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A Theoretical Model for Utilizing Mammalian

Pharmacology and Safety Data to Prioritize Potential
Impacts of Human Pharmaceuticals to Fish
a

D. B. Huggett , J. C. Cook , J. F. Ericson & R. T. Williams

a

Pfizer Global Research and Development, Groton, Connecticut, USA

Available online: 18 Jun 2010

To cite this article: D. B. Huggett, J. C. Cook, J. F. Ericson & R. T. Williams (2003): A Theoretical Model for Utilizing
Mammalian Pharmacology and Safety Data to Prioritize Potential Impacts of Human Pharmaceuticals to Fish, Human and
Ecological Risk Assessment: An International Journal, 9:7, 1789-1799
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Human and Ecological Risk Assessment, 9: 17891799, 2003

Copyright C ASP
ISSN: 1080-7039 print
DOI: 10.1080/10807030390260498

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A Theoretical Model for Utilizing Mammalian

Pharmacology and Safety Data to Prioritize Potential
Impacts of Human Pharmaceuticals to Fish
D. B. Huggett, J. C. Cook, J. F. Ericson, and R. T. Williams
Pfizer Global Research and Development, Groton, Connecticut, USA
ABSTRACT
Due to the potential for long-term, low-level exposure of environmental species to
pharmaceuticals in the environment, concerns over chronic ecotoxicity have been
raised. Pharmaceuticals typically have specific enzyme and receptor-based modes of
action, which are extensively studied in mammals during drug development. A survey
of the literature demonstrated that there is conservation of many enzyme/receptor
systems between mammalian and teleost systems. Based on this conservation of
enzyme/receptor systems across teleost species, a model has been developed to utilize
the information from mammalian pharmacology and toxicology studies to evaluate the potential for chronic receptor mediated responses in fish. In this model, a
measured human therapeutic plasma concentration (HT PC) is compared to a predicted steady state plasma concentration (FSS PC) in fish, and an effect ratio (ER =
HT PC/FSS PC) is computed. The lower the ER, the greater the potential for a pharmacological response in fish. Data collection and model validation will strengthen
the applicability of this approach as a viable tool for prioritizing research initiatives
that examine the potential impact of pharmaceuticals on fish.
Key Words:

environmental assessment, pharmaceuticals, ecotoxicity, drug safety.

INTRODUCTION
The presence and potential hazards of pharmaceuticals in the aquatic environment have received increased attention recently (Daughton and Ternes 1999;
Kummerer 2001). This attention is largely the result of a growing number of peerreviewed papers reporting trace levels of pharmaceuticals in municipal effluent, surface water, groundwater and to a lesser extent, drinking water. For instance, a national
reconnaissance by the United States Geological Survey (USGS) detected 82 pharmaceutical and personal care products in surface waters of the United States (Kolpin
et al. 2002). While pharmaceuticals are being detected in the aquatic environment,
Received 11 November 2002; revised manuscript accepted 11 April 2003.
Address correspondence to Duane B. Huggett, Pfizer Global Research and Development,
Eastern Point Road, Mailstop 8118D-4050, Groton, CT 06340, USA. E-mail: duane huggett@
groton.pfizer.com
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D. B. Huggett et al.

the levels are low and the science is generally not yet developed enough to determine
whether these low-levels pose a risk to environmental species.
Environmental hazard assessment frameworks based on an initial evaluation of
acute ecotoxicity endpoints have been developed to evaluate the potential impact
of pharmaceuticals on aquatic species (FDA 1998; EMA 2001). The science of these
evaluations centers on: 1) the development of a predicted environmental concentration (PEC) to estimate potential aquatic organism exposure, 2) determining acute
toxicity concentrations using algae, Daphnia sp. and fish, and 3) application of multipliers (safety or assessment factors) to account for uncertainties such as inter- or
intra-species variability or extrapolation from acute to chronic toxicity. Hazard assessment frameworks may trigger investigation of increasingly complex and difficult
to measure chronic toxicity endpoints. Depending upon the properties of the drug
under investigation, these tiered evaluation frameworks may result in a conclusion
that no further action is required or the development of a detailed risk characterization with recommendations for potential precautionary measures as needed. In
addition, properties of the compound such as Log P and bioaccumulation potential
may trigger chronic toxicity evaluation outside of the standard acute toxicity/safety
factor framework.
Acute toxicity tests assess toxicity following short exposures (96 hr) using the
primary endpoint of death. Hence, these tests generally do not assess the potential
of a test substance to produce effects following chronic, low level exposures. Acute
toxicity of pharmaceuticals at environmentally relevant concentrations has been
suggested to not be a significant issue because the acute effects concentrations historically determined are generally well above predicted environmental levels (Webb
2001). The biologically active nature of pharmaceuticals, however, has led some
to suggest that aquatic organisms may be impacted in ways that are not being detected using current test methodologies and data evaluation strategies (Daughton
and Ternes 1999).
The mammalian nonclinical safety profile developed to support registration of
a pharmaceutical and use in a patient population is based on data from a series
of in vitro and in vivo studies (Table 1). In vitro studies may be used to determine
key characteristics of a pharmaceutical such as metabolism and stability, bioavailability, mutagenicity and other selected endpoints (e.g., toxic effects to specific tissues).
In vivo safety studies are typically performed in rodent and non-rodent animal species
using standard, non-clinical toxicology study designs and the intended clinical dosing route. A battery of genotoxicity tests are also conducted. Safety pharmacology
studies are conducted to evaluate potential adverse effects at high dosages of the
drug on critical organs such as the heart, lungs, brain, and other organ systems based
on the pharmacology of the drug. The safety assessment studies typically increase
in duration as development of a drug continues. Testing includes acute through
sub-chronic (up to three month) studies in two species prior to Phase I clinical investigation, where initial human exposure in small populations of healthy subjects
occurs.
In conjunction with the progressive phases of clinical development, non-clinical
studies of increasing duration are performed. During Phase II clinical trials, fertility,
developmental toxicity, and pre/postnatal studies (commonly referred to as Segments or Studies I-III) are conducted to assess the safety of the drug in women of
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Table 1.

Summary of pharmacology and toxicology studies conducted to support

drug registration.

Test

Test System / Species

Pharmacology tests to establish activity

Genotoxicity
Safety pharmacology

In vitro and in vivo studies based on target system

Ames, In vitro/In vivo cytogenetics
CNS (rat) pulmonary (rat), renal (rat),
cardiovascular (rat, primate), gastric
emptying (rat)
Rat, dog/primate

Absorption, distribution, excretion

and metabolism (ADME)
Acute toxicity
Sub-chronic toxicity (1-3 months)
Chronic toxicity
Carcinogenicity
Fertility
Developmental toxicity
Pre/Postnatal toxicity
Juvenile animal

Rat, dog/primate
Rat, dog/primate
6-month rat, 9-12 month dog/primate
Mouse, rat
Rat
Rat, rabbit
Rat
Rat, dog

Source: USFDA 2002.

child bearing potential and chronic studies (6-month duration in rodent, one-year
duration in non-rodent) are conducted to assess safety during long-term use. During Phase III, carcinogenicity studies are conducted in mice and rats if the drug is
intended for chronic usage in patients.
In many ways, fish are not that biochemically different from mammals. Aquatic
vertebrates appear to have very similar enzyme and receptor systems as humans
(Table 2) (Evans 1993). The National Institutes of Health recognize the zebrafish
(D. reno) as a biomedical model to elucidate an understanding of vertebrate development and disease (NIH 2002). A large number of receptors in fish have been
identified through cloning and sequencing techniques in both the central and peripheral organs of fish (Table 2). Sequence homologies for receptors and enzymes
in fish range from 31 to 88 %. For example, -adrenergic receptors, peroxisome proliferation activated receptors (PPAR ), and serotonin receptors (5-HT) have been
identified in representative fish species with sequence homologies of 63, 47 and
72%, respectively (Nickerson et al. 2001; Yamaguchi and Brenner 1997; Andersen
et al. 2000). Since fish have many similar enzyme and receptor systems, this potentially makes them susceptible to similar biochemical and physiological mechanisms
of activation/inactivation. Because of the conservation of enzyme and receptor systems between mammals and these fish, chronic and target organ toxicity identified
in mammalian safety assessments is likely to be useful in predicting the need for
additional toxicity evaluation in teleosts.
While reports indicate that vertebrates may have receptors similar to those in humans (Evans 1993), the data set for invertebrates is much more limited. In addition,
receptor and enzyme systems in invertebrates may be structurally similar to those in
mammals, but be physiologically distinct with regard to functionality. For instance,
17-ethinyl estradiol regulates ovarian activity in mammals, but it does not appear
to elicit aquatic invertebrate reproductive responses through arthropod hormonal
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Summary of select receptor and enzyme expression in teleost species.

Table 2.

Species

Homology1

Reference

Receptor
PPAR
ER/ER
AR
5-HT1A /5-HT1D
2 -adrenoceptor
A1A -adrenoceptor
NMDA (NR1 subunit)
GlyR ( subunit)
GluR (R3 subunit)
IGF
IR
AH
OR1
NPY
BK2
IL1
GnRH
FSH
LH
TSH
GH
RAR
SST

S. salar
D. rerio
P. major
F. rubripes
O. mykiss
O. latipes
A.leptorhychus
D. rerio
Oreochromis
D. rerio
Salmon
D. rerio
D. rerio
Cod
D. rerio
S. salar
O. mykiss
I. punctatus
I. punctatus
M. saxatilis
C. auratus
F. rubripes
C. auratus

47
47/47
45
72/71
63
61
88
77
87
63
83
43
66
50
35
31
45
53
47
57
42
58
62

Andersen et al. 2000

Menuet et al. 2002
Touhata et al. 1999
Yamaguchi and Brenner 1997
Nickerson et al. 2001
Yasuoka et al. 1996
Dunn et al. 1999
David-Watine et al. 1999
Chang et al. (1998)
Maures et al. 2002
Chan et al. 1997
Andreasen et al. 2002
Rodriguez et al. 2000
Sharma et al. 1999
Duner et al. 2002
Subramaniam et al. 2002
Madigou et al. 2000
Kumar et al. 2001
Kumar et al. 2001b
Kumar et al. 2000
Lee et al. 2001
Wentworth et al. 1999
Lin et al. 2000

Enzyme
Lipoprotein Lipase
P450 1beta-hydroxylase
COX
P450 Aromatase
Creatine Kinase
Caspase-3
Stearoyl-CoA desaturase
ADH
iNOS
AChE

O. mykiss
O. mykiss
D. rerio
D. labrax
D. rerio
D. rerio
C. chanos
D. rerio
O. mykiss
D. rerio

56
33
67
50
86
60
63
81
62
62

Lindberg and Olivecrona 2002

Kusakabe et al. 2002
Grosser et al. 2002
Valle et al. 2002
Dickmeis et al. 2001.
Yabu et al. 2001
Hsieh al. 2001
Dasmahapatra et al. 2001
Wang et al. 2001
Bertrand et al. 2001

System

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Percent Homology to Mammalian Receptor/Enzyme.

PPAR = peroxisome proliferator activated receptor, ER = estrogen receptor, AR = androgen
receptor, 5-HT = serotonin receptor, B-AR = beta adrenoceptor, A-AR = alpha adrenoceptor, NMDA = N-methyl-D-aspartate receptors, GlyR = glycine receptor, GluR = glutamate
receptor, IGF = insulin like growth factor, IR = insulin receptor, AH = aryl hydrocarbon,
OR = opiate receptor, NPY = neuropeptide Y, BK = bradykinin, IL = interleukin, GnRH =
gonadotropin releasing hormone, FSH = follicle stimulating hormone, LSH = leutinizing
hormone, TSH = thyroid stimulating hormone, GH = growth hormone, RAR = retinoic
asid receptor, SST = somatostatin , COX = cyclooxygenase, ADH = alcohol dehydrogenase,
iNOS = inducible nitric oxide synthase, AChE = acetylcholinesterase.

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Environmental Safety of Pharmaceuticals

systems (Hutchinson 2002). As another example, HMG-COA reductase is an important enzyme in invertebrate juvenile hormone production, compared to being a
mediator in cholesterol formation in mammals (Debernard et al. 1994). The most
extensive comparative dataset with the best homology exists between mammals and
fish (Table 2). Therefore, use of this model to target areas worthy of additional study
initially should be limited to fish.
The purpose of this paper is to present a theoretical model which utilizes the extensive scientific and mechanistic understanding of pharmaceuticals in mammalian
systems to aid in prioritizing which pharmaceuticals may require additional chronic
ecotoxicity testing in teleosts. The premise for the model described in this paper is
that the drugs with the highest potential for additional chronic testing in fish are
those where the anticipated plasma concentration in fish approaches the plasma concentration in humans at which therapeutic effects are observed. It is assumed that
a pharmacological response would occur prior to a toxicological response, due to
the specificity of a compound for its respective target. Much like in human pharmacology, the dose determines the response, whether it be pharmacological or toxicological. Therefore, a receptor/enzyme mediated therapeutic response in non-target
organisms may be viewed as a precursor or surrogate response when compared to
traditional toxicological effects (survival, growth, or reproduction).
The European Agency for the Evaluation of Medicinal Products guidance on environmental risk assessment for medicinal products (EMA 2001) specifically requires
that the assessment utilize toxicology, mechanism of action, and other information
provided in the marketing application to determine if there is any potential for adverse ecotoxicological effects. This model is not intended to change the conditions
that trigger an environmental assessment for pharmaceuticals either in the United
States or Europe. However, with validation and further development, the model has
the potential to be a valuable component of the environmental impact assessment
process.

MODEL DESCRIPTION
The key assumption for the model is that many receptors and enzyme systems
are conserved across mammalian and non-mammalian species, thus making mechanism of action extrapolations possible (Table 2). The model requires comparison
of human and aquatic vertebrate plasma concentrations for a given pharmaceutical. Ideally, the model would be based on the highest human plasma steady state
concentration measured that corresponds to a No Observable Effect Concentration
(NOEC). Such a human plasma concentration at the NOEC (HSS PC NOEC) for
a given pharmaceutical could then be compared to a measured fish steady state
plasma concentration (FSS PC ) derived from an environmental exposure. If these
data were available, the ratio of these two plasma concentrations could be expressed
as a plasma based margin of safety:
Margin of SafetyPB = HSS PC NOEC/FSS PC
Ideally, anthropogenic compounds would have a margin of safety that is as large as
possible. Unfortunately, drug levels in humans and/or mammalian test species are
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Figure 1.

Conceptual derivation of a safety ratio based on mammalian pharmacological and environmental concentration data.

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traditionally reported at the recommended human dose rather than at the NOEC.
As would be expected, there is a very limited amount of published steady state fish
plasma data for pharmaceuticals linked to an actual aqueous exposure. These two
situations of limited data availability mean that this margin of safety approach for
comparing human and environmental species plasma levels is currently not feasible.
As an alternative to the margin of safety approach, an effect ratio (ER) can be
calculated (Figure 1). The human therapeutic plasma concentration (HT PC) at the
maximum dose of a drug is commonly available and can be utilized instead of a
HSS PC NOEC. For the fish plasma level, a predicted or measured environmental
concentration can be used in conjunction with its respective Log Kow to calculate a
FSS PC. These values can then be used to derive an ER:
ER = HT PC/FSS PC
As in the case with a margin of safety, the ER ratio would ideally be as large as possible
under environmental exposure conditions. An ER 1 indicates that the predicted
drug concentration in the fish plasma is equal to or greater than the drug concentration in human plasma that elicits a therapeutic effect. This plasma concentration
relationship would indicate that there is a potential for receptor mediated responses
in fish, especially if the target enzyme or receptor is expressed in the fish. Therefore,
the lower the ER, the greater the potential need for additional chronic investigation
in fish. Conversely, if the ER > 1 then the predicted fish plasma drug concentration
is less than the human therapeutic plasma drug concentration, indicating a lower
likelihood of receptor mediated responses in fish.
HT PC Derivation
Human therapeutic plasma concentrations (HT PC) are determined as a standard
part of the drug development process and are expressed as Cmax (maximum concentration) or AUC (area under the curve) values. HT PC describes the presence of
a drug in the systemic circulation system at either a single point in time (Cmax) or
as a function of time (AUC) (Hardman et al. 1996). For the ER model, the Cmax
and AUC values can be obtained from the Online Physicians Desk Reference (2002)
or DrugDex Drug Evaluations (2002).
FSS PC Derivation
Fish steady state plasma concentrations (FSS PC) can be predicted utilizing a
Log Kow value and a measured or predicted environmental concentration (EC).
In general, Log Kow values and measured ECs for drugs can be obtained from the
literature or calculated (ACD/LogD software, ACD Labs, Ontario Canada; Huang
and Sedlak 2001; Ternes 1998; Synder et al. 1999; Koplin et al. 2002).
For the purpose of this model, fish steady state blood concentrations were based
solely on calculations using hydrophobicity . Accumulation via the food chain was
not considered (i.e ., trophic transfer). Partitioning between the aqueous phase and
arterial blood in trout has been described with the equation (Fitzsimmons et al.
2001):
Log PBlood:Water = 0.73 Log Kow 0.88.
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A very similar relationship was described by Veith et al. (1979), indicating that fish
bioconcentration was related to the following relationship:
LogBCF = 0.85(logKow) 0.70.
Utilizing these relationships, drug partitioning between blood and water for a
given predicted/measured EC can then be equated to a fish steady state plasma
concentration:

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FSS PC = EC (PBlood:Water ).
The major assumption in FSS PC derivation is that the driving force behind a
compound crossing from the aquatic media into the blood stream of a fish is its
hydrophobicity (i.e ., Log Kow ). The reasonableness of this assumption for estimation
purposes has been demonstrated (Veith et al. 1979; Fitzsimmons et al. 2001). The
proposed model further assumes that no metabolism, excretion or protein binding
occurs in the fish (this is a worst case scenario for producing and maintaining a
maximum blood level). Once a compound crosses the gill into the arterial blood,
we assume that it stays in the blood plasma fully dissolved and bioavailable.
ER Derivation
The outcome of the model will be an effect ratio (ER). The ER will be defined as:
ER = HT PC/FSS PC.
An ER 1 indicates that the predicted drug concentration in the fish plasma is equal
to or greater than the drug concentration in human plasma that elicits a therapeutic
effect, while an ER > 1 occurs when the fish drug plasma concentration is lower
than the plasma drug concentration in humans that elicits a therapeutic effect. The
lower the ER, the greater the potential need for additional chronic fish testing to
determine if there are concerns.
Safety Factor Analysis
Safety factors should be used to refine the degree of uncertainty expressed in the
model. Using standard U.S. Environmental Protection Agency (USEPA) guidelines,
a safety factor of 1000 could be used as an initial approach (USEPA 1989). This
factor is derived by applying a 10-fold factor for extrapolation of humans to animals,
a 10-fold factor for sensitivity differences, and 10-fold factor for extrapolating from
mammalian to non-mammalian species. Based on these assumptions, compounds
with an ER < 1000 might warrant additional assessment in fish. The SF of 1000 should
be viewed as an initial assessment, since no fish uptake and receptor activation data
has been developed with human pharmaceutical compounds. As data are developed
in support of this model, the SF can be further refined.

CONCLUSIONS
This model approach has begun to leverage the extensive mammalian pharmacological and toxicological data that are available on pharmaceutical products. The
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crux of this approach lies in the understanding of pharmaceutical mechanisms of

action in humans and the homology between enzyme and receptor expression across
mammalian and teleost species. Further data collection, model validation and refinement will strengthen the applicability of this approach.

ACKNOWLEDGMENTS

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The authors thank Dr. Robert Chapin, Pfizer Inc, for review and constructive
comments made regarding this manuscript.

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