Regulatory Toxicology and Pharmacology 99 (2018) 142–158

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Distinguishing between endocrine disruption and non-specific effects on T

endocrine systems
M. Sue Martya,∗, Chris Borgertb, Katie Coadya, Richard Greenc, Steven L. Levined, Ellen Mihaiche,
Lisa Ortegof, James R. Wheelerc, Kun Don Yig, Leah M. Zorrillaf
a
The Dow Chemical Company, Toxicology & Environmental Research and Consulting, 1803 Building, Midland, MI, 48674, USA
b
Applied Pharmacology and Toxicology, Inc., C.E.H.T. Dept. Physiological Sciences, University of FL College of Veterinary Medicine, 2250 NW 24th Avenue, Gainesville,
FL, 32605, USA
c
Dow AgroSciences, 3b Park Square, Milton Park, Abingdon, Oxfordshire, OX14 4RN, United Kingdom
d
Monsanto Company, Global Regulatory Science, 700 Chesterfield Parkway W, Chesterfield, MO, 63017, USA
e
Environmental and Regulatory Resources, LLC, 6807 Lipscomb Drive, Durham, NC, 27712, USA
f
Bayer CropScience, 2 TW Alexander Dr, Research Triangle Park, NC, 27709, USA
g
Syngenta Crop Protection, LLC, 410 S Wing Rd, Greensboro, NC, 27409, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The endocrine system is responsible for growth, development, maintaining homeostasis and for the control of
Endocrine disruption many physiological processes. Due to the integral nature of its signaling pathways, it can be difficult to dis-
Endocrine system tinguish endocrine-mediated adverse effects from transient fluctuations, adaptive/compensatory responses, or
Systemic toxicity adverse effects on the endocrine system that are caused by mechanisms outside the endocrine system. This is
Stress
particularly true in toxicological studies that require generation of effects through the use of Maximum Tolerated
Weight of evidence
Endocrine screening
Doses (or Concentrations). Endocrine-mediated adverse effects are those that occur as a consequence of the
Maximum tolerated dose interaction of a chemical with a specific molecular component of the endocrine system, for example, a hormone
receptor. Non-endocrine-mediated adverse effects on the endocrine system are those that occur by other me-
chanisms. For example, systemic toxicity, which perturbs homeostasis and affects the general well-being of an
organism, can affect endocrine signaling. Some organs/tissues can be affected by both endocrine and non-en-
docrine signals, which must be distinguished. This paper examines in vitro and in vivo endocrine endpoints that
can be altered by non-endocrine processes. It recommends an evaluation of these issues in the assessment of
effects for the determination of endocrine disrupting properties of chemicals. This underscores the importance of
using a formal weight of evidence (WoE) process to evaluate potential endocrine activity.

1. Introduction molecules (hormones) with their conjugate receptors operating in lar-

Biological functions are regulated in higher organisms by complex
signaling networks within the neuroendocrine system. The endocrine
branch of this system is critically important in two broad classes of
biological functions – 1) regulation of growth and development, and 2)
maintenance of internal homeostasis and normal physiological pro-
cesses (reproduction, temperature regulation, metabolism, etc). Some
of these functions are critical at a single point in the life cycle of an
organism, while others must be repeated continually throughout life.
Although often studied separately, the endocrine system serves
these two major physiological roles using the same set of signaling
gely the same set of hormonally responsive tissues. This simple fact
makes it difficult to design sensitive, hormone-specific eco/tox-
icological assays that clearly distinguish permanent changes from
transient fluctuations, or adverse alterations from adaptive (and com-
pensatory) responses (Keller et al., 2012). The difficulty is increased by
the fact that all organs/tissues can be affected by physiological stimuli,
xenobiotic chemicals or natural substances, which may lead to tox-
icological processes and events outside the control of the endocrine
system, but which in turn, indirectly affect or trigger an endocrine re-
sponse.
This paper will identify and explain some of the non-endocrine

∗
Corresponding author.
E-mail addresses: msmarty@dow.com (M.S. Marty), cjborgert@apt-pharmatox.com (C. Borgert), kcoady@dow.com (K. Coady),
Richard_Green@vrtx.com (R. Green), steven.l.levine@monsanto.com (S.L. Levine), emihaich@nc.rr.com (E. Mihaich), lisa.ortego@bayer.com (L. Ortego),
jrwheeler@dow.com (J.R. Wheeler), sue.yi@syngenta.com (K.D. Yi), leah.zorrilla@bayer.com (L.M. Zorrilla).

https://doi.org/10.1016/j.yrtph.2018.09.002
Received 13 July 2018; Accepted 4 September 2018
Available online 12 September 2018
0273-2300/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.S. Marty et al.
pathways and processes that can affect endpoint measurements and
interpretation of endocrine-mediated responses in endocrine screening
assays and eco/toxicological studies. Factors such as cytotoxicity,
general systemic toxicity (including overt toxicity), stress, and experi-
mental conditions can influence endocrine responses, which may ap-
pear as either reduced or enhanced endocrine signaling. This paper
describes influential variables that can result in altered estrogen, an-
drogen or thyroid signaling in vitro and in vivo. This information is
useful to consider when conducting weight of evidence (WoE) evalua-
tions and can reduce the degree of uncertainty in the overall assessment
or in the interpretation of various endpoints. This may also assist with
dose/concentration level selection and/or the inclusion of additional
clarifying endpoints when designing studies to evaluate endocrine-
sensitive endpoints.
2. Criteria for distinguishing endocrine-mediated from NON-
endocrine-mediated effects on estrogen, androgen or thyroid
signaling
Endocrine-mediated effects are those that occur as a consequence of
the interaction of a chemical with a specific molecular component of
the endocrine system, such as a hormone receptor or transporter, a
transcriptional enhancer or repressor of steroid-responsive genes, an
enzyme specific to the endocrine system, etc. The use of the potent
pharmaceutical estrogen, ethinylestradiol in oral contraceptives is a
clear example of intentional and safe use of an endocrine-mediated
adverse effect (i.e., endocrine disruption) to achieve control of a spe-
cific reproductive process. Adverse effects on male reproductive per-
formance and secondary sex characteristics in response to potent
pharmaceutical estrogens is also endocrine disruptive. Both occur as a
consequence of interaction with estrogen receptors (ERs) in target tis-
sues and in areas of the brain that mediate feedback control of hormone
production.
Non-endocrine-mediated adverse effects on the endocrine system
are those that occur by mechanisms other than direct interaction of a
chemical with a specific component of the endocrine system. Such non-
endocrine-mediated modes of actions (MoAs) could include toxicity to a
tissue or organ of the endocrine system or to physiological processes
that either control or are controlled by the endocrine system. For ex-
ample, multiple changes in hormone levels occur and contribute to
parturition; however, this process also requires muscle contraction. If a
toxicant blocks muscle contraction, this is not an endocrine MoA. A
second example is the reduction of testosterone levels caused by abuse
of ethyl alcohol, which is due to altered metabolism of steroid hor-
mones that arises due to chronic liver toxicity rather than to a direct
interaction of ethanol with steroidogenesis.
Endocrine- versus non-endocrine-mediated adverse effects can often
be distinguished using the lead toxic effect approach (Bars et al., 2011,
2012), where the adverse effect that occurs at the lowest dose/con-
centration is considered the lead toxic effect. This approach describes
the most sensitive effect, which may assist in the determination of
whether the apparent endocrine effect is mediated by an interaction
with a component specific to the endocrine system or is instead pro-
duced as a result of the lead toxic effect itself. This concept is critically
important as it can help determine the necessity for conducting addi-
tional tests to describe the hazard of a substance. For instance, in the
EPA's Endocrine Disruptor Screening Program (EDSP), a true endocrine
disruptive effect may require additional higher tier testing to fully de-
scribe the dose/concentration relationship before risk assessment can
be performed. On the contrary, if the effect is due to a non-endocrine
MoA, the existing data package may already fully describe the tox-
icological response for the lead effect, making additional testing re-
dundant. Further, in some regions it may be extremely important to
separate endocrine-mediated from non-endocrine-mediated toxicities as
some legal frameworks will regulate these differently (e.g. the endo-
crine hazard-based cut-off criterion in the EU) (Wheeler and Coady,
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Regulatory Toxicology and Pharmacology 99 (2018) 142–158
2016).
Consideration of dose and realistic exposures is important for un-
derstanding toxicity responses that have an endocrine component. The
likelihood of altered homeostasis affecting endocrine-related endpoints
is increased in toxicological studies requiring the use of a maximum
tolerated dose or concentration (MTD or MTC), which aims to produce
an adverse effect in the test system. Even with endocrine-mediated ef-
fects, it is important to consider whether effective dose levels are close
to realistic exposure levels or whether there are large margins of ex-
posure.
Addressing these and other challenges has been central to devel-
opment and continued refinement of the US EPA's EDSP and the de-
velopment of the OECD's Conceptual Framework for the testing and
assessment of endocrine disrupters (OECD, 2012a). During a review of
the EDSP, the FIFRA Scientific Advisory Panel (SAP) was asked to
comment on the validity of including treatments that demonstrated
overt systemic toxicity to assess effects of potential endocrine interac-
tion. On the issue of overt systemic toxicity, the SAP in May of 2013
stated: “In summary, the Panel agreed that little, if any, weight should be
placed on signs of endocrine disruption in the presence of overt systemic
toxicity. All effects in endocrine sensitive tissues should be evaluated in terms
of primary interactions with the endocrine system vs. secondary effects re-
lated to toxicity in non-endocrine organs or overall disruptions in home-
ostasis”. The US EPA (2009a, b) recommended that an approximate ten
percent decrease in terminal body weight (with a statistically sig-
nificant decrease in body weight gain) should be used as a general
guideline for mammalian assays because body weight decreases greater
than ten percent have been shown to confound the interpretation of
reproductive system-related endpoints in the pubertal assays (Laws
et al., 2007). Furthermore, a 6% decrease in terminal body weight can
affect pubertal assay thyroid endpoints. Similarly, clinical pathology
alterations like increased blood urea nitrogen and creatinine in the
absence of body weight changes indicate that the MTD has been ex-
ceeded. Physiological changes associated with doses that produce sys-
temic or overt toxicity are often the consequence of activation of the
hypothalamic-pituitary-adrenal axis, which in turn affects all endocrine
systems including those that are the focus of the EDSP. This thinking
supports the regulatory approach in the US, but acceptance at the OECD
level is less clear. Greater emphasis should be placed on using these
concepts in the design and interpretation of eco/toxicological assays.
3. In vitro endocrine screening assays – responses confounded by
description of normal cellular function and cytotoxicity
New toxicity testing strategies increasingly incorporate in vitro as-
says, either to replace traditional animal studies or to aid in identifying
the key biochemical perturbations that produce adverse effects in those
studies (NRC, 2007; Judson et al., 2010). In vitro assays can be useful
screening tools and provide mechanistic information on initiating or
intermediate events in adverse outcome pathways (AOPs) (Judson
et al., 2013; Ankley et al., 2010). However, to reliably use results from
in vitro endocrine assays, assessors must be able to distinguish between
endocrine-mediated and non-endocrine-mediated effects. The analog in
in vitro assays for overt chemical toxicity observed in whole animal
assays is cell stress and cytotoxicity-mediated processes that disrupt
specific biomolecular functions. Cell-disruptive processes include pro-
tein, DNA or lipid reactivity, physico-chemical disruption of proteins or
membranes (e.g., by surfactants), or processes such as apoptosis, oxi-
dative stress response, mitochondrial disruption, endoplasmic re-
ticulum stress, microtubule disruption, or a heat shock response among
others. There are also confounding factors that can alter assay stability
in cell free systems that can include changes in buffer pH, receptor
denaturation, protein-protein and protein-membrane interactions, dis-
ruption/enhancement of binding kinetics, test substance precipitation,
aggregation of individual molecules and potentially other mechanisms.
Recently, some limitations of in vitro screening assays in ToxCast
M.S. Marty et al.
were identified by examining a diverse chemical and assay space
(Judson et al., 2016). ToxCast is a large battery of assays with over 800
in vitro assay endpoints from seven high-throughput platforms. Ap-
proximately 15% of the assays in ToxCast assess endocrine endpoints
related to the estrogen, androgen and thyroid pathways. To aid in the
interpretation of results, ToxCast includes numerous cell stress and
cytotoxicity assays but not all assays are evaluated for cytotoxicity or
impacts to cell-free systems. Cytotoxicity has been defined within
ToxCast as a positive call in two or more cytotoxicity assays or cyto-
static effects in a proliferation assay. The threshold of two was selected
based on the observation that most chemicals with activity in two or
more of these cytotoxicity assays displayed a concentration response.
Not surprisingly, cell-free assays in ToxCast showed a rapid increase in
the frequency of responses at concentrations where cell stress/cyto-
toxicity responses were observed in the cell-based assays. The observed
activity could be divided into two categories: (1) specific biomolecular
interactions against one or more target concentrations below the
threshold for overt cell stress/cytotoxicity, and (2) non-specific bio-
molecular interactions associated with cell stress or cytotoxicity re-
sulting from triggering specific cell stress pathways, disruption of pro-
teins or membranes, or broad low-affinity non-covalent interactions.
Several trends were identified in the ToxCast analysis (Judson et al.,
2016). Chemicals showing a greater number of specific biomolecular
interactions were generally designed to be bioactive (e.g., pharmaceu-
ticals) whereas intentional food-use chemicals or pesticides targeting
systems that are phylogenetically distant from humans tended to show
the fewest specific interactions (e.g., herbicides). For many chemicals, a
disproportionately large number of positive assay responses was ob-
served in the concentration range where cytotoxicity was observed.
This phenomenon, where there was a steep increase in cytotoxicity was
termed the “burst region”. A metric termed the Z-score was developed
to assess specificity by indicating whether true activity or cytotoxicity
has occurred, with a high Z-score representing high specificity and a
low Z-score representing low specificity. A class of compounds that
consistently showed non-specific responses was surfactants and this
non-specific activity reflects the ability of surfactants to disrupt cell
membrane structure and function and protein–protein interactions.
Graphical analyses comparing the concentration where cytotoxicity
occurs versus responses for endocrine and non-endocrine screening
assays in ToxCast is presented in Mihaich et al. (2017a, b) for propi-
conazole and triclosan.
Recently, high-content imaging (HCI), which applies automated
image analysis to capture multiple cytological features, has been eval-
uated within ToxCast to determine a “tipping point” or point of de-
parture for adverse impacts in cell line-based assays (Shah et al., 2016).
The aim of this work was to integrate an analysis of impacts on normal
cellular function to identify transition points between adaptive changes
and adverse effects that will allow for the appropriate interpretation of
ToxCast data. The results of Shah et al. (2016) underscore re-
commendations by Judson et al. (2016) regarding the importance of
characterizing concentration- and time-dependent effects on cytotoxi-
city and MoA endpoints to accurately characterize the sequence of ef-
fects and to support or negate a possible MoA for an adverse endocrine
outcome. More development is required to implement HCI metho-
dology into in vitro platforms to understand the role of adaptive cellular
responses and pathways that lead to adverse in vivo outcomes
(Boekelheide and Andersen, 2010).
The EDSP battery used to evaluate List 1 compounds included five in
vitro assays that screened estrogen and androgen endpoints (Table 1)
and these assays are included in Level 2 of the OECD Conceptual Fra-
mework (OECD, 2012a). The androgen receptor transcriptional acti-
vation assay (ARTA; OECD 458; OECD, 2016, Table 2) was not vali-
dated at the time List 1 chemicals were screened but if it was available,
it likely would have been included in the in vitro battery. These in vitro
assays screen for potential endocrine modulation through a specific
endocrine mechanism. Therefore, it is critical to include a
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Regulatory Toxicology and Pharmacology 99 (2018) 142–158
comprehensive assessment of non-specific factors that could potentially
confound assay results and interpretation and it is incumbent on the
investigators to find the maximum allowable in vitro concentration for
these diverse biological systems. The National Toxicology Program In-
teragency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM) and the Interagency Coordinating Committee on
the Validation of Alternative Methods (ICCVAM) recommended for cell
line assays that only concentrations that do not cause greater than 10%
cytotoxicity should be included in an analysis (NICEATM and ICCVAM,
2011). Allowing for high levels of cytotoxicity (i.e., > 10%), without a
correction for cytotoxicity, greatly increases the likelihood of producing
a false-positive or false-negative response depending on the test system.
Although the ER binding assay has been used as a research tool for
decades, several factors may affect assay performance and interpret-
ability (Laws et al., 2006). In an assessment of the performance of the
ER binding assay (OCSPP 890.1250), seven out of 50 chemicals that
covered a broad range of structural and physiochemical diversity re-
quired diagnostic secondary Ki experiments to demonstrate that these
chemicals were not true competitive inhibitors of ER binding (Laws
et al., 2006). From these secondary plots, it was shown that the seven
chemicals altered the stability of the assay by either changing buffer
pH, denaturing ER and changing its conformation, or disrupting the ER
binding kinetics. If one of these factors is at play, it is not uncommon to
see a large change in ER binding (i.e., steep slope) over a single log unit
(e.g., 0% inhibition to 90% inhibition). Therefore, if a response is ob-
served in the ER binding assays, it is important to evaluate whether the
response represents a competitive or non-competitive interaction and is
not merely an artifact of one of the above-mentioned factors. The an-
drogen receptor (AR) binding assay, like the ER binding assay, has also
been used as a research tool for decades and the EPA test guideline for
the AR binding assay outlined in OCSPP 890.1150 is based on the es-
tablished methodology of Kelce et al. (1995). The same factors dis-
cussed above that impact the ER binding assay apply to the perfor-
mance and interpretability of the AR binding assay.
In addition to the ER and AR binding assays, the Tier 1 EDSP battery
also required screening for potential estrogen agonist activity with a
cell-based ER transcriptional activation (ERTA) assay (OCSPP
890.1300). However, since the time of the initial compounds going
through the EDSP, the ERTA has been validated to also assess antag-
onism (OECD, 2012b) and OECD has recently developed a guideline for
an ARTA assay that can assess agonism and antagonism (OECD 455;
OECD, 2016). For both assays, cytotoxicity is evaluated with a con-
stitutive luciferase reporter construct and cannot exceed 20%. An as-
sessment of cytotoxicity is arguably more important for the assessment
of antagonism because cytotoxicity can impact reporter activity by
disturbing normal cellular function. A positive response for antagonism
is achieved with ≥30% inhibition of induced ER or AR reporter ac-
tivity. Therefore, it is recommended that when cytotoxicity is ap-
proaching a significant level (e.g., ≥10%), ER and AR reporter activity
should be corrected for the level of observed cytotoxicity (i.e., signal
adjusted for the proportion of viable cells remaining).
The aromatase assay (OCSPP 890.1200) measures the ability of a
test substance to inhibit conversion of testosterone to estrogen. The EPA
test guideline recommends conducting the assay using commercially-
available human recombinant microsomes containing CYP19 plus re-
ductase, and as such, may be less susceptible to inconsistency compared
to cytosolic preparations from animal tissue. Overall, the method has
been shown to be sensitive and specific. However, like the ER and AR
competitive binding assays, chemicals that denature or affect the
structure of the enzyme in vitro could produce a false positive result.
This in vitro assay can be especially susceptible to factors that denature
or affect the structure of the enzyme because the total protein con-
centration in the assay is extremely low. For this reason, cytosolic
preparations could be less sensitive to chemicals that denature or affect
the structure of the enzyme. Evidence of this assay's potential sus-
ceptibility to confounding effects from test chemicals is foreshadowed
M.S. Marty et al. Regulatory Toxicology and Pharmacology 99 (2018) 142–158

Table 1
EPA's EDSP Tier I in vitro Screening Battery.
Assay Type Assay Description

ER binding assay An ER binding assay that utilizes rat uterine cytosol to examine the ability of a test chemical to bind with estrogen receptors. Assesses ER
agonism and antagonism.
ER transcriptional activation assay An ER binding assay that uses a human cell line to examine the ability of a test chemical to bind with one of the estrogen receptors. Assesses
ER agonism but can also assess and differentiate antagonism.
AR binding assay An AR binding assay that utilizes rat prostate cytosol to examine the ability of a test chemical to bind with androgen receptors. Assesses AR
agonism and antagonism.
Aromatase assay Aromatase is an enzyme responsible for estrogen biosynthesis that converts androgens into estrogens, estradiol, and estrone. The aromatase
assay uses a human recombinant form of the protein and focuses on this portion of the steroidogenic pathway to detect substances that
inhibit aromatase activity.
H295R Steroidogenesis assay The Steroidogenesis assay utilizes the H295R human adrenocortical carcinoma cell line to detect interference and induction with the body's
production of male and female steroid sex hormones (estrogen and testosterone).

Table 2 measured in the assay are concentrations of testosterone and 17β-es-

Referenced test guideline (TG) studies to evaluate potential endocrine activity. tradiol in the cell culture media after 48 h of exposure. This assay re-
Test Guideline U.S. EPA TG No. OECD TG
quires an evaluation of cytotoxicity that is preferably run parallel and
No. cytotoxicity should not exceed 20%. The test guideline recommends
performing either a visual categorical examination of the cells, per-
In Vitro Assays forming the Live/Dead Assay® or performing the MTT assay to assess
ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 OECD 493
cytotoxicity. On mechanistic grounds, the MTT assay is probably the
ERα Transcriptional Activation Assay OCSPP 890.1300 OECD 455 most appropriate assay because it largely measures mitochondrial
AR Binding Assay OCSPP 890.1150 function (Berridge et al., 2005). A reliable evaluation of mitochondrial
AR Transcriptional Activation Assay OECD 458 function is critical because the mitochondrial electrochemical gradient
Steroidogenesis Assay (H295R cells) OCSPP 890.1550 OECD 456
is required for the steroidogenic acute regulatory protein to transfer
Aromatase Assay OCSPP 890.1200
cholesterol to the inner membrane of mitochondria and initiate ster-
In Vivo Assays oidogenesis (King and Stocco, 1996). Therefore, to properly assess
mitochondrial function and potential impacts to steroidogenesis, a
Uterotrophic Assay (Rat) OCSPP 890.1600 OECD 440
more sensitive indicator of mitochondrial function than the MTT assay
Hershberger Assay (Rat) OCSPP 890.1400 OECD 441
Pubertal Female Assay (Rat) OCSPP 890.1450
may be required and the MitoTracker® Red CMXRos assay that directly
Pubertal Male Assay (Rat) OCSPP 890.1500 assesses mitochondrial potential is a consideration for ToxCast assays
Reproduction/Developmental Toxicity OCSPP OECD 421; (Levine et al., 2007; Shah et al., 2016). A positive call in the test
Screening Test (RDTS) (Rat); Combined 870.3550; 422 guideline is detecting a statistically significant difference from the
Repeated Dose Toxicity Study with RDTS 870.3650
control, which could be less than a 20% mean difference from control
Prenatal Developmental Toxicity Study (Rat, OCSPP 870.3700 OECD 414
Rabbit) levels. Consequently, it is recommended that when cytotoxicity is ap-
Two-Generation Reproductive Toxicity Study OCSPP 870.3800 OECD 416 proaching a significant level, but still less than 20%, reporter activity
(Rat) should be corrected for this cytotoxicity.
Extended One-Generation Reproductive OECD 443 Recently, the H295R steroidogenesis assay has become one of the
Toxicity Study (Rat)
Fish Short-term Reproduction Assay OCSPP 890.1350 OECD 229
high-throughput assays included in ToxCast and these data could be
Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100 OECD 231 used to prioritize testing under the EDSP (Karmaus et al., 2016; US EPA,
Medaka Extended One Generation OCSPP 890.2200 2017a). Modifications to the OECD 456 test guideline by ToxCast in-
Reproduction Test clude raising the level of basal steroidogenesis with a 48-h pre-induc-
tion with forskolin. Measurement of 13 steroidogenic hormones from
Test guidelines available at:
four hormone classes (glucocorticoids, progesterones, androgens, es-
OCSPP: https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/
series-890-endocrine-disruptor-screening-program and https://www.epa.gov/ trogens) is performed using a novel high throughput HPLC-MS/MS
test-guidelines-pesticides-and-toxic-substances/series-870-health-effects-test- (high-performance liquid chromatography followed by tandem mass
guidelines. spectrometry) method. The permissible level of cytotoxicity using the
OECD: https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the- MTT assay, before a concentration is rejected from analysis, is currently
testing-of-chemicals-section-4-health-effects_20745788. ≥30%. A concern with using this criterion to set a maximally tolerated
concentration is that a ≥30% effect on mitochondrial function could
in the guideline, which cautions that the presence of any detergent artificially produce an unacceptably large number of false positive re-
residue on glassware can impact activity. A chemical that has detergent sults. In addition, the MitoTracker assay discussed above could be a
like properties can denature proteins or impact protein-protein inter- replacement for the MTT assay to improve sensitivity and specificity.
actions (i.e., CYP19 interactions with its associated reductase), affecting Therefore, either a correction for cytotoxicity or lowering the criterion
assay results and would not be representative of potential effects in vivo. for allowable toxicity may be required to minimize false positives.
This cautionary statement is consistent with the findings of Judson et al.
(2016), which pointed out low Z-scores for surfactants in ToxCast. 4. In vivo endocrine screening assays: non-endocrine target organ
The steroidogenesis assay (OECD 456; OECD, 2011), measures the effects from chemicals
ability of a chemical to stimulate or inhibit the production of steroid
hormones with a unique steroid hormone-producing human cell line. In intact animals, the endocrine system integrates activity across
The EDSP initially intended to use a minced testes assay, but that multiple organ systems to maintain homeostasis and interpret whether
method was found to be unsuitable because it could not adequately external environmental conditions are suitable for reproduction.
distinguish between cytotoxicity and steroid synthesis inhibition However, not only does the endocrine system affect the functioning of
(Powlin et al., 1998). The EPA, in coordination with OECD, subse- multiple organ systems, multiple organ systems also can affect endo-
quently developed and validated the H295R assay. The endpoints crine system function. Consequently, when a substance is evaluated for

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M.S. Marty et al.
potential endocrine activity, careful characterization of target organ
toxicity should be included. These data can provide information on
potential non-endocrine-mediated effects on endocrine endpoints and
provide some regulatory context to interpret changes in endocrine-
sensitive tissues. If endocrine endpoints are affected at treatment levels
higher than those required to produce target organ toxicity, regulatory
exposure limits are likely to be protective against potential effects on
the endocrine system.
Many aspects of the endocrine system are conserved across species,
including, in some cases, the response to systemic (general) toxicity.
While the endocrine system is still being characterized, some interesting
parallels between mammalian and non-mammalian responses to sys-
temic toxicity have come to light in recent years. In both mammalian
and non-mammalian species, systemic (e.g., stress) or target organ
toxicity (e.g., toxicity in the liver, kidneys, nervous system) can alter
endocrine endpoints as described below. These examples highlight the
importance of a WoE approach to differentiate specific vs. non-specific
endocrine findings.
4.1. Liver effects
4.1.1. Mammals
While not the primary source of estrogen, androgen or thyroid
hormone production, the liver can affect serum levels of these hor-
mones. The liver plays an important role in synthesizing transport
proteins for hormone binding, as well as hormone metabolism and
excretion (DePeyster and Mihaich, 2014; Khan et al., 1981, 2002).
Thus, liver effects, which are a relatively common response in MTD/
MTC toxicity studies, can cause secondary endocrine effects in mam-
mals through: (a) Direct damage or degenerative changes to the liver
leading to reduced functional capacity; or (b) Enlargement of the liver
accompanied by induction of biotransformation enzymes leading to
increased hormone clearance, resulting in secondary effects on circu-
lating hormone levels. Regulations that protect against liver toxicity
may be sufficient to protect against indirect effects on the endocrine
system.
In human cases of marked liver dysfunction, altered steroid hor-
mone levels have been reported, possibly due to alterations in binding
proteins and/or enhanced hormone catabolism. Sex hormone altera-
tions have been demonstrated in males and females with chronic liver
disease. Men with liver disease are often hypogonadal with gyneco-
mastia, testicular atrophy, decreased testosterone and androstenedione
levels and increased dehydroepiandrostenedione sulphate levels
(Bannister et al., 1987; Gluud, 1988). Both men and women were re-
ported to have increased levels of estrogens and sex hormone binding
globulin (SHBG), which can affect hormone signaling. While total
hormone levels may appear increased for a period with increased
SHBG, free hormone levels may be decreased when liver dysfunction is
coupled with elevated SHBG. In women, liver disease can alter sex
hormone metabolism, resulting in decreased serum levels of steroid
sulphates and 5-alpha-dihydrotestosterone (Becker, 1993). Chronic
liver disease may lead to early menopause and has been associated with
lower levels of hepatic ER in females (Becker, 1993; Shimizu, 2003).
Burra (2013) has reviewed the relationship between liver abnormalities
and endocrine diseases, including altered thyroid hormone levels in
some patients with liver disease.
Liver dysfunction may result in decreased hepatic enzyme activity,
which can have secondary effects on the endocrine system. A good
example of this is inhibition of hepatic hydroxyphenolpyruvate dehy-
drogenase (HPPD), an enzyme involved in tyrosine catabolism.
Inhibition of HPPD results in accumulation of tyrosine (tyrosinaemia)
which has been shown to result in non-endocrine-mediated effects on
the thyroid in the rat. For example, in their recent review of mesotrione
(an HPPD-inhibiting herbicide), the European Food Safety Authority
(EFSA) stated that: “Increased incidence of thyroid follicular adenomas was
also considered a secondary effect of increased levels of tyrosinaemia in rats
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upon long term exposure” (EFSA, 2016).
Conversely, hepatic enzyme induction is often seen in toxicity stu-
dies with rodents. Some test substances may induce liver enzymes with
enhanced testosterone metabolism and clearance (e.g., Freyberger and
Schladt, 2009; Marty et al., 2015). In general, metabolism markedly
decreases or inactivates testosterone's androgenic activity with the ex-
ception of dihydrotestosterone (DHT). Prepubertal animals may be
particularly sensitive to this MoA, because, unlike the negative feed-
back loop in adult male rats, where higher testosterone levels have an
inhibitory effect on GnRH/LH release, in prepubescent male rats, in-
creasing testosterone levels are stimulatory, potentiating the pituitary's
response to GnRH, resulting in maximum LH release between 35 and 45
days of age (Stoker et al., 2000b). The increase in LH further increases
testosterone secretion, resulting in puberty onset and increased acces-
sory sex tissue (AST) size. With hepatic enzyme induction prior to
puberty, testosterone may be metabolized at a greater rate, delaying
maximum LH release and attainment of sufficient testosterone levels to
drive puberty onset and/or AST weight increases, which are marked
between 40 and 55 days of age; (Monosson et al., 1999; Ashby and
Lefevre, 2000). There is some evidence for this MoA in a recent series of
studies with pronamide (Marty et al., 2015; Rasoulpour et al., 2015).
Liver effects in rats also can lead to activation of the hypothalamic-
pituitary-thyroid (HPT) axis and secondary alterations in thyroid end-
points, including serum hormone levels, thyroid weight and histo-
pathology. Enzyme induction in the liver may enhance thyroid hor-
mone (thyroxine, T4) conjugation and clearance, particularly in rats
that, as a species, are sensitive to this MoA (e.g., McClain, 1995; Jahnke
et al., 2004). Liver enzyme induction is typically accompanied by in-
creases in liver weights, as well as changes in liver histopathology. This
decrease in serum T4 will activate the HPT axis and subsequently can
affect thyroid-related endpoints. However, humans are quantitatively
less sensitive to this MoA (Dellarco et al., 2006), because: 1) transient
decreases in T4 do not readily stimulate TSH increases in humans; and
2) humans have thyroid binding globulin (TBG) with a higher binding
affinity for thyroid hormone transport in the blood, making it less likely
to release T4 for hepatic metabolism and clearance than albumin, the
primary transporter in adult rats (Choksi et al., 2003).
In contrast to rats, humans are likely to be less sensitive to hormone
metabolism secondary to enzyme induction due to the presence of a
high-affinity steroid hormone transport protein, SHBG and TBG (Dunn
et al., 1981). In humans, most testosterone/estrogen and thyroid hor-
mones travel tightly bound to SHBG or TBG, respectively, with only a
small fraction (1–2%) unbound as the biologically active fraction. These
proteins regulate access of hormones into target cells (Hammond et al.,
2012; Siiteri et al., 1982). Conversely, peripubescent and adult rodents
rely heavily on albumin to transport hormones in blood, which has a
much lower dissociation constant (Kd) for steroids (10−6 to 10−4 M)
than SHBG (10−10 to 10−8 M). A similar difference is seen with thyroid
hormone, where the T4 association constant (Ka) is 1010 for TBG vs.
2 × 108 for transthyretin and 1.5 × 106 for human serum albumin
(Refetoff, 2015). Free serum hormones are more readily metabolized
and cleared from the body; thus, the concentration and affinity of
hormones for transport proteins has been directly related to metabolic
clearance rates (Siiteri et al., 1982).
These data raise the question as to whether liver toxicity with en-
zyme induction, increased liver weight and centrilobular hypertrophy is
considered systemic toxicity when endocrine changes occur secondary
to these effects (Maronpot et al., 2010). EPA Guidance (US EPA, 2003)
provides that a WoE for hepatic centrilobular hypertrophy may be
“considered adverse if associated with biologically relevant increases in
clinical chemistry parameters, particularly those indicative of effects on
hepatocyte function, hepatocellular membrane leakage, …” For ex-
ample, a quantitative associated clinical chemistry indicator for hepa-
tocellular membrane injury in the liver of rats and mice is at least a 2-
fold increase in alanine aminotransferase (ALT) enzyme levels as
compared to controls. Thus, liver hypertrophy and a subsequent
M.S. Marty et al.
increase in hormone clearance could occur without meeting a definition
of systemic toxicity. This “non-adverse” designation is consistent with a
variety of natural dietary constituents that induce phase I and phase II
enzymes (e.g., cruciferous vegetables, coffee/tea, herbs/spices like
garlic, etc.; Manson et al., 1997) and could increase hormone catabo-
lism without inducing systemic toxicity.
4.1.2. Fish
Similarly to mammals, fish undergo alterations in some endocrine-
sensitive endpoints in response to alterations in liver function. One
primary endocrine endpoint monitored in fish, vitellogenin (Vtg), is
sensitive to modulation by both endocrine effects and altered liver
function. Vtg is the primary egg yolk protein in fish. Vtg is under es-
trogenic control via free estradiol (E2) interaction with the ER in the
liver leading to increased hepatic Vtg synthesis (Wheeler et al., 2005;
Hutchinson et al., 2006). Decreased circulating Vtg in female fish
plasma may indicate lower E2 production due to steroidogenesis in-
hibition (Ankley and Gray, 2013) or a direct anti-estrogenic interaction
with ER (Smeets et al., 1999; Panter et al., 2002). However, other
factors that may influence the expression of Vtg also should be con-
sidered as they could lead to a false positive conclusion for endocrine
activity from fish endocrine assays (e.g., Fish Short-Term Reproduction
Assay [FSTRA]; OCSPP 890.1350) (Wheeler and Coady, 2016).
A number of specific, but non-endocrine mechanisms can lead to
reduced Vtg in female fish (Wheeler and Coady, 2016). Liver effects are
a relatively common response (particularly for pesticidal active sub-
stances). Similar to mammals, both mechanisms (liver dysfunction and
enzyme induction) could potentially occur in fish, leading to a reduc-
tion in circulating female Vtg. Direct toxicity to the liver could reduce
its capacity to produce Vtg or non-endocrine-mediated liver effects
could reduce circulating levels of E2 (via induction of biotransforma-
tion enzymes) available to bind the ER and so transcribe Vtg mRNA. For
example, the triazole fungicide, tebuconazole, has been shown to cause
reduced yolk accumulation in oocytes (histopathologically) of fathead
minnow exposed over sexual development (EU DAR, 2012). However,
this was only concomitant with liver cell degenerative changes in both
sexes, indicating it did not have a primary endocrine mechanism but
was secondary to liver toxicity. Additionally, in a tebuconazole FSTRA,
blood plasma Vtg was reduced at the high treatment level, and again
histopathological changes in the liver (necrosis) were concurrent at the
reduced Vtg and next lower concentration (US EPA, 2015a), further
supporting that endocrine-related changes may be secondary to liver
toxicity.
4.2. Kidney effects
4.2.1. Mammals
The kidneys play a critical role in fluid and electrolyte balance,
detoxification and excretion in mammals, and alterations in kidney
function can have secondary effects that can be misinterpreted as direct
effects on steroidogenesis. For example, kidney alterations in electro-
lytes or blood volume could result in secondary effects on the adrenal
gland through the renin-angiotensin system, which can result in in-
creased adrenal weight and adrenal histopathology (Haschek et al.,
2010). A change in adrenal weight is a potential indicator of ster-
oidogenesis inhibition; however, it is important to distinguish this
kidney-adrenal response from primary effects on steroidogenesis or
effects on the hypothalamic-pituitary-adrenal axis, which could be
mediated by stress. In cases of electrolyte or blood volume changes, the
pituitary may not exhibit histopathological changes, because the hy-
pothalamic-pituitary-adrenal axis is not involved in these adrenal glo-
merulosa-kidney interactions. Under other circumstances, renin can
affect the gonadal hormone pathways (see below). Another example of
how kidney toxicity can result in secondary effects on the endocrine
system is renal osteodystrophy (ROD) resulting in secondary hy-
perparathyroidism. In ROD, imbalances in calcium, phosphorous and
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vitamin D levels as a result of impaired kidney function trigger a
compensatory increase in parathyroid hormone synthesis and para-
thyroid hyperplasia (Lewin et al., 2005).
The role of the kidney in health maintenance and its ability to im-
pact the endocrine system is acknowledged by the US EPA by including
renal function as part of the MTD criteria for the male and female
pubertal assays (OCSPP 890.1450 and 890.1500). These test guidelines
state, “… abnormal blood chemistry values at termination (particularly
creatinine and blood urea nitrogen (BUN)) may indicate that MTD was
exceeded, even in the absence of a reduction in terminal body weight
compared to controls”. However, creatinine and BUN are insensitive
biomarkers of kidney damage; and some kidney injury can progress
undetected until damage is advanced. One option when testing com-
pounds that target the kidney is to better characterize kidney toxicity
during the range-finding study by using newer urinary biomarkers (e.g.,
α-glutathione-S-transferase (α-GST), clusterin, and/or renal papillary
antigen-1 (RPA-1)) in conjunction with BUN and creatinine (Harpur
et al., 2011; Fuchs and Hewitt, 2011). These new biomarkers, which
have been accepted by the US Food and Drug Administration and the
European Medicines Agency, are more sensitive and specific for the
detection of early stage renal damage and may localize the site of da-
mage. These data may be useful to document functional kidney
changes, coupled to renal pathology, in selecting an MTD based on the
kidney effects for assays with endocrine-sensitive endpoints (e.g.,
pubertal assays).
4.2.2. Fish
The kidneys in fish also play important roles in ion and water bal-
ance as well as the excretion of endogenous and exogenous solutes.
While kidney structure and function varies among different fish species,
most fish kidneys are composed of an encapsulated capillary network
called the glomerulus and a tubule lined with epithelial cells.
Freshwater fish, since they are hyperosmotic relative to their environ-
ment, have a higher glomular filtration rate as compared to saltwater
fish, which are hypoosmotic. In fish, up to 80% of the cardiac output
drains (via the caudal vein) to the kidneys, compared to ∼20% of
cardiac output flowing to the kidneys in mammals. Thus, along with the
gills, the kidneys are important for the elimination of exogenous com-
pounds, and fish kidneys receive a large amount of blood flow, making
them targets of chemical exposure (Kleinow et al., 2008).
Fish kidney histopathology has been evaluated in toxicity studies,
particularly in response to metal exposure (Lemly, 2002; Mishra and
Mohanty, 2008). Renal toxicity has reportedly been the result of in-
terference with ion exchange activity at lower concentrations, and as
the result of lethal cell damage at higher concentrations (Bonga and
Lock, 2008). Histopathology of the kidney of the medaka fish at 15
weeks post fertilization is included in the OECD and USEPA test
guidelines for the Medaka Extended One Generation Reproduction Test
(MEOGRT; US EPA, 2015b; OECD, 2015). Renal impairment can be the
result of exposure to estrogenic chemicals resulting in increased pro-
duction of Vtg which can damage the kidney via protein overload. This
is expected to be more pronounced in male fish with estrogenically
induced high levels of Vtg protein which have no outlet via deposition
in developing oocytes. However, histopathological findings in the
kidney of exposed fish may also reflect the influence of systemic toxi-
city. Limited guidance on differentiation of kidney histopathology that
results from systemic toxicity versus exposure to estrogenic substances
is provided in Appendix 12 of the US EPA MEOGRT test guideline
(OCSPP 890.2200; US EPA, 2015b). As experience progresses with the
evaluation of kidney toxicity in fish, it will be important to summarize
histopathological findings and their connection to either endocrine
MoA or in response to more general, systemic toxicity that can interfere
with ion transport or result in kidney cell damage and/or death.
M.S. Marty et al.
Table 3
High dose levels for EDSP tier 1 assays compared with points of departure for neurotoxicity or biomarkers.
Chemical Uterotrophic High Dose Hershberger High Dose Male Pubertal High
Level (mg/kg/day) Level (mg/kg/day) Dose Level (mg/kg/
day)
Permethrin NA 150 120
Bifenthrin 15 10 10
Cyfluthrin 20 40 40
Chlorpyrifos 4 12 2 2
Imidacloprid 100 80 100
Abbreviations: POD - Point of Departure; NOAEL - No Observed Adverse Effect Level.
a
Benchmark Dose (BMD) is lower based on red blood cell cholinesterase inhibition; BMD10 ≥ 0.15 mg/kg/day.
4.3. Nervous system effects
4.3.1. Mammals
Toxicants may target the nervous system, resulting in primary
neurotoxicity, which at higher dose levels can progress to secondary
alterations in endocrine signaling pathways. One example of target
organ toxicity in the nervous system, which has been associated with
potential endocrine activity, is acetylcholinesterase (AChE) inhibition
by organophosphates and carbamates. Chlorpyrifos, an organopho-
sphate, can be used as one case study. In one published study with
chlorpyrifos, Joshi et al. (2007) reported that male Wistar rats exposed
to 7.5, 12.5 or 17.5 mg/kg bw/day chlorpyrifos by oral gavage had
decreased testis weights, reduced spermatid/sperm counts, decreased
serum testosterone levels, degenerative changes in the seminiferous
tubules of the testis, and decreased fertility. Rats in all dose groups
exhibited significant clinical signs, which were likely due to substantial
inhibition of brain AChE. Thus, these doses were confounded by overt
toxicity as defined by the EDSP test guidelines (e.g., US EPA, 2009a, b).
A second study by De Angelis et al. (2009) reported decreased serum T4
and altered thyroid histopathology (exfoliated necrotic follicular cells)
at postnatal day (PND) 150 in CD1 mice exposed to ≥ 3 mg/kg bw/kg
chlorpyrifos (0, 3 or 6 mg/kg bw/day on gestation day 15–18 by oral
gavage and 0, 1 and 3 mg/kg/day on PND 11–14 by subcutaneous ex-
posure). Neither the Joshi et al. (2007) nor De Angelis et al. (2009)
studies included concurrent measurements of AChE activity in various
tissues, although both studies were clearly within dose ranges where
significant brain AChE inhibition would be expected (Marty et al.,
2012).
Chlorpyrifos was included on the US EPA's Priority List 1 for en-
docrine screening using the eleven assays in the EDSP Tier 1 battery.
Dose ranges used for in vitro assays were based on test guideline re-
quirements, whereas in vivo assays were conducted at dose levels
causing significant AChE inhibition. The US EPA (US EPA, 2017b)
concluded that cholinesterase inhibition was relevant for chlorpyrifos.
The conclusion of the subsequent WoE analysis, which included Tier 1
assay results, regulatory guideline studies and published literature, was
that chlorpyrifos had no interactions with the estrogen, androgen or
thyroid pathways at doses below the dose levels that cause significant
brain AChE inhibition (Juberg et al., 2013; US EPA, 2015a). Regulatory
exposure limits are sufficient to protect against potential endocrine
activity as exposure limits are based on red blood cell cholinesterase
inhibition, which is more sensitive (affected more quickly and at lower
concentrations than brain cholinesterase inhibition).
With sufficient brain AChE inhibition, alteration of other endocrine
pathways may occur, including effects on anterior pituitary hormone
secretion and the hypothalamic-pituitary-adrenal (HPA) axis, which
can be stimulated by central cholinergic signaling (Rhodes et al., 2001).
Brain cholinesterase inhibition also has been associated with decreased
serum levels of TSH, prolactin (PRL), luteinizing hormone (LH), growth
hormone (GH) and thyroid hormone in rats (Smallridge et al., 1991;
Rocksén et al., 2008). Therefore, when assessing potential endocrine
effects from AChE inhibitors, it is useful to document: 1) the endocrine
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Female Pubertal High POD NOAEL (mg/kg/day)- POD NOAEL (mg/kg/day)-
Dose Level (mg/kg/ Chronic (EPA Risk Subchronic (EPA Risk
day) Assessment) Assessment)
150 25 11
10 3.1 3.1
40 2.4
≥0.78a
100 8 8
endpoints that may be altered; 2) the magnitude of brain AChE in-
hibition that is occurring; and 3) whether regulating these compounds
for protection against inhibition of brain AChE (or the more sensitive
endpoint, RBC cholinesterase) will be protective for endocrine-related
effects.
The pyrethroid insecticides are another class of chemicals that act
on the nervous system, but also have been reported to alter endocrine
function in some studies. The pyrethroids are synthetic derivatives of
the natural pyrethrins, which are designed to be more photostable than
the pyrethrins, and therefore suited for crop protection and other
commercial uses. Pyrethroids act on voltage-sensitive sodium channels
in the insect and in mammals to produce transient manifestations of
acute neurotoxicity (Soderlund et al., 2002). The effects observed fol-
lowing oral administration of pyrethroids, the preferred route in the
EDSP studies, are well defined following administration in rodents, and
the two subclasses are based upon chemical structure and the produc-
tion of either tremors (T-syndrome) with Type I pyrethroids or chor-
eoathetosis with salivation (CS-syndrome) with Type II pyrethroids.
One of the criteria used for dose selection for the mammalian EDSP tests
is evidence of acute neurotoxicity. With cyfluthrin, dose levels for the
Uterotrophic, Hershberger and Male and Female Pubertal Assays were
largely based upon transient neurotoxic signs.
The dose levels chosen for the Uterotrophic Assay (5, 10 and 20 mg/
kg/day; OCSPP 890.1600) and for the Male and Female Pubertal Assays
and the Hershberger Assays (10 and 20 mg/kg/day; OCSPP 890.1400)
with cyfluthrin are approximately 10–20 times higher than the point of
departure dose for chronic exposures (2.4 mg/kg/day, Table 3), which
is based on acute neurotoxic signs in the one-year dog study. Thus,
these data indicate that the nervous system is more sensitive to per-
turbation by pyrethroids than the endocrine system; and protection
against neurotoxicity would therefore protect against any endocrine-
related effects due to systemic toxicity, as the EPA concluded in the List
1 EDSP WoE assessments. Therefore, it may be reasonable to evaluate
other scientifically relevant information to determine if the existing
data and points of departure are protective for potential endocrine ef-
fects prior to initiating any additional screening studies on new com-
pounds.
4.3.2. Fish and frogs
Similar effects also can be seen in other species when neurotoxicity
is the lead toxic effect. AChE inhibition can cause secondary effects on
endocrine function in wildlife species. In fish, fecundity decreases in
response to brain AChE inhibition have been reported (Beauvais et al.,
2001; Beyers and Sikoski, 1994; Zinkl et al., 1987a; b). For example,
when chlorpyrifos was tested in the FSTRA, concentrations resulting in
0.251–3.02 μg/L did not affect wet weight, length, gonado-somatic
index, tubercle score, plasma concentrations of Vtg, 17β-estradiol,
testosterone, or testicular or ovarian histopathology; however, brain
AChE was affected at all concentrations in female fish (male brain AChE
affected at the mid- and high concentrations) and fecundity was re-
duced (Juberg et al., 2013). Thus, it was concluded that brain AChE
inhibition was a more sensitive marker of chlorpyrifos exposure in the
M.S. Marty et al.
FSTRA than endocrine-sensitive endpoints.
In the AMA, decreased tadpole length and weight, as well as delayed
development and decreased normalized hind limb length, were ob-
served at 13.6 μg/L chlorpyrifos (Juberg et al., 2013). These effects
were seen in the presence of cholinesterase inhibition in tadpole tissues,
which occurred at concentrations ≥3.68 μg/L chlorpyrifos. Thyroid
histopathology was unaffected with chlorpyrifos exposure. Once again,
cholinesterase inhibition was a more sensitive target than endocrine
effects. To characterize the relative sensitivity of these endpoints,
cholinesterase activity could be included as an endpoint in these assays
when evaluating compounds that operate via this MoA.
4.4. Altered muscle contractility
4.4.1. Mammals
Some endpoints that are commonly associated with endocrine
function can be mediated by other factors as well. For example, par-
turition is a time of altered endocrine signaling, resulting in cervical
ripening and uterine contractility for successful delivery of offspring.
Conversely, dystocia, a state of difficult/protracted delivery, may be
based on alterations in the initiation or progression of parturition.
While dystocia may be related to altered endocrine signaling, labor is a
complex process with many physiological pathways involved. For ex-
ample, calcium channel blockers (e.g., nifedipine, verapamil, diltiazem)
have been used to stop delivery of fetuses in a rat model of preterm
labor (i.e., pregnant rats that were ovariectomized on gestation day 16)
(Sharma and Gupta, 1994). Similar to the therapeutic role of these
compounds (i.e., reducing impulse conduction in cardiac muscle or
relaxing the smooth muscle in blood vessels), calcium channel blockers
decrease uterine contractility. Thus, the origins of dystocia also may
include myogenic factors from agents like calcium channel blockers
that alter myometrial contractility by affecting calcium entry
(Blackburn, 2014).
5. In vivo assays: non-specific effects on endocrine organs
5.1. Adrenal
The adrenal gland is one of the most common target organs and
endocrine organs for chemical toxicity (Rosol et al., 2001). Observing
non-specific cytotoxic adrenal effects by a hydrophobic chemical at
high dose levels in the adrenal cortex is not uncommon or surprising
(Ribelin, 1984; Frith et al., 2000). The adrenal cortex is divided into
three layers, the zona glomerulosa that synthesizes mineralocorticoids,
the zona fasciculata that synthesizes glucocorticoids and the zona re-
ticularis that synthesizes glucocorticoids/androgens (Rosol et al.,
2001). The adrenal gland is vulnerable to toxicity because of its high
perfusion rates, unique anatomical features of the adrenal blood supply,
and its ability to accumulate hydrophobic chemicals in its lipid-rich
cells (Inomata and Sasano, 2015; Rosol et al., 2001). This accumulation
can disrupt normal cellular function of adrenal cortical cells leading to
degeneration, reduction in cell volume, and subsequently atrophy
(Ribelin, 1984; Rosol et al., 2001). The zona fasciculata and zona re-
ticularis are the most frequently affected adrenal cortical zones by
toxicants (Ribelin, 1984, 2010; Suttie and Sutcliffe, 2018). Effects to the
zona fasciculata/reticularis are characterized as degeneration and/or
reduction in cell volume and vacuolization (Rosol et al., 2001). In the
zona fasciculata/reticularis, degeneration leading to atrophy is gen-
erally initiated with cytotoxic effects to the mitochondria and the
smooth endoplasmic reticulum (SER) and is followed by vacuolization
of neutral lipids and cholesterol. As previously discussed, the initial and
rate limiting steps of steroidogenesis take place in the mitochondria and
subsequent steps in the steroidogenic pathway are performed by cyto-
chrome P450s anchored in the SER (Stocco, 2001; Rosol et al., 2001).
Disruption of the mitochondrial electrochemical gradient by cytotoxi-
city blocks the rate limiting steps in steroidogenesis and stress to the
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SER can alter the activity of cytochrome P450s that are embedded in it
and responsible for carrying out the final transformations of steroid
biosynthesis leading to vacuolization and potentially necrosis, cell lysis
or apoptosis (King and Stocco, 1996; Rosol et al., 2001). Therefore, a
visual assessment of mitochondrial and SER ultrastructure can be used
as a sensitive and specific indicator of mitochondrial and SER function
and can be used to differentiate between direct and indirect non-spe-
cific cytotoxic effects on steroidogenesis (Ribelin, 1984; Levine et al.,
2007).
5.2. Thyroid
5.2.1. Mammals
Serum thyroid hormones undergo endogenous fluctuations and
there are numerous factors that can alter thyroid hormone levels, in-
cluding decreased growth rate, stress and study-specific parameters. For
example, serum T4 levels can be decreased secondary to effects on body
weight gain in growing animals. In the male pubertal assay, doses
causing decreases of approximately 6% or 9% resulted in a 14% or 23%
decrease in serum T4 levels (Laws et al., 2007). Thus, a careful WoE
approach and/or additional studies may be necessary to properly in-
terpret thyroid endpoints in the presence of decreased growth rate.
Stress also can influence thyroid hormone status. This may be due to
housing (i.e., isolation), generalized stress (e.g., cage transport, hand-
ling, nutrition) or stress at study termination related to the method of
blood collection, euthanasia and/or anesthesia (Dӧhler et al., 1979).
Liver effects that result in altered transporter protein synthesis or in-
creased hormone catabolism also can affect thyroid endpoints, as dis-
cussed in the section on Liver.
Age also can influence thyroid hormone signaling. For example,
during the change from a light to dark period, serum TSH exhibited a
short duration, rapid increase in adult male rats, a longer duration and
slower increase in pubertal male rats and no change in prepubertal male
rats (Dӧhler et al., 1979; Wong and Dӧhler, 1978). T4 levels followed a
similar pattern. Pregnancy status also can affect thyroid functioning as
dams must make sufficient T4 to support fetal neurodevelopment for
nearly all of gestation (Perez-Castillo et al., 1985; Grijota-Martinez
et al., 2011). Lastly, the stage of the estrous cycle also can affect thyroid
signaling (Dӧhler et al., 1979), which may make these data more dif-
ficult to interpret in the female pubertal assay.
5.2.2. Frogs
As stated in the section on Stress, amphibian metamorphosis is not
solely dependent on thyroid hormone. Instead, there is a second sig-
naling pathway involving hypothalamic corticotropin releasing factor
(CRF) that can accelerate metamorphosis in response to changing en-
vironmental conditions, such as decreased water levels or high popu-
lation density (Denver, 1997a; b; Okada et al., 2007; Kühn et al., 1998;
Rose, 2005; Coady et al., 2010). Furthermore, developmental/growth
delays can hinder metamorphosis. Given the potential contribution of
multiple factors, it is important to integrate all available information
when determining whether effects on metamorphosis are specific to
altered thyroid function. Thyroid histopathology provides a more spe-
cific endpoint to assess thyroid involvement.
6. Stressors
Stress has been known to impact steroidogenesis in both mammals
and fish (e.g., DePeyster et al., 2014; Marić et al., 1996; Almeida et al.,
1998). Thus, stress responses should be considered when evaluating the
potential endocrine activity of test substances, particularly when toxi-
city tests are conducted at MTD/MTC levels. Sometimes stress re-
sponses, which may include increases in adrenal weights and histo-
pathology, may be difficult to discern from broad spectrum
steroidogenesis inhibitors (e.g., ketoconazole), which can alter these
same endpoints.
M.S. Marty et al.
6.1. Generalized stress response
6.1.1. Mammals
Excellent review papers on stress responses and their interpretation
in toxicology studies has been published (e.g., Everds et al., 2013;
Witorsch, 2016). In Everds et al. (2013), the term “stress” is used to
encompass physiological responses to perturbations of homeostasis.
Stress commonly affects functioning of the immune, reproductive and/
or endocrine systems. Common effects that can occur secondary to
stress involve multiple systems and include changes in serum hormone
levels (e.g., increased corticosterone and progesterone, LH suppression,
decreased testosterone and androstenedione), decreases in body
weight/gain, food consumption, activity, estrous cyclicity, organ
weights (thymus, spleen and reproductive organs, including weights of
the epididymides, seminal vesicles, prostate, ovaries and uterus), lym-
phocyte depletion in the thymus and spleen, leukocyte counts and al-
tered reproductive functions (Witorsch, 2016; Everds et al., 2013).
Adrenal weights are generally increased and may be accompanied by
hypertrophy/hyperplasia in the cortex. Histopathologically, decreased
thymic and splenic cellularity may be seen, as well as lipid depletion of
the zona fasciculata of the adrenal gland and atrophy or inactivity in
the epididymides, prostate, seminal vesicles, ovary, uterus and vagina.
Testicular effects may include disorganized seminiferous tubular epi-
thelium and enlargement of the interstitial spaces (Everds et al., 2013;
DePeyster and Mihaich, 2014; Pellegrini et al., 1998).
Everds et al. (2013) reported that stress responses should not be
interpreted as primary endocrine-mediated effects, but rather as non-
endocrine-mediated effects of toxicant exposure. Stress responses re-
flect activation of the sympathetic nervous system or the hypothalamic-
pituitary-adrenal axis in an effort to restore normal physiological con-
ditions. Rats also can reflexively induce physiological adaptations that
include decreased metabolic demand (e.g., decreased cardiac output,
blood pressure/placental perfusion, acid-base status, hemoglobin con-
centrations and oxygen saturation) and hypothermia. The presence of
stress responses in toxicological studies is not surprising and may ap-
pear as toxicant-induced adverse effects. For example, decreased feto-
placental oxygenation, secondary to a maternal stress response, was
considered to be a likely cause of decreased fetal and placental growth
and developmental delays in a cyfluthrin inhalation developmental
toxicity study (Pauluhn, 2018; OECD 414). In this study, cyfluthrin
exposures were above the chemosensory threshold and triggered a
stress response in pregnant dams. Thus, a WoE approach is needed to
differentiate stress-mediated effects from direct toxicity. The evaluation
of clinical pathology parameters, body temperature, adrenal function
and/or immunotoxicity may be needed to determine the contribution of
stress to the results of toxicity studies.
As noted in Everds et al. (2013), the response to stress can be
gender- and age-dependent, a point that is especially relevant for the
male and female pubertal assays. In adults, chronic stress has been
shown to decrease circulating gonadal hormones and may reduce fer-
tility (Cameron, 1997; Kalantaridou et al., 2004). However, in some
cases, repeated stress may result in a decreased stress response due to
negative feedback by glucocorticoids. Stress-related parameters can
return to baseline levels (habituation) even in the continued presence of
stressors. Female rats exhibit a greater peak corticosterone response to
stress than males, but females return to baseline more quickly (Romeo
et al., 2004a; b).
Age differences include increased responsiveness of the HPA axis to
stress during puberty and adolescence which has been demonstrated in
several studies (reviewed in Klein and Romeo, 2013). This includes a
greater release of adrenocorticotropic hormone (ACTH) from the
anterior pituitary in prepubescent rats (Klein and Romeo, 2013). Be-
tween 30 and 40 days of age, prepubertal male and female rats begin
showing a protracted hormone response to acute and heterotypic
stresses (Foilb et al., 2011; Lui et al., 2012), releasing more corticos-
terone in response to stress than adults. In addition, the rate for
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corticosterone to return to baseline is considerably slower in pre-
pubescent rats, requiring twice as long as adults. Pubertal rats transition
rapidly to adult responsiveness between 50 and 60 days of age (Klein
and Romeo, 2013). Thus, changes in stress-responsiveness overlap with
changes in the production and secretion of sex steroid from the hy-
pothalamic-pituitary-gonadal (HPG) axis. While testosterone generally
reduces stress responsiveness in adult males (i.e., male rats castrated
after puberty show slightly higher corticosterone responses to stress)
(Handa et al., 1994; McCormick et al., 2002; Viau and Meaney, 1996),
prepubertal male rats continued to show a protracted stress response
even when given adult testosterone levels (Romeo et al., 2004a). In
prepubescent male rats, stress can decrease levels of testosterone even
at the stage when only low baseline levels of testosterone are present
(Foilb et al., 2011). In adult females, estradiol levels increase stress
responsiveness; however, regardless of estrogen status, prepubertal fe-
male rats showed enhanced ACTH/corticosterone responses to stress
(Klein and Romeo, 2013). Thus, while testosterone and estrogen can
influence adult programmed stress response, these hormones do not
mediate enhanced prepubertal stress responses, which appear to be age
dependent.
These examples illustrate that stress responses are complex. The
endpoints that are altered and the magnitude of the response will de-
pend on the type, duration and magnitude of the stress, as well as
species, strain, gender, age, physiological state and individual animal
variability. Thus, discerning specific endocrine-mediated effects from
secondary, stress-induced changes in endocrine-sensitive endpoints can
be challenging.
6.1.2. Fish
Stress responses are important to understanding the specificity of
any observed reduced Vtg response. At the most basic level, stress can
lead to a trade-off between energy allocation for survival (e.g. detxifi-
cation processes) and non-essential activities (i.e., reproduction and
somatic growth) (i.e., the energy allocation principle) (Schreck et al.,
2001). Indeed, non-specific decreases in Vtg have been observed in the
validation studies for presumed non-endocrine active materials for the
FSTRA (US EPA, 2007). The test concentration setting guidance for the
FSTRA (US EPA, 2009c) requires that the high treatment should be
approximately one third of the fish acute 96-h LC50; therefore, it is quite
likely systemic stress will be encountered (Wheeler et al., 2013). Thus,
decreases in Vtg from systemic stress may be common, at least, in the
high treatment levels of regulatory studies.
Another example is the impact of stress on the hypothalamus-pi-
tuitary-inter-renal axis (HPI) of fish (essentially equivalent to HPA in
mammals). Stress can lead to increased synthesis of cortisol, and cor-
tisol can act as an inhibitory hormone of both male and female fish
reproductive physiology over the whole reproductive cycle (Milla et al.,
2010). Cortisol can lower sex steroid levels, leading to decreased go-
nado-somatic index (GSI), decreased Vtg levels and decreased sec-
ondary sex characteristics (tubercle score) (Foo and Lam, 1993;
Carragher and Sumpter, 1990; Aluru and Vijayan, 2009; Milla et al.,
2010; Pankhurst and Van der Kraak, 2000; Haddy and Pankhurst, 1999;
Wu et al., 2003; Lethimonier et al., 2000). These results have been
confirmed using in vivo studies of fish exposed to confinement stress
(Pankhurst and Van der Kraak, 2000) or implanted with cortisol (Foo
and Lam, 1993) leading to reduced sex hormone levels.
In addition, stress mediated through the glucocorticoid pathway can
delay fish gonadal development and lower gamete quality (Aluru and
Vijayan, 2009). The effects of cortisol on sex steroids (Schreck et al.,
2001) can potentially impact gonadal histopathological evaluations.
For instance oocyte size, atresia and reabsorption have been shown to
respond to actual or simulated stress (cortisol treatment) (Schreck et al.,
2001; Clearwater and Pankhurst, 1997).
Therefore, it is clear that stress can influence the very measures or
endpoints included in fish endocrine screening assays to detect endo-
crine activity. It is, therefore, very important to distinguish responses in
M.S. Marty et al.
these parameters from general stress and specific endocrine interac-
tions. Further regulatory guidance is required to develop this assess-
ment and interpretation of effects. For example, there is limited gui-
dance for the reporting of clinical signs in ecotoxicology assays and it is
likely there is great variation amongst performing laboratories. This
will undoubtedly limit evaluators' ability to contextualize endocrine
and stress responses in cases where anything less than overt toxicity is
observed.
6.1.3. Frogs
Generalized stress, for example the presence of predators (Laurila
et al., 1998), can delay growth in tadpoles, affecting weight and length
measurements in the amphibian metamorphosis assay (Wilbur and
Collins, 1973; OECD, 2007). Delays in growth also can result in delays
in the progression of metamorphosis (Wilbur and Collins, 1973; OECD,
2007). Chemical stressors can result in delayed development including
reductions in wet weight, snout-vent length and hind limb length in the
absence of specific thyroid activity. In an evaluation of amphibian
metamorphosis test results by the US EPA, myclobutanil resulted in
significant decreases in all of these morphological parameters at test
levels in which amphibians exhibited transient abnormal behaviors
(floating on the surface, lying on the bottom of the tank, inverted or
irregular swimming, lack of surfacing activity, and being nonresponsive
to stimulus) but no increase in mortality. There was also no notable
thyroid pathology. In the same review, isophorone also caused de-
creases in wet weight and snout-vent length in the absence of delayed
developmental stage or treatment-related thyroid histopathological
findings (US EPA, 2009b). No signs of generalized toxicity were noted,
so it is important to consider that either the generalized toxicity was of
a subclinical level or these endpoints are susceptible to spurious out-
comes, both scenarios potentially confounding interpretation. In the
cases of the chemical stressors, it becomes critical to correlate mor-
phological findings with thyroid histopathogy (stage-matched samples)
in order to maximize an accurate interpretation of potential thyroid
effects.
While stress generally delays responses in mammals, stress also can
advance developmental stage in amphibians, since increased stress can
cause premature metamorphosis via increased CRF from the hypotha-
lamus (Denver, 1997a; b; Okada et al., 2007; Kühn et al., 1998; Rose,
2005; Coady et al., 2010). An acceleration of metamorphosis was ob-
served as a result of water-level manipulation in Rana temporaria
(Loman, 1999; Laurila and Kujasalo, 1999) independent of tempera-
ture. The proposed mechanism involves increased CRF from the hy-
pothalamus that reduces appetite and foraging behavior and stimulates
the hormonal system responsible for metamorphosis (Denver, 1997a; b;
Okada et al., 2007; Kühn et al., 1998; Rose, 2005; Coady et al., 2010).
This phenomenon was also observed for other species (Hyla pseudo-
puma, Crump, 1989; Scaphiopus couchii, Newman, 1988, 1989; Sca-
phipus multiplicatus, Pfennig, 1990; Bufo calamita, Tejedo and Reques,
1994; Scaphiopus hammondi, Denver and Denver, 1995). Development
rate can also be increased by competition density (Wilbur, 1976, 1977;
Dash and Hota, 1980; Morin, 1986), probably due to reduced food re-
sources. Of course, what this represents is a very plastic approach to
metamorphosis that may be affected by different stressors. This makes
it critical that changes in the endpoint are interpreted using all avail-
able information and in the context of the exposure conditions and
general toxic response that may result from the chemical exposure.
6.2. Nutrition/energy status and altered rate of growth
6.2.1. Mammals
Numerous factors can influence the onset of puberty in mammalian
species, including humans. Nutritional status in childhood (postnatal
period) can have significant effects on pubertal development, such as
timing of the onset of puberty. Both over-nutrition and under-nutrition
have been associated with altered puberty onset in humans (Soliman
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et al., 2014). There is evidence indicating that obesity in girls can ac-
celerate pubertal signs and alter progression, possibly due to altered
hormone levels; however, the evidence in boys is inconsistent
(Marcovecchio and Chiarelli, 2013; Soliman et al., 2014). Under-nu-
trition or a restricted nutrition has been shown to impair growth that
can lead to a delay and/or prolongation of puberty (Plant, 1994). In
humans, it has been demonstrated that food availability/consumption,
the balance between energy intake and expenditure, is a major en-
vironmental factor modulating the timing of sexual maturation
(Villamor and Jansen, 2016; Ellis, 2004; Soliman et al., 2014). The
tremendous metabolic demand placed by intense athletic training like
long distance running, gymnastics, and ballet are often associated with
delayed pubertal development or interrupted menstrual cycles
(Georgopoulos et al., 2010). These effects are normally reversible
within months after return to “normal”. In toto, experimental data
suggests that effects on food intake (thus on body weight gain) are
expected to have a major impact on sexual maturation.
Rats show similar effects on puberty onset as humans in response to
over- and under-nutrition. Cumulative dietary intake has been shown to
determine the onset of puberty in female rats following guideline pro-
tocols for pubertal assays whereby higher metabolizable energy intake
was associated with earlier puberty onset. In fact, energy intake of 2300
kJ/rat beginning at weaning was identified as the trigger for puberty
onset as measured by vaginal opening (Odum et al., 2004). In addition,
Odum et al. (2004) demonstrated that guideline uterotrophic assays
(i.e., uterine weight) using immature rats also were affected by cumu-
lative dietary intake, because uterine weights are more closely related
to body weight in weanling rats than adult ovariectomized rats (OECD,
2003).
In contrast, decreased growth rates have been associated with de-
layed puberty onset in both male and female rats (e.g., Engelbregt et al.,
2000; Ashby and Lefevre, 2000; Stump et al., 2014). Several peer-re-
viewed publications report studies that have been conducted utilizing
feed restriction in rats, resulting in varying degrees of decreased weight
gain, to determine the influence of body weight reduction on puberty
onset. In most toxicity studies, depressed body weight gain is a common
sign of systemic toxicity. Thus, effects observed after chemical treat-
ment and unrelated to the endocrine system can confound the inter-
pretation of results of endpoints evaluated in standard reproductive
toxicology studies.
The effects of decreased growth rate on puberty onset and other
endocrine-related endpoints has been examined for decades; however,
newer studies have examined the effects of decreased growth rates
using similar study designs and body weight decreases of relevant
magnitude for regulatory toxicity studies (e.g., OECD 416, OCSPP
890.1450, 890.1500; Carney et al., 2004; Chapin et al., 1993; Laws
et al., 2000, 2007; Marty et al., 2003; Stoker et al., 2000a). Feed-re-
striction studies using study designs similar to the Tier 1 male and fe-
male pubertal assays were summarized by Stump et al. (2014). For the
female pubertal assay, there was no apparent effect of feed restriction
on age at puberty onset with terminal body weight effects ranging from
2.1 to 18.9% less than ad libitum-fed controls on PND 42. The greatest
difference was a 1.6-day delay in age at vaginal opening with an 8.6%
change in terminal body weight; this level of feed restriction also af-
fected the number of 4–5 day estrous cycles and pituitary weight. Pi-
tuitary, adrenal and ovarian weights were affected at higher feed re-
striction levels. For the male pubertal assay, Ashby and Lefevre (2000)
demonstrated a relationship between initial body weight on PND 22
and age/body weight at preputial separation and also concluded that
there appears to be a continuum between body weight and age at
preputial separation in pubertal male rats. Whereas one study reported
no effects on preputial separation with feed restriction resulting in
terminal body weights ranging from 1.8 to 19.2% less than ad libitum-
fed controls (Laws et al., 2007), two other studies reported a 1.8-day
and 2.1-day delay in age at preputial separation with feed restriction
resulting in an 11.3% and 15.0% decrease in terminal body weight,
M.S. Marty et al.
respectively (Marty et al., 2003; Stoker et al., 2000b). Other endpoints
were more sensitive to growth rate changes than age at preputial se-
paration as pituitary weights were affected when terminal body weights
were decreased by ≥ 5.9% and seminal vesicle, ventral prostate and
epididymal weights affected with a ≥11.3% decrease in terminal body
weights (Stump et al., 2014). Testes weights were not altered. These
results are similar to the study by Rehm et al. (2008), where Sprague-
Dawley rats were feed restricted by 20% from six to eight weeks of age
(14.6% lower terminal body weight than ad libitum-fed control rats).
These animals exhibited decreased absolute epididymal and relative
seminal vesicle weights, a 72% decrease in plasma testosterone and
‘minimal’ grade pachytene spermatocyte degeneration in Stage VII of
the spermatogenic cycle.
The potential effect of growth rate/body weight on pubertal assay
endpoints has been recognized in the male and female pubertal assay
test guidelines (OCSPP 890.1450, 890.1500; US EPA, 2009a, b), where
a difference in terminal body weight of > 10% is considered to exceed
the MTD (although the male pubertal test guideline also states that a
6% decrease in body weight gain at termination can complicate assay
interpretation). Therefore, careful interpretation of age at puberty
onset, estrous cyclicity, and endocrine-sensitive tissue weights is
needed when body weight gain is decreased relative to control animals.
Maternal under-nutrition during pregnancy and/or lactation has
been associated with reduced primordial, secondary and antral follicle
numbers in rat offspring (Bernal et al., 2010); notably, the timing and
magnitude at which under-nutrition occurs impacts outcome. In addi-
tion, maternal toxicity/under-nutrition in lactating rats has been shown
to result in an increased period of lactational diestrous when nursing
their litters (Woodside, 1991). In these rats, ovarian pathology, follicle
numbers and corpora lutea of dosed animals experiencing toxicity may
appear different from those of control animals as they have not returned
to normal cycling.
Alterations in nutritional status also may affect steroidogenesis. For
example, hypercholesterolemia has been associated with decreased
testosterone levels in humans, rats and mice (Tanaka et al., 2001;
Martínez-Martos et al., 2011). Cholesterol serves as a precursor for
steroid hormone synthesis. Diet-induced hypercholesterolemia in male
mice resulted in decreased circulating testosterone levels, hypotheti-
cally through inhibition of testosterone synthesis by the renin-angio-
tensin system in the testis (Martínez-Martos et al., 2011; Khanum and
Dufau, 1988). The renin-angiotensin system also may play a role in
progesterone synthesis in the rat ovary (De la Chica et al., 2007).
Thus, in instances where systemic toxicity affects body weight/rate
of growth, a WoE analysis of endpoints within and across studies may
be needed to determine the specificity of effects on the endocrine
system.
6.2.2. Fish
Similar to mammals, effects on body weight/size can affect endo-
crine-sensitive endpoints in fish. For example, male fat pad weight, and
in both sexes gonad and liver weights were found to be proportional to
body weight (Watanabe et al., 2007). Thus, if there are systemic effects
on body weight (reduced feeding, etc.) then other tissue weights would
be impacted also, affecting study metrics such as Gonadal Somatic
Index (GSI). Tubercle score also has been correlated with fish size
(Watanabe et al., 2007). Hutchinson et al. (2009) reported that de-
creases in the expression of male secondary sex characteristics may not
be endocrine specific. Therefore, changes in GSI or gonad stage/histo-
pathology should not be regarded as solely or specifically indicative of
exposure to an endocrine disruptor in the FSTRA.
Energy status in the presence of systemic toxicity also impacts en-
docrine endpoints in environmental species. As stated in Wheeler et al.
(2013): “The response of these (endocrine) endpoints to systemic
toxicity is not surprising, since the amount of energy that an individual
can invest in the processes supporting maintenance, growth and re-
production is limited (Sibly and Calow, 1986). Reductions in energy
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acquisition and/or increasing energy demand, in response to a toxicant,
will result in a decreased ability to maintain non-essential processes
such as expression of vitellogenin”. Thus, effects on the well-being/
energy status of fish can confound endocrine endpoints too. As in-
dicated above, assessing such effects in fish can be problematic due to
an inability to accurately measure food consumption and assess growth
parameters in screening studies such as the FSTRA. This further un-
derlines the importance of using all available data where other tests not
specifically designed to assess endocrine effects may provide better
information on the general health status and growth effects of the or-
ganism under similar exposure levels (e.g. the fish early life stage test).
7. Other factors (non-test article variables)
When conducting toxicological studies in general, or the EDSP
screening assays in particular, some aspects of study design, animal
welfare/husbandry, etc., can affect study outcome. Some factors to
consider include:
7.1. Dose Administration
Oral gavage (bolus) dosing of a test material at MTD dose levels is a
significant source of stress in rodent toxicity studies. While daily dose
administration via gavage of animals is a common and accepted method
in toxicology studies, it still causes stress even if done properly (Brown
et al., 2000; Gonzales et al., 2014). Oral gavage can be difficult de-
pending upon the experience and skill of the technician, and there is a
potential for increased morbidity or stress due to repeated incorrect
dose administrations (Atcha et al., 2010; Gonzales et al., 2014). In-
correct intubations in which the gavage tube enters the trachea instead
of the esophagus or causes reflux due to rapid dose administration can
lead to significant stress, abnormal clinical observations, or death.
Gavage errors are not always distinguished from chemically caused
deaths in study reports. Proper scruffing of the animal is also important
to ensure it cannot move its head during gavage or alternatively be-
come distressed from being too tightly scruffed (Atcha et al., 2010).
Vehicle-treated animals are also gavaged to control for stress, but some
test compounds have a very pungent taste and/or odor and could irri-
tate the stomach or produce damage due to gavage-related reflux.
Therefore, one can imagine an experimental animal gavaged with some
test materials, regardless of the dose, might find that experience more
stressful than being gavaged only with vehicle (e.g., DePeyster and
Mihaich, 2014).
Damsch et al. (2011) reviewed the limited literature database on
gavage-related reflux in regards to risk factors, interpretation of histo-
logical changes, and toxicological relevance. Gavage-related reflux can
occur and cause aspiration of irritant material, resulting in respiratory
effects as well as irritation in the larynx, pharynx, sinuses, middle ear,
and nose (Damsch et al., 2011). Many factors can cause gavage-related
reflux including feeding status, high volume administered (> 10 ml/
kg/day), increased gastric acid production, delayed gastric emptying,
and decreased muscular tone or dysfunction of the esophagus. In ad-
dition, high viscosity formulations, or those with irritant properties can
cause technical issues as discussed above due to increased reactions of
the animals. Gonzales et al. (2014) evaluated plasma corticosterone
concentrations after oral administration of a drug formulation using a
peanut butter pellet compared to oral gavage in mice. As expected, the
plasma levels of corticosterone in animals which were treated with drug
by oral gavage were significantly increased above those given the drug
by pellet, or animals not treated by any method. Furthermore, the an-
imals treated for 2 days by oral gavage had corticosterone levels as high
as those animals which were restrained (and not administered drug).
7.2. Terminal estrous stage and female reproductive endpoints
The stage of terminal estrous influences hormone levels and
M.S. Marty et al.
reproductive organ weights at necropsy in cycling female animals (e.g.,
the uterus weighs more during proestrus than diestrus). In most assays,
there is no requirement to necropsy animals at the same stage of es-
trous; therefore, stage of terminal estrous is an uncontrolled variable.
Females, which were undergoing normal estrous cycles, could be in
proestrus, estrus, metestrus or diestrus at the time of necropsy. Thus, it
is important to evaluate terminal estrous stage across treatment groups
to provide context for organ weight and hormone data. In studies where
(anti)estrogenicity or steroidogenesis inhibition is suggested, a more
thorough evaluation of estrous cycle length and estrous cycle pattern
may be useful. As stated in the pubertal assay Standard Evaluation
Procedures document (US EPA, 2011), regularity of the estrous cycle
should be given more weight in the evaluation of potential endocrine
effects, whereas ovarian and uterine weights should be interpreted with
caution due to normal variability in cycling animals. Pathologists
should determine whether uterine and ovarian histopathology appears
normal for various stages of the estrous cycle. It is important to note
that aside from estrogenicity/antiestrogenicity and altered ster-
oidogenesis, stress also can alter the regularity of estrous cycles;
therefore, a WoE is needed to interpret differences in female estrous
cyclicity and reproductive organ weights relative to other endpoints
and other available data.
7.3. Testicular histopathology in juvenile dogs
Rehm (2000) and Goedken et al. (2008) evaluated spontaneous
testicular and epididymal histopathological findings in control beagle
dogs. These researchers reported that young dogs (e.g., < 8 months)
routinely have numerous testicular observations, including lower tes-
ticular weights, hypospermatogenesis, atrophy/hypoplasia in semi-
niferous tubules and incomplete filling of epididymal tails with sperm.
These findings, which can mimic those seen in studies with testicular
toxicants, were attributed to sexual immaturity. Thus, male beagles
should not be necropsied at less than ten months or older if sperma-
togenesis evaluations are needed. Some observations (e.g., atrophy/
hypoplasia of seminiferous tubules) were elevated in dogs under 12
months of age.
7.4. Parasites
7.4.1. Fish
Parasitic infections (microsporidia, mycobacteria, tapeworm) in the
FSTRA can affect gonadal histopathology and inhibit reproduction
(Coady et al., 2014). Histopathological changes in fish ovaries caused
by parasites commonly found in aquarium fish such as Pleistophora
infestation in fathead minnows, Pimephales promelas (Rafinesque), have
been reported (Ruehl-Fehlert et al., 2005). Microsporidia also can cause
the degeneration of ovarian tissue and the appearance of testicular
tissue in infected fish (Wiklund et al., 1996). In addition, the tapeworm,
Ligula intestinalis, has been shown to inhibit reproduction by interfering
with the pituitary–gonadal axis of its fish host (Arme, 1997; Williams
et al., 1998).
7.4.2. Frogs
Bent tails (i.e. scoliosis) have been commonly observed in laboratory
populations of African-clawed frog tadpoles (Xenopus laevis); an in-
cidence of ∼20% bent tail across both control and treated tadpoles was
not an uncommon observation in some tadpole cohorts during testing
efforts for the AMA (OCSPP 890.1100; Coady et al., 2014). Since, in this
case, bent tails among X. laevis tadpoles were reported to occur at the
same rate in control and test material-exposed populations, the cause of
the bent tails was not attributable to specific chemical exposures
(Coady et al., 2014). However, it should be noted that effects on tadpole
axial skeleton development has been noted in response to some che-
mical stressors (Dumpert and Zietz, 1984; Bacchetta et al., 2008). The
cause of bent tail amongst X. laevis control populations is not known;
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however, genetic factors as well as nutritional factors (i.e. deficiencies
in the amino acid, tryptophan; phospholipid deficiencies) have been
discussed as potential causes for bent tail in tadpole and/or fish larvae
(Droin, 1992; Cahu et al., 2003). Since length measurements are made
from snout-to-vent in the AMA, the presence of bent tails is not ex-
pected to hamper data collection in this particular assay, especially if
the incidences of bent tail are relatively minor and do not translate into
spinal deformities in subsequent metamorphs.
8. Discussion/conclusion
While not exhaustive, this paper provides some examples of how
specific target organ toxicity, systemic toxicity and/or altered home-
ostasis can affect endocrine-sensitive endpoints by non-endocrine
MoAs. This illustrates some of the challenges of distinguishing endo-
crine from non-endocrine MoAs, which can have critical impacts on
regulatory decisions. To support accurate determination of the endo-
crine-disrupting potential of substances under evaluation, it is im-
portant to consider several factors:
1) The endocrine system maintains various aspects of dynamic
homeostasis; therefore, environmental conditions, animal
well-being, and perturbations of other organ systems affect
endocrine function. These effects can be observed across species
and can be related to lifestyle factors (e.g., high intensity exercise,
nutritional status) or environment (e.g., accelerated development in
frogs due to habitat constriction or increased population density).
Alterations in endocrine system function may not necessarily in-
dicate an adverse effect, but may instead indicate an adaptive re-
sponse to different perturbations. Because the endocrine system has
a primary role in maintaining normal physiology to manage stress,
the apparent endocrine-disrupting effects that may be observed in
toxicity studies may be difficult to discern from effects triggered by
alterations in another target organ.
2) The lead toxic effect drives the hazard profile and can be pro-
tective of endocrine-related effects using established risk as-
sessment procedures. The lead toxic effect approach (Bars et al.,
2012) involves considering all available toxicity data for a substance
and identifying the adverse effect that occurs at the lowest dose.
This adverse effect is considered the lead toxic effect and describes
the most sensitive toxicological response and drives the hazard
profile of a substance. Any risk management measures based on the
lead toxic effect is likely to be protective of other toxic effects (in-
cluding endocrine effects) occurring at higher treatment levels.
Therefore, endocrine disruption is only of high regulatory concern
for a substance if it is the lead toxic effect.
3) Dose/concentration selection is important for lower and higher
tier screening/testing. Treatment level (dose or concentration)
selection takes on enormous importance in studies screening or
testing for potential endocrine activity/disruption. These assays are
not simply testing for an adverse effect but rather an adverse effect
through an endocrine mechanism. The hypotheses that are being
tested in screening assays are whether there is the potential for
endocrine modulation through a specific endocrine mechanism
(e.g., estrogen, androgen, thyroid or steroidogenic pathway). The
purpose of higher tier testing is to further examine a potential en-
docrine effect observed in screening assays and then put the findings
in the context of a human or ecological risk assessment (or definitive
hazard assessment for endocrine disrupting properties for EU hazard
based cut-off). Therefore, results of endocrine screens and tests
should include a careful assessment of systemic toxicity to not
confound the interpretation of potential impacts on endocrine me-
chanisms. As discussed, the use of the MTD and MTC approaches
means that this evaluation and separation of endocrine-mediated
from non-endocrine-mediated effects is extremely important in
regulatory application.
M.S. Marty et al.
In mammalian screening assays, the MTD approach has been
adopted to set the highest dose level. The MTD is not a biological
constant, therefore, the MTD can vary between range-finding assays
and definitive assays. For example, a response to a given dose level
in the range-finding assay may only cause a 5% bodyweight loss but
in the definitive study, the effect could result in a 10% loss.
Consequently, there is a likelihood that the MTD based on range-
finding assays can result in systemic toxicity and confound study
interpretation due to toxicity to non-endocrine targets and/or acti-
vation of the hypothalamic-pituitary-adrenal axis, which in turn
affects all endocrine systems (US EPA, 2013).
For aquatic testing, the approach is termed the MTC which is cal-
culated as a function of median lethal concentrations. The current
MTC guidance in the test guideline is to set the highest tested con-
centration for the 21-day AMA and 21-day FSTRA at 1/3rd of the
96-h LC50 value. This approach may be acceptable for a small group
of compounds that exhibit very steep concentration-effect relation-
ships. However, for classes of compounds that do not have steep
concentration-effect relationships, this approach will likely produce
overt toxicity in a 21-day continuous exposure. Therefore, flexibility
in the concentration setting approach is required and concentration
setting needs to be handled on a substance-by-substance basis. One
recommendation is to perform 14–21-day range-finding assays that
include sensitive indicators of sub-lethal toxicity and provide a
predictive assessment of systemic toxicity. Examples of sub-lethal
endpoints that could be included in these extended range-finding
assays include gill, liver, and kidney structure and/or function,
presence of edema, as well as other indicators of systemic toxicity.
A toxicokinetic (TK) approach could also be employed to set doses
for endocrine screening and testing. The goal of this approach is to
identify the highest dose level that does not exceed linear pharma-
cokinetics in plasma. The use of excessively high doses or con-
centrations can create TK processes that result in a systemic ex-
posure to either parent compound or metabolites that is not dose
proportional (i.e., no longer increases with increasing dose, or a
systemic exposure that hugely increases with a small increase in
dose). If there is a sufficient margin of exposure, exceedingly high
doses and associated non-linear TK will likely produce results that
are not relevant for hazard or risk assessments (Saghir et al., 2012).
Therefore, the driver for adoption of the TK approach for dose set-
ting in mammalian toxicology studies has been to improve testing
and assessment methodologies and not confound the interpretation
for human relevance (Boobis et al., 2008). Creton et al. (2012)
discuss practical examples that underscore the role TK can play in
improving study design and interpretation through selection of ap-
propriate high doses (e.g., use of OECD 421 to establish kinetically
derived maximum dose levels for an OECD 443; Saghir et al., 2013;
Marty et al., 2013). Application of TK approaches for dose setting in
ecotoxicology is rare. One of the barriers to the application of TK in
ecotoxicology would be development of improved sampling
methods and analytical capabilities, but this would be a highly
productive area of research for the aquatic and wildlife toxicology
community in general.
4) In an effort to distinguish endocrine-mediated from non-endo-
crine-mediated effects on the endocrine system, it is useful to
adopt a WoE approach when evaluating endocrine data. In this
paper, there are numerous examples that different types of systemic
toxicity and/or stress can affect endocrine-sensitive endpoints.
Ideally, eco/toxicological data across studies should be examined in
a systematic manner for evidence of potential endocrine activity or
altered endpoints indicative of other types of toxicity. For the EDSP
Tier 1 assays, the US EPA proposed a WoE approach (US EPA, 2013).
A more formalized, structured WoE approach also has been pro-
posed by Borgert et al. (2014). In this approach, it is recognized that
some EDSP Tier 1 endpoints can be specific and sensitive indicators
154
Regulatory Toxicology and Pharmacology 99 (2018) 142–158
of endocrine effects (e.g., Vtg in male fish as an indicator of estro-
genicity, tubercles in female fish as an indicator of androgenicity).
Borgert et al. (2014) designated these endpoints as Rank 1 and ap-
plied greater weight to these effects in the WoE process. Conversely,
less specific endpoints (e.g., estrous cyclicity in female pubertal rats,
fecundity in fish) were assigned Rank 3 and were used to provide
supporting data for a potential endocrine MoA; Rank 3 endpoints
alone were considered ‘insufficient’ evidence of endocrine activity.
Rank 2 endpoints are intermediate in their specificity and support
for an endocrine MoA. In this way, relevance weighting can be
considered in a WoE assessment.
Across the EDSP Tier 1 assays, there is sufficient redundancy (i.e.,
endpoints sensitive to estrogen, androgen or thyroid perturbations)
to allow for the detection of endocrine activity in multiple assays
(US EPA, 2013); however, this pattern of effects may be complicated
by route of exposure, treatment level and species differences. For
example, a compound that shows ER binding and transactivation
may be negative in a uterotrophic assay via the oral route, but may
be detected in the Fish Short Term Reproduction test. Furthermore,
compounds may be detected as bioactive in in vitro assays at doses
that are either not achieved systemically in animal models or that
result in marked systemic toxicity. In these cases, in vitro-to-in vivo
pharmacokinetic models may provide some context for these dose
levels. Lastly, differences in mammalian and non-mammalian re-
sponses may suggest a species difference in response or sensitivity to
the endocrine activity being tested (Ankley and Gray, 2013).
5) To distinguish direct from non-endocrine-mediated effects on
endocrine-sensitive endpoints, there may be additional end-
points or other data that can be useful to better characterize
stress and systemic toxicity. These suggested endpoints should be
considered in light of the particular substance being tested and its
known toxicity.In vitro
• Consider whether the class of chemistry is amenable to in vitro
screening or whether false positive results are possible (e.g., sur-
factants, denaturing agents, etc.).
• For cell systems, consider whether effects are specific or potentially
non-specific. Include assays to characterize cell cytotoxicity and
stress, particularly for “loss of signal assays” where a decrease in
signal indicates potential endocrine activity. Note that more than
one assay may be needed to adequately characterize cell status.In
vivo
• Clinical signs and frequent body weight measurements can provide
useful information on overall well-being of test animals. However in
fish and frogs, descriptors and evaluations are not well standardized
and there is variability across laboratories as to what is recorded and
how it is interpreted. A better understanding of body weight and
length changes may be useful in fish and frog endocrine screens, but
often these data are difficult to collect in a meaningful manner (see
Wheeler et al., 2014) and interpret in relation to screening assay
outcomes; further work to standardize assessments is encouraged.
Clinical signs in rodents and ecological species may be particularly
important for neuroactive compounds or systemic toxicity. Food
and/or water consumption also provide useful information.
• Characterize the toxicity of known target organs or biochemical
markers in eco/toxicology studies designed to examine potential
endocrine effects. As indicated above, significant target organ toxi-
city can affect endocrine endpoints. Target organ evaluation should
include a more routine evaluation of liver tissue from female fish
showing decreased Vtg production (Wolf and Wheeler, 2018).
• Include clinical chemistry parameters in mammalian studies to
evaluate physiological state, paying particular attention to target-
organ-related parameters. Body temperature also may provide
useful information as rodents can induce a hypothermic response
M.S. Marty et al.
(decreased core temperature and heart rate) to toxicant exposure
(e.g., Watkinson et al., 2003).
•
Evaluate hepatic enzyme induction, particularly in cases where de-
creased endocrine responses are seen in the presence of liver weight
increases and/or liver hypertrophy or hyperplasia.
• If kidney and adrenal changes are seen in rodents, examine clinical
pathology parameters related to electrolytes, BUN, creatinine or
other more sensitive biomarkers (see Kidney section above). If re-
levant clinical pathology parameters are affected, consider including
an evaluation of the pitutiary to determine whether there is evi-
dence of hypothalamic-pituitary activation. Kidney histopathology
may be a relevant endpoint to characterize systemic toxicity in fish
studies so further research should be encouraged.
• Consider whether corticosterone measurements in rodents or cor-
tisol measurements in fish would be useful, particularly in cases
where adrenal weights are increased and/or the adrenal gland ex-
hibits hypertrophy or hyperplasia.
• Consider whether the route of exposure also might contribute to
stress (e.g., gavage). Include an evaluation of lung and trachea as
part of these evaluations to rule out problems with dose adminis-
tration.
• Evaluate TK to determine if a kinetically derived maximum dose
(KMD) level may be used instead of MTD/MTC (e.g., Saghir et al.,
2012).
• Include histopathology of affected endocrine pathways (e.g.,
thyroid, ovary, testis, pituitary, etc.) as this may establish a pattern
of endocrine changes. When evaluating potential thyroid changes in
female rats, stage of estrous cycle also should be monitored.
• Include estrous stage when evaluating female rodent reproductive
organ weights.
• Fish should be screened for parasitic infections before use in endo-
crine tests. If observed in test animals, caution should be taken with
data interpretation.
Most importantly, a WOE approach across all relevant studies is needed
to place potentially endocrine-related effects in the context of other
toxicity produced by the chemical in question.
Acknowledgements
There was no specific grant associated with this work. This paper
was supported by each authors' employer and The Endocrine Policy
Forum.
Transparency document
Transparency document related to this article can be found online at
https://doi.org/10.1016/j.yrtph.2018.09.002.
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