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1 Unclassified ENV/JM/MONO(2010)15 ENV/JM/MONO(2010)15 Unclassified Organisation de Coopération et de Développement Économiques Organisation for Economic Co-operation and Development 08-Jun-2010 English - Or. English ENVIRONMENT DIRECTORATE JOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY Cancels & replaces the same document of 07 June 2010 Series on Testing and Assessment No. 124 GUIDANCE FOR THE DERIVATION OF AN ACUTE REFERENCE DOSE English - Or. English JT Document complet disponible sur OLIS dans son format d'origine Complete document available on OLIS in its original format

2 OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 124 GUIDANCE FOR THE DERIVATION OF AN ACUTE REFERENCE DOSE Environment Directorate ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT Paris

3 Also published in the Series on Testing and Assessment: No. 1, Guidance Document for the Development of OECD Guidelines for Testing of Chemicals (1993; reformatted 1995, revised 2006 and 2009) No. 2, Detailed Review Paper on Biodegradability Testing (1995) No. 3, Guidance Document for Aquatic Effects Assessment (1995) No. 4, Report of the OECD Workshop on Environmental Hazard/Risk Assessment (1995) No. 5, Report of the SETAC/OECD Workshop on Avian Toxicity Testing (1996) No. 6, Report of the Final Ring-test of the Daphnia magna Reproduction Test (1997) No. 7, Guidance Document on Direct Phototransformation of Chemicals in Water (1997) No. 8, Report of the OECD Workshop on Sharing Information about New Industrial Chemicals Assessment (1997) No. 9, Guidance Document for the Conduct of Studies of Occupational Exposure to Pesticides during Agricultural Application (1997) No. 10, Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (1998) No. 11, Detailed Review Paper on Aquatic Testing Methods for Pesticides and industrial Chemicals (1998) No. 12, Detailed Review Document on Classification Systems for Germ Cell Mutagenicity in OECD Member Countries (1998) No. 13, Detailed Review Document on Classification Systems for Sensitising Substances in OECD Member Countries 1998) No. 14, Detailed Review Document on Classification Systems for Eye Irritation/Corrosion in OECD Member Countries (1998) No. 15, Detailed Review Document on Classification Systems for Reproductive Toxicity in OECD Member Countries (1998) No. 16, Detailed Review Document on Classification Systems for Skin Irritation/Corrosion in OECD Member Countries (1998) No. 17, Environmental Exposure Assessment Strategies for Existing Industrial Chemicals in OECD Member Countries (1999) ENV/JM/MONO(2010)15 3

4 No. 18, Report of the OECD Workshop on Improving the Use of Monitoring Data in the Exposure Assessment of Industrial Chemicals (2000) No. 19, Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals used in Safety Evaluation (1999) No. 20, Revised Draft Guidance Document for Neurotoxicity Testing (2004) No. 21, Detailed Review Paper: Appraisal of Test Methods for Sex Hormone Disrupting Chemicals (2000) No. 22, Guidance Document for the Performance of Out-door Monolith Lysimeter Studies (2000) No. 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000) No. 24, (2001) Guidance Document on Acute Oral Toxicity Testing No. 25, Detailed Review Document on Hazard Classification Systems for Specifics Target Organ Systemic Toxicity Repeated Exposure in OECD Member Countries (2001) No. 26, Revised Analysis of Responses Received from Member Countries to the Questionnaire on Regulatory Acute Toxicity Data Needs (2001) No 27, Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment (2001) No 28, Guidance Document for the Conduct of Skin Absorption Studies (2004) No 29, Guidance Document on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (2001) No 30, Detailed Review Document on Hazard Classification Systems for Mixtures (2001) No 31, Detailed Review Paper on Non-Genotoxic Carcinogens Detection: The Performance of In-Vitro Cell Transformation Assays (2007) No. 32, Guidance Notes for Analysis and Evaluation of Repeat- Dose Toxicity Studies (2000) 4

5 No. 33, Harmonised Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures (2001) No. 34, Guidance Document on the Development, Validation and Regulatory Acceptance of New and Updated Internationally Acceptable Test Methods in Hazard Assessment (2005) No. 35, Guidance notes for analysis and evaluation of chronic toxicity and carcinogenicity studies (2002) No. 36, Report of the OECD/UNEP Workshop on the use of Multimedia Models for estimating overall Environmental Persistence and long range Transport in the context of PBTS/POPS Assessment (2002) No. 37, Detailed Review Document on Classification Systems for Substances Which Pose an Aspiration Hazard (2002) No. 38, Detailed Background Review of the Uterotrophic Assay Summary of the Available Literature in Support of the Project of the OECD Task Force on Endocrine Disrupters Testing and Assessment (EDTA) to Standardise and Validate the Uterotrophic Assay (2003) No. 39, Guidance Document on Acute Inhalation Toxicity Testing (2009) No. 40, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures Which Cause Respiratory Tract Irritation and Corrosion (2003) No. 41, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures which in Contact with Water Release Toxic Gases (2003) No. 42, Guidance Document on Reporting Summary Information on Environmental, Occupational and Consumer Exposure (2003) No. 43, Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment (2008) No. 44, Description of Selected Key Generic Terms Used in Chemical Hazard/Risk Assessment (2003) No. 45, Guidance Document on the Use of Multimedia Models for Estimating Overall Environmental Persistence and Long-range Transport (2004) No. 46, Detailed Review Paper on Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances (2004) ENV/JM/MONO(2010)15 5

6 No. 47, Detailed Review Paper on Fish Screening Assays for the Detection of Endocrine Active Substances (2004) No. 48, New Chemical Assessment Comparisons and Implications for Work Sharing (2004) No. 49, Report from the Expert Group on (Quantitative) Structure-Activity Relationships [(Q)SARs] on the Principles for the Validation of (Q)SARs (2004) No. 50, Report of the OECD/IPCS Workshop on Toxicogenomics (2005) No. 51, Approaches to Exposure Assessment in OECD Member Countries: Report from the Policy Dialogue on Exposure Assessment in June 2005 (2006) No. 52, Comparison of emission estimation methods used in Pollutant Release and Transfer Registers (PRTRs) and Emission Scenario Documents (ESDs): Case study of pulp and paper and textile sectors (2006) No. 53, Guidance Document on Simulated Freshwater Lentic Field Tests (Outdoor Microcosms and Mesocosms) (2006) No. 54, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (2006) No. 55, Detailed Review Paper on Aquatic Arthropods in Life Cycle Toxicity Tests with an Emphasis on Developmental, Reproductive and Endocrine Disruptive Effects (2006) No. 56, Guidance Document on the Breakdown of Organic Matter in Litter Bags (2006) No. 57, Detailed Review Paper on Thyroid Hormone Disruption Assays (2006) No. 58, Report on the Regulatory Uses and Applications in OECD Member Countries of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models in the Assessment of New and Existing Chemicals (2006) No. 59, Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats (2006) No. 60, Report of the Initial Work Towards the Validation of the 21-Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1A) (2006) 6

7 No. 61, Report of the Validation of the 21-Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1B) (2006) No. 62, Final OECD Report of the Initial Work Towards the Validation of the Rat Hershberger Assay: Phase-1, Androgenic Response to Testosterone Propionate, and Anti-Androgenic Effects of Flutamide (2006) No. 63, Guidance Document on the Definition of Residue (2006, revised 2009) No. 64, Guidance Document on Overview of Residue Chemistry Studies (2006, revised 2009) No. 65, OECD Report of the Initial Work Towards the Validation of the Rodent Utertrophic Assay - Phase 1 (2006) No. 66, OECD Report of the Validation of the Rodent Uterotrophic Bioassay: Phase 2. Testing of Potent and Weak Oestrogen Agonists by Multiple Laboratories (2006) No. 67, Additional data supporting the Test Guideline on the Uterotrophic Bioassay in rodents (2007) No. 68, Summary Report of the Uterotrophic Bioassay Peer Review Panel, including Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the follow up of this report (2006) No. 69, Guidance Document on the Validation of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models (2007) No. 70, Report on the Preparation of GHS Implementation by the OECD Countries (2007) No. 71, Guidance Document on the Uterotrophic Bioassay - Procedure to Test for Antioestrogenicity (2007) No. 72, Guidance Document on Pesticide Residue Analytical Methods (2007) No. 73, Report of the Validation of the Rat Hershberger Assay: Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists and Negative Reference Chemicals by Multiple Laboratories. Surgical Castrate Model Protocol (2007) No. 74, Detailed Review Paper for Avian Two-generation Toxicity Testing (2007) No. 75, Guidance Document on the Honey Bee (Apis Mellifera L.) Brood test Under Semi-field Conditions (2007) ENV/JM/MONO(2010)15 7

8 No. 76, Final Report of the Validation of the Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances: Phase 1 - Optimisation of the Test Protocol (2007) No. 77, Final Report of the Validation of the Amphibian Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study (2007) No. 78, Final Report of the Validation of the 21-day Fish Screening Assay for the Detection of Endocrine Active Substances. Phase 2: Testing Negative Substances (2007) No. 79, Validation Report of the Full Life-cycle Test with the Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007) No. 80, Guidance on Grouping of Chemicals (2007) No. 81, Summary Report of the Validation Peer Review for the Updated Test Guideline 407, and Agreement of the Working Group of National Coordinators of the Test Guidelines Programme on the follow-up of this report (2007) No. 82, (2007) Guidance Document on Amphibian Thyroid Histology No. 83, Summary Report of the Peer Review Panel on the Stably Transfected Transcriptional Activation Assay for Detecting Estrogenic Activity of Chemicals, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007) No. 84, Report on the Workshop on the Application of the GHS Classification Criteria to HPV Chemicals, 5-6 July Bern Switzerland (2007) No. 85, Report of the Validation Peer Review for the Hershberger Bioassay, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007) No. 86, Report of the OECD Validation of the Rodent Hershberger Bioassay: Phase 2: Testing of Androgen Agonists, Androgen Antagonists and a 5 α-reductase Inhibitor in Dose Response Studies by Multiple Laboratories (2008) No. 87, Report of the Ring Test and Statistical Analysis of Performance of the Guidance on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (Transformation/ Dissolution Protocol) (2008) 8

9 No.88 Workshop on Integrated Approaches to Testing and Assessment (2008) No.89 Retrospective Performance Assessment of the Test Guideline 426 on Developmental Neurotoxicity (2008) No.90 Background Review Document on the Rodent Hershberger Bioassay (2008) No.91 Report of the Validation of the Amphibian Metamorphosis Assay (Phase 3) (2008) No.92 Report of the Validation Peer Review for the Amphibian Metamorphosis Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-Up of this Report (2008) No.93 Report of the Validation of an Enhancement of OECD TG 211: Daphnia Magna Reproduction Test (2008) No.94 Report of the Validation Peer Review for the 21-Day Fish Endocrine Screening Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2008) No.95 Detailed Review Paper on Fish Life-Cycle Tests (2008) No.96 Guidance Document on Magnitude of Pesticide Residues in Processed Commodities (2008) No.97 Detailed Review Paper on the use of Metabolising Systems for In Vitro Testing of Endocrine Disruptors (2008) No. 98 Considerations Regarding Applicability of the Guidance on Transformation/Dissolution of Metals Compounds in Aqueous Media (Transformation/Dissolution Protocol) (2008) No. 99 Comparison between OECD Test Guidelines and ISO Standards in the Areas of Ecotoxicology and Health Effects (2008) No.100 Report of the Second Survey on Available Omics Tools (2009) No.101 Report on the Workshop on Structural Alerts for the OECD (Q)SAR Application Toolbox (2009) No.102 Guidance Document for using the OECD (Q)SAR Application Toolbox to Develop Chemical Categories According to the OECD Guidance on Grouping of Chemicals (2009) No.103 Detailed Review Paper on Transgenic Rodent Mutation Assays (2009) ENV/JM/MONO(2010)15 9

10 No.104 Performance Assessment: Conparsion of 403 and CxT Protocols via Simulation and for Selected Real Data Sets (2009) No. 105 Report on Biostatistical Performance Assessment of the draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation Toxicity (2009) No.106 Guidance Document for Histologic Evaluation of Endocrine and Reproductive Test in Rodents (2009) No.107 Preservative treated wood to the environment for wood held in storage after treatment and for wooden commodities that are not cover and are not in contact with ground. (2009) No.108 Report of the validation of the Hershberger Bioassay (weanling model) (2009) No. 109 Literature review on the 21-Day Fish Assay and the Fish Short-Term Reproduction Assay (2009) No. 110 Report of the validation peer review for the weanling Hershberger Bioassay and agreement of the working of national coordinators of the test guidelines programme on the follow-up of this report (2009) No. 111 Report of the Expert Consultation to Evaluate an Estrogen Receptor Binding Affinity Model for Hazard Identification (2009) No. 112 The 2007 OECD List of High Production Volume Chemicals (2009) No. 113 Report of The Focus Session On Current And Forthcoming Approaches For Chemical Safety And Animal Welfare (2010) No. 114 Performance Assessment of Different Cytotoxic and Cytostatic Measures for the In Vitro Micronucleus Test (MNVIT): Summary of results in the collaborative trial (2010) No. 115 Guidance Document on the Weanling Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties (2009) No. 116 Guidance Document 116 on the Conduct and Design of Chronic Toxicity and Carcinogenicity Studies, Supporting Test Guidelines 451, 452 and 453. First edition including the general introduction and the section on dose selection (2010) No. 118 Workshop Report on OECD Countries Activities Regarding Testing, Assessment and Management of Endocrine Disrupters Part I and Part II (2010) 10

11 No. 119 Classification and Labelling of chemicals according to the UN Globally Harmonized System: Outcome of the Analysis of Classification of Selected Chemicals listed in Annex III of the Rotterdam Convention (2010) No. 120 Explanatory Background Document to the OECD Draft Test Guideline on in vitro Skin Irritation Testing (2010) No. 121 Detailed review paper (DRP) on Molluscs life-cycle Toxicity Testing (2010) No. 122 Guidance Document on the determination of the Toxicity of a Test Chemical to the Dung Beetle Aphodius Constans (2010) No. 123 Guidance Document on the Diagnosis of Endocrinerelated Histopathology in Fish Gonads (2010) No. 124 Draft Guidance for the Derivation of an Acute Reference Dose (2010) ENV/JM/MONO(2010)15 OECD 2010 Applications for permission to reproduce or translate all or part of this material should be made to: Head of Publications Service, RIGHTS@oecd.org, OECD, 2 rue André-Pascal, Paris Cedex 16, France 11

12 ABOUT THE OECD The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental organisation in which representatives of 31 industrialised countries in North and South America, Europe and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise policies, discuss issues of mutual concern, and work together to respond to international problems. Most of the OECD s work is carried out by more than 200 specialised committees and working groups composed of member country delegates. Observers from several countries with special status at the OECD, and from interested international organisations, attend many of the OECD s workshops and other meetings. Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is organised into directorates and divisions. The Environment, Health and Safety Division publishes free-of-charge documents in ten different series: Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides and Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of Novel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission Scenario Documents; and Safety of Manufactured Nanomaterials. More information about the Environment, Health and Safety Programme and EHS publications is available on the OECD s World Wide Web site ( This publication was developed in the IOMC context. The contents do not necessarily reflect the views or stated policies of individual IOMC Participating Organizations. The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was established in 1995 following recommendations made by the 1992 UN Conference on Environment and Development to strengthen co-operation and increase international coordination in the field of chemical safety. The Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO and OECD. The World Bank and UNDP are observers. The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by the Participating Organisations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. 12

13 This publication is available electronically, at no charge. For this and many other Environment, Health and Safety publications, consult the OECD s World Wide Web site ( or contact: OECD Environment Directorate, Environment, Health and Safety Division 2 rue André-Pascal Paris Cedex 16 France Fax: (33-1) ehscont@oecd.org 13

14 FOREWORD The objective of this document is to provide guidance on how to derive an Acute Reference Dose (ARfD). It outlines a stepwise approach on how to best use all available toxicological data, and what to do if more data are needed on the refinement of the toxicological data base with regard to the acute effects of the compound being investigated or an advanced exposure assessment, if the risk assessment indicates a human health concern. It is not intended that this guidance provide a new Test Guideline or to encourage additional animal testing. The results of a recent retrospective analysis confirmed that the development of a design for an acute study that produces more comprehensive toxicological data for setting ARfDs would be valuable but that such a special ARfD study would become necessary for very few pesticides only. Therefore, guidance on the design and performance of a single dose test is provided on the basis that if a single exposure study is necessary, it should be performed according to a harmonised OECD test procedure. This guidance document is primarily intended for pesticides, biocides and veterinary drugs, but could be used for all categories of chemical substances which may be ingested by human beings in food and/or drinking water as well as non-dietary oral exposure. The general principles and concepts can also be applied to dermal and inhalation exposure routes. However, these routes would more appropriately be addressed in separate OECD guidance documents. An OECD Guidance Document is under development for setting acute reference concentrations for the inhalation exposure route. The proposal to develop this guidance document was submitted by Germany in The Working Group of National Coordinators of the Test Guidelines Programme (WNT) was requested to comment on successive drafts in November 2007, February 2009, and November The draft document was also submitted to the Working Group on Pesticides for comments. A small drafting group meeting was held in Germany in October 2008, and an expert group meeting was hosted in Geneva in September 2009, hosted by the WHO. The WNT slightly revised the draft guidance document and approved the revised draft guidance document at its meeting held on March The Joint Meeting of Chemicals Committee and Working Party on Chemicals, Pesticides and Biotechnology agreed to its declassification on 1 June This document is published under the responsibility of the Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology. 14

15 TABLE OF CONTENTS ABOUT THE OECD FOREWORD BACKGROUND Purpose Basic Considerations Tiered-Approach for the Derivation of an appropriate ARfD Step One Step Two (Annex 1) Step Three (Annex 2) Specific Guidance on the Derivation of ARfDs Haematotoxicity Immunotoxicity Neurotoxicity Kidney and liver effects Endocrine effects Developmental effects: Maternal-toxic effects in developmental toxicity studies Direct effects on Gastrointestinal (GI) tract: Other findings relevant for setting an ARfD Animal Welfare Consideration G. Consideration of Human Data H. Consideration of Different Subpopulations References ANNEX REFINEMENT OF THE EXPOSURE CALCULATION FOR THE ACUTE RISK ASSESSMENT Case Case Case ANNEX GUIDANCE FOR CONDUCTING A SINGLE EXPOSURE TOXICITY STUDY INITIAL CONSIDERATIONS PRINCIPLE OF THE TEST DESCRIPTION OF THE METHOD Selection of Animal Species Housing and Feeding Conditions Preparation of Animals Preparation of Doses PROCEDURE

16 Study Duration Number and Sex of Animals Dose Selection Administration of Doses Clinical Observations Body Weight and Food/Water Consumption Toxicokinetics Functional Observations Haematology Clinical Biochemistry Urinalysis Pathology DATA AND REPORTING Test Report ANNEX LIST OF ACRONYMS

17 Background 1. Regulatory requirements or legislation relating to the protection of human health have led to the establishment of Acute Reference Values (ARVs) for substances which would cause toxic effect after acute human exposure. This approach applies primarily to pesticide, biocide, and veterinary drug residues in food and drinking water for which Acute Reference Doses (ARfDs) have to be considered (1) (2) (3) (4) (5). Regulatory authorities are required to protect the general population including susceptible groups against effects induced by acute oral exposure to hazardous substances, if the Tolerable/Acceptable Daily Intake (TDI/ADI) is likely to be substantially exceeded for short periods of time (6) (7). 2. The ARfD of a chemical is an estimate of the amount of a substance in food and/or drinking water, normally expressed on a body weight basis that can be ingested in a period of 24 hours or less, without appreciable health risk to consumer, on the basis of all the known facts at the time of the evaluation (2). 3. Various guidance documents are available for setting ARVs (1) (2) (3) (4) (7) (8). The WHO panel of the Joint Meeting on Pesticide Residues (JMPR) adopted general considerations in setting of ARfDs for pesticide chemicals (1) (2). Solecki et al. (1) described in detail a stepwise process for establishing ARfDs, as well as specific considerations and guidance regarding the identification of the most appropriate critical effects for selected toxicological endpoints. The general principles and methods for setting an ARfD for chemicals in food have been recently updated by IPCS (9) (10). 4. For a critical effect, a no-observed-adverse-effect-level (NOAEL) that is typically determined from animal studies has been traditionally used as a Point of Departure (PoD) when deriving an ARfD. An alternative method to derive a PoD is the use of the Benchmark Dose (BMD) modelling approach response (11). The BMD is defined as the dose producing a predetermined level of change in response (such as a 10% increase in the incidence of a particular toxic effect) compared with the background. A BMD is derived by fitting a mathematical model to the dose-response data, and is often accompanied by an estimate of the statistical lower confidence limit (BMDL) on the BMD. 5. The JMPR ARfD guidance acknowledged that endpoints from a repeat dose toxicity study could be used for setting an ARfD if the critical effect of the compound has not been adequately evaluated in a single exposure study. The JMPR recognised that this approach is likely to be conservative. 6. A retrospective analysis of ARfD values was conducted on 198 pesticides, which have been evaluated and peer-reviewed in Europe and were included in Annex I of EU directive 91/414/EEC in the time period between the years 2000 and 2008 (9) (12). For 48 % of all substances, no ARfD was considered necessary because of the low acute toxicity of these pesticides. The majority of ARfDs was based on studies which are regularly required for pesticides and in which specific acute alerts were investigated. In less than 10 % of cases, conservatively established ARfD values were based on repeated dose toxicity or multigeneration studies. For 4 of these pesticides (i.e. 2 %) a refinement of the ARfDs using an additional toxicity study would be justified because refinement on the exposure side was not sufficient. In the analysed database, such special studies for ARfD refinement were submitted for 4 % of the 198 pesticides. They were mostly performed in addition to the basic acute toxicity data requirements, if it was apparent that the acute intake estimation exceeded a conservatively established ARfD. However, in some cases such studies were not accepted by the authorities because of quality deficiencies. 7. The results of this recent analysis confirmed that the development of a harmonised acute study design that produces more comprehensive toxicological data for setting ARfDs would be valuable. 17

18 8. In a few cases, the results from monitoring programs may provide residue data showing that a conservatively derived ARfD is exceeded. To estimate if there is a real human health risk, a refinement of the risk assessment might be performed first on the basis of exposure calculation and second on the basis of a PoD from a more appropriate special single exposure study. Such a study could be undertaken as a last resort to perform a more realistic human health risk assessment or to justify the lowering of Maximum Residue Levels (MRLs) or the withdrawal of an authorisation of a plant protection product. 9. The ILSI Health and Environmental Sciences Institute (HESI), through its Agricultural Chemical Safety Assessment (ACSA) Committee, designed a toxicity testing scheme for agricultural chemicals that uses a tiered approach. It moves away from paradigms that involve extensive animal testing for every possible adverse outcome to an approach which focuses more on the needs of the risk assessment (13). The HESI ACSA Task Force devised a set of studies in a tiered approach which could provide information for the most relevant human exposure periods and outlined a draft protocol for a single exposure test in dogs or rodents as an optional step five (14). Doe et al. (14) proposed that the ARfD could be based on 28-day rat and 90-day dog studies with additional parameters and if the ARfD derived from the repeat dose study indicates an adequate margin of exposure, then the performance of a single exposure study would not be necessary. The authors emphasized that if a single exposure study is considered necessary, existing data/knowledge should be considered to determine the relevant endpoints and the most appropriate species (rat or dog). 10. The tiered approach proposed by HESI ACSA to determine the need for a single exposure study is consistent with the stepwise approach outlined by Solecki et al. (1). As emphasized by Solecki et al. (1) and by Doe et al. (14), results of existing toxicity data, combined with knowledge of potential human exposure, should be used to determine the need for an acute exposure study. The available toxicity studies may also guide whether the dog or the rat should be used. 11. The single exposure study design proposed by the JMPR (2) and Solecki et al. (1) and adopted by the HESI ACSA Task Force (14) forms the basis of the guidance on how to perform and tailor a single exposure test (Annex 2). 12. The results of the single exposure study should (i) clarify whether a substance poses an unacceptable acute risk and (ii) allow the derivation of a refined ARfD for acute intake of residues in food and/or drinking water. 13. At the 13 th Meeting of the OECD Working Group on Pesticides in 2002, the JMPR presented a proposal for an OECD Test Guideline animal study, designed to support the JMPR-Guidance to derive an ARfD for dietary risk assessment for human health (15). The EC, Spain, and Crop Life International supported this proposal, suggested improvements and recommended that JMPR should proceed by approaching the Working Group of National Co-ordinators to the Test Guidelines Programme (WNT). 14. The 19 th WNT meeting then agreed that an improved stepwise approach for a harmonised guidance document, but not an OECD Test Guideline animal study, should be developed for the derivation of an ARfD. The WNT agreed to include the project in the work plan for 2007 (16). Purpose 15. The objective of this document is to provide a stepwise approach for a harmonized guidance on how to set an ARfD based on all appropriate existing toxicological and exposure data. The general considerations in the setting of an ARfD in an enhanced stepwise process, as well as specific considerations and guidance regarding the identification of the most appropriate critical effects for selected toxicological endpoints are described. The general biological background and the data available through standard toxicological testing for regulatory purposes, interpretation of the data, 18

19 conclusions and recommendations for future improvements are described for these selected relevant endpoints. Special emphasis is placed on evaluating whether toxic effects observed in the standard package of repeat dose toxicity studies may also occur after single doses. 16. Data are not often available for many types of effects under acute exposure conditions and it is possible that the PoDs and endpoints that will be critical for setting an ARfD may differ from those for setting chronic RfDs, or ADIs. The general principle is agreed that the ARfD should be equal to or greater than other long-term reference values of the same chemical (i.e. an individual can generally tolerate a higher amount of a substance with an acute exposure than with a repeat exposure). This is important because ARfDs are typically coupled with high-end exposure values rather than the average exposure values that are employed in risk assessments involving repeat exposures. 17. This guidance document is intended to promote a harmonised scientific basis for the derivation of ARfDs suitable for refined acute risk assessment in a range of acute human oral exposure scenarios. 18. This proposed guidance document will Replace the need to conduct unnecessary tests on animals by introducing a tiered approach for human health risk assessment, including a refined exposure assessment, Reduce the need to repeat animal tests which have not been performed in a way which adequately satisfy the requirements of different regulatory agencies, and Refine a harmonised procedure for determining an ARfD of a compound in infrequent exposure situations where it is necessary to adequately characterise the acute hazard. 19. This document presents specific guidance on How to select relevant endpoints and PoDs from the existing database, How to refine the exposure calculation for the acute risk assessment in Annex 1, and How to perform a tailored single exposure study (Annex 2), including the minimum parameters that should be examined depending on all existing data to allow the derivation of a PoD for the most relevant acute effect(s) in the most appropriate species; however, this is not intended to become a routine data requirement. Basic Considerations 20. The derivation of an ARfD should be done based on following the tiered approach which is specified in more detail in Section D. 21. The appropriateness of all available endpoints from all existing oral toxicity studies to establish ARfDs needs to be carefully considered as a first step. The pertinent biology of the system affected should be considered to determine whether an acute exposure may compromise the ability of the organ to compensate and maintain homeostasis. Particular weight should be given to observations and investigations at the beginning of repeat dose studies. However, this approach should also consider such acute effects involving C max -dependent and rapidly reversible effects to ensure that acute effects from repeat dose dietary studies are not underestimated. Isolated findings, showing no specificity or clear pattern are not necessarily indications of toxicity. In the absence of information to the contrary, all toxic effects seen in repeat dose studies should be evaluated for their relevance in establishing an ARfD. 19

20 22. For determination of the PoD, the most sensitive endpoint in the most sensitive species must be selected with appropriate toxicological expertise to avoid selecting an endpoint in a given species particularly sensitive to that endpoint under normal conditions. 23. After reviewing the available toxicological database, the possible exposure scenarios should be considered as a second step, based on the guidance in Annex 1. A tiered human health risk assessment should be conducted that includes a comparison of the ARfD with the potential acute oral exposure (or internal body burden), based on a worst-case assumption. If this worst-case assessment does not indicate unacceptable health risks, no further refinement of the acute risk assessment may be warranted. However, if this risk assessment indicates a borderline or a clear concern, then the next tier should focus on a further refinement of the exposure assessment (from refined acute intake estimation). Models used for the calculation of dietary exposure are based on the premise that intake is a function of the concentration of pesticide in food and the amount of food consumed (9). For that reason a more elaborated analysis of the actual acute dietary exposure as well as toxicological assessments do allow for a more realistic approach for determining the actual risk in quite a number of cases. If the health risks are now acceptable, no further refinement is warranted. 24. If the refined exposure assessment still shows unacceptable health risks and if conservative assumptions were used in setting the ARfD, then as a third step consideration may be given to conducting a single exposure study to establish a refined ARfD and how to perform and tailor this single exposure test, based on the guidance in Annex 2. This will only be necessary for a very limited number of substances according to the retrospective analysis (11). Tiered-Approach for the Derivation of an appropriate ARfD Step One 1. Evaluate the total database of the substance, establish a toxicological profile for the relevant exposure periods to this substance and determine if there is an adverse effect occurring as a result of a single oral dose up to the limit dose. 2. Consider the principles for not setting an ARfD up to the limit dose 25. The upper limit for a relevant ARfD was considered with reference to the potential range of dietary exposures to acutely toxic pesticides. The estimated maximum exposure of 1 mg/kg bw is conservatively based on a 50 kg person consuming in a single sitting a large food item (e.g., 500 g) treated with pesticide having a MRL of 20 mg/kg. A variability factor of 5 was applied. The cut-off of 500 mg/kg bw/day would allow the derivation of an ARfD of 5 mg/kg bw with an assessment factor of 100 and an additional Margin of Exposure (MoE) of 5. No findings indicative of adverse effects elicited by an acute exposure are seen at doses which are relevant for the acute risk assessment, i.e. up to about 500 mg/kg bw/day for residues of pesticides, justification see (1). However, establishment of ARfD should be considered if mortalities are observed at doses up to 1000 mg/kg bw in single oral exposure studies and the observed mortalities are relevant to human exposures. 26. If the above criteria do not preclude the setting of an ARfD up to the limit doses, then further consideration should be given to setting a value, using the most appropriate endpoint in the most relevant species. 3. Selection of appropriate endpoints for setting an ARfD 20

21 Select the toxicological endpoints most relevant for a single (day) exposure in the most relevant species. Select the most relevant or adequate study in which these endpoints have been adequately determined. Identify the PoD for these endpoints. Select the most relevant endpoint providing the lowest PoD. 27. An endpoint from a repeat dose toxicity study should be used if the critical effect of the compound has not been adequately evaluated in a single exposure study and the effects in the repeat dose study are observed early on (e.g., early resorptions, etc). This is likely to be a more conservative approach and should be stated. 28. If after consideration of all the endpoints in appropriate available studies, an ARfD is not set, then the reasons should be justified and explained. 4. Selection of appropriate assessment factors for setting an ARfD. 29. The selection of appropriate assessment factors for inter-species and human inter-individual variability should be considered based on available data. To allow for the quantitative incorporation of specific information on toxicokinetic/toxicodynamic differences for a chemical, the IPCS recommended (17) (18) that the two 10X factors (for inter-species and inter-individual) each be divided into toxicokinetic/toxicodynamic sub factor for inter-species and human inter-individual differences as shown below, where available information on one or more specific sources of variability could be replaced. 30. A combined assessment factor may be based on (AKAF or AKUF) (ADAF or ADUF) (HKAF or HKUF) (HDAF or HDUF) where AK represents inter-species toxicokinetic variability AD represents inter-species toxicodynamic variability HK represents human interindividual toxicokinetic variability HD represents human interindividual toxicodynamic variability AF represents a chemical-specific adjustment factor (CSAF) UF represents a default assessment sub-factor 31. In determining the appropriate assessment factor, a stepwise approach is proposed. - Determine whether the database is adequate to support the derivation of a CSAF (17). IPCS recommended default sub factors, i.e. 4-fold and 2.5-fold for inter-species toxicokinetic and toxicodynamic differences, respectively, and 3.16 for each of human interindividual toxicokinetic and toxicodynamic differences. - Some reduction for human toxicokinetic differences from its default value of 3.16, may be justified. An example where the factor was reduced based on considerations relating to C max and rate of elimination was recently documented by JMPR (18). 21

22 - If chemical specific toxicokinetic and toxicodynamic data are inadequate to justify data based assessment factors, consider if there is any information (e.g., QSAR, mode of action, on closely related compounds) that would indicate reduced or increased uncertainty. - If no refinement is justified, the 100-fold (or 10-fold) default should be used. When using data obtained from animals, the default assessment factor is 100. This comprises a factor of 10 to allow for inter-species differences and a factor of 10 for intra-species (human interindividual) differences. The overall assessment factor is the product of these two factors, i.e Further to consideration of inter-species and human inter-individual variability, additional factors may be applied to address other uncertainties such as relating to completeness of the database and steepness of the dose-response curve, or to address an additional precaution in the case of especially severe effects. 33. Whenever an assessment factor other than a default is used, a clear explanation of the derivation of the factor should be provided. Individual countries may select assessment factors dependent on their specific regulatory requirements or national legislation. Step Two (Annex 1) 5. Application of the ARfD for the acute risk assessment - Determine whether the acute exposure estimate is exceeding the ARfD. - If the acute intake estimation does not exceed the ARfD, no further refinement is necessary. 34. If the risk assessment indicates a borderline or a clear concern, then a refinement of the exposure assessment should be performed. 6. Refinement of the exposure calculation for the acute risk assessment - In determining a refined exposure calculation, a stepwise approach is proposed. 35. If the risk assessment indicates still a clear concern, then a refinement of the ARfD could be performed. Step Three (Annex 2) 7. Experimental refinement of the ARfD derivation - As a last resort a single exposure study according to the test design in Annex 2 should be considered for the generation of data to establish and refine more appropriate ARfD. Specific Guidance on the Derivation of ARfDs 36. Particular toxicology endpoints that are relevant to ARfD establishment are considered in the JMPR publication (2) and by Solecki et al. (1). Note that these documents are not intended to comprehensively cover all potentially relevant endpoints but focus on the interpretation of a number of selected endpoints which have proved to be problematic in reaching a decision as to whether an effect is relevant to an acute exposure. 22

23 Haematotoxicity ENV/JM/MONO(2010) The induction of methaemoglobinaemia is considered to be a critical effect in consideration of acute responses to chemical exposure. For acute exposure to methaemoglobin-inducing xenobiotics, a level of 4% methaemoglobin (or higher) above background in dogs or a statistically-significant increase in rodents cf. background is considered to be relevant to set an ARfD (1). Haemolytic anaemias induced by mechanical damage, immune mediated anaemia, oxidative injury to RBCs and non-oxidative damage are considered to be less relevant for ARfD derivation since the severity of such effects appear to generally depend on prolonged exposure. If changes in haematological parameters are observed early in a repeat dose study and do not appear to progress during the course of the study, then such effects should be considered as relating to acute exposure to the substance. In assessing whether effects observed in repeat dose studies should be used for setting an ARfD, one should evaluate the mode of action. If known, this could provide arguments for selecting or not selecting the endpoint for setting an ARfD. Immunotoxicity 38. Immunotoxicity data derived from currently available standard repeat exposure studies are not likely to be adequate for setting an ARfD due to lack of specific immunotoxic parameters and/or early observations. Even though effects on the immune system can be induced by a single exposure, typically a high dose may be required to cause immunotoxicity following a single exposure, except for chemicals that are eliminated slowly. Changes in immune function may be the result of overt toxicity (decreased food intake, irritation or inflammation, increased glucocorticoid release, or a general decline in fitness) rather than a direct effect on the immune system. Single dose effects on immune function are considered to be unlikely because the immune cells and mediators are constantly replaced and because of the inherent redundancy in the system (e.g., alternative mechanisms to resist infection). Potential concerns for acute effects would include auto-immune responses as well as effects on the immune system during development and hypersensitivity. As knowledge and methodologies evolve on immunotoxicity, this guidance may be reconsidered in the future. Neurotoxicity 39. The nervous system has limited capacity for repair and regeneration. Therefore, any neurotoxicity seen in repeat dose studies could be the result of a single exposure that is not reparable, i.e. any evidence of neurotoxicity should be considered relevant to an ARfD assessment unless it can be demonstrated that the effects are produced only after repeat exposures. In addition to long-term or irreversible effects associated with acute exposure, attention should be paid to transient effects, as these could be considered as adverse under some circumstances. 40. Delayed neurotoxicity following single chemical exposures can occur and thus any acute exposure study should have an adequate period of investigation In functional observation batteries (FOB) a large amount of data is produced; interpretation of such studies should include a consideration not only of the statistical significance of results but the nature, severity, persistence, dose-relationship and pattern of the effects. Isolated findings showing no specificity or clear pattern do not necessarily indicate neurotoxicity. A common neurotoxic endpoint used to date in the derivation of ARfDs for insecticides is inhibition of acetylcholinesterase. The JMPR has previously defined criteria for the assessment of cholinesterase inhibition; these apply equally to the setting of ADIs and ARfDs. 23

24 Kidney and liver effects 42. If effects on these organs cannot be discounted as being either adaptive or as the result of prolonged exposure, an ARfD can be derived on the basis of these effects. Such an ARfD is likely to be conservative and it may be possible to subsequently refine it using an appropriately designed single exposure study. When interpreting data on liver and kidney toxicity in repeat dose studies, one should consider two important aspects, firstly, the type of effect observed and secondly, any information on correlations between exposure duration and effect. 43. For liver toxicity it is considered that findings of increased serum cholesterol, cirrhosis, induced activity of metabolising enzymes, regenerative hyperplasia, hepatocyte hypertrophy, fibrosis, or sclerosis in repeat dose studies are, in isolation, either adaptive or the result of prolonged exposure and therefore are not applicable for deriving an ARfD. 44. For kidney toxicity it is considered that the following findings of kidney toxicity in repeat dose studies are, in isolation, the result of prolonged exposure and are not applicable for deriving an ARfD: changes in organ weight; regenerative hyperplasia; altered serum calcium and phosphate. Endocrine effects 45. In general, analysis should consider the mode of action of endocrine toxicity including human relevance, dynamics/kinetics, redundancy in the system, the ability of the organism to compensate and critical windows of sensitivity. If treatment- related adverse effects affecting development of the offspring, female reproduction function, or resulting in male germ Leydig or Sertoli cell toxicity were observed, it should be considered if These effects were caused by acute toxic key events directly related to interaction/disruption of hormonal system, and These effects are relevant for establishing ARfD, e.g. the adverse effects are not considered to be secondary effects such as lesions via oxidative stress that cause deficit of hormone production/imbalance. 46. The nature of the effects, including their potential to occur following a single dose, as well as the dose-relationship and pattern of the findings should be considered in order to best ascertain whether endocrine findings are relevant for ARfD consideration. For example, an effect such as vacuolation in an endocrine tissue that is considered to be secondary to sustained alterations in steroidogenesis would be expected to be a consequence of repeated exposure, and thus not relevant as the basis of an ARfD. Further, as with any toxicological endpoint, isolated findings in endocrine tissues with no clear pattern or dose-response trend are not necessarily indications of toxicity. In most cases, thyroid hormonal effects in humans are unlikely to result from an acute exposure given the buffering capacity of the human thyroid system. It should be noted that endocrine toxicity is an evolving area and any guidance given is considered interim. Developmental effects: 47. It is important to consider critical or sensitive windows of exposure that impact on the developing organism. Thus, any treatment-related adverse effect on fetuses or offspring which has resulted from exposure during any phase of development should be considered as potentially appropriate to use in acute dietary risk assessment, despite the fact that the treatment period typically consists of repeat dosing. Therefore, ARfD values that were derived from embryo/feto toxicity in rats or rabbits are considered appropriate to sufficiently protect women of childbearing age including the developing organism. But our knowledge on the mode of action of an acute exposure during a sensitive window 24