EFSA Journal 2013;11(12):3472
SCIENTIFIC OPINION
Scientific Opinion on the relevance of dissimilar mode of action and its
appropriate application for cumulative risk assessment of pesticides
residues in food1
EFSA Panel on Plant Protection Products and their Residues (PPR)2, 3
European Food Safety Authority (EFSA), Parma, Italy
ABSTRACT
The European Food Safety Authority (EFSA) asked the Panel on Plant Protection Products and their Residues
(PPR Panel) to develop a Scientific Opinion on the relevance of dissimilar mode of action and its appropriate
application for cumulative risk assessment of pesticides residues in food. The present opinion was preceded by
three previous opinions of the PPR Panel (EFSA, 2008, 2009, 2013). The purpose of the present opinion was to
assess the relevance of dissimilar modes of action (MoA) for cumulative risk assessment, to evaluate the existing
methods for assessing chemicals acting by dissimilar MoA and to identify which methods need to be considered.
The PPR Panel restricted its considerations of pesticide combinations with dissimilar modes of action to
substances that produce a common adverse effect on the same organ/system. The PPR Panel noted that there is
no case documented in the scientific literature where independent action provided more conservative predictions
of combination effects than dose addition, and where at the same time independent action also produced accurate
predictions. The use of independent action as an assessment concept for combination effects requires
demonstration that modes of action of individual substances in a mixture are strictly independent, a condition
that can rarely be met in practice. The PPR Panel also noted that there is no cumulative risk assessment method
derived from independent action. The PPR Panel therefore recommends using cumulative risk assessment
methods derived from dose addition also for the assessment of mixtures of pesticides with dissimilar modes of
action, provided they produce a common adverse outcome. Pesticides that produce common adverse outcomes
on the same target organ/system should be grouped together in CAGs, and their combined effects assessed by
using the concept of dose addition as a pragmatic and conservative default approach for the purpose of assessing
cumulative risk in relation to MRL setting or risk assessment of chemical mixtures in practice.
© European Food Safety Authority, 2013
1
2
3
On request from EFSA, Question No EFSA-Q-2011-01020, adopted on 20 November 2013.
Panel members: Alf Aagaard, Theo Brock, Ettore Capri, Sabine Duquesne, Metka Filipic, Antonio F. Hernandez-Jerez,
Karen I. Hirsch-Ernst, Susanne Hougaard Bennekou, Michael Klein, Thomas Kuhl, Ryszard Laskowski, Matthias Liess,
Alberto Mantovani, Colin Ockleford, Bernadette Ossendorp, Daniel Pickford, Robert Smith, Paulo Sousa, Ingvar Sundh,
Aaldrik Tiktak, Ton Van Der Linden. Correspondence: pesticides.ppr@efsa.europa.eu
Acknowledgement: The Panel wishes to thank the members of the Working Group on the Relevance of Dissimilar Mode of
Action and its Appropriate Application for Cumulative Risk Assessment Karen Ildico Hirsch-Ernst, Susanne Hougaard
Bennekou, Alberto Mantovani, Antonio Hernandez Jerez, Andreas Kortenkamp, Diane Benford and the EFSA staff Andrea
Terron, Federica Crivellente, Luc Mohimont and Hans Steinkellner for the support provided to this Scientific Opinion.
Suggested citation: EFSA PPR Panel (EFSA Panel on Plant Protection Products and their Residues), 2013. Scientific Opinion
on relevance of dissimilar mode of action and its appropriate application for cumulative risk assessment of pesticides residues
in food. EFSA Journal 2013;11(12): 3472, 40 pp. doi:10.2903/j.efsa.2013.3472
Available online: www.efsa.europa.eu/efsajournal
© European Food Safety Authority, 2013
Relevance of dissimilar mode of action
KEY WORDS
Cumulative risk assessment, pesticides, dissimilar mode of action, independent action, dose addition.
EFSA Journal 2013;11(12):3472
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SUMMARY
The European Food Safety Authority (EFSA) asked the Panel on Plant Protection Products and their
Residues (PPR Panel) to develop a Scientific Opinion on the relevance of dissimilar mode of action
and its appropriate application for cumulative risk assessment of pesticides residues in food.
The present opinion was preceded by three previous Opinions of the PPR Panel (EFSA, 2008, 2009,
2013) that dealt with the development of methodology for the purpose of performing cumulative risk
assessment (CRA) for pesticide residues in food. The first Opinion (EFSA, 2008) focused on the
evaluation of existing methodologies for their suitability for assessment of risks resulting from dietary
exposure to multiple pesticide residues, and covered both aspects of hazard and exposure assessment.
Criteria for grouping active substances into cumulative assessment groups (CAGs) were proposed,
based on chemical structure, mechanism of pesticidal action, mode/mechanism of mammalian toxicity
and common toxic effects. In particular, it was assumed that mixture effects for substances acting via a
common mode of action (MoA) might be predicted by the dose addition (DA) concept. In a next step,
the PPR Panel tested the methodology proposed in the previous Opinion by preparing a worked
example of a dietary risk assessment for a group of triazole pesticides, a well-defined group in terms
of structure, pesticidal mode of action and common toxic effects (EFSA, 2009).
For the third Opinion (EFSA PPR Panel, 2013), the PPR Panel further elaborated general methodology
and criteria for the establishment of CAGs of pesticides, based on their toxicological profiles. The
adopted methodology follows a phenomenological approach based on organ or system toxicity, which
consists of including all pesticides causing a specific effect in a CAG. Since mechanistic data
supporting decisions regarding similarity of action are rarely available for toxicity endpoints on active
substances, this approach takes common organ/system toxicity as an approximation of similar or
interrelated modes of action. It was considered that the cumulative effects of pesticides within a CAG
could then be assessed by a method derived from the dose addition concept. Nevertheless, the Panel
acknowledged that CAGs based on phenomenological effects would be expected to include substances
acting via different modes of action, but contributing to a common adverse outcome. Although the
Panel noted that “…further refinement of grouping may be achieved when data on the precise
toxicological mode of action may be available”, it pointed out that “information that justifies any
deviation from dose-addition might also be necessary to consider for such a refinement”.
There is good experimental evidence that combination effects can arise from co-exposure to chemicals
which produce common (adverse) outcomes through different mechanisms and modes of action. This
also applies to combinations of pesticides. The PPR Panel therefore concludes that dissimilar modes of
action are relevant to the assessment of cumulative risks from pesticides residues in food. In addition,
a number of cases have been documented for which joint effects of substance combinations in
mammalian test systems were approximated by dose addition.
The purpose of the present Opinion was to assess the relevance of dissimilar modes of action for
assessing cumulative risks from pesticide residues in food, to evaluate the applicability of the existing
methods for assessing cumulative risks resulting from dissimilar modes of action, and to provide
general criteria to identify which type and when dissimilar modes of action need to be considered in
CRA. Thus, this Opinion represents a further step in hazard assessment for dietary cumulative risk
assessment in the context of maximum residue levels (MRL) setting. In line with its previous Opinion
on cumulative assessment groups (EFSA PPR Panel, 2013), the Panel acknowledges that CRA is a
process that involves several steps and multiple considerations, including availability of occurrence
data and the scientific and technical capacity of exposure assessment methodologies. However, many
of these considerations are beyond the scope and Terms of Reference of the present opinion.
Substances exhibiting dissimilar modes of action can potentially produce a variety of different, non
overlapping toxic effects in different organs/systems. In such cases, it is difficult to identify a
combination effect. Therefore, the PPR Panel restricted its considerations of pesticide combinations
EFSA Journal 2013;11(12):3472
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Relevance of dissimilar mode of action
with dissimilar modes of action to substances that produce a common adverse effect on the same
organ/system.
Furthermore, the available empirical evidence, although limited, suggests that synergisms at dietary
exposure levels are rather rare. Synergisms cannot be predicted quantitatively on the basis of the
toxicity of mixture components. Therefore, the Opinion is also not dealing with the topic of
synergisms.
In elaboration of the present Opinion, the Panel considered information compiled within the preceding
EFSA Opinions dealing with cumulative risk assessment methodology (EFSA, 2008, 2009, 2013),
within the supporting project commissioned by EFSA on “Investigation of the state of the science on
combined actions of chemicals in food through dissimilar modes of action and proposal for sciencebased approach for performing related cumulative risk assessment” (Kortenkamp et al., 2012), as well
as the current further scientific literature.
With respect to the existing methods for assessing cumulative risks, the Panel recognised that the
approaches developed to date for performing CRA of chemicals, including pesticides, are based on the
dose addition concept. The applicability of this approach has been established for groups of chemicals
acting by a similar mode of action. However, in case of performing CRA for chemicals having a
dissimilar mode of action and eliciting the same phenomenological effect, it remains to be answered
whether a method based on dose addition (DA) or independent action (IA) would be more appropriate.
After analysing the premise for applying an approach based on IA, the PPR Panel noted that to apply
this method the chemicals in question would need to act independently. However, it was pointed out
that this is not necessarily equivalent to chemicals having a dissimilar mode of action. Further, it was
concluded that widely used definitions of mode or mechanism of action are not helpful in deciding
when a mode/mechanism of action is sufficiently separate or distinct from another mode to support an
assessment in favour of independent action. Mode of action definitions, as useful as they may be for
the analysis of single chemicals, are generally not informative in terms of making the case for
independence of action. In principle, the case can be made empirically, by investigating whether
experimentally observed mixture effects are approximated better by dose addition or independent
action. The usefulness of such empirical approaches for CRA in a regulatory context is, however,
limited, because it requires experimental data for the mixture of concern, which is usually not
available.
The Panel noted that there is to date no empirical evidence for the validity of independent action as a
prediction concept for multi-component mixtures in the mammalian toxicological literature, and
concluded that this supports the default use of dose addition for the approximation of mixture effects.
Available empirical evidence, together with mathematical analyses and findings from simulation
studies, indicate that the quantitative differences under realistic exposure conditions between predicted
mixture effects derived from dose addition and those derived from independent action are likely to be
small (under one order of magnitude), considering the variability and sources of error normally
encountered in realistic assessment situations. Furthermore, no case could be identified in which
independent action provided a more conservative mixture effect prediction than dose addition, which
at the same time was also more accurate. Thus, the Panel concludes that dose addition is a sufficiently
conservative approach to protect consumers´ health.
There are several CRA methods in practical use that are derived from the concept of dose addition,
e.g. the Hazard Index and Point of Departure Index methods or the Toxicity Equivalency Factor
approach. In contrast, there is no established method based on independent action. The PPR Panel
emphasised that the application of independent action in risk assessment practice is further
complicated by the fact that it places demands on the data quality of the required input values that
cannot be met in practice. In particular, it requires detailed descriptions of effects in the low dose
range, which are rarely available.
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The Panel recognised that the lack of data about the mode for pesticide active substances in relation to
health effects represents a scientific uncertainty and that the absence of a generally accepted
classification scheme based on sufficiently different MoAs is problematic. This complicates the
grouping of chemicals for CRA according to the principles of independent action. To obtain a basis for
potential further improvement of criteria for the grouping of substances for CRA, the PPR Panel
recommends to enrich key knowledge on the importance of mode of action in CRA. In particular, the
Panel recommends more research on the characterisation of adverse outcome pathways.
The PPR Panel identified some uncertainties with respect to the default use of assessment methods
derived from dose addition. The validity of independent action in prediction of combination effects
from multi-component mixtures of dissimilarly acting substances has to date only been experimentally
demonstrated in bacteria and algae. Even in such cases in which independent action yielded the more
accurate prediction, dose addition provided a more conservative estimate than independent action,
supporting the use of dose addition as the default concept in mixture risk assessment. A comparable
example for the validity of independent action, has however not been identified for mammalian cells
or multi-cellular organisms. The Panel pointed out that more research is needed in mammalian
systems to investigate the applicability of independent action. In particular, further research is required
to clarify whether a proof-of-principle example of validity of independent action exists for a
mammalian test system.
In tests and systems with relevance to ecotoxicity, dose addition usually provided the more
conservative mixture toxicity estimate. It remains to be confirmed whether dose addition tends to
provide the more conservative prediction for endpoints relevant to mammalian toxicology. In theory, a
more conservative prediction might be produced by the independent action model as compared to the
dose addition model in the case when all components of the mixture exhibit very shallow doseresponse curves. Nevertheless, the Panel recognised that this is a rather theoretical situation and that
the “increasingly flat” dose responses are unlikely to be biologically plausible. More knowledge about
the distribution of slopes of dose response curves in mammalian systems is required to distinguish any
cases where independent action might provide more conservative predictions of mixture effects.
The PPR Panel recognised that there are uncertainties regarding the possible occurrence of interactions
and that the presence of such interactions could call into question the use of any additivity concept as a
default. However, the Panel concluded that current evidence indicates that synergisms are less likely to
occur at low doses/concentrations corresponding to the dietary exposure to mixtures of pesticides and
are thus less relevant for cumulative risk assessment of pesticide residues in food. This would provide
a degree of reassurance that risks arising from interactions might be rather rare. The Panel
recommended that, to address uncertainties as to whether default application of dose addition is
generally sufficiently conservative, it is important to better define determinants of synergisms by
researching the possibilities of toxicokinetic and toxicodynamic interactions.
Based on the current state of knowledge and taking into account the above mentioned uncertainties,
the PPR Panel comes to the overall conclusion that distinctions between similar and dissimilar MoA,
are fraught with great conceptual and practical difficulties, when it comes to deciding which concept
(dose addition or independent action) should be used for risk assessment practice. It is therefore
recommended to use cumulative risk assessment methods derived from dose addition also for the
assessment of mixtures of pesticides with dissimilar modes of action, provided they produce a
common adverse outcome. Pesticides that produce common adverse outcomes on the same target
organ/system should be grouped together in CAGs, and their combined effects assessed by using the
concept of dose addition as a pragmatic and conservative default approach for the purpose of assessing
cumulative risk in relation to MRL setting or risk assessment of chemical mixtures in practice as
proposed in the 2013 EFSA Opinion on CAGs.
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TABLE OF CONTENTS
Abstract .................................................................................................................................................... 1
Summary .................................................................................................................................................. 3
Table of contents ...................................................................................................................................... 6
Background as provided by EFSA ........................................................................................................... 7
Terms of reference as provided by EFSA ................................................................................................ 8
Assessment ............................................................................................................................................... 9
1. Interpretation of the Terms of Reference by the PPR Panel ............................................................. 9
2. Introduction .................................................................................................................................... 10
2.1.
General considerations on combined toxicity ....................................................................... 10
2.2.
Summary of the previous EFSA opinions on cumulative risk assessment ........................... 12
2.3.
Summary of the supporting project ....................................................................................... 15
3. Combined effects of chemicals and the relevance of dissimilar modes of action .......................... 18
4. Analysis of empirical evidence for the applicability of independent action to the prediction and
assessment of chemical mixtures ....................................................................................................... 19
4.1.
The applicability of independent action as a prediction concept for mixtures composed of
chemicals with dissimilar modes of action ........................................................................................ 19
4.1.1 Literature survey on empirical evidence in support of the validity of independent action19
4.2.
The role of mode of action information in guiding a priori choices between independent
action and dose addition for evaluating mixture effects .................................................................... 20
4.3.
Conservatism of dose addition compared to independent action .......................................... 22
4.4.
Dissimilarly acting mixtures at low doses ............................................................................ 22
4.5.
Relevance of interactions for cumulative risk assessment .................................................... 23
4.6.
Summary of chapter 4 ........................................................................................................... 23
5. Quantitative differences between mixture effect predictions derived from independent action and
dose addition ...................................................................................................................................... 24
5.1.
Empirical findings of prediction differences......................................................................... 24
5.2.
Mathematical analysis ........................................................................................................... 24
5.3.
Summary of chapter 5 ........................................................................................................... 26
6. Difficulties in applying independent action in regulatory practice ................................................. 27
6.1.
Simplified applications of independent action ...................................................................... 27
6.2.
Data requirements for using independent action................................................................... 27
6.3.
Data requirements for utilising cumulative risk assessment methods based on dose addition28
6.4.
The relevance of dissimilar MoA for grouping of pesticides in CAGs ................................ 29
6.5.
Summary of chapter 6 ........................................................................................................... 29
7. Uncertainties ................................................................................................................................... 30
Conclusions and Recommendations ....................................................................................................... 31
References .............................................................................................................................................. 34
Appendix A. Mathematical calculation supporting the example described in 6.2 “Data requirements
for the use of independent action”. ......................................................................................................... 38
Glossary and abbreviations .................................................................................................................... 39
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BACKGROUND AS PROVIDED BY EFSA
Regulation (EC) No 396/20054 of the European Council and the European Parliament on maximum
residue levels of pesticides in food and feed of plant and animal origin and amending Council
Directive 91/414/EEC5 stresses the importance of “carrying out further work to develop a
methodology to take into account cumulative and synergistic effects” for the setting of maximum
residue levels (MRLs). In order to initiate development of such methodologies, EFSA organised a
colloquium in 2006 on “Cumulative risk assessment of pesticides to human health: the way forward”.
The results and conclusions from this workshop were published in the form of a report (EFSA, 2007).
Following this workshop EFSA’s PPR Panel has, as a first step in its work on cumulative risk
assessment, drafted an opinion in which the suitability of the currently existing methodologies for the
assessment of cumulative risks was explored and evaluated and which was published in 2008 (EFSA,
2008).
As a second step, in order to find out if the established principles could also be applied in practice, the
Panel tested the proposed methodologies on a selected group of compounds (i.e. triazole fungicides).
The outcome of this work, together with further refinements for the approach proposed in the first
opinion, have been published as a second scientific opinion on cumulative risk assessment (EFSA,
2009).
To complete the work on cumulative risk assessment of pesticides for the setting of MRLs of
pesticides in food and feed the Panel adopted a third opinion to identify pesticides that can, based on
their toxicological profile, be grouped together for risk assessment in so called “cumulative assessment
groups” (EFSA PPR Panel, 2013).
In the first opinion (EFSA, 2008), where the PPR Panel gave recommendations on the methodologies
to be used for cumulative risk assessments, it was concluded that “from the available data there is no
evidence that exposure to a mixture of pesticides with different modes of action poses any substantially
greater risk than that of exposure to the individual pesticides in the mixture, when exposure occurs
below the respective reference value” adding that “the PPR Panel did not further address the
combined risk of exposure to pesticides with different targets and different modes of action, when they
occur as residues in food.”
It is notable in this context that in the first opinion (EFSA, 2008) reference was made to scientific
work showing that cumulative effects can also occur by different modes of action, as it was stated that
“an additional consideration arises from evidence in the literature that certain endocrine disruptors
show dose-additivity even if they do not share the same primary molecular target (Kortenkamp, 2007
and other papers there reviewed)” and that therefore, “it appears that in these cases the criterion for
grouping should rather be that of a common phenomenological effect (e.g. altered ano-genital
distance for antiandrogens) (Kortenkamp, 2007)”.
Since then, further scientific evidence has been obtained corroborating the relevance of a dissimilar
mode of action for cumulative risk assessment, in particular for substances having effects on or acting
via the endocrine system (Christiansen et al., 2008; Kortenkamp, 2008; Moretto, 2008; Kortenkamp
and Hass, 2009; Kortenkamp et al., 2009; Kortenkamp and Faust, 2010; Jacobsen et al., 2010;
Reffstrup et al., 2010).
Therefore EFSA’s PPR Unit launched a call for tender in 2010 on the “Investigation of the state of the
science on combined actions of chemicals in food through dissimilar modes of action and proposal for
4
Regulation (EC) No396/2005 of the European Parliament and of the Council of 23 February 2005 on maximum residue
levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. Official
Journal L 70, 1-16. 16 March 2005.
5
Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. Official
Journal L 230, 1-290. 19 August 1991
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science-based approach for performing related cumulative risk assessment” in order to provide
scientific information on different aspects of combined actions of chemicals in food acting through
dissimilar modes of action and to define criteria regarding the elaboration of cumulative assessment
groups of pesticides which do not necessarily share a common mechanism or mode of action.
The final report from this project was published on the 31st of January 2012, providing support to the
development of a scientific opinion of the PPR Panel specifically focusing on the relevance of
response addition for cumulative risk assessment.
TERMS OF REFERENCE AS PROVIDED BY EFSA
The Scientific Panel on Plant Protection Products and their Residues is asked by EFSA to prepare a
scientific opinion on the relevance of dissimilar modes of action and its appropriate application for
cumulative risk assessment of pesticides residues in food.
In particular the PPR Panel is asked to:
assess the relevance of dissimilar modes of action for assessing cumulative risks from
pesticide residues in food,
evaluate the applicability of the existing methodologies for assessing cumulative risks
resulting from dissimilar modes of action, and
provide general criteria to identify which type and when dissimilar modes of action need to be
considered and combined in cumulative risk assessment
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ASSESSMENT
1.
Interpretation of the Terms of Reference by the PPR Panel
In the Terms of Reference EFSA has requested a scientific opinion on the relevance of dissimilar
modes of action and its appropriate application for cumulative risk assessment of pesticides residues in
food.
In the explanatory background provided by EFSA in 2011, the request was triggered by published
literature indicating that cumulative effects can also be anticipated by substances acting through
dissimilar modes of action on the same target organ/system. In June 2013, the PPR Panel adopted the
opinion on the identification of pesticides to be included in cumulative assessment groups (CAGs) on
the basis of their common toxicological profile (EFSA PPR Panel, 2013). The adopted methodology
follows a phenomenological approach based on organ or system toxicity, which consists of including
all pesticides causing the effect under consideration in a CAG for a specific effect. Because
mechanistic data supporting decisions about similarity of action are rarely available for toxicity
endpoints of active substances, this approach takes common organ toxicity as an approximation of
similar common modes of action. The cumulative effects of pesticides in a CAG can then be assessed
by using methods derived from the dose addition concept.
The PPR Panel recommended in its CAGs opinion that further refinement of grouping may be
achieved when data on the precise toxicological mode of action are available; however, information
that justifies any deviation from dose addition might also be necessary to consider such a refinement.
Thus, it is important to envisage when and how such refinement may be achieved in such a way that
conducting cumulative risk assessments with the refined cumulative assessment groups does not
underestimate the actual combined toxicity of pesticides.
The CAG opinion acknowledged that conducting CRA is a process that involves several steps and
multiple considerations, many of which go also beyond the scope and Terms of Reference of this
opinion.
There are indications that even with a mixture of many pesticides affecting the same organ system the
majority of them might not contribute significantly to a given combination effect, either because
exposure is very low, and/or because potency in relation to the effect considered is weak. This would
mean that cumulative risks from actual exposures are likely to be driven mainly by a few pesticides of
a mixture.
Therefore, taking into account its opinion on CAGs for pesticides (EFSA PPR Panel, 2013), the PPR
Panel included the following specification as a matter of clarification of the original Terms of
References:
When information is available and indicates that active pesticide substances causing the same
phenomenological effect are acting with different modes/mechanisms of action, under what
conditions would it be justified to deviate from a default application of the concept of dose
addition, as was proposed in the opinion on identification of cumulative assessment groups of
pesticides (EFSA PPR Panel, 2013)?
Are there conditions where application of dose addition to pesticides that exhibit toxicity
through dissimilar modes of action would lead to an underestimation of cumulative effects?
Which alternative methodology would be appropriate in such cases?
Substances exhibiting dissimilar modes of action can potentially produce a variety of different, non
overlapping toxic effects in different organs/systems In such cases, it is difficult to identify a
combination effect. Therefore, the PPR Panel restricted its considerations of pesticide combinations
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Relevance of dissimilar mode of action
with dissimilar modes of action to substances that produce a common adverse effect on the same
organ/system.
2.
2.1.
Introduction
General considerations on combined toxicity
Apart from synergism and antagonism (interactive effects, see chapter 4.5 in this opinion) the
combined effects of several chemicals are often referenced in terms of two fundamentally different
modes: similar action and dissimilar action (sometimes also called independent joint action). These
distinctions were first introduced by Bliss (1939) and Hewlett and Plackett (1952), on the basis of
statistical principles. They have gained wide acceptance for the interpretation of mixture effects and
are allied to the assessment concepts of dose addition (DA, linked with similar action) and
independent action or response addition (IA, linked with dissimilar action). Many regulatory bodies
and competent authorities have used these terms in broadly identical ways, although there are
differences in the level of detail specified for each of the two modes (summarised in Kortenkamp et al.
2012). There is a consensus that similar action “occurs when chemicals in a mixture act in the same
way, by the same mechanism/mode of action, and differ only in their potencies” (EFSA, 2008).
Conversely, “dissimilar action” occurs with combinations of chemicals that produce a common effect
by action through different modes of action, or at different sites. In its opinion on triazoles, the EFSA
PPR panel has defined the term as a synonym for response-addition (“response-addition, also referred
to as simple dissimilar action”) and stated that it “occurs where the modes of action and possibly, but
not necessarily, the nature and sites of toxic effects differ between the chemicals in a mixture, and one
chemical does not influence the toxicity of another” (EFSA, 2008).
While these definitions are clear-cut in principle, in practice it is often not straight-forward to
distinguish between dissimilar action and similar action. In many cases, the mechanistic information
needed to differentiate between the two types of combination effect is not available. Clear distinctions
are further complicated by ambiguities concerning the precise meaning of the terms “mode of action”
and “site of action” and its implications for assessments of combination effects. For example, two
chemicals might affect different sites of an effector chain leading to an adverse outcome, in agreement
with a key feature of simple dissimilar action. However, if the same key metabolite or intermediate is
affected, the toxicological consequences could be better described in terms of similar action.
The distinction between similar action and dissimilar action goes back to two fundamentally different,
but theoretically equally legitimate, conceptualisations of the joint action of chemicals, dose addition
and independent action.
Dose addition is based on the idea that all components in a mixture behave as if they were dilutions of
one another (Loewe and Muischnek, 1926). Examples would be combinations of chemicals that all
exert their toxicity by binding to the same receptor, e.g. the Ah receptor (polychlorinated dioxins) or
the active centre of acetylcholinesterase (organophosphates, carbamates). In these cases, similar action
applies because one chemical can be replaced by an equal fraction of an equi-effective concentration
(e.g. an EC50) of another, without diminishing the overall combined effect. Dose addition implies that
every toxicant in the mixture contributes to the combination effect in proportion to its dose and
individual potency. Whether the individual doses are also effective on their own does not matter. Thus,
under dose addition, combination effects can be expected when toxicants are present at levels below
effect thresholds, but only if the number of components sums up to a total mixture dose sufficiently
large to produce effects. For example, two chemicals combined at 1/10 of their threshold concentration
are not expected to produce a combination effect according to dose addition.
Independent action (response addition) conceptualises mixture effects in a different way. It assumes
that a combination effect can be calculated from the responses of the individual mixture components
by following the statistical concept of independent random events (Bliss, 1939). The idea of
independent random events also applies to the same chemical administered in a sequential fashion
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within time frames when recovery phenomena do not occur. In this situation, the mode of action is
identical, but the randomness of events is introduced by exposures that occur in sequence. The overall
effect is then accessible by multiplication of the likelihood of independent events (administrations). In
the case of simultaneous exposure to several chemicals, the principle of independence of effects is
only applicable when all the chemicals in the mixture act through strictly dissimilar modes by
affecting strictly different targets (simple dissimilar action). Examples would be combinations of
chemicals that affect algal reproduction by disrupting photosynthesis, DNA synthesis and multiple
other sub-systems. The principles of independence of effects also imply that components present at
doses below thresholds and thus associated with zero effects will not contribute to the joint effect of
the mixture. If this condition is fulfilled for all mixture components, combination effects are not
expected under independent action.
By making the assumption that each mixture component exerts its effects in the same way as it would
do when present on its own, i.e. without diminishing or enhancing the toxicity of other components
(antagonism or synergy), it becomes possible to formulate a quantitative expectation of the joint
effects of multiple chemicals. These so-called additivity expectations are calculated on the basis of the
effects of each individual chemical in the mixture. Additivity expectations can be derived from
independent action or dose addition and serve as points of reference for the experimental identification
of synergisms or antagonisms. This idea is only workable when both the mixture and all its
components produce the same toxic effect. In cases when some chemicals in the mixture do not affect
the outcome of interest, it is not possible to predict quantitatively a joint effect from the effects of
mixture components, unless the chemicals not showing the effect of interest are excluded from the
assessment. It follows that independent action is only applicable to combinations of chemicals that
produce a common toxic effect (and the same applies when additivity expectations are derived from
dose addition). Chemicals with entirely different non overlapping toxicity profiles can also be classed
as dissimilarly acting, but this is not relevant to the application of independent action because a joint
effect cannot be defined. Mathematical concepts for the (quantitative) calculation of expected
combined effects cannot be used in these cases.
When it comes to the evaluation of experimental data, algorithms derived from both concepts are used
to translate effect doses (in the case of dose addition) or effects (in the case of independent action) of
the individual mixture components into expected combined effects. Often, but not always, the two
concepts produce different predictions for additive mixture effects, on the basis of the same doseresponse information for the single mixture components. This can give rise to an assessment dilemma:
if the observed mixture effects fall between the window defined by the dose addition and independent
action predictions, they can be evaluated as synergisms (effects stronger than anticipated assuming
additivity) in relation to one, but as antagonism (effects weaker than predicted) in relation to the other
concept. This unsatisfactory situation can then only be resolved by using additional information
regarding the modes of action of each of the mixture components, but quite frequently, such
information is not available. While distinctions between similar and dissimilar action may have
importance for the evaluation of experimental data, the implications for cumulative risk assessment are
less clear-cut. Two aspects are of relevance:
First, it deserves consideration whether cumulative risk assessment is warranted at all. With reference
to the apparent diversity of chemicals that occur together, it has often been argued that independent
action is likely to apply generally to the evaluation of realistic exposure scenarios (COT 2002).
Considering the low exposure levels to single chemicals normally encountered by the general
population, together with the central tenet of independent action that combination effects are not
expected when all components stay below zero-effect doses, and under the assumption that healthbased guidance values for individual threshold substances correspond to zero-effect levels, the
assessment of joint effect has been regarded as unnecessary, at least for human health effects
(Scientific Committees, 2012). Other international bodies have proposed to use dose addition as a
default assessment concept, as long as evidence supporting the case for dissimilar action, and hence
the use of independent action, is not available (IPCS, 2009). This can be supported in the light of
evidence that combination effects are well described by dose addition, even though the mixture
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Relevance of dissimilar mode of action
components involved do not share the same molecular target or mechanism (Christiansen et al., 2008;
Kortenkamp, 2008; Moretto, 2008; Flippin et al., 2009; Kortenkamp and Hass, 2009; Kortenkamp et
al., 2009; Kortenkamp and Faust, 2010; Jacobsen et al., 2010; Jacobsen et al., 2012; Reffstrup et al.,
2010).
The second aspect of relevance concerns the criteria used for deciding on the grouping of chemicals to
be subjected to cumulative risk assessment. Especially in the USA there has been a tradition of
employing criteria derived from features of chemical structural similarity for the assessment of
combinations of pesticides (USEPA, 2002). Only active substances that are structurally similar are
assessed together, with the implicit assumption that they must meet the criteria of similar action, and
therefore warrant the use of dose addition. Without further evidence, chemicals that fall outside the
chemical space of interest are regarded as acting together according to the principles of independent
action. With the additional assumption that exposures are below threshold doses, assessments of
cumulative risks are then considered unnecessary. However, fuelled by a concern that real existing
risks might be underestimated, this practice has increasingly come under criticism, with reference to
empirical evidence of combination effects with mixtures composed of chemicals showing widely
varying structural features (USNRC, 2008). As a more appropriate alternative, grouping criteria
emphasising common adverse outcomes have been recommended (USNRC, 2008; EFSA PPR Panel,
2013).
In light of the practical difficulties encountered when using considerations of similar or dissimilar
action as the starting point for cumulative risk assessment (lack of mechanistic data, ambiguities
associated with key terms such as mode of action, mechanism of action), the Panel considered whether
it would be possible to abandon the dichotomous approach to cumulative risk assessment and instead
develop a common approach for the assessment of similarly and dissimilarly acting chemicals based
on the pragmatic use of the concept of dose addition. In the arena of human risk assessment, such a
departure from current practice would be scientifically credible if it can be shown that one or more of
the following conditions are fulfilled:
There is no case documented in the scientific literature where independent action provided
more conservative predictions of combination effects than dose addition, and where at the
same time independent action also produced accurate predictions,
The prediction differences between independent action and dose addition encountered during
the evaluation of multi-component mixtures are relatively small and therefore of limited
relevance to cumulative risk assessment practice
There is no practicable cumulative risk assessment concept based on independent action.
In this scientific opinion the Panel evaluates whether cumulative risk assessment methods derived
from dose addition can also be applied to the assessment of mixtures composed of dissimilarly acting
chemicals. Should this be the case, the way would be open for the development of a unified approach
to cumulative risk assessment, irrespective of the mode of action of mixture components. This could
successfully overcome difficulties that currently prevent continuation of cumulative risk assessment,
especially in situations where uncertainties about the mode of action of mixture components blocks
further analysis.
2.2.
Summary of the previous EFSA opinions on cumulative risk assessment
Regulation (EC) No 396/2005 on maximum residue levels of pesticides in or on food and feed of plant
and animal origin emphasises the importance “to carry out further work to develop a methodology to
take into account cumulative and synergistic effects” of pesticides.
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In 2006, EFSA hosted a colloquium on “Cumulative risk assessment of pesticides to human health:
The way forward” (EFSA, 2007). A further International Workshop on Aggregate/Cumulative Risk
Assessment was organised by WHO/IPCS in 2007 (WHO, 2009).
Taking into account the outcome of the discussions held at these international meetings, the PPR Panel
self-tasked to evaluate existing methodologies for their suitability for assessment of risks resulting
from combined exposure to two or more compounds, focussing on dietary risk assessment for
pesticide residues, particularly in the context of setting MRLs according to Regulation (EC) 396/2005
(EFSA, 2008). The opinion elaborated by the Panel covered both hazard and exposure assessment and
recommended that a tiered approach for both toxicological evaluation and intake estimations should be
followed to make best use of available resources.
Concerning toxicological evaluation, the Panel considered different forms of combined toxicity (doseaddition, response-addition, or interaction) with respect to their potential relevance to risk assessment
for pesticide residues occurring in food. Based on the available empirical evidence (as referenced in
EFSA, 2008), it was concluded that toxicologically/biologically significant interactions (synergism,
antagonism) between chemicals were much less likely to occur at doses below the effect levels for
individual compounds than at higher doses, although it could not be excluded that in some particular
cases, interactions might result in combination effects, even if exposure to pesticide residues were to
correspond to levels below individual pesticide No Observed Adverse Effect Levels (NOAELs). Since
dietary exposure of consumers to pesticide residues is expected to be low (substantially below the
NOAELs), interactions were considered by the Panel to be less relevant to risk assessment for
pesticide residues in food. Based on the concept that toxic effects resulting from response addition
would not be expected if no toxicity would occur from any of the single components of the mixture,
and given the low levels of pesticide residues in food, it was assumed that “… response-additive
toxicity will rarely if ever occur from pesticide residues in food”.
Considering empirical and mechanistic evidence that dose addition might occur for mixtures of
substances administered at relatively low individual doses, this form of combined toxicity was
regarded by the Panel as being of potential relevance for risk assessment of pesticide residues in food.
Consequently, the opinion was further limited to the possible impact of dose addition in cases of
consumer exposure to combinations of pesticide residues, and part of the opinion was devoted to the
identification of CAGs for which dose addition might be anticipated. In particular, it was assumed that
mixture effects for substances acting via a common mode of action might be predicted by dose
addition. The PPR Panel endorsed a step-wise approach for grouping of substances within cumulative
assessment groups, as outlined in the following:
1.
2.
Preliminary identification of a candidate set of substances that might cause a common toxic
effect by a common mode of action. This preliminary grouping is based on one or more of the
following criteria:
a.
Chemical structure
b.
Mechanism of pesticidal action
c.
General mode/mechanism of action
d.
Particular toxic effect. It is conceivable that similar toxic effects by different compounds
might be caused via a common mode/mechanism. This criterion might allow the
identification of structurally unrelated substances that act by the same mode of action.
Identify those substances from Step 1 that cause a common toxic effect. This step allows a first
refinement of the preliminary grouping described above.
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Relevance of dissimilar mode of action
3.
Determine the toxic mode/mechanism of action by which each substance causes a common
toxic effect.
4.
Compare the mechanisms of toxicity/modes of action of the different substances
5.
Refine grouping by excluding substances that cause a common toxic effect by a different
mechanism/mode of action.
The Panel agreed that full consideration of all of these criteria would provide the most robust
grouping. However, since such a detailed evaluation up to the last step might not be necessary or
possible in all cases, for the purpose of cumulative risk assessment, substances might be grouped on
the basis of a less refined evaluation of the mode of action, e.g. based only on target organ toxicity. In
addition, it was acknowledged by the Panel that there was evidence in the literature, particularly for
some substances interacting with the endocrine system, that dose additivity might occur even if the
individual substances in the mixture do not share the same molecular target, but contribute to a
common adverse outcome (e.g. substances acting by different anti-androgenic modes of action
(Kortenkamp et al., 2007 (and papers reviewed therein: Gray et al. 2001; Hotchkiss et al., 2004)). It
was considered that in such cases, “the criterion for grouping should rather be that of a common
phenomenological effect” (EFSA, 2008).
In a next step, the PPR Panel tested the methodology proposed in the preceding opinion (EFSA, 2008)
by preparing a worked example of a dietary risk assessment for the group of triazole pesticides (EFSA,
2009). This exercise dealt with both hazard and exposure assessment, yet it was not intended to
provide a complete assessment of cumulative risks to human health for the combined triazole group.
Regarding establishment of cumulative assessment groups, 26 compounds were initially identified as
sharing a common toxicophore, the triazole ring, which is responsible for a common mechanism of
pesticidal action (inhibition of the fungal cyp51 enzyme and thus inhibition of ergosterol synthesis). A
subgroup of triazoles causing craniofacial/brain malformations was used for further acute cumulative
risk assessment, assuming a common mode of action for these effects. Since liver is a common target
for triazoles, hepatotoxicity, involving for example liver hypertrophy or changes in activities of
cytochrome P450 enzymes, was used as an endpoint for chronic cumulative risk assessment on a
subset of triazoles. It was noted, however, that for a full regulatory assessment, all of the triazoles
causing hepatotoxicity would need to be considered in the CAG. It is worth noting that, although
mechanisms of hepatic toxicity had not yet been fully characterised and multiple effects had been
observed, the Panel concluded that there was at that time “no evidence that these compounds would
not act in a dose-additive way regarding hepatotoxicity”. Thus, for the purpose of this particular
exercise, cumulative assessment groups were defined by common chemical structure, common
pesticidal mode of action and common toxicological effect, and thus by applying a selection of the
criteria proposed by the previous opinion (EFSA, 2008) for grouping.
In the triazole opinion, the PPR Panel regarded the establishment of relevant CAGs as the starting
point for all dietary cumulative risk assessments. One of the conclusions of the opinion was that the
basis for and establishment of relevant CAGs at the European level should be resolved.
The issue of establishing CAGs was further pursued in the Scientific Opinion of the PPR Panel on the
identification of pesticides to be included in cumulative assessment groups on the basis of their
toxicological profile (EFSA PPR Panel, 2013).
Recognising that grouping based on common chemical structure or on definite information on modes
of action might miss substances acting via a similar mode of action and contributing to a common
effect, the opinion elaborated a general methodology and criteria for establishment of cumulative
assessment groups for pesticides, based on phenomenological effects. The methodology was
developed to support assessment of cumulative risk resulting from dietary exposure and was thus
prepared on the basis of toxicological data sets of oral toxicity studies evaluated in draft assessment
reports (DARs). The approach was initially applied to establish CAGs for pesticides having effects on
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Relevance of dissimilar mode of action
the thyroid and nervous system, but was deemed to be generally applicable to all organs and organ
systems. Further preparatory work was also carried out to provide a basis for defining CAGs for other
organs/organ systems in the future.
Key steps for grouping on a phenomenological basis involved the identification and characterisation of
common specific and unambiguous toxicological effects for an organ or organ system. It was explored
whether further refinement of grouping might be possible, based on the actual information on modes
of action. In the context of toxicological data evaluation by the PPR Working Group, however, it
became evident that many compounds might affect the same target organ and/or cell population, but
that often only few or no data were available on modes of action. As there appeared to be only a
limited opportunity for refinement of CAGs on the basis of current mode of action information, all
pesticides were included in a specific effect CAG, for which dose additivity would be assumed for
cumulative risk assessment, even if the underlying mechanisms of toxicity/modes of action were
unknown. It follows that such CAGs would be expected to also include pesticides acting via dissimilar
modes of action. In line with the previous opinion (EFSA, 2008), the Panel recognised that there may
also be cases in which different substances may act via different molecular mechanisms, but may have
a common joint effect in target organs/organ systems which might be approximated by dose addition
(Kortenkamp, 2007 and papers reviewed therein: Gray et al., 2001, Hotchkiss et al., 2004; Hass et al.,
2012; Jacobsen et al., 2012). Although the PPR Panel noted in its CAGs opinion (EFSA PPR Panel,
2013) that “… further refinement of grouping may be achieved when data on the precise toxicological
mode of action may be available”, it pointed out that “information that justifies any deviation from
dose-addition might also be necessary to consider for such a refinement”.
As further progress in hazard assessment for cumulative risk assessment, and taking into account
lessons learnt from the previous EFSA opinions (EFSA, 2008, 2009, 2013), the present opinion
focuses on the relevance of dissimilar mode of action and its implications for cumulative risk
assessment of pesticides residues in food.
2.3.
Summary of the supporting project
In support of refining hazard assessment for dietary cumulative risk assessment and of preparation of
the present opinion, EFSA commissioned a project on the “Investigation of the state of the science on
combined actions of chemicals in food through dissimilar modes of action and proposal for sciencebased approach for performing related cumulative risk assessment”. The final report to the project was
published in January 2012 (Kortenkamp et al., 2012). The specific objectives of the project were to:
Gather information and to provide a review on the state of the science on combined actions of
chemicals acting via dissimilar mode of action. This review was to include endocrine active
substances, since a number of previous papers in the literature have dealt with combination
effects of substances interacting with parts of the endocrine system. In order to obtain wider
information, the review was to be applied to chemicals present in food in general, and not
restricted to pesticides.
Elaborate general criteria for establishment of CAGs of pesticides and chemicals in or on food
acting through dissimilar modes of action leading to a common effect.
Propose a science-based approach for performing cumulative risk assessment of chemicals in
food acting through dissimilar modes of action.
Identify research needs to improve the understanding of the relevance of independent action
for mixture toxicity.
Accordingly, the authors of the report performed a systematic literature search to identify relevant
experimental studies on combined actions of chemicals, focussing on “independent action” and
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Relevance of dissimilar mode of action
synonyms. This initial search was complemented by further ad hoc searches. The literature retrieved
up to May 2011 was compiled within a database (CRADIS, Cumulative Risk Assessment of
Dissimilarly Acting Chemicals), which was also populated with relevant articles from previous key
reports, e.g. from the State of the Art Report on Mixture Toxicity (Kortenkamp, 2009).
The database served as a basis for a summary and assessment of the state of the science. It included
174 experimental studies dealing with mixtures, of which 94 studies were classified as being relevant
for dissimilarly acting substances. 27 studies were classified as having relevance for mixture effects at
low doses. Although different definitions of low dose were used in the literature, low dose was related
to doses/concentrations at or below the NOAEL in the majority of studies. Fourteen mixture studies
were identified as being of dual relevance to both chemical dissimilarity and mixture effects at low
doses and were analysed in more detail concerning mixture design and results.
In this context, the ecotoxicology literature was found to contain some examples for which the
experimentally demonstrated effects of mixtures validated the independent action (IA) model,
including a study of 16 biocides whose combination effect on algal toxicity was accurately predicted
by IA (Faust et al., 2003). In this case, the effect predicted by the dose addition (DA) model was
greater than that under IA, and so DA could be considered the more conservative risk estimate,
supporting the use of DA as the default concept in assessment of mixtures for CRA. By contrast, no
example for validity of a prediction following IA was identified in the mammalian toxicology
literature, which was deemed to be partially due to the difficulties in optimising mammalian studies to
allow the predictions of IA and DA to be distinguished from one another. Overall, the authors
concluded that a situation when IA provided an accurate prediction that was also more conservative
than that provided by DA could not be identified in the literature. Further analysis of the quantitative
differences between DA and IA predictions, based mainly on ecotoxicological test data and involving
mathematical analyses as well as simulation studies, revealed that such differences might be expected
to be relatively small, generally not exceeding one order of magnitude.
In addition to evaluation of DA and IA models for the prediction of mixture effects, a number of new
approaches, including mixed models, were summarised in the report. Although the mixed models
combine both DA and IA as underlying concepts, it was concluded that data and method gaps
currently limit implementation of such mixed models in practice.
It was considered that the presence of significant interactions, particularly synergisms, could question
the use of any additivity concept, including DA, as a default in cumulative risk assessment. However,
based on the evidence analysed within a critical literature review (Boobis et al., 2011), indicating that
significant synergisms can be considered “not likely to occur at the low doses permitted under existing
exposure standards”, the suitability of DA as a conservative default was regarded as being unaffected.
Grouping of chemicals for cumulative risk assessment based on the narrow criterion of chemical
similarity was regarded to potentially underestimate risks by excluding substances from the analysis
that might nevertheless contribute to common effects, even by different modes of action. With respect
to mode/mechanism of action, it was recognised that only limited information on mode of action was
included in the studies analysed in the supporting project. In fact, it was noted that concepts such as
mode of action were not framed to guide the choice between different mixture concepts, since even for
different modes of action, information on actual or plausible independence of action, which would be
the key criterion for selecting IA over DA as a prediction model, was generally not available. In
addition, the authors referred to experimental evidence that DA produces reliable estimations of
combination effects for mixtures of chemicals with diverse modes of action (Crofton et al., 2005;
Christiansen et al., 2009; Rider et al., 2010). Accordingly, for the purpose of grouping of substances
based on their toxicological profile, the authors of the supporting project recommended to apply
phenomenological grouping criteria based on a sound understanding of physiological processes, and to
consider all substances contributing to a common adverse outcome for inclusion within a cumulative
assessment group.
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Relevance of dissimilar mode of action
In the context of a review of approaches to cumulative risk assessment methodology, it was noted that
almost all cumulative risk assessment methods in current use are applications of the concept of dose
addition, while methods explicitly derived from independent action are not developed. Thus, the only
practical approach for the use of IA for CRA would appear to be the pragmatic assumption that
mixture effects will not occur if each component is below its individual effect level. However, the
authors noted that a prerequisite for this application would be the identification of dose/concentration
levels without effect, which might be difficult. In particular, it was considered that precursor effects,
e.g. biochemical changes on the molecular level at low doses, might accumulate to exceed
homeostasis, and thereby contribute to overt apical effects in certain cases of exposure to mixtures.
The overall conclusion of the project was that it is feasible and justified to utilise CRA methods and
tiered framework analyses originally developed for similarly acting mixtures also for combinations of
dissimilarly acting substances, and that DA could be regarded as being sufficiently conservative to
serve as a default concept also for the evaluation of mixtures of dissimilarly acting chemicals.
Consequently, one unifying approach was proposed by the authors of the report for dealing with
mixtures in regulatory practice, irrespective of (often presumed) modes of action.
This approach involved a tiered framework. It was suggested that at lower tiers, the grouping of
chemicals for the purpose of cumulative risk assessment should be driven by their co-occurrence in the
exposure scenario under investigation, while at higher tiers, the substances leading to a common
adverse outcome should be grouped together. In the course of the review of the report of the
supporting project, and during elaboration of the opinion on CAGs (EFSA PPR Panel, 2013), the PPR
Panel noted that a tiered, exposure driven approach, would be difficult to apply in the context of
establishing MRLs, based on the consideration that in principle, every authorized pesticide may occur
in a food item or within the diet. It was therefore concluded by the Panel that grouping of pesticides in
CAGs should be based on intrinsic properties of the substances, rather than being driven by first tier
exposure assessment.
Concerning the identification of data gaps and further research needs, the authors of the report pointed
out that it should be explored whether a reference case demonstrating the validity of IA for the
prediction of experimental mixture effects could also be established for endpoints relevant for
mammalian and human toxicology, as has been done in some ecotoxicological models. Furthermore, it
was recommended that databases comprising concentration/dose-response relationships for
ecotoxicological and mammalian toxicological endpoints should be established to evaluate and verify
prediction differences between IA and DA models. Finally, it was noted that information requirements
detailed in important pieces of regulation are currently not geared towards meeting the standards
necessary for the conduct of higher tier CRA analyses, and that this concerned particularly a lack of
data for adverse endpoints that are less sensitive than the endpoints critical for establishing the
reference values, e.g. Acceptable Daily Intakes (ADIs).
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Relevance of dissimilar mode of action
3.
Combined effects of chemicals and the relevance of dissimilar modes of action
It is intuitively obvious that combinations of chemicals which act through identical mechanisms will
produce combined effects. Well-studied examples include the polychlorinated dioxins, furans and
biphenyls which exert their toxicity by binding to the Ah receptor (USEPA, 2008; Van den Berg et al.,
2006), Similar examples include substances that can activate or antagonise steroid hormone receptors
(Kortenkamp, 2007) or chemicals that interfere with the action of specific enzymes such as
acetylcholinesterase (USEPA, 2002). A more recent example is the induction of micronuclei by
substances that disrupt microtubule polymerisation by the same mechanism, binding to microtubulin
monomers (Ermler et al., 2013a). In most of these cases, the need for considering combination effects
in risk assessment and regulation is widely recognised and has led to quite elaborate risk assessment
approaches, for example the TCDD equivalence factor concept (Van den Berg et al., 2006).
Perhaps more difficult to judge is whether combinations of chemicals with different modes of action
will produce joint effects. This is not helped by difficulties distinguishing between mode of action and
mechanism of action. It is sometimes thought that joint effects are not to be expected from substances
with different mode of actions. As briefly discussed above, this may be correct for mixtures composed
of substances that affect entirely different physiological systems, with common adverse outcomes
either not materialising or not becoming obvious. However, scientific evidence has accumulated and
shows that combination effects can also arise from multiple chemicals that produce common adverse
outcomes by interfering with a variety of different cellular systems, target organs or physiological
processes. Early examples include studies with algal toxicants and their effects on algal reproduction
through interaction with a multitude of cellular targets and processes (Faust et al., 2003). In
mammalians, disruption of male sexual differentiation can be induced by combinations of chemicals
that act on a variety of molecular targets, including the androgen receptor and various steroid
metabolising enzymes (Christiansen et al., 2009; Rider et al., 2010). Maintenance of sufficient levels
of foetal androgens is key to proper sexual differentiation which depends on the programming effects
of the hormone. Androgen action can be disrupted in many different ways and by a variety of modes
of action which then result in common adverse outcomes (USNRC, 2008). Similar inter-relations
between important physiological systems exist during processes leading to thyroid hormone system
disruption, which includes interactions not only with thyroid hormone receptors, but also with various
cellular transporters and metabolising enzymes in the liver (Crofton et al., 2005). Another example
includes chemicals that produce micronuclei by diverse mechanisms of action including blocking of
microtubule polymerisation, interference with the depolymerisation of microtubuli, by forming DNA
adducts or by inducing chromosome breaks. All these different mechanisms can contribute to produce
joint effects on micronuclei (Ermler et al., 2013b).
There is good evidence that combination effects can arise from co-exposure to chemicals that produce
common (adverse) outcomes through entirely different modes of action and mechanisms, and this also
applies to combinations of pesticides (Faust et al., 2003; Crofton et al., 2005; Christiansen et al., 2009,
2012; Rider et al., 2010). Restricting regulatory approaches solely to combinations of chemicals that
produce mixture effects by affecting the same molecular domains or targets would therefore ignore a
wide variety of ways in which combination effects can arise. Thus, in response to the first item of the
terms of reference provided by EFSA, the PPR Panel states that dissimilar modes of action are highly
relevant to the assessment of cumulative risks from pesticide residues in food.
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4.
Analysis of empirical evidence for the applicability of independent action to the prediction
and assessment of chemical mixtures
The Panel analysed the scientific literature on combination effects of chemicals with respect to four
questions:
Is there empirical evidence that independent action provides accurate predictions of the effects
of mixtures composed of dissimilarly acting chemicals, in situations where dose addition and
independent action yield separable predictions?
Are there clear cut criteria based on MoA information that can guide the choice between
independent action and dose addition for evaluating mixture effects?
Are there cases where independent action produced predictions that were more conservative
than those derived from dose addition, and were accurate at the same time?
Is there empirical evidence for combination effects of dissimilarly acting mixtures at low
doses, around no-observed-effect-levels and below?
In view of the generic nature of these issues, the Panel considered the relevant literature, not only
papers dealing with pesticide residues in food. This also included the ecotoxicological literature
(Kortenkamp et al., 2009, 2012).
4.1.
4.1.1
The applicability of independent action as a prediction concept for mixtures composed
of chemicals with dissimilar modes of action
Literature survey on empirical evidence in support of the validity of independent action
The applicability of independent action for predicting the effects of mixtures composed of dissimilarly
acting chemicals has been demonstrated in experiments with multi-component mixtures (up to 16
components) in bacteria and algae (Backhaus et al., 2000; Walter et al., 2002; Faust et al., 2003). In
these studies, substances with a wide variety of chemical structural features, and established dissimilar
modes of action were combined. These experiments were decisive, because predictions of additive
combination effects derived from dose addition were evaluated in parallel. The experimentally
observed combination effects agreed with the responses derived from independent action. Dose
addition anticipated higher effects than independent action and was therefore the more conservative
model. If these findings were generally applicable there would be reassurance that even in cases when
independent action is valid, dose addition can still provide a conservative risk estimate, supporting the
use of dose addition as the default concept in mixture risk assessment.
The Panel was unable to locate a proof-of-principle example of the validity of independent action with
mammals, mammalian cells (in vitro) or with multi-cellular organisms (see also Kortenkamp et al.,
2009, 2012). The reasons for this can at present only be speculated on, but one factor can be seen in
the difficulties of performing studies with mammals in vivo with a sufficiently large number of
mixture components (which is required to allow the predictions of independent action and dose
addition to be distinguished from each other). Another difficulty is in selecting an appropriate
common specific effect for which a sufficiently large number of chemicals with well-characterised and
strictly different specific modes of action is known. The Panel also notes that the number of chemicals
relevant to cumulative exposures is likely to be larger than the number of distinct, strictly independent
modes of action, such that there are likely to be several chemicals operating via the same mode of
action or interrelated modes of action, thereby violating the principles of strict independence of action.
Another point to consider relates to chemicals that affect different elements of signal pathways.
Especially with pathways that converge on a nodal point, independence of action may not apply even
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Relevance of dissimilar mode of action
though all chemicals in the mixture affect different steps of the pathway up-stream of a nodal point.
All these issues may be reasons why a proof-of-principle example of independent action has not yet
come to light with mammalian test species.
4.2.
The role of mode of action information in guiding a priori choices between independent
action and dose addition for evaluating mixture effects
The mixture assessment concept of independent action conceptualises combination effects of
chemicals in terms of random independent effects. The idea of independence of effects becomes
accessible by considering first the case of sequential administration of one and the same chemical. The
overall effect of sequential administrations (which are independent events) can be calculated by
multiplying the probabilities of effect of each individual application, as is familiar from stochastics
(Bliss, 1939; Hewlett and Plackett, 1952). Berenbaum has popularised these principles by choosing the
analogy with throwing pebbles at eggs (Berenbaum, 1985). If we assume that throwing one handful
(dose unit) of pebbles destroys 50% of the eggs, and assume further that eggs and pebbles are
distributed perfectly randomly, then administration of a second dose unit of pebbles will eliminate
50% of the eggs that remained intact after the first throw. Thus, after two administrations, 25% of the
original number of eggs will be left. The two throws are separate from each other and therefore
independent events. It is important to realise, that in this example the principles of independence are
fulfilled, despite the fact that the “mode of action” of pebbles is exactly the same during sequential
administration. Thus, independent action is not necessarily always associated with dissimilarity of
modes of action. For scenarios involving simultaneous exposure to multiple chemicals, however, the
principles of independence of action can only be met with combinations that elicit common effects
through strictly dissimilar modes of action. This has been demonstrated for combinations of chemicals
that operate by dissimilar mechanisms, and at different sites (Faust et al., 2003). If some chemicals
produce the effect by similar mechanisms, the principles of independence of action are not met
anymore, and it is likely that observed mixture effects deviate somewhat from those predicted by the
independent action concept. A recent paper examining the joint effects of several substances
producing micronuclei through different modes of action has given indications that the number of
different mechanisms available for the induction of this effect is smaller than the number of chemicals
capable of producing this kind of toxicity (Ermler et al., 2013b).
The Panel analysed several sources of guidance on mixture risk assessment, with the aim of
identifying criteria that might be used to establish whether dissimilar or similar action should apply to
a mixture of interest. The two key concepts that can feature in guiding decisions about the
applicability of independent action or dose addition are mode of action and mechanism of action.
Although a consistent and uniform use of the terms is not always practiced, these concepts have been
defined as follows:
Mode of action (MoA): a “biologically plausible sequence of key events leading to an observed effect
supported by robust experimental observations and mechanistic data” (Boobis et al., 2006).
Mechanism of action: “a sufficient understanding of the molecular basis for an effect and its detailed
description so causation can be established in molecular terms” (Boobis et al., 2006) or “a detailed
explanation of the individual biochemical and physiological events leading to a toxic effect” (EFSA,
2008).
Preston and Williams (2005) have provided an example of a MoA definition, mapping out the key
events for a DNA-reactive chemical leading to cancer. These include exposure of target cells to DNA
reactive species, reaction with DNA to produce adducts or other damage, misreplication or misrepair
of the DNA damage, mutations in critical genes, clonal expansion of mutant cells with further
mutations in critical genes, imbalanced and uncontrolled clonal growth of mutant cells leading to
preneoplastic lesions, progression of preneoplastic cells and emergence of overt neoplasms.
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Relevance of dissimilar mode of action
This example shows that even a MoA can contain a significant amount of detail, and illustrates the
data demands that would have to be met to systematically analyse multiple chemicals with multiple
modes of action. If mechanisms of action are used as a basis for consideration, the situation may
become even more demanding. A fully elucidated mechanism of toxicity is only available for a few
chemicals, one example being cyanide, whilst more chemicals have a known MoA, with key events
that are “known, measurable, necessary and consistent” (Carmichael et al., 2011). The studies
necessary for an identification of a MoA, let alone a mechanism of action, are not currently a
regulatory requirement for pesticide active substances or industrial chemicals. The Panel predicts that
this situation is unlikely to change in the foreseeable future, thus compromising the practicability of
using MoA information as the starting point for cumulative risk assessment, with a high likelihood of
encountering situations where lack of information will block further assessment.
Frameworks for assessing the relevance of modes of action for cancer (Boobis et al., 2006) and noncancer endpoints (Boobis et al., 2008) have been developed by IPCS. These frameworks apply a
weight of evidence approach based on the Bradford Hill criteria for causality to evaluate a proposed
MoA and its relevance to humans. Such frameworks provide a mechanism to establish whether the
MoA of a chemical has been described previously for other chemicals or whether it is distinct from
known MoAs. Of relevance to cumulative risk assessment, the systematic use of such frameworks, and
MoAs arising from, or validated by them, might provide the basis for developing a set of MoAs
founded on the same principles. This might facilitate systematic comparisons, with a view to using
sets of MoA as a more reliable basis for the grouping of chemicals for cumulative risk assessment. It
was suggested that a database of generally accepted MoAs and informative cases should be
constructed and maintained (Boobis et al., 2006). This need has been recognised by others, for
example McCarty and Borgert considered that “the absence of any generally accepted classification
scheme for either modes/mechanisms of toxic action or of mechanisms of toxicity interactions is
problematic as it produces a cycle in which research and policy are interdependent and mutually
limiting” (McCarty and Borgert, 2006). The Panel recognises that this issue constitutes a substantial
knowledge gap.
In any case, it has to be emphasised that the above considerations of MoAs, as useful as they are in
other regulatory contexts, are not geared towards concluding on whether independent action would be
the appropiate assessment concept. This raises the question as to whether mode of action frameworks
as detailed by Boobis et al (2006) and EFSA (2008) are suitable for the purpose of deciding on the
applicability of independent action and dose addition. One complication is that the widely used
definitions of mode of action do not define when a mode is sufficiently separate or distinct from
another mode to support an assessment in favour of using independent action. However, this aspect is
critical for the selection of a mixture assessment concept, independent action or dose addition. It
therefore appears that the MoA definitions are not informative in terms of making the case for
independent action. This is further complicated by the fact that a single chemical can have different
modes of actions. It is therefore conceivable that the same combination of chemicals can act similarly
and dissimilarly.
In view of these conceptual difficulties, the Panel considered whether the choice between the two
mixture assessment concepts can be supported empirically, by performing a mixture experiment, with
the aim of making inferences about the similarity or dissimilarity of the components of a mixture on
the basis of whether its results are closer to the independent action prediction (infer dissimilarity) or
the dose addition prediction (infer similarity). Some authors have taken this approach (reviewed in
Kortenkamp et al., 2012), which illustrates one possible response to the difficulties in assigning
similarity and dissimilarity a priori, namely that the assignment is done post hoc. The usefulness of
this empirical approach for cumulative risk assessment in a regulatory context is however limited,
because it requires experimental data for the mixture of concern, which is usually not available.
Of particular relevance to the issue of making choices between independent action and dose addition
are cases where the applicability of independent action could have plausibly been expected, but which
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were shown to follow dose addition or hybrid models combining dose addition and independent action
(Christiansen et al., 2009; Rider et al. 2008, 2010; Ermler et al., 2013b).
4.3.
Conservatism of dose addition compared to independent action
No situation when independent action was both more conservative than dose addition, and also
provided accurate predictions of mixture effects was identified in the literature. The factors that
determine prediction differences between independent action and dose addition, and the conditions
when independent action predicts stronger effects than dose addition, are well understood and are
discussed further in section 5. The Panel regards the putative situation where independent action yields
predictions that are accurate and more conservative than dose addition as an important one to consider,
because of the obvious implications for the use of dose addition as a conservative default.
Several papers have begun to use hybrid approaches to evaluating the combined effects of
experimental mixtures, where subgroups of chemicals with similar modes of action were assessed
according to dose addition, and the group-wise predictions finally aggregated by using independent
action (see for example Ermler et al., 2013b and Rider et al., 2005). This has often successfully
resolved situations where the observed combination effects fell between the prediction window
defined by dose addition and independent action. However, the applicability of such hybrid
approaches to risk assessment practice will be severely constrained by the associated data demands
which are rarely met in practice (see section 6 below).
4.4.
Dissimilarly acting mixtures at low doses
The mathematical formulations of independent action (listed in Kortenkamp et al., 2012) imply that
agents present at doses associated with zero effects will not contribute to the joint effect of the
mixture, provided they act in a strictly dissimilar fashion. If this condition is fulfilled for all
components in the mixture, combination effects are not expected according to the principles of
independent action.
However, attempts to prove this notion experimentally are complicated by significant practical
difficulties. This stems from the fact that zero effect levels cannot be established empirically, on
grounds of principle (discussed e.g. by Slob, 1999) which means that claims of absence of mixture
responses are limited by the statistical power of the experiment, i.e. by the ability to demonstrate very
small effects. Especially with mixtures composed of large numbers of components, this demand
usually exceeds the power of toxicological studies which normally struggle to resolve effects of a
magnitude in the order of 5-10%. The issue can be illustrated by considering the mathematical
expression of independent action as it applies to simultaneous exposures to several chemicals that
show ascending dose-response curves (equation 1):
n
E(c mixture ) 1
(1 E(ci ))
i 1
Here, E(cmixture) is the total effect of the mixture when all chemicals are present at a mixture
concentration (or dose) cmixture which is the sum of all individual concentrations (doses) of all mixture
components. Π is the multiplication sign and E(ci) denotes the effect caused by the individual
compound i present at concentration (dose) ci . The individual effects of mixture compounds E(ci) are
calculated from the concentration-response relationships of each mixture component.
According to this formula, 10 components at doses associated with 5% effect will already produce a
combination effect of 45%. Correspondingly, 100 agents with a 1% effect are expected to produce a
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mixture effect of 63%, and with 100 chemicals each producing a 0.1% effect the expected joint
response under independent action will still be 9.5 %. Such small effects can only be demonstrated
with astronomically large numbers of animals (see for example Williams et al., 2009). There have
been attempts with bacterial and algal tests to empirically determine joint effects according to
independent action when all mixture components are present at effect doses associated with very small
effects, in the order of 1% (Backhaus et al., 2000; Walter et al., 2002; Faust et al., 2003). In all these
cases significant mixture effects occurred, accurately predicted by independent action, of a magnitude
of up to 20% effect.
4.5.
Relevance of interactions for cumulative risk assessment
The Panel is aware that this Opinion is not dealing with interactions which represent situations where
some or all components present in a mixture influence each other’s toxicity, e.g. by inducing
metabolic steps that toxify (or detoxify) another component. These so-called toxicological interactions
may give rise to deviations from expected additivity, either in terms of synergisms or antagonisms.
Especially potential synergisms are of concern during cumulative risk assessment. If overlooked, the
actual combined toxicity may be greater than that expected on the basis of the effects of the mixture
components that form the basis of the assessment. Based on reviews by Kortenkamp et al. (2009) and
Boobis et al. (2011), it can be assumed that synergism is less likely to occur at low doses
corresponding to dietary exposure. In particular, Boobis et al. (2011) identified studies demonstrating
synergism in mammalian test systems. Most of the studies used individual doses/concentrations
eliciting toxic effects, though some studies were identified that showed synergism at
doses/concentrations of mixture components close to (at or near) the individual points of departure
(e.g. NOAEL) (Crofton et al., 2005; Christiansen et al., 2009). For the studies that allowed quantitative
estimates of synergy, “the magnitude of synergy at low doses did not exceed the levels predicted by
additive models by more than a factor of 4”. It was also noted that “in a number of the positive studies,
the occurrence of synergy was dose dependent and observed only at the higher doses in the study”.
Finally, it was concluded that “…there is probably merit in the default regulatory approaches that
assume toxicological interactions are not likely to occur at the low doses permitted under existing
exposure standards. However, it is too early to draw firm conclusions, particularly for cumulative and
low level chronic exposures.” (Boobis et al., 2011). This provides a degree of reassurance that risks
arising from interactions might be rather rare. Nevertheless, the Panel notes a deficit in understanding
comprehensively the determinants of interactions which in turn demands more research efforts.
Concepts that would allow quantitative predictions of magnitudes of interactions are currently not
available.
4.6.
Summary of chapter 4
There are currently no examples where independent action produced experimentally confirmed
predictions that were more conservative and accurate than those derived from dose addition. Available
data on MoA are not sufficient to guide the choice between dose addition and independent action
models. The use of the assessment concept of dose addition is thus considered by the Panel as the most
appropriate for cumulative risk assessment even when dealing with substances acting with different
modes of action. The Panel also notes that dissimilarity cannot be inferred from a lack of compliance
with criteria for similarity. Taken together, these difficulties lead to the questions: What is the
importance of making choices between independent action and dose addition, considering that such
choices are often difficult to make? What is the impact on regulatory decisions of having to use one or
the other concept?
These questions have led the Panel to consider the issue of quantitative differences in mixture effect
predictions derived from independent action and dose addition.
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5.
Quantitative differences between mixture effect predictions derived from independent
action and dose addition
The WHO/IPCS framework on cumulative risk assessment (Meek et al., 2011) has proposed the use of
dose addition as a default tier zero assessment concept for all mixture components that occur together
in an exposure scenario of concern and may contribute to a common adverse health outcome. This is
supported by the notion that the assumption of dose additivity generally provides a more conservative
estimate of mixture toxicity than the alternative assumption of independent action. Synergistic effects
that exceed the dose addition expectation are exceptions and not the rule, at least for multi-component
mixtures (Kortenkamp et al., 2009; EFSA, 2008).
There are concerns that the general application of dose addition may be conceptually flawed because it
could result in overly conservative mixture toxicity assessments, not scientifically justified and in
conflict with the principle of proportionality in the regulatory management of chemicals risks.
These concerns can only be addressed by analysing the quantitative prediction differences between
independent action and dose addition. Such an analysis has to determine the maximal quantitative
error that may result, if dose addition is applied in a situation where in fact independent action would
provide the correct mixture toxicity estimate.
5.1.
Empirical findings of prediction differences
In published experimental studies with multi-component mixtures, the quantitative differences
between independent action and dose addition predictions were found to be remarkably small. For
different types of mixtures with up to 20 components, predictions of EC50 or ED50 values derived from
the two models differed by no more than a factor of 5 (Kortenkamp et al., 2009). This would support
the idea that the quantitative differences between independent action and dose addition are of little
relevance in a regulatory context.
However, this position can be challenged on grounds of principle: The reported (small) differences
may not represent realistic exposure and assessment situations, and they may be restricted to the
mixtures, conditions and toxicity endpoints that have been investigated, and therefore cannot be
generalised.
Thus, a consensus about the general application of dose addition as a default in cumulative risk
assessment will have to be based on robust arguments that can dispel concerns that dose addition is
overly conservative. The Panel therefore considered whether the empirical evidence of relatively small
prediction differences between independent action and dose addition represents a general rule or
whether it is applicable only to special situations. The Panel recognised that the issue cannot be
resolved by experimentation, but requires conceptual approaches founded in the mathematics behind
the two assessment concepts (Kortenkamp et al., 2012).
5.2.
Mathematical analysis
As established by Drescher and Boedeker (1995), the prediction differences between independent
action and dose addition are influenced by four factors: the number of mixture components, the
mixture ratio, the slope of the dose-response curves of the individual components, and the magnitude
of the combination effect considered for analysis.
Faust (1999) has addressed the question whether there are factors that limit the theoretically possible
prediction differences, here defined as the ratio of mixture effect dose according to independent action,
EDxIA and according to dose addition, EDxDA (see also Kortenkamp et al., 2012). Faust established that
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for a mixture with a given number of components n, the ratio EDxIA / EDxDA cannot take any value.
The effect doses predicted by independent action can never exceed the corresponding dose addition
prediction by a factor that is greater than n, i.e. the number of mixture components. Thus, for a twocomponent mixture a maximal difference of 2 applies, and for mixtures with ten components it can
never exceed 10. This means that for multi-component mixtures with very large numbers of
components the difference may still be quite large, if no restrictions apply to the other determining
factors.
Further analysis reveals that the maximum ratio EDxIA / EDxDA of n can only occur when all
components of the mixture are present at equal fractions of equi-effective doses, in other words,
contribute in equal measure to the overall mixture effect. In any other situation the ratio will always be
smaller than n. It can be shown that if one mixture component already contributes 50% to the total
sum of toxic units, the quotient ECxIA / ECxDA can never exceed a value of 2, no matter what the total
number of components and their toxic units may be (Junghans et al., 2006; Kortenkamp et al., 2012).
Independent of the limiting effect of a specific mixture ratio, the slopes of individual dose response
curves of a given set of components in a mixture also limit the possible range of the prediction ratio
EDxIA / EDxDA. The ratio EDxIA / EDxDA takes the value of 1, i.e. both concepts give exactly the same
prediction, if the dose response curves of all mixture components can be described by a specific form
of the Weibull function (Drescher and Bodeker, 1995. See equation 2), with a specific value for the
slope parameter. If all dose response curves are steeper than expressed by this parameter, dose
addition always predicts effects larger than independent action. The maximum possible ratio
EDxIA / EDxDA tends towards the maximum value of n (= number of components) if all curves become
infinitely steep. Conversely, dose addition predicts smaller mixture effects than IA, and the lowest
possible ratio EDxIA / EDxDA tends towards zero, if the individual dose response curves of all mixture
components become increasingly flat. This situation is of particular interest in relation to situations
where independent action might produce predictions that are more conservative than dose addition. It
can only happen when all components exhibit very shallow dose-response curves. However, the Panel
recognised that the “infinitely steep” and “increasingly flat” dose responses are unlikely to be
biologically plausible.
Equation 2, Weibull function:
E(di)=1-exp(-exp(αi + ln(10)●log(di)))
In which the general slope parameter βi has the special value of ln(10) (=2.3025...), while αi is a
location parameter that has no effect on the ratio EDxIA / ECxDA.. E(di)= effect dose
In situations where the mixture components exhibit curves of varied steepness, large prediction
differences are unlikely to occur. Whether independent action or dose addition predict the larger
combination effect depends strongly on the mixture ratio and the effect level considered for analysis
(for further details and illustrative examples see Kortenkamp et al., 2012).
The finding that the steepness of individual dose response curves is a crucial limiting factor for the
possible prediction differences raises the question: what distributions of slope values for dose response
curves are encountered empirically?
To address this question, simulation studies based on empirical dose-response data for algal toxicity
were conducted by Kortenkamp et al. (2012). This was done because dose-response data of
comparable quality are not available in the literature for endpoints relevant to mammalian toxicity.
These analyses revealed that for any mixture that could be generated from a set of 106 chemicals the
ratio EDxIA / EDxDA never exceeded 8.3, considerably smaller than the theoretical maximum value of
106.
Further probabilistic analyses showed that dose addition usually provides the more conservative
mixture toxicity estimate. The likelihood for the alternative situation, i.e. that IA predicts higher
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combined toxicity than DA, was found to be low. It tended towards zero as the number of mixture
components increased. Furthermore, a ratio EDxIA / EDxDA greater than 4.2 was never observed in
these simulations.
The Panel noted that these analyses were based on ecotoxicological test data. Data of comparable
quality, required to conduct such analyses, are not available for mammalian toxicity endpoints.
However, the few available data from mammalian toxicity studies (Christiansen et al., 2008, 2009;
Hass et al., 2007; Metzdorff et al., 2007) show that these dose response curves show ranges of slopes
also found in ecotoxicological data. Thus, there are good reasons to assume that small prediction
differences are also to be expected with mammalian toxicology test data.
5.3.
Summary of chapter 5
The available empirical evidence with mixtures of up to 20 components shows that prediction
differences between IA and DA both for mammalian and ecotoxicological studies were not greater
than a factor of 5. Mathematical analyses and findings from simulation studies based on
ecotoxicological endpoints show that quantitative differences between predictions of the combined
effects of multi-component mixtures derived from independent action and dose addition typically only
differ by a factor of less than 5 for mixtures with up to 100 components. Considering the variability
and sources of error normally encountered in realistic assessment situations, this difference is rather
small, less than one order of magnitude.
The current state of the science of mixture toxicology, and prediction differences between independent
action and dose addition supports the use of dose additivity as a pragmatic and conservative default
approach to the assessment of chemical mixtures.
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6.
Difficulties in applying independent action in regulatory practice
The evaluation of experimental data describing the combined effects of chemicals has to be
distinguished from approaches employed for conducting cumulative risk assessment in practice, here
termed cumulative risk assessment methods.
The application of cumulative risk assessment methods requires clarity about the goal of the
assessment. The aim can be to arrive at a risk estimate, an estimation of safe levels, of margins of
exposure, or can consist of ways of prioritizing certain mixtures, for further study or for regulatory
interventions. Cumulative risk assessment methods can also be applied to decisions relevant to the
authorisation of pesticides, such as decisions about Maximum Residue Levels (MRL) (EFSA, 2008).
Almost all cumulative risk assessment methods in current use are applications of the concept of dose
addition. These include the Hazard Index (HI), Toxic Unit Summation (TUS), Point of Departure
Index (PODI), Relative Potency Factors and the Toxic Equivalency (TEQ) concept. The features and
use of these methods has been summarised in Kortenkamp et al., 2009, 2012 and in EFSA, 2008,
2009).
Cumulative risk assessment methods explicitly derived from independent action have not been
developed to date. An implicit assumption of using independent action is that mixture effects will not
arise when all the chemicals in question are present at levels below their zero-effect levels. Verifying
the validity of this assumption, however, will always be hampered by difficulties in defining zeroeffect levels. The Panel notes that NOAELs from experimental studies cannot implicitly be seen as
zero-effect levels, but may actually be associated with effect levels that are only poorly defined in
terms of effect magnitudes (e.g. 1%, 5% or 20%). Although combination effects may be predicted by
the independent action approach for combined exposure to substances at low individual doses (and
thus also principally for substances producing minimal effects at or below their NOAELs), a
calculation of the combined effect requires the information on effect magnitudes. Although a
benchmark dose (BMD) approach would be expected to enable a more robust estimation of doses
associated with certain effect magnitudes to be used for calculation of joint effects, it does not solve
the problem of defining a zero-effect dose level.
6.1.
Simplified applications of independent action
The Panel considered simplified approaches to cumulative risk assessment based on independent
action that have been mooted. These are based on the notion that the mixture effect is equal to the
effect of the most potent component or that the mixture effect is equal to the summation of the effects
of the components. These approaches appear to rely on assumptions about the correlation of
susceptibility to the mixture components (Kortenkamp et al., 2012). Although these assumptions are
rarely stated explicitly and are hard to substantiate, the Panel analysed whether the simple use of effect
summation would be a practicable alternative to applications of dose addition in risk assessment
practice. To this end, the Panel evaluated the data requirements for such an approach.
6.2.
Data requirements for using independent action
The mathematical formulation of independent action uses single substance effects for predicting a
mixture effect. For small effects, this can be approximated by calculating the simple sum of individual
effects. However, this means that the data quality required for utilising this approach increases
substantially as the number of mixture components rises, as the following simple examples show:
According to independent action (see equation 1 and Appendix A), a binary mixture of two agents that
individually produce a 30% effect will lead to a 50% mixture effect (the effect summation
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Relevance of dissimilar mode of action
approximation predicts 60%). To be able to apply independent action in this way, the evaluator has to
use reliable measurement of 30% effects as input values, which usually does not present problems.
However, with a 10-component mixture that produces a 50% mixture effect, each component is
present at a dose associated with an effect of 6.7% (5% according to effect summation). Effects of that
magnitude are already at the borderline of what can be measured reliably in many in vivo toxicological
experiments. Furthermore, such assessments have to rely on dose-response data with good descriptions
of small effects. Such data are not available, not even for pesticidal active substances and are not
likely to be forthcoming in the foreseeable future.
The situation becomes increasingly demanding the more compounds are present in a mixture, because
ever smaller effects for the individual mixture components are required as input values e.g. for
estimating a 50% mixture effect. The assessment of mixture effects smaller than 50% increases the
data demands even further. The fact that increasingly lower individual effects for each component
need to be measured for calculating combination effects based on independent action (or effect
summation) is a serious drawback, as it increases experimental demands beyond what is technically
achievable with the number of animals per does group normally used in toxicity studies.
NOAELs are also not suitable as input data for independent action. NOAELs denote the highest tested
doses that do not produce statistically significantly different relevant adverse effects as compared to
untreated controls but they do not describe the magnitude of possible unobserved effects. Depending
on the resolving power of the chosen experimental arrangement, effects associated with NOAELs may
be quite large, but cannot be measured directly, and are only accessible through regression modelling
in dose-response analyses. However, the number of doses tested in studies that establish a NOAEL is
often rather limited and does not permit regression analysis. As a result, it is normally not possible to
establish whether a NOAEL is associated with a 5%, 10% or 20% effect. Consequently, the input data
required for using independent action are not accessible through reporting a NOAEL.
These difficulties do not arise with BMDs, because these are associated with pre-determined effect
magnitudes. For this reason, the use of BMDs is generally preferable as inputs to cumulative risk
assessments. The PPR Panel refers to the Scientific Opinion on the use benchmark doses which was
prepared by EFSA’s Scientific Committee (EFSA, 2009).
6.3.
Data requirements for utilising cumulative risk assessment methods based on dose
addition
To predict mixture effects on the basis of dose addition for an evaluation of experimental data,
information about the doses that induce the same specific effect are required for both the mixture and
all single components. For example, if the effect dose of a mixture leading to a 50% effect is known,
then the equivalent effect doses (ED50) for all mixture components need to be available to reach
decisions whether the combined effect is dose additive. The same requirements need to be met for any
other effect level. Usually, information about effect doses is accessible through dose-response analyses
of the individual components in a mixture.
As with independent action, NOAELs are strictly speaking not suited as input values for using dose
addition, because NOAELs represent different (but unknown) effect doses. However, unlike
independent action, the measurement precision required for using the concept does not change with
increases in the number of mixture components. This feature makes dose addition generally easier to
use in most situations, and the error associated with using NOAELs as input values is rather small.
However, more appropriate would be to use BMDs associated with the same effect magnitude (e.g.
5%).
It is obvious from the mathematical descriptions of dose addition that these represent the weighted
harmonic mean of the individual ECx values, with the weights being related to the mixture ratio (for
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Relevance of dissimilar mode of action
details see Kortenkamp et al., 2009, 2012). This has important favourable consequences for the
statistical uncertainty of combined toxicity predicted by dose addition. As the statistical uncertainty of
the dose addition-predicted ECx for the mixture is the result of averaging the uncertainties of the
single substance ECx-values, the stochastic uncertainty of the dose addition prediction is always
smaller than the highest uncertainty found in all individual ECx-values. Perhaps counter to intuition,
the consideration of mixtures composed of a large number of agents actually reduces the overall
stochastic uncertainty. This feature renders dose addition predictions quite reliable and robust.
Cumulative risk assessment methods based on dose addition rely on input values that are readily
available for many chemicals, such as Acceptable Daily Intake (ADI’s), Tolerable Daily Intake
(TDI’s), BMDs or NOAELs.
6.4.
The relevance of dissimilar MoA for grouping of pesticides in CAGs
In its previous opinion on CAGs (EFSA PPR Panel, 2013), the PPR Panel defined dose addition as a
default approach to be used for pesticides having the same phenomenological effect on a given target
organ/system, providing that a set of criteria is satisfied. The PPR Panel considered that the MoA(s) of
relevance to human toxicity are insufficiently defined for the majority of pesticides and that evidence
changing this situation is unlikely to be forthcoming in the foreseeable future.
The Panel recognizes the scientific relevance of considering dissimilar mode of action in cumulative
risk assessment. However, for the practical purpose of defining CAGs for MRL setting, the following
assumptions have to be made:
1) if pesticides do not elicit the same effect on a target organ/system, it is assumed that the compounds
are considered not to produce a common adverse outcome at dietary exposure levels and are not
grouped in the same CAG;
2) if pesticides do elicit the same effect on a target organ/system, it is assumed that the joint effect of
compounds is approximated by using dose addition. Accordingly, based on our present limited
understanding of mode of action, they should be grouped in the same CAG, until empirical evidence
becomes available that they do not produce a combination effect.
In summary, based on the available knowledge, the PPR Panel recommends to use the
phenomenological effect approach, as defined in its previous opinion, for pesticides, and to use dose
addition as a default method.
As a consequence, all pesticides exerting the same phenomenological effect in a given organ/tissue
should be included in the same CAG until and unless further relevant information about MoAs
becomes available.
6.5.
Summary of chapter 6
Cumulative risk assessment methods explicitly derived from independent action have not been
developed. The application of independent action in risk assessment practice places demands on the
data quality of the required input values that cannot be met. Furthermore, the required data quality
increases with rising numbers of mixture components.
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7.
Uncertainties
The Panel noted that some uncertainties exist, although a quantitative estimation of these uncertainties
was not possible due to the paucity or absence of comparative empirical data.
The applicability of independent action to mammalian organisms and cells has yet to be demonstrated.
The validity of independent action has been demonstrated for reference mixtures of dissimilarly acting
chemicals in algae and bacteria. Nevertheless, in tests and systems with endpoints relevant to
ecotoxicology, dose addition usually provided the more conservative mixture toxicity estimate, and
independent action often underestimated the combined effects of various chemicals with unknown
MoA. It remains to be seen whether IA tends to underestimate combination effects for endpoints
relevant to mammalian toxicology, and this represents an uncertainty regarding the conservatism of
default use of assessment methods derived from dose addition. Mathematical analyses show that
prediction differences between independent action and dose addition depend on the number of mixture
components, the mixture ratio, the effect level considered for the analysis, and the steepness of the
individual dose response curves. When all components in the mixture exhibit very shallow doseresponse curves, a more conservative prediction is produced by the independent action prediction,
ceteris paribus. However, the Panel recognised that this is a rather theoretical situation and that the
“increasingly flat” dose responses are unlikely to be biologically plausible.
There are uncertainties as to whether different components of a mixture are acting in a strictly
independent manner: The Panel recognises that the lack of data about the mode of action of pesticide
active substances in relation to unwanted toxic effects represents a scientific uncertainty and that
absence of a generally accepted classification scheme based on sufficiently different modes of action
is problematic. This complicates the grouping of chemicals according to the principles of independent
action for cumulative risk assessment. Even when data about modes of action are available, the level
of detail in information may be insufficient to support decisions as to whether different substances act
together according to independent action. It follows that careful considerations should be made when
using mode of action as criteria of deciding on the use of assessment concepts in cumulative risk
assessment.
There are uncertainties regarding the possible occurrence of interactions. The occurrence of relevant
interactions, synergisms or antagonisms, could call into question the use of any additivity concept as a
default. In particular, the prospect of synergism would imply that an additivity model might be
underestimating mixture effects. However, the Panel acknowledges that for the studies that allowed
quantitative estimates of synergy, the magnitude of synergy at low doses did not exceed the levels
predicted by additive models by more than a factor of 4 and that in a number of studies providing
evidence of synergism, its occurrence was dose dependent and only observed at the higher doses used
in the studies (Boobis et al., 2011). This provides a degree of reassurance that risks arising from
interactions might be rather rare. Following these considerations, the Panel agreed that toxicological
synergisms are not likely to occur for mixtures of pesticides at dietary exposure levels.
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CONCLUSIONS AND RECOMMENDATIONS
Following a mandate issued by EFSA, the PPR Panel has developed a Scientific Opinion on the
relevance of dissimilar modes of action and its appropriate application for cumulative risk assessment
of pesticides residues in food. In line with its previous opinion on cumulative assessment groups, the
Panel acknowledges that conducting CRA is a process that involves several steps and multiple
considerations, many of which go also beyond the scope and Terms of Reference of this opinion.
It is further recognised that even with a mixture of many pesticides affecting the same organ system
the majority of them might not contribute significantly to a given combination effect, either because
exposure is very low, and/or because potency in relation to the effect considered is weak. Instead,
cumulative risks from actual exposure are likely to be driven mainly by a few pesticides of a mixture.
Since there is good evidence that combination effects can arise from co-exposure to chemicals which
produce common (adverse) outcomes through entirely different modes of action and mechanisms, this
also applies to combinations of pesticides. Therefore, in response to the first item of the terms of
reference provided by EFSA, the PPR Panel states that dissimilar modes of action are relevant to the
assessment of cumulative risks from pesticide residues in food.
In this opinion similar and dissimilar MoA are understood to result in a common adverse outcome.
The approaches developed to date for conducting cumulative risk assessment of chemicals, including
pesticides, are based on dose addition. The applicability of DA has been established for groups of
chemicals acting by a similar MoA. However, in the case of performing cumulative risk assessment
for chemicals having a dissimilar MoA and eliciting the same effect, it remained to be answered
whether dose addition would also be the appropriate method or whether independent action would be
more relevant.
The Panel comes to the overall conclusion that distinctions between similar and dissimilar MoA
are fraught with great conceptual and practical difficulties and are of limited relevance in
cumulative risk assessment practice. Pesticides that produce common adverse outcomes on the
same target organ/system should be grouped together in CAGs, and their combined effects
assessed by using the concept of dose addition as a pragmatic and conservative default approach
for the purpose of assessing cumulative risk in relation to MRL setting or risk assessment of
chemical mixtures in practice, as already proposed in the 2013 EFSA Opinion on CAGs (EFSA
PPR Panel, 2013).
The Panel based these conclusions on the following considerations:
After analysing the premise for applying independent action, the Panel notes that to apply this
methodology the chemicals in question need to act independently. However, importantly, the
Panel notes that this is not equivalent to chemicals having a dissimilar MoA. Widely used
definitions of mode or mechanism of action are not helpful in deciding when a
mode/mechanism is sufficiently separate or distinct from another mode/mechanism to support
an assessment in favour of independent action. MoA and mechanism of action definitions, as
useful as they may be for the analysis of single chemicals, are generally not informative in
terms of making the case for independence of action. In principle, the case can be made
empirically, by investigating whether experimentally observed mixture effects are
approximated better by dose addition or independent action. The usefulness of such empirical
approaches for cumulative risk assessment in a regulatory context is however limited, because
it requires experimental data for the mixture of concern, which is usually not available.
There is to date no empirical evidence for the validity of independent action as a prediction
concept for multi-component mixtures in the mammalian toxicological literature. It seems that
the number of chemicals relevant to cumulative exposures is larger than the number of
distinct, strictly independent modes of action, such that there are likely to be several chemicals
EFSA Journal 2013;11(12):3472
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Relevance of dissimilar mode of action
in a particular mixture operating via the same mode of action or interrelated modes of action,
thereby violating the principles of strict independence of action. The Panel concludes that this
supports the default use of dose addition for the approximation of mixture effects. Available
empirical evidence, together with mathematical analyses and findings from simulation studies,
indicate that the quantitative differences under realistic exposure conditions between predicted
mixture effects derived from dose addition and independent action are likely to be small,
considering the variability and sources of error normally encountered in realistic assessment
situations. There is no case documented in the scientific literature where independent action
provided more conservative predictions of combination effects than dose addition, and where
at the same time independent action also produced accurate predictions. Thus, the Panel
concludes that dose addition is a sufficiently conservative approach to protect consumers’
health.
There are several cumulative risk assessment methods in practical use that are derived from
the concept of dose addition. These include the Hazard Index and Point of Departure Index
methods, and the Toxicity Equivalency Factor approach. In contrast, there is no method based
on independent action. The application of independent action in risk assessment practice is
further complicated by the fact that it places demands on the data quality of the required input
values that cannot be met in practice. In particular, it requires detailed descriptions of effects
in the low dose range which are rarely available. Furthermore, the required data quality
increases as the number of components in the mixture rises. Taken together, this makes the
application of independent action in cumulative risk assessment impossible under realistic
conditions.
The Panel emphasises that this conclusion does not disregard the relevance of considering
dissimilar/similar MoA for a biological understanding of adverse effects and characterisation of
adverse outcome pathways. The use of independent action also has merit for the analysis of
experimental data. However, in the light of the current understanding of assessing mixture toxicity this
is of little help when it comes to decisions supporting the use of dose addition or independent action
for the purpose of assessing cumulative risk in relation to MRL setting or cumulative risk assessment
in practice. Should refined knowledge about dissimilar mode of action produce evidence that
combined effects do not arise, despite the fact that pesticides affect the same target organ/system, the
pesticides can be excluded from a CAG. However, importantly, independence of action has to be
confirmed in order to apply an alternative methodology to dose addition.
The Panel makes the following recommendations for further potential cumulative risk assessment
activities in the future:
In order to develop better criteria for the grouping of substances or to further refine CAGs, the
Panel recommends to enrich key knowledge of the importance of MoA in cumulative risk
assessment. In particular, the Panel recommends more research on characterising adverse
outcome pathways.
More research is needed in mammalian systems with the aim of investigating the applicability
of independent action.
To put the case for sufficient conservatism of dose addition on a sound footing, more
knowledge about the distribution of slopes of dose response curves in mammalian systems is
needed. This may help to distinguish cases where independent action might provide more
conservative predictions of mixture effects. An analysis could be carried out on the basis of
toxicological data from existing pesticide dossiers.
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Relevance of dissimilar mode of action
To address residual uncertainties as to whether default application of dose addition is
generally sufficiently conservative, it is important to better define determinants of synergisms,
by researching the possibility of toxicokinetic and toxicodynamic interactions.
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Relevance of dissimilar mode of action
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APPENDIX A: MATHEMATICAL CALCULATION SUPPORTING THE EXAMPLE DESCRIBED IN 6.2
“DATA REQUIREMENTS FOR THE USE OF INDEPENDENT ACTION”
According to independent action a binary mixture of two agents that individually produce a
30% effect will lead to a 50% mixture effect (the effect summation approximation predicts
60%). To be able to apply independent action in this way, the evaluator has to use reliable
measurement of 30% effects as input values, which usually does not present problems.
E = 1– (1 – 0.3) (1 – 0.3)
0.3 is 30%
E = 1 – (0.7) (0.7)
E = 1 – 0.49
E = 0.51
0.51 is 51%
However, with a 10-component mixture that produces a 50% mixture effect, each component
is present at a dose associated with an effect of 6.7% (5% according to effect summation).
In this case, E (mixture effect) is 50% (equalling 0.5), and the individual effect of each
component of the mixture is calculated. Thus,
0.5 = 1 – (1– x) (1– x) (1– x) (1– x) (1– x) (1– x) (1– x) (1– x) (1– x) (1– x)
0.5 = 1 – (1– x)10
(1 – x)10 = 1 – 0.5
(1 – x)10 = 0.5
(1 – x) = 0.51/10
(1 – x) = 0.50.1
(1 – x) = 0.933
x = 1 – 0.933
x = 0.067
0.067 is 6.7%
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GLOSSARY AND ABBREVIATIONS
ADI (Acceptable daily intake): Estimate of the amount of substance in food expressed on a body
weight basis, that can be ingested daily over a lifetime, without appreciable risk to any consumer on
the basis of all known facts at the time of evaluation, taking into account sensitive groups within the
population (e.g. children and the unborn).
Adverse effect: Change in the morphology, physiology, growth, reproduction, development or
lifespan of an organism which results in impairment of functional capacity to compensate for
additional stress or increased susceptibility to the harmful effects of other environmental influences.
Adverse outcome pathway: An AOP is a sequence of events from the exposure of an individual or
population to a chemical substance to a final adverse (toxic) effect at the individual level (for human
health) or population level (for ecotoxicological endpoints). The key events in an AOP should be
definable and make sense from a physiological and biochemical perspective. AOPs incorporate the
toxicity pathway and mode of action for an adverse effect. AOPs may be related to other mechanisms
and pathways as well as to detoxification routes (OECD 2012)
Antagonism: Pharmacological or toxicological interaction in which the combined biological effect of
two or more substances is less than expected on the basis of the simple summation of the toxicity of
each of the individual substances
Dissimilar action: Occurs where the modes of action and possibly, but not necessarily, the nature and
sites of toxic effects differ between the chemicals in a mixture, and one chemical does not influence
the toxicity of another.
Dose addition (DA): All components in a mixture behave as if they were dilutions of one another.
One chemical can be replaced by an equal fraction of an equi-effective concentration (e.g. an EC50) of
another, without diminishing the overall combined effect, This implies that every toxicant in the
mixture contributes to the combination effect in proportion to its dose and individual potency.
Cumulative Assessment Grouping (CAG): Group of active substances that could plausibly act by a
common mode of action, not all of which will necessarily do so.
Hazard index (HI): Sum of Hazard Quotients, i.e. ratios between exposure and the reference value
for the common toxic effect of each component in the CAG.
Independent action (response addition): A combination effect that can be calculated from the
responses of the individual mixture components by following the statistical concept of independent
random events. In the case of simultaneous exposure to several chemicals, the principle of
independence of effects is only applicable when all the chemicals in the mixture act through strictly
independent modes by affecting strictly different targets.
Maximum residue level (MRL): Upper legal level of a concentration for a pesticide residue in or on
food or feed set in accordance with Regulation 396/2005, based on good agricultural practice and the
lowest consumer exposure necessary to protect vulnerable consumers.
Mechanism of action: Detailed explanation of the individual biochemical and physiological events
leading to a toxic effect.
Mode of action (MoA): Biologically plausible sequence of key events leading to an observed effect
supported by robust experimental observations and mechanistic data. It refers to the major steps
leading to an adverse health effect following interaction of the compound with biological targets. It
does not imply full understanding of mechanism of action at the molecular level.
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Similar action: Occurs when chemicals in a mixture act in the same way, by the same
mechanism/mode of action, and differ only in their potencies.
Synergism: Pharmacological or toxicological interaction in which the combined biological effect of
two or more substances is greater than expected on the basis of the simple summation of the toxicity of
each of the individual substances.
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