Setting of new MRLs for isopyrazam in several cereals and food commodities of animal origin 1

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1 REASOED OPIIO Setting of new MRLs for isopyrazam in several cereals and food commodities of animal origin 1 European ood Safety Authority 2 European ood Safety Authority (ESA), Parma, Italy SUMMARY According to Article 6 of the Regulation (EC) o 396/2005, the United Kingdom, herewith referred to as the Evaluating Member State (EMS), received an application from Syngenta Crop Protection AG to set the MRLs for a new active substance isopyrazam in wheat, rye, barley, oats and in bovine fat, kidney, liver, meat and milk. The United Kingdom drafted an evaluation report according to Article 8 of Regulation (EC) o 396/2005 which was submitted to the European Commission and forwarded to ESA on 7 June Isopyrazam is a new active substance according to Directive 91/414/EEC. The Draft Assessment Report (DAR) has been submitted to ESA in May 2010, but is not yet peer reviewed and therefore all conclusions derived in this reasoned opinion are provisional and might be revised in the light of the outcome of the peer review under Directive 91/414/EEC. ESA derived the following conclusions based on the submitted evaluation report as well as the Draft Assessment Report (DAR) prepared by the Rapporteur Member State (RMS) United Kingdom under Directive 91/414/EEC. The toxicological profile of was assessed by the RMS United Kingdom who proposes the ADI value of mg/kg bw/day and an ARfD of mg/kg bw. The metabolism of isopyrazam was investigated in three crop groups (cereals, leafy vegetables and fruit and fruiting vegetables) where a similar metabolic pathway was observed. The EMS proposed to establish the enforcement residue definition as isopyrazam and the risk assessment residue definition as the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam. ESA provisionally agrees with the proposed residue definition. o additional metabolism studies on cereals 1 On request from the European Commission, Question o ESA-Q , issued on 16 September Correspondence: praper.mrl@efsa.europa.eu Suggested citation: European ood Safety Authority; Setting of new MRLs for isopyrazam in several cereals and food commodities of animal origin.. [34 pp.] doi: /j.efsa Available online: European ood Safety Authority,

2 are required. Sufficiently validated analytical method with the validated LOQ of 0.01 mg/kg in dry matrices can be proposed for the enforcement. The submitted residue trials indicate that a MRL of 0.6 mg/kg for barley and oats and a MRL of 0.2 mg/kg for wheat and rye would be required to support the proposed use on these crops in the Southern and orthern regions of Europe. The effect of processing on the magnitude of isopyrazam residues was investigated in a hydrolysis study indicating that isopyrazam does not undergo degradation under core processing conditions. The effect of processing on the nature of the plant metabolite CSCD has not been investigated. The effect of processing on the magnitude of isopyrazam residues was investigated in the processed fractions of wheat (flour and bread) and barley (pot barley and beer). A reduction of residues was observed in all processed fractions, except in wheat bran. The details of the processing conditions were not provided to verify the study and therefore the derived processing factors are not proposed for the inclusion in Annex VI of Regulation (EC) o 396/2005. According to soil degradation studies, isopyrazam and its plant metabolite CSCD and soil metabolite CSCD are considered as persistent in the soil. Metabolism of isopyrazam in rotational crops indicates that metabolite CSCD465008, even though detected in very low concentrations, is the main residue in several rotational crops. This substance was considered as of no toxicological relevance by the EMS. The EMS assumes that metabolic pathway of isopyrazam in rotational crops proceeds in a similar pathway as in primary plants and that the same residue definitions are applicable. or the crops under consideration ESA provisionally agrees with the proposed residue definitions. Considering the intended application rate and interception by the treated crop, it can be assumed that isopyrazam residues will not occur above 0.01 mg/kg in the edible parts of rotational crops. However, as ispoyrazam is persistent in the soil, a consideration has to be given to a possible accumulation of residues in the soil over the years and the subsequent residue uptake in rotational crops. Therefore Member States granting authorisations for isopyrazam should consider appropriate risk mitigation measures. Cereals and by-products can be used as livestock feed and therefore the livestock exposure to isopyrazam residues was assessed. The calculated livestock dietary burden exceeds the trigger value of 0.1 mg/kg DM for dairy and meat ruminants and is driven by the intake of wheat straw. The metabolism of isopyrazam and hydroxyl isopyrazam (CSCD459488) was investigated in lactating goats and laying hens. Metabolism studies indicate that parent isopyrazam and dihydroxy isopyrazam (CDCD656800) are the relevant residues in livestock. The EMS proposed to establish the enforcement residue definition for products of animal origin as parent isopyrazam and the risk assessment residue definition as the sum of isopyrazam and dihydroxy isopyrazam (CDCD656800), expressed as isopyrazam. ESA provisionally agrees with the proposed residue definitions. The magnitude of isopyrazam residues was investigated in the feeding study with lactating goats. or estimating the MRLs in the food commodities of bovine, sheep and goat, ESA took into account the calculated dietary burdens for ruminants and the results of the livestock feeding study. It is proposed to set the MRLs at the LOQ of mg/kg in milk, ruminant meat, fat, kidney and liver. An adequate analytical enforcement method is available to control the proposed MRLs at the validated LOQ of mg/kg. The provisional consumer exposure assessment was performed with revision 2 of the ESA PRIMo. or the calculation of the chronic exposure ESA used the STMR values as derived from the residue trials on cereals, converted by the residue definition conversion factor. or the animal commodities under consideration, the STMR values as derived from the feeding study were used as input values. or the acute exposure assessment the input values were the STMR values as derived from the residue trials on cereals, corrected by the residue definition conversion factor. or animal commodities under 2

3 consideration, the HR values (except milk for which STMR value was used) as derived from the feeding study were used as input values. The calculated long-term and short-term exposure was compared with the ADI and ARfD, respectively, as derived for isopyrazam. o long-term consumer intake concerns were identified for any of the European diets. The total calculated intake values accounted for a maximum of 1.2 % of the ADI. o short-term consumer intake concerns were identified with regard to the exposure from isopyrazam residues (including the relevant metabolites) from the intake of cereals and animal commodities. The highest acute exposure results from the intake of bovine liver (2.3% of the ARfD) and wheat (1.5% of the ARfD). The acute exposure to isopyrazam residues from the intake of other commodities accounts for less than 1% of the ARfD. rom the provisional risk assessment it is concluded that the intended use of ispoyrazam on cereals will not result in consumer exposure exceeding the toxicological reference values. It has to be noted that in order to demonstrate the full validity of the studies, the applicant is requested to perform additional storage stability studies with longer storage intervals of metabolite CSCD (in dry matrices of plant origin) and CSAA moiety (in animal matrices) as both are relevant metabolites for the risk assessment. The following recommendations have been derived by ESA: Code number a Commodity Existing EC MRL Proposed EC MRL Justification for the proposal Enforcement residue definition: Isopyrazam Barley 0.01* 0.6 The MRL proposals are Oats 0.01* 0.6 sufficiently supported by data and no risk for consumers was Wheat 0.01* 0.2 identified for the intended use Rye 0.01* 0.2 The applicant has to provide relevant storage stability studies. Enforcement residue definition: Isopyrazam () , , Bovine, sheep, 0.01* 0.005* The MRL proposals are goat meat sufficiently supported by data , , Bovine, sheep, 0.01* 0.005* and no risk for consumers was goat fat identified for the proposed , , Bovine, sheep, 0.01* 0.005* MRLs. The applicant has to goat liver provide relevant storage , , Bovine, sheep, 0.01* 0.005* stability studies goat kidney , , Bovine, sheep, 0.01* 0.005* goat milk a according to Annex I of Regulation (EC) o 396/2005 (*): Indicates that the MRL is set at the limit of analytical quantification. () at-soluble KEY WORDS Isopyrazam, barley, oats, wheat, rye, triticale, commodities of animal origin, MRL application, Regulation (EC) o 396/2005, consumer risk assessment, carboxamide fungicide, syn/anti isomers 3

4 TABLE O COTETS Summary... 1 Table of contents... 4 Background... 5 Terms of reference... 5 The active substance and its use pattern... 6 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 ature and magnitude of residues in plant Primary crops Rotational crops ature and magnitude of residues in livestock Dietary burden of livestock ature of residues Magnitude of residues Consumer risk assessment Conclusions and recommendations References Appendix A Good Agricultural Practices (GAPs) Appendix B Pesticide Residues Intake Model (PRIMo) Abbreviations

5 BACKGROUD Commission Regulation (EC) o 396/ establishes the rules governing the setting of pesticide MRLs at Community level. Article 6 of that regulation lays down that a party requesting an authorisation for the use of a plant protection product in accordance with Council Directive 91/414/EEC 4 shall submit to a Member State, when appropriate, an application to set or modify an MRL 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 Syngenta Crop Protection AG 5 to set a new MRLs for the active substance isopyrazam in wheat, rye, barley, oats and in ruminant fat, kidney, liver and meat. This application was notified to the European Commission and ESA and subsequently evaluated by the EMS in accordance with Article 8 of the Regulation. After completion, the evaluation report of the EMS was submitted to the European Commission who forwarded the application, the evaluation report and the supporting dossier to ESA on 7 June The application was included in the ESA Register of Questions with the reference number ESA-Q and the following subject: Isopyrazam - Application to set the MRLs in various commodities of plant and animal origin. The applicant proposes to establish the following MRLs for isopyrazam: 0.2 mg/kg in wheat and rye, 1 mg/kg in barley and oats, mg/kg for bovine meat, fat, kidney, liver and milk. ESA then proceeded with the assessment of the application as required by Article 10 of the Regulation. TERMS O REERECE According to Article 10 of Regulation (EC) o 396/2005, ESA 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. According to Article 11 of that Regulation, the reasoned opinion shall be provided as soon as possible and at the latest within 3 months from the date of receipt of the application. Where ESA requests supplementary information, the time limit laid down shall be suspended until that information has been provided. In this particular case the calculated deadline for providing the reasoned opinion is 7 September Commission Regulation (EC) o 396/2005 of 23 ebruary OJ L 70, , p Council Directive 91/414/EEC of 15 July 1991, OJ L 230, , p Syngenta Crop Protection AG, WRO , CH-4058 Basel, Switzerland 5

6 THE ACTIVE SUBSTACE AD ITS USE PATTER Isopyrazam is the ISO common name for mixture of 2 syn-isomers 3-(difluoromethyl)-1-methyl-- [(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and 2 anti-isomers 3-(difluoromethyl)-1-methyl--[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl- 1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide (IUPAC). The isomers have the following chemical structure: Molecular weight: g /mol Isopyrazam contains two diastereoisomers, designated as syn- and anti-isomers, identified as SY and SY534968, which are contained in technical isopyrazam in a syn:anti isomer ratios between 70:30 and 100:0. Each of the isomers is a racemate of two enantiomers. Both isomers are likely to be biologically active. As the LogP ow for syn-isomer is 4.1 and for anti-isomer is 4.4, isopyrazam can be considered as fat-soluble. Isopyrazam is a substance belonging to a chemical class of carboxamides. It is a broad spectrum fungicide with protectant properties and is to be used as a foliar spray to control cereal diseases (leaf blotch, brown rust, yellow rust in wheat and net blotch, biotic leaf spots in barley). It inhibits the succinate dehydrogenase, which is a functional part of the tricarboxylic acid cycle (TCA) and the mitochondrial electron transport chain. Isopyrazam has some curative activity through reduction of intercellular mycelial growth but its major effects on the pathogen occur when it is applied protectively. Isopyrazam is a new active substance under Directive 91/414/EEC for which the peer review is at an early stage. The Draft Assessment Report (DAR) has been submitted to ESA in May By Commission Decision 2010/132/EU of 2 March 2010, the submitted dossier on isopyrazam was considered as satisfying the data requirements of Directive 91/414/EEC. The representative uses evaluated in the DAR are foliar applications of isopyrazam on cereals. There are currently no uses of isopyrazam in Europe and therefore no EU MRLs have been established for this active substance in Regulation (EC) o 396/2005. Codex Alimentarius Commission also has not established CXLs for isopyrazam and the JMPR evaluation is planned in

7 The applicant now intends to apply for the provisional authorisation of isopyrazam on wheat, barley, oats, triticale and rye in EU and SEU according to a GAP which envisages 2 applications of the active substance on cereals at an application rate of kg a.s./ha. The application of isopyrazam on barley and oats is intended at the growth stages of BBCH The application of isopyrazam on wheat, rye and triticale is intended at the growth stages of BBCH The details of the GAPs are given in Appendix A. As the DAR is not yet peer reviewed by ESA, all conclusions derived in this reasoned opinion are provisional and might be revised in the light of the outcome of the peer review under Directive 91/414/EEC. 6 BBCH 30: Beginning of stem elongation; pseudostem and tillers erect, first internode begins to elongate, top of influorescence at least 1 cm above tillering mode 7 BBCH 59: End of heading: inflorescence fully emerged 8 BBCH 69: End of flowering: all spikelets have completed flowering but some dehydrated anthers may remain 7

8 ASSESSMET ESA bases its assessment on the evaluation report submitted by the United Kingdom (2010) and the Draft Assessment Report (DAR) prepared under Directive 91/414/EEC (United Kingdom, 2010). The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation of the Authorization of Plant Protection Products set out in Annex VI to Directive 91/414/EEC and the currently valid EU guidance documents for consumer risk assessment (European Commission, 1996, 1997a, 1997b, 1997c, 1997d, 1997e, 1997f, 1997g, 2008). 1. Methods of analysis 1.1. Methods for enforcement of residues in food of plant origin The analytical enforcement methods for the determination of isopyrazam residues in plant commodities have been evaluated in the DAR by the EMS United Kingdom (United Kingdom, 2010). The analytical method which is proposed as an enforcement method is a modified version of a German multi-residue method S19. The method applies the HPLC-MS/MS principle and was sufficiently validated at the LOQ of 0.01 mg/kg (0.005 mg/kg for each of isomers) for the determination of isopyrazam residues in dry matrices. An ILV was performed. It is concluded that an adequate analytical enforcement method is available to control the proposed MRLs in cereals Methods for enforcement of residues in food of animal origin The analytical enforcement methods for the determination of isopyrazam residues in commodities of animal origin have been evaluated in the DAR by the EMS United Kingdom (The United Kingdom, 2010). The method applies the HPLC-MS/MS principle and was sufficiently validated at the LOQ of mg/kg ( mg/kg for each of isomers) for the determination of isopyrazam residues in milk, eggs, bovine liver, kidney, fat and muscle. The method was validated by an independent laboratory for the determination of isopyrazam in milk and liver. It is concluded that an adequate analytical enforcement method is available to control the proposed MRLs in bovine meat, fat, liver, kidney and milk. 2. Mammalian toxicology The toxicological properties of isopyrazam have been evaluated in the DAR prepared under Directive 91/414/EEC (The United Kingdom, 2010) and the relevant reference values have been derived. These values are summarized in Table 2-1. Currently no other toxicological reference values have been set for this active substance in Europe or by other international bodies. Most of the mammalian toxicity studies were conducted with a test material containing syn/anti isomers in a ratio of 92.8:7.2. Some studies were performed with test material with different ratios of syn/anti isomers which gave an indication that the anti isomer is likely to be more toxic. 8

9 Table 2-1. Overview of the toxicological reference values Isopyrazam Source Year Value (mg/kg bw/d) Study relied upon Safety factor ADI DAR Rabbit developmental 4300 ARfD DAR Rabbit developmental 4300 The safety factor of 4300 was applied to the OAEL of 150 mg/kg/day from the rabbit developmental toxicity study which was performed with test material containing a ratio of 93% syn- and 7% of the anti-isomer. This safety factor comprises the default safety factor of 100, a factor of 4.3 to account for a possible higher acute toxicity of anti-isomer and the additional factor of 10 which was used by the EMS to account for the severe nature of the key endpoint in teratogenicity studies (United Kingdom, 2010). ESA is of the opinion that the choice of the most appropriate OAEL needs to be further discussed by experts. However, since the proposed toxicological reference values are derived by applying a sufficiently conservative approach, they are accepted for the provisional risk assessment in the framework of this evaluation. The relevant isopyrazam plant metabolite CSCD and animal metabolite CSCD have also been tested for their mammalian toxicity. Metabolite CSCD was characterized to be of a low acute oral toxicity and low sub-acute toxicity and tested negative in genotoxicity tests. This metabolite, however, was not identified in the rat metabolism. Metabolite CSCD was characterized to be of a low acute oral and sub-acute toxicity and tested negative in genotoxicity tests. The metabolite was assumed to have quantitatively and qualitatively similar sub-acute toxicity to the parent, due to similar OAELs observed in the sub-acute toxicity study in rats as well as due to a common target organ (liver) in the body (United Kingdom, 2010) It has to be noted that the derived toxicological reference values for isopyrazam has to be considered as provisional pending the outcome of the peer review under Directive 91/414/EEC. H O O H 9 CSCD459488: hydroxyl isopyrazam O OH 10 CSCD H 9

10 3. Residues 3.1. ature and magnitude of residues in plant Primary crops ature of residues The studies investigating the metabolism of isopyrazam in primary plants are reported in the DAR (United Kingdom, 2010): Crop group Crop Position of radiolabel Cereals wheat Phenyl and pyrazole rings Phenyl ring ruits and fruiting vegetables Leafy vegetables grapes Phenyl and pyrazole rings lettuce Phenyl and pyrazole rings Type of application Use (/ G) Application data Rate o Sampling (kg a.s/ha) Remarks oliar G maturity syn:anti 96:4 syn:anti 70:30 oliar G maturity syn:anti (PHI 21 70:30 days) oliar G DAT and at maturity (PHI 14 d) syn:anti 70:30 In the metabolism study with wheat the ratio of syn/anti isomers was 96:4 in phenyl and pyrazole study and 70:30 in additional phenyl study. Wheat grain and straw samples were taken at maturity. The total TRR at harvest (expressed as isopyrazam equivalents) in the grain and straw were 0.06 mg/kg and mg/kg, respectively (in study with syn/anti ratio 96:4). Lower levels of the TRR (0.03 mg/kg of TRR in grain and 14 mg/kg TRR in straw) were observed in the phenyl study where syn/anti ratio was 70:30. The major component of the TRR was parent isopyrazam, which accounted for 53-69% TRR in the grain and straw. Only one of the identified metabolites CSCD accounted for up to 10% (1.9 mg/kg) of the TRR in the straw. The highest amount of non-extractable residues (21% of the TRR) were found in the study with the syn/anti isomer ratio of 70:30. The possible racemisation was also investigated in forage samples from the phenyl study (syn/anti 70:30), indicating that the syn/anti ratio has not changed. In the metabolism study with grapes, the ratio of syn/anti isomers in phenyl and pyrazole labelled 14 C isopyrazam was 70:30. Grape samples were taken at maturity- 21 days following the last application. The total TRR at harvest (expressed as isopyrazam equivalents) in grapes were 0.15 mg/kg and 0.16 mg/kg in the pyrazole and phenyl studies, respectively. The major component of the TRR in grapes was parent isopyrazam, which accounted for 89-90% ( mg/kg). In leaves isopyrazam accounted for % TRR ( mg/kg). o other metabolites were present in grape fruits or leaves at levels exceeding 10 % of the TRR. The possibility of racemisation was also observed in grape and leaf samples from both studies, indicating an insignificant change in syn/anti ratio - 72:28 in grape fruit and in grape leaves (21 d PHI). In the metabolism study with lettuce, the ratio of syn/anti isomers in phenyl and pyrazole labelled 14 C isopyrazam was 70:30. Samples were taken 3 days after the final application and at maturity (14 10

11 DAT). The TRR in lettuce at harvest (expressed as isopyrazam equivalents) were mg/kg. Parent isopyrazam was the major component of the TRR accounting for 35-45% (0.1 mg/kg) in mature lettuce. Among all metabolites identified, only metabolite CSCD accounted for 17% of the TRR (0.06 mg/kg). Several other unknown conjugates were also present representing 16-19% (0.05 mg/kg) of the TRR. The metabolism of isopyrazam has been investigated in three crop groups. A similar metabolic pathway was observed. ollowing the assessment of these studies the EMS proposed to establish the enforcement residue definition as isopyrazam and the risk assessment residue definition as the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam. ESA provisionally agrees with the proposed residue definition which might be reconsidered at a later stage when the peer review of isopyrazam under Directive 91/414/EEC is finalized Magnitude of residues or the proposed EU and SEU uses of isopyrazam on wheat, rye, triticale, barley and oats, the applicant submitted residue trials on wheat and barley. The extrapolation of residue data from wheat to rye and triticale and from barley to oats is proposed. In some of the residue trials the same trial plot was treated with formulation having different syn/anti ratios (70:30 and 95:5). Other trial plots were treated either with formulations with syn/anti ratio of 95:5 or 70:30. All trial samples were analyzed separately for syn isomer, anti isomer and metabolite CSCD (and its isomer CSCD459489). The results were provided as the total sum of these compounds. Individual levels of metabolite CSCD were derived as a difference between the total sum and isomers. The sum of both isomers was used to express the residue data according to the enforcement residue definition. The residue data in straw have also been reported. Wheat In total 13 residue trials from the EU (orthern rance, Germany, The United Kingdom) and 9 residue trials from the SEU (Spain, Southern rance, Italy) were submitted by the applicant. Trials have been performed over two seasons in 2006 and All EU trials were incompliant with the intended GAP in terms of the number of applications (3 instead of 2 applications), but this was considered as not having a major impact on the final residue levels. 9 residue trials were designed as residue decline studies, demonstrating the residue behaviour on the day of the last application and after the last application until the harvest. Residues of parent isopyrazam in grain were within a range of < mg/kg; residues according to the risk assessment residue definition were within a range of < mg/kg. All SEU trials were incompliant with the intended GAP in terms of the number of applications (3 instead of 2 applications), but this was considered as not having a major impact on the final residue levels in the grain. 7 residue trials were designed as residue decline studies, demonstrating the residue behaviour on the day of the last application and after the last application until the harvest. Residues of parent isopyrazam in grain were within a range of < mg/kg; residues according to the risk assessment residue definition were within a range of < mg/kg. Bareley or the EU use on barley, the applicant submitted 12 GAP compliant supervised residue trials as performed in 2006 and 2007 in Switzerland, orthern rance, Germany, The United Kingdom. 8 of these trials were designed as residue decline studies. Residue levels of parent isopyrazam in grain were 11

12 within a range of mg/kg; residues according to risk assessment residue definition accounted for mg/kg. or the intended SEU use on barley, the applicant submitted 9 GAP compliant residue trials as performed in 2006 and 2007 in Italy, Southern rance and Spain. 7 of these trials were designed as residue decline studies. Residue levels of parent isopyrazam in grain were within a range of < mg/kg; residues according to risk assessment residue definition accounted for mg/kg. The storage stability of isopyrazam residues in plant matrices with high water, high oil content, in dry commodities, in ryegrass forage and barley straw was investigated for the peer review process and is reported in the DAR (United Kingdom, 2010). Crop samples were fortified with 1 mg/kg of isopyrazam and stored deep frozen (-18ºC) for 24 months. Demonstrated analytical recoveries indicate that isopyrazam residues in dry matrices are stable for 24 months when deep frozen. The storage stability of metabolite CSCD and its isomer CSCD in plant matrices with high water-, high acid-, high oil content and in dry matrices and in barley straw was investigated for the peer review process and is reported in the DAR (United Kingdom, 2010). The crop samples were fortified with 0.5 mg/kg of the substance and stored deep frozen for up to 11 months. Recovery data indicate that residues of metabolite CSCD and its isomer CSCD are stable in all investigated matrices for 11 months under deep freezer conditions. The supervised residue field trial samples prior to analysis were stored deep frozen for a period not exceeding 25 months. As good recoveries for the storage stability of parent isopyrazam were demonstrated at the storage period of 24 months, it can be assumed that when stored slightly longer, the degradation of isopyrazam residues did not occur. However, the residue trial samples prior to analysis of metabolite CSCD were stored for a period exceeding the demonstrated storage stability. To demonstrate full validity of the study the applicant is requested to perform additional storage stability studies of metabolite CSCD for dry matrices covering longer storage periods. The analytical methods using LC-MS-MS or GC-MS-MS detection were used to analyse the residue trials samples. The analytical methods were sufficiently validated at the overall LOQ of 0.01 mg/kg for isopyrazam (0.005 mg/kg for syn-isomer and mg/kg for anti- isomer) and at the overall LOQ of 0.01 mg/kg for metabolite CSCD and its isomer CSCD The submitted number of residue trials is sufficient to support the proposed residue data extrapolation from barley to oats and from wheat to rye and triticale (European Commission, 2008). Residue data indicate that the ratio of isomers in the applied formulation does not result in differences in residue levels. The use of isopyrazam in Southern regions of Europe results in a more critical residue situation both in wheat and barley and therefore was used to derive the MRL proposals and risk assessment values. The risk assessment values have to be reviewed with regard to storage stability of metabolite CSCD ESA provisionally accepted the residue data for the metabolite CSCD as the compound was present in the residue trial samples of grain (indicating that a full degradation has not occurred) as well as because the calculated consumer exposure to ispoyrazam residues is low and contributes insignificantly to the overall exhaustion of the ADI. The submitted residue data indicate that a MRL of 0.2 mg/kg would be required for wheat, rye and triticale and a MRL of 0.6 mg/kg for barley and oats in order to support the intended use. ESA derived also the MRL proposal for wheat straw of 15 m/kg and for barley straw of 10 mg/kg in case MRLs for livestock feed will be set in the future. 12

13 Table 3-1. Overview of the available residues trials data Commodity Wheat Rye, Triticale Barley Oats Region (a) Outdoor /Indoor Individual trial results Enforcement (isopyrazam (provisional)) EU Outdoor Grain: 6 x <0.01; 0.01; 0.011; 2 x 0.012; 0.013; 0.014; Straw: 0.11; 0.16; 0.26; 0.29; 0.83; 0.92; 2 x 0.95; 0.98; 2 x 1.1; 1.4; 1.5 SEU Outdoor Grain: 2 x <0.01; 0.018; 0.019; 0.028; 0.03; 0.041; 0.086; 0.12 Straw: <0.01; 0.2; 1.3; 2.8; 3.6; 5.1; 6.7; 7.1; 8.4 EU Outdoor Grain: 2 x 0.014; 0.015; 0.016; 0.017; 0.02; 0.022; 0.024; 2 x 0.026; 0.028; Straw: 0.076; 0.12; 0.13; 0.18; 0.33; 0.47; 0.5; 0.53; 0.68; 0.72; 0.84; 1.1 Risk assessment (the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam(provisional)) Grain: 6 x <0.015; 0.015; 0.016; 0.018; 3 x 0.019; Straw: 0.17; 0.33; 0.49; 0.85; 0.98; 1.2; 1.6; 1.8; 2 x 2.1; 2.3; 2.4; 2.6 Grain: 2 x <0.015; 0.023; 0.025; 2 x 0.036; 0.062; 0.091; 0.15 Straw: 0.015; 0.43; 1.7; 3.6; 5.1; 2 x 7.2; 7.6; 9.3 Grain: 2 x 0.02; 0.022; 0.026; 0.029; 0.032; 0.042; 0.043; 0.046; 2 x 0.048; Straw: 0.097; 0.25; 0.26; 0.31; 0.41; 2 x 0.59; 0.6; 0.71; 2 x 1.1; 1.3 STMR (b) HR (c) MRL proposal Median C (d) Comments Rber=0.025 Rmax= Rber=2.2 Rmax= Rber=0.13 Rmax= Rber=13.8 Rmax= Rber=0.05 Rmax= Rber=1.42 Rmax=

14 Commodity Region (a) Outdoor /Indoor Individual trial results Enforcement (isopyrazam (provisional)) SEU Outdoor Grain: 2 x <0.01; 0.016; 0.024; 0.046; 2 x 0.17; 0.23; 0.5 Straw: 0.13; 0.15; 0.26; 0.45; 1.1; 2.5; 3.5; 5.9; 6.9 Risk assessment (the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam(provisional)) Grain: 0.016; 0.018; 0.032; 0.052; 0.062; 0.21; 0.22; 0.36; 0.53 Straw: 0.19; 0.31; 0.33; 0.59; 1.8; 3.0; 4.03; 6.7; 7.2 STMR (b) HR (c) MRL proposal Median C (d) Comments Rber=0.40 Rmax= Rber=6.4 Rmax=8.8 (a): EU, SEU, EU or Import (country code). In the case of indoor uses there is no necessity to differentiate between EU and SEU. (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 residues trial. 14

15 Effect of industrial processing and/or household preparation The effect of processing on the nature of isopyrazam residues was investigated for the peer review in a hydrolysis study and is reported in the DAR (United Kingdom, 2010). Phenyl and pyrazole 14 C isopyrazam spiked buffer solution was treated under conditions simulating baking/brewing/boiling (ph 4, 90 ºC), pasteurisation (ph 5, 100 ºC) and sterilisation (ph 6, 120 ºC). According to the demonstrated analytical recoveries for ispoyrazam (>95% after the treatment), it was concluded that isopyrazam does not undergo degradation under core processing conditions. o data are available on the effects of processing on the nature of relevant plant metabolite CSCD The effects of processing on the magnitude of isopyrazam residues was investigated in the processing of wheat grain (processed into wheat flour and bread) and barley grain (processed into beer and pearl barley) (United Kingdom, 2010). or the processing of wheat and barley, residue trials samples from trials carried out in orthern rance were used. The initial residue levels in wheat grain were mg/kg for the total isopyrazam residues (parent plus metabolite CSCD459488) and mg/kg for parent isopyrazam. The initial residue levels in barley grain were mg/kg for the total isopyrazam residues (parent plus metabolite CSCD459488) and mg/kg for parent isopyrazam. The residue data in processed commodities were provided for the total isopyrazam residues (parent plus metabolite CSCD459488) and for the parent isopyrazam. In none of the processed fractions, except wheat bran, a concentration of residues was observed. The residues in wheat products (except bran) and pot barley accounted for 20-70% compared with the unprocessed cereal grain. In beer the residues were below the LOQ in all samples. Wheat and barley grain samples prior to analysis were stored deep frozen for 2 and 4 months, respectively; processed wheat and barley product samples prior to analysis were stored deep frozen for 1 and 4 months, respectively. The analytical methods used for analyzing samples for processing products were sufficiently validated. Table 3-2. Overview of the available processing studies Processed commodity umber of studies Median P (a) Median C (b) Comments Enforcement residue definition: Isopyrazam Wheat, flour type In two samples residues were below the LOQ. Wheat, wholemeal flour Wheat, wholemeal bread Wheat, bran Barley, pot barley Barley, beer 4 < Residues in all samples were below the LOQ. (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. 15

16 The details of the processing study (processing conditions) were not provided to verify the study and therefore the derived processing factors are not proposed for the inclusion in Annex VI of Regulation (EC) o 396/ Rotational crops Preliminary considerations The degradation of isopyrazam in soil was investigated for the peer review and the relevant studies and results are reported in the DAR (United Kingdom, 2010). According to laboratory and field degradation studies, the DT 90lab and DT 90f for isopyrazam accounts for 132 to >1000 days and for 63 to 2089 days, respectively. The rate of degradation in soil was investigated also for isopyrazam plant metabolite CSCD and soil metabolite CSCD and the relevant DT 90lab values for these compounds account for a maximum of > 1000 days. As ispoyrazam and its metabolites can be classified as highly persistent in the soil, the occurrence of isopyrazam residues in rotational crops has to be investigated ature of residues The metabolism of ispoyrazam in rotational crops was investigated for the peer review and the relevant studies are reported in the DAR (United Kingdom, 2010). The pyrazole and phenyl ring labelled 14 C isopyrazam was applied on a bare soil at an application rate of 0.36 kg a.s./ha. 30, 90 and 300 days after treatment (DAT), lettuce, wheat and turnip were planted. Immature and mature crop samples were taken for analysis. The maximum TRR (expressed as isopyrazam equivalents) was identified in the pyrazole study. In the rotational crops planted 30, 90 and 300 DAT, the TRR accounted, respectively, for 0.02, 0.03 and 0.02 mg/kg in lettuce, 0.02 mg/kg for all DAT intervals in wheat grain, 0.05 mg/kg (30 and 90 DAT) and 0.04 mg/kg in turnip tops and 0.02 and <0.01 mg/kg (90 and 300 DAT) in turnip roots. In wheat straw 30, 90 and 300 DAT the TRR accounted for a maximum of 0.92, 0.88 and 0.71 mg/kg. The characterisation of the TRR was performed for both phenyl and pyrazole studies and differences in identified metabolites were observed. In crops planted 30 DAT, parent isopyrazam was the main component in turnip roots (26-34% TRR, <0.01 mg/kg; phenyl and pyrazole study) and lettuce (13% TRR; <0.01 mg/kg; phenyl study). Metabolite CSCD and its conjugate were the main compounds in wheat forage (13-18% TRR) and wheat straw (ca. 16% TRR) in both phenyl and pyrazole studies. Metabolite CSCD and its conjugate were present in lettuce accounting for 31 % TRR (0.01 mg/kg) and in turnip tops accounting for 30% TRR (0.01 mg/kg), but were identified in the pyrazole study only. In crops planted 90 DAT, the parent compound isopyrazam in pyrazole study accounted in all crops for less than 3% TRR. Metabolite CSCD and its conjugate accounted the highest for 9.6% O OH 11 CSCD465008: H 16

17 TRR in wheat forage, 13.8% TRR in wheat hay and 17.6% TRR (0.17 mg/kg) in wheat straw. In the pyrazole study metabolite CSCD and its conjugate were the main components of the TRR in lettuce (35%; 0.01 mg/kg), turnip tops (47%; 0.02 mg/kg) and in wheat forage (21.7%; 0.03 mg/kg). In the phenyl study no characterisation of the TRR was further done for wheat grain, lettuce, turnip top and root samples, because samples contained less than 0.01 mg/kg. In crops planted 300 DAT, metabolite CSCD and its conjugate were the only compounds accounting for >10% of the TRR (in pyrazole study) in wheat forage (12%; 0.01 mg/kg), in lettuce (24%; <0.01 mg/kg) and in turnip tops (39%; 0.01 mg/kg). In the phenyl study no characterisation of the TRR was further done for wheat grain, lettuce, turnip top and root samples, because samples contained less than 0.01 mg/kg. The identification of metabolite CSCD in the pyrazole study indicates that the bridge between phenyl and pyrazole rings had been broken and metabolite CSCD465008, even though detected in very low concentrations, is the main residue in the some parts of the rotational crops (lettuce, forage, turnip tops). This substance has not been identified as a metabolite in rat. It was, however, considered as of no toxicological relevance by the EMS. The EMS proposes that metabolic pathway of isopyrazam in rotational crops proceeds in a similar pathway as in primary plants and that the same residue definitions are applicable. With regard to grain (and straw) as the crop under consideration in the current application, ESA provisionally agrees with the proposed residue definitions. or other crops a final decision on the residue definition in rotational crops is to be considered in the framework of the peer review under Directive 91/414/EEC Magnitude of residues The magnitude of isopyrazam residues in rotational crops was investigated for the peer review in four field studies (United Kingdom, 2010). Trials were conducted in Germany and rance growing barley, spinach and carrots in soil which was previously used for growing winter wheat, which had been treated with 3 foliar applications of isopyrazam (syn:anti ratio 70:30) at a combined rate of kg a.s./ha (1.5 in comparison to intended use). With the last application on wheat at a growth stage of BBCH 30-32, the crop was destroyed and rotational crops were planted 28-32, and 365 days (results not yet available) after the last application on wheat. Residues of isopyrazam in rotational crops at harvest were below the LOQ of 0.01 mg/kg, except in carrot root sample (30 day plant back interval (PBI)) which contained residues at 0.01 mg/kg. Total isopyrazam residues (parent isopyrazam plus metabolite CSCD459488) in crops at harvest were below the LOQ of mg/kg, except in spinach where no parent compound but low concentrations of CSCD (up to mg/kg) were measured. In barley straw the total residues accounted for up to 0.06 mg/kg at PBI. The residues of metabolite CSCD accounted the highest in spinach at 0.06 mg/kg (59-63 PBI), in carrot tops at 0.15 mg/kg (59-63 PBI) and in barley straw at 0.04 mg/kg (59-63 PBI) (United Kingdom, 2010). The RMS concluded that with the proposed application rates of isopyrazam on cereals, residues above 0.01 mg/kg will not occur in the edible parts of rotational crops. With regard to residues in cereal straw, their additional contribution to residues present from the direct treatment is considered to be insignificant. The presence of metabolite CSCD in spinach, carrots tops and barley straw is considered of no relevance as this substance is, according to the EMS, of a low toxicological concern. Considering that the intended application rate is lower than the application rate tested in the rotational crop studies, and that the active substance is intercepted by the treated crop, ESA agrees that total isopyrazam residues (parent isopyrazam and metabolite CSCD459488) will not occur above

18 mg/kg in the edible parts of rotational crops. However, as ispoyrazam is persistent in the soil, a consideration has to be given to a possible accumulation of residues in the soil over the years and the subsequent residue uptake in rotational crops. Therefore Member States granting authorisations for isopyrazam should consider appropriate risk mitigation measures ature and magnitude of residues in livestock Dietary burden of livestock Cereals and by products can be used as livestock feed for dairy and meet ruminants, pigs and poultry and therefore the livestock exposure to isopyrazam residues has to be assessed (European Commission, 1996). o other uses of isopyrazam on feed crops are currently intended to be authorized in Europe and therefore ESA calculated the livestock dietary burden considering the intake of cereals only. The STMR and HR values which were derived from the supervised residue field trials data (Table 3-1.) multiplied by the conversion factors for enforcement residue definition to risk assessment residue definition, were used as input values. or wheat bran the processing factor of 4 as derived from processing studies was applied. The input values for dietary burden calculation are given in the Table 3-3. Table 3-3. Input values for the dietary burden calculation Commodity Median dietary burden Maximum dietary burden Input value Comment Input value Comment Risk assessment residue definition: the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam Wheat, rye grain STMR SEU * C STMR SEU * C Barley, oat grain 0.07 STMR SEU * C 0.07 STMR SEU * C Wheat, rye bran 0.14 STMR SEUgrain *C*P (4) 0.14 STMR SEUgrain *C*P (4) Wheat, rye straw 4.72 STMR SEU * C 11 HR SEU *C Barley, oat straw 1.32 STMR SEU * C 8.28 HR SEU *C The results of the dietary burden calculation are reported in the table below. Table 3-4. Results of the dietary burden calculation Maximum dietary burden (mg/kg bw/d) Median dietary burden (mg/kg bw/d) Highest contributing commodity Max dietary burden (mg/kg DM) Trigger exceeded? Risk assessment residue definition: the sum of isopyrazam and hydroxyl isopyrazam (CSCD459488), expressed as isopyrazam 18

19 Maximum dietary burden (mg/kg bw/d) Median dietary burden (mg/kg bw/d) Highest contributing commodity Max dietary burden (mg/kg DM) Trigger exceeded? Dairy ruminants Wheat straw 2.59 Yes Meat ruminants Wheat straw 6.44 Yes Poultry Barley grain 0.05 o Pigs Barley grain 0.06 o The calculated livestock dietary burden exceeds the trigger value of 0.1 mg/kg DM for dairy and meat ruminants and is driven by the potential livestock intake of wheat straw. Consequently the possible occurrence of isopyrazam residues in commodities of animal origin has to be investigated ature of residues The metabolism of parent compound isopyrazam and its plant metabolite CSCD in livestock was investigated for the peer review and the relevant studies are reported in the DAR (United Kingdom, 2010). Metabolism of isopyrazam was studied in goats and poultry; metabolism of CSCD was investigated in goats only. The studies were performed using phenyl and pyrazole 14 C isopyrazam. The ratio of syn/anti isomers in 14 C isopyrazam was 95:5 and 70:30. Three lactating goats each received ispoyrazam at a dose rate of mg/kg in feed (DM). The TRR reached a plateau in milk after 4 days regardless of the position of the radiolabel or ratio of isomers. In tissues the TRR (expressed as isopyrazam equivalents) were less than 0.04 mg/kg with the exception of liver ( mg/kg) and kidney ( mg/kg). The characterisation of the TRR identified that parent isopyrazam and metabolite CDCD , respectively, accounted the highest for 8.6% (<0.01 mg/kg) and 44% (<0.01 mg/kg) in muscle, 40-51% (0.01 mg/kg) in fat (isopyrazam only), 1.9 % (<0.01 mg/kg) and 17% (0.1 mg/kg (free and conjugated)) in liver, 0.6% (<0.01 mg/kg) and 25% (0.04 mg/kg (free and conjugated)) in kidney and 1.4% (<0.01 mg/kg) and 32% (0.02 mg/kg) in milk. An additional metabolite CSCD was present in liver (12-21% TRR ( mg/kg)). In the OH O OH H 12 CDCD656800: dihydroxy isopyrazam OH O H 13 CSCD563692: 19

20 phenyl study (syn/anti ratio 95:5) and pyrazole study metabolite CSCD (syn isomer) in liver accounted for 20% TRR (0.12 mg/kg) and 11% TRR (0.07 mg/kg). One lactating goat received seven daily doses of pyrazole ring labelled 14 C hydroxyl isopyrazam CSCD at a dose rate of 19 mg/kg feed DM. TRR in tissues (expressed as isopyrazam equivalents) were less than 0.05 mg/kg, with exception of liver (0.44 mg/kg) and kidney (0.25 mg/kg). Metabolite CDCD was identified as the major component of the TRR in muscle (56%; <0.01 mg/kg), milk (33%; 0.04 mg/kg), fat (36%; <0.01 mg/kg), liver (36%; 0.16 mg/kg) and in kidney (38%; 0.02 mg/kg). Unchanged hydroxyl isopyrazam accounted the highest in fat (6.2%; <0.01 mg/kg) and were below 1.5% in other matrices. ifteen laying hens each received daily dose of isopyrazam at a dose rate of 11 mg/kg feed DM. Total TRR in eggs (expressed as isopyrazam equivalents) reached a plateau after 7 days and accounted for a maximum of 0.03 mg/kg in phenyl study (syn/anti ratio 70:30). In eggs two major components of the TRR were identified as parent isopyrazam (not identified in egg whites; % TRR in egg yolks) and metabolite CDCD which accounted for 7-29% (<0.01 mg/kg) in egg white and for % (<0.01 mg/kg) in egg yolk. TRR in poultry tissues (expressed as isopyrazam equivalents) were less than 0.03 mg/kg, with the exception of liver ( mg/kg). In poultry fat parent isopyrazam was the major compound of the TRR (5.9-18%). In liver parent isopyrazam and metabolite CDCD accounted for 1-2% TRR only. The metabolites observed in the livestock metabolism studies have been also observed in rat metabolism. The RMS proposes to establish the enforcement residue definition for products of animal origin as parent isopyrazam only; the risk assessment residue definition is proposed as the sum of isopyrazam and its metabolite CDCD (dihydroxy isopyrazam), expressed as isopyrazam 15. The RMS states that metabolite CDCD was not included in the enforcement residue definition due to the complex nature of the molecule and availability of the analytical standard to monitoring laboratories. ESA provisionally agrees with the proposed risk assessment and enforcement residue definitions in food commodities of animal origin Magnitude of residues The magnitude of isopyrazam residues in livestock was investigated for the peer review in lactating cows and the relevant studies are reported in the DAR (United Kingdom, 2010). Twelve lactating cows (three per dose group) each received 28 daily doses of isopyrazam (syn/anti ratio 70:30) at rates of 15, 42 and 140 mg/kg feed DM. In order to provide results according to the proposed risk assessment and enforcement residue definitions, samples were analysed, respectively, for the total isopyrazam residues (isopyrazam plus all metabolites containing CSAA moiety, expressed as OH O H 14 CSCD658108: 15 Method of analysis determines parent isopyrazam and all its metabolites containing the CSAA moiety O OH [MW= g/mol ], expressed as parent isopyrazam (multiplying results by the MW conversion factor of 2.04 to convert from CSAA to parent isopyrazam) 20