Dessi-Fulgheri and F. Farabollini (2005). Bisphenol-A exposure during pregnancy and lactation affects maternal behavior in rats. Brain Res Bull 65(3): 255-260.

Dessi-Fulgheri, F., S. Porrini and F. Farabollini (2002). Effects of perinatal exposure to bisphenol A on play behavior of female and male juvenile rats. Environmental Health Perspectives 110 Suppl 3: 403-7.

Farabollini, F., S. Porrini, D. Della Seta, F. Bianchi and F. Dessi-Fulgheri (2002). Effects of perinatal exposure to bisphenol A on sociosexual behavior of female and male rats. Environ Health Perspect 110 Suppl 3: 409-14.

Farabollini, F., S. Porrini and F. Dessi-Fulgheri (1999). Perinatal exposure to the estrogenic pollutant bisphenol A affects behavior in male and female rats. Pharmacol. Biochem. Behav. 64: 687-694.

Fujimoto, T., K. Kubo and S. Aou (2006). Prenatal exposure to bisphenol A impairs sexual differentiation of exploratory behavior and increases depression-like behavior in rats. Brain Res 1068(1): 49-55.

Gupta, C. (2000). Reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals. Proc Soc Exp Biol Med 224(2): 61-68.

Howdeshell, K. L., A. K. Hotchkiss, K. A. Thayer, J. G. Vandenbergh and F. S. vom Saal (1999). Exposure to bisphenol A advances puberty. Nature 401: 763-764.

Hunt, P. A., K. E. Koehler, M. Susiarjo, C. A. Hodges, A. Hagan, R. C. Voigt, S. Thomas, B. F. Thomas and T. J. Hassold (2003). Bisphenol A causes meiotic aneuploidy in the female mouse. Current Biology 13: 546-553.

Kabuto, H., M. Amakawa and T. Shishibori (2004). Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice. Life Sci 74(24): 2931-40.

Kawai, K., N. Takehiro, H. Nishikata, S. Aou, M. Takii and C. Kubo (2003). Aggressive behavior and serum testosterone concentration during the maturation process of male mice: The effects of fetal exposure to bisphenol A. Environ. Health Perspect. 111: 175-178.

Kawato, S. (2004). Endocrine disrupters as disrupters of brain function: a neurosteroid viewpoint. Environ Sci 11(1): 1-14.

Kubo, K., O. Arai, M. Omura, R. Watanabe, R. Ogata and S. Aou (2003). Low dose effects of bisphenol A on sexual differentiation of the brain and behavior in rats. Neurosci Res 45(3): 345-56.

Laviolaa, G., L. Gioiosa, W. Adriania and P. Palanza (2005). d-Amphetamine-related reinforcing effects are reduced in mice exposed prenatally to estrogenic endocrine disruptors. Brain Research Bulletin 65: 235-240.

Mizuo, K., M. Narita, K. Miyagawa, E. Okuno and T. Suzuki (2004). Prenatal and neonatal exposure to bisphenol-A affects the morphine-induced rewarding effect and hyperlocomotion in mice. Neurosci Lett 356(2): 95-8.

Nagel, S. C., J. L. Hagelbarger and D. P. McDonnell (2001). Development of an ER action indicator mouse for the study of estrogens, selective ER modulators (SERMs), and Xenobiotics. Endocrinology 142(11): 4721-4628.

Nagel, S. C., F. S. vom Saal, K. A. Thayer, M. G. Dhar, M. Boechler and W. V. Welshons (1997). Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105(1): 70-6.

Nishizawa, H., N. Manabe, M. Morita, M. Sugimoto, S. Imanishi and H. Miyamoto (2003). Effects of in utero exposure to bisphenol A on expression of RARalpha and RXRalpha mRNAs in murine embryos. J Reprod Dev 49(6): 539-45.

Nishizawa, H., M. Morita, M. Sugimoto, S. Imanishi and N. Manabe (2005). Effects of in utero exposure to bisphenol a on mRNA expression of arylhydrocarbon and retinoid receptors in murine embryos. J Reprod Dev 51(3): 315-324.

Palanza, P., K. L. Howdeshell, S. Parmigiani and F. S. vom Saal (2002). Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior in mice. Environ. Health Perspect. 110: 415-422.

Porrini, S., V. Belloni, D. Della Seta, F. Farabollini, G. Giannelli and F. Dessi-Fulgheri (2005). Early exposure to a low dose of bisphenol A affects socio-sexual behavior of juvenile female rats. Brain Res. Bull. 65(3): 261-266.

Razzoli, M., P. Valsecchi and P. Palanza (2005). Chronic exposure to low doses bisphenol A interferes with pair-bonding and exploration in female Mongolian gerbils. Brain Res Bull 65(3): 249-254.

Ryan, B. C. and J. G. Vandenbergh (2006). Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice. Horm Behav 50(1): 85-93.

Sakaue, M., S. Ohsako, R. Ishimura, S. Kurosawa, M. Kurohmaru, Y. Hayashi, Y. Aoki, J. Yonemoto and C. Tohyama (2001). Bisphenol A affects spermatogenesis in the adult rat even at a low dose. Journal of Occupational Health 43: 185-190.

Sawai, C., K. Anderson and D. Walser-Kuntz (2003). Effect of bisphenol A on murine immune function: modulation of interferon-gamma, IgG2a, and disease symptoms in NZB X NZW F1 mice. Environ Health Perspect 111(16): 1883-1887.

Timms, B. G., K. L. Howdeshell, L. Barton, S. Bradley, C. A. Richter and F. S. vom Saal (2005). Estrogenic chemicals in plastic and oral contraceptives disrupt development of the mouse prostate and urethra. Proc. Natl. Acad. Sci. 102: 7014-7019.

vom Saal, F. S., P. S. Cooke, D. L. Buchanan, P. Palanza, K. A. Thayer, S. C. Nagel, S. Parmigiani and W. V. Welshons (1998). A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior. Toxicol. Ind. Health 14(1-2): 239-260.

Yoshino, S., K. Yamaki, X. Li, T. Sai, R. Yanagisawa, H. Takano, S. Taneda, H. Hayashi and Y. Mori (2004). Prenatal exposure to bisphenol A up-regulates immune responses, including T helper 1 and T helper 2 responses, in mice. Immunology 112(3): 489-495.

Yoshino, S., K. Yamaki, R. Yanagisawa, H. Takano, H. Hayashi and Y. Mori (2003). Effects of bisphenol A on antigen-specific antibody production, proliferative responses of lymphoid cells, and TH1 and TH2 immune responses in mice. Br J Pharmacol 138(7): 1271-6.

 

 

 

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