Exploration of alternative methods for toxicity assessment of pesticide

metabolites

Alternative in vitro methods to characterize the role of endocrine active substances (EAS) in hormone-targeted tissues, Istituto Superiore di Sanita` , Rome, 17th December 2012

Background

Regulation (EC) No 1107/2009 concerning the placing of PPPs on the market

requires:

• identification of metabolites

• determination of their “toxicological relevance” for in/exclusion in the residue definition

Background

OECD Guidance Document on the Definition of Residue

• Proposes residue definition for risk assessment, reflecting the actual toxicological burden consisting of active substance and relevant metabolites

• Requires quantitative and qualitative assessment of metabolites

Pesticide metabolites as residues

• In contrast to active substances for which a full toxicological dossier needs to be provided usually very limited or no toxicological data are available although their toxic potency and toxicological profile might differ substantially from the parent compound

• Reference values of parent compounds are applied for metabolites present in food commodities

Assessment tools

Conventional testing of metabolites?

• Number of metabolites

• Difficulties in synthesising sufficient amounts

• Costs

• Research capacities

• Animal welfare concerns

Preparatory Work for the Toxicology Part of the Opinion

• Project: Impact of Metabolism on Toxicity

(AGES, Austria)

• Project on Threshold of Toxicological Concern (TTC) Concept (CRD, UK)

• 2 projects on (Quantitative) Structure-Activity Relationships (Q)SARs (JRC, EC)

Assessment tools

Impact of metabolic and degradation processes on the toxicological properties of residues of pesticides in food commodities

(published 6 May 2010)

http://www.efsa.europa.eu/en/supporting/pub/49e.htm

Assessment tools – Impact of Metabolism

• Review of transformation pathways (11 pesticide classes) and comparisonwith toxicity data (DARs, public literature)

• Based on lack of other information analyses largely based onacute toxstudies

• In total more than 140 chemical changes identified and analysed

Conclusions:

• Toxification/detoxification cannot be attributed to certain metabolic steps(Conjugates not necessary less toxic due to cleavage)

• Metabolite > 10% to be considered as contributing significantly totoxicity

• Improvement of ADME studies (more accuracy, more information)necessary

Assessment tools – Impact of Metabolism

Applicability of thresholds of toxicological concern in the dietary risk assessment of metabolites, degradation and reaction products of pesticides

(published 24 March 2010)

http://www.efsa.europa.eu/en/supporting/pub/44e.htm

Assessment tools - TTC

Threshold of Toxicological Concern (TTC) Concept

• Level of exposure below which no appreciable risk is expected

• Based on comparison of chemical structures and chronic data (CPDB data base - Gold et al. 1984, 1989)

• So far only applicable for chronic exposures

• Already in use for assessment of food flavourings

Assessment tools – TTC

Threshold of Toxicological Concern (TTC) Concept

Classification TTC threshold (µg/person/d)

TTC threshold (µg/kg bw/d)

Structural features

Cramer class I 1800 30 Simple structure + Metabolism

Cramer class II 540 9 Less innocuous than class I

Cramer Class III 90 1.5 Suggestive of significant toxicity – Functional groups

Cramer class III + Neurotoxicity

18 0.3 Parent compound neurotoxic –OP + Carbamate structure

Cramer class III + Genotoxicity

0.15 0.0025 Parent compound genotoxicIdentification with QSAR

Assessment tools - TTC

• Review on the TTC concept and current use

• Validation of TTC for pesticides: Comparison of 100 pesticide ADIs with TTC classification

• Case study with 79 metabolites of 15 selected pesticides

Conclusions:

• TTC appropriate for assessment of metabolites

• Exposure to metabolites considered to be covered by TTC thresholds allocated – critical is the genotoxicity threshold

• QSARs predictions for genotoxicity considered reliable

Assessment tools - TTC

Applicability of QSAR analysis to the evaluation of the toxicological relevance of metabolites and degradates of pesticide active substances for dietary risk assessment

(published 7 May 2010)

http://www.efsa.europa.eu/en/supporting/pub/50e.htm

Assessment tools – QSAR I

• Reviewon the use of QSARfor regulatory purposes

• Extensive evaluation of potentially useful QSARs

(e.g. Toxtree, Lazar, Derek, Hazard Expert , Caesar, Topkat) using different data sets (Pesticide metabolites, EU classified substances, CPDB)

• Case studies focused on genotoxicity based on the outcome ofthe TTC project

Conclusions:

• Currently limited use of QSARs for regulatory purposes

• Recommendations for the application of different modelscovering all relevant toxicological endpoints - i.e.combination of models recommended to optimise sensitivityand specificity in regard to genotoxicity

Assessment tools – QSAR I

Applicability of QSAR analysis in the evaluation ofdevelopmental and neurotoxicity effects for theassessment of the toxicological relevance ofmetabolites and degradates of pesticide activesubstances for dietary risk assessment

(published 16 June 2011)

http://www.efsa.europa.eu/en/supporting/pub/169e.htm

Assessment tools – QSAR II

• Evaluation of QSAR models (Derek, Caesar, Topkat,Leadscope, Hazard Expert, PASS, ADME predictor, Accord)and read across (OECDtoolbox) for assessment ofdevelopmental effects and neurotoxicity

Conclusions:

• No appropriate QSAR models for neurotoxicity identified

• Stepwise approach for developmental effects combiningread across and QSARmight be promising

Assessment tools – QSAR II

Extension of the mandate including assessment of isomers (September 2011)

In order to:

• investigate if the approaches proposed for assessment of metabolites are applicable for isomers

• identify specific issues, suitable methodologies, research needs for isomers

Scientific Opinion on Evaluation of the Toxicological Relevance of Pesticide Metabolites for Dietary Risk Assessment (published 26 July 2012)

Available from:

http://www.efsa.europa.eu/en/efsajournal/pub/2799.htm

Scientific Opinion – Toxicology Part

Chapters:

• Presentation and evaluation of projects on Impact of Metabolism, TTC and QSARs

• Development of a single TTC value for acute exposures (based on short term NOAELs)

• Proposal for toxicity assessment and for acuteand chronic exposures (decision trees)

• Evaluation of the assessment tools for isomerassessment

• Critical Issues/Uncertainties

Compounds predicted negative

Compounds predicted negative/inconclusive

Is an ARfD assigned to parent substance

NO

Has the parent compound a neurotoxic mechanism of action

YES QSAR analysis for developmental toxicitySee chapter 7.2

Compounds predicted positive

Read-across See chapter 6.3

Compounds predicted positive /inconclusive

Acute exposure estimate of metaboliteSee chapter 8

NO

OP,carbamate toxicophoreSee chapter 5

Exposure >5 µg/kg bw/d(modified acute value)See chapter 5.3.1 No acute

RA necessary

YES

Nofurther testing

or**

The metabol

ite is not

relevantNo

No

YES

Non TTC approachsee chronic

Exposure >0.3 µg/kg bw/dSee chapter 5.3.1

No

YES

Is the metabolite in Cramer Class I?

Exposure >30 µg/kg bw/d (modified acute value)See chapter 5.3.1

Is the metabolite in CramerClass II or III?

No

No

YES

YES

YES

• Toxification/detoxification not attributable to specific metabolic steps

• Use of PBPK models recommended for assessment of ADME

• TTC concept is an appropriate and sufficiently conservative evaluation tool for metabolites , but should not be used for parent compounds

• For acute exposures an acute TTC threshold of 5 µg/kg bw/d can be applied

• Where exposure exceeds TTC values, targeted testing can be applied

• TTC can in principle used for evaluation of endocrine active substances (except steroids) but should preferably be based on testing if data already indicate that a compound has endocrine mediated adverse effects

Conclusions/Recommendations

• Once an EU agreed approach for defining and assessing of endocrine active substances is available, the use of the TTC approach for EAS needs to be revisited

• In principle the TTC concept and QSARs could be used for assessment of stereoisomers present in mixtures, however, in practice information on stereochemistry is not provided in the data sets underlying these tools

• QSARs should not be used as a stand alone tool for assessments but can together with the application of read-across complement the TTC approach

• The usability of QSARs for the assessment of endocrine active active substance has not been investigated based on lack of a definition and appropriate data bases

• A guidance should be developed based on the scientific opinion

Conclusions/Recommendations

Thank you!