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Europaudvalget 2008-09
EUU Alm.del Bilag 443
Offentligt

Expert workshop on combination effects of chemicals, 28-30

January 2009, Hornbæk, Denmark

Organized under the auspices of the Danish Ministry of the Environment and the Danish

Environmental Protection Agency

Workshop Report

Professor Andreas Kortenkamp

The School of Pharmacy, University of London

Centre for Toxicology

29-39 Brunswick Square

London WC1N 1AX

Dr Ulla Hass

National Food Institute

Danish Technical University

Mørkhoj Bygade

DK 2860 Søborg

June 2009

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Summary

An expert workshop on effects of combined exposure to chemicals, with special

emphasis on chemicals with endocrine activity was held under the auspices of the Danish

Ministry of the Environment. The aim of the workshop was to examine existing scientific

knowledge on combination effects of endocrine disrupters, with a focus on regulatory

aspects. The workshop participants considered the state of the science of mixtures risk

assessment for endocrine disrupters, and discussed the feasibility of approaches to

cumulative risk assessment.

A consensus about a number of important issues could be formulated, and this included a

series of recommendations:

Cumulative risk assessment (CRA) for endocrine disrupters was seen as both necessary

and feasible. The predominant chemical-by-chemical approach in risk assessment was

regarded as insufficiently protective against the possibility of mixture effects/ effects of

combined exposure.

The application of dose (or concentration) addition as an assessment method was

recommended as a default, until evidence as to the suitability of alternative assessment

concepts emerges.

A pre-occupation with mechanisms or modes of action as the starting point for the

grouping of endocrine disrupters into classes to be subjected to mixtures risk assessment

was seen as not practical and scientifically hard to justify. Instead, grouping criteria

should focus on common health related effects and the likelihood of co-exposures.

The full potential of CRA for endocrine disrupters cannot be reached without filling a

number of data gaps, most importantly in the area of mixtures exposure assessment.

An enhancement of the legal framework in Europe with a view to mandating CRA should

be given serious consideration.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Abbreviations

ADI

AhR

BBP

CERCLA

CMG

CRA

DBP

DEHP

DEPA

FQPA

GHS

MOE

NOEC

NOEL

NOAEL

NRC

PBDE

PCDD/F

PODI

REACH

RfD

TCDD

TDI

TEF

TEQ

UVBC

Acceptable daily intake

Aryl hydrocarbon receptor

Benzyl butyl phthalate

Concentration addition

Comprehensive Environmental Response Compensation and Liability Act

Common mechanism group

Cumulative risk assessment

Dose addition

Dibutyl phthalate

Diethyl hexyl phthalate

Danish Environmental Protection Agency

Estrogen receptor

Food Quality Protection Act

Global Harmonisation System

Hazard index

Independent action

Margin of exposure

No observed effect concentration

No observed effect level

No observed adverse effect level

National Research Council

Polybrominated diphenyl ether

Polychlorinated dibenzo-p-dioxin/furan

Point of departure index

Registration Evaluation and Authorisation of Chemicals

Reference dose

Tetra chloro dibenzo-p-dioxin

Tolerable daily intake

TCDD equivalency factor

TCDD equivalent

Uncertainty factor

Unknown or variable composition complex reaction products or biological

materials

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Table of contents

1. Terms of reference and workshop aims

2. The workshop programme, resource materials

3. State of the science on combination effects of endocrine disrupters

4. Cumulative risk assessment – is it necessary?

5. Approaches to Cumulative Risk Assessment

6. Consensus formulation and recommendations

7. References

Appendix

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

1. Terms of reference and workshop aims

The Danish Environment Minister authorized the Danish Environmental Protection

Agency (DEPA) to host an expert workshop on combination effects of chemicals, with

special emphasis on endocrine disrupters. This workshop took place on 28 – 30 January

2009 in Hornbæk, Denmark.

The aim of the workshop was to examine existing scientific knowledge on combination

effects of endocrine disrupters, with a focus on regulatory aspects. The following

questions were to be addressed:

•

What is the state-of-the-science on combination effects at present – for chemicals

in general and specifically for endocrine disrupters?

Which problems can be identified on the basis of the existing knowledge – in

relation to health and in relation to the environment?

What are the challenges the regulatory authorities have to face?

How can these challenges be met and the existing knowledge be taken into

account within the existing regulation?

What are suggestions for actions with a focus on regulatory aspects on a global, a

regional (EU) and national (DK) level?

2. The workshop programme, resource materials

To realize the workshop aims, five different sessions were set up.

Session 1,

“Mixtures risk assessment – is it necessary?” was intended as a first step

towards defining the issues of the workshop. A second goal was to review the

experimental evidence for mixture effects when chemicals are combined at low doses,

close to levels that are “points of departure” for risk assessment and regulation (e.g.

benchmark doses or NOAELs).

The plan for

Session 2,

“A basis for combined risk assessment – case study: phthalates

and other anti-androgens” was to summarize the experimental evidence for combination

effects of antiandrogens, to review criteria for grouping these substances for purposes of

mixtures risk assessment and to gain an overview of risk assessment methods for

mixtures.

Session 3,

“The basis of combined risk assessment for other classes of endocrine

disrupters and other chemicals” aimed to consider topics for mixture risk assessment

relevant to other endocrine disrupting chemicals, such as: What are effect outcomes or

mechanisms on which mixtures risk assessment should be based? What is the evidence

for combination effects?

Session 4,

“From mixtures risk assessment to regulation” was set up for a more general

treatment of the mixtures risk assessment, relevant to other groups of chemicals, by

summarizing approaches to mixtures regulation, also in ecotoxicology, including an

analysis of uncertainty factors and their suitability for dealing with mixture effects.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Sessions 1 – 4 consisted of a series of formal talks, followed by discussions. The talks

were based on resource material which was distributed in advance to all participants.

Finally,

Session 5

“Looking forward – what can/should be done?” was conducted in the

form of a structured discussion among workshop participants, with the aim of drawing up

recommendations for risk assessment, regulation and research.

The workshop programme together with bibliographic references for the resource

material, and the list of participants can be found in the appendix. Since most formal talks

during the workshop were based on published scientific articles, their content is

accessible through the resource list. For this reason, the workshop talks will not be

summarized in chronological order in this report. Rather, a structured digest of the

presentations, discussions and recommendations of the workshop will be given.

3. State of the science on combination effects of endocrine

disrupters

Over the past decade, mixture toxicology has undergone a remarkable and productive

development. While earlier experimental studies focused mainly on mixtures composed

of only two chemicals, the planning, conduct and assessment of multi-component

mixtures with up to 50 chemicals is now state of the art. This has extended from in vitro

assays to in vivo studies, although scientific data about in vivo combination effects are

less prevalent than in vitro studies.

Most mixture studies with endocrine disrupters published in the peer-reviewed literature

have been conducted with the aim of explaining the joint action of selected pure

compounds in terms of their individual effects (component-based approach).

3.1 Definitions and terms

It is noted that the terms “mixture effects” and “effects of combined exposure” (to more

chemicals) are used without discrimination here and that the term “mixture” thus has a

broader meaning in this context than when used in chemicals legislation including

guidance (e.g. REACH and GHS). The field of mixture toxicology is notorious for its use

of poorly defined terms. Depending on context, there are many synonyms, and some

terms are uncritically used with entirely different meanings. For this reason, workshop

participants agreed on tentative definitions for a number of frequently used terms:

Mixture:

A mixture is a combination of several chemicals with which organisms come

into contact, either simultaneously, or sequentially. A binary mixture is a combination of

two agents. The term “complex mixture” is used to denote a mixture of unknown

composition, isolated from environmental media or other sources. “Complex mixture” is

sometimes used to describe combinations composed of three or more chemicals, but for

the purposes of this review, the term “multi-component mixture” is preferred.

Mixture effect, combination effect, joint effects:

The response of a biological system to

several chemicals, either after simultaneous or sequential exposure. The terms are used

synonymously.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Additivity:

In the context of mixture toxicology, additivity cannot be equated with

“additivity” in the mathematical sense. It refers to a situation, termed “non-interaction”

(and often used synonymously with ”additivity”), where the toxicity of a mixture

resembles the effects expected to occur when all mixture components act without

diminishing or enhancing each others effects. Additivity expectations for mixtures can be

derived from the concepts of dose (or concentration) addition and independent action (see

3.2.1 and 3.2.2). In certain situations, valid expectations for additive combination effects

can also be calculated by building the arithmetic sum of the individual effects of all

mixture components (“effect summation”).

Synergism, antagonism:

When an observed combination effect is larger or smaller than

expected according to an additivity assumption (based either on dose addition or

independent action), there is synergism or antagonism, respectively.

Mechanism of action:

Molecular sequence of events that produce a specific biological

response.

Mode of action:

A sequence of key cellular and biochemical events with measurable

parameters that result in a toxic effect. Mode of action considerations are used to decide

whether an effect observed after administration of a chemical in animals has relevance

for humans. Mode of action is not intended to build a comprehensive model of a

chemical’s actions. It is often confused with mechanism of action, or used in overlapping

ways. Mode of action can include mechanisms of action, but is considered to be broader.

Cumulative risk assessment (CRA), mixtures risk assessment:

The terms are used

synonymously. They denote risk assessment approaches that consider the impact of

multiple chemical exposures, from multiple sources, routes and pathways, over multiple

time frames. It is worth noting that the European use of the term “cumulative risk

assessment” encompasses multiple sources, routes and pathways, but restricts

considerations to one chemical, not multiple chemicals. For the purposes of this report,

the European use of the term is ignored. Toxicity assessments of multi-constituent

substances (e.g. technical solvents) or UVBC (unknown or variable composition,

complex reaction products or biological materials) also do generally not fall under

mixtures risk assessments of the kind discussed during the workshop. The reason is that

multi-constituent substances and UVBCs often are treated in the same way as a single

chemical entity would be dealt with; no attempts are made to explain mixture effects in

terms of the activity of the constituents.

There are various approaches to chemicals risk assessment (Suter and Cormier 2008), and

these also impact on CRA. First, risk assessment can be carried out in order to provide

trigger values for regulatory action to protect humans or wild life from harm

(“protective” risk assessment). In this case, a bias towards conservatism and worst case

assumptions is essential. Second, there is risk assessment aimed at quantifying the

magnitude of impact resulting from certain exposures to chemicals. Such approaches

(“quantitative” risk assessment) need to be as accurate as possible in their risk estimates

and tend to utilize probabilistic methods. This report is mainly concerned with protective

risk assessment, and less so with quantitative risk assessment.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

3.2 Prediction and assessment of mixture effects

When several chemicals occur together in a mixture, they may influence each others

effects by enhancing or diminishing their action. In mixture toxicology, such situations

are described as toxic interactions. More frequently however, chemicals act together

without influencing each others actions. In such cases, it is possible to anticipate

quantitatively the effects of a mixture from knowledge about the effects of its individual

components. This phenomenon is called non-interaction or additivity. Two concepts are

available for the formulation of the null hypothesis of additivity:

dose (or concentration)

addition

and

independent action.

These concepts are based on two entirely different ideas about how the joint action of

chemicals can be perceived.

3.2.1 Dose addition

Dose addition (DA) is based on the idea that all components in the mixture behave as if

they are simple dilutions of one another, which is often taken to mean that the concept

describes the joint action of compounds with an identical mechanism of action. When

these chemicals interact with an identical, well-defined molecular target, it is thought that

one chemical can be replaced totally or in part by an equal fraction of an equi-effective

concentration (e.g. an EC50) of another, without changing the overall combined effect.

A widely used application of this approach is the “toxic equivalence factor” (TEF)

concept for the assessment of mixtures of polychlorinated dioxins and furans (PCDD/F)

(van den Berg et al. 1998). Here, doses of specific PCDD/F isomers are all expressed in

terms of the dose of a reference chemical, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),

needed to induce the same effect (“equivalent” or “equi-effective” dose). The assessment

of the resulting combined effect is obtained simply by adding up all equivalent TCDD

doses. The application of TEF only holds when the underlying dose-effect relationships

are linear. If this pre-condition is violated, TEFs vary with the effect level that is

considered for analysis.

DA implies that every toxicant in the mixtures contributes in proportion to its toxic unit

(i.e. its concentration and individual potency) to the mixture toxicity. Whether the

individual doses are also effective alone does not matter. Thus, combination effects

should also result from toxicants at or below effect thresholds, provided sufficiently large

numbers of components sum up to a sufficiently high total dose. In view of the exposure

situation in many environmental compartments, the verification or falsification of this

conclusion has been a major topic in recent mixture toxicity studies (see below). An

overview of mixture studies that focused on this issue is given by Kortenkamp and co-

workers (Kortenkamp et al. 2007).

3.2.2 Independent action (response addition, effect multiplication)

Independent action (IA) conceptualises mixture effects in a different way. It assumes that

the joint effect of a combination of agents can be calculated from the responses of

individual mixture components by adopting the statistical concept of independent events.

The resulting combined effect can be calculated from the effects caused by the individual

mixture components by following the statistical concept of independent random events

(Bliss, 1939).

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

As IA uses the individual effects of the mixture components to calculate the expected

mixture effect, this concept implies that agents present at doses below their individual

effect thresholds (i.e. at zero effect levels) will not contribute to the joint effect of the

mixture. Hence if this condition is fulfilled for all components there will be no

combination effect. This central tenet of IA is commonly taken to mean that exposed

subjects are protected from mixture effects as long as the doses of all agents in the

combination do not exceed their no-observed-effect-levels or –concentrations (NOEL or

NOECs) (see below).

3.2.3 Choosing between dose addition and independent action for the purpose of

assessment and prediction

A question of fundamental importance to risk assessment and regulation is which of the

two concepts, DA or IA, should be exclusively chosen for the interpretation of empirical

data, or for anticipating mixture effects of untested combinations. As a way of resolving

the issue, DA and IA have been allied to broad mechanisms of combination toxicity, with

DA thought to be applicable to mixtures composed of chemicals with a similar mode of

action, with the corresponding mechanistic model of “simple similar action”, and IA for

chemicals with diverse modes of action, and the mechanistic model of “independent joint

action”.

The issue of distinguishing between these mechanistic models becomes especially

important, when DA and IA predict different mixture toxicities. In such cases it is

important to realize that the prediction differences or similarties stem from the

mathematical features that form the basis of DA and IA (Drescher and Boedeker 1995).

Prediction differences are not driven by the biology or toxicology of combinations of

chemicals with similar or diverse mode of actions.

Dose addition is thought to be applicable to mixtures composed of chemicals that act

through a similar or common mode of action (US EPA 1986, 1999, 2000). Although the

original paper by Loewe and Muischneck (1926) contains little that roots dose addition in

mechanistic considerations, the idea of similar action probably derives from the

“dilution” principle which forms the basis of this concept. Because chemicals are viewed

as dilutions of each other, it is implicitly assumed that they must act via common or

similar mechanisms.

Conversely, IA is widely held to be appropriate for mixtures of agents with diverse or

“dissimilar” modes of action. Although rarely stated explicitly, this presumably stems

from the stochastic principles that underpin this concept. The idea that chemicals act

independently is equated with the notion of action through different mechanisms. By

activating differing effector chains, so goes the argument, every component of a mixture

of dissimilarly acting chemicals provokes effects independent of all other agents that

might also be present, and this feature appears to lend itself to statistical concepts of

independent events. However, theoretically, the stochastic principles of IA are also valid

when one and the same agent is administered sequentially. This can be illustrated by

using cytotoxicity as an example. Because cells cannot die twice, the probabilistic

principle of IA applies, even though the precise mechanisms that underlie the cytotoxic

action of the chemical are identical in sequential administration. In the case of

simultaneous administration of many chemicals however, the principle of independent

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

events only applies when the additional assumption is made that all mixture components

act strictly independently, through different mechanisms.

The practical relevance of IA for the assessment of mixture effects has been called into

question on the basis of considerations of biological organisation. The principle of strictly

independent events may rarely apply due to converging signalling pathways and inter-

linked subsystems. For these reasons, DA is seen as more broadly applicable, and has

been termed the “general solution” for mixture toxicity assessment (Berenbaum 1985).

However, the few studies that were specifically designed for a comparative evaluation of

both concepts for mixtures composed of strictly dissimilarly acting substances,

demonstrated that IA provides a better prediction of the observed mixture toxicities

(Backhaus et al. 2000; Faust et al. 2003). These observations argue against DA as the

“general solution” for mixture assessments.

It appears that theoretical considerations are not decisive in answering the question of

choice between DA and IA as assessment concepts for endocrine disrupter mixtures. To

resolve the issue, it is therefore necessary to consider the empirical evidence.

3.3 Dose addition or independent action? - Experimental evidence with mixtures of

endocrine disrupters

The study of mixtures composed of chemically pure endocrine disrupters, in laboratory

settings, has yielded a considerable body of evidence showing that concentration (dose)

addition provides a sound approximation of experimentally observed additive

combination effects (see the review by Kortenkamp 2007). However, due to a

predilection of researchers to combine endocrine disrupters of the same type (e.g.

estrogenic, antiandrogenic or thyroid-disrupting chemicals), in many of the published

studies IA could not have been expected to produce valid additivity expectations.

Even so, there are recent indications that DA gives better approximations of combination

effects of endocrine disrupters with diverse modes of action. For example, Rider et al.

(2008) conducted mixture experiments with the three phthalates BBP, DBP, and DEHP in

combination with the antiandrogens vinclozolin, procymidone, linuron, and prochloraz.

Its components have a variety of antiandrogenic modes of action. Vinclozolin and

procymidone are AR antagonists, and linuron and prochloraz exhibit a mixed mechanism

of action: inhibiting steroid synthesis and blocking the steroid receptor. DA gave

predictions of combined effects of the mixed-mode antiandrogens that agreed better with

the observed responses than did the expectations derived from IA.

Mixtures of thyroid disrupting chemicals with diverse modes of action also showed

combination effects that were approximated better by DA, not IA (Kevin Crofton,

workshop presentation of unpublished data).

No case has yet been identified, where IA yielded predictions of endocrine disrupter

combination effects larger than those derived from DA, and at the same time were in

agreement with experimental data. Taken together, the determinants of additive joint

action of endocrine disrupters are fairly well established, and it appears that DA provides

good approximation of combination effects. Therefore, until evidence to the contrary

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

emerges, DA can be adopted as the default concept for the assessment and prediction of

endocrine disrupter mixture effects.

Factors that might lead to deviations from expected additive effects, indicative of

synergisms or antagonisms, are beginning to emerge and require further research. The

magnitude of such deviations cannot be predicted quantitatively. Toxicokinetic

interactions are one established cause of deviations from additivity. A notable example of

such deviations is the synergism that was observed with a mixture of vinclozolin,

prochloraz, finasteride and DEHP with respect to hypospadias and genital malformations

among male offspring of female rats (Ulla Hass, workshop presentation of unpublished

data).

4. Cumulative risk assessment – is it necessary?

Many experimental studies of mixture effects have been motivated by understanding

determinants of additivity and predictability. Inevitably, this has meant that chemicals

had to be combined at doses considerably higher than those encountered by the general

population. Two issues need to be addressed to judge the relevance of combination

effects for risk assessment: Do combination effects occur when chemicals are combined

at low doses? Are the uncertainty factors used to translate apparently safe dose levels

derived from animal experiments into acceptable exposures for humans insufficiently

protective to take account of mixture effects?

4.1 Mixture effects at low doses of mixture components

Certain experimental mixture studies have been designed to assess whether combination

effects occur when chemicals are combined at low doses, here defined as being

sufficiently low to be without observable effects when tested on their own (i.e. below the

sensitivity of the chosen experiment to be measurable). Often, these doses were in the

range of those commonly used to derive estimates of safe human exposures (so-called

points of departure, usually no-observed-adverse-effect-levels, NOAELs, or benchmark

doses). The review by Kortenkamp et al. (2007) summarizes the evidence for endocrine

disrupters and other types of chemicals, and an update was provided by Michael Faust

(workshop presentation).

For combinations composed of chemicals that interact with the same molecular receptor

or molecular target in an organism, there is good evidence that mixture effects can arise

at doses around, or below, points of departure. Considering the main assumptions

underlying the concept of dose addition, this is to be expected (see 3.1.1).

In contrast, theory predicts that mixtures which follow IA should not yield a combination

effect as long as all components are present at doses associated with zero responses. This

is widely held to mean that mixtures of dissimilarly acting chemicals are safe, as long as

exposure to each component does not exceed its individual point of departure (COT

2002, VKM 2008). With reference to the apparent diversity of chemical exposures in the

“real world”, IA is taken as the default assessment concept in human toxicology, when

strict similarity criteria of dose addition appear to be violated or if specific evidence for

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

the compounds of a given mixture is lacking. Implicitly taking “dissimilar action” or

“independent joint action” as the negation of “simple similar action” it is then assumed

that IA must hold, even without further proof that the underlying mechanisms indeed

satisfy any explicit dissimilarity criterion. This is then taken to mean that combined

exposures are without risk as long as all components stay below their points of departure.

Consequently, possible mixture effects are considered an irrelevance for chemicals risk

assessment.

In apparent contradiction to this view, there is good evidence that combinations

composed of chemicals with diverse modes of action also exhibit mixture effects when

each component is present at doses equal to, or below points of departure (Kortenkamp et

al. 2007, and updates in Michael Faust’s workshop presentation).

The flaw in the above line of thinking is two-fold:

First, when chemicals cannot be shown to interact with the same molecular targets, it

does not follow, that they must act in a dissimilar fashion. It is conceivable that diverse

modes of action lead to similar adverse outcomes – dissimilar action is not the simple

negation of similar action.

Second, points of departure, and particularly NOAELs, are confused with with true zero

effect levels. Under IA, combination effects cannot arise when the individual responses

of each component in the mixture are zero. With large numbers of chemicals however,

even very small individual effects will lead to considerable combined responses. For

example, 100 chemicals that each produce 0.1% of a maximal effect, are expected to

yield a response of 9%, according to IA. However, the resolving power of most testing

methods in regulatory use is far too low to demonstrate such small effects. Far from

signifying zero effect levels, NOAELs describe a grey zone, where the presence of

effects can neither be proven, nor ruled out with confidence. NOAELs are frequently

associated with effects of between 5 and 10% (Kortenkamp et al. 2007, Scholze and

Kortenkamp 2007).

Taken together, there is good evidence to show that the implicit null-model of many

regulatory assessments, namely, that only the most potent compound determines the

toxicity of the mixture, is usually wrong. Instead, more than one chemical in the mixture

contributes to the observed effects (either according to DA or IA) in contradiction to the

regulatory default model of “only the most toxic compound counts”.

The demonstration that mixtures of dissimilarly acting chemicals are not without effect

when they are combined at doses around points of departure, does say little about

whether or not risks are present in “real world” exposure settings. The decisive factor for

such risks to occur lies in the number of chemicals, and their levels: Only if sufficient

numbers of chemicals of sufficient potency and at sufficiently high exposure levels are

present, are combination effects to be expected. The issue can only be decided on the

basis of better information about relevant combined exposures of human populations and

wild life. This information is currently missing, and this knowledge gap presents a major

challenge to risk assessment.

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4.2 Uncertainty factors in risk assessment and standard setting – do they allow for

the possibility of mixture effects?

Although observations of combination effects of endocrine disrupters at low doses have

lent urgency to calls to account for such effects in chemicals risk assessment and

regulation, the need for doing so is often disputed with the argument that the

conventional chemical-by-chemical risk assessment is sufficiently protective. The

Uncertainty Factors (UF) usually applied to translate apparently safe dose levels derived

from animal experiments into acceptable exposures for humans, so goes the argument,

already cover the possibility of combination effects. The issue was examined by Martin

Scholze (workshop presentation).

Uncertainty factors are used in two different ways: Either to assess the health risks

associated with certain chemical exposures by deriving Margins of Exposure (MOE) or

Margins of Safety (MOS), or with the aim of establishing recommended health-based

guidance values, such as Acceptable (or Tolerable) Daily Intakes (ADI, TDI), Reference

Doses (RfD) and such like. Depending on context and goals, they are also referred to as

Assessment Factors.

The widely used UF of 100 is obtained by multiplication of two factors, one to allow for

intra-species sensitivity differences (10), the other for species-species extrapolations from

animal to human (10). Additional factors may be used to compensate for uncertainties

due to lack of information. For example, in the absence of data for chronic toxicity, an

(additional) default factor of 10 can be employed. Similarly, if test data do not allow the

estimation of a NOAEL, an additional factor of 10 may be brought into play. The various

assessment factors are multiplied, and this can yield a very large overall UF. The largest

reported overall UF in USEPA’s Integrated Risk Information System is 10,000. A

specific factor intended to allow for possible mixture effects is not in use.

Nevertheless, the common practice of combining different types of assessment factors by

multiplication has led to the idea that many overall UF’s are overly conservative. By

implication, this is taken to mean that mixture effects are covered. This idea appears to be

based on a mistaken interpretation of the multiplication rule of probabilities for rare

events. While is it clear that the occurrence of two rare independent events together tends

towards zero, assessment factors cannot be equated with probabilities. A direct

translation of UF’s into probabilities is not possible.

There is evidence that the common practice of using a factor of 10 to deal with animal-to-

human extrapolations may lead to underestimations of risk. The same applies to the factor

of 10 to allow for between-human differences in sensitivity. These considerations force

the conclusion that an UF of 100 offers insufficient room to allow for mixture effects for

all possible realistic mixtures.

Finally, the issue of UF’s and mixture effects can be approached from a different

direction by asking the question: how large would an additional assessment factor have to

be to take account of mixture effects? For a combination of chemicals that follows dose

addition, it can be shown that the RfD’s for each individual chemical would have to be

divided by the number of chemicals that contribute to an overall mixture effect. For

example, if a combined effect from simultaneous exposure is due to 5 chemicals, then the

RfD of every chemical has to be divided by 5, which is equivalent to saying that an

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

additional assessment factor of 5 is needed to cover mixture effects (NRC 2008).

Correspondingly larger factors are needed if more chemicals can be shown to contribute

to a common adverse outcome. However, choices about sufficiently protective factors

cannot be made without better information about the number of relevant chemicals, their

levels and potency, and how they contribute to human exposures.

To summarize, a specific “mixtures assessment factor” is currently not employed in the

traditional chemical-by-chemical risk assessment, and there is little to suggest that

commonly used UF are overly protective. There is not much “room” to allow for mixture

effects.

5. Approaches to Cumulative Risk Assessment

The practice of Cumulative Risk Assessment (CRA) is furthest developed in the USA,

where the US EPA is by far the most important authority for mixtures risk assessment

and regulation. Until recently, a common application of mixtures risk assessment in the

USA was to Superfund waste sites. The Comprehensive Environmental Response

Compensation and Liability Act (CERCLA) which came into force in 1980 specifically

calls for mixture risk assessment during the evaluation of risks that stem from hazardous

waste sites and chemical accidents. An additional stimulus for CRA was the passage of

the Food Quality Protection Act (FQPA) in 1996 which required the estimation of health

risks from combinations of pesticides with a common mode of action, from any exposure

source.

Several workshop presentations have dealt with existing approaches and practices of

CRA (presentations by Linda Teuschler, Rolf Altenburger and Henrik Tyle), and one

workshop aim was to evaluate whether these approaches can be used productively to deal

with endocrine disrupter mixtures.

5.1 The grouping of chemicals for the purpose of cumulative risk assessment

CRA begins with the identification of chemicals that should be grouped together and

subjected to joint risk assessment. In Superfund site assessments this is driven by

considerations of joint exposures. In contrast, CRA for pesticides begins with the

identification of a group of chemicals that are considered to induce a common toxic effect

by a common mechanism, a so-called common mechanism group (CMG). The criterion

proposed by US EPA (2000) for grouping chemicals for cumulative risk assessment is

“toxicological similarity”.

Extensive guidance exists about how this should be implemented (US EPA 2000).

Pesticides and other chemicals are considered to qualify for inclusion in a CMG when

their mechanism of toxicity shows similarities in both nature and sequence of major

biochemical events (workshop presentations by Linda Teuschler and Rolf Altenburger).

The use of toxicological similarity based on mechanisms, however, may lead to overly

narrow groupings. For example, organophosphate pesticides and carbamates inhibit

acetylcholinesterase, and this is shown to be a relevant step in the manifestation of

toxicity. Because the mechanism of inhibition by carbamates is via carbamylation, and

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that of organophosphates by phosphorylation, and because this is judged to represent

different molecular mechanisms, the two types of pesticides are not assessed together, but

included in separate CMGs for the purpose of mixtures risk assessment. Such narrow

groupings ignore that joint effects can also occur from combined exposures with other

than common mechanisms (workshop presentation by Rolf Altenburger).

5.1.1 Grouping for antiandrogens

An exaggerated focus on mechanisms of toxicity may lack plausibility and credibility

when it is applied as a grouping criterion for endocrine disrupters. With a recent report by

the National Research Council (NRC) of the US National Academy of Sciences the issue

came to a head with antiandrogens, including phthalates. The NRC advised that a

cumulative risk assessment should not only consider certain phthalates, but also other

chemicals that could potentially cause the same health effects as phthalates (NRC 2008).

It was recommended that phthalates and other chemicals that affect male reproductive

development in animals, including antiandrogens, be considered in the cumulative risk

assessment. Solely mechanism-based criteria may lead into a dilemma: Because there are

subtle differences in the precise molecular details by which phthalates can act as

endocrine disrupters, not even all antiandrogenic phthalates would be subjected to CRA,

when mechanistic considerations are the sole grouping criterion.

The NRC therefore recommended a broader based move towards establishing grouping

criteria for phthalates and other antiandrogens. With this type of endocrine disrupter, a

case can be made for adopting a physiological approach to analyzing toxic mechanisms

of action with respect to similarity or dissimilarity. If it is recognized that the driver of

male sexual differentiation during development is the effect of androgen action, it is

irrelevant whether the hormones’ effects are disrupted by interference with steroid

synthesis, by antagonism of the androgen receptor, or by some other mechanism (for

example, affecting consequences of androgen receptor activation). The resulting

biological effects with all their consequences for male sexual differentiation are similar,

although the molecular details of toxic mechanisms - including metabolism, distribution

and elimination - differ profoundly in many respects. Judged from such a perspective, a

focus on phthalates to the exclusion of other antiandrogens not only would be artificial

and lack credibility, but could imply serious underestimation of cumulative risks posed

by agents for which there is simultaneous exposure (workshop presentations by Ulla Hass

and Andreas Kortenkamp).

5.1.2 Thyroid disrupting chemicals

Similar considerations may apply to the group of thyroid disrupting chemicals which

affect multiple targets through a variety of mechanisms. In an echo of the situation with

antiandrogenic chemical mixtures, the question is: which level of biological complexity

should be used to cumulate joint effects? If an endpoint representative of a specific mode

of action is chosen (e.g. variations in T4 levels), then certain chemicals might be left out

of a common grouping. On the other hand, if the endpoint chosen for integration is at a

very high level of complexity (e.g. changes in cognitive function), not only a very large

number of chemicals but also a variety of non-chemical stressors will have to be taken

into account. This may become difficult to handle in risk assessment settings (workshop

presentation by Kevin Crofton).

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5.1.3 Dioxin-like endocrine disrupters

Dioxins and dioxin-like compounds represent a group of endocrine disrupters where key

events of toxicity are thought to be mediated by binding to the arylhydrocarbon receptor

(AhR). These chemicals were first grouped according to descriptors of chemical structure

(to include only polychlorinated dibenzo-p-dioxins and –furans, PCDD, PCDF), but

insights into their biological activity led to the incorporation of co-planar PCBs and other

poly-halogenated polycyclic hydrocarbons (workshop presentation by Martin van den

Berg). By using the criterion of AhR activation, polybrominated diphenyl ethers (PBDE)

were not included in the group of dioxin-like chemicals. It turned out that pure PBDE

were devoid of AhR activity, and that earlier reports of AhR activation could be ascribed

to contamination with dioxin-like chemicals, most importantly polybrominated

dibenzodioxins and –furans. The most potent PCDD, 2,3,7,8 TCDD, is selected as the

reference chemical, and the potency of all other dioxin-like chemicals is expressed in

terms of TCDD effect concentrations, so-called TCDD equivalents, with TCDD

equivalency factors (TEF) (van den Berg et al 2006). The use of TEF for the assessment

of mixtures of dioxin-like chemicals is an application of the concept of dose addition, and

is a widely accepted risk assessment method.

5.1.4 Estrogenic chemicals

In many ways, estrogenic chemicals resemble dioxin-like chemicals: Their activity is

thought to be mediated by binding to estrogen receptors (ER alpha or beta), which

suggests itself as a straightforward grouping criterion (workshop presentation by Andreas

Kortenkamp). Furthermore, there are reference agents of high potency (17-beta-estradiol,

DES) and there is good evidence that mixtures of estrogenic chemicals follow dose

addition when the assessment is based on events relatively close to receptor activation

(Kortenkamp 2007). Consequently, it has been suggested that this group of endocrine

disrupters should be assessed just like dioxin-like chemicals, by using the toxicity

equivalency concept. However, this suggestion has been called into question by Safe,

with reference to the complexity of estrogen signaling (discussed in Kortenkamp 2007).

Nearly 20 years ago, evidence has emerged that ER activation is possible without binding

to the binding pocket of the steroid hormone, e.g. by phosphorylation through activation

by growth factors. This opens the possibility that estrogen action can be substantially

modulated by chemicals interfering with other phosphorylation events. Should such

agents be subjected to CRA with estrogenic chemicals? Furthermore, more research is

needed to elucidate the toxicological relevance of ER activation. Although chemicals

such as DES are potent disrupters of male and female sexual differentiation, it remains to

be seen whether these effects are mediated by ER activation. Similarly, the mechanisms

by which estrogens play a role in breast cancer are not entirely resolved.

Considerations of mode of action as a grouping criterion are often of little use in

ecotoxicological mixtures risk assessment, because each chemical usually exhibits

multiple modes of action. The solution to this problem is to take account of sensitivity

differences in various receptors and species (Leo Posthuma, workshop presentation).

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5.2 Mixtures risk assessment methods

The application of mixtures 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 in ways to prioritize certain mixtures (Linda

Teuschler and Leo Posthuma, workshop presentations). Estimations of safe levels or

margins of exposure may be based on worst-case-assumptions, but the prioritization of

mixtures (or affected sites) has to rely on fairly accurate quantitations of risk.

Considering that experimental studies with endocrine disrupters showed that dose

addition is a useful concept for the approximation of combination effects, component-

based methods derived from dose addition suggest themselves as risk assessment

approaches. These include the Hazard Index (HI), Point of Departure Index (PODI) and

the TEQ concept (Linda Teuschler and Henrik Tyle, workshop presentations).

All these methods require dose-response information of mixture components as input

values. The HI sums up ratios of exposure levels and reference doses over chemicals. The

reference doses can be arrived at by utilizing different UF for each mixture component. If

this is perceived to be a problem, the PODI method can be used. PODI is based not on

reference doses, but on points of departure (NOAELs, benchmark doses). Extrapolation

issues (e.g. animal to human) are then dealt with by using one overall UF. Finally, the

TEQ concept is predicated on the choice of a reference chemical and requires parallel

dose-response curves for all components. Both these requirements are often not met by

endocrine disrupters, but the method has been validated for dioxin-like endocrine

disrupters.

5.2.1 Tiering

Depending on the quality of the data that are available for CRA (data poor or data rich),

tiering methods might be very productive to explore the problem, and refine (with more

sophisticated models and associated supporting data) when needed (Leo Posthuma,

workshop presentation). At the lowest tier (tier 0), it may become apparent that the

situation to be evaluated does in fact not present an issue for mixtures risk assessment. In

the next higher tier (tier 1), termed “simple generic”, data about mixed exposures may not

be present, but it may be deemed desirable to safeguard against the possibility of joint

effects by adopting a specific mixtures assessment factor. In tier 2, “moderately simple

generic”, sufficient data may be available to warrant the assumption of dose addition

throughout, in which case variants of this concept could be applied, even though

independent action may produce less conservative estimates. In a quite data rich situation

(tier 3, “complex specific”) sufficient information about various modes of action may be

available, such that mixed mixtures assessment models (DA within groups of compounds

perceived to follow simple similar action, followed by IA across groups) can be applied.

Finally, in the highest tier 4 (“highly specific”) it would be possible to address both issues

of modes of action and differences in the vulnerability of various species or risk

receptors.

In the light of the data situation typical for many endocrine disrupters, it would appear

that assessments at tier 1 and tier 2 are currently possible.

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5.2.2 Validation

With the aim of putting CRA methods on a sound footing, it is important to seek

situations where the outcome of specific assessments can be validated. While this is

achievable in ecotoxicology (presentation Leo Posthuma), the situation is much more

complicated in the arena of human toxicology.

5.3 Regulation and risk management

CRA for endocrine disrupters and other chemicals can yield important stimuli for

regulation and risk management, by providing the basis for a procedure of relative

ranking, e.g. according to the most potent chemicals. This would offer the possibility of

strictly regulating, or even eliminating those chemicals that are shown to have the

greatest impact on a combination effect. Other rankings could be performed in terms of

the most problematic exposure settings, or the most vulnerable population subgroups

(Leo Posthuma, workshop presentation).

6. Consensus formulation and recommendations

The workshop participants reached a consensus on a number of specific issues relevant to

CRA of endocrine disrupters. The participants also made certain recommendations

concerning risk assessment methods, research needs, and legislative requirements.

6.1 Mixtures risk assessment is necessary

In view of the evidence about mixture effects at low experimental doses (see 4.1) and the

uncertainty of commonly employed UF in single-chemical risk assessment (see 4.2) a

disregard for combination effects was considered undesirable and not in line with

currently available empirical evidence. Any CRA method, even one that employs the

narrowest possible toxicological grouping criteria, was deemed to represent an

improvement compared to the current pre-occupation of conventional risk assessment

with chemical-by-chemical approaches. Moreover, an extended look at simultaneous or

sequential exposure issues was deemed crucial, to add to the classical toxicological

approaches.

6.2 The assumption of independent action as a default for “real world” mixtures is

not tenable

As discussed in 4.1, the absence of proof of “similarity” in the mode of action of mixture

components cannot be taken to indicate applicability of IA as a default, with the implicit

assumption that combination effects are not to be expected if all chemicals are present at

doses below their individual points of departure or NOAELs. The workshop participants

recognized that the available empirical data do not support this widely held view. Instead,

there is good evidence that mixtures that follow IA exert effects even when all mixture

components are present at doses below their NOAELs.

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6.3 The application of dose addition is recommended as a default, until evidence to

the contrary appears

The empirical evidence with endocrine disrupter mixtures (see 3.3) shows that DA yields

reasonable approximations of observed combination effects. There are many examples

where IA has produced underestimations of observed joint effects. Crucially, no case

could be identified, where IA afforded a more conservative mixture effect prediction that

was at the same time in agreement with the experimental mixture effects. It is

conceivable that such evidence appears in the future, but until this is the case, DA was

recommended as the default assessment method, irrespective of presumed modes of

action. This modus operandi has the additional advantage of requiring fewer data than the

alternative concept of IA, with the consequence that it can serve as lower-tier approach in

many circumstances.

6.4 Steps towards CRA: criteria for the grouping of chemicals, assessment methods

Grouping criteria that are driven exclusively by thoughts about mechanisms or the key

events for a mode of action were seen as problematic by the workshop participants (see

5.1), and it was recommended that grouping should be dissociated from mechanistic

considerations. For risk assessment, phenomenological grouping criteria, based on

common adverse health outcomes, were seen as a more useful starting point for

groupings. Nevertheless, it was recognised that toxic effects become less specific for the

initiating event, as one moves further down-stream of an effector chain. This loss of

specificity may lead to the inclusion of an ever wider array of chemicals into a grouping

for CRA, ultimately blurring all distinctions, with the need to include all chemicals.

However, this was not perceived to be a critical problem for endocrine disrupting

chemicals.

Another useful criterion for groupings is the likelihood with which simultaneous

exposures to several chemicals occurs.

A tiered assessment, depending on the extent and quality of existing data about hazards

and exposures is recommended. For example, to alleviate concerns about mixture effects,

it would be possible to adopt a specific assessment factor in the traditional chemical-by-

chemical risk assessment, even without any further data. In more data-rich situations, it is

feasible to utilize applications of the dose addition concept to define margins of exposure

or other indicators of risk.

6.5 CRA for endocrine disrupters, although feasible, is hampered by important data

gaps

Due to significant experimental advances in the last five years, determinants of additive

mixture effects of classes of endocrine disrupters are now quite well understood. The

prospect of CRA for endocrine disrupters is limited by incomplete information about

relevant exposure scenarios. This is particularly critical for human risk assessment: it is

not even possible to say with confidence whether there are only a few chemicals that

contribute significantly to an overall mixture effect, or whether the number of relevant

chemicals is likely to be high. Better knowledge about this aspect of the problem would

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have an enormous impact on the prospects of CRA. The issue can only be resolved

through dedicated mixtures exposure assessment approaches, where scores of chemicals

are measured in one and the same sample. This would also provide information about the

feasibility of using certain index chemicals as surrogates for exposure measurements.

Furthermore, it was recommended to identify exposure “hot spots” with the aim of using

those for targeted monitoring (with associated exposure ‘cold spots’ as points of

reference for interpretation).

Another challenge concerns the issue of dose metrics. The usefulness of data from animal

experiments would be enhanced greatly if the internal tissue levels resulting from

experimental exposures were known. This would enable a read-across to readily

accessible data about human tissue levels.

Further research needs are in the following areas:

The joint effects of different classes of endocrine disrupters need to be evaluated, and a

better understanding of hormone systems other than estrogens, androgens and thyroid

hormones is urgently required.

Finally, determinants that lead to synergisms between endocrine disrupters need to be

investigated.

6.6 A better legislative basis for CRA is needed in Europe

Without the legal mandates laid down in the US American CERCLA and FQPA,

cumulative risk assessment would not have been implemented in the USA. With the

exception of the recent changes in European pesticides regulations, where mixture risk

assessment is mandated, comparative legal frameworks that clearly address CRA do

currently not exist in Europe. In REACH for example, CRA for multiple chemicals from

multiple sources, routes and pathways is only addressed to a very limited extent in the

current guidance. Other relevant European legislations do not contain a mandate for CRA

for multiple chemicals from multiple sources, routes and pathways.

Development of a comprehensive implementation of CRA should be given serious

consideration in all relevant legislation and guidance dealing with chemicals safety

assessment and the establishment of safe emission and exposure levels. It is essential to

assess the scope of existing laws and guidance in order to define better whether existing

regulation can be amended to accommodate CRA, or whether tailor-made regulations

need to be developed.

6.7 Prioritisation

The workshop participants were asked to distill their recommendations into a few main

points and to prioritize. Consensus on the following was reached:

•

CRA for endocrine disrupters can start immediately – important information

necessary to make decisions about groupings of chemicals to be subjected to

mixture risk assessment is available.

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•

Dose addition should be used as the default lower-tier assessment method. It

should replace the current risk assessment paradigm that is focused on single

chemicals, with its erroneous implicit assumption of “only the most toxic

compound counts”.

The legal basis and/or guidance for CRA in Europe needs to be enhanced further.

•

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

7. References

Backhaus T, Scholze M, Grimme LH. 2000. The single substance and mixture toxicity of

quinolones to the bioluminescent bacterium

Vibrio fischeri.

Aquat Toxicol 49:49-

61.

Berenbaum MC. 1985, "The expected effect of a combination of agents: the general

solution",

Journal of Theoretical Biology,

114: 413-431.

Bliss CI. 1939. The toxicity of poisons applied jointly.

Ann Appl Biol

26:585-615.

COT (Committee on Toxicity of Chemicals in Food, Consumer Products and the

Environment). 2002. Risk assessment of mixtures of pesticides and similar

substances. Her Majesty’s Stationary Office, London, United Kingdom. Available:

http://www.food.gov.uk/science/ouradvisors/toxicity/COTwg/wigramp/

[accessed 7

September 2005].

Drescher K, Boedeker W. 1995. Concepts for the assessment of combined effects of

substances: the relationship between concentration addition and independent action.

Biometrics

51:716-730.

Faust M. et al. 2003, "Joint algal toxicity of 16 dissimilarly acting chemicals is

predictable by the concept of independent action.",

Aquatic Toxicology,

63: 43-63.

Kortenkamp A. 2007, "Ten years of mixing cocktails: a review of combination effects of

endocrine-disrupting chemicals",

Environ.Health Perspect.,

115 (Suppl 1): 98-105.

Kortenkamp A, Faust M, Scholze M,Backhaus T. 2007, "Low-level exposure to multiple

chemicals: reason for human health concerns?",

Environ.Health Perspect.,

115

(Suppl 1):106-114.

Loewe S, Muischnek H. 1926. Über Kombinationswirkungen. 1. Mitteilung: Hilfsmittel

der Fragestellung [in German].

Naunyn-Schmiedebergs Arch Exp Pathol

Pharmakol

114:313-326.

NRC, 2008. Phthalates Cumulative Risk Assessment – The Tasks Ahead. Committee on

Phthalates Health Risks, National Research Council, National Academy of

Sciences, Board on Environmental Science and Technology, National Academy

Press, Washington, DC.

Rider CV, Furr J, Wilson VS, Gray LE., Jr. 2008, "A mixture of seven antiandrogens

induces reproductive malformations in rats",

Int. J. Androl,

31: 249-262.

Scholze M, Kortenkamp A. 2007. Statistical power considerations show the endocrine

disrupter low dose issue in a new light.

Environ. Health Perspect.

115 (Suppl.

1):84-90.

Suter GW, Cormier SM. 2008. What is meant by risk-based environmental quality

criteria.

Integrated Environ Assess Monitoring

4: 486-489.

U.S. EPA. 1986. Guidelines for health risk assessment of chemical mixtures. Fed. Reg.

51(185):34014-34025.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

U.S. EPA. 1989. Risk assessment guidance for superfund. Vol. 1. Human health

evaluation manual (Part A). EPA/540/1-89/002. Washington, DC:U.S.

Environmental Protection Agency.

U.S. EPA. 2000. Supplementary guidance for conducting health risk assessment of

chemical mixtures. EPA/630/R-00/002. Washington, DC:U.S. Environmental

Protection Agency.

Van den Berg M, Birnbaum L, Bosveld ATC, Brunstrom B, Cook P, Feeley M, et al.

1998. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and

wildlife.

Environ Health Perspect

106:775-792.

Van den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,

Fiedler, H., Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,

Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., and Peterson,

R.E., 2006. The 2005 World Health Organization Reevaluation of Human and

Mammalian Toxic Equivalency Factors for Dioxins and Dioxin-Like Compounds.

Toxicol. Sci.

93: 223–241.

VKM. 2008. Combined toxic effects of multiple chemical exposures. Opinion of the

Scientific Steering Committee of the Norwegian Scientific Committee for Food

Safety. Oslo. ISBN (printed version) 978-82-8082-232-1

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Appendix

Expert workshop on combination effects of chemicals, 28-30

January 2009, Hornbæk, Denmark

Programme outline

Wednesday, 28 January 2009

12:00

13:30

Lunch

Henrik Soren Larsen, Andreas Kortenkamp

Welcome and introductory remarks

Session 1: Mixtures risk assessment – is it necessary?

13:45

Round table opening discussion: Are there examples where

combined exposures have proven to pose risks?

This discussion is intended as a first attempt at defining issues: workshop participants

are invited to give their opinions about what, if any, they regard as important examples

where mixtures are a problem, in human and/or ecotoxicology.

14:30

Michael Faust

Low dose mixture effects – a review of experimental evidence

This presentation will review the experimental evidence for mixture effects when

chemicals are combined at low doses, close to levels that are “points of departure” for

risk assessment (i.e. benchmark doses or NOAELs).

Resource:

Kortenkamp et al. 2007 EHP 115 Suppl 1 : 106

15:00

15:30

Discussion

Coffee break

Session 2: A basis for combined risk assessment – case study: phthalates

and other anti-androgens

Beginning with a fairly well-researched group of chemicals, this session is a first attempt

at crystallizing issues for mixtures regulation: What is the experimental evidence for

combination effects of phthalates and other antiandrogens? How can these data be

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

assessed? What are criteria for grouping these substances for purposes of mixtures risk

assessment?

15:45

Ulla Hass

Combination effects of phthalates and other anti-androgens after

gestational exposure

Resource:

Hass et al. 2007 EHP 115, Suppl 1 : 122, Metzdorff et

al. 2007 Toxicol Sci 98 : 87, Christiansen et al. 2008 Int. J.

Androl. 31: 241

16:15

16:30

Discussion

Andreas Kortenkamp

Which chemicals should be grouped to protect against combination

effects resulting in disruption of male sexual differentiation? – a

discussion of grouping criteria

Resource:

Summary chapter of US NRC report “Cumulative risk

assessment for phthalates – the tasks ahead”

17:00

17:15

Discussion

Linda Teuschler

An overview of chemical mixtures risk assessment methods

Resource:

Teuschler 2007 TAP 223: 139

Having discussed the specifics of antiandrogen mixtures in some detail, this presentation

is intended to broaden the discussion and will summarize the methods that have been

used to group other substances for the purpose of mixtures risk assessment. Is it possible

to derive generally applicable criteria?

17:45

18:00

Discussion

Drinks and dinner

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Thursday, 29 January 2009

Session 3: The basis of combined risk assessment for other classes of

endocrine disrupters and other chemicals

The following series of talks will consider topics for mixture risk assessment relevant to

other endocrine disrupting chemicals, such as: What are effect outcomes or mechanisms

on which mixtures risk assessment should be based? What is the evidence for

combination effects?

9:00

Kevin Crofton

Effect profiles of thyroid-disrupting chemicals and experimental

evidence of mixture effects

Resource:

Crofton 2008 IJA, Crofton et al. 2005 EHP

Discussion

9:30

Martin van den Berg

Dioxins, PCBs and related chemicals – an update on the TEF

approach

Resource:

Van den Berg et al. 2006 Tox Sci 93 : 223

Discussion

10:00

Andreas Kortenkamp

Estrogens and estrogen-like chemicals – an update on combined

effects

Resource:

Kortenkamp 2007EHP 115 Suppl 1: 98

Discussion

10:30

11:00

Coffee break

Rolf Altenburger

A brief overview of other efforts of mixtures risk assessment:

organophosphates, carbamates, chloroacetanilides, triazines…

Resource:

EPA guidance documents

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

11:30

General discussion: Is mixtures risk assessment for endocrine

disrupters and other chemicals a viable prospect? What are

barriers? What are opportunities?

Suggested topics for discussion include: Are toxicologically relevant endpoints

sufficiently well characterized to provide a basis for mixtures risk assessment? What are

major sources of uncertainty? Knowledge gaps?

12:30

Lunch

Session 4: From mixtures risk assessment to regulation

The scene is set for a more general treatment of the mixtures risk assessment, relevant to

other groups of chemicals. The session begins with a brief summary of approaches to

mixtures regulation, considers practice in ecotoxicology, and what can be derived for

human toxicology and ends with an analysis of uncertainty factors and their suitability

for covering mixture effects.

14:00

Henrik Tyle

Synopsis of approaches to mixtures regulation (top n, PODI, HI,

TEF, relative potency factors, etc)

Resource: VKM report 2008, p 38 – 51, Feron et al 2004, ETAP

18, 215

14:30

Leo Posthuma

Practical approaches in ecotoxicological mixture risk assessment in

support of urgent policy questions

Martin Scholze

Uncertainty factors in standard setting – are mixture effects

covered?

Coffee break

General discussion – focus: can existing chemicals regulation

be modified to take account of mixtures effects?

Break-out group: Formulation of theses and summary

15:00

15:30

16:00

17:00

Here we are looking for volunteers with extreme stamina: Three to four participants are

wanted who are willing to take it upon themselves to distill the discussions so far into a

few theses/summary, to be presented the following day.

18:00

Drinks and dinner

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Friday, 30 January 2008

Session 5: Looking forward – what can/should be done?

9:30

Break-out group

Presentation of theses and summary

The break-out group will present their theses and summary for discussion and comment.

10:00

General discussion and conclusion

At this stage, this discussion is deliberately left a little unstructured, but the intention is to

reflect on the insights from a science perspective with practical steps for risk assessment

and regulation in mind.

10:30

10:45

12:30

14:00

15:30

Coffee break

General discussion (continued)

Lunch

General discussion (continued)

Andreas Kortenkamp

Summing up, conclusion, recommendation and outlook

16:00

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Resources references

(in the order of the talks)

Michael Faust

Kortenkamp, A., Faust, M., Scholze, M., & Backhaus, T. 2007, "Low-level exposure to

multiple chemicals: reason for human health concerns?",

Environ.Health Perspect.,

vol.

115 Suppl 1, pp. 106-114.

Ulla Hass

Hass, U., Scholze, M., Christiansen, S., Dalgaard, M., Vinggaard, A. M., Axelstad, M.,

Metzdorff, S. B., & Kortenkamp, A. 2007, "Combined exposure to anti-androgens

exacerbates disruption of sexual differentiation in the rat",

Environ.Health Perspect.,

vol.

115 Suppl 1, pp. 122-128.

Metzdorff, S. B., Dalgaard, M., Christiansen, S., Axelstad, M., Hass, U., Kiersgaard, M.

K., Scholze, M., Kortenkamp, A., & Vinggaard, A. M. 2007, "Dysgenesis and

histological changes of genitals and perturbations of gene expression in male rats after in

utero exposure to antiandrogen mixtures",

Toxicological Sciences,

vol. 98, no. 1, pp. 87-

98.

Christiansen, S., M. Scholze, M. Axelstad, J. Boberg, A. Kortenkamp, & U. Hass. 2008.

Combined exposure to anti-androgens causes markedly increased frequencies of

hypospadias in the rat.

Int. J. Androl.

Vol 31, no 2, pp.241-248.

Andreas Kortenkamp

NRC, 2008. Phthalates Cumulative Risk Assessment – The Tasks Ahead. Committee on

Phthalates Health Risks, National Research Council, National Academy of Sciences,

Board on Environmental Science and Technology, National Academy Press, Washington,

DC.

Linda Teuschler

Teuschler, L.K. 2007, Deciding which chemical mixtures risk assessment methods work

best for what mixtures.

Toxicology and Applied Pharmacology,

vol 223, pp 139-147.

Kevin Crofton

Crofton, K. M., Craft, E. S., Hedge, J. M., Gennings, C., Simmons, J. E., Carchman, R.

A., Carter, W. H., & Devito, M. J. 2005, "Thyroid-hormone-disrupting chemicals:

Evidence for dose-dependent additivity or synergism",

Environmental Health

Perspectives,

vol. 113, no. 11, pp. 1549-1554.

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Martin van den Berg

Van den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,

Fiedler, H., Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,

Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., & Peterson, R.E.,

2006. The 2005 World Health Organization Reevaluation of Human and Mammalian

Toxic Equivalency Factors for Dioxins and Dioxin-Like Compounds.

Toxicol. Sci.

vol

93 no. 2, pp 223–241

Andreas Kortenkamp

Kortenkamp, A. 2007, "Ten years of mixing cocktails: a review of combination effects of

endocrine-disrupting chemicals",

Environ.Health Perspect.,

vol. 115 Suppl 1, pp. 98-105.

Rolf Altenburger

US EPA, 1999. Guidance for Indentifying Pesticide Chemicals and Other Substances that

Have a Common Mechanism of Toxicity. Office of Pesticide Programs, US

Environmental Protection Agency, Washington, DC

US EPA 2006a. Cumulative Risk from Triazine Pesticides. Office of Pesticide Programs,

US Environmental Protection Agency, Washington, DC., March 2006

US EPA 2006b. Cumulative Risk from Chloroacetanilide Pesticides. Office of Pesticide

Programs, US Environmental Protection Agency, Washington, DC., March 2006

Henrik Tyle

VKM. 2008. Combined toxic effects of multiple chemical exposures. Opinion of the

Scientific Steering Committee of the Norwegian Scientific Committee for Food Safety.

Oslo. ISBN (printed version) 978-82-8082-232-1

Feron, V.J., van Vliet, P.W. & Notten, R.F., 2004. Exposure to combinations of

substances: a system for assessing health risks.

Environ Toxicol Pharmacol

vol 18, pp.

215-222.

There were no resources circulated for Leo Posthuma’s and Martin Scholze’s

presentations

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

List of Participants

Expert Workshop on Combinations Effects 28-30 January 2009

Name

USA

Linda Teuschler

Kevin Crofton

EU excl. DK

Martin van den Berg

Michael Faust

Affiliation

e-mail

U.S. EPA/NCEA-Cin

US EPA/RTC-NC

[email protected]

University of Utrecht, NL

[email protected]

Faust & Backhaus Environmental

[email protected]

Consulting, DE

University of London, UK

[email protected]

Martin Scholze

Leo Posthuma

National Institute for Public

Health and the Environment

(RIVM), NL

Helmholtz Centre for

Environmental Research – UFZ,

Göteborg University, SE

European Chemicals Agency,

FIN

University of London, UK

Rolf Altenburger

[email protected]

Thomas Backhaus

(absent)

Gabriele Schöning

[email protected]

pa.eu

andreas.kortenkamp@pharmacy

.ac.uk

Andreas Kortenkamp

Scientific organiser

Jens-Jørgen Larsen

Ulla Hass

Practical and co-scientific

organiser

National Food Institute, Danish

Technical University, DK

National Food Institute, Danish

Technical University, DK

[email protected]

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Expert workshop on combination effects of chemicals, Hornbæk, Denmark

Nina Cedergreen

University of Copenhagen, Dep.

of Agricultural Sciences, DK

Danish EPA, Chemicals

Division, DK

Danish EPA, Chemicals

Division, DK

Danish EPA, Chemicals

Division, DK

[email protected]

Henrik Tyle

[email protected]

Marie Louise Holmer

(attended the last day)

Pia Juul Nielsen

Workshop co-coordinator

[email protected]

Location:

Sauntehus slotshotel,

e-mail:

[email protected]