Reasoned opinion on the modification of the existing MRL for guazatine in citrus fruits 1

Transcription

1 EFSA Journal 2014;12(8):3818 REASONED OPINION Reasoned opinion on the modification of the existing MRL for guazatine in citrus fruits 1 ABSTRACT European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy In accordance with Article 6 of Regulation (EC) No 396/2005, the United Kingdom, hereafter referred to as the evaluating Member State (EMS), received an application from Exponent International Ltd., on behalf of ICA International Chemicals (PTY) Ltd., to set an import tolerance for the active substance guazatine in citrus fruits. In order to accommodate for the reported post-harvest use of guazatine acetates in South Africa, the United Kingdom proposed the MRL of 4 mg/kg. The United Kingdom drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005, which was submitted to the European Commission and forwarded to EFSA. According to EFSA the metabolism of guazatine acetates in citrus fruits is not fully elucidated. A tentative MRL proposal of 5 mg/kg for guazatine acetates (corresponding to 4 mg/kg for residues expressed as guazatine) was derived. This MRL proposal should be considered as tentative. The MRL set in the country of origin is 5 mg/kg for guazatine. Although the indicative consumer risk assessment did not identify a consumer health risk for the reported post-harvest use of guazatine acetates on citrus fruits imported from South Africa, EFSA does not propose to set a MRL in citrus since the risk assessment is affected by a high level of scientific uncertainties resulting from the validity of the toxicological reference values, the lack of elucidation of the metabolic pathway in plants and livestock and the validity of the supervised residue trials analysed with an analytical method which is based on the analysis of one constituent of the technical material (marker substance GG-diacetate) only. Thus, EFSA is of the opinion that the available data are not sufficient to demonstrate that the residues of guazatine acetates on citrus fruits resulting from the South African GAP are safe for European consumers. European Food Safety Authority, 2014 KEY WORDS guazatine, citrus fruits, MRL application, Regulation (EC) No 396/2005, consumer risk assessment, guanidine fungicide, GG-diacetate 1 On request from the European Commission, Question No EFSA-Q , approved on 25 August Correspondence: pesticides.mrl@efsa.europa.eu Suggested citation: EFSA (European Food Safety Authority), Reasoned opinion on the modification of the existing MRL for guazatine in citrus fruits. EFSA Journal 2014;12(8):3818, 29 pp. doi: /j.efsa Available online: European Food Safety Authority, 2014

2 SUMMARY In accordance with Article 6 of Regulation (EC) No 396/2005, the United Kingdom, hereafter referred to as the evaluating Member State (EMS), received an application from Exponent International Ltd., on behalf of ICA International Chemicals (PTY) Ltd., to set an import tolerance for the active substance guazatine in citrus fruits. In order to accommodate for the reported post-harvest use of guazatine acetates in South Africa, the United Kingdom proposed the MRL of 4 mg/kg. The United Kingdom drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005, which was submitted to the European Commission and forwarded EFSA on 30 August Guazatine is a mixture of products resulting from the amidination of technical iminodi(octamethylene)diamine, containing numerous guanidines and polyamines. It is no longer authorised for use in plant protection products within the EU. EFSA reviewed the existing MRLs in the framework of the MRL review under Article 12 of Regulation (EC) No 396/2005 and proposed to lower all MRLs to the appropriate LOQ as no import tolerances were notified. This assessment is based on the evaluation report and the additional information and clarifications submitted by the EMS upon requests of EFSA, the Draft Assessment Report (DAR) and its addendum prepared under Council Directive 91/414/EEC, the EFSA conclusion on the peer review of the pesticide risk assessment of the active substance guazatine as well as the reasoned opinion on the review of all existing MRLs and the JMPR evaluation report. The toxicological profile of guazatine was assessed in the framework of the peer review under Council Directive 91/414/EEC. Based on the available information an ADI of mg/kg bw per day and an ARfD of 0.04 mg/kg bw were proposed for guazatine acetates, which is the variant used in plant protection products. Since a clear conclusion on whether the active substance used in the toxicological studies is representative for the active substance guazatine acetates contained in the South African plant protection product for which the import tolerance is requested can not be drawn, the toxicological reference values should be considered as indicative. Metabolism studies in orange fruits under post-harvest storage conditions were provided in the framework of the import tolerance request. The studies were conducted using individually the GGdiacetate or the GGG-triacetate, two of the constituents of technical guazatine. EFSA is of the opinion that the studies are not sufficient to fully elucidate the metabolism of guazatine acetates in citrus fruits. EFSA does not agree with the position of the applicant that GG-diacetate and GGG-triacetate are fully representative for the metabolism of guazatine acetates in citrus fruits and that the results for these two components can be extrapolated to the other components of the mixture. The assumption that the metabolic behaviour of all constituents contained in guazatine is comparable with the metabolism of GG-diacetate and GGG-triacetate needs to be confirmed with appropriate studies. Consequently, a final decision on the residue definition cannot be taken and further information is required. Tentatively and in order to perform an indicative risk assessment, EFSA used the residue definition for enforcement and risk assessment as guazatine acetates. Based on the results of eight residue trials reflecting the GAP, a tentative MRL proposal of 5 mg/kg for guazatine acetates (corresponding to 4 mg/kg for residues expressed as guazatine) was derived. The MRL set in the country of origin is 5 mg/kg for guazatine. This MRL proposal should be considered as tentative given the uncertainty with regard to the residue definition and the validity of the supervised residue trials analysed with an analytical method which needs to be demonstrated to be fit for the purpose. Studies conducted using either the GG-diacetate or the GGG-triacetate components of guazatine acetates showed that these compounds were hydrolytically stable under pasteurisation conditions. The behaviour of the other guazatine constituents was not investigated. To conclude on the residue definition for processed citrus products a hydrolysis study conducted with other components representative for guazatine acetates is required, including other test conditions like sterilisation. An indicative peeling factor was derived from residue trials where the distribution between pulp and peel EFSA Journal 2014;12(8):3818 2

3 was measured. Processing studies on the magnitude of residues conducted according to the current standard would be required as the residue levels in raw agricultural commodities (RAC) exceed the trigger value of 0.1 mg/kg. Since the use of guazatine is on imported permanent crops treated post-harvest, investigations of residues in rotational crops are not required. Considering that citrus pomace obtained from the processing of the imported fruits may be fed to animals, the potential carry-over into food of animal origin was assessed. The indicative calculated livestock dietary burdens significantly exceed the trigger value of 0.1 mg/kg (dry matter) for ruminants. The nature of guazatine residues was investigated in lactating cows. Since a full characterisation of the residues was not carried out, this study is not adequate to elucidate the metabolism of guazatine in livestock and to conclude on a residue definition for animal products. Further information is necessary to conclude on the need to set MRLs in bovine, sheep and goat edible tissues and milk resulting from the post-harvest treatment on citrus fruits. The consumer risk assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). EFSA updated the long-term risk assessment performed in the framework of the review of the existing MRLs for guazatine acetates according to Article 12 of Regulation (EC) No 396/2005 with the tentative median residue value for citrus fruits derived from the submitted supervised residue trials. The acute exposure assessment was performed only with regard to citrus fruits using the MRL established in the country of origin, as worst case. The input values were corrected by the peeling factor to take into account the lower residues expected in the peeled citrus. Based on the above calculation, no long-term consumer intake concerns were identified for any of the European diets incorporated in the EFSA PRIMo. The total calculated intake accounted for up to 74 % of the ADI (Dutch child diet). The contribution of residues in citrus fruits to the total consumer exposure accounted for a maximum of 7.9 % of the ADI for oranges, 1.5 % of the ADI for mandarins and less for grapefruits, lemons, limes and the other citrus fruits. No acute consumer risk was identified in relation to the MRL set for citrus fruits in the country of origin. The calculated maximum exposure in percentage of the ARfD was 99.5 % for oranges, 67 % for grapefruits, 42 % for mandarins, 26 % for lemons and 15 % for limes. Although the indicative consumer risk assessment did not identify a consumer health risk for the reported post-harvest use of guazatine acetates on citrus fruits imported from South Africa, EFSA does not propose to set a MRL in citrus since the risk assessment is affected by a high level of scientific uncertainties resulting from the validity of the toxicological reference values, the lack of elucidation of the metabolic pathway in plants and livestock and the validity of the supervised residue trials analysed with an analytical method which is based on the analysis of one constituent of the technical material (marker substance GG-diacetate) only. Thus, EFSA is of the opinion that the available data are not sufficient to demonstrate that the residues of guazatine acetates on citrus fruits resulting from the South African GAP are safe for European consumers. EFSA Journal 2014;12(8):3818 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background... 5 Terms of reference... 5 The active substance and its use pattern... 6 Assessment Method of analysis Methods for enforcement of residues in food of plant origin Methods for enforcement of residues in food of animal origin Mammalian toxicology Residues Nature and magnitude of residues in plant Primary crops Rotational crops Nature and magnitude of residues in livestock Dietary burden of livestock Nature and magnitude of residues Consumer risk assessment Conclusions References Appendices Appendix A. Good Agricultural Practice (GAPs) Appendix B. Pesticide Residue Intake Model (PRIMo) Appendix C. Existing EU maximum residue levels (MRLs) Appendix D. List of components and metabolites and related structural formula Abbreviations EFSA Journal 2014;12(8):3818 4

5 BACKGROUND Regulation (EC) No 396/ establishes the rules governing the setting of pesticide MRLs at European Union level. Article 6 of that Regulation lays down that any party having a legitimate interest or requesting an authorisation for the use of a plant protection product in accordance with Council Directive 91/414/EEC 4, repealed by Regulation (EC) No 1107/2009 5, shall submit to a Member State, when appropriate, an application to set an import tolerance in accordance with the provisions of Article 7 of that Regulation. The United Kingdom, hereafter referred to as the evaluating Member State (EMS), received an application from the company Exponent International Ltd. 6, on behalf of ICA International Chemicals (PTY) Ltd., to modify the existing MRL for the active substance guazatine in citrus fruits. This application was notified to the European Commission and EFSA and was subsequently evaluated by the EMS in accordance with Article 8 of the Regulation. After completion, the evaluation report was submitted to the European Commission who forwarded the application, the evaluation report and the supporting dossier to EFSA on 30 August The application was included in the EFSA Register of Questions with the reference number EFSA-Q and the following subject: Guazatine - Application to modify the existing MRLs in citrus fruit. The United Kingdom proposed to set an import tolerance for guazatine in citrus fruit at 4 mg/kg. EFSA proceeded with the assessment of the application and the evaluation report as required by Article 10 of the Regulation. EFSA identified some data requirements which prevented to conclude on the consumer risk assessment and the evaluation of the import tolerance application was stopped. The EMS provided the response on 26 March 2014 (United Kingdom, 2014a) with further clarifications on 7 July 2014 (United Kingdom, 2014b), that were taken into consideration by EFSA for the finalisation of this reasoned opinion. TERMS OF REFERENCE In accordance with Article 10 of Regulation (EC) No 396/2005, EFSA shall, based on the evaluation report provided by the evaluating Member State, provide a reasoned opinion on the risks to the consumer associated with the application. In accordance with Article 11 of that Regulation, the reasoned opinion shall be provided as soon as possible and at the latest within three months (which may be extended to six months where more detailed evaluations need to be carried out) from the date of receipt of the application. Where EFSA requests supplementary information, the time limit laid down shall be suspended until that information has been provided. The recalculated deadline for providing the reasoned opinion was 27 March Regulation (EC) No 396/2005 of the Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. OJ L 70, , p Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230, , p Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, , p Exponet International Ltd., The Lenz, Hornbeam Business Park, Harrogate, HG2, North Yorkshire, United Kingdom. EFSA Journal 2014;12(8):3818 5

6 THE ACTIVE SUBSTANCE AND ITS USE PATTERN Guazatine is the ISO common name for a mixture of the reaction products from polyamines, comprising mainly octamethylenediamine, iminodi(octamethylene)diamine and octamethylenebis (imino-octamethylene)diamine, and carbamonitrile. The technical material used in plant protection product comprises ca. 70 % w/w of guazatine acetates in water (United Kingdom, 2007). A basic formula describing the constituents of guazatine is reported below. R R R (CH 2 ) 8 N [(CH 2 ) 8 N] n H n may be 0 or 1 or 2 etc. and any R substituent may be H (17-23%) or 2 (77-83%) The chemical structure and molecular weight of some constituents of guazatine are reported in Appendix D. There are no FAO specifications for guazatine. Guazatine is a non-systemic contact fungicide belonging to the guanidine family. Guazatine affects the membrane function of fungi and controls a wide range of seed-borne diseases of cereals and citrus moulds. Guazatine was evaluated in the framework of Council Directive 91/414/EEC. The United Kingdom designated as rapporteur Member State (RMS) prepared a Draft Assessment Report (DAR). The variant assessed was guazatine acetates and the representative use assessed in the DAR was the seed treatment of cereals. Since the notifier voluntarily withdrew the application, a non-inclusion decision was taken by Commission Decision 2008/934/EC 7. Guazatine was then subject to a resubmission application with supplementary information. The initial DAR and additional data assessed by the RMS were peer reviewed by EFSA. It was concluded that the specification of the technical material could not be finalised since the available batch analysis did not identify all compounds occurring in concentrations at or above 1 g/kg. EFSA highlighted that a clear conclusion could not be derived whether the batches used in the toxicological studies were representative for the technical material assessed in the peer review. Critical areas of concerns and data gaps were identified on the environmental and fate and ecotoxicology sections as well and a list of studies to be generated was proposed (EFSA, 2010). Following the peer review, the non-inclusion decision was maintained. According to Regulation (EC) No 1107/2009, repealing Council Directive 91/414/EEC guazatine is a non-approved active substance. The EU MRLs for guazatine are established in Annex IIIA of Regulation (EC) No 396/2005 (Appendix C). The existing EU MRL for guazatine on citrus fruits is set at 5 mg/kg. In 2013, EFSA issued a reasoned opinion on the review of the existing MRLs for guazatine according to Article 12 of Regulation (EC) No 396/2005 (EFSA, 2013). EFSA recommendation to have the residue definition as guazatine acetates and to lower all the MRLs to the LOQ have not yet been voted by the Standing Committee on Plants, Animals, Food and Feed (SCoPAFF). Codex Alimentarius has established a guideline level for citrus fruits at 5 mg/kg; no full CXL was derived because of substantial data gaps that led to the withdrawal of the ADI (FAO, 1997). The MRL for citrus established in South Africa is 5 mg/kg 8 ; the residue definition is set as free guazatine (United Kingdom, 2014a). C 7 Commission Decision 2008/934/EC of 5 December 2008 concerning the non-inclusion of certain active substances in Annex I to Council Directive. 91/414/EEC and the and the withdrawal of authorisations for plant protection products containing these substances. OJ L 333, , p South Africa. Regulations governing the maximum limits for pesticide residues that may be present in foodstuffs. GNR No 246 of 1994 and amendments. Full text avaialble online: saf73495.pdf EFSA Journal 2014;12(8):3818 6

7 The details of the GAP reported for South Africa (ZA) are given in Appendix A. The application refers to a single post-harvest treatment on citrus by dipping at the dose of 100 g/hl of solution, equivalent to 1000 mg/kg of guazatine acetates. ASSESSMENT EFSA bases its assessment on the evaluation report and the additional information and clarifications submitted by the EMS (United Kingdom, 2013, 2014a, b), the Draft Assessment Report (DAR) and its addendum prepared under Council Directive 91/414/EEC (United Kingdom, 2007, 2009), the conclusion on the peer review of the pesticide risk assessment of the active substance guazatine (EFSA, 2010), the reasoned opinion on the review of existing MRLs according to Article 12 of Regulation (EC) No 396/2005 (EFSA, 2013) and the JMPR Evaluation report (FAO, 1997). The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation and the Authorisation of Plant Protection Products adopted by Commission Regulation (EU) No 546/20119 and the currently applicable guidance documents relevant for the consumer risk assessment of pesticide residues (EC, 1996, 1997a-g, 2000, 2010a,b, 2011; OECD, 2011). 1. Method of analysis 1.1. Methods for enforcement of residues in food of plant origin Two analytical methods for the determination of guazatine residues in high acid content commodities and respective validation data have been submitted in the framework of the import tolerance application (United Kingdom, 2013). In both methods the extraction and derivatization steps are designed to determine guazatine free basis using GG-diacetate 10 (1-8-diguanidino-octane) as marker compound and GG-C6 (1,6-diguanidino-hexane) 11 as internal standard. The quantification is based on the assumption that the analytical standard and the technical material used have the same GG-diacetate concentration (United Kingdom, 2013, 2014b). The first method is based on GC-MS/MS while in the second method the determination is performed with LC-MS/MS. The limits of quantification (LOQs) for citrus matrices (whole fruit, peel, flesh/pulp, juice and jam 12 ) were mg/kg as free guazatine. An ILV on citrus whole fruit was provided only for the latter method. EFSA concludes that the proposed LC-MS/MS analytical method is sufficiently validated to enforce residues of guazatine on citrus fruits at the LOQ of 0.05 mg/kg, provided that the ratio between the marker residue (GG-diacetate) and the total residue is known and constant. The LOQ expressed as guazatine acetates is calculated as 0.07 mg/kg Methods for enforcement of residues in food of animal origin Fully validated analytical methods for the enforcement of residues of guazatine acetates and the residue definition in commodities of animal origin are currently not available. There are indications that guazatine can be enforced (GC-MSD method using GG-diacetate as marker compound) with an LOQ of 0.05 mg/kg in meat, fat, liver, kidney and eggs and an LOQ of 0.02 mg/kg in milk (EFSA, 2013). 9 Commission Regulation (EU) No 546/2011 of 10 June 2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards uniform principles for evaluation and authorisation of plant protection products. OJ L 155, , p GG-diacetate. See Appendix D. 11 GG-C6. See Appendix D. 12 The number of samples per fortification level for orange jam and orange juice were lower than those specified in the current guidance document EC, 2010b). Considering the overall results, the deviation is acceptable. 13 A conversion factor of 1.47 was obtained from the ratio between (guazatine + acetates) and guazatine based on the data reported in the certificate of analysis of the analytical standard (United Kingdom, 2014b). EFSA Journal 2014;12(8):3818 7

8 2. Mammalian toxicology Modification of the existing MRL for guazatine in citrus fruits The toxicological profile of the active substance guazatine was assessed in the framework of the peer review under Council Directive 91/414/EEC (EFSA, 2010). Based on the available information the following toxicological reference values were proposed for guazatine acetates and are compiled in Table 2-1. Table 2-1: Overview of the toxicological reference values Guazatine acetates Source Year Value Study relied upon Safety factor ADI EFSA mg/kg bw per day Dog, 52-wk study 500 (b) ARfD EFSA mg/kg bw Rat, development study 500 (b) : Toxicological reference values were derived from the tested technical material (containing approximately 70 % guazatine acetates) adjusted to 100 % guazatine acetates pure active substance (EFSA, 2010). (b): An extra safety factor of 5 was applied in order to maintain an adequate margin of safety with regard to the observed increased incidence of hemangiosarcomas and broncus-associated lymphoid tissues (EFSA, 2010). The identity of the active substance and the compliance of the batches tested in the toxicological studies were a critical area of concern during the peer review. The technical material assessed in the peer review contained impurities in significant amounts ( 1 g/kg) which were requested to be identified. The available batch analysis demonstrated a large variability in the composition of the technical material in terms of the nature and the amount of the constituents comprising guazatine acetates. Thus, a clear conclusion on whether the toxicological studies were representative for the technical material assessed could not be drawn as adequate data were not available. Upon establishment of a technical specification for guazatine acetates, the notifier should provide the assessment of the equivalence of the batches tested in the key toxicological studies (EFSA, 2010). The data gaps related to the specification of the technical material and the validity of the toxicological reference values derived during the peer review also apply to the technical material which is used in the South African plant protection product. On the subject, the EMS informed EFSA that the technical material used in South Africa is produced by the same manufacturer that supported the EU review. This argument is not sufficient as it does not demonstrate unequivocally that the batches are comparable; for example, the manufacturing process may have been changed over the years leading to a different composition of constituents and/or impurities. In fact, the typical composition of free guazatine reported for the peer review contained an amount in GGG-triacetate 14 significantly higher than the amount of 10 % declared by the applicant (United Kingdom, 2013, 2014a,b). Valid data need to be provided to demonstrate that the active substance used in the toxicological studies assessed in the peer review are representative for the active substance used in South Africa. EFSA concludes that the toxicological reference values for guazatine acetates should be considered as indicative only for the active substance guazatine acetates contained in the plant protection product marketed in South Africa for which an import tolerance is requested. It is noted that in 1997 JMPR withdrew the previously established ADI value of 0.03 mg/kg bw per day. In its assessment JMPR highlighted that some data were provided to show that the composition of the batches used in the key toxicity studies were similar to that of the batches produced at the same time ( ), but there were no data to confirm that the batches used in the studies on toxicity were representative for those produced at the time JMPR performed the assessment (FAO, 1997). 14 GGG-triacetate. See Appendix D. EFSA Journal 2014;12(8):3818 8

9 3. Residues 3.1. Nature and magnitude of residues in plant Primary crops Nature of residues Metabolism studies in orange fruits under post-harvest storage conditions were provided in the framework of the import tolerance request. The studies were conducted applying radiolabelled GGdiacetate (GG) or GGG-triacetate (GGG) components of guazatine. The overview of the metabolism study designs is presented in the table below. Table 3-1: Summary of available metabolism studies in plants Group Crop Label position Application details Fruits and fruiting vegetable Orange fruits [ 14 C]-GGG triacetate Post-harvest dipping, for [ 14 C]-GG diacetate 1 min., G [ 14 C] guazatine Post-harvest drench, for s. (b), G Method, Rate No Sampling Remarks F or G 1 mg/ml 1 0, 7, 30, 90 DAT 1 mg/ml 2 mg/ml [ 3 H] guazatine 1 mg/ml 2 mg/ml 1 0, 1, 8, 24, 50 DAT Samples stored at about 5ºC. Supportive. Pre-GLP. Samples stored at 4ºC. : Outdoor/field application (F) or glasshouse/protected crops/indoor application (G). (b): Part of the oranges was de-greened with gaseous ethylene for 24 hours. Both non-degreened and de-greened oranges were then dried and treated with a wax spray containing thiabendazole at 4 g/kg (United Kingdom, 2013). In the first metabolism study (GLP-study) mature orange fruits were treated with radiolabelled GG or GGG applied individually at 1 mg/ml (corresponding to 1N the nominal application rate for total guazatine acetates) and stored at about 5 ºC; the total radioactive residue (TRR) as sum of surface rinse, peel and pulp residues, remained stable during the storage period of 90 days (d) (GG: from 2.6 to 2.5 mg eq/kg; GGG: from 2.8 to 2.7 mg eq/kg). The extraction efficiency accounted for more than 86 % and 74 % of the TRR for the GG-study and GGG-study, respectively. On the day of the treatment (0 DAT) the majority of the total residues present on the surface of the oranges at application was removed in the surface rinse; the concentrations declined over the time, showing a gradual translocation into the peel. During the 90-d storage period, residues in the surface rinse decreased from 71 to 26 % of the TRR (GG-study) or 68 to 12 % of the TRR (GGG-study), whereas the TRR in the peel increased from 28 to 73 % of the TRR (GG-study) or 30 to 88 % of the TRR (GGG-study). In the pulp, total residues decreased from 1.2 to 1 % of the TRR (GG-study) or 1.3 to 0.6 % of the TRR (GGG-study) but the concentrations were stable over the storage time (GG: 0.03 mg a.s. eq/kg; GGG: 0.04 to 0.02 mg a.s. eq/kg). In the pulp, unchanged GG or GGG were the only compounds detected at day 0 in each study (about mg/kg; % of the TRR) and represented the majority of the radioactive residues up to day 90 (0.01 mg/kg; 0.4 % of the TRR). No metabolite was detected in significant concentrations (>0.01 mg/kg): metabolite Met 2 15 accounted for 0.01 mg/kg at day 30 and 90, but represented no more than the 0.7 % of the TRR (GG-study); only trace amounts of metabolite Met 1, unknown 1 and 2 (GG-study) and metabolite Met 3 16 and unknown 1 (GGG-study) were observed. Further investigations suggested that Met 1 being a mixture of three compounds: 1-(8-aminooctyl)guanidine 15 Met 2. See Appendix D. 16 Met 3: See Appendix D EFSA Journal 2014;12(8):3818 9

10 (NGN) 17, N-(8-guanidino-octyl)guanidine (GG) and N-(8-guanidinooctyl)carbamimidic acid (Met 2) (United Kingdom, 2014a). In the peel, unchanged GG or GGG represented the majority of the radioactive residues, except at day 90 of the GG-study, where Met 1 was about three times the concentration of parent GG. Apart Met 2 at day 30 (GG-study), few minor metabolites were present, none of them in relevant amounts and concentrations. A number of minor peaks designated as unknown progressively increased over the time and represented the 11 % (up to 0.3 mg/kg) at day 90. The distribution of residues over a 90-d period in orange peel after the post-harvest application of radiolabelled GG and GGG to oranges at 1 mg/ml is reported in Table 3-2. Table 3-2: Distribution of radiolabelled residues in orange peel after post-harvest application of 14 C- GG or 14 C-GGG. Days 14 C-GG study 14 C-GGG study after Po GG GGG Met 1 Met 2 GGG GG Met 1 Met 2 Met 3 treatment mg/kg (% TRR) mg/kg (% TRR) Day ND ND ND (24 %) (0.2 %) (0.4 %) (19 %) (3 %) Day (25 %) ND ND 0.14 (5 %) 1.78 (56 %) 0.04 (1.4 %) - ND 0.03 (0.9 %) Day (28 %) 0.02 (1.5 %) ND 0.17 (11 %) 1.15 (47 %) 0.04 (1.7 %) - ND 0.13 (5.5 %) Day (14 %) ND 0.97 (39 %) 0.14 (6 %) 0.76 (28 %) 0.02 (0.8 %) 0.23 (8.3 %) 0.09 (3.5 %) 0.16 (5.9 %) Unknown 1 : max 0.03 mg/kg (1.9 %) at day 30; Unknown 2 (b) : max 0.01 mg/kg (0.6 %) at day 30; Unknown 1: max 0.06 mg/kg (2.3 %) at day 90; Unknown 2: max 0.06 mg/kg (2 %) at day 90; Unassigned: 0.17 mg/kg (11 %) at day 30: ND: not detected. Po: post-harvest. : Observed at retention time of 8 min. (b): Observed at retention time of 19 min. Unassigned: max 0.3 mg/kg (11 %) at day 90. In the surface rinse, unchanged GG or GGG represented the majority of the radioactive residues. Only metabolite Met 2 was detected at day 0 (GG-study) and day 90 (GGG-study). The second metabolism study on oranges (pre-glp study) showed rather stable TRR levels over the time. In the pulp only % of the applied radioactivity was found. De-greening had no observable effects on the residues found in any of the fruit parts (United Kingdom, 2013; FAO, 1997). Overall, the results of the metabolism studies indicated that during the 90-d storage under refrigeration conditions the degradation of residues in the peel and their transfer from the external peel into the orange pulp was limited. Although unchanged parent GG or GGG represented the major component of the residues up to day 30 in the peel, a progressive increase of metabolites Met 1, Met 2 and unassigned metabolite (no further specified) was observed at later treatment intervals (day 30 and day 90). The EMS proposed a residue definition in citrus fruits for monitoring and risk assessment as guazatine acetates (United Kingdom, 2013). The metabolic pathway over the time of the whole guazatine acetates and the guazatine polymers different than GG and GGG has not been investigated. According to the EMS, GG and GGG represent 30 % and 10 % of free guazatine, respectively; the other constituents are present individually at less than 10 %. The EMS is of the opinion that considering the structure of the components of guazatine 17 NGN. See Appendix D. EFSA Journal 2014;12(8):

11 acetates, the results of the metabolism studies derived for the components GG and GGG, which were individually applied at the rate reported for guazatine acetates (1N), are adequate to address the metabolism for all constituents of guazatine technical material in terms of composition/structure (United Kingdom, 2014a). The EMS also noted that the dietary intake of the metabolites observed in relevant amounts in the peel from the metabolism studies is expected to be low (less than 1.5 µg/kg bw per day, below the threshold of toxicological concern for a Crammer class III compound). Since there is a large difference in the magnitude of residues between the peel and the pulp of oranges, further consideration of the potential toxicity of the metabolites observed and their inclusion in the residue definition for risk assessment is not necessary (United Kingdom, 2014a). EFSA acknowledges that the available metabolism studies provide evidence that the major part of the residues resulting from GG and GGG is expected in and on the peel with low transfer to the pulp. The studies also give an indication on the nature of residues resulting from these two constituents. Since the identified metabolites of GG and GGG were found in low concentrations in the edible part of the crop, they are considered to be of minor relevance for the overall dietary exposure of consumers. However, EFSA is of the opinion that the metabolism of guazatine acetates in citrus fruits is not fully elucidated and is based on several assumptions, which need to be confirmed by experimental data. EFSA does not agree with the statement that GG and GGG (about 30 % and 10 % of guazatine) applied at 1N are fully representative for the metabolism of guazatine acetates in citrus fruits and that the results from these two components can be extrapolated to the other components of the mixture, for the following reasons. No experimental data were provided on the composition of the batches used in the plant protection product marketed in South Africa to conclude that the selected GG and GGG are valid marker substances; No information on the metabolic behaviour of guazatine constituents that are not completely guanidated (containing the amino-group 18 ) is available. Thus, metabolism studies with representative constituents should be provided to conclude that the nature and amount of metabolites from the incompletely guanidated polymers are comparable with the metabolites identified from GG and GGG; The residues which were extracted from the oranges at 0 DAT in the metabolism studies conducted with either GG or GGG individually applied at 1N the nominal application rate for total guazatine acetates (sum of peel plus pulp: about 0.66 mg/kg) are significantly lower than the residues measured in oranges at PHI 0 day in the supervised residue trials (1.1 to 1.9 mg/kg). These findings give an indication that the metabolism studies might not be fully representative. Consequently, based on the available information a final decision on the residue definition can not be taken. Tentatively and in order to perform an indicative consumer risk assessment, EFSA used the residue definition for enforcement and risk assessment as guazatine acetates Magnitude of residues In support of the import tolerance the applicant submitted eight independent GAP-compliant residue trials conducted on oranges (4 trials) and mandarins/clementines (4 trials) in South Africa in 2009 and The fruits were treated by dipping for a minimum of 40 seconds, allowed to dried and coated with wax. All samples were collected after the application (PHI 0 day), pulp and peel were analysed separately and residues recalculated to the whole fruits. At the application rate of the GAP no residues were found in the pulp. In two trials samples were analysed at PHI 30 d: Since higher residues (about +40 %) were observed in the whole fruit of one of these two samples, additional data investigating the 18 i.e. constituents coded as GN-diacetate. See Appendix D. EFSA Journal 2014;12(8):

12 residues at PHI 30 d and beyond would be desirable to conclude on the decline of the residues from PHI 0 day on. According to EU guideline the extrapolation from oranges and mandarins treated post-harvest to the group of citrus fruits is acceptable (EC, 2011). The results of the residue trials, the related tentative risk assessment input values (highest residue, median residue) and the MRLs calculated based on this dataset are summarised in Table 3-3. As the supervised residue trial samples were analysed within 30 d (United Kingdom, 2013), it is concluded that the residue data are valid with regard to storage stability. According to the EMS, the LC-MS/MS analytical method described in Section 1.1. with residue expressed as guazatine was used to analyse the supervised residue trial samples and was proven to be fit for the purpose (United Kingdom, 2013). In order to conclude on the validity of the residue trials EFSA is of the opinion that the assumption that the marker substance GG-diacetate can be used to estimate the total residues resulting from guazatine needs to be confirmed. Given the uncertainty with regard to the residue definition (see Section ) and the analytical method (see Section 1.1.) together with the minor deficiency identified in the residue trials, the MRLs of 5 mg/kg for guazatine acetates (corresponding to 4 mg/kg for residues expressed as guazatine) should be regarded as tentative. The MRL set in the country of origin is 5 mg/kg for guazatine. EFSA Journal 2014;12(8):

13 Table 3-3: Overview of the available residues trials data Commodity Residue region Outdoor /Indoor Individual trial results (mg/kg) Enforcement Risk assessment Median residue (mg/kg) (b) Highest residue (mg/kg) (c) MRL proposal (mg/kg) Median CF (d) Comments (e) Residue definition for enforcement and risk assessment: Guazatine acetates (tentative) Oranges, mandarins citrus fruits Import (ZA) Outdoor Oranges: 1.1; 1.2; 1.9; 2.6 (f) Mandarins/clementines: 2 1.5; 1.8; 1.9 Residue definition for enforcement: Guazatine (Regulation (EC) No 396/2005) Residue definition for risk assessment: Guazatine acetates (tentative) Oranges, mandarins citrus fruits Import (ZA) Outdoor Oranges: 0.77; 0.83; 1.3; 1.8 (f) Mandarins/clementines: 0.99; 1.0; 1.2; ; 1.2; 2 1.5; 1.8; ; 2.6 (f) (tentative) 1.1; 1.2; 1.9; 2.6 (f) 2 1.5; 1.8; (tentative) 1 Combined dataset. R ber = 3.8 R max = 3.21 MRL OECD = 5.06/ Combined dataset. R ber = 2.6 R max = 2.2 MRL OECD = 3.45/4 : NEU (Northern and Central Europe), SEU (Southern Europe and Mediterranean), EU (i.e. indoor use) or Import (country code) (EC, 2011). (b): Median value of the individual trial results according to the enforcement residue definition. (c): Highest value of the individual trial results according to the enforcement residue definition. (d): The conversion factor for enforcement to risk assessment is obtained from the information on the purity of the analytical standard (see footnote Section 1.1.). (e): Statistical estimation of MRLs according to the EU methodology (R ber, R max ; EC, 1997g) and unrounded/rounded values according to the OECD methodology (OECD, 2011). (f): Highest values measured at PHI 30 d. EFSA Journal 2014;12(8):

14 Effect of industrial processing and/or household preparation Modification of the existing MRL for guazatine in citrus fruits The effect of processing on the nature of guazatine residues was investigated with radiolabelled GG and GGG in a study performed at a single test condition representing pasteurisation (United Kingdom, 2013). No significant hydrolysis was observed for GGG and GG under pasteurisation conditions (20 minutes at 90 C, ph 4). The behaviour of the other guazatine constituents, such as those not completely guanidated, was not investigated. To conclude on the residue definition for processed citrus products a hydrolysis study conducted with other components representative for guazatine acetates is required. In addition, considering that citrus products may be canned, it would be desirable to investigate the nature of guazatine related residues under sterilisation conditions. Specific studies to assess the magnitude of guazatine acetates residues during the processing of citrus fruits are required as the residue levels in raw agricultural commodities (RAC) exceed the trigger value of 0.1 mg/kg (EC, 1997d). The results of a processing study conducted on oranges, lemons and grapefruits at 1 and 2 times the reported post-harvest use were provided (United Kingdom, 2013). The fruits were processed into cold-pressed oil, juice and dried peel. The study gives indications that guazatine acetates does not accumulate in citrus oil and juice, whereas a concentration of residues was observed in dried peel. Since it is not compliant with current standard (pre-glp, relevant information on nature of the processed used to generate the processed fractions and the validity of the method of analysis were missing), the study can only provide supportive information. It is noted that the applicant and the EMS are of the opinion that imported citrus are intended for direct consumption and not for processing (and the by-products for animal feeding). However, it cannot be excluded that citrus imported into Europe will never be processed. Therefore, data on the magnitude of residues in processed citrus fruits (juice, marmalade/jam, oil, pomace) are required. The distribution of residues in peel and pulp of citrus fruits was investigated in the residue trials submitted in support to the current import tolerance application. Oranges and mandarins treated postharvest with guazatine acetates at different concentration rates (0.5, 1 and 2 times the intended rate) and coated with a wax were analysed separately for residues in the pulp and the peel at PHI 0 d and PHI 30 d (2 trials). Guazatine could be measured in the pulp (0.08 to 0.2 mg/kg) only at the higher dose tested. Different processing factors (PFs) were derived at PHI 0 and 30 d (<0.06 and <0.04, respectively). The findings confirmed that residues are predominantly found in the peel and support the assumption of the EMS that guazatine is not expected to concentrate in citrus juice produced without the peel (United Kingdom, 2013). Table 3-4: Overview of the available processing studies Processed commodity Number of analysis Median PF Median CF (b) Individual peeling factors Tentative enforcement residue definition: Guazatine (pasteurisation) Tentative risk assessment residue definition: Guazatine acetate Orange, peeled 4 < Range: <0.02 to 0.11 PHI 0 d :<0.06; PHI 30 d: <0.04 Mandarin, peeled 4 < Range <0.04 to <0.09 Citrus, peeled 8 < Combined dataset. : The median processing factor is obtained by calculating the median of the individual processing factors of each processing study. (b): The median conversion factor for enforcement to risk assessment is obtained from the information on the purity of the analytical standard (see footnote Section 1.1.). EFSA Journal 2014;12(8):

15 Rotational crops Since the use of guazatine is on imported permanent crops treated post-harvest, investigations of residues in rotational crops are not required Nature and magnitude of residues in livestock Although the EMS argued that the imported fruits treated with guazatine acetates will mainly be used for human consumption as fresh fruits, it cannot be excluded that pomace obtained from the imported and processed fruit may be potentially fed to livestock. Thus, the nature and magnitude of guazatine residues in livestock is assessed in the framework of this import tolerance application (EC, 1996) Dietary burden of livestock Considering the open questions regarding the residue definition (see Section ), only an indicative dietary burden for livestock calculation could be conducted, using the agreed European methodology (EC, 1996). The input values for this indicative calculation were selected according to the latest FAO recommendations (FAO, 2009) considering the livestock intake from citrus assessed in this import tolerance application (see Table 3-2) expressed as citrus pomace by applying a default processing factor of 2.5. The input values for the indicative dietary burden calculation are summarised in Table 3-5. The default processing factor has been added to the table in brackets. Table 3-5: Input values for the indicative dietary burden calculation Commodity Median dietary burden Maximum dietary burden Input value (mg/kg) Comment Risk assessment residue definition: Guazatine acetates (tentative) Citrus, pomace ( ) Median residue PF(2.5) Input value (mg/kg) ( ) The results of the dietary burden calculation are summarised in the following table. Comment Median residue PF(2.5) Table 3-6: Results of the indicative dietary burden calculation Maximum dietary burden (mg/kg bw per d) Median dietary burden (mg/kg bw per d) Risk assessment residue definition: Guazatine acetates(tentative) Highest contributing commodity Max dietary burden (mg/kg DM) Trigger exceeded (Y/N) Dairy ruminants Orange pomace 1.79 Y Meat ruminants Orange pomace 5.38 Y Poultry, Pigs N : Calculated for the maximum dietary burden The calculated dietary burden indicated that the trigger value of 0.1 mg/kg dry matter (DM) was exceeded for ruminants, meaning that the occurrence of guazatine residues in commodities from ruminant origin has to be investigated. EFSA Journal 2014;12(8):

16 Nature and magnitude of residues Modification of the existing MRL for guazatine in citrus fruits The metabolism in ruminants was investigated using 14 C labelled guazatine (71.7 % purity, FAO, 1997). The details of the study and the results are reported in a previous EFSA reasoned opinion (EFSA, 2013). The study, which was designed in two phases (single intraruminal dose interval of 168 hours 21 intraruminal doses administered twice daily), was assessed by the EMS. Lactating cows were dosed with either 0.1 mg kg bw or 1 mg/kg bw (corresponding to about 0.4 and 4 times the maximum dietary burden in beef cattle, respectively) in phase one and at the same two doses 19 in phase two (United Kingdom, 2013). No details on the composition of the test substance were reported for this study (United Kingdom, 2014a). About 93 % of the radiolabelled guazatine was excreted unchanged in the faeces with poor oral absorption and limited transfer in blood, milk and tissues. Liver and kidneys of the high dose group were found to contain the highest levels of absorbed radioactivity, however the extraction efficiency was less than 50 % (United Kingdom, 2013). Since a full characterisation of the TRR was not carried out, this study is not adequate to elucidate the metabolism of guazatine in livestock. Thus, no residue definition in commodities of animal origin can be proposed. JMPR reached the same conclusion (FAO, 2007). No livestock feeding studies was provided. The indicative maximum dietary burden for ruminants taking into account the use on citrus authorised in South Africa (0.23 mg/kg bw) is lower than the highest dose rate reported by the EMS to be tested in the metabolism study in cows (1 mg/kg). At this dose level total residues as guazatine equivalents were found in kidney and liver (0.08 mg/kg) and milk (0.02 mg/l at day 3) but not in muscle and fat (<0.02 mg/kg) (EFSA, 2013; FAO, 1997). Based on the available information and the indicative dietary burden calculation, it cannot be excluded that low but quantifiable residues of guazatine acetates may occur in the edible tissues (kidney and liver) of ruminants. Further information on the nature and magnitude of residues of guazatine acetates would be required to conclude on the need to set MRLs in commodities of ruminant origin resulting from the post-harvest treatment on citrus fruits. 4. Consumer risk assessment As guazatine is no longer authorised within the EU and no CXLs or specific import tolerances were notified, in the framework of the MRL review under Article 12 of Regulation (EC) No 396/2005 a consumer exposure assessment was performed to assess whether the proposed LOQ of 0.05 mg/kg for guazatine acetates in all commodities was sufficiently protective for European consumers (EFSA, 2013). EFSA updated this long-term risk assessment with the tentative median residue value for citrus fruits derived from the submitted supervised residue trials. The indicative consumer risk assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). This exposure assessment model contains the relevant European food consumption data for different sub-groups of the EU population 20 (EFSA, 2007). It is noted that this risk assessment is affected by uncertainties related to the open questions on the residue definition and the appropriateness of the toxicological reference value. The acute exposure assessment was performed only with regard to citrus fruits assuming the consumption of a large portion of the food items as reported in the national food surveys and that these items contained residues at the level of the MRL established in the country of origin as worst case. 19 It is noted that for this study discrepant information regarding the dose rates applied during the second animal phase are reported in the study report assessed by the EMS (United Kingdom, 2013) and the JMPR evaluation report, where lower doses (0.5 mg/kg and 0.05 mg/kg bw) are reported (FAO, 2007; EFSA, 2013). 20 The calculation of the long-term exposure (chronic exposure) is based on the mean consumption data representative for 22 national diets collected from MS surveys plus 1 regional and 4 cluster diets from the WHO GEMS Food database; for the acute exposure assessment the most critical large portion consumption data from 19 national diets collected from MS surveys is used. The complete list of diets incorporated in EFSA PRIMo is given in its reference section (EFSA, 2007). EFSA Journal 2014;12(8):