Reasoned opinion on the modification of the MRL for dicamba in genetically modified soybean 1

Transcription

1 EFSA Journal 2013;11(10):3440 ABSTRACT REASONED OPINION Reasoned opinion on the modification of the MRL for dicamba in genetically modified soybean 1 European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy In accordance with Article 6 of Regulation (EC) No 396/2005, Denmark, hereafter referred to as the evaluating Member State (EMS), received an application from MONSANTO Europe NV to review the residue definition for the active substance dicamba, in order to accommodate the use of dicamba on dicamba-tolerant soybean in the USA. Denmark drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005, which was submitted to the European Commission and forwarded to EFSA. Since the relevant component of the residues in dicamba-tolerant soybean was identified as the metabolite 3,6-dichlorosalicylic acid (DCSA) while dicamba was not detected at harvest, EFSA proposed to set a specific import tolerance of 0.4 mg/kg for the metabolite DCSA in soybean, and not to change the current MRL of 0.05* mg/kg set for dicamba under Regulation (EC) No 396/2005 in conventional plants. Based on the risk assessment results, EFSA concludes that the proposed use of dicamba on dicamba-tolerant soybean will not result in a consumer exposure exceeding the toxicological reference values proposed for the metabolite DCSA and therefore, is unlikely to pose a consumer health risk. European Food Safety Authority, 2013 KEY WORDS dicamba, soybean, MON 87708, MRL application, Regulation (EC) No 396/2005, consumer risk assessment, benzoic acid herbicide 1 On request from the European Commission, Question No EFSA-Q , approved on 17 October Correspondence: pesticides.mrl@efsa.europa.eu Suggested citation: European Food Safety Authority, Reasoned opinion on the modification of the MRL for dicamba in genetically modified soybean. EFSA Journal 2013;11(10):3440, 38 pp. doi: /j.efsa Available online: European Food Safety Authority, 2013

2 SUMMARY In accordance with Article 6 of Regulation (EC) No 396/2005, Denmark, hereafter referred as the evaluating Member State (EMS), received an application from MONSANTO Europe NV to review the residue definition for the active substance dicamba, in order to accommodate the use of dicamba on dicamba-tolerant soybean in the USA. Denmark drafted an evaluation report according to Article 8 of Regulation (EC) No 396/2005, which was submitted to the European Commission and forwarded to EFSA on 9 November EFSA bases its assessment on the evaluation report submitted by the EMS, the Draft Assessment Report (DAR) prepared under Council Directive 91/414/EEC and its addenda, the Commission Review Report, the conclusion on the peer review of the pesticide risk assessment of the active substance dicamba and the JMPR evaluation reports. The toxicological profile of dicamba was assessed in the framework of the peer review under Directive 91/414/EEC and the data were sufficient to propose an ADI of 0.3 mg/kg bw per day and an ARfD of 0.3 mg/kg bw. It was concluded that these toxicological reference values are also applicable to its major metabolite 5-OH-dicamba in conventional plants. Since the additional plant metabolism study provided in the framework of this MRL application has shown the metabolites 3,6-dichloro-salicylic acid (DCSA) and 3,6-dichloro-gentisic acid (DCGA) to be the major components of the residues in dicamba-tolerant soybean, further toxicological studies to assess the toxicological relevance of these metabolites were provided. The data were sufficient to propose an ADI of 0.04 mg/kg per day and an ARfD of 0.3 mg/kg for DCSA, these values being also applicable to DCGA. The metabolism of dicamba in conventional crops was investigated in wheat, sugar cane, soybean and cotton. From these studies, the peer review established the residue definition for risk assessment as "sum of dicamba and 5-OH-dicamba and their conjugates expressed as dicamba" and as "dicamba and its conjugates" for enforcement. It is noted that the current residue definition for enforcement in Regulation (EC) No 396/2005 is limited to dicamba only. These residue definitions are not applicable to the use of dicamba on dicamba-tolerant soybean, as the metabolism pattern of the active substance in genetically modified plants was shown to be different. In addition, as the available data do not allow to conclude whether dicamba and DCSA act through the same toxicological mode of action, EFSA proposes to set the following additional residue definition for soybean, in order to cover the use of dicamba on dicamba-tolerant soybean: Enforcement Risk assessment Sum of DCSA and its conjugates, expressed as DCSA Sum of DCSA, DGSA and their conjugates, expressed as DCSA For the other plants and plant groups for which no dicamba-tolerant plants have been developed, the existing residue definitions remain applicable. A total of 22 supervised residue trials conducted in the USA on dicamba-tolerant soybean according to the reported US GAP were provided and considered sufficient to derive an import tolerance proposal of 0.4 mg/kg for DCSA and its conjugates. As dicamba and its metabolite 5-OH-dicamba were not detected in the trials conducted on dicamba-tolerant soybean, no change is proposed to the current MRL value set for dicamba at 0.05* mg/kg under Regulation (EC) No 396/2005. Confirmation is however required that the analytical method used to generate the residue data and the method proposed for enforcement purpose, are efficient in analysing the DCSA conjugates included in the residue definitions. Studies on the nature of DCSA and DCGA residues in processed commodities were not provided. Two studies conducted in the USA investigating the effect of processing on the magnitude of DCSA and DCGA residues in processed soybean products were submitted. Provisional processing factors were derived for different processed fractions. EFSA Journal 2013;11(10):3440 2

3 The residues of dicamba in rotational crops were not investigated as the current MRL application refers to the use of dicamba in the USA. Since soybean meal is used as feed product, a potential carry-over of DCSA and DCGA residues into food of animal origin has to be assessed. The expected livestock dietary exposure to DCSA and DCGA was calculated to be below the trigger value of 0.1 mg/kg (dry matter) for all relevant animals. Residues in products of animal origin are therefore not expected to occur and no MRLs were proposed for DCSA or DCGA in animal matrices. The consumer risk assessment was conducted for the proposed risk assessment residue definition (sum of DCSA, DCGA and their conjugates expressed as DCSA) using the revision 2 of the EFSA Pesticide Residue Intake Model (PRIMo) and the STMR and HR values derived from the supervised residue trials. No long term or short term intake concerns were identified. The TMDI was calculated to be less than 0.1% of the ADI for all European diets incorporated in the EFSA PRIMo, and the IESTI only 0.4% of the ARfD. Consequently, EFSA concludes that the occurrence of DCSA residues (including its conjugates) at the proposed MRL of 0.4 mg/kg on imported soybean will not result in a consumer exposure leading to a consumer health risk. Thus EFSA proposes to include a new entry as reported in the summary table, in Regulation (EC) No 396/2005 in order to cover the use of dicamba in the USA on dicamba-tolerant soybean. SUMMARY TABLE Code number (a) Commodity Existing EU MRL (mg/kg) Proposed EU MRL (mg/kg) Justification for the proposal Enforcement residue definition: DCSA (3,6-dichloro-salicylic acid) and its conjugates, expressed as DCSA Soya bean The import tolerance proposal is sufficiently supported by data and no risk for consumers was identified for the intended uses. Confirmation is required that the analytical method used to generate the residue data and the method proposed for enforcement purpose cover the conjugates of DCSA. Enforcement residue definition: dicamba (according Reg. (EC) No 396/2005) Soya bean 0.05* No change No change, as dicamba is not a relevant component of the residues in dicamba-tolerant soybean. (a): According to Annex I of Regulation (EC) No 396/2005. (*): Indicates that the MRL is set at the limit of analytical quantification. EFSA Journal 2013;11(10):3440 3

4 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 4 Background... 5 Terms of reference... 5 The active substance and its use pattern... 6 Assessment 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 Toxicological assessment of parent dicamba Toxicological assessment of DCSA and DCGA 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 References Appendix A. Good Agricultural Practices (GAPs) Appendix B. Pesticide Residues Intake Model (PRIMo) Appendix C. Existing EU maximum residue limits (MRLs) for dicamba Appendix D. List of metabolites and related structural formula Abbreviations EFSA Journal 2013;11(10):3440 4

5 BACKGROUND Regulation (EC) No 396/ establishes the rules governing the setting of pesticide MRLs at European Union level. Article 6 of that Regulation lays down that any party having a legitimate commercial interest may submit to the rapporteur Member State designated pursuant to Council Directive 91/414/EEC 4, repealed by Regulation (EC) No 1107/ an application to set an import tolerance in accordance with the provisions of Article 7 of that Regulation. Denmark, hereafter referred to as the evaluating Member State (EMS), received from the company MONSANTO Europe NV 6 an application to set an import tolerance for the active substance dicamba in genetically modified soybeans containing the dmo gene which codes for the dicamba monooxygenase enzyme. This application was notified to the European Commission and EFSA 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 EFSA on 29 November The application was included in the EFSA Register of Questions with the reference number EFSA-Q and the following subject: Dicamba Application to modify the existing MRL in soya bean In the evaluation report, Denmark noted that the use of dicamba on genetically modified soybeans does not require a modification of the existing MRL of 0.05* mg/kg. However, the EMS identified the need to reconsider the residue definition. Denmark also noted that in the framework of the MRL review under Article 12(1) of Regulation (EC) No 396/2005 it was proposed to raise the MRL for dicamba on conventional soybeans to 3 mg/kg considering a desiccation use in the USA. The use as desiccant was not assessed in the framework of this MRL application and EFSA proceeded with the assessment considering the use defined in the USA on dicamba-tolerant soybean. During the assessment, EFSA identified a need for further clarifications from the applicant and data requirements were sent on August Additional data were submitted to the EMS on October 2012 and July 2013 and assessed in a form of an updated evaluation report (Denmark, 2013). TERMS OF REFERENCE In accordance with Article 10 of Regulation (EC) No 396/2005, EFSA shall, based on the evaluation report provided by the evaluating Member State, provide a reasoned opinion on the risks to the consumer associated with the application. In accordance with Article 11 of that Regulation, the reasoned opinion shall be provided as soon as possible and at the latest within three months (which may be extended to six months where more detailed evaluations need to be carried out) from the date of receipt of the application. Where EFSA requests supplementary information, the time limit laid down shall be suspended until that information has been provided. In this particular case the calculated deadline for providing the reasoned opinion was 5 July Regulation (EC) No 396/2005 of the Parliament and of the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. OJ L 70, , p Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230, , p Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, , p MONSANTO EUROPE NV Tervurenlaan , 1150 Brussels, Belgium EFSA Journal 2013;11(10):3440 5

6 THE ACTIVE SUBSTANCE AND ITS USE PATTERN Dicamba is the ISO common name for 3,6-dichloro-2-methoxybenzoic acid (IUPAC). The chemical structure of the compound is herewith reported. Molecular weight: Dicamba is a synthetic auxin which belongs to the benzoic acid herbicide family. It is generally used as post-emergent herbicide by foliar spray in maize, sorghum, cereals and pastures, as well as in noncrop land and non-agricultural uses for the post-emergence control of annual and perennial broadleaved weeds. It is absorbed through leaves, shoots and roots and then rapidly translocated acropetally and basipetally throughout the plant and accumulates in the meristematic tissues, where it mimics indol acetic acid (IAA), promoting axial prolongation in sensitive weeds. Dicamba is an active substance which was evaluated according to Directive 91/414/EEC with Denmark designated as rapporteur Member State (RMS). It was included in Annex I of this Directive by Directive 2008/69/EC 7 which entered into force on 01 January 2009 for use as herbicide only. Dicamba is deemed to be approved under Regulation (EC) No 1107/ in accordance to Commission Implementing Regulation (EU) No 540/2011 9, as amended by Commission Implementing Regulation (EU) No 541/ In 2011 the approval has been amended by Regulation (EU) No 1100/ , following the peer review performed by EFSA (EFSA, 2011). The representative uses evaluated in the peer review were foliar applications on maize and pastures. The EU MRLs for dicamba are established in Annex IIIA of Regulation (EC) No 396/2005 (Appendix C); the current residue definition for enforcement comprises only the parent compound. The existing EU MRL for dicamba on soybean is set at the LOQ of 0.05* mg/kg. Dicamba has been reviewed by the Codex Alimentarius (FAO, 2010a, 2010b) and CXLs have been established for some plant and animal commodities. A proposal for soybean seeds to cover a desiccant use as pre-harvest application on conventional soybean was assessed by JMPR (FAO, 2011), but no agreement was achieved in the 2013 CCPR meeting to amend the CXL level (FAO, 2013). The GAP authorised in the USA for which an import tolerance is requested in the EU refers to outdoor foliar applications on a genetically modified dicamba-tolerant soybean. A dmo expression cassette, which contains a derivative of the coding sequence of the dicamba mono-oxygenase (DMO) protein 7 Commission Directive 2008/69/EC of 1 July 2008, amending Council Directive 91/414/EEC to include clofentezine, dicamba, difenoconazole, diflubenzuron, imazaquin, lenacil, oxadiazon, picloram and pyriproxyfen as active substances. OJ L 172, , p Regulation (EC) No 1107/2009 of European Parliament and Council of 21 October 2009 concerning the placing of plant protection products on the market and repealing Council Directives 79/117/EEC and 91/414/EEC. OJ L 309, , p Commission Implementing Regulation (EU) No 540/2011 of 23 May 2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 541/2011 of 1 June 2011 amending Implementing Regulation (EU) No 540/2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 1100/2011 of 31 October 2011 amending Implementing Regulation (EU) No 540/2011 as regards the conditions of approval of the active substances dicamba, difenoconazole, and imazaquin. OJ L 285, , p EFSA Journal 2013;11(10):3440 6

7 from Stenotrophomanas maltophilia was introduce in the soybean genome. Dicamba tolerance is achieved by the expression of the DMO protein, an enzyme which demethylates dicamba to produce formaldehyde and 3,6-dichlorosalicyl acid (DCSA) which denotes no herbicidal activities. The US GAP for which an import tolerance is requested in the EU is defined with a total of 3 applications of a SL formulation containing 480 g dicamba/l, the first as pre-emergence at a rate of 1120 kg a.s./ha and the other two, as post-emergence at 560 g a.s./ha at growth stages BBCH 13 and BBCH 60-61, respectively. The details of the GAP are given in Appendix A. It should be noted that in the framework of the review under Art of the regulation (EC) No 396/2005, an import tolerance of 3 mg/kg has been requested in the EU in order to accommodate the pre-harvest use of dicamba as desiccant on conventional soybean in the USA, with a single application at 2240 g a.s./ha, 7 days before harvest. This use is not assessed in the current evaluation. It is noted that in parallel, an application for the placing on the market of the herbicide-tolerant genetically modified soybean MON 87708, containing a single insert of the dmo expression cassette, was submitted to EFSA and evaluated by the EFSA GMO Panel under the reference number EFSA-Q The scientific opinion related to this application was published on 3 October 2013 (EFSA, 2013). EFSA Journal 2013;11(10):3440 7

8 ASSESSMENT EFSA bases its assessment on the updated evaluation report submitted by the EMS (Denmark, 2013), the Draft Assessment Report (DAR) and its addendum prepared under Council Directive 91/414/EEC (Denmark, 2007, 2010), the Commission Review Report on dicamba (EC, 2008), the conclusion on the peer review of the pesticide risk assessment of the active substance dicamba (EFSA, 2011) and the JMPR Evaluation reports (FAO, 2010a, 2010b, 2011). The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation of the Authorization 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, 2000, 2004, 2010, 2011; OECD 2011). It is noted that the assessment of the application regarding the authorisation of the genetically modified soybean MON containing the insert of the dmo expression cassette, for food and feed uses, import and processing in accordance with Regulation (EC) No 1829/ was conducted by the EFSA GMO Panel (EFSA, 2013). It is highlighted that the compliance of the product with the provisions of this Regulation is not assessed in the framework of the import tolerance request under Regulation (EC) No 396/ Methods of analysis 1.1. Methods for enforcement of residues in food of plant origin An analytical method referenced REM was assessed in the DAR and in the conclusion on the peer review under Directive 91/414/EEC (Denmark, 2007; EFSA, 2011). As the conclusion of the peer review was to consider dicamba conjugates in the residue definition, further information was requested to confirm whether the hydrolysis step included in the analytical method is efficient to release conjugates. The REM method should however be considered validated to analyse dicamba alone, which is corresponding to the current enforcement residue definition established in Regulation (EC) No 396/2005. This analytical method is however not appropriate to support the use of dicamba on dicamba-tolerant soybean, as the proposed residue definition derived for dicamba tolerant soybeans also comprises the metabolite 3,6-dichlorosalycilic acid (DCSA) 14 (Denmark, 2007; EFSA, 2011). In the framework of this MRL application, the applicant submitted an analytical method for the determination of dicamba and its metabolites DCSA and DCGA 15 included in the residue definition for risk assessment. This analytical method was considered not suitable by EFSA for routine analyses, since it requires the use of 13 C labelled internal standards for the quantification by LC-MS/MS to correct for matrix effects observed in some plant commodities and the applicant was asked to provide an alternative method. In October 2012 and June 2013 a new method referenced R0035, its validation data and its ILV were provided and evaluated by the EMS. The method involves an extraction and hydrolysis by heating in 1N HCl at 95 C, followed by a hexane/ethyl acetate partition and quantification by LC-MS/MS using two mass transitions. The method was correctly validated on soybean seeds to analyze dicamba and its metabolites 5-hydroxy-dicamba and DCSA, achieving a LOQ of 0.01 mg/kg per analyte. However, no information was provided to confirm whether the hydrolysis step is fully appropriate to release and analyse conjugates. Although the hydrolysis conditions involved in the method are considered to be rather harsh, further confirmation of this point is needed. 12 Commission Regulation (EU) No 546/2011 of 10 June 2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards uniform principles for evaluation and authorisation of plant protection products. OJ L 155, , p Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, , p ,6-dichlorosalycilic acid (DCSA): See Appendix D 15 3,6-dichlorogentisic acid (DCGA): see appendix D EFSA Journal 2013;11(10):3440 8

9 1.2. Methods for enforcement of residues in food of animal origin An analytical method for the determination of DCSA residues in food of animal origin was not provided and is not required as no significant residues of DCSA are expected in animal products (see section 3.2). Currently there are no analytical methods available to analyse food of animal origin for residues of dicamba. For the current application such a method is not be required since no residues of dicamba are expected on dicamba tolerant soybeans. However, it should be mentioned that the submission of a method for dicamba in products of animal origin was required in the conclusion on the peer review under Directive 91/414/EEC (Denmark, 2007; EFSA, 2011), since based on the supported uses on maize and pasture, MRLs were proposed for ruminant products. 2. Mammalian toxicology 2.1. Toxicological assessment of parent dicamba The toxicological profile of the active substance dicamba was assessed by the RMS in the framework of the peer review under Directive 91/414/EEC. The data were sufficient to derive toxicological reference values for dicamba (EFSA, 2011) which are compiled in Table 2-1 hereafter. Table 2-1. Toxicological reference values for dicamba Source Year Value Study relied upon Uncertainty factor Dicamba ADI EFSA mg/kg bw per day Rat, 2-generation study 100 ARfD EFSA mg/kg bw Rabbit, teratology study 100 The Acceptable Daily Intake (ADI) was set at 0.3 mg/kg bw per day, based on the offspring No Observed Adverse Effect Level (NOAEL) of 35 mg/kg bw per day in the multi-generation study and applying an uncertainty factor of 100. The Acute Reference Dose (ARfD) was set at the level of 0.3 mg/kg bw, based on the rabbit maternal NOAEL of 30 mg/kg bw per day and applying an uncertainty factor of 100. During the peer review it was concluded that the toxicological endpoints set for dicamba are also applicable to its main metabolite 5-OH-dicamba observed in conventional plants (EFSA, 2011) Toxicological assessment of DCSA and DCGA The toxicological profile of metabolite DCSA observed in the ruminant metabolism studies (liver and kidney), was also discussed during the peer review of dicamba. Based on a limited toxicological dataset, an ADI of 0.01 mg/kg bw per day was provisionally agreed for this metabolite (EFSA, 2011). Considering that DCSA and DCGA are the major components of the residues in dicamba-tolerant soybean, the applicant submitted additional toxicological data in support of this MRL application. The following toxicological studies were submitted to the EMS and evaluated (Denmark, 2011): DCSA: - ADME - Acute oral toxicity, rats - 90-day, rats - 90-day, dogs - In vitro bacterial cell gene mutation - In vitro mammalian cell gene mutation - In vitro human cell chromosomal aberration - In vivo chromosomal aberrations rat bone marrow EFSA Journal 2013;11(10):3440 9

10 - In vivo mammalian erythrocyte micronucleus test - Two-generation, rats - Prenatal developmental toxicity range-finding, rats - Prenatal developmental toxicity, rats - Prenatal developmental toxicity range-finding, rabbits - Prenatal developmental toxicity, rabbits - Combined chronic toxicity/carcinogenicity, rats DCGA: - ADME - Acute oral toxicity, rats - 28-day, rats - In vitro bacterial cell gene mutation - In vivo chromosomal aberrations rat bone marrow - Prenatal developmental toxicity range-finding, rats - Prenatal developmental toxicity, rats DCSA showed low acute oral toxicity (LD mg/kg bw); in repeated dose studies (subchronic) the most sensitive species was the dog, with a relevant NOAEL of 15 mg/kg bw per day based on liver effects (increased ALP and GGT and relative liver weight). In a chronic toxicity study in rats, the NOAEL was set at 48.8 mg/kg bw per day (males) based on increased mononuclear infiltrates, inflammation in the prostate, in the nasal cavity and cardiomyopathy. No carcinogenic potential was shown. DCSA was found to be a weak clastogen in an in vitro human cell chromosomal aberrations test, but negative in two in vivo studies. In a 2-generation study, the maternal NOAEL was 37 mg/kg bw per day (based on decreased bodyweight gain), the reproductive NOAEL was 362 mg/kg bw per day (highest dose tested) and the offspring NOAEL was 4 mg/kg bw per day (based on decreased bodyweight and relative thymus weight). DCSA did not show any potential for developmental toxicity in the absence of maternal toxicity, with a maternal NOAEL of 50 mg/kg bw per day (rat) and 25 mg/kg bw per day (rabbit) and a developmental toxicity NOAEL of 50 mg/kg bw per day (rat) and 65 mg/kg bw per day (rabbit). The acute oral LD 50 of DCGA is 1460 mg/kg bw. The relevant short term toxicity NOAEL is 474 mg/kg bw per day from a 28-day study in rat (the dog was not studied), based on effects in males (decreased white blood cells, decreased body weight and increased relative weight of brain, kidney and testes). DCGA did not show any indications of mutagenicity. The NOAEL for DCGA for maternal toxicity was 50 mg/kg/day whereas; NOAEL for developmental toxicity was 1000 mg/kg/day, the highest dose tested. EFSA Journal 2013;11(10):

11 Table 2-2 Summary of the acute toxicity studies Type of test/species Test substance/ Purity of test substance Results Acceptability Report references (a) DCSA Acute, oral, Rats DCSA (MON52708); Lot no. GLP T 97.9 % LD mg/kg bw Yes CRO /EUF00137 DCGA Acute, oral, Rats DCGA (MON52724); Lot no. GLP T, 96.3 % LD mg/kg bw Yes EPS /27301 Table 2-3 Summary of the short term toxicity studies Type of test/ Species Dose levels NOAEL (mg/kg bw/d) Effects at LOAEL and higher doses (mg/kg bw/d) Acceptability of the study Report references (a) DCSA 90 day oral (dietary). Rats 0, 500, 3000, 6000 and ppm Females: 222 mg/kg bw per day (3000 ppm) Males: 363 mg/kg bw per day (6000 ppm) Females: decreased body weight, changes in clinical parameters, increased relative kidney weight, one incidence of stomach erosion. Males: decreased body weight and food consumption, changes in haematological and clinical parameters, increased relative liver and brain weight, bone marrow depletion, stomach hyperplasia and stomach erosion Yes WI / WIL day oral (capsule). Dogs 0, 15, 50 and 150 mg/kg bw per day 15 mg/kg bw per day Increased ALP and GGT, increased relative liver weight. Yes WI / WIL DCGA 28 day oral (dietary) study using rats. 0, 500, 3000, 6000 and ppm Females: 1063 mg/kg bw per day (12000 ppm) Males: 474 mg/kg bw per day (6000 ppm) Females: No LOAEL Males: decreased body weight, decreased white blood cells, lymphocyte levels, increased relative brain, kidney and testes weight. Yes WI / WIL EFSA Journal 2013;11(10):

12 Table 2-4 Summary of the genotoxicity studies Test substance (batch and purity) In vitro studies DCSA DCSA (MON52708) Lot no. GLP T, 97.9 % DCSA (MON52708) Lot no. GLP T 97.9 % Test system Concentrations/doses Results Bacterial reverse mutation assay Test strain: TA98, TA100, TA1535, TA1537 & WP2uvrA CHO HGPRT forward mutation assay with a confirmatory assay and duplicate cultures (+S9) (-S9): 33.3, 100, 333, 1000, 2500 and 5000 μg/plate Conc. (-S9) Ini.: 400, 600, 800, 1000, 1200 and 1400 μg/ml Conc. (-S9) Con.: 1100, 1200, 1300, 1400, 1500 and 1600 μg/ml Conc. (+S9) Ini.: 400, 500, 600, 700, 800 and 1000 μg/ml Conc. (+S9) Con.: 700, 800, 900, 1000, 1100 and 1200 μg/ml DCSA did not induce gene mutations in bacteria No evidence of mutagenic potential either with or without metabolic activation. Acceptability of the study Yes Yes Report (a) references CV / CV / DCSA (MON52708) Lot no. GLP T, 97.9 % Chromosomal aberrations in cultured human peripheral blood lymphocytes Conc. (-S9) Ini.: 17.0, 24.2, 34.6, 49.4, 70.6, 101, 144, 206, 294, 420, 600, 858, 1230, 1750 and 2500 µg/ml, Conc. (-S9) Con. 1: 11.3, 22.5, 45.0, 90.0, 125, 188, 250, 375, 500, 700, 850, 1000 and 1200 μg/ml Conc. (-S9) Con. 2: 62.5, 125, 250, 350, 400, 425, 450, 475, 500, 600 and 700 μg/ml. Conc. (+S9)* Ini.: 17.0, 24.2, 34.6, 49.4, 70.6, 101, 144, 206, 294, 420, 600, 858, 1230, 1750 and 2500 µg/ml, Conc. (+S9) Con.: 250, 375, 500, 700, 750, 800, 850, 925 and 1000 μg/ml Weak clastogenicity both with and without metabolic activation Yes CV / EFSA Journal 2013;11(10):

13 Test substance (batch and purity) In vitro studies DCGA DCGA (MON52724) Lot no. GLP T, 96.3 % In vivo studies DCSA DCSA (MON52708) Lot no. GLP T, 97.9 % In vivo studies DCGA DCGA (MON52724) Lot no. GLP T, 96.3 % Test system Concentrations/doses Results Bacterial reverse mutation assay Test strain: TA98, TA100, TA1535, TA1537 & WP2uvrA Chromosomal aberrations in vivo in rat bone marrow cells In vivo mouse bone marrow micronucleus assay Chromosomal aberrations in vivo in rat bone marrow cells (-S9): 33.3, 100, 333, 1000, 2500, and 5000* μg/plate, * only WP2uvrA (+S9): 33.3, 100, 333, 1000, 2500, and 5000 μg/plate Males: 0, 400, 800 and 1600 mg/kg. Females: 0, 300, 600 and 1200 mg/kg DCGA did not induce gene mutations in bacteria (the partly positive study results for TA1537 (-S9) have insufficient strength and are therefore not considered biological relevant). No signs of clastogenicity 0, 250, 500 and 1000 mg/kg No clastogenic or aneugenic potenial 0, 375, 750 and 1500 mg/kg No signs of clastogenicity Acceptability of the study Yes Yes Yes Limited Report (a) references CV / CV / CV / CV / Table 2-5 Summary of the long term toxicity studies Type of test/species Dose levels (mg/kg) NOAEL (mg/kg bw/d) Effects at LOAEL and higher doses (mg/kg bw/d) Acceptability of the study Report (a) references DCSA Combined chronic toxicity/carcinogenicity study using rats 0, 10, 100, 300, 1000 and 3000 ppm Females: mg/kg bw per day (3000 ppm) Males: 48.8 mg/kg bw per day (1000 ppm) Females : no LOAEL Males: prostate inflammation, inflammation in the nasal cavity, cardiomyopathy Yes WI / WIL EFSA Journal 2013;11(10):

14 Table 2-6. Summary of the reproductive toxicity studies Type of test/species Multigenerational DCSA Two-generation, Rats Developmental DCSA Prenatal developmental toxicity oral (gavage) range-finding study. Rats Prenatal developmental toxicity oral (gavage). Rats Prenatal developmental toxicity oral (gavage) range-finding study, Rabbits Prenatal developmental toxicity oral (gavage) study. Rabbits Developmental DCGA Prenatal developmental toxicity oral (gavage) range-finding study Rats Dose levels (mg/kg bw/d) 0, 50, 500 and 5000 ppm 0, 50, 200, 500 and 1000 mg/kg bw/ day 0, 10, 30 and 100 mg/kg bw/day 0, 10, 30, 100 and 300 mg/kg bw/day 0, 10, 25 and 65 mg/kg bw/day 0, 50, 200, 500 and 1000 mg/kg bw/ day NOAEL (mg/kg bw/d) Parental: (500 ppm) - male: 37 mg/kg bw/day - female: 43 (premating), 34 (gestation) and 78 (lactation) mg/kg bw/day Reproduction: 362 mg/kg bw/day (males), 414 mg/kg bw/day (females) (5000 ppm) Offspring: 4 mg/kg bw/day for males and for females (50 ppm) Maternal: Developmental: Maternal: 50 mg/kg bw per day 50 mg/kg bw per day 100 mg/kg bw per day Developmental: 100 mg/kg bw per day Maternal: Developmental: Maternal: Developmental: Maternal: 30 mg/kg bw per day 30 mg/kg bw per day 25 mg/kg bw per day 65 mg/kg bw per day 50 mg/kg bw per day Developmental: 1000 mg/kg bw per day Effects at LOAEL and higher doses (mg/kg bw/d) Parental: decreased bodyweight (both sexes) increased relative brain and kidney weight (females). Reproductive toxicity: no LOAEL Offspring: decreased bodyweight (both sexes, F1 generation) decreased relative thymus weight (F1 males) Maternal: clinical signs of toxicity, decreased absolute liver weight Developmental: decreased foetal body weights Maternal toxicity: no LOAEL Developmental toxicity: No LOAEL Maternal: increased mortality, decreased defecation, food consumption, bodyweight. Developmental: decreased number viable foetuses, implantation sites, corpora lutea. Maternal: one mortality, decreased food consumption, decreased bodyweight reductions. Developmental: no LOAEL in study Maternal: clinical signs of toxicity. Developmental: no LOAEL (a) : References in Toxicological assessment rapport of: 3,6-dichlorosalicylic acid and 3,6-dichlorogentisic acid (Denmark, 2011) Acceptability of the study Yes Yes Yes Yes Yes Yes Report ref. (a) WI /WIL WI /WIL WI /WIL WI /WIL WI /WIL WI /WIL EFSA Journal 2013;11(10):

15 Reference values for metabolites DCSA and DCGA Modification of the MRL for dicamba in genetically modified soybean The available data do not allow to show whether dicamba and metabolites DCSA and DCGA act through the same mode of action: some of the target organs are the same, but some are not, as well as some toxicological effects are not in common. No mechanistic studies are available. Therefore, based on the available data, it is considered appropriate to set specific reference values for DCSA and DCGA. The EMS proposed an ADI for DCSA of 0.04 mg/kg bw per day, based on the offspring NOAEL of 4 mg/kg bw per day (from the 2-generation study in rats), applying an uncertainty factor of 100. An ARfD of 0.3 mg/kg bw was derived based on effects on pups in the 2-generation study in rats (in the highest dosed group there were signs of severe neonatal toxicity, a generally poor condition and statistically significant reduced postnatal survival), which were considered as a potential result of an acute effect. The NOAEL of the mothers during gestation was 34 mg/kg bw per day. An uncertainty factor of 100 is applied. The EMS concluded that there is insufficient toxicological data to set a specific ADI and ARfD for DCGA; however DCGA is a metabolite of DSCA and the conducted tests do not indicate higher toxicity than DCSA. It was therefore concluded that the ADI and ARfD proposed for DCSA will also cover DCGA. These conclusions are agreed. An overview of the dietary toxicological values proposed DCSA and DCGA are summarized in the table 2-7 below. Table 2-7. Overview of the toxicological reference values for metabolites DCSA Source Year Value Study relied upon Uncertainty factor ADI EMS DK mg/kg bw per day 2-generation study in rats 100 ARfD EMS DK mg/kg bw 2-generation study in rats 100 DCGA ADI EMS DK mg/kg bw per day DCSA 2-generation study in rats ARfD EMS DK mg/kg bw DCSA 2-generation study in rats EFSA Journal 2013;11(10):

16 3. Residues 3.1. Nature and magnitude of residues in plant Primary crops The metabolism of dicamba was evaluated in primary crops (conventional crops) by the RMS (Denmark, 2007) and reviewed by EFSA (EFSA, 2011) in the framework of the peer review under Directive 91/414/EEC. In support of this application, a metabolism study on dicamba-tolerant soybean was submitted. The reference of the variety tested was not reported. The overview of the study designs is presented in the table below. Table 3-1: Summary of metabolism studies in conventional and dicamba-tolerant plants Group Pulses and oilseeds Cereals Crop Soybean (conventional) Soybean (dicamba tolerant) Cotton (conventional) Wheat (conventional) Sugar cane (conventional) Method, F or G (a) F Rate ca. 1.7 b g a.s./ha G 2860 g a.s./ha F 2820 g a.s./ha ca. 5.9 b g a.s./ha F 144 g a.s./ha F ca b g a.s./ha Application details No/ Interval 1, Early growth stage 1, Late growth stage 1, preemergence 1, at BBCH 13 1, Green boll stage 1, BBCH leaves Sampling 0, 1, 6, 7 and 14 DAT 0, 1 and 5 DAT 36, 56 and 112 DAT 7, 27 and 83 DAT 0, 7 and 14 DAT 18 & 85 DAT 0, 1 2, 5, 12, 21 and 28 DAT Remarks Local leaf application using micro-syringe Soil application Foliar application Local leaf application using micro-syringe Foliar spray Local leaf application using micro-syringe (a): Outdoor/field use (F) or glasshouse/protected crops/indoor application (G) (b): Rates per ha should be considered as rough estimates since derived from local application on leaves using microsyringe Nature of residues in conventional crops Metabolism in conventional plants was investigated in cereals (wheat, sugar cane) and in the pulses/oilseeds plant group (soya, cotton), using 14 C-dicamba labelled on the phenyl moiety applied by foliar spraying (wheat), or by droplet applications by means of a micro-syringe to a limited number of leaves (sugar cane, soya, cotton). In sugar cane, soya and cotton, where the characterisation of the residues was investigated shortly after the application (6 to 28 days) dicamba was shown to represent the major component of the residues, accounting for % TRR in sugar cane leaves, % TRR in soya beans and 72 % TRR in cotton seed. 5-OH-dicamba represented 47 % and 20 % TRR in sugar cane leaves, 12 and 28 days after application, respectively. Other identified metabolites, among them DCSA and DCGA, were observed only in low proportions (<2 % TRR). In wheat, dicamba was more extensively metabolised, accounting for only 10 % TRR in immature plant (forage), and 2 % and 16 % TRR in straw and grain at harvest. The metabolite 5-OH-dicamba was detected as the major component in wheat forage collected 18 days after the application (65 % TRR), with a steady decrease to less than 4 % TRR in grain and straw at harvest. Both dicamba and 5-OH-dicamba, were observed in free and conjugated forms. EFSA Journal 2013;11(10):

17 Based on these studies a metabolic pathway was proposed for conventional plants. The metabolism proceeds first by hydroxylation of dicamba to form 5-OH-dicamba. A minor route consists of the demethylation of the parent compound to DCSA which is further degraded to DCGA. Having regard to the very low proportions and levels the metabolites DCSA and DGSA were observed, the residue definition for monitoring for conventional plants was limited to dicamba and its conjugates. For risk assessment the residue was defined as the "sum of dicamba, 5-OH-dicamba and their conjugates expressed as dicamba" (EFSA, 2011). It is noted that the current enforcement residue definition established in Regulation (EC) No 396/2005 comprises only the parent compound dicamba Nature of residues in dicamba-tolerant soybean An additional metabolism study conducted on dicamba-tolerant soybean containing the dmo expression cassette was provided and evaluated by the EMS in the framework of this MRL application. The study was conducted using 14 C-dicamba labelled on the phenyl moiety, following a single pre-emergence application at 2860 g/ha or a single post-emergence (BBCH 13) application at 2820 g/ha on dicamba-tolerant soybean (ca. 1N rate when considering the total annual rate). EFSA considers the study conducted at stage BBCH 13 as appropriate to cover the post-emergence uses of dicamba up to the stage BBCH as proposed in the US GAP (see appendix A), since a less extensive metabolism is expected in plants from applications done beyond the stage BBCH 13. Following pre-emergence application, the radioactive residues were mostly composed of the glucoside conjugated of DCSA that accounted for more than 70% TRR (1.0 and 0.7 mg eq/kg) in the forage and hay samples collected 36 and 56 days after application. At harvest, after 112 days, DCSA conjugates remained the major component in seeds but representing ca. 20% TRR (0.05 mg eq/kg). The parent dicamba was almost not detected (<2% TRR) and 5-OH-dicamba was not identified. A similar picture was observed following post-emergence application. DCSA-glucoside was ca. 60% TRR in forage and hay (80 and 26 mg eq./kg) and 15% TRR in seeds (0.06 mg eq./kg) at harvest. The parent dicamba was however observed in higher proportions in the forage and hay samples collected 7 and 27 days after application, accounting for 24% TRR 12% TRR, respectively. At harvest however, dicamba accounted for only 0.6% TRR (0.003 mg/kg) in seeds. In the dicamba-tolerant soybean variety tested, the metabolism proceeds by an initial demethylation of dicamba to form DCSA which is further hydroxylated at the 5-position to give the DCGA metabolite, both being mainly observed as conjugates. This preferential metabolic pathway though the demethylation of dicamba to DCSA results from the action of the DMO protein, as a consequence of the introduction of the dicamba mono-oxygenase (dmo) expression cassette. Parent dicamba was detected in significant levels only shortly after the post-emergence application and was present at harvest in traces only. 5-OH-dicamba was not identified in dicamba-tolerant soybean. EFSA requested additional information to demonstrate that the mono-oxygenase enzyme expressed in the genetically modified soybeans does not catalyse the demethylation of other active substances commonly applied on soybeans, resulting in the formation of metabolites that are not occurring in conventional plants. In order to demonstrate the specificity of the dicamba-mono-oxygenase protein the applicant submitted crystallographic studies which confirmed that only compounds structurally related to dicamba (molecules containing a phenyl ring with carboxylic acid and methoxy moieties) are potential substrates of the DMO protein. Five endogenous soybean compounds structurally similar to dicamba and identified as potential substrate to the DMO protein (o-anisic acid, vanilllic acid, syringic acid, ferulic acid and sinapic acid) were tested. As none of these structures were catabolised, these results give some evidence of the specificity of the DMO protein. In case active substances used in plant protection products on soybean or their related metabolites are identified to contain a structure which might be a potential substrate to the DMO protein, further data on the specificity may be requested. EFSA Journal 2013;11(10):

18 Conclusion, residue definitions Qualitatively, the metabolism in conventional and dicamba-tolerant soybean is different. In conventional plants, the main metabolic pathway starts with the hydroxylation of the parent compound at the 5-position to give the 5-OH-dicamba, while in dicamba-tolerant soybean, this route is not identified and the metabolism proceeds by the demethylation of dicamba to give the metabolite DCSA which is further hydroxylated to DCGA, both being mostly observed in their conjugated forms. The residue definitions for monitoring agreed during the peer review for conventional crops and limited to the parent dicamba is therefore not applicable to dicamba-tolerant soybean where the marker of the residues is the metabolite DCSA. Having regard to the different metabolic profiles observed in conventional plants and dicamba-tolerant soybean, the EMS proposed for soybean the following to residue definitions (Denmark, 2013): - Enforcement: Sum of dicamba, DCSA and their conjugates expressed as dicamba - Risk assessment: Sum of dicamba + 5-OH-dicamba + DCSA* + DGSA*, expressed as dicamba (*: a correction factor of 7.5 is proposed for DCSA and DCGA to conduct the chronic risk assessment, considering the ratio of the ADI proposed for dicamba and DCSA respectively). Considering that the ADI set for DCSA is significantly lower than that proposed for dicamba and, since the available data do not allow to conclude whether dicamba and DCSA act through the same toxicological mode of action, EFSA proposes to set two separate residue definitions for soybeans and to conducted two separate risk assessments (Table 3-2). It is noted that the enforcement residue definition currently established in Regulation (EC) No 396/2005 comprises the parent compound dicamba only. Since a modification of the enforcement residue definition might have an impact on existing MRLs, EFSA proposes to implement the new enforcement residue definition in the framework of the comprehensive MRL review under Article 12 of the mentioned regulation. Thus, for the time being, the current enforcement residue definition is applicable. Table 3-2: Residue definitions for dicamba-tolerant soybeans Residue definitions for soybean 1 2 Dicamba Enforcement (Sum dicamba and its conjugates, expressed as dicamba; to be implemented in the framework of the MRL review under Article 12 of Regulation (EC) No 396/2005). Sum of DCSA and its conjugates, expressed as DCSA Risk assessment Sum dicamba, 5-OH-dicamba and their conjugates, expressed as dicamba Sum DCSA, DGSA and their conjugates, expressed as DCSA For all other plants the residue definitions remain unchanged as proposed in the conclusions of the peer review under Directive 91/414/EEC (EFSA. 2011): - Enforcement: dicamba and its conjugates, expressed as dicamba - Risk assessment: sum dicamba, 5-OH-dicamba and their conjugates, expressed as dicamba EFSA Journal 2013;11(10):

19 Magnitude of residues Modification of the MRL for dicamba in genetically modified soybean Twenty two residue trials conducted in the USA in the year 2008 in 15 different states were provided. It should be noted that the residue trials were performed on soybean varieties that contain the dmo expression cassette conferring tolerance against dicamba, stacked with a cp4 epsps cassette conferring tolerance against glyphosate. Considering that the cp4 epsps event is not expected to affect the dmo expression activity, EFSA considers the residue trials conducted on varieties containing both events, as relevant to address the residue levels in seeds produced from the variety MON and containing the single dmo expression cassette. Trials were conducted in compliance with the notified US GAP. Dicamba was applied on dicambatolerant soybean as pre-emergence at a dose of 1050 to 1150 g a.s./ha and post-emergence at growth stage BBCH 13 and BBCH at a dose rate of 510 to 580 g a.s./ha. Samples were collected 73 to 98 days after the last application and analysed for dicamba, 5-OH-dicamba, DCSA and DCGA. The results of the residue trials, the related risk assessment input values (STMR, HR) and the MRL proposals for the two residue definitions proposed by EFSA are summarized in Table 3-4. The residue trials confirm the findings of the metabolism study on dicamba-tolerant soybean. Only metabolites DCSA (up to mg/kg) and DCGA (up to mg/kg) were detected in dicambatolerant soybean, and dicamba and its metabolite 5-OH-dicamba are not relevant residues, since never detected above the LOQ. A median conversion factor for risk assessment of 1.7 was derived from these residue trials. The storage stability of dicamba and its metabolite 5-OH-dicamba was investigated in the DAR under Directive 91/414/EEC (Denmark, 2007). Residues of dicamba and 5-OH-dicamba were found to be stable at -17 C for up to 3 years in matrices with high water content (maize forage and fodder) and in high starch containing matrices (maize grain), but no information was provided for matrices with high oil content and on the stability of the metabolites DCSA and DCGA. Further information was therefore provided in the framework of this MRL application. Storage stability data for dicamba, DCSA and DCGA in soybean matrices were generated using the samples collected in the metabolism study conducted on dicamba-tolerant soybean and stored at ca. -20 C over several months. HPLC analyses on pre-emergence and post emergence samples were conducted a different time points, and compared to the initial analytical values. Stability data are summarised in the table 3-3 below. Table 3-3: Summary of quantitative storage stability analyses Matrix Forage (pre-emergence application) Hay (pre-emergence application) Hay (post-emergence application) Soybean seeds (post-emergence application) Analytes mg/kg dicamba equivalents Initial intermediate final Interval a 5 months 28 months 38 months Dicamba DCSA DCGA Interval a 1 month 28 months 37 months Dicamba DCSA DCGA Interval a 1 month 28 months 37 months Dicamba DCSA DCGA Interval a 6 months 26 months 35 months Dicamba DCSA DCGA (a): length of time (month) between the collection of the samples and HPLC analyses. EFSA Journal 2013;11(10):

20 These additional data confirm the stability of dicamba in high water containing matrices (soybean forage) and dry matrices (hay) up to ca. 3 years. However, the initial low residue concentration of dicamba in seeds close to the LOQ, do not allow to adequately assess the stability of dicamba in matrices with high oil content. Further stability data should therefore be provided. This data gap is of minor relevance for this MRL application, as no significant residues of dicamba are expected to be present in dicamba-tolerant soybean. DCSA is shown to be stable up to 3 years in the different soybean matrices, the residue levels measured at the different time points being comparable to initial values. In contrast, for DCGA, a significant decrease is observed at the final time point in hay. The stability of DCGA in dry matrices should be limited to 2 years only. As the soybean seeds collected in the residue trials performed in the USA were analysed 29 to 177 days after sampling, it is concluded that the residue data are valid with regard to storage stability data. Additional information to confirm the stability of dicamba in high oil containing matrices would be desirable. Validation data were provided for the analytical method used to generate the residue trial data. The fortification levels for DCSA, DCGA, dicamba and 5-OH-dicamba ranged from 0.01 mg/kg to 5 mg/kg. All mean recoveries were in the range of 74% to 111% with acceptable RSD below 20%. For DCGA, mean recoveries were however slightly above 120% at the highest fortification levels. No information was provided to confirm that the hydrolysis step (1N HCl, 95 C) included in the analytical method was fully appropriate to release and analyse conjugates. Although the hydrolysis conditions are rather extreme, confirmatory data should be provided to demonstrate that the analytical method used to generate the residues data is efficient in analysing the DCSA conjugates. EFSA concludes that the data are sufficient to confirm that residues of dicamba are not expected to be present in dicamba-tolerant soybean and therefore, there is no need to modify the current MRL of 0.05* mg/kg set for dicamba under Regulation (EC) No 396/2005 in conventional soybeans. In contrast, an MRL proposal of 0.4 mg/kg has been derived for the specific residue definition proposed for soybean (sum of DCSA and its conjugates, expressed as DCSA), to cover the use of dicamba on dicamba-tolerant soybean in the USA. EFSA Journal 2013;11(10):

21 Table 3-4: Overview of the available residues trials data Commodity Soybean (trials on dicamba tolerant soybean) Region (a) Outdoor /Indoor Individual trial results (mg/kg) Enforcement Risk assessment Median Residue & STMR (mg/kg) (b) Highest Residue & HR (mg/kg) (c) Enforcement residue definition: Sum of DCSA and its conjugates, expressed as DCSA Risk assessment residue definition: Sum of DCSA, DGSA and their conjugates, expressed as DCSA USA Outdoor 0.011, 0.011, 0.012, 0.013, 0.014, 0.018, 0.019, 0.021, 0.022, 0.026, 0.029, 0.038, 0.041, 0.045, 0.046, 0.051, 0.052, 0.068, 0.072, 0.087, 0.111, , 0.026, 0.024, 0.027, 0.029, 0.028, 0.029, 0.031, 0.036, 0.054, 0.044, 0.048, 0.083, 0.055, 0.069, 0.092, 0.094, 0.115, 0.136, 0.142, 0.161, MRL proposal (mg/kg) Median CF (d) Comments (e) R ber = 0.11 R max = 0.25 MRL OECD = 0.41/0.40 Enforcement residue definition: Sum of dicamba and its conjugates expressed as dicamba (proposed in the peer review, EFSA, 2011) Risk assessment residue definition: Sum of dicamba, 5-OH-dicamba and their conjugates, expressed as dicamba USA 22x < x <0.033 <0.013 <0.033 <0.013 < * - uses on dicamba tolerant soybean covered by the current MRL of 0.05* mg/kg (a): NEU (Northern and Central Europe), SEU (Southern Europe and Mediterranean), EU (i.e. outdoor use) or Import (country code). (b): Median Residue: value of the individual trial results according to the enforcement residue definition. STMR: Supervised Trial Median Residue. Median value of the individual trial results according to the residue definition for risk assessment (values underlined in the table) (c): Highest Residue: value of the individual trial results according to the enforcement residue definition and risk assessment residue definition (between brackets). HR: Highest Residue value of the individual trial results according to residue definition for risk assessment (values underlined in the table). (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. (e): Statistical calculation of the MRLs according to the EU methodolgy (Rber, Rmax; EC, 1997g) and unrounded/rounded values according to the OECD methodology (OECD, 2011) (*): Indicates that the MRL is set at the limit of analytical quantification. EFSA Journal 2013;11(10):

22 Effect of industrial processing and/or household preparation Modification of the MRL for dicamba in genetically modified soybean Studies on the effect of processing on the nature of dicamba residues were not reported in the DAR (Denmark, 2007) and no information was provided in the framework of this application. Two studies investigating the effect of processing in the magnitude of DCSA and DCGA residues on processed soybean products were submitted in support of this MRL application (Denmark, 2013). Soybean seeds collected in two US trials from plots treated with an exaggerated dose rate of 3360 g a.s./ha (1.5N) were processed and the different fractions analyzed for DCSA and DCGA. The results are summarized in the table 3-5 below. Indicative processing factors and conversion factors for risk assessment were calculated for each processed fractions. Table 3-5: Summary of the available processing studies on soybean Processed fractions USA (Nebraska, 2008) USA (Wisconsin, 2008) Residues (a) (mg/kg) PF Residues (a) (mg/kg) CF DCSA DCGA Total (DCSA) DCSA DCGA total Soybean seed (RAC) PF (DCSA) Hulls Meal (toasted, defatted) Oil (degummed) <0.01 <0.01 <0.02 < <0.01 <0.01 <0.02 < Oil (refined) <0.01 <0.01 <0.02 < <0.01 <0.01 <0.02 < Crude lecithin <0.01 <0.01 <0.02 < < Flour (deflated) Protein isolate <0.01 <0.01 <0.02 < Protein concentrate <0.01 <0.01 <0.02 < Soymilk <0.01 <0.01 <0.02 < Tofu <0.01 <0.01 <0.02 < < (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. DCSA and DGSA follow the same behaviour under processing and the individual processing factors calculated for DCGA are similar to the values calculated for DCGA and listed in the table 3-5 above. A concentration was observed for DCSA and DCGA in the hulls, meal and flour while transfer in the other fractions was calculated to be less than 20%. A mean processing factor of 1.35 for soya meal is proposed for the calculation the animal burden (see section 3.2) EFSA proposes not to include the derived indicative processing factors for soybean products in Annex VI of Regulation (EC) No 396/2005, since only 2 studies were provided Rotational crops Preliminary considerations As the use supported in the framework of this application is authorised outside the European Union, possible occurrence of residues in rotational crops is not considered relevant for the European consumer exposure and the possible residues of dicamba in rotational crops were not considered. CF EFSA Journal 2013;11(10):

23 3.2. Nature and magnitude of residues in livestock Modification of the MRL for dicamba in genetically modified soybean Since the use of dicamba on genetically modified soybean does not require a modification of the current MRL which is established at the limit of quantification (0.05 mg/kg), there is no need to assess the expected residues of dicamba in products of animal origin resulting from feed produced from genetically modified soybean (MON 87708) are not considered in the framework of this application. The MRLs for dicamba currently into force at EU level for animal products and listed under Regulation (EC) No 396/2005 apply. Therefore, only the possible transfer of DCSA and DCGA residues in animal matrices was assessed Dietary burden of livestock The dietary burden for livestock was calculated using the agreed European methodology (EC, 1996). The input values for the dietary burden calculation were selected according to the latest FAO recommendations (FAO, 2009) considering the livestock intake from soybean meal only, as DCSA and DCGA are deemed not to be present as residues on conventional crops on which the use of dicamba is authorized in the EU and for which existing EU MRLs have been proposed above the LOQ under Regulation (EC) No 396/2005. The input values for the dietary burden calculation are summarized in Table 3-6 and the results of the dietary burden calculations in Table 3-7 hereafter. Table 3-6: Input values for the dietary burden calculation Commodity Median dietary burden Input value (mg/kg) Comment Risk assessment residue definition: sum DCSA, DCGA expressed as DCSA Soybean meal (from dicamba-tolerant soybean) (0.051*1.35) Maximum dietary burden Input value (mg/kg) STMR*PF (0.051*1.35) Comment STMR*PF Table 3-7: Results of the dietary burden calculation Maximum dietary burden (mg/kg bw/d) Median dietary burden (mg/kg bw/d) Highest contributing commodity Risk assessment residue definition: (DCSA + DCGA expressed as DCSA) Max dietary burden (mg/kg DM) (a) Trigger exceeded (Y/N) Dairy ruminants Soybean Meal N Meat ruminants Soybean Meal N Poultry Soybean Meal N Pigs Soybean Meal N (a): Dry matter feed The calculated dietary burden indicated that the trigger value of 0.1 mg/kg dry matter (DM) was not exceeded, therefore further considerations regarding residues in livestock and the setting of MRLs in commodities of animal origin are not necessary Nature of residues The metabolism in livestock of dicamba was considered in the DAR prepared under Directive 91/414/EEC (Denmark, 2007) and in the conclusion of the peer review (EFSA, 2011). Metabolism studies in lactating goat, lactating cow and hen using 14 C-dicamba and feeding studies on cow using dicamba and 5-OH-dicamba were assessed. These evaluations are however not relevant considering the use of dicamba on dicamba-tolerant-soybean, since livestock fed with dicamba-tolerant soybean will not be exposed to dicamba and 5-OH-dicamba residues, but to DCSA and DCGA. EFSA Journal 2013;11(10):

24 It should be noted that the animal metabolism studies have shown dicamba (free and conjugated) to be the major component of the residues accounting for more than 50% TRR in all animal matrices. However, DCSA was also identified as a significant metabolite in ruminant kidney and liver, up to 21% TRR. Nonetheless, the conclusion of the peer review was not to include DCSA in the animal residue definition since low residues of DCSA are expected to be present in ruminant liver and kidney, leading to insignificant contribution to the consumer intakes (<0.2% ADI). The residue definition for enforcement and risk assessment was therefore proposed as dicamba alone and the applicant was required to provide a validated analytical method to enforce dicamba in animal matrices Magnitude of residues Based on the supported use on dicamba-tolerant soybean, the setting of MRL for DCSA in animal matrices is not required. 4. Consumer risk assessment As the metabolism studies and the supervised residue trials have shown dicamba and 5-OH-dicamba not to be the relevant components of the residues in dicamba-tolerant soybean and since no changes have been proposed to the current MRLs listed for dicamba under Regulation (EC) No 396/2005 (see appendix C), the consumer risk assessment was conducted for DCSA and DCGA only. The assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). This exposure assessment model contains the relevant European food consumption data for different sub-groups of the EU population 16 (EFSA, 2007). For the calculation of the chronic exposure, EFSA used the median residue value of mg/kg as derived from the residue trials on soybean (see Table 3-2) and taking into account DCSA and DCGA residues in seeds. No values were considered for the remaining commodities of plant and animal origin, as DCSA and DCGA are not expected to be present in conventional plants for which authorisations have been granted in EU. The acute exposure assessment was performed assuming the consumption of a large portion of the food item as reported in the national food surveys and containing residues at the highest level as observed in supervised field trials (0.533 mg/kg). In addition, a variability factor accounting for the inhomogeneous distribution on the individual items consumed was included in the calculation. The input values used for the dietary exposure calculation are summarized in Table 4-1. Table 4-1: Input values for the consumer dietary exposure assessment Commodity Chronic exposure assessment Input value (mg/kg) Comment Acute exposure assessment Input value (mg/kg) Comment Risk assessment residue definition: sum DCSA + DCGA expressed as DCSA Soybean seeds STMR HR The estimated exposure was then compared with the toxicological reference value derived for DCSA (see Table 2-6). The results of the intake calculation are presented in Appendix B to this reasoned opinion. 16 The calculation of the long-term exposure (chronic exposure) is based on the mean consumption data representative for 22 national diets collected from MS surveys plus 1 regional and 4 cluster diets from the WHO GEMS Food database; for the acute exposure assessment the most critical large portion consumption data from 19 national diets collected from MS surveys is used. The complete list of diets incorporated in EFSA PRIMo is given in its reference section (EFSA, 2007). EFSA Journal 2013;11(10):

25 No long-term consumer intake concerns were identified for any of the European diets incorporated in the EFSA PRIMo. All calculated chronic intake values resulting from the contribution of DCSA and DCGA residues in soya seeds were less than 0.1 % of the ADI. No acute consumer risk was identified in relation to the MRL proposal for soya seeds. The calculated maximum exposure in percentage of the ARfD was 0.4 %, considering the maximum residue level observed seeds. Consequently EFSA concludes that the intended use of dicamba on dicamba-tolerant soybean will not result in a consumer exposure exceeding the toxicological reference values and therefore, will not pose a public health concern. EFSA Journal 2013;11(10):

26 CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The toxicological profile of dicamba was assessed in the framework of the peer review under Directive 91/414/EEC and the data were sufficient to propose an ADI of 0.3 mg/kg bw per day and an ARfD of 0.3 mg/kg bw. It was concluded that these toxicological reference values are also applicable to its major metabolite 5-OH-dicamba in conventional plants. Since the additional plant metabolism study provided in the framework of this MRL application has shown the metabolites 3,6-dichloro-salicylic acid (DCSA) and 3,6-dichloro-gentisic acid (DCGA) to be the major components of the residues in dicamba-tolerant soybean, further toxicological studies to assess the toxicological relevance of these metabolites were provided. The data were sufficient to propose an ADI of 0.04 mg/kg per day and an ARfD of 0.3 mg/kg for DCSA, these values being also applicable to DCGA. The metabolism of dicamba in conventional crops was investigated in wheat, sugar cane, soybean and cotton. From these studies, the peer review established the residue definition for risk assessment as "sum of dicamba and 5-OH-dicamba and their conjugates expressed as dicamba" and as "dicamba and its conjugates" for enforcement. It is noted that the current residue definition for enforcement in Regulation (EC) No 396/2005 is limited to dicamba only. These residue definitions are not applicable to the use of dicamba on dicamba-tolerant soybean, as the metabolism pattern of the active substance in genetically modified plants was shown to be different. In addition, as the available data do not allow to conclude whether dicamba and DCSA act through the same toxicological mode of action, EFSA proposes to set the following additional residue definition for soybean, in order to cover the use of dicamba on dicamba-tolerant soybean: Enforcement Risk assessment Sum of DCSA and its conjugates, expressed as DCSA Sum of DCSA, DGSA and their conjugates, expressed as DCSA For the other plants and plant groups for which no dicamba-tolerant plants have been developed, the existing residue definitions remain applicable. A total of 22 supervised residue trials conducted in the USA on dicamba-tolerant soybean according to the reported US GAP were provided and considered sufficient to derive an import tolerance proposal of 0.4 mg/kg for DCSA and its conjugates. As dicamba and its metabolite 5-OH-dicamba were not detected in the trials conducted on dicamba-tolerant soybean, no change is proposed to the current MRL value set for dicamba at 0.05* mg/kg under Regulation (EC) No 396/2005. Confirmation is however required that the analytical method used to generate the residue data and the method proposed for enforcement purpose, are efficient in analysing the DCSA conjugates included in the residue definitions. Studies on the nature of DCSA and DCGA residues in processed commodities were not provided. Two studies conducted in the USA investigating the effect of processing on the magnitude of DCSA and DCGA residues in processed soybean products were submitted. Provisional processing factors were derived for different processed fractions. The residues of dicamba in rotational crops were not investigated as the current MRL application refers to the use of dicamba in the USA. Since soybean meal is used as feed product, a potential carry-over of DCSA and DCGA residues into food of animal origin has to be assessed. The expected livestock dietary exposure to DCSA and DCGA was calculated to be below the trigger value of 0.1 mg/kg (dry matter) for all relevant animals. Residues in products of animal origin are therefore not expected to occur and no MRLs were proposed for DCSA or DCGA in animal matrices. The consumer risk assessment was conducted for the proposed risk assessment residue definition (sum of DCSA, DCGA and their conjugates expressed as DCSA) using the revision 2 of the EFSA Pesticide EFSA Journal 2013;11(10):

27 Residue Intake Model (PRIMo) and the STMR and HR values derived from the supervised residue trials. No long term or short term intake concerns were identified. The TMDI was calculated to be less than 0.1% of the ADI for all European diets incorporated in the EFSA PRIMo, and the IESTI only 0.4% of the ARfD. Consequently, EFSA concludes that the occurrence of DCSA residues (including its conjugates) at the proposed MRL of 0.4 mg/kg on imported soybean will not result in a consumer exposure leading to a consumer health risk. Thus EFSA proposes to include a new entry as reported in the summary table, in Regulation (EC) No 396/2005 in order to cover the use of dicamba in the USA on dicamba-tolerant soybean. Summary table Code number (a) Commodity Existing EU MRL (mg/kg) Proposed EU MRL (mg/kg) Justification for the proposal Enforcement residue definition: DCSA (3,6-dichloro-salicylic acid) and its conjugates, expressed as DCSA Soya bean The import tolerance proposal is sufficiently supported by data and no risk for consumers was identified for the intended uses. Confirmation is required that the analytical method used to generate the residue data and the method proposed for enforcement purpose cover the conjugates of DCSA. Enforcement residue definition: dicamba (according Reg. (EC) No 396/2005) Soya bean 0.05* No change No change, as dicamba is not a relevant component of the residues in dicamba-tolerant soybean. (a): According to Annex I of Regulation (EC) No 396/2005. (*): Indicates that the MRL is set at the limit of analytical quantification. REFERENCES Denmark, Draft assessment report on the active substance dicamba prepared by the rapporteur Member State Denmark in the framework of Council Directive 91/414/EEC, February Denmark, Addendum to section B.7 in DAR for dicamba, September Denmark, Toxicological assessment rapport of: 3,6-dichlorosalicylic acid and 3,6- dichlorogentisic acid prepared by the evaluating Member State Denmark under Article 8 of Regulation (EC) No 396/2005, November 2011, 83 pp. Denmark, Evaluation report on the setting of MRL for dicamba in genmodified soybean prepared by the evaluating Member State Denmark under Article 8 of Regulation (EC) No 396/2005, 15 November 2011 updated July 2013, 37 pp. EC (European Commission), Appendix G. Livestock Feeding Studies. 7031/VI/95-rev.4. EC (European Commission), 1997a. Appendix A. Metabolism and distribution in plants. 7028/IV/95- rev.3. EC (European Commission), 1997b. Appendix B. General recommendations for the design, preparation and realisation of residue trials. Annex 2. Classification of (minor) crops not listed in the Appendix of Council Directive 90/642/EEC. 7029/VI/95-rev.6. EC (European Commission), 1997c. Appendix C. Testing of plant protection products in rotational crops. 7524/VI/95-rev.2. EFSA Journal 2013;11(10):

28 EC (European Commission), 1997d. Appendix E. Processing studies. 7035/VI/95-rev.5. EC (European Commission), 1997e. Appendix F. Metabolism and distribution in domestic animals. 7030/VI/95-rev.3. EC (European Commission), 1997f. Appendix H. Storage stability of residue samples. 7032/VI/95- rev.5. EC (European Commission), 1997g. Appendix I. Calculation of maximum residue level and safety intervals. 7039/VI/95. EC (European Commission), Residue analytical methods. For pre-registration data requirement for Annex II (part A, section 4) and Annex III (part A, section 5 of Directive 91/414). SANCO/3029/99-rev.4. EC (European Commission), Review report for the active substance dicamba. Finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 14 March 2008 in view of the inclusion of dicamba in Annex I of Council Directive 91/414/EEC. SANCO/829/08- rev.2, 07 March 2008, 8 pp. EFSA (European Food Safety Authority), Pesticide Residues Intake Model for assessment of acute and chronic consumer exposure to pesticide residues-rev.2. EC (European Commission), 2010a. Classes to be used for the setting of EU pesticide Maximum Residue Levels (MRLs). SANCO 10634/2010 Rev. 0, finalised in the Standing Committee on the Food Chain and Animal Health at its meeting of March EC (European Commission), 2010b. Residue analytical methods. For post-registration control. SANCO/825/00-rev.8.1. EC (European Commission), Appendix D. Guidelines on comparability, extrapolation, group tolerances and data requirements for setting MRLs. 7525/VI/95-rev.9. EFSA (European Food Safety Authority), Conclusion on the peer review of the pesticide risk assessment of the active substance dicamba. EFSA Journal 2011; 9(1):1965, 52 pp. doi: /j.efsa EFSA (European Food Safety Authority), Scientific Opinion on application EFSA-GMO-NL for the placing on the market of the herbicide-tolerant genetically modified soybean MON for food and feed uses, import and processing under Regulation (EC) No 1829/2003, from Monsanto. EFSA Journal 2013;11(10):3355, 30 pp. doi: /j.efsa FAO (Food and Agriculture Organization of the United Nations), Submission and evaluation of pesticide residues data for the estimation of Maximum Residue Levels in food and feed. Pesticide Residues. 2 nd Ed. FAO Plant Production and Protection Paper 197, 264 pp. FAO (Food and Agriculture Organization of the United Nations), 2010a. Dicamba. In: Pesticide residues in food Evaluations. Part I. Residues. FAO Plant Production and Protection Paper 206, 151 pp. FAO (Food and Agriculture Organization of the United Nations), 2010b.. Dicamba. In: Pesticide residues in food Evaluations. Part II. Toxicology. World Health Organization. FAO (Food and Agriculture Organisation of the United Nations), Dicamba. In: Pesticide residues in food Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 211, 3 pp. FAO (Food and Agriculture Organisation of the United Nations), Report of the 45 th session of the codex committee on pesticide residues. Beijing, China, 6-11 May 2013, 140 pp. Meier U, Growth Stages of mono- and dicotyledonous plants. BBCH Monograph, 2nd Ed., Federal Biological Research Centre of Agriculture and Forest. Braunschweig, Germany. EFSA Journal 2013;11(10):

29 OECD (Organization for Economic Co-operation and Development), OECD MRL Calculator: spreadsheet for single data set and spreadsheet for multiple data set, 2 March In: Pesticide Publications/Publications on Pesticide Residues. EFSA Journal 2013;11(10):

30 Appendix A. GOOD AGRICULTURAL PRACTICES (GAPS) Crop and/or situation (a) Soybean (dicamba tolerant variety MON 87708) Member State or Country F G or I (b) Pest or group of pests controlled (c) Formulation Application Application rate per treatment type (d-f) conc. a.s. (i) method kind (f - h) growth stage & season (j) USA F Herbicide SL 480 spraying Pre-emergence & Post-emergence at BBCH 13 and BBCH number min-max (k) interval min max kg as/hl min-max Water L/ha min-max kg a.s./ha min-max PHI (days) (l) 90 Remarks (m) Remarks: (a) (b) (c) (d) (e) (f) (g) For crops, EU or other classifications, e.g.codex, should be used; where relevant, the use situation should be described (e.g. fumigation of a structure) Outdoor or field use (F), glasshouse application (G) or indoor application (I) e.g. biting and suckling insects, soil born insects, foliar fungi, weeds e.g. wettable powder (WP), emulsifiable concentrate (EC), granule (GR) GCPF Technical Monograph No 2, 4 th Ed., 1999 or other codes, e.g. OECD/CIPAC, should be used All abbreviations used must be explained Method, e.g. high volume spraying, low volume spraying, spreading, dusting, drench (h) (i) (j) (k) (l) (m) Kind, e.g. overall, broadcast, aerial spraying, row, individual plant, between the plants - type of equipment used must be indicated g/kg or g/l Growth stage at last treatment (Growth stages of mono-and dicotyledonous plants. BBCH Monograph, 2 nd Ed., 2001), including where relevant, information on season at time of application The minimum and maximum number of application possible under practical conditions of use must be provided PHI - minimum pre-harvest interval Remarks may include: Extent of use/economic importance/restrictions (i.e. feeding, grazing) EFSA Journal 2013;11(10):

31 Appendix B. PESTICIDE RESIDUES INTAKE MODEL (PRIMO) DCSA Status of the active substance: Code no. LOQ (mg/kg bw): proposed LOQ: Toxicological end points ADI (mg/kg bw/day): 0.04 ARfD (mg/kg bw): 0.3 Source of ADI: EMS (DK) Source of ARfD: EMS (DK) Year of evaluation: 2012 Year of evaluation: 2012 Prepare workbook for refined calculations Undo refined calculations Chronic risk assessment - refined calculations TMDI (range) in % of ADI minimum - maximum No of diets exceeding ADI: --- Highest calculated TMDI values in % of ADI MS Diet Highest contributor to MS diet (in % of ADI) Commodity / group of commodities 2nd contributor to MS diet (in % of ADI) Commodity / group of commodities 3rd contributor to MS diet (in % of ADI) Commodity / group of commodities 0.08 WHO Cluster diet F 0.1 Soya bean 0.08 WHO Cluster diet B 0.1 Soya bean 0.07 WHO cluster diet E 0.1 Soya bean 0.0 WHO cluster diet D 0.0 Soya bean 0.0 PT General population 0.0 Soya bean 0.0 WHO regional European diet 0.0 Soya bean 0.0 NL child 0.0 Soya bean 0.0 DE child 0.0 Soya bean 0.0 NL general 0.0 Soya bean 0.0 FI adult 0.0 Soya bean 0.0 IT kids/toddler 0.0 Soya bean 0.0 ES child 0.0 Soya bean 0.0 IT adult 0.0 Soya bean 0.0 ES adult 0.0 Soya bean 0.0 PL general population 0.0 Soya bean DK adult DK adult DK adult DK adult DK adult DK adult DK adult DK adult DK adult DK adult DK adult DK adult ptmrls at LOQ (in % of ADI) Conclusion: The estimated Theoretical Maximum Daily Intakes (TMDI), based on ptmrls were below the ADI. A long-term intake of residues of DCSA is unlikely to present a public health concern. EFSA Journal 2013;11(10):

32 Processed commodities Unprocessed commodities Modification of the MRL for dicamba in genetically modified soybean Acute risk assessment /children - refined calculations Acute risk assessment / adults / general population - refined calculations The acute risk assessment is based on the ARfD. For each commodity the calculation is based on the highest reported MS consumption per kg bw and the corresponding unit weight from the MS with the critical consumption. If no data on the unit weight was available from that MS an average European unit weight was used for the IESTI calculation. In the IESTI 1 calculation, the variability factors were 10, 7 or 5 (according to JMPR manual 2002), for lettuce a variability factor of 5 was used. In the IESTI 2 calculations, the variability factors of 10 and 7 were replaced by 5. For lettuce the calculation was performed with a variabilty factor of 3. Threshold MRL is the calculated residue level which would leads to an exposure equivalent to 100 % of the ARfD. No of commodities for which ARfD/ADI is exceeded (IESTI 1): No of commodities for which No of commodities for which No of commodities for which ARfD/ADI is --- ARfD/ADI is exceeded (IESTI 2): --- ARfD/ADI is exceeded (IESTI 1): --- exceeded (IESTI 2): --- IESTI 1 *) **) IESTI 2 *) **) IESTI 1 *) **) IESTI 2 *) **) Highest % of ARfD/ADI Commodities ptmrl/ threshold MRL (mg/kg) Highest % of ARfD/ADI Commodities ptmrl/ threshold MRL (mg/kg) Highest % of ARfD/ADI Commodities ptmrl/ threshold MRL (mg/kg) Highest % of ARfD/ADI Commodities ptmrl/ threshold MRL (mg/kg) 0.4 Soya bean / Soya bean / Soya bean / Soya bean / - No of critical MRLs (IESTI 1) --- No of critical MRLs (IESTI 2) --- No of commodities for which ARfD/ADI is exceeded: Highest % of ARfD/ADI Processed commodities No of commodities for which --- ARfD/ADI is exceeded: --- ***) ***) ptmrl/ ptmrl/ threshold MRL Highest % of Processed threshold MRL (mg/kg) ARfD/ADI commodities (mg/kg) *) The results of the IESTI calculations are reported for at least 5 commodities. If the ARfD is exceeded for more than 5 commodities, all IESTI values > 90% of ARfD are reported. **) ptmrl: provisional temporary MRL ***) ptmrl: provisional temporary MRL for unprocessed commodity Conclusion: For DCSA IESTI 1 and IESTI 2 were calculated for food commodities for which ptmrls were submitted and for which consumption data are available. No exceedance of the ARfD/ADI was identified for any unprocessed commodity. For processed commodities, no exceedance of the ARfD/ADI was identified. EFSA Journal 2013;11(10):

33 Appendix C. EXISTING EU MAXIMUM RESIDUE LIMITS (MRLS) FOR DICAMBA (Pesticides - Web Version - EU MRLs (File created on 24/09/ :13) Code number Groups and examples of individual products to which the MRLs apply dicamba FRUIT FRESH OR FROZEN; NUTS (i) Citrus fruit 0.05* Grapefruit (Shaddocks, pomelos, 0.05* sweeties, tangelo, ugli and other hybrids) Oranges (Bergamot, bitter 0.05* orange, chinotto and other hybrids) Lemons (Citron, lemon ) 0.05* Limes 0.05* Mandarins (Clementine, 0.05* tangerine and other hybrids) Others 0.05* (ii) Tree nuts (shelled or 0.05* unshelled) Almonds 0.05* Brazil nuts 0.05* Cashew nuts 0.05* Chestnuts 0.05* Coconuts 0.05* Hazelnuts (Filbert) 0.05* Macadamia 0.05* Pecans 0.05* Pine nuts 0.05* Pistachios 0.05* Walnuts 0.05* Others (iii) Pome fruit Apples (Crab apple) 0.05* Pears (Oriental pear) 0.05* Quinces 0.05* Medlar 0.05* Loquat 0.05* Others 0.05* (iv) Stone fruit 0.05* Apricots 0.05* Cherries (sweet cherries, sour 0.05* cherries) Peaches (Nectarines and similar 0.05* hybrids) Plums (Damson, greengage, 0.05* mirabelle) Others 0.05* (v) Berries & small fruit 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba (a) Table and wine grapes 0.05* Table grapes 0.05* Wine grapes 0.05* (b) Strawberries 0.05* (c) Cane fruit 0.05* Blackberries 0.05* Dewberries (Loganberries, 0.05* Boysenberries, and cloudberries) Raspberries (Wineberries ) 0.05* Others 0.05* (d) Other small fruit & berries 0.05* Blueberries (Bilberries 0.05* cowberries (red bilberries)) Cranberries 0.05* Currants (red, black and white) 0.05* Gooseberries (Including hybrids 0.05* with other ribes species) Rose hips 0.05* Mulberries (arbutus berry) 0.05* Azarole (mediteranean medlar) 0.05* Elderberries (Black chokeberry 0.05* (appleberry), mountain ash, azarole, buckthorn (sea sallowthorn), hawthorn, service berries, and other treeberries) Others 0.05* (vi) Miscellaneous fruit 0.05* (a) Edible peel 0.05* Dates 0.05* Figs 0.05* Table olives 0.05* Kumquats (Marumi kumquats, 0.05* nagami kumquats) Carambola (Bilimbi) 0.05* Persimmon 0.05* Jambolan (java plum) (Java apple 0.05* (water apple), pomerac, rose apple, Brazilean cherry (grumichama), Surinam cherry) Others 0.05* (b) Inedible peel, small 0.05* Kiwi 0.05* Lychee (Litchi) (Pulasan, 0.05* rambutan (hairy litchi)) Passion fruit 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba Prickly pear (cactus fruit) 0.05* Star apple 0.05* American persimmon (Virginia 0.05* kaki) (Black sapote, white sapote, green sapote, canistel (yellow sapote), and mammey sapote) Others 0.05* (c) Inedible peel, large 0.05* Avocados 0.05* Bananas (Dwarf banana, plantain, 0.05* apple banana) Mangoes 0.05* Papaya 0.05* Pomegranate 0.05* Cherimoya (Custard apple, sugar 0.05* apple (sweetsop), llama and other medium sized Annonaceae) Guava 0.05* Pineapples 0.05* Bread fruit (Jackfruit) 0.05* Durian 0.05* Soursop (guanabana) 0.05* Others 0.05* VEGETABLES FRESH OR FROZEN (i) Root and tuber vegetables 0.05* (a) Potatoes 0.05* (b) Tropical root and tuber 0.05* vegetables Cassava (Dasheen, eddoe 0.05* (Japanese taro), tannia) Sweet potatoes 0.05* Yams (Potato bean (yam bean), 0.05* Mexican yam bean) Arrowroot 0.05* Others 0.05* (c) Other root and tuber 0.05* vegetables except sugar beet Beetroot 0.05* Carrots 0.05* Celeriac 0.05* Horseradish 0.05* Jerusalem artichokes 0.05* Parsnips 0.05* Parsley root 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba Radishes (Black radish, Japanese 0.05* radish, small radish and similar varieties) Salsify (Scorzonera, Spanish 0.05* salsify (Spanish oysterplant)) Swedes 0.05* Turnips 0.05* Others 0.05* (ii) Bulb vegetables 0.05* Garlic 0.05* Onions (Silverskin onions) 0.05* Shallots 0.05* Spring onions (Welsh onion and 0.05* similar varieties) Others 0.05* (iii) Fruiting vegetables (a) Solanacea 0.05* Tomatoes (Cherry tomatoes, ) 0.05* Peppers (Chilli peppers) 0.05* Aubergines (egg plants) (Pepino) 0.05* Okra, lady s fingers 0.05* Others 0.05* (b) Cucurbits - edible peel 0.05* Cucumbers 0.05* Gherkins 0.05* Courgettes (Summer squash, 0.05* marrow (patisson)) Others 0.05* (c) Cucurbits-inedible peel 0.05* Melons (Kiwano ) 0.05* Pumpkins (Winter squash) 0.05* Watermelons 0.05* Others 0.05* (d) Sweet corn (e) Other fruiting vegetables 0.05* (iv) Brassica vegetables 0.05* (a) Flowering brassica 0.05* Broccoli (Calabrese, Chinese 0.05* broccoli, Broccoli raab) Cauliflower 0.05* Others 0.05* (b) Head brassica 0.05* Brussels sprouts 0.05* Head cabbage (Pointed head cabbage, red cabbage, savoy 0.05* EFSA Journal 2013;11(10):

34 Code number Groups and examples of individual products to which the MRLs apply dicamba cabbage, white cabbage) Others 0.05* (c) Leafy brassica 0.05* Chinese cabbage (Indian 0.05* (Chinese) mustard, pak choi, Chinese flat cabbage (tai goo choi), peking cabbage (pe-tsai), cow cabbage) Kale (Borecole (curly kale), 0.05* collards) Others 0.05* (d) Kohlrabi 0.05* (v) Leaf vegetables & fresh herbs 0.05* (a) Lettuce and other salad plants 0.05* including Brassicacea Lamb s lettuce (Italian cornsalad) 0.05* Lettuce (Head lettuce, lollo rosso 0.05* (cutting lettuce), iceberg lettuce, romaine (cos) lettuce) Scarole (broad-leaf endive) (Wild 0.05* chicory, red-leaved chicory, radicchio, curld leave endive, sugar loaf) Cress 0.05* Land cress 0.05* Rocket, Rucola (Wild rocket) 0.05* Red mustard 0.05* Leaves and sprouts of Brassica 0.05* spp (Mizuna) Others 0.05* (b) Spinach & similar (leaves) 0.05* Spinach (New Zealand spinach, 0.05* turnip greens (turnip tops)) Purslane (Winter purslane 0.05* (miner s lettuce), garden purslane, common purslane, sorrel, glassworth) Beet leaves (chard) (Leaves of 0.05* beetroot) Others 0.05* (c) Vine leaves (grape leaves) 0.05* (d) Water cress 0.05* (e) Witloof 0.05* (f) Herbs 0.05* Chervil 0.05* Chives 0.05* Celery leaves (fennel leaves, Coriander leaves, dill leaves, Caraway leaves, lovage, angelica, sweet cisely and other Apiacea) 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba Parsley 0.05* Sage (Winter savory, summer 0.05* savory, ) Rosemary 0.05* Thyme ( marjoram, oregano) 0.05* Basil (Balm leaves, mint, 0.05* peppermint) Bay leaves (laurel) 0.05* Tarragon (Hyssop) 0.05* Others 0.05* (vi) Legume vegetables (fresh) 0.05* Beans (with pods) (Green bean 0.05* (french beans, snap beans), scarlet runner bean, slicing bean, yardlong beans) Beans (without pods) (Broad 0.05* beans, Flageolets, jack bean, lima bean, cowpea) Peas (with pods) (Mangetout 0.05* (sugar peas)) Peas (without pods) (Garden pea, 0.05* green pea, chickpea) Lentils 0.05* Others 0.05* (vii) Stem vegetables (fresh) Asparagus Cardoons 0.05* Celery 0.05* Fennel 0.05* Globe artichokes 0.05* Leek 0.05* Rhubarb 0.05* Bamboo shoots 0.05* Palm hearts 0.05* Others 0.05* (viii) Fungi 0.05* Cultivated (Common mushroom, 0.05* Oyster mushroom, Shi-take) Wild (Chanterelle, Truffle, Morel 0.05*,) Others 0.05* (ix) Sea weeds 0.05* PULSES, DRY 0.05* Beans (Broad beans, navy beans, 0.05* flageolets, jack beans, lima beans, field beans, cowpeas) Lentils 0.05* Peas (Chickpeas, field peas, 0.05* chickling vetch) Lupins 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba Others 0.05* OILSEEDS AND 0.05* OILFRUITS (i) Oilseeds 0.05* Linseed 0.05* Peanuts 0.05* Poppy seed 0.05* Sesame seed 0.05* Sunflower seed 0.05* Rape seed (Bird rapeseed, turnip 0.05* rape) Soya bean 0.05* Mustard seed 0.05* Cotton seed 0.05* Pumpkin seeds 0.05* Safflower 0.05* Borage 0.05* Gold of pleasure 0.05* Hempseed 0.05* Castor bean 0.05* Others 0.05* (ii) Oilfruits 0.05* Olives for oil production 0.05* Palm nuts (palmoil kernels) 0.05* Palmfruit 0.05* Kapok 0.05* Others 0.05* CEREALS Barley Buckwheat Maize Millet (Foxtail millet, teff) Oats Rice Rye Sorghum Wheat (Spelt Triticale) Others TEA, COFFEE, HERBAL 0.05* INFUSIONS AND COCOA (i) Tea (dried leaves and stalks, 0.05* fermented or otherwise of Camellia sinensis) (ii) Coffee beans 0.05* (iii) Herbal infusions (dried) 0.05* (a) Flowers 0.05* Camomille flowers 0.05* Hybiscus flowers 0.05* Rose petals 0.05* Jasmine flowers 0.05* Code number Groups and examples of individual products to which the MRLs apply dicamba Lime (linden) 0.05* Others 0.05* (b) Leaves 0.05* Strawberry leaves 0.05* Rooibos leaves 0.05* Maté 0.05* Others 0.05* (c) Roots 0.05* Valerian root 0.05* Ginseng root 0.05* Others 0.05* (d) Other herbal infusions 0.05* (iv) Cocoa (fermented beans) 0.05* (v) Carob (st johns bread) 0.05* HOPS (dried), including hop 0.05* pellets and unconcentrated powder SPICES 0.05* (i) Seeds 0.05* Anise 0.05* Black caraway 0.05* Celery seed (Lovage seed) 0.05* Coriander seed 0.05* Cumin seed 0.05* Dill seed 0.05* Fennel seed 0.05* Fenugreek 0.05* Nutmeg 0.05* Others 0.05* (ii) Fruits and berries 0.05* Allspice 0.05* Anise pepper (Japan pepper) 0.05* Caraway 0.05* Cardamom 0.05* Juniper berries 0.05* Pepper, black and white (Long 0.05* pepper, pink pepper) Vanilla pods 0.05* Tamarind 0.05* Others 0.05* (iii) Bark 0.05* Cinnamon (Cassia ) 0.05* Others 0.05* (iv) Roots or rhizome 0.05* Liquorice 0.05* Ginger 0.05* Turmeric (Curcuma) 0.05* Horseradish 0.05* Others 0.05* EFSA Journal 2013;11(10):

35 Code number Groups and examples of individual products to which the MRLs apply dicamba (v) Buds 0.05* Cloves 0.05* Capers 0.05* Others 0.05* (vi) Flower stigma 0.05* Saffron 0.05* Others 0.05* (vii) Aril 0.05* Mace 0.05* Others 0.05* SUGAR PLANTS Sugar beet (root) 0.05* Sugar cane Chicory roots 0.05* Others 0.05* PRODUCTS OF ANIMAL ORIGIN-TERRESTRIAL ANIMALS (i) Meat, preparations of meat, offals, blood, animal fats fresh chilled or frozen, salted, in brine, dried or smoked or processed as flours or meals other processed products such as sausages and food preparations based on these (a) Swine Meat 0.05* Fat free of lean meat Liver 0.7 Code number Groups and examples of individual products to which the MRLs apply dicamba Kidney Edible offal Others 0.05* (b) Bovine Meat Fat Liver Kidney Edible offal Others (c) Sheep Meat 0.05* Fat Liver Kidney Edible offal Others 0.05* (d) Goat Meat 0.05* Fat Liver Kidney Edible offal Others 0.05* (e) Horses, asses, mules or hinnies Meat 0.05* Fat Liver 0.7 Code number Groups and examples of individual products to which the MRLs apply dicamba Kidney Edible offal Others 0.05* (f) Poultry -chicken, geese, duck, turkey and Guinea fowl-, ostrich, pigeon Meat Fat Liver Kidney Edible offal Others 0.05* (g) Other farm animals (Rabbit, Kangaroo) Meat 0.05* Fat Liver Kidney Edible offal Others 0.05* (ii) Milk and cream, not concentrated, nor containing added sugar or sweetening matter, butter and other fats derived from milk, cheese and curd Cattle Sheep Goat 0.2 Code number Groups and examples of individual products to which the MRLs apply dicamba Horse Others (iii) Birds eggs, fresh preserved 0.05* or cooked Shelled eggs and egg yolks fresh, dried, cooked by steaming or boiling in water, moulded, frozen or otherwise preserved whether or not containing added sugar or sweetening matter Chicken 0.05* Duck 0.05* Goose 0.05* Quail 0.05* Others 0.05* (iv) Honey (Royal jelly, pollen) 0.05* (v) Amphibians and reptiles 0.05* (Frog legs, crocodiles) (vi) Snails 0.05* (vii) Other terrestrial animal products 0.05* (*) Indicates lower limit of analytical determination EFSA Journal 2013;11(10):

36 Appendix D. LIST OF METABOLITES AND RELATED STRUCTURAL FORMULA Common name IUPAC name Structure 5-OH-dicamba 2,5-dichloro-3-hydroxy-6-methoxybenzoic acid DCSA (3,6-dichlorosalicylic Acid) 3,6-dichloro-2-hydroxybenzoic acid DCGA (3,6-dichlorogentisic Acid) 3,6-dichloro-2,5-dihydroxybenzoic acid o-anisic acid 2-methoxybenzoic acid Vanillic acid 4-hydroxy-3-methoxybenzoic acid Syringic Acid 4-hydroxy-3,5-dimethoxybenzoic acid Ferulic acid 3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid Sinapic acid 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2- enoic acid EFSA Journal 2013;11(10):