chandra

1. ATLA 28, 119–131, 2000 119
Biomarkers as Predictive Tools in Toxicity
Testing
The Report and Recommendations of ECVAM Workshop 40 1,2
Diane J. Benford,3 A. Bryan Hanley,4 Krys Bottrill,5 Sarah Oehlschlager,4
Michael Balls, 6 Francesco Branca,7 Jean Jaques Castegnaro,8 Jaques
Descotes, 9 Kari Hemminiki,10 David Lindsay11 and Benoit Schilter12
3School of Biological Science, University of
Surrey, Guildford, Surrey GU2 5XH, UK;
4Central Science Laboratory, Sand Hutton, York Y041 1LZ, UK; 5FRAME, Russell and
Burch House, 96–98 North Sherwood Street, Nottingham NG1 4EE, UK; 6ECVAM, Institute
for Health & Consumer Protection, Joint Research Centre, European Commission, 21020
Ispra (VA), Italy; 7Unitadi Nutrizione Umana, Instituto Nazionale della Nutrizione, Rome,
Italy; 8Unit of Gene Environment Interactions, IARC, 150 Cours Albert Thomas, 69008
Lyon, France; 9Department of Pharmacology, Medical Toxicology and Medicine, INSERM U
98-X, Faculté de Médecine Lyon RTH Laennec, 69372 Lyon Cedex 98, France; 10Department
of Molecular Epidemiology, Centre of Nutritional Toxicology, Karolinska Institute, Novum,
141 57 Huddinge, Sweden; 11Euro Science Perspectives Ltd, 3 Patcham Grange, Brighton
BN1 8UR, UK; 12Nestle Research Safety Centre, Verschezles Blanc, 1000 Lausanne 26,
Switzerland.
Preface tion of alternative tests into regulatory pro-
cedures. It was decided that this would be
This is the report of the fortieth of a series of best achieved by the organisation of ECVAM
workshops organised by the European Cen- workshops on specific topics, at which small
tre for the Validation of Alternative Methods groups of invited experts would review the
(ECVAM). ECVAM’s main goal, as defined in current status of various types of in vitro
1993 by its Scientific Advisory Committee, is tests and their potential uses, and make rec-
to promote the scientific and regulatory ommendations about the best ways forward
acceptance of alternative methods which are (1). In addition, other topics relevant to the
of importance to the biosciences and which Three Rs concept of alternatives to animal
reduce, refine or replace the use of labora- experiments have been considered in several
tory animals. One of the first priorities set by ECVAM workshops.
ECVAM was the implementation of proce- This ECVAM workshop on Biomarkers as
dures which would enable it to become well- Predictive Tools in Toxicity Testing was held
informed about the state-of-the-art of in Burnham Market (Norfolk, UK) on 5–8
non-animal test development and validation, October 1998, under the co-chairmanship of
and the potential for the possible incorpora- Diane Benford (University of Surrey, UK)
Address for correspondence: Diane Benford, School of Biological Science, University of Surrey, Guildford, Sur-
rey GU2 5XH, UK.
Address for reprints: ECVAM, TP 580, JRC Institute for Health & Consumer Protection, 21020 Ispra (Va), Italy.
1European Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the
participants as individual scientists.

2. 120 D.J. Benford et al.
and Bryan Hanley (Central Science Labora- system that can be related to an expo-
tory, UK). The participants, whose expertise sure to, or effect from, a specific xenobi-
included toxicology, biomarker research, the otic or type of toxic material.” (4)
Three Rs and methods of validation, came
4. “Biomarkers, broadly defined, are indi-
from industry, universities and other
cators of variation in cellular or bio-
research institutions. The aim of the work-
chemical components or processes,
shop was to provide a forum for discussing
structures, or functions, that are mea-
the utility of biomarkers as predictive tools
surable in biological systems or sam-
for toxicity testing. A major focus of the
workshop was to examine the possibilities ples.” (5)
afforded by the use of biomarkers for the 5. A biomarker is “an indicator that sig-
reduction, refinement or replacement of ani- nals events in biological systems or sam-
mal use in toxicity testing. This report sum- ples, and it is generally taken to be any
marises the outcome of the discussions and biochemical, genetic, or immunological
includes a number of conclusions and recom- indicator that can be measured in a bio-
mendations. logical specimen.” (6)
Introduction
6. “The term biomarker has been used to
The term biomarker has grown in popularity describe measurements in the sequence
in recent years. With this growth has come a of events leading from exposure . . . to
great increase in applications and a corre- disease. At each step, persons may differ
spondingly increased diffuseness in the in susceptibility, thus a biomarker may
meaning of the term, to the extent that one also refer to an indicator of susceptibil-
of the major difficulties in the area of bio- ity.” (7)
marker research is reconciling differing 7. “A biomarker is a xenobiotically
views on what constitutes an acceptable def- induced variation in cellular or bio-
inition. This is reflected in the large number chemical components or processes,
of definitions appearing in the literature, a structures, or functions, that is measur-
selection of which are given below in chrono- able in a biological system or sample.”
logical order. This is not designed to be (J.F. McCarthy, R.S. Halbrook & L.R.
either comprehensive or a critically evalu- Shugart, 1991, Conceptual strategy for
ated collection, but it does represent the design, implementation and validation
range of uses to which biomarkers are put of a biomarker-based biomonitoring
and the justifications for the use of the word capability. Internal document of the
in different applications. They range from Environmental Sciences Division of the
exposure measures, and biological indices US EPA, used as the discussion docu-
which support a mechanistic postulate, to ment for the NATO Advanced Research
clinical markers with diagnostic implica- Workshop on Strategy for Biomarker
tions. Research and Application in the Assess-
1. “Biological markers are indicators sig- ment of Environmental Health).
nalling events in biological systems or 8. “. . . the biomarkers are any of a series
samples”, and “Biological markers are of biochemical or molecular responses
measurements of body fluids, cells or to compounds that have entered an
tissues that indicate in biochemical or organism, reached sites of toxic action,
cellular terms the presence and magni- and are exerting an effect on the organ-
tude of toxicants or of host responses.” ism.” (Proposal document for the
(2) NATO Advanced Research Workshop
2. Biomarkers are “cellular, biochemical, on Strategy for Biomarker Research
or molecular alterations which are mea - and Application in the Assessment of
surable in biological media such as Environmental Health, Texel, NL, May
human tissues, cells or fluids.” (3) 1991)
3. “The term biomarker refers to the use 9. Biomarkers are “parameters that puta-
made of a piece of information, rather tively represent some step along the
than to a specific type of information. A causal pathway between exposure and
biomarker is a change in a biological effect.” (8)

3. ECVAM Workshop 40: biomarkers 121
10. Biomarkers are “markers of biologic It is apparent from the breadth of these def-
activity that reflect not evidence of, but initions that any use of the term “bio-
the potential for, neoplastic progression marker” must also include a definition of
. . . Unlike tumour markers, which are what is meant by it within the context of a
biological indicators found in neo- specific discussion. This workshop high-
plasms, prevention biomarkers are lighted the great difficulties associated with
specifically related to earlier stages of the imprecise use of this word. A realistic
carcinogenesis. These intermediate end- appraisal of the biomarker area is required
points can be defined as measurements in order to clarify between scientists and
of a particular biologic factor associated clinicians the different ways in which the
with the evolution of neoplasia and term may be used.
occurring with increased frequency in
abnormal cells.” (9)
Genesis/Taxonomy of Biomarkers
11. “The term biomarker is used in a broad
sense to include almost any measure-
The term biomarker has a significant and
ment reflecting an interaction between
lengthy history. The meaning for which it is
a biological system and an environmen-
used clearly depends upon the context and
tal agent, which may be chemical, phys-
this is reflected most clearly in the parame-
ical or biological.” (10)
ters of the database which is used as the
12. “A biomarker is a measurable event basis for any search. Since the topic of this
occurring in a biological system, such workshop was the use of biomarkers as pre-
as the human body. In environmental dictive tools in toxicity testing, the focus was
epidemiology, a biomarker represents on biological, medical and toxicological uses
a sub-clinical and reversible change; it of the term. Therefore, an appropriate data-
is not a diagnostic test, but an indica- base set was likely to be found in Medline
tor that an early change has occurred and Toxline, so these databases were
that could later lead to clinical dis- searched for the term “biomarker”, with the
ease.” (11) following results:
13. A biomarker is “a measurement made 1966–1975: Only one record, which related
on body tissue, body fluid or excretion to the measurement of porphyrin in soil sam-
to give a quantitative indication of expo- ples (16).
sure to a chemical and which may give
1976–1985: 51 records, of which the majority
an estimate of the risks consequent on
were concerned with the use of tumour bio-
the exposure.” (12)
markers to diagnose cancer, obtain a progno-
14. “Biomarkers are observable endpoints sis or monitor therapy. Other topics included
in a continuum of events leading from ageing, monitoring wildlife rabies vaccina-
exposure to toxic agents to diseases that tion, rat pituitary enzymes, ecotoxicology,
ultimately result from exposure.” (13) assessment of toxic exposure, and the molec-
ular epidemiology of cancer.
15. “The term biomarker is used in a broad
sense to describe parameters reflecting 1986–1990: 308 records, in which the major
an interaction between a biological sys- subjects were the use of tumour biomarkers
tem and a potential hazard of chemical, for diagnosis, prognosis, monitoring of ther-
biological and physical nature. The apy and chemoprevention programmes,
measured response may be functional, assessment of toxic effects, and ecotoxicol-
physiological and biochemical at a cellu- ogy. Other topics included ageing, assess-
lar or molecular level.” (14) ment of toxic exposure, use of biomarkers in
risk assessment, assessment of susceptibility
16. “A biomarker is a parameter which can
to toxic insult, monitoring wildlife rabies
be evaluated quantitatively, semi-quan-
vaccination, and studies on other diseases.
titatively or qualitatively, and which
provides information on exposure to a 1991–1995: 3579 records, which included the
xenobiotic, or on the actual or potential first references to validation (17, 18). The sub-
effects of that exposure in an individual jects covered included all those mentioned
or in a group.” (15) above, but with far less emphasis on ageing.

4. 122 D.J. Benford et al.
There was increased use of biomarkers for the ageing. These investigations have little over-
monitoring of chemoprevention programmes, all relevance to classical toxicity testing,
the monitoring of specific groups of workers though they may contribute to the assess-
for exposure, and the assessment of suscepti- ment of cumulative toxic insult in humans, if
bility. There was a new subject for discussion, ageing is the result of a predominantly
namely, the ethical aspects associated with exogenous process.
the biomonitoring of individuals, especially In the period 1986–1990, the total number
with respect to their susceptibility to disease. of references to biomarkers increased sub-
Other new topics included the use of bio- stantially, particularly in relation to toxicol-
markers in epidemiology and in the assess- ogy. There was a gradual increase in studies
ment of health risks within the general on biomarkers of occupational exposure,
population, the use of biomarkers in studies which is likely to reflect a change in termi-
on the mechanisms of carcinogenesis, and the nology from “biological monitoring” to “bio-
identification of biomarkers of dietary intake. markers”, as well as an actual increase in
There was more theoretical consideration of research. The primary use of biomarkers of
methods to be used in the analysis of bio- exposure is to establish the adequacy of con-
marker data, possible sources of bias, and the trol measures with respect to chemical expo-
validation of biomarkers. sure in the workplace, i.e. occupational
exposure. They are not intended to be pre-
1996–November 1998: 3107 records covering
dictive of an adverse effect, but since occupa-
topics very similar to those of the previous
tional exposure limits are intended to protect
period.
the workforce from a known effect, infringe-
This breakdown of the uses of the term ment of the control measures clearly has the
shows a clear progression in the ideas associ- potential to cause harm. (Studies relating to
ated with biomarkers. The earliest reference smoking aimed to investigate specific causal
given above refers to the measurement of a agents and mechanisms of carcinogenicity.)
biological compound (porphyrin) in a non- A further area of development was that of
biological matrix (shale), and as such, has no biomarkers of effect. These are parameters
toxicological relevance. that change in response to exposure and
Between 1976 and 1985, many of the stud- reflect a biological consequence to that expo-
ies in which the term biomarker was used sure. For the first time, some studies which
were concerned with tumour biomarkers as related biomarkers to risk assessment were
diagnostic/prognostic tools. This use of the carried out.
term remains current. Most of these diag- In the period 1991–1995, a number of def-
nostic biomarkers are concerned with indi- initions of biomarkers were published, five of
cating current status and measuring the which are listed above (11–15). A number of
effectiveness of treatment. Such biomarkers papers referred to biomarkers as a means of
are not strictly predictive of outcome, monitoring the effects of interventions. This
although they may contribute to an overall usage is based on the hypothesis that a par-
prognosis given by the physician. ticular exposure is associated with a particu-
A second use of the term biomarker in this lar disease. Intervention methods may be
period was in the detection of exposure to used to modify the exposure under the
toxic agents. In some cases, biomarkers were assumption that this will reduce the risk of
measured in humans. But a major usage at that disease. Biomarkers of exposure are
this time was related to the use of biological used to verify that the intervention has the
systems as bioindicators of environmental anticipated effect on the internal exposure;
pollutants. For the purposes of such studies, biomarkers of effect, if available, may give an
the most biologically relevant systems were earlier indication of whether the interven-
those which were most sensitive in relation tion is likely to result in reduced incidence of
to the pollutants of interest. This type of the disease. If such relationships are estab-
study is not intended to be predictive of lished, this helps to strengthen the evidence
effects in other species, but is indicative of of causality. Because of their increasing
exposure that may represent a potential haz- availability and sensitivity, biomarkers are
ard to our species. increasingly being used to explore the links
The final area of active research in this between exposure and effect in order to
period was the development of biomarkers of establish causality. Because biomarkers can

5. ECVAM Workshop 40: biomarkers 123
measure internal exposure in an individual, animal use in toxicology studies. In particu-
the importance of individual susceptibility lar, the ultimate aim of toxicology is to eval-
and vulnerability become more clearly uate the potential risk to humans, where the
defined. Biomarkers that reflect these indi- risk is a function of exposure as well as the
vidual differences are referred to as bio- hazard.
markers of susceptibility; they need not Biomarkers of exposure could be predic-
relate to specific exposures. In addition, tive of risk, if sufficient information were
some awareness of the ethical issues involved available on the dose-response relationship.
in biomarker research and monitoring popu- Biomarkers of effect cover a range of mea-
lations began to emerge. surements which may be indicative of expo-
In the most recent period (1996–Novem- sure to a particular agent, although the
ber 1998), the numbers of biomarker-related specificity is generally less than for biomark-
records in Medline and Toxline increased ers of exposure. They may also show correla-
dramatically, although the topics being dealt tions with possible clinical outcomes, but
with were broadly similar to those noted in cannot be assumed to be predictive unless
the previous period. they are shown to reflect a significant step
In conclusion, work on biomarkers over along the causal pathway between exposure
the past 30 years or so has progressed, such and effect. The extreme example of a bio-
that, currently, a range of definitions have marker that reflects a current clinical condi-
been adopted, and this diversity is repre- tion, is actually a diagnostic marker and,
sented in Figure 1. However the most com- because it is rarely related to a chemical
monly used terms are biomarkers of exposure, it does not meet the above defini-
exposure, biomarkers of effect and biomark- tion of a biomarker. Biomarkers of suscepti-
ers of susceptibility. bility most notably include genetic
For the context of this report, we will susceptibility. The concept of susceptibility
define a biomarker as “a parameter which will not be dealt with in this report per se,
can be measured in a biological sample, and but it must be recognised that individual sus-
which provides information on an exposure, ceptibility and population susceptibility are
or on the actual or potential effects of that important issues, which should be taken into
exposure in an individual or in a group”. account and may be appropriate for a future
This report aims to consider the use of bio- workshop, once the establishment of a coher-
markers as predictive tools in toxicity test- ent meaning to the use of biomarkers in pre-
ing, so we must also consider what is meant dictive toxicology has been better defined.
by predictivity. The purpose of regulatory It is important to distinguish between use
toxicity testing is to define the inherent haz- of biomarkers in toxicological studies in
ardous properties of a substance. Previous experimental animals and in humans. Tradi-
ECVAM workshops have considered the tional toxicological endpoints are generally
value of in vitro test systems for the predic- empirical observations of the consequences
tion of various hazardous properties, and of exposure to specific agents. However, pro-
important elements of the discussion include vided that a study is conducted under strictly
the required level of accuracy of predictivity, controlled conditions, with appropriate sta-
in terms of false negatives and false posi- tistical analysis, it is reasonable to assume
tives, and how to achieve acceptance as that the observed effects were caused by the
replacement tests for regulatory toxicity exposure under investigation. Exposure to
testing. The point of reference is usually the the animal is well defined but, nevertheless,
results produced by in vivo animal toxicity there may be requirements for internal expo-
studies; comparison with human data would sure to be verified by toxicokinetic measure-
be preferable, but sufficient human data of ments, such as blood levels of test material,
sufficiently high quality are rarely available. in guidelines for some types of toxicity test.
Clearly, the uses of biomarkers listed above These are not normally considered to be bio-
do not include prediction of hazardous prop- marker studies, although they clearly meet
erties or of “toxicity”. We therefore need to many of the definitions of a biomarker. Bio-
consider the broader aspects of toxicology markers of effect (although again not termed
(rather than toxicity testing) and the value of as such) are frequently included in a battery
biomarkers as predictive tools that might of clinical chemistry assays on blood and
also allow for refinement and reduction of liver, and possibly other tissues, depending

6. 124 D.J. Benford et al.
upon the nature of the effect under investi- assay is chromosomal damage, this could be
gation. In contrast, human studies, even considered to be the use of a biomarker of (a
apparently well controlled volunteer studies, secondary) effect as a biomarker of exposure.
involve many other variables and inter-indi- In principle, biomarkers of effect could be
vidual differences that mean effects cannot valuable tools in toxicity testing protocols.
be so readily ascribed to the exposure under Biomarkers that develop early in the course
investigation. With investigations on “free- of repeat exposure, but that can be reliably
living” human populations, the potential for correlated to the subsequent development of
confounding factors increases enormously. It a lesion or an effect, would allow animal test-
therefore becomes essential to establish the ing protocols to be refined and animal testing
specificity of a “biomarker” for the exposure to be reduced. However, the mechanistic link
and/or effect of interest. For a biomarker of to the lesion needs to be understood in order
exposure, this relates to the measurement to define the uses and limitations of a partic-
techniques and the toxicokinetics of the sub- ular biomarker. At the current time, there is
stance but, for a biomarker of effect, it must insufficient understanding of mechanisms of
be based on understanding of the mechanism toxicity to permit the identification of mean-
of action. This leads to two key questions. ingful biomarkers for inclusion in routine
1. What are the current status and future toxicity testing protocols. For a very small
prospects for the use of biomarker number of compounds, some associations are
methodology in toxicity testing? known, such as liver glutathione content as
an indicator of paracetamol hepatotoxicity
2. What are the prospects for use of bio- (19), but these cannot be used in a general
markers leading to refinement, reduction sense. Some of the “biomarkers” now used in
and replacement of animal procedures in clinical chemistry, such as serum levels of
toxicology? liver transaminases, are more appropriately
Answers to these questions form the basis of termed “organ function tests”, and arguably
the rest of this report. should be defined as diagnostic markers
rather than as biomarkers. Nevertheless,
they are valuable because they demonstrate
Biomarkers in Toxicity Testing an important link between in vivo and in
vitro testing, i.e. that the same endpoints can
Toxicity tests are designed to identify the be used.
hazardous properties of a chemical sub- Biomarkers have the potential either to be
stance, tested in an isolated form in labora- used as indicators of events at various stages
tory animals. Because they are primarily in the progression of a toxic lesion, or to pro-
intended for the testing of novel substances, vide tools to test the relevance of proposed
the protocols have to be generic and cannot toxic mechanisms. The extent to which a bio-
be related to specific mechanisms of action. marker is a predictive tool depends on its
There is little opportunity for biomarkers of connection to the causal pathway leading
exposure to be included in such studies. As from exposure to effect, and on the quantita-
noted above, there are some exceptions. One tive assessment of the consequences of
example is the requirement for plasma levels changes in the biomarker in terms of toxico-
of compound in carcinogenicity studies of logical endpoints.Our current understanding
pharmaceutical agents, which are compared of mechanisms of toxicity is generally not
with data on human plasma levels at the sufficient to support development of bio-
therapeutic dose, in order to demonstrate markers that could be early predictors of
that sufficiently high doses of drug have been toxic effect. However, as our understanding
tested. A second example is the mouse bone increases, and with the enormous growth in
marrow micronucleus test, for which a nega- the use of new techniques such as genomics
tive result requires evidence that the com- and proteomics, which seek to determine the
pound is available to the bone marrow, link between gene expression, gene function,
either by specific chemical analysis (bio- protein synthesis and protein function and
marker of exposure), or by changes in the exposure to environmental and chemical
ratio of normal to polychromatic erythro- agents, there is the potential to develop such
cytes. Because this reflects toxicity to the predictive biomarkers in the future. The suc-
bone marrow, whereas the endpoint of the cess of a genomics/proteomics based

7. ECVAM Workshop 40: biomarkers 125
approach to biomarkers relies on the use of enzymes at much lower (and probably more
intelligent data manipulation, and on corre- physiologically relevant) levels. At normal
lation between measurable changes in the consumption levels, the benefits of the latter
functional properties of biological macromol- effect may outweigh the risks associated
ecules and the properties of the cell, organ or with the former; however, until directly com-
organism. parable human studies are carried out, it is
In addition to traditional toxicity tests, not possible to make a net toxicity/benefit
predictive models are available for risk prediction. The dose responsiveness of bioac-
assessment, ranging from structure/activity tive constituents, the way that a biomarker
models (i.e. QSAR) and knowledge based sys- can reflect this, and the choice of appropriate
tems (for example, DEREK) to in vitro test systems, are clearly keys to establishing
assays. The relevance, reliability and overall the validity of biomarkers as predictive tools.
value of any predictive model system is cru-
cially dependent on the quality of the data-
base used in its development and the method Exposure and Effect
used for assessing the reliability of various
pieces of information. It is possible that any The relationship between exposure and
biomarker which has been shown to fit into effect is crucial to any consideration of the
an established mechanistic pathway could risk associated with a given chemical. When
provide information which could be used by considering experimental toxicology, it has
an established predictive model. Such predic- been assumed that there is a linear progres-
tive systems should also be capable of assess- sion from exposure to effect, with the likeli-
ing the effects of substances which may hood of increasing severity of toxicity with
reduce or limit toxicity (in dietary terms, the increasing exposure. Therefore, it is possible
concept of “protective factors”). to envisage a number of biomarkers indica-
Clearly, the expression of a toxic effect is tive of the magnitude of exposure/response
determined by the magnitude of exposure, and/or the progression along the pathway
i.e. the dose. There are a growing number of (Figure 2).
examples where the validity of extrapolating The control exerted over the process in the
from high doses to low doses can be ques- laboratory helps to assure of the link
tioned, especially where this extrapolation is between exposure and effect. In contrast,
not based on detailed knowledge of the mech- when considering the majority of human
anisms of toxicity. (However, the concepts of health problems, it is possible to identify a
minimum effective dose and biological differ- large number of potential exposures and a
ences between high and low dose scenarios large number of diseases for a given popula-
must also be included. The dose level at tion, but the links between them are poorly
which a biomarker is measured in response defined, if at all. If, on the basis of knowledge
to a chemical challenge must be realistic in of mechanisms of toxicity, biomarkers can be
terms of “normal” human exposure. There is developed that match those defined in Figure
little purpose in developing effective bio- 2, there is the potential to:
markers (in terms of their relevance to a
1. unravel the links between exposure and
toxic endpoint), if they are of little relevance effect, i.e. establish causality;
to quantifiable measurements at human
exposure levels, or cannot be measured at 2. use biomarkers of internal exposure and
these levels. In addition, it is important to be early biomarkers of effect to determine
able to compare conflicting data on the the efficacy of interventionary measures;
effects of compounds by working in similar and
test systems. For example, consumption of
3. use biomarkers of effect as predictive
over-boiled coffee has been found to increase
tools in investigating agents that may
blood cholesterol (20) in a human study;
exert a similar toxic effect.
however, the level of exposure was higher
than would normally be encountered outside Biomarkers of exposure can be defined
a testing regime. The same test material has strictly by chemical analysis. The simplest
been demonstrated to have a chemoprotec- forms involve measurement either of the
tive effect against genotoxicity (21) in a non- agent in question, or of a metabolite; how-
human test system by stimulating phase 2 ever, they must be specific to the exposure of

9. ECVAM Workshop 40: biomarkers 127
Figure 2: The pathway from exposure to overt clinical effect
Exposure
Internal
Effective exposure
Dose
Cumulative
exposure
Early
marker
of effect
Late
marker
of effect
Overt Diagnostic
toxicity markers
interest. For example, hippuric acid has been half-life of lead in the blood is 36 days, and it
used as a urinary biomarker of exposure to is therefore most useful as a measure of
toluene. While hippuric acid is a metabolite recent exposure (24). Estimates of cumula-
of toluene, and therefore could be expected tive lead exposure can be made by measuring
to reflect exposure in a coherent way, it is bone lead, where the half-life of lead in corti-
non-specific, because it may also be derived cal bone is more than ten years, while that in
from a number of food constituents (22). The the more metabolically active trabecular
value of urinary hippuric acid as a biomarker bone is less. However, although techniques
therefore depends upon the relative sources that permit in vivo measurement of lead in
from dietary or occupational origins in a par- bone are available, they are not suitable for
ticular individual. In contrast, the presence routine monitoring purposes. A further com-
of the urinary metabolite, equol, is com- plication is introduced by the fact that bone
pletely specific for the ingestion of lead can be mobilised in various circum-
isoflavones. However, some individuals are stances, such as pregnancy, and may there-
unable to produce this metabolite, so its fore be a significant contributor to blood lead
absence in urine may not be reflective of levels (24). Urinary lead levels may also be
non-exposure (23). Therefore, knowledge of used as a marker of recent exposure, but are
the toxicokinetics of the agent is required, in subject to considerable inter-individual vari-
order to determine the most appropriate ation.
metabolite, tissue, and sampling times, and Once considerations go beyond a direct
the relevance of the results obtained. In measurement of chemical exposure, they
some instances, it may be possible to estab- begin to include an assessment of effect and
lish different biomarkers as measures of lose specificity for the chemical in question.
recent and cumulative exposure. An example For example, other biomarkers that have
of this can be seen in studies on lead. The been used in measuring lead exposure

10. 128 D.J. Benford et al.
involve the monitoring of effects upon the in investigations of causal links and into
haem synthesis pathway. This is based on mechanisms of toxicity. Once causality has
the known haematotoxicity of lead, which is been established, a biomarker should fulfil
mediated via the inhibition of enzymes of the criteria discussed below, if it is to be
haem synthesis, leading to the accumulation used in a predictive sense, whether in exper-
of the intermediates δ-aminolaevulinic acid imental toxicology, or in human populations.
(ALA), zinc protoporphyrin (ZPP) and copro-
porphorin (24). These biomarkers of effect
are not specific to lead exposure, as they may Validation Issues
be influenced by other haematotoxic agents
or by nutritional or sub-clinical disease sta- The validation of biomarkers is not directly
tus. In addition, they are not connected to comparable to validation of in vitro tests, but
the disease endpoints now considered to be must follow the basic principle of demon-
of most concern (because haematoxicity strating reproducibility, reliability and fit-
occurs at higher levels than are now likely to ness-for-purpose. The actual process will
occur, whereas reproductive and neurobe- vary, depending on the type of biomarker
havioural effects may occur at lower levels of and its intended use. It was beyond the scope
exposure). of this workshop to consider this issue in
Provided there is sufficient evidence to detail, but some general comments can be
assume that a given disease is associated made.
with a specific exposure, it is not necessary Validation of biomarkers of exposure
for the mechanism of effect to be understood should include consideration of: a) the toxi-
in order for biomarkers of exposure to be of cokinetics of the substance, i.e. the quantita-
value. Thus, it can be assumed that increas- tive and time relationship of levels in body
ing exposure increases risk, and decreasing fluids and tissues when exposure is of short,
exposure decreases risk, even though these medium or long duration; b) specificity, i.e.
increases or decreases may not be quantifi- whether the substance or its metabolite may
able. However, in order to identify meaning- result from sources other than the exposure
ful biomarkers of effect, as indicated in of interest; c) analytical quality control, i.e.
Figure 2, it is necessary to have an estab- the use of appropriate reference materials to
lished, mechanistic causal pathway which establish the accuracy of results; d) stability
links measurements of exposure, intermedi- in appropriate biological fluids; and e) poten-
ate effects and the final outcome. At the far tial confounding factors.
end of the chain of events are diagnostic It is notable that a number of quality con-
markers. Just as exposure markers are tied trol schemes are in operation, at regional,
to specific chemical exposures, diagnostic national and international levels, for meth-
markers relate to specific clinical events. It is ods used in biological monitoring of occupa-
axiomatic that the further along the pathway tional exposure (25). Such schemes could
from exposure to effect, the more difficult it provide a model for other biomarker applica-
is to define precisely the extent to which the tions.
exposure contributes to a specific clinical Validation of biomarkers of effect should
outcome, and vice versa. However, measure- include consideration of: a) relevance to the
ments of biological indices which are indica- effect of interest; b) reliability of prediction
tive of a causal pathway between exposure of the associated endpoint; c) interlaboratory
and outcome (i.e. biomarkers of effect) may reproducibility; d) analytical quality control;
be considered to have a degree of predictiv- e) stability in appropriate biological fluids;
ity. Following the definition of the scope and and f) potential confounding factors.
limitations of such biomarkers, they could be
incorporated into in vivo and in vitro toxicity
studies. Future Considerations
In conclusion, biomarkers of exposure
alone cannot be predictive of a toxic effect, Although biomarkers currently cannot
but have established value in monitoring and replace or reduce the use of animals in toxic-
controlling exposure to chemicals that are ity testing, future technological develop-
recognised or assumed hazards to health. ments show great promise for making this
Biomarkers of effect might be able to be used possible. In vitro techniques may play an

11. ECVAM Workshop 40: biomarkers 129
important role in the development of bio- decreased risk of developing particular dis-
markers of effect, as the greater control eases) are potentially of great value.
which is possible in in vitro studies facilitates The importance of the establishment of a
investigation of mechanisms of toxicity, and causal pathway was a recurring theme of the
therefore the identification of key events. A workshop. Currently, there are few examples
mechanistic understanding of the progres- of situations where this has been achieved.
sion of a toxic effect is a necessary prerequi- However, this reflects the lack of informa-
site for the use of biomarker approaches for tion on the mechanisms which underpin tox-
the prediction of toxicity. It is possible to icity. At present, too little emphasis is placed
envisage a natural progression, in which a on this issue in toxicological studies.
potential biomarker is identified during in
vitro mechanistic studies, and its value
tested in animal models, is then incorporated Conclusions and Recommendations
in in vivo testing protocols (refinement of
animal testing), and is subsequently vali- 1. The term “biomarkers” is employed very
dated for use in in vitro toxicity tests (lead- loosely. There are many differences of
ing to reduction and replacement of animal opinion about the definition, uses and
testing). Once an acceptable level of use has potential value of biomarkers.
been established for a new chemical, there is 2. Most controversy relates to the use of the
a potential for biomarkers to provide the term “predictive biomarker”. A bio-
continuity between initial toxicity testing marker is, by definition, a reflection of
and post-marketing surveillance, to ensure the current status of the biological sam-
that the acceptable levels are adequate to ple or system at the time of analysis. For
protect the human population against a biomarker of exposure, that status
adverse effects. could reflect either recent or longer term
Biomarkers of exposure are not directly exposure, i.e. a past or present situation.
relevant to toxicity testing per se, but they Provided there is a known association
may become relevant outside the laboratory with a disease, the biomarker of exposure
environment in the determination of expo- can predict a change in risk of that dis-
sure and dose (molecular dosimetry) and in ease, but it cannot predict a toxic effect. A
monitoring to check that acceptable expo- biomarker of effect might reflect an early
sure levels are not exceeded. They are also stage in the development of a disease, and
relevant to risk assessment and to investi- therefore may be predictive of eventual
gating mechanistic causes of human dis- disease. However, the development of
eases. such biomarkers is dependent upon
If biomarker techniques can be readily understanding of the aetiology of the dis-
automated, they have the potential to facili- ease in question, and few examples are
tate the development of high throughput available as yet.
assays, which permit the testing of com-
pounds which have not already been tested, 3. The use of biomarkers as research tools
but to which populations are exposed. This is to illuminate a postulated pathway or to
particularly relevant to dietary exposure, measure imprecise endpoints is an impor-
partly because of the vast number of chemi- tant aspect of their role.
cal components of food, but also because the 4. Traditional toxicity testing is not predic-
complex nature of whole foods makes delin- tive of the range of effects which may
eation of the effects of individual compo- occur in a real-life exposure situation.
nents both difficult and misleading. For This incomplete picture of the effects of
example, dietary oestrogens interact with exposure may be clarified by the develop-
each other and with the consumer in unpre- ment of biomarkers which have specific
dictable ways. Therefore, markers which can relevance to human disease. Such rele-
combine the different active and protective vant biomarkers could ultimately be used
components into an overall biological respon- to help establish the causal links between
siveness (for example, increases in levels of exposure and outcome and/or be indica-
sex hormone-binding protein or changes in tive of a potential effect in humans.
the expression of specific genes) linked to There is a need to establish the criteria
clinical endpoints (for example, increased or by which biomarkers can be considered to

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