Reasoned opinion on the review of the existing maximum residue levels (MRLs) for spiroxamine according to Article 12 of Regulation (EC) No 396/2005 1

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1 EFSA Journal 215;13(1):3992 REASNED PININ Reasoned opinion on the review of the existing maximum residue levels (MRLs) for spiroxamine according to Article 12 of Regulation (EC) No 396/25 1 ABSTRACT European Food Safety Authority 2, 3 European Food Safety Authority (EFSA), Parma, Italy According to Article 12 of Regulation (EC) No 396/25, the European Food Safety Authority (EFSA) has reviewed the Maximum Residue Levels (MRLs) currently established at European level for the pesticide active substance spiroxamine. In order to assess the occurrence of spiroxamine residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Directive 91/414/EEC as well as the import tolerance and European authorisations reported by Member States (incl. the supporting residues data). Based on the assessment of the available data, MRL proposals were derived and a consumer risk assessment was carried out. Although no apparent risk to consumers was identified, some information required by the regulatory framework was found to be missing. Hence, the consumer risk assessment is considered indicative only and all MRL proposals derived by EFSA still require further consideration by risk managers. European Food Safety Authority, 215 KEY WRDS spiroxamine, MRL review, Regulation (EC) No 396/25, consumer risk assessment, fungicide 1 n request from EFSA, Question No EFSA-Q , approved on 18 December Correspondence: pesticides.mrl@efsa.europa.eu 3 Acknowledgement: EFSA wishes to thank the rapporteur Member State, Germany, for the preparatory work on this scientific output. Suggested citation: EFSA (European Food Safety Authority), 215. Reasoned opinion on the review of the existing maximum residue levels (MRLs) for spiroxamine according to Article 12 of Regulation (EC) No 396/25. EFSA Journal 215;13(1):3992, 48 pp. doi:1.293/j.efsa Available online: European Food Safety Authority, 215

2 SUMMARY Spiroxamine was included in Annex I to Directive 91/414/EEC on 1 September 1999, which is before the entry into force of Regulation (EC) No 396/25 on 2 September 28. EFSA is therefore required to provide a reasoned opinion on the review of the existing MRLs for that active substance in compliance with Article 12(2) of the aforementioned regulation. In order to collect the relevant pesticide residues data, EFSA asked Germany, as the designated rapporteur Member State (RMS), to complete the Pesticide Residues verview File (PRFile) and to prepare a supporting evaluation report. The requested information was submitted to EFSA on 2 November 28 and, after having considered several comments made by EFSA, the RMS provided on 13 May 213 a revised PRFile and evaluation report. Based on the conclusions derived by EFSA in the framework of Directive 91/414/EEC, and the additional information provided by the RMS, EFSA issued on 21 July 214 a draft reasoned opinion that was circulated to Member States experts for consultation. Comments received by 26 September 214 were considered in the finalisation of this reasoned opinion. The following conclusions are derived. The toxicological profile of spiroxamine was evaluated in the framework of Council Directive 91/414/EEC, which resulted in an ADI and an ARfD being established at.25 mg/kg bw per day and.1 mg/kg bw, respectively. The metabolism of spiroxamine was investigated in fruits (grapes, banana) and in cereals (wheat). Spiroxamine was found to be the major compound of the total residues in all crop parts. The residue definition for enforcement following foliar treatment was therefore defined as spiroxamine (sum of isomers) both in fruit crops and cereals. Significant differences were however observed in the metabolism of spiroxamine in the two crop groups. In cereals, no cleavage of the parent molecule was observed and metabolites structurally related to the parent compound (group A metabolites) were mainly formed. In fruit crops, cleavage of the parent molecule at the dioxalane moiety followed by successive steps of de-alkylation and oxidation resulted in two distinct groups of metabolite structures containing either the tert-butyl-cyclohexanol moiety (group B metabolites) or the aminodiol moiety (group C metabolites). These groups of B and C metabolites were globally observed in similar proportions compared to the parent spiroxamine. EFSA therefore proposed two separate residue definitions for the risk assessment: Cereals: sum of spiroxamine and all metabolites containing the tert-butyl-cyclohexanone moiety, expressed as spiroxamine (sum of isomers), Fruit crops: sum of spiroxamine and all metabolites containing the N-ethyl-N-propyl-1,2- dihydroxy-3-amino-propane moiety, expressed as spiroxamine (sum of isomers). Since the toxicological properties of the groups B and C metabolites were not provided, the proposed residue definition for risk assessment in fruit crops has to be regarded as tentative. Validated analytical methods are available to enforce the proposed enforcement residue definition. Regarding the magnitude of residues in primary crops, the available residues data are considered sufficient to derive MRL proposals as well as risk assessment values for all commodities under evaluation. Considering however that the residue definition for risk assessment is tentative in fruit crops, that further storage stability data are required in acidic and dry commodities and that further residues trials are required in cereals, all MRL proposals should be considered tentative. The nature of the residues in processed commodities has been sufficiently investigated and it is proposed to apply the same residue definition for enforcement and risk assessment as for primary crops. A robust processing factor for enforcement and risk assessment could be derived only for peeled banana. Further processing studies are not required as they are not expected to affect the outcome of the risk assessment. However, if more robust processing factors were to be required by risk managers, in particular for enforcement purposes, additional processing studies would be needed. EFSA Journal 215;13(1):3992 2

3 The metabolism of spiroxamine in rotational crops was evaluated in confined and field rotational crop studies. A specific residue definition for rotational crops is not deemed necessary and it was concluded that significant residues in rotational crops are not expected provided that spiroxamine is applied in compliance with the authorized uses. Livestock metabolism studies were submitted. In ruminants, the main metabolic routes of degradation consisted of oxidation in the tert-butyl moiety and N-desalkylation leading to the formation of metabolites spiroxamine carboxylic acid (M6) and hydroxy acid spiroxamine (M7) followed by a further glucuronide conjugation of M6 to compound M19. The general metabolic pathways in rodents and ruminants were found to be comparable; the findings in ruminants can therefore be extrapolated to pigs. In poultry, the metabolism of spiroxamine proceeds either via oxidation of the tert-butyl moiety to form metabolite M6 or via de-alkylation of the amino group to yield desethyl-spiroxamine (M1) and despropyl-spiroxamine (M2). None of the identified metabolites was of toxicological concern. Hence, the residue definition for enforcement is proposed as the sum of spiroxamine and spiroxamine carboxylic acid metabolite M6, expressed as spiroxamine (sum of isomers) for livestock matrices. For risk assessment purposes, two different residue definitions are proposed: Ruminants matrices: sum of spiroxamine, spiroxamine carboxylic acid (M6), its glucuronide conjugate (M19) and hydroxy acid spiroxamine (M7), expressed as spiroxamine (sum of isomers) Poultry matrices: sum of spiroxamine, desethyl-spiroxamine (M1), despropyl-spiroxamine (M2) and spiroxamine carboxylic acid (M6) expressed as spiroxamine (sum of isomers). The submitted feeding study also permitted to derive MRLs in ruminants and pigs. Considering however that an analytical method is still required for enforcement of spiroxamine in commodities of animal origin, these MRLs are tentative only. For poultry, no feeding study was provided and MRL proposals could not be derived by EFSA. Chronic and acute consumer exposure resulting from the authorised uses reported in the framework of this review was calculated using revision 2 of the EFSA PRIMo. For those commodities where data were insufficient to derive an MRL, EFSA considered the existing EU MRL for an indicative calculation. The highest chronic exposure was calculated for WH Cluster diet B, representing 13 % of the ADI, and the highest acute exposure was calculated for table grapes, representing 32 % of the ARfD. Based on the above assessment, EFSA does not recommend inclusion of this active substance in Annex IV to Regulation (EC) No 396/25. MRL recommendations were derived in compliance with the decision tree reported in Appendix D of the reasoned opinion (see summary table). None of the MRL values listed in the table are recommended for inclusion in Annex II to the Regulation as they are not sufficiently supported by data. In particular, all tentative MRLs and existing EU MRLs need to be confirmed by the following data: a validated analytical method for the determination of spiroxamine in animal commodities; data on the toxicological properties of plant metabolites of group B (tert-butyl-cyclohexanol group) and group C (aminodiol group); 4 additional residue trials on barley compliant with the critical northern outdoor GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d) with a possible extrapolation to oats; 8 residue trials on barley compliant with the southern outdoor GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 35 d) investigating residues according to the risk assessment residue definition with a possible extrapolation to oats; 8 residue trials on wheat compliant with the northern outdoor GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d) with a possible extrapolation to rye and 8 residue trials on wheat compliant with the southern outdoor GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 35 d), all investigating residues according to the risk assessment residue definition; EFSA Journal 215;13(1):3992 3

4 Review of the existing MRLs for spiroxamine a storage stability study in acidic and dry commodities where samples are analysed for parent spiroxamine; a poultry feeding study where samples are analysed according to the proposed residue definition for enforcement and risk assessment. It is highlighted, however, that the MRLs derived for wheat and rye result from a fall-back GAP in northern Europe, while a more critical GAP was reported but not supported by data. EFSA therefore identified the following data gap for national authorisation: 8 residue trials on wheat compliant with the northern outdoor GAP (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d) with a possible extrapolation to rye. If the above reported data gaps are not addressed in the future, Member States are recommended to withdraw or modify the relevant authorisations at national level. Furthermore, it is highlighted that the MRL proposal for barley and oats was derived from an outdoor GAP which according to Belgium, France and Germany will no longer be supported following the reregistration procedure of the plant protection products containing spiroxamine. In this case, the MRL proposals for oats and barley might be reduced to.5 mg/kg on the basis of a fall-back GAP, but this would still need to be confirmed by the following data: 8 residue trials on barley compliant with the northern fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42d) investigating residues according to the risk assessment residue definition. EFSA emphasises that the above assessment does not consider the possible impact of plant and livestock metabolism on the isomer ratio of spiroxamine and further investigation on this matter would in principle be required. Since guidance on the consideration of isomer ratios in the consumer risk assessment is not yet available, EFSA recommends that this issue is reconsidered when such guidance is available. SUMMARY TABLE Code number Commodity Existing EU MRL MRL Enforcement residue definition (existing): spiroxamine Enforcement residue definition (proposed): spiroxamine (sum of isomers) utcome of the review Comment 1511 Table grapes 1.6 Further consideration needed (a) 1512 Wine grapes 1.5 Further consideration needed (a) 1632 Banana 3 3 Further consideration needed (a) 51 Barley.3.4 Further consideration needed (a) 55 ats.3.4 Further consideration needed (a) 57 Rye.5*.5 Further consideration needed (a) 59 Wheat.5*.5 Further consideration needed (a) - ther products of plant origin See Appendix C1 - Further consideration needed (c) Enforcement residue definition (existing): spiroxamine carboxylic acid metabolite M6, expressed as spiroxamine Enforcement residue definition (proposed): sum of spiroxamine and spiroxamine carboxylic acid metabolite M6, expressed as spiroxamine (sum of isomers) 1111 Swine muscle.5*.2* Further consideration needed (a) EFSA Journal 215;13(1):3992 4

5 Code number Commodity Existing EU MRL MRL utcome of the review Comment 1112 Swine fat.5*.2* Further consideration needed (a) 1113 Swine liver.2.2* Further consideration needed (a) 1114 Swine kidney.2.2* Further consideration needed (a) 1121 Bovine muscle.5*.3 Further consideration needed (a) 1122 Bovine fat.5*.5 Further consideration needed (a) 1123 Bovine liver.2.3 Further consideration needed (a) 1124 Bovine kidney.2.15 Further consideration needed (a) 1131 Sheep muscle.5*.3 Further consideration needed (a) 1132 Sheep fat.5*.5 Further consideration needed (a) 1133 Sheep liver.2.3 Further consideration needed (a) 1134 Sheep kidney.2.15 Further consideration needed (a) 1141 Goat muscle.5*.3 Further consideration needed (a) 1142 Goat fat.5*.5 Further consideration needed (a) 1143 Goat liver.2.3 Further consideration needed (a) 1144 Goat kidney.2.15 Further consideration needed (a) 1161 Poultry muscle.5*.5 Further consideration needed (b) 1162 Poultry fat.5*.5 Further consideration needed (b) 1163 Poultry liver.2.2 Further consideration needed (b) 121 Cattle milk.2.15 Further consideration needed (a) 122 Sheep milk.2.15 Further consideration needed (a) 123 Goat milk.2.15 Further consideration needed (a) 13 Birds' eggs.5*.5 Further consideration needed (b) - ther products of animal origin See Appendix C - Further consideration needed (c) (*): Indicates that the MRL is set at the limit of analytical quantification. (a): Tentative MRL is derived from a GAP evaluated at EU level, which is not fully supported by data but for which no risk to consumers was identified; no CXL is available (combination E-I in Appendix D). (b): GAP evaluated at EU level is not supported by data but no risk to consumers was identified for the existing EU MRL; no CXL is available (combination C-I in Appendix D). (c): There are no relevant authorisations or import tolerances reported at EU level; no CXL is available. Either a specific LQ or the default MRL of.1 mg/kg may be considered (combination A-I in Appendix D). EFSA Journal 215;13(1):3992 5

6 TABLE F CNTENTS Abstract... 1 Summary... 2 Background... 7 Terms of reference... 8 The active substance and its use pattern... 8 Assessment Methods 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 of residues Magnitude of residues Consumer risk assessment Conclusions and recommendations Documentation provided to EFSA References Appendix A Good Agricultural Practices (GAPs) Appendix B Pesticide Residues Intake Model (PRIMo) Appendix C Existing EU maximum residue limits (MRLs) Appendix D Decision tree for deriving MRL recommendations Appendix E List of metabolites and related structural formula Abbreviations EFSA Journal 215;13(1):3992 6

7 BACKGRUND Regulation (EC) No 396/25 4 establishes the rules governing the setting and the review of pesticide MRLs at European level. Article 12(2) of that regulation stipulates that EFSA shall provide by 1 September 29 a reasoned opinion on the review of the existing MRLs for all active substances included in Annex I to Directive 91/414/EEC 5 before 2 September 28. As spiroxamine was included in Annex I to the above mentioned directive on 1 September 1999, EFSA initiated the review of all existing MRLs for that active substance and a task with the reference number EFSA-Q was included in the EFSA Register of Questions. According to the legal provisions, EFSA shall base its reasoned opinion in particular on the relevant assessment report prepared under Directive 91/414/EEC. It should be noted, however, that in the framework of Directive 91/414/EEC only a few representative uses are evaluated, while MRLs set out in Regulation (EC) No 396/25 should accommodate all uses authorised within the EU, and uses authorised in third countries that have a significant impact on international trade. The information included in the assessment report prepared under Directive 91/414/EEC is therefore insufficient for the assessment of all existing MRLs for a given active substance. In order to gain an overview of the pesticide residues data that have been considered for the setting of the existing MRLs, EFSA developed the Pesticide Residues verview File (PRFile). The PRFile is an inventory of all pesticide residues data relevant to the risk assessment and MRL setting for a given active substance. This includes data on: the nature and magnitude of residues in primary crops; the nature and magnitude of residues in processed commodities; the nature and magnitude of residues in rotational crops; the nature and magnitude of residues in livestock commodities and; the analytical methods for enforcement of the proposed MRLs. Germany, the designated rapporteur Member State (RMS) in the framework of Directive 91/414/EEC, was asked to complete the PRFile for spiroxamine. The requested information was submitted to EFSA on 2 November 28 and subsequently checked for completeness. n 13 May 213, after having clarified some issues with EFSA, the RMS provided a revised PRFile. A draft reasoned opinion was issued by EFSA on 21 July 214 and submitted to Member States (MS) for commenting. All MS comments received by 26 September 214 were considered by EFSA in the finalisation of the reasoned opinion. 4 Regulation (EC) No 396/25 of the European Parliament and of the Council of 23 February 25 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. J L 7, , p Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. J L 23, , p EFSA Journal 215;13(1):3992 7

8 TERMS F REFERENCE According to Article 12 of Regulation (EC) No 396/25, EFSA shall provide a reasoned opinion on: the inclusion of the active substance in Annex IV to the Regulation, when appropriate; the necessity of setting new MRLs for the active substance or deleting/modifying existing MRLs set out in Annex II or III of the Regulation; the inclusion of the recommended MRLs in Annex II or III to the Regulation; the setting of specific processing factors as referred to in Article 2(2) of the Regulation. THE ACTIVE SUBSTANCE AND ITS USE PATTERN Spiroxamine is the IS common name for 8-tert-butyl-1,4-dioxaspiro[4.5]decan-2-ylmethyl(ethyl) (propyl)amine (IUPAC). N Figure 1: Structure of Spiroxamine (MW: 297.5) Spiroxamine is a fungicide that acts as a sterol biosynthesis inhibitor (SBI). Following spiroxamine application before infection, the spores continue to germinate but the growth of germ tubes is inhibited. Spiroxamine acts in a physical-chemical manner on the cell wall (burning effects) and can quickly lead to a loss of cell turgidity and death of mildew spores. With a curative application of spiroxamine, application between infection and appearance of symptoms, the fungal structures are significantly damaged and die. Spiroxamine was first evaluated in the framework of the Council Directive 91/414/EEC with Germany being the designated rapporteur Member State (RMS). The representative uses supported for the peer review process were 2 foliar outdoor spray applications on cereals (spring and winter barley, winter wheat) (BBCH 61 to 69) at a dose rate of.75 kg a.s./ha in northern Europe. Following the peer review, a decision on inclusion of the active substance in Annex I to Directive 91/414/EEC was published by means of Commission Directive 1999/73/EC 6, which entered into force on 1 September The expiry date for inclusion in Annex I of Directive 91/414/EEC of spiroxamine was then amended by Commission Directive 27/21/EC 7 and was established as 31 December 211. In accordance with Commission Regulation (EC) No 737/27 8, the renewal of the approval of spiroxamine was evaluated with Germany and Hungary being the designated RMS and co-rms respectively. The representative uses supported for this second peer review process, which was carried out by EFSA, were foliar outdoor spray treatments on cereals (wheat, barley, oats, rye, triticale), 2 applications at a dose of.375 kg a.s./ha (BB to 69) and on grapes, with 2 applications at a dose of.2 kg a.s./ha followed by up to 3 applications at a dose of.4 kg a.s./ha, with PHIs of 35 days (wine grapes) and 14 days (table grapes), both in northern and southern Europe. According to 6 Commission Directive 1999/73/EC of 19 July 1999 including an active substance (spiroxamine) in Annex I to Council Directive 91/414/EEC concerning the placing of plant protection products on the market, J L 26, , p Commission Directive 27/21/EC of 1 April 27 amending Council Directive 91/414/EEC as regards the expiry dates for inclusion in Annex I of the active substances azoxystrobin, imazalil, kresoxim-methyl, spiroxamine, azimsulfuron, prohexadion-calcium and fluroxypyr, J L 97, , p Commission Regulation (EC) No 737/27 of 27 June 27 on laying down the procedure for the renewal of the inclusion of a first group of active substances in Annex I to Council Directive 91/414/EEC and establishing the list of those substances, J L 169, , p EFSA Journal 215;13(1):3992 8

9 Commission Implementing Regulation (EU) No 797/211 9, amending Regulation (EU) No 54/211 1, spiroxamine is deemed to have been approved under Regulation (EC) No 117/ This approval is restricted to uses as fungicide only. The EU MRLs for spiroxamine are established in Annexes II and IIIB of Regulation (EC) No 396/25. All existing EU MRLs, which are established for the parent compound in products of plant origin and for spiroxamine carboxylic acid (M6) (expressed as spiroxamine) in products of animal origin, are summarised in Appendix C to this document. CXLs for spiroxamine are not available. For the purpose of this MRL review, the critical uses of spiroxamine currently authorised within the EU as well as uses authorised in third countries that might have a significant impact on international trade, have been collected by the RMS and reported in the PRFile. The additional GAPs reported during the consultation of Member States were also considered (see Appendix A). These GAPs include foliar spray applications on table and wine grapes, and on cereals (barley, oats, wheat, rye, triticale) in northern and southern Europe. An import tolerance use was also reported including foliar spray applications on banana. During the Member States consultation, Belgium, France and Germany also highlighted that the northern outdoor GAPs on barley and oats (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d) and on rye and wheat (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d) as reported in this reasoned opinion will no longer be supported following the re-registration procedure of the plant protection products containing spiroxamine. The new northern outdoor GAPs that will be supported in the future consist of 2 applications at a dose of.375 kg a.s./ha, up to BBCH 61 (barley, oats) and up to BBCH 69 (wheat, rye). Since these GAPs are already authorized in France and in order to facilitate future risk management decisions, EFSA reported these GAPs as fall-back GAPs in this reasoned opinion. ASSESSMENT EFSA bases its assessment on the PRFile submitted by the RMS, the conclusion on the peer review of the pesticide risk assessment of the active substance spiroxamine (EFSA, 21), as well as the evaluation reports submitted during the consultation of Member States (France, 214; Germany, 214a and 214b). The assessment is performed in accordance with the legal provisions of the Uniform Principles for Evaluation and Authorisation of Plant Protection Products adopted by Commission Regulation (EU) No 546/ and the currently applicable guidance documents relevant for the consumer risk assessment of pesticide residues (EC, 1996, 1997a, 1997b, 1997c, 1997d, 1997e, 1997f, 1997g, 2, 21a, 21b, 211 and ECD, 211). 1. Methods of analysis 1.1. Methods for enforcement of residues in food of plant origin During the peer review under Council Directive 91/414/EEC, an analytical method using HPLC- MS/MS, confirmed by a second ion transition, and its ILV were evaluated and validated for the determination of spiroxamine in plant matrices with an LQ of.5 mg/kg in high water content 9 Commission Implementing Regulation (EU) No 797/211 of 9 August 211 approving the active substance spiroxamine, in accordance with Regulation (EC) No 117/29 of the European Parliament and of the Council concerning the placing of plant protection products on the market, and amending the Annex to Commission Implementing Regulation (EU) No 54/211, J L 25, , p Commission Implementing Regulation (EU) No 54/211 of 25 May 211 implementing Regulation (EC) No 117/29 of the European Parliament and of the Council as regards the list of approved active substances, J L 153, , p Regulation (EC) No 117/29 of the European Parliament and of the Council of 21 ctober 29 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC, J 39, , p Commission Regulation (EU) No 546/211 of 1 June 211 implementing Regulation (EC) No 117/29 of the European Parliament and of the Council as regards uniform principles for evaluation and authorisation of plant protection products. J L 155, , p EFSA Journal 215;13(1):3992 9

10 (tomato, banana), high oil content (rape seed), acidic (orange) and dry (barley grain) commodities, and in hops (Germany, 29, EFSA, 21). Furthermore, the multi-residue QuEChERS method in combination with LC-MS/MS is reported for analysis of spiroxamine with an LQ of.1 mg/kg in high water content (cucumber), in acidic (lemon) and in dry (wheat flour) commodities (CEN, 28). Table 1-1: Recovery data for the analysis of spiroxamine in different crop groups using the multiresidue QuEChERS method in combination with LC-MS/MS (CEN, 28) Commodity group Spiking levels Acidic (lemon).1.1 High water (cucumber).1.1 Dry (Wheat flour).1.1 Recoveries Mean (%) RSD (%) n No of labs Hence it can be concluded that spiroxamine can be enforced in food of plant origin with an LQ of.1 mg/kg in high water content, acidic and dry commodities, and with an LQ of.5 mg/kg in high oil content commodities and hops. Available methods are however not stereoselective Methods for enforcement of residues in food of animal origin During the peer review under Council Directive 91/414/EEC, an analytical method using LC-MS/MS, confirmed by GC-MS, was evaluated and considered as sufficiently validated for the determination of spiroxamine carboxylic acid (M6) with an LQ of.1 mg/kg in milk and an LQ of.2 mg/kg in muscle, liver and kidney (Germany, 29; EFSA, 21). An ILV was provided for this method (Germany, 214b). Furthermore, a GC-MS method was evaluated and validated for the determination of spiroxamine carboxylic acid (M6) in fat and eggs with an LQ of.2 mg/kg. For confirmatory purposes, the GC-MS analysis was conducted on a silylated derivative (Germany, 29; EFSA, 21). An ILV was also provided for this method (Germany, 214b). The available methods are not stereoselective. Hence it is concluded that spiroxamine carboxylic acid (M6) can be enforced in food of animal origin with an LQ of.2 mg/kg in muscle, fat, liver, kidney and eggs and with an LQ of.1 mg/kg in milk, but a fully validated analytical method for the determination of parent spiroxamine in livestock matrices was not reported and is still required. 2. Mammalian toxicology The toxicological assessment of spiroxamine was peer reviewed under Council Directive 91/414/EEC and toxicological reference values were established by EFSA (21). These toxicological reference values are summarised in Table 2-1. EFSA Journal 215;13(1):3992 1

11 Table 2-1: verview of the toxicological reference values Spiroxamine Source Year Value Study relied upon Safety factor ADI EFSA mg/kg bw per day 1-year dog study 1 ARfD EFSA 21.1 mg/kg bw Acute neurotoxicity study in rat 1 3. Residues 3.1. Nature and magnitude of residues in plant Primary crops Nature of residues Metabolism of spiroxamine was investigated for foliar application on cereals (wheat) and on fruit crops (grapes, banana), using [1-14 C-cyclohexyl] and [4-14 C-dioxolane]-labelled spiroxamine (Germany, 29). The characteristics of these studies are summarised in Table 3-1. Table 3-1: Summary of available metabolism studies in plants Group Crop Label position Fruit crops Grape vines 1-14 C- cyclohexyl and 4-14 C- dioxolane Method, F or G (a) Foliar spray, F Application and sampling details Rate (kg a.s./ha) No Sampling (DAT) Remarks 1 + 1, 35 (berries, stalks) nly bunches of grapes were sprayed. Banana 1-14 C- cyclohexyl and 4-14 C- dioxolane Foliar spray, G (small fruit), 17 (medium size), 71 (at harvest) ne tree was used for each labelling form. Cereals Spring wheat Winter wheat 1-14 C- cyclohexyl 4-14 C- dioxolane Foliar spray, F Foliar spray, F (a): utdoor/field application (F) or glasshouse/protected/indoor application (G) 1 + 1, 14 (forage), 61 (straw, grain) , 14 (forage), 56 (straw, grain) Applications at BB & 51 Applications at BB & 51 In both crop groups, spiroxamine was found to be the major compound of the total residues, accounting in mature crops for 3 25 % TRR in wheat grain and straw and 25 6 % TRR in grapes and banana for both the labelling forms. Significant differences were however observed in the metabolism of spiroxamine in the two crop groups. EFSA Journal 215;13(1):

12 In cereals, the metabolism proceeds mainly by oxidation and de-alkylation at the amine group and by hydroxylation at the tert-butyl group of the parent molecule with the formation of metabolites structurally related to the parent compound (group A metabolites) identified mainly as M3 13 (18 % 22 % TRR; mg eq/kg) in wheat grain and straw. No cleavage of the parent molecule occurred. In fruits, the cleavage of the parent molecule at the dioxalane moiety followed by successive steps of de-alkylation and oxidation resulted in two distinct groups of metabolites structures containing either the tert-butyl-cyclohexanol moiety (group B metabolites) (mainly metabolites M13 14, M14 15 and their sugar conjugates) accounting for 55.5 % TRR and 22.8 % TRR respectively in grapes and banana, or the aminodiol moiety (group C metabolites) (mainly metabolite M28 16 ), that occurs in grapes and banana at a level of 4 % TRR and 33.6 % TRR, respectively. In fruit crops at harvest, group B and C metabolites were globally observed in similar proportions to the parent spiroxamine. Since the parent compound was shown to be a relevant marker substance of the total residues in all the crops, the residue definition for enforcement was defined as spiroxamine for fruit crops and cereals following foliar treatment. Furthermore, since analytical methods were developed where residues were quantified after hydrolysis as the tert-butylcyclohexanone moiety (M15 17 ) in cereals and as the aminodiol moiety (M28) in grapes and banana, EFSA proposed the two following residue definitions for the risk assessment: Cereals: sum of spiroxamine and all metabolites containing the tert-butyl-cyclohexanone moiety, expressed as spiroxamine, Fruit crops: sum of spiroxamine and all metabolites containing the N-ethyl-N-propyl-1,2- dihydroxy-3-amino-propane moiety, expressed as spiroxamine. Validated analytical methods for enforcement of the proposed residue definition are available (see also Section 1.1). However, since the toxicological properties of the metabolites containing either the tertbutyl-cyclohexanol moiety (group B metabolites) or the aminodiol moiety (group C metabolites) were not provided, the proposed residue definitions for risk assessment for fruit crops have to be regarded as tentative. It is highlighted that confirmatory data on the toxicity of the plant metabolites formed in fruit crops have been submitted in the framework of the renewal of the approval of this active substance under Regulation (EC) No 117/29 and are currently under assessment. In addition, EFSA emphasises that the above studies do not investigate the possible impact of plant metabolism on the isomer ratio of spiroxamine and further investigation on this matter would in principle be required. Since guidance on the consideration of isomer ratios in the consumer risk assessment is not yet available, EFSA recommends that this issue is reconsidered when such guidance is available. Meanwhile, since the available analytical methods are not stereoselective, the proposed residue definitions for enforcement and risk assessment are derived for the sum of isomers Magnitude of residues According to the RMS, spiroxamine is authorised in northern and southern Europe for foliar application in table and wine grapes, banana, barley, oats, wheat and rye (northern Europe only) under outdoor conditions (see Appendix A). To assess the magnitude of spiroxamine residues resulting from these GAPs, EFSA considered all residue trials reported in the PRFile, including residue trials evaluated in the framework of the peer review (Germany, 29, EFSA, 21) and additional data submitted during the consultation of Member States (France, 214; Germany, 214a). However, only the residue trials where samples were analysed according to both the enforcement and risk assessment residue definitions were considered by EFSA in order to derive conversion factors for risk assessment, 13 M3: [(8-tert-butyl-1,4-dioxaspiro[4.5]dec-2-yl)methyl]ethyl(propyl)amine oxide; see Appendix E 14 M13: 4-tert-butylcyclohexanol; see Appendix E 15 M14: 4-(1-hydroxy-2-methylpropan-2-yl)cyclohexanol; see Appendix E 16 M28: 3-[ethyl(propyl)amino]propane-1,2-diol; see Appendix E 17 M15: 4-(1-hydroxy-2-methylpropan-2-yl)cyclohexanol; see Appendix E EFSA Journal 215;13(1):

13 except for the southern outdoor GAPs and for the northern fall-back GAPs on cereals where no residue trials analysing the residue definition for risk assessment were available. All residue trials considered by EFSA that comply with the authorised GAPs, are summarised in Table 3-2. The number of residue trials and extrapolations were evaluated in accordance with the European guidelines on comparability, extrapolation, group tolerances and data requirements for setting MRLs (EC, 211a). A sufficient number of trials complying with the GAP was reported by the RMS for all crops under assessment, except in the following cases: Barley and oats: The number of residue trials supporting the critical northern outdoor GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d) is not compliant with the data requirements for this major crop (4 trials instead of 8) and residue trials supporting the southern outdoor GAP did not investigate residues according to the residue definition for risk assessment. Consequently, although tentative MRL and risk assessment values can be derived from the northern outdoor data, 4 additional trials on barley complying with the northern outdoor GAP and 8 residue trials on barley compliant with the southern outdoor GAP (investigating residues according to the risk assessment residue definition) are still required with a possible extrapolation to oats. Furthermore, it is highlighted that in case the critical northern outdoor GAP is being withdrawn in the future, the northern fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42d) is supported by a sufficient number of residues trials to derive an MRL proposal for enforcement purposes. However, as for the southern outdoor GAP, residue trials supporting the southern outdoor GAP did not investigate residues according to the residue definition for risk assessment. Hence, in case the critical northern outdoor GAP is no longer supported in future, 8 residue trials on barley compliant with the northern fall-back GAP (investigating residues according to the risk assessment residue definition), would still be required. Wheat and rye: Residue trials are not available to support the northern outdoor use (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d). Considering that it is a major crop in northern Europe, 8 residue trials compliant with this GAP are in principle still required. Furthermore, residue trials supporting the southern outdoor GAP and the northern fall-back GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d) did not investigate residues according to the residue definition for risk assessment, Consequently, although tentative MRL and risk assessment values can be derived from the northern fall-back data, 8 residue trials on wheat compliant with the northern fall-back GAP (with a possible extrapolation to rye) and 8 residue trials on wheat compliant with the southern outdoor GAP (all investigating residues according to the risk assessment residue definition) are still required. The potential degradation of residues during storage of the residue trials samples was also assessed. In the framework of the peer review, the storage stability of the total spiroxamine residues under frozen conditions was demonstrated in dry commodities (barley straw, grain) for 14 months, in acidic matrices (grapes) for 18 months and in high water content matrices (banana) for 24 months. According to the RMS, all residue trials samples reported in the PRFile were stored in compliance with the storage conditions reported above. Degradation of the total spiroxamine residues during storage of the trial samples is therefore not expected. However, stability of parent spiroxamine is only confirmed in high water content matrices (banana) over 24 months (EFSA, 21). In dry and acidic commodities, the analysis was performed only using the common moiety methods and a possible degradation of spiroxamine to the metabolites covered by the analytical methods used is not excluded. Therefore, a new storage stability study where samples are specifically analysed for spiroxamine in acidic and dry commodities and covering the maximum storage time interval of the residue samples from the reported trials is required. EFSA Journal 215;13(1):

14 Table 3-2: verview of the available residue trials data Commodity Residue region (a) utdoor /Indoor Individual trial results Enforcement Risk assessment Median residue (b) Highest residue (c) MRL proposal Median CF (d) Comments Residue definition for enforcement: spiroxamine (sum of isomers) Residue definition for risk assessment: sum of spiroxamine and all metabolites containing the N-ethyl-N-propyl-1,2-dihydroxy-3-amino-propane moiety, expressed as spiroxamine (sum of isomers) Table grapes SEU utdoor.21;.2;.24;.31;.13;.19;.9;.13 Wine grapes NEU utdoor.15;.12;.6;.33;.1;.12;.17;.22;.13;.17;.2;.1;.2 Banana SEU utdoor.17;.2;.9;.14;.7;.1; <.5; <.5 Import tolerance utdoor.44;.97;.34;.33;.8; 1.2; 2.;.82;.7;.35;.4;.91.3;.33;.46;.47;.2;.16;.18;.35.4;.34;.29;.49;.52;.79;.29;.48;.27;.37;.57;.21;.47.27;.31;.32;.29;.16;.12;.12;.21.81; 1.3;.72;.65; 1.2; 1.7; 2.4; 1.3;.14;.56; 1.2; (tentative) (e), (f) (tentative) (e), (f) (tentative) (e), (f) (tentative) (e) 1.6 GAP-compliant residue trials (Germany, 29). MRL ECD =.56 R ber =.47 R max = GAP-compliant residue trials (Germany, 29). MRL ECD =.48 R ber =.4 R max = GAP-compliant residue trials (Germany, 29). MRL ECD =.33 R ber =.33 R max = GAP-compliant residue trials (Germany, 29). MRL ECD = 2.82 R ber = 1.91 R max = 2.15 EFSA Journal 215;13(1):

15 Commodity Residue region (a) utdoor /Indoor Individual trial results Enforcement Risk assessment Median residue (b) Highest residue (c) MRL proposal Median CF (d) Comments Residue definition for enforcement: spiroxamine (sum of isomers) Residue definition for risk assessment: sum of spiroxamine and all metabolites containing the tert-butyl-cyclohexanone moiety, expressed as spiroxamine (sum of isomers) Barley, oats grain NEU utdoor Critical GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d):.16;.127;.17;.172 Critical GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d):.5;.91;.312; (tentative) (f), (g) 5 GAP-compliant residue trials with extrapolation to oats (Germany, 29, 213). MRL ECD =.38 R ber =.32 R max =.29 Fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42 d): 1 <.5;.1;.2;.2 Fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42 d): (tentative) (f), (h) - GAP-compliant residue trials with extrapolation to oats (Germany, 29, 214a). SEU utdoor <.5; <.5; <.5; <.5;.1;.1;.1;.1; (tentative) (f), (h) - GAP-compliant residue trials with extrapolation to oats (Germany, 29; France, 214). EFSA Journal 215;13(1):

16 Commodity Residue region (a) utdoor /Indoor Individual trial results Enforcement Risk assessment Median residue (b) Highest residue (c) MRL proposal Median CF (d) Comments Barley, oats straw NEU utdoor Critical GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d):.416;.635;.65;.669 Critical GAP (2.75 kg a.s./ha, up to BBCH 61, PHI: 35 d): 2.58; 3.99; 4.4; (tentative) 6.2 GAP-compliant residue trials with extrapolation to oats (Germany, 29, 213). MRL ECD = 1.78 R ber = 1.33 R max = 1.2 Fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42 d):.61;.59;.23;.41;.47;.36;.54;.49;.12;.9;.57;.59; 1.1 Fall-back GAP (2.375 kg a.s./ha, up to BBCH 61, PHI: 42 d): (tentative) - GAP-compliant residue trials with extrapolation to oats (Germany, 29, 214a). MRL ECD = 1.51 R ber = 1.18 R max = 1.16 SEU utdoor.7;.71;.32;.44;.73;.42;.55;.7; (tentative) - GAP-compliant residue trials with extrapolation to oats (Germany, 29; France, 214). MRL ECD = 1.45 R ber = 1.41 R max = 1.21 EFSA Journal 215;13(1):

17 Commodity Residue region (a) utdoor /Indoor Individual trial results Enforcement Risk assessment Median residue (b) Highest residue (c) MRL proposal Median CF (d) Comments Wheat, rye grain NEU utdoor Critical GAP (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d): - Critical GAP (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d): No trials available to support the critical GAP. Fall-back GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d): 1 <.5; <.1; <.1;.2;.3 Fall-back GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d): (tentative) (f), (h) - GAP-compliant residue trials with extrapolation to rye (Germany, 29, 214a). SEU utdoor 4 <.5; <.1; <.1; <.1; (tentative) (f), (h) - GAP-compliant residue trials (Germany, 29; France, 214); no authorized use on rye in southern Europe. EFSA Journal 215;13(1):

18 Commodity Residue region (a) utdoor /Indoor Individual trial results Enforcement Risk assessment Median residue (b) Highest residue (c) MRL proposal Median CF (d) Comments Wheat, rye straw NEU utdoor Critical GAP (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d): - Fall-back GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d):.47;.69; 1.1; 1.1;.69;.31; 1.2;.4;.24;.39;.7;.81;.99;.5 SEU utdoor.36; 2;.71;.31;.45;.55;.65;.51 Critical GAP (2.75 kg a.s./ha, up to BBCH 71, PHI: 35 d): - Fall-back GAP (2.375 kg a.s./ha, up to BBCH 69, PHI: 42 d): No trials available to support the critical GAP (tentative) (tentative) (a): NEU (Northern and Central Europe), SEU (Southern Europe and Mediterranean), EU (i.e. outdoor use) or Import (country code) (EC, 211). (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 median conversion factor for enforcement to risk assessment is obtained by calculating the median of the individual conversion factors for each residue trial. (e): Tentative residue definition for risk assessment proposed in fruit crops. (f): Storage stability data on parent spiroxamine in acidic and dry commodities are required. (g): Additional residue trials on barley with a possible extrapolation to oats are required. (h): Residue trials analysing the residue for risk assessment are required. - GAP-compliant residue trials with extrapolation to rye (Germany, 29, 214a) MRL ECD = 2.6 R ber = 2.4 R max = GAP-compliant residue trials (Germany, 29; France, 214); no authorized use on rye in southern Europe. MRL ECD = 2.87 R ber = 1.39 R max = 2.43 EFSA Journal 215;13(1):

19 Consequently, the available residues data are considered sufficient to derive MRL proposals as well as risk assessment values for all commodities under evaluation. Considering however that the residue definition for risk assessment is tentative in fruit crops, that further storage stability data are required in acidic and dry commodities and that further residues trials are required in cereals, all MRL proposals should be considered tentative. Where several uses are authorised for one commodity, the final MRL proposal was derived from the most critical use and indicated in bold in Table 3-2. Tentative MRLs were also derived for feed crops (barley and wheat straw) in view of the future need to set MRLs in feed items Effect of industrial processing and/or household preparation The effect of processing on the nature of spiroxamine has not been investigated in the framework of the peer review (Germany, 29, EFSA, 21). However, a processing study simulating representative hydrolytic conditions for pasteurisation (2 minutes at 9 C, ph 4), boiling/brewing/baking (6 minutes at 1 C, ph 5) and sterilisation (2 minutes at 12 C, ph 6) was submitted in the framework of this assessment (Germany, 214b). Parent spiroxamine constituted the predominant compound of the total applied radioactivity (AR) with levels ranging between 74 % and 94 % of AR, for both the dioxolane and the cyclohexyl labels. The hydrolysis compounds were identified as spiroxamine aminodiol (M28) (4.4 % to 16.5 % AR) for the dioxolane labelling and spiroxamine cyclohexanone (M15) (3.2 % to 23.3 % AR) for the cyclohexyl labelling. Since the metabolic pattern of spiroxamine was shown to be similar in primary crops and in processed products, it is proposed to apply the same residue definition for enforcement and risk assessment as for primary crops. Studies investigating the magnitude of residues in peeled bananas were assessed in the framework of the peer review (Germany, 29, EFSA, 21) and are reported in Table 3-3. A robust processing factor for enforcement and risk assessment could therefore be derived only for peeled banana. Further processing studies are not required as they are not expected to affect the outcome of the risk assessment. However, if more robust processing factors were to be required by risk managers, in particular for enforcement purposes, additional processing studies would be needed. Table 3-3: verview of the available processing studies Processed commodity Number of studies Median PF (a) Median CF (b) Comments Enforcement residue definition: spiroxamine (sum of isomers) Risk assessment residue definition: sum of spiroxamine and all metabolites containing the N-ethyl-Npropyl-1,2-dihydroxy-3-amino-propane moiety, expressed as spiroxamine (sum of isomers) Processing factors recommended (sufficiently supported by data) Banana, peeled (Germany, 29) (a): 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 by calculating the median of the individual conversion factors of each processing study Rotational crops Preliminary considerations Spiroxamine is authorised for use on cereals that might be grown in rotation. According to the soil degradation studies evaluated in the framework of the peer review, the DT 9field value of spiroxamine is 466 days which is higher than the trigger value of 1 days (EFSA, 21). The major soil metabolites EFSA Journal 215;13(1):

20 were identified as M1 18 and M2 19 (DT 9field values of 321 and 318 days, respectively). According to the European guidelines on rotational crops (EC, 1997b), further investigation of residues in rotational crops is relevant Nature of residues The metabolism of spiroxamine in rotational crops (Swiss chard, turnips, wheat) has been evaluated (Germany, 29, EFSA, 21). A field and a confined rotational crop studies were conducted both with [1-14 C-cyclohexyl] and [4-14 C-dioxolane]-labelled spiroxamine and investigated the nature of residues at different plant back intervals. The characteristics of these studies are summarised in Table 3-4. Table 3-4: Summary of available metabolism studies in rotational crops Crop group Crop Label position Leafy vegetables Root and tuber vegetables Swiss chard 1-14 C- cyclohexyl and 4-14 C- dioxolane Turnips 1-14 C- cyclohexyl and 4-14 C- dioxolane Cereals Wheat 1-14 C- cyclohexyl and 4-14 C- dioxolane Method, F or G (a) F G F G F G Application and sampling details Rate (kg a.s./ha) (a): utdoor/field application (F) or glasshouse/protected/indoor application (G) Sowing intervals (DAT) 3, 161 3, 193, 294 3, 161 3, 193, 294 3, 161 3, 193, 294 Harvest Intervals (DAT) Remarks - Bare soil application - - Plants sown after soil ageing for 3, 161, 193 and 294 days Crop interception: % The metabolic pathway of spiroxamine in the edible parts of the rotational crops was considered as sufficiently investigated and was found to be similar to that observed in cereals as primary crops. The main degradation routes involved the desalkylation of the parent compound yielding the metabolites M1 and M2, and the oxidation of the tert-butyl group leading to hydroxy and carboxylic acid derivatives, especially present as hexose conjugates. A minor degradation pathway consisted of parent molecule cleavage. This metabolic pathway was also found to be similar to the metabolism of spiroxamine depicted in soil suggesting a potential uptake by these plants of major soil metabolites M1 and M2. The residue definition proposed for cereals as primary crops and relying on the tertbutylcyclohexanone moiety for risk assessment is also appropriate to cover the residues in rotational crops. Finally, based on this metabolism study and considering that the total annual application rate of spiroxamine within the EU is 1.5 kg a.s./ha (cereals) and the fact that bare soil treatment was applied (interception of spiroxamine residues by the primary crops is in practice expected at a rate of up to 9 %), lower levels of total residues may be expected in the edible parts of the rotated crops, provided that spiroxamine is applied in compliance with the GAPs reported in Appendix A. 18 M1: N-[(8-tert-butyl-1,4-dioxaspiro[4.5]dec-2-yl)methyl]propan-1-amine; see Appendix E 19 M2: N-[(8-tert-butyl-1,4-dioxaspiro[4.5]dec-2-yl) methyl]ethanamine; see Appendix E EFSA Journal 215;13(1):3992 2