Risk Policy Report 7:47-50, 2000.
NIH PANEL CONFIRMS THAT ENDOCRINE DISRUPTING CHEMICALS
CAUSE EFFECTS AT VERY LOW DOSES
Frederick S. vom Saal
Wade V. Welshons
Frederick S. vom Saal
114 Lefevre Hall
Division of Biological Sciences
University of Missouri
Columbia, MO 65211
TEL: 573-882-4367
FAX: 573-884-5020
EMAIL: vomsaafl@missouri.edu
Dr. Wade V. Welshons
Department of Veterinary Biomedical Sciences
E102 Veterinary Medicine
University of Missouri-Columbia
Columbia, MO 65211
TEL: 573-882-3347
DEPT OFFICE FAX: 573-884-6890
LAB FAX 884-7186
WelshonsW@missouri.edu
A Paradigm Shift in Regulatory Toxicology and Risk Assessment
During the 1990s toxicologists and federal regulatory agencies responsible for assessing
the safety of environmental chemicals became aware of a previously unknown issue,
namely, that numerous chemicals used in pesticides, plastics, and other industrial
products and by-products could mimic or interfere with the normal functioning of the
endocrine system. These chemicals are called endocrine disruptors, and the major
concern regarding these chemicals was with the permanent, irreversible damage that
could occur due to exposure during critical periods in organ development in fetuses,
infants and children [1, 2]. An issue that generated considerable controversy was whether
adverse developmental effects of endocrine disruptors were occurring in humans and
wildlife below exposure levels previously considered to be “safe” based on traditional
methods used in tests conducted by industry and regulatory agencies to test chemicals
for toxicity.
Here we report an important development concerning endocrine disruptor research that
directly challenges the validity of current estimates of levels of environmental chemicals
that have been declared “safe” for human exposure. This represents a major milestone in
the ongoing “paradigm shift” that has been occurring in toxicology due to the discovery of
endocrine disruption as a mechanism of toxicity and of published research findings that
there are adverse effects of endocrine disruptors at doses below those previously
declared “safe”. This milestone concerns the conclusion by a blue ribbon panel of
scientists brought together by the NIH to determine whether chemicals can cause harm at
“low doses”. For purposes of discussion at the meeting, “low dose effects” were defined
as follows:
"low-dose effects" referred to biological changes that occur in the range of human
exposures or at doses that are lower than those typically used in the U.S. EPA's standard
testing paradigm for evaluating reproductive and developmental toxicity [3].
The NIH panel concluded that:
“Low-dose effects have been clearly demonstrated for estradiol and some
estrogenic compounds” [3].
This statement directly challenges the fundamental premise of risk assessment that
studies in which only high doses of chemicals were tested could be used by risk
assessors to accurately predict that “low doses”, within the range of human exposure,
would not produce effects. The statement by the panel acknowledges that unpredicted
effects are now being reported based on directly testing low doses. Paradigm shifts have
been identified as moving through a series of predictable stages, and are generally
unsettling to those wedded to the old paradigms [4]. We propose that once the NIH
panel’s final report is forwarded to the EPA, a complete reevaluation of the assumptions
governing risk assessment for systemic toxicants, which includes endocrine disrupting
chemicals, should be considered.
A related issue is that dioxin is an endocrine disruptor that causes effects via binding to
receptors located in cells, similar to the mechanism of action of the endocrine disrupting
estrogenic chemicals we will discuss here. A reassessment by the EPA of the health
hazards posed by dioxin has recently been posted on the internet [5], and findings
concerning low-dose effects and dose-response models for dioxin are presented.
However, the published information about low-dose effects and the shape of the
dose-response curve for dioxin are apparently not being applied to other endocrine
disruptors by regulatory agencies. The separation by regulatory agencies of low-dose
effects of dioxin and low-dose effects of all other endocrine disrupting chemicals is
obviously a political decision that ignores the fact that at the mechanistic level, such a
separation is unjustified.
Are the Doses of Environmental Chemicals that Humans are Exposed to Really
Safe?
The public and the great majority of public officials likely are unaware that the doses of
chemicals determined to be “safe” for humans by regulatory agencies are only rarely
directly tested for effects in animals or humans. Instead, “safe doses” are based on
assumptions that, until very recently, have not been directly challenged by conducting
experiments to determine whether the doses are, in fact, really safe. Those who conduct
risk assessments should carefully consider the appropriateness of continuing to use
assumptions that are being challenged by research findings as invalid, particularly when
the research findings are from the United States National Toxicology Program.
The dose issue has become of central importance since one of the greatest challenges
posed by findings from experiments with endocrine disruptors has been to the long-held
conviction by many toxicologists (and by regulatory agencies that use this information to
assess human risk) that “the dose makes the poison”. Basically, the premise by
regulators regarding dose is that exposure to a small dose of a chemical, below some
theoretical “threshold” would be “safe”, and as dose increased, exposure to more of the
chemical would lead to a greater and greater adverse response. Thus, the dose-response
curve was assumed to be monotonic (response always continuously increases or
decreases with dose but never changes directions). Risk assessment for endocrine
disruptors and other systemic toxicants is based on the premise that other dose-response
relationships would not occur, such as response first increasing with dose and then
decreasing at higher doses, forming an inverted U. Although a fundamental tenet of risk
assessment is a monotonic dose-response curve, non-monotonic dose-response curves
are, in fact, common for hormones and would thus be expected for hormone-mimicking
environmental chemicals [6, 7]. In contrast, because of the erroneous assumption of a
monotonic dose-response curve for toxicants such as endocrine disruptors, it was
accepted that testing only a very few high doses of a chemical in an animal study would
provide risk assessors with adequate information to predict (based on montonic
dose-response models) “safe” levels for daily human exposure to the chemical.
When adverse effects are found at the lowest dose tested in a study, this dose is referred
to as the lowest adverse effect level (LOAEL) and this LOAEL dose is then used to
calculate a “safe” dose (in fact. the LOAEL is typically a very high dose with regard to
human exposure levels). The LOAEL is then often divided by a “safety factor” of 1000,
providing a calculated, but untested, dose that can then be declared “safe” for human
consumption [8]. This method of extrapolation (dividing the high LOAEL dose by a
constant, the safety factor) can only provide accurate safety data for exposure to lower
doses if the dose-response curve is linear. One obvious basis for testing only high doses
is the need to establish a dose that causes toxicity but not death. Generally, a no adverse
effect dose (NOAEL) for gross malformations and some other adverse effects is expected
to be found within the dose range of 50-fold below the dose that causes acute toxicity.
Toxicological studies conducted for regulatory purposes have thus not historically been
designed to test for any effects of chemicals outside of this narrow range of very high
doses [9]. Importantly, these studies have not been designed to detect endocrine
disruption. By way of comparison, effects of the endocrine disrupting estrogenic drug
diethylstilbestrol (DES) occur at doses 100,000-times lower than the acutely toxic dose
[10, 11]. The low-dose effects of DES being observed in these recent experiments are
clearly not predicted to occur based on the current models governing the design of
toxicological tests for determining “safe” exposure levels for environmental chemicals.
As indicated above, the primary conclusion of the NIH panel was that “low-dose effects
have been clearly identified for estradiol and some estrogenic compounds.” The
panel determined that the natural hormone, estradiol, acted at extremely low doses (in
the low part per trillion range). This is important since estradiol is the reference hormone
against which estrogenic endocrine disruptors are compared for potency. Most
estrogenic endocrine disruptors of concern appear to be within the range of
1,000-10,000-times weaker than estradiol. However, potency relative to estradiol can
change depending on many factors. For example, recent evidence suggests that
chemicals that appear weak in cell culture experiments or when tested in adults, can have
very potent effects in fetuses [12]. In addition, the panel determined that other estrogenic
chemicals to which humans are routinely exposed, such as genistein, the natural
estrogenic compound found in plants such as soy, and nonylphenol, an antioxidant used
in many plastics, in hand creams, detergents and a host of other products, had been
shown to produce effects in the defined “low-dose” range, that is, at doses lower than had
previously been tested based on traditional high-dose testing methods in regulatory
toxicology. These “low dose” effects were considered as being particularly important
since they were conducted by NIH and FDA scientists in the National Toxicology Program
using rigorous quality control protocols.
For chemicals that interfere with the endocrine system via other mechanisms, such as
interfering with androgens during masculinization of males during fetal life, the panel
found that no studies had been conducted to test whether the predicted “safe” doses of
these chemicals were, in fact, safe; the panel called for appropriate studies to be
conducted. For another chemical, bisphenol A, which is the monomer used to make
polycarbonate plastic, such as baby bottles, food storage containers, carboys, CDs, the
resin lining of food and beverage cans, and many other products, the panel could not
reach a decision concerning the certainty of low-dose effects. We will address below in
greater detail the issue of replication in science and the nature of positive and negative
findings, particularly concerning bisphenol A, that were referred to by the panel.
Bisphenol A as a Model for Low-Dose Effects of Endocrine Disrupting Chemicals
Bisphenol A was singled out for analysis by the EPA, since it is one of the top 50
chemicals in commerce, represents a multi-billion dollar industry, and conflicting data
exist for replication of low dose effects. Specifically, concern about bisphenol A has been
raised by published reports of effects in rats and mice due to exposure to doses of
bisphenol A below the dose previously reported by industry to have no effect based on
studies which only examined very high doses relative to human exposure [13]. Bisphenol
A was reported in the 1930s by Dodds and co-workers to elicit a complete estrogenic
response in rats [14]. Years later, polymer chemists discovered how to polymerize
bisphenol A molecules to make polycarbonate plastic and resins. Today, over 2 billion
pounds of bisphenol A are produced per year for use in a wide variety of products, which
are thus made using an estrogenic chemical. There is now evidence that there is
significant human exposure to bisphenol A due to use of food and beverage containers
made with this chemical [15-17].
Bisphenol A effects in the “low dose range” have been reported for the pituitary [18, 19],
vagina [20], prostate [21-24], testicular sperm production [25] and sexual behavior [26].
Several other effects of low doses of bisphenol A in a wide variety of animals have been
presented at international meetings, but have not yet appeared in peer reviewed,
scientific journals. Some of the published findings and most of the unpublished studies
were not discussed by the NIH panel.
There are two industry-funded studies [27, 28] that were conducted with the intention of
determining the reliability of the initial finding of Nagel [21], a former graduate student in
Dr. vom Saal’s laboratory, who reported that fetal exposure to low doses of bisphenol A,
within the range of human exposure due to using products made from bisphenol A,
resulted in permanent enlargement of the prostate in male CF-1 mice. The finding by
Nagel was independently replicated in a series of studies by Gupta, who conducted her
studies with a different stock of mice (CD-1 mice), and also reported that bisphenol A
significantly increased prostatic androgen receptors [22, 23]. In each of the
industry-funded studies, no effects of bisphenol A were found.
A critical issue is that the two research teams that industry chose to conduct the bisphenol
A replication study had no prior experience conducting similar experiments with the male
reproductive system in mice. In fact, technicians from both research groups asked for and
received a few hours of training from members of Dr. vom Saal’s laboratory; in contrast,
graduate students typically receive five years of training. The review panel did not directly
address the issue of demonstrated competence with regard to the Ashby or Cagen
studies. Their summary stated: “Although attempts were made to replicate early results,
the Panel concluded that in most cases both the positive and negative study findings are
credible and sound within the context of the experimental design. Study design
differences (e.g., species/strain, animal husbandry, diet, chemical purity, dosing regime,
endpoints evaluated, genetic factors, etc.) were identified among laboratories that might
account for discrepancies in experimental outcomes.” However, there are virtually
unlimited reasons for an experiment to fail, particularly when conducted by investigators
without prior experience with the animal model or effects being examined. The issue of
demonstrated competence was addressed as a general issue in the following manner:
“The Panel also recommended that laboratories demonstrate effects with positive
controls before initiating new studies”.
The importance of the above admonition concerning the need to demonstrate positive
effects with a positive control chemical before testing other chemicals (such as bisphenol
A) for estrogenic activity is that the panel made special note of the fact that in both the
Cagen and Ashby studies, not only did these investigators not replicate earlier results for
bisphenol A reported by Nagel and Gupta, but also failed to obtain positive results with
DES. DES was the positive control chemical to demonstrate competence to find a positive
effect used by the vom Saal laboratory and by Gupta, and both groups reported effects
with DES that were virtually identical to those produced by bisphenol A [10, 21-23]. With
regard to the study conducted by both Cagen and Ashby, the plastic industry had publicly
reported that the study design included DES as a positive control chemical. In science,
success or failure of a positive control (quality control) procedure determines success or
failure of the experiment. In both the Ashby and Cagen studies, the positive control
chemical, DES, did not produce a positive effect, even though the dose of DES used by
Ashby and Cagen was 10-times higher than a dose that other research (including studies
conducted by the National Toxicology Program) showed to result in developmental
effects. Of considerable importance, in neither of the publications by Ashby or Cagen
describing their results did the authors acknowledge that DES had been initially included
in the study as a positive control chemical. These authors thus did not address the
significance of their failure to find effects of DES, which was clearly inappropriate. But, as
shown above, the NIH panel emphasized the importance of first finding effects with
positive control chemicals prior to attempting to test for effects of other chemicals.
An interesting aspect of the NIH meeting was that the issue of the animal model used to
test chemicals emerged as a central topic of discussion and concern. This had particular
relevance to the debate about positive and negative findings in studies with bisphenol A.
It has been known for many years that there can be marked species and strain
differences in sensitivity to chemicals. As a result, regulatory agencies propose that with
regard to testing for health effects of environmental chemicals or drugs, the most
sensitive outcome in the most sensitive species or strain be used in the risk assessment
process. At the meeting, a number of recent papers showing marked differences in the
response to estrogen between different strains of mice were discussed [29, 30]. In
addition, a series of studies investigating the estrogenic effects and potency of bisphenol
A in different rat strains was reviewed [18-20]. These studies showed that one of the most
commonly used animals in toxicological studies, the Sprague-Dawley rat, had an
impaired ability to respond to bisphenol A, but not to estradiol, relative to another strain,
the Fischer rat. These findings would prompt investigators not to use the
Sprague-Dawley rat to investigate the health effects of bisphenol A.
Concern about the decision by the chemical industry to fund a recently completed large
study that involved the use the model animal (the Sprague Dawley rat) that had been
reported to be insensitive to bisphenol A relative to other rats or mice was raised by the
panel. As a result the panel “advocated the use of species and strains that are highly
responsive to endocrine active compounds”, a position consistent with existing regulatory
policy. For example, the vom Saal laboratory recently switched from using the CF-1
mouse, which is not commonly used in toxicological studies, to the CD-1 mouse, which is
the mouse strain used by the National Toxicology Program as well as by many
toxicologists. The basis for choosing the CD-1 mouse as a model to study the
developmental effects of estrogen was based on the common use of the animal in
toxicological research, and decades of research by the National Toxicology Program and
other investigators with the CD-1 mouse showing that it was highly sensitive to the effects
of estrogenic chemicals on development [31, 32]. We have now reported that CD-1 mice
show the exact same types of differences in postnatal traits, including and increase in
prostate size in males and uterine size in females, that had previously been identified in
CF-1 mice due to very low doses of estrogenic endocrine disruptors. In this experiment
with CD-1 mice we used the doses of DES and bisphenol A that had previously resulted in
effects in CF-1 mice [33]. We thus replicated the findings of Gupta using CD-1 mice [22,
23].
Increased attention needs to be paid to the appropriateness of the model animal being
used to provide data that will be evaluated by regulatory agencies to set “safe” intake
levels for humans. The consequences of using the wrong model animal were tragically
revealed when thalidomide was tested for developmental effects in rabbits, which turned
out to not show the devastating malformations, such as severe malformations of the arms
and legs, that occurred in thousands of infants whose mothers took this drug during early
pregnancy.
The panel also “supported collaborations among laboratories in standardizing study
protocols for the purpose of replicating findings.” It will obviously take more time and
experiments to achieve a final resolution regarding the health effects of bisphenol A. In
order to achieve consensus we are going to have to conduct a collaborative round-robin
study involving a number of laboratories. These studies should include experienced
investigators who have published extensively in the area of the effects of estrogenic
chemicals on the development of the reproductive system. In addition, the experiment
must include appropriate positive controls. Four years ago, we participated in an attempt
to initiate this type of collaborative study, funded in part by industry and government.
Unfortunately, the chemical industry withdrew its support for this study and instead
invested millions of dollars in the studies that the NIH Panel determined, for reasons
discussed above, to not have provided a clear answer to the issue of low-dose effects of
bisphenol A. For a chemical such as bisphenol A that generates huge revenues for
industry, there is an obvious advantage to tactics that delay a resolution of concerns
about safety, particularly when this could lead to the establishment of “safe” intake levels
for humans below those currently in place, thus potentially resulting in restrictions in the
use of the chemical.
LITERATURE CITED
1.
Colborn, T., vom Saal, F.S., and Soto, A.M. 1993. Developmental Effects of
Endoocrine Disrupting Chemicals in Wildlife and Humans. Environ. Health Persp.
101:378-384.
2.
Colborn, T., Dumanoski, D., and Myers, J.P., 1996. Our Stolen Future. New York:
Penguin.
3.
NTP, N.T.P. . in Endocrine Disruptors Low Dose Peer Review. 2000. Raleigh, NC:
http://ntp-server.niehs.nih.gov/htdocs/liason/LowDoseWebPage.html.
4.
Kuhn, T., 1996. The Structure of Scientific Revolutions. Chicago: University of
Chicago Press.
5.
EPA, Draft Dioxin Reassessment, . 2000:
http://www.epa.gov/ncea/pdfs/dioxin/dioxreass.htm.
6.
Medlock, K.L., Lyttle, C.R., Kelepouris, N., Newman, E.D., and Sheehan, D.M.
1991. Estradiol down-regulation of the rat uterine estrogen receptor. Proc Soc Exp Biol
Med 196:293-300.
7.
NRC, 1999. Hormonally Active Agents in the Environment. Washington, D.C.:
National Academy Press.
8.
vom Saal, F.S. and Sheehan, D.M. 1998. Challenging risk assessment. Forum for
Applied Research and Public Policy 13:11-18.
9.
Calabrese, E.J. and Baldwin, L.A. 1997. The dose determines the stimulationa
(and poison): Development of a chemical hormesis database. International Journal of
Toxicology 16:545-559.
10.
vom Saal, F.S., Timms, B.G., Montano, M.M., Palanza, P., Thayer, K.A., Nagel,
S.C., Dhar, M.D., Ganjam, V.K., Parmigiani, S., and Welshons, W.V. 1997. Prostate
enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol
and opposite effects at high doses. Proceedings of the National Academy of Sciences
94:2056-61.
11.
Newbold, R.R., Jefferson, W.N., and Banks, E.P. Developmental exposure to low
doses of diethylstilbestrol (DES) results in permanent alterations in the reproductive tract.
in Endocrine Society. 1999. San Diego, CA.
12.
Welshons, W.V., Taylor, J.A., Judy, B.M., and vom Saal, F.S. Why bisphenol A is
weak in the adult yet strong in the fetus. in Bisphenol A: low dose effects-high dose
effects. November, 2000. Freie Universitat, Berlin.
13.
Society of the Plastics Industry. Report on the potential exposures to bisphenol A
from epoxy can coatings. 1996, Washington, D.C.: Society of the Plastics Industry.
14.
Dodds, E.C. and Lawson, W. 1936. Synthetic oestrogenic agents without the
phenanthrene nucleus. Nature 137:996.
15.
Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedraza, V., and Olea, N. 1995.
Xenoestrogens released from lacquer coating in food cans. Environmental Health
Perspectives 103:608-612.
16.
Olea, N., Pulgar, R., Perez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A.,
Pedraza, V., Soto, A.M., and Sonnenschein, C. 1996. Estrogenicity of resin-based
composites and sealants used in dentistry. Environ Health Perspect 104:298-305.
17.
Takao, Y., Chul Lee, H., Ishibashi, Y., Kohra, S., Tominaga, N., and Arizono, K.
1999. Fast screening method for bisphenol A in environmental water and in food by
solid-phase microextraction (SPME). Journal of Health Science 45:39.
18.
Steinmetz, R., Brown, N.G., Allen, D.L., Bigsby, R.M., and Ben-Jonathan, N. 1997.
The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo
[see comments]. Endocrinology 138:1780-6.
19.
Khurana, S., Ranmal, S., and Ben-Jonathan, N. 2000. Exposure of newborn male
and female rats to environmental estrogens: Delayed and sustained hyperprolactinemia
and alterations in estrogen receptor expression. Endocrinol. in press.
20.
Long, X., Steinmetz, R., Ben-Jonathan, N., Caperell-Grant, A., Young, P.C.M.,
Nephew, K.P., and Bigsby, R.M. 2000. Strain diffrences in vaginal responses to te
xenoestrogen bisphenol A. Environ. Health Perspect. 108:243-247.
21.
Nagel, S.C., vom Saal, F.S., Thayer, K.A., Dhar, M.G., Boechler, M., and
Welshons, W.V. 1997. Relative binding affinity-serum modified access (RBA-SMA) assay
predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol.
Environmental Health Perspectives 105:70-6.
22.
Gupta, C. 2000a. Reproductive malformation of the male offspring following
maternal exposure to estrogenic chemicals. Proc. Soc. Exp. Biol. Med. 224:61-68.
23.
Gupta, C. 2000b. The role of estrogen receptor, androgen receptor and growth
factors in diethylstilbestrol-induced programming of prostate differentiation. Urological
Research 28:223-229.
24.
Elswick, B.A., Welsch, F., and Janszen, D.B. 2000. Effect of different sampling
designs on outcome of endocrine disruptor studies. Reprod. Toxicol. in press.
25.
vom Saal, F.S., Cooke, P.S., Buchanan, D.L., Palanza, P., Thayer, K.A., Nagel,
S.C., Parmigiani, S., and Welshons, W.V. 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 14:239-60.
26.
Farabollini, F., Porrini, S., and Dessi-Fulgheri, F. 1999. Perinatal exposure to the
estrogenic pollutant bisphenol A affects behavior in male and female rats. Pharmacol.
Biochem. Behav. 64:687-694.
27.
Ashby, J., Tinwell, H., and Haseman, J. 1999. Lack of effects for low dose levels of
bisphenol A (BPA) and diethylstilbestrol (DES) on the prostate gland of CF1 mice
exposed in utero. Reg Tox Pharm. 30:156-166.
28.
Cagen, S.Z., Waechter, J.M., Dimond, S.S., Breslin, W.J., Butala, J.H., Jekat,
F.W., Joiner, R.L., Shiotsuka, R.N., Veenstra, G.E., and Harris, L.R. 1999. Normal
reproductive organ development in CF-1 mice following prenatal exposure to Bisphenol
A. Tox. Sci. 11:15-29.
29.
Roper, R.J., Griffith, J.S., Lyttle, C.R., Doerge, R.W., McNabb, A.W., Broadbent,
R.E., and Teuscher, C. 1999. Interacting quantitative trait loci control phenotypic variation
in murine estradiol-regulated responses. Endocrinology 140:556-61.
30.
Spearow, J.L., Doemeny, P., Sera, R., Leffler, R., and Barkley, M. 1999. Genetic
Variation in Susceptibility to Endocrine Disruption by Estrogen in Mice. Science
285:1259-1261.
31.
Vandenbergh, J.G. and Huggett, C.L. The effects of intrauterine position on
subsequent sex ratio in house mice. in Estrogens in the Environment III: Global Health
Implications. 1994. Washington, D.C.
32.
Newbold, R. 1995. Cellular and molecular effects of developmental exposure to
diethylstilbestrol: implications for other environmental estrogens. Environmental Health
Perspectives 103:83-87.
33.
vom Saal, F.S., Howdeshell, K.L., Ruhlen, R.L., Taylor, J.A., Timms, B.G., and
Welshons, W.V. High sensitivity of the fetal prostate to endogenous and environmental
estrogens. in Bisphenol A: Low dose effects-high dose effects. November, 2000. Freie
Universitat, Berlin.