20120201 - Acropolis stakeholder conference -...

Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano

The contribution of The contribution of

toxicologytoxicology

Angelo MorettoDepartment of Occupational Health, Università di Milan o

International Centre for Pesticides (ICPS), Azienda Ospedaliera Luigi Sacco, Milano

Italy

ACROPOLIS Stakeholder Conference Bruxelles, 1 February 2012


OutlineOutline

�State of the art of cumulative risk assessment (for pesticides)�How is this addressed in ACROPOLIS

� In vitro studies � In vivo (in-the-field) studies� PBPK modelling

�Multiple routes of exposure (aggregate exposure)�Some preliminary results�Conclusions


The state of the art in EUThe state of the art in EU

(EFSA PPR Panel Opinions)(EFSA PPR Panel Opinions)


Opinion of the Scientific Panel on Plant Protection Products

and their Residues to evaluate the suitability of e xisting

methodologies and, if appropriate, the identificati on of new

approaches to asses cumulative and synergistic risk s from

pesticides to human health with a view to set MRLs for

those pesticides in the frame of Regulation (EC) 39 6/2005

The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1The EFSA Journal (2008) 704, 1--------8484848484848484


Scientific opinion on risk assessment for a Selecte d Group

of Pesticides from the Triazole Group to test possi ble

methodologies to assess cumulative effects from exp osure

through food from these pesticides on human health

The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167The EFSA Journal(2009); 7(9)1167


Types of combined actionsTypes of combined actions

� Dose additivity (Simple similar action)

� Response additivity (Simple dissimilar action)

� Interaction

� Stronger than expected effect

� Weaker than expected effect


Available evidence is that interaction

either does not occur or it is of

low relevance at doses that are at

or below the No -Observable -Adverse -

Effect-Level (NOAEL)

(EFSA, 2008; Boobis et al., 2011)

CONCLUSIONCONCLUSION


Only dose additivity seems to be a

priority in risk assessment

i.e. only exposure to substances

sharing a common Mode Of

Action (MOA) needs to be

cumulated


The problemThe problem

Definition of the Cumulative Assessment

Groups (CAG)


1.1. Preliminary grouping based on:Preliminary grouping based on:

� Structural similarity

� Mechanism of pesticidal action

� General mechanism of mammalian toxicity

� A particular toxic effect


2.Refinement of the preliminary

grouping by detailed evaluation

of available toxicology data

3.Determination of the MOA by

which each substance causes

a common toxic effect


Issues on definition CAGIssues on definition CAG

�Refinement might not be

� Feasible (lack of information)

� Necessary (e.g. too time-consuming vsexpected outcome)

�Care is needed on definition of common mode of action (e.g.: anti-androgens and estrogen -like)


CUMULATIVE CUMULATIVE

ASSESSMENT ASSESSMENT GROUP GROUP

VSVS

COMMONCOMMON

MODE/MECHANISM OF MODE/MECHANISM OF

ACTIONACTION GROUPGROUP


Objective of ACROPOLISObjective of ACROPOLIS

““Test a strategy for Refinement Test a strategy for Refinement

of CAGof CAG””

� In vitro approaches

� In-the-field approach

� Use of PBPK modelling

� Existing models

� Development of a modelling strategy


In vitro approachesIn vitro approaches

� CONAZOLES (UMIL)

� NEUROTOXICANTS (IRAS)


ConazolesConazoles

Teratogenic effects (basis for EFSA PPR Panel opini on on triazoles): use of the rat embryo in vitro model

�Treatment with single conazoles (both teratogenic and non-teratogenic)

�Treatment with mixtures of conazoles (as above)

�Treatment with compounds, not conazoles, causing teratogenic effects

�Treatment with mixtures of conazoles and non-conazoles


In vitro tests allow for: In vitro tests allow for:

�Testing many compounds to identify a common hazard

�Testing many doses to establish an accurate dose-response curve

�Testing many mixture combinations to establish additivity, synergy, antagonism

�Testing unrelated chemicals

�Measurements of several relevant parameters, if needed


In vitro approachIn vitro approach

Consequently, the number of in vivo tests:

� can be reduced

� focused on relevant combinations

� conducted with doses relevant to derive Reference Values or close to human exposure


Fig. 1. Percentage of branchial arch abnormalities (partial or total fusion between I and II branchial

arches) and plurimalformed embryos in controls and in embryos exposed in vitro to tebuconazole or

cyproconazole.


triadimefon (FON) and imazalil (IMA) (MIX2) or triadimefon, imazalil and fluconazole (FLUCO) (MIX3)

%of abnormal embryos

Effects of a mixture of two agrochemical azoles with or without the

co-exposure to a clinical azole in in vitro whole embryo cultures


In vivo tests

RRR Approach

Verification of the correctness of the in

vitro approach


Considerations from these initial resultsConsiderations from these initial results

The tested azoles exert a common and additive activity

and presumably should be included in a CAG (inhibition

of the embryonic CYP26?).

The observed effects indicates the need of a

cumulative and aggregate risk assessment for the azole

fungicides, used in agriculture and in human medicine.

The post-implantation rat whole embryo culture method

is a simple, rapid and sensitive method to determine the

effects of mixtures on embryo development.


NeurotoxicantsNeurotoxicants

�Measurements of several parameters in vitro after treatment with a single compound

�Measurements as above after multiple compounds

�Development of a predictive model(s)


NeurotoxicityNeurotoxicity

� Neuronal communication is electroNeuronal communication is electroNeuronal communication is electroNeuronal communication is electro----chemical signalingchemical signalingchemical signalingchemical signaling

� Neuronal system is strictly regulated to avoid miscommunicationNeuronal system is strictly regulated to avoid miscommunicationNeuronal system is strictly regulated to avoid miscommunicationNeuronal system is strictly regulated to avoid miscommunication

� Neurotoxicity = dysfunction of neuronal communicationNeurotoxicity = dysfunction of neuronal communicationNeurotoxicity = dysfunction of neuronal communicationNeurotoxicity = dysfunction of neuronal communication


Aim & RationaleAim & Rationale

� Cell ViabilityCell ViabilityCell ViabilityCell Viability• Reduced cell viability is a clear measure of toxicityReduced cell viability is a clear measure of toxicityReduced cell viability is a clear measure of toxicityReduced cell viability is a clear measure of toxicity

� Oxidative StressOxidative StressOxidative StressOxidative Stress• Early marker of cell deathEarly marker of cell deathEarly marker of cell deathEarly marker of cell death

• Oxidative stress is related to accelerated ageing of the neuronaOxidative stress is related to accelerated ageing of the neuronaOxidative stress is related to accelerated ageing of the neuronaOxidative stress is related to accelerated ageing of the neuronal cells and l cells and l cells and l cells and implicated in various neurological disordersimplicated in various neurological disordersimplicated in various neurological disordersimplicated in various neurological disorders

� Changes in intracellular calcium concentrations ([CaChanges in intracellular calcium concentrations ([CaChanges in intracellular calcium concentrations ([CaChanges in intracellular calcium concentrations ([Ca2+2+2+2+]]]]iiii

• Early marker of cell deathEarly marker of cell deathEarly marker of cell deathEarly marker of cell death

• CaCaCaCa2+2+2+2+ plays an important role in intraplays an important role in intraplays an important role in intraplays an important role in intra---- and intercellular signaling pathwaysand intercellular signaling pathwaysand intercellular signaling pathwaysand intercellular signaling pathways

• To test for mixture (neuro-)toxicity using a battery of in vitro tests


MethodsMethods

� Dopaminergic and nonDopaminergic and nonDopaminergic and nonDopaminergic and non----dopaminergic cell linesdopaminergic cell linesdopaminergic cell linesdopaminergic cell lines

� 6 Fungicides (Conazole), 2 Insecticides (Organochlorine)6 Fungicides (Conazole), 2 Insecticides (Organochlorine)6 Fungicides (Conazole), 2 Insecticides (Organochlorine)6 Fungicides (Conazole), 2 Insecticides (Organochlorine)

� Mixtures Mixtures Mixtures Mixtures

� Parameters investigated:Parameters investigated:Parameters investigated:Parameters investigated:

• Lysosomal function, Membrane integrity & Mitochondrial Lysosomal function, Membrane integrity & Mitochondrial Lysosomal function, Membrane integrity & Mitochondrial Lysosomal function, Membrane integrity & Mitochondrial metabolism as measure of cell viabilitymetabolism as measure of cell viabilitymetabolism as measure of cell viabilitymetabolism as measure of cell viability

• Production of Reactive Oxygen Species as measure of Production of Reactive Oxygen Species as measure of Production of Reactive Oxygen Species as measure of Production of Reactive Oxygen Species as measure of oxidative stressoxidative stressoxidative stressoxidative stress

• Intracellular CaIntracellular CaIntracellular CaIntracellular Ca2+2+2+2+ homeostasis (homeostasis (homeostasis (homeostasis (““““RestingRestingRestingResting”””” CaCaCaCa2+2+2+2+, Stimulus, Stimulus, Stimulus, Stimulus----evoked Caevoked Caevoked Caevoked Ca2+2+2+2+, Fluorescence microscopy) as measure for , Fluorescence microscopy) as measure for , Fluorescence microscopy) as measure for , Fluorescence microscopy) as measure for neuronal signalingneuronal signalingneuronal signalingneuronal signaling


CaCa2+2+ imaging, an example:imaging, an example:StimulusStimulus--evoked [Caevoked [Ca2+2+]]ii

� Left: Example recordings of [CaLeft: Example recordings of [CaLeft: Example recordings of [CaLeft: Example recordings of [Ca2+2+2+2+]]]]iiii. Both lindane and dieldrin . Both lindane and dieldrin . Both lindane and dieldrin . Both lindane and dieldrin

decrease stimulusdecrease stimulusdecrease stimulusdecrease stimulus----evoked [Caevoked [Caevoked [Caevoked [Ca2+2+2+2+]]]]iiii(K2).(K2).(K2).(K2).

� Right: Quantification of treatment ratio (K2/K1).Right: Quantification of treatment ratio (K2/K1).Right: Quantification of treatment ratio (K2/K1).Right: Quantification of treatment ratio (K2/K1). Exposure to a binary Exposure to a binary Exposure to a binary Exposure to a binary mixture reveals effectmixture reveals effectmixture reveals effectmixture reveals effect----addition on the inhibition of stimulusaddition on the inhibition of stimulusaddition on the inhibition of stimulusaddition on the inhibition of stimulus----evoked evoked evoked evoked [Ca[Ca[Ca[Ca2+2+2+2+]]]]

iiii

Heusinkveld and Westerink 2012


Field study on tebuconazole: outlineField study on tebuconazole: outline

�Assess potential and actual dermal exposure

�Assess dietary exposure

�Assess time-course of urinary excretion of tebuconazole and its relevant metabolites


Pilot studyPilot study

�5 farmers applied TEB on vineyards �2 applied for 1 day (one twice in different months)�2 applied for 2 consecutive days�1 applied for 3 consecutive days

Total of 9 working days

�Farmer’s coverall, underwear and head cover (12+3+1 samples) and hand washes (1-3 per day)�On-cloth and under-cloth sampling pads (8 per day)�Duplicate diets on day -1 and day of application�Pre- and post-application urine (fractional mincturi tions)

Samples collected


Example of work activityExample of work activity

Application of fungicide by manual spraying

Application of fungicide from tractor


Working clothes and sampling Working clothes and sampling

pointspoints1. Right calf2. Left calf3. Right forearm4. Left forearm5. Right thigh6. Left thigh7. Right arm8. Left arm9. Paunch10.Lower back11.Chest12.Upper back13.T-shirt front14.T-shirt back15.Boxer trunks16.Head cover

1 2

3 4

6 5

8 7

9-10

11-12

15

13-14

ba

dc

164 samplingpads underthe clothes

4 samplingpads overthe clothes


TRAINING OF FARMERS ON:•USE OF MATERIALS SUPPLIED

•OPERATIONS TO DUPLICATE THEIR DIET

FARMER RECRUITMENT

COLLECTION OF FOOD THE DAY OF PESTICIDE

APPLICATION

COLLECTION OF DUPLICATE DIET IN REFRIGERATED BOXES

TRANSPORTATION TO LABORATORY

PROVISION OF MATERIALS (SEALABLE PET CONTAINERS

AND COOL BOXES) FOR COLLECTION OF FOODS

AND LABELLING FILLING OF QUESTIONNAIRE ON DIETARY INTAKE THROUGH INTERVIEW

COLLECTION OF FOOD THE DAY BEFORE PESTICIDE

APPLICATION (CONTROL)

EXPERIMENTAL PROTOCOL


FRUIT AND VEGETABLES

FOOD RECORDING AND CREATION OF DATABASE

SAMPLING

HOMOGENIZATION

DELIVERY OF SAMPLES FOR ANALYSIS

PARTITION OF FOOD ITEMS CEREAL BASED

FOODS

FREEZING

OTHER FOODS

EXPERIMENTAL PROTOCOL

LIQUIDS

Total of 64 samples


Physiologically Based Pharmaco Physiologically Based Pharmaco

Kinetic (PBPK) ModellingKinetic (PBPK) Modelling

� Development of models from experimental studies

� Application of models to “exposure data”(conazoles)


Provisional conclusionsProvisional conclusions

� In vitro studies can provide the basis for defining CAG

� In vitro studies can provide indication of (lack of ) synergistic effects

� In vivo studies in animals might be required for confirmation of in vitro findings

�PBPK modelling will help in understanding the toxicological significance of findings in workers ( or other exposed population)


University of Milan, ItalyField study

M. Brusamolino, C. Colosio, S. Fustinoni, S. Mandic- Rajcevic,R. Mercadante, E. Polledri, M. Porrini, F.M. Rubino, P. Ri so, R. Rizzo, G. Vianello

In vitro conazole studyE. Giavini, E. Menegola, F. Di Renzo

INRAN, ItalyField study

L. Gambelli, M. Oggioni, A. TurriniUtrecht University, The Netherlands

In vitro neurotox studyH. Heusinkveld, R. Westerink

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