APPENDIX II-CA:  Myers, et al, “Does the Dose Make the Poison?”  Environmental Health News, April 30, 2007. [Permethrin is an endocrine disruptor; only one small dose can cause serious health problems].


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April 30, 2007.

Does 'the dose make the poison?'

Toxicology testing assumes 'the dose makes the poison.'

Photograph from Retha Newbold, NIEHS.

Measuring how much of a compound, called its dose, produces a response, usually some kind

of health effect, is difficult and time consuming. To understand how dose and effects are

linked, toxicologists expose animals, tissues, or cells to pollutants. They then examine how

the subject responds to the exposure.

The "dose makes the poison" is a

common adage in toxicology. It

implies that larger doses have greater

effects than smaller doses. That

makes common sense and it is the

core assumption underpinning all

regulatory testing. When "the dose

makes the poison," toxicologists can

safely assume that high dose tests will

reveal health problems that low dose

exposures might cause. High dose

tests are desirable because, the logic

goes, they not only will reveal low

dose effects, they will do so faster and

with greater reliability. Greater

reliability and speed also mean less


by Pete Myers, Ph.D. and Wendy Hessler While exposure in the womb to 100 parts

per billion of the estrogenic drug

diethylstilbestrol (DES) causes mice to

become scrawny as adults, exposure to a

much lower amount, 1 ppb, causes

grotesque obesity. This photograph

compares a control animal (left) to an

animal exposed to a very small amount of

DES in the womb (right).

The trouble is, some pollutants, drugs and natural substances don't adhere to this logic, as can

be seen in the photograph above. Instead, they cause different effects at different levels,

including impacts at low levels that do not occur at high doses. Sometimes the effects can even

be precisely the opposite at high vs. low. Because all regulatory testing has been designed

assuming that "the dose makes the poison," it is highly likely to have missed low dose effects,

and led to health standards that are too weak.

Extensive results challenge a core assumption in toxicology

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In standard toxicology, as the dose increases, so does the effect. Conversely as dose

decreases, so does its impact. This relationship is called a monotonic dose-response curve

because effects are either increasing or decreasing. In a monotonic curve, they never reverse

direction. It is akin to a dimmer switch and a lightbulb. The more electricity you let through by

turning the knob, the brighter the bulb gets.

The diagrams to the right present idealized forms of

monotonic (left) andnon-monotonic (right) doseresponse

curves. Monotonic can either be linear ornonlinear.

The key point is that the direction of the curve

never changes from positive to negative or vice-versa.

A monotonic curve can flatten, i.e., reach an


Non-monotonic curves, in contrast, change direction.

Over part of the curve, response increases with dose,

while over another portion it decreases as dose

increases. Non-monotonic curves are often called

'inverted-U' (upper) or 'U'(lower).

How toxicology tests are used to

develop health standards

Government agencies identify and

regulate dangerous substances assuming

that 'the dose makes the poison.'

To set exposure limits, three to five doses

of a substance are tested in the

laboratory. Toxicologist start at the

highest dose chosen and continue to lower

doses until they find the point where

effects are no longer detectable, that is,

the dose at which experimental animals

no longer differ from controls. This safe

dose - the lowest amount that poses an

acceptable risk - is called the 'no

observed adverse effect level,' or NOAEL.

Traditional toxicology guiding health

regulations rarely tests doses lower than

NOAEL due the 'dose makes the poison'


The final acceptable level for human

exposure--called the 'reference dose'—is

calculated from the NOAEL by adding a

series of safety factors. These safety

factors take into account uncertainties in

extrapolating animal research to human,

as well as differences insensitivity among

groups of people, and between kids and

adults. Thus if the NOAEL is found to be 1

milligram per kilogram of bodyweight per

day (which corresponds to apart per

million), then the reference dose might be

1 part per billion per day.

Blindsided by hormonally-active


While toxicologists have traditionally assumed

that the dose makes the poison,

endocrinologists --scientists who study the

action of hormones--have long known that

hormones can have different effects at

different doses.

The graph to the right comes from a simple

study looking at the response of a gene inside

a cell as it is exposed to different amounts of

estradiol, the common form of the natural

human hormone, estrogen.

In the experiment, the scientists experimented

over an extremely wide range of doses, from

around 10 parts per quadrillion (ppq) to 10

parts per million (ppm).

Figure adapted from Welshons et al. 2003

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Most estradiol in human blood is bound up by

special proteins. When bound, it can't interact with

hormone receptors. Because that interaction is a

crucial step in the process that turns on estrogenresponsive

genes, bound estrogen doesn't turn on

genes. Only the unbound estrogen can, and its

concentration in human blood is normally in the

green zone of the graph, parts per quadrillion to low

parts per trillion.

As the dose of estradiol rises through the green

zone of the graph, the response increases. This

green zone is the range of concentrations over

which unbound estradiol is found in blood.

Initially, at just above 1 part per quadrillion, there's

no difference between the control (0 estradiol) and

the response to estradiol. As dose increases up to

just above 1 part per trillion, the response

increases. It then flattens out, over a wide range of

doses, all the way to 100 parts per billion. But once

it gets into the high-dose range, it drops, and by

just over 10 parts per million the system shuts

down, with no response whatsoever.

Could this mean higher

doses are safer than lower


A frequent response from people

seeing a

dose-response curve like that

above for the first time is to ask

'Does this mean higher doses are


Emphatically, no. At the highest

doses used in this experiment, the

system was no longer able to

respond to estrogen signaling. That

means that crucial events under

the control of estrogen would not

occur. The consequences, for

example, of shutting off estrogen

signaling responses during

development would most likely be

catastrophic for the organism


What's happening? As estradiol increases in the low dose range, it is binding with receptors and

stimulating the responsive gene. This is what is supposed to happen over this dose range, the

range found naturally in people. However, as receptor occupancy increases above 10%, a

feedback loop cuts in, leading to a reduction in the availability of additional receptors.

As dose increases further, the effect of the feedback loop grows until no amount of additional

estradiol can increase the system's response. That produces the long flat portion of the graph,

from just over 1 ppt to 100 ppb.

As doses rise above 100 ppb, estradiol becomes overtly toxic to the cell and the system stops

responding completely, dropping even below the control level.

This dose-response curve dramatically violates the assumption that high dose experiments can

be used to predict low dose results. At high doses, estradiol shuts the system down. At low

doses it turns the system up. Over part of the dose range, response increases, while over

another part, it decreases. This curve is called a non-monotonic dose-response curve.

Consider this 'thought experiment.' Think again about that light bulb hooked up to a dimmer

switch, but instead of running it through your normal wiring (110 volts), plug it into the circuit

for the dryer (220 volts). When the dimmer is turned down, there's very little light coming

through. Turn it up and the light gets brighter. Turn it all the way up and the light bulb blows

up. All of a sudden, it's dark again. There was more voltage and current than the system was

designed for.

With 'dose makes the poison' thinking dominating toxicology, traditional toxicologists didn't

pursue the possibility that there might be effects at levels far beneath those used in standard

experiments. No health standards incorporated the possibility. Over the past 15 years,

however, as scientists began to explore the impacts of endocrine disrupting compounds--

compounds that behave like hormones or interfere with hormone actions-- many examples of

non-monotonic dose response began to be published in scientific journals.

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In 2006, a team of German researchers published a vivid example of how traditional testing to

set health standards can miss low dose effects. Their work examined the effect of a phthalate

on the activity of an enzyme in the brain of developing male rats. This enzyme, aromatase,

converts testosterone to estrogen. Counter-intuitively, estrogen early in the life is necessary to

masculinize the brain of male mammals. If they don’t get enough, key parts of the brain that

normally differ between males and females will be more similar to the female form than the

male form.

In their experiment they exposed pregnant females to the phthalate DEHP, with different

groups exposed to an extremely wide range of doses. The highest dose used is one known to

cause reproductive damage to developing males without obviously harming the mother. The

lowest dose, 19,000 times beneath the high dose, was set at a level commonly observed in

people in Germany.

Many cases of non-monotonic dose-response curves have now been published in research on

endocrine disruption. Below follow some recent examples. Because they are now being reported

frequently in research on the effects of endocrine-disrupting chemicals, it is clear that

regulatory toxicology can no longer safely assume that 'the dose makes the poison.' It is also

clear that the standard approaches used to develop estimates of safe exposure levels, by

basing their design on a false assumption, are likely to have set safety standards that are not

strong enough to protect public health.

Narita et al. report that a key step in

immune reactions, the release of

histamine and cytokines by mast cells,

is exacerbated by very low levels of

environmental contaminants, similar to

the effect of estradiol. These

experiments, done in cell culture, used

levels of the contaminants well within

the range of human exposure. The peak

response was seen at approximately 0.1

parts per billion (10


molar). By the

time the dose rose to 10 parts per

billion (10


molar), the response

disappeared. This experiment was done

with mouse and human cells in culture.

Graph adapted from Narita et al.

Their results, seen to the right, show that

doses from 15 mg/kg/day to 405 mg/kg/day

(statistically significant in purple) cause an

increase in aromatase activity. Intermediate

doses (1.215 and 5 mg/kg/day) do not differ

from control (the blue horizontal line) But

lower doses suppress aromatase activity

(statistically significant in red, 0.134 and

0.405 mg/kg/day). As the research team

point out in their article, a regulatory test for

DEHP effects would not have gone below 5

mg/kg/day and therefore would have missed

the significant aromatase suppression at

lower levels.

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At doses far beneath the current EPA safe level,

Takano et al. found that the phthalate DEHP

increases the immune response of mice to a

common allergen. Clinical scores of an allergic

reaction were strongest at intermediate doses(4

and 20 µg). A dose of 100 µg (yellow line) was

no different than the control (purplish blue line).

Graph adapted from Takano et al.

Working with a suite of compounds that bind to

a newly discovered estrogen receptor on the

surface of the cell membrane, Wozniak et al.

found that cells and prolactin release (graphs to

left) follow markedly non-monotonic patterns.

Bisphenol A provoked responses at the lowest

dose tested, 0.23 parts per trillion. Bisphenol A

has been considered a weak estrogen because

its relatively binding affinity with the estrogen

receptor in the cell nucleus is much lower than

that of estradiol. In contrast, with this cell

membrane receptor, bisphenol A is just as

powerful as estradiol.

Graphs adapted from Wozniak et al.

Ralph et al. discovered that prostate cells

respond in anon-monotonic fashion to exposure

to the organochlorine pesticide

hexachlorobenzene (HCB). High levels suppress

androgenic activity of the cells relative to

controls (redline), whereas low levels enhance

androgenic activity. Their experiments with live

mice revealed that prostate weight in adult mice

also showed that high doses produced the

opposite effect of low doses.

Wetherill et al. found that a one nanomolar dose

of bisphenol A yields the strongest proliferation

response by prostate tumors in experiments

with cells. The impact of a dose 100-times

higher didn't differ from control.

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Andrade, AJM, SW Grande, CE Talsness, K Grote and I Chahoud. 2006. A dose–response study

following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): Nonmonotonic

dose–response and lowdose effects on rat brain aromatase activity. Toxicology 227:


Narita, S, RM Goldblum, CS Watson, EG Brooks, DM Estes, EM Curran and T Midoro-Horiuti.

2007. Environmental Estrogens Induce Mast Cell Degranulation and Enhance IgE-mediated

Release of Allergic Mediators.Environmental Health Perspectives 115:48–52

Newbold, RR, E Padilla-Banks, RJ Snyder and WN Jefferson. 2005. Developmental Exposure to

Estrogenic Compounds and Obesity. Birth Defects Research (Part A) 73:478–480.

Ralph, JL, M-C Orgebin-Crist, J-J Lareyre and CC Nelson. 2003. Disruption of androgen

regulation in the prostate by the environmental contaminant hexachlorobenzene. Environmental

Health Perspectives 111:461-466

Takano, H, R Yanagisawa, K-I Inoue, T Ichinose, K Sadakano, and T Yoshikawa. 2006. Di-(2-

ehylhexyl) Phthalate Enhances Atopic Dermatitis-Like Skin Lesions in Mice. Environmental Health

Perspectives 114: 1266-1269.

Welshons, WV, KA Thayer, BM Judy, JA Taylor, EM Curran and FS vom Saal. 2003. Large effects

from small exposures. I. Mechanisms for endocrine disrupting chemicals with estrogenic

activity. Environmental Health Perspectives 111:994-1006.

Wetherill, YB, CE Petre, KR Monk, A Puga, and KE Knudsen. 2002. The Xenoestrogen Bisphenol

A Induces Inappropriate Androgen Receptor Activation and Mitogenesis in Prostatic

Adenocarcinoma Cells. Molecular Cancer Therapeutics 1: 515–524.

Wozniak, AL, NN Bulayeva and CS Watson. 2005. Xenoestrogens at Picomolar to Nanomolar

Concentrations Trigger Membrane Estrogen Receptor-alpha-Mediated Ca++ Fluxes and

Prolactin Release in GH3/B6

Pituitary Tumor Cells. Environmental Health Perspectives 113:431-439.

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