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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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
The state of the art in EUThe state of the art in EU
(EFSA PPR Panel Opinions)(EFSA PPR Panel Opinions)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
The problemThe problem
Definition of the Cumulative Assessment
Groups (CAG)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
CUMULATIVE CUMULATIVE
ASSESSMENT ASSESSMENT GROUP GROUP
VSVS
COMMONCOMMON
MODE/MECHANISM OF MODE/MECHANISM OF
ACTIONACTION GROUPGROUP
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
In vitro approachesIn vitro approaches
� CONAZOLES (UMIL)
� NEUROTOXICANTS (IRAS)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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.
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
In vivo tests
RRR Approach
Verification of the correctness of the in
vitro approach
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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.
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
Example of work activityExample of work activity
Application of fungicide by manual spraying
Application of fungicide from tractor
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
Physiologically Based Pharmaco Physiologically Based Pharmaco
Kinetic (PBPK) ModellingKinetic (PBPK) Modelling
� Development of models from experimental studies
� Application of models to “exposure data”(conazoles)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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)
Centro Internazionale per gli Antiparassitari e la Prevenzione SanitariaAzienda Ospedaliera “Luigi Sacco” - Milano
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