Reasoned opinion on the setting of MRLs for imazapyr in genetically modified soya bean and other oilseeds and in lentils 1

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1 EFSA Journal 2014;12(6):3743 REASONED OPINION Reasoned opinion on the setting of MRLs for imazapyr in genetically modified soya bean and other oilseeds and in lentils 1 ABSTRACT European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy In accordance with Article 6 of Regulation (EC) No 396/2005, the United Kingdom, hereafter referred to as the evaluating Member State (EMS), received an import tolerance application from Exponent International Ltd., on behalf of BASF Corporation, to set MRLs for the use of the active substance imazapyr in genetically modified soya bean grown in Brazil, imidazolinone-tolerant lentils, rape and mustard plants grown in Canada and sunflowers in Argentina. The United Kingdom proposed to set MRLs of 5 mg/kg in soya bean, 0.08 mg/kg in sunflower seed, 0.05 mg/kg in rape seed and mustard seed and 0.2 mg/kg in lentils. The United Kingdom drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005 which was submitted to the European Commission and forwarded to EFSA. According to EFSA the data are sufficient to derive MRL proposals of 5 mg/kg on soya bean, 0.08 mg/kg on sunflower seed, the limit of quantification (LOQ) of 0.05 mg/kg on rape and mustard seed and 0.3 mg/kg on lentils. In the countries of origin the MRLs are set at 3 mg/kg on soya bean, 0.05 mg/kg on sunflower, rape and mustard seed and 0.2 mg/kg on lentils. Adequate analytical enforcement methods are available to control the residues of imazapyr in the crops under consideration. Based on the risk assessment results, EFSA concludes that the proposed uses of imazapyr in soya bean, sunflower seed, rape seed, mustard seed and lentils will not result in a consumer exposure exceeding the toxicological reference value and therefore are unlikely to pose a consumer health risk. European Food Safety Authority, 2014 KEY WORDS imazapyr, oilseeds and lentils, MRL application, Regulation (EC) No 396/2005, consumer risk assessment, imidazolinone herbicide, imidazolinone-tolerant crops 1 On request from the European Commission, Question No EFSA-Q , approved on 23 June Correspondence: pesticides.mrl@efsa.europa.eu Suggested citation: EFSA (European Food Safety Authority), Reasoned opinion on the setting of MRLs for imazapyr in genetically modified soya bean and other oilseeds and in lentils. EFSA Journal 2014;12(6):3743, 31 pp. doi: /j.efsa Available online: European Food Safety Authority, 2014

2 SUMMARY In accordance with Article 6 of Regulation (EC) No 396/2005, the United Kingdom, hereafter referred to as the evaluating Member State (EMS), received an import tolerance application from Exponent International Ltd., on behalf of BASF Corporation, to set MRLs for the use of the active substance imazapyr in genetically modified soya bean grown in Brazil, imidazolinone-tolerant lentils, rape and mustard plants grown in Canada and sunflowers in Argentina. The United Kingdom proposed to set MRLs of 5 mg/kg in soya bean, 0.08 mg/kg in sunflower seed, 0.05 mg/kg in rape seed and mustard seed and 0.2 mg/kg in lentils. The United Kingdom drafted an evaluation report in accordance with Article 8 of Regulation (EC) No 396/2005 which was submitted to the European Commission and forwarded to EFSA on 30 January EFSA bases its assessment on the evaluation report and the response to the EFSA request for additional information, the original data (dossier) submitted by the applicant, the comments received during the Member State consultation of the draft reasoned opinion and the JMPR evaluation report. The toxicological profile of imazapyr was assessed in the framework of the import tolerance application. From these data, the United Kingdom proposed an ADI of 2.5 mg/kg bw per day, whereas no ARfD was deemed necessary. The proposal was accepted by EFSA and by Member States during the consultation. The metabolism of imazapyr in primary crops was investigated in genetically modified soya bean and imidazolinone-tolerant maize labelled in the pyridine ring after foliar application. From these studies the EMS proposed to establish the residue definition for enforcement and risk assessment as imazapyr. EFSA agrees with the proposed residue definitions for the crops under consideration (group of pulses and oilseeds). EFSA concludes that the submitted supervised residue trials are sufficient to derive MRL proposals of 5 mg/kg on soya bean, 0.08 mg/kg on sunflower seed, the limit of quantification (LOQ) of 0.05 mg/kg on rape and mustard seed and 0.3 mg/kg on lentils. In the countries of origin the MRLs are set at 3 mg/kg on soya bean, 0.05 mg/kg on sunflower, rape and mustard seed and 0.2 mg/kg on lentils. The validity of the residue data on rape seeds with regard to storage stability should be confirmed. Adequate analytical enforcement methods are available to control the residues of imazapyr in the crops under consideration at the validated LOQ of 0.01 mg/kg. Imazapyr was found to be hydrolytically stable under processing conditions representative of pasteurisation, boiling/cooking and sterilisation. The applicant submitted processing studies in genetically modified soya beans. The data demonstrated that imazapyr residues are not transferred to the oil, but remained in the soya bean meal. The data were sufficient to derive the following processing factors, which are recommended to be included in Annex VI of Regulation (EC) No 396/2005. Soya bean, oil: <0.01 Soya bean, meal: 1.3 The residues of imazapyr in rotational crops are of no relevance for the import tolerance application. Oilseeds and/or their by products are used as feed products. Since the calculated dietary burdens exceeded the trigger value of 0.1 mg/kg dry matter (DM) for all livestock species, the metabolism in livestock and the potential carry-over into food of animal origin was assessed. Based on the submitted metabolism studies on goats and hens, the EMS proposed to establish the residue definition for enforcement and for risk assessment as imazapyr. Taking into account the results from the metabolism studies, which were conducted at significantly higher dose rates, and the expected livestock dietary burdens, the setting of MRLs above the LOQ in products of animal origin is not required in the framework of the current import tolerance application. EFSA Journal 2014;12(6):3743 2

3 The consumer risk assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). For the calculation of the chronic exposure EFSA used the median residue values as derived from the residue trials on oilseeds and lentils and the LOQ value for tissues of animal origin. For the remaining products of plant and animal origin the MRL of 0.01 mg/kg was used as input value. No long-term consumer intake concerns were identified for any of the European diets incorporated in the EFSA PRIMo. The total calculated intake accounted for up to 0.05 % of the ADI (WHO Cluster diet B). No acute consumer exposure assessment was performed, as an ARfD has not been proposed for the active substance. EFSA concludes that the proposed uses of imazapyr in soya bean, sunflower seed, rape seed, mustard seed and lentils will not result in a consumer exposure exceeding the toxicological reference value and therefore are unlikely to pose a consumer health risk. Thus EFSA proposes to amend the existing MRLs as reported in the summary table. SUMMARY TABLE Code Commodity Existing EU number (a) MRL (mg/kg) Enforcement residue definition: imazapyr Lentils Proposed EU MRL (mg/kg) Justification for the proposal 0.3 or 0.2 The import tolerance request is sufficiently supported by data and no consumer intake concerns were identified for the use of imazapyr on imidazolinonetolerant lentils. Two MRL options are proposed to risk managers: 0.3 mg/kg, derived with OECD methodology or 0.2 mg/kg, obtained using the previous EU methodology and corresponding to the MRL established in Canada, Sunflower seed 0.08 or 0.05 The import tolerance request is sufficiently supported by data and no consumer intake concerns were identified for the use of imazapyr on imidazolinonetolerant oilseeds. Two MRL options are proposed to risk managers: 0.08 mg/kg 0.01*mg/kg (default MRL derived with OECD methodology or 0.05 mg/kg as established in the country according to of origin Rape seed Art. 18(1)(b)) 0.05* The import tolerance request is sufficiently supported by data and no consumer intake concerns were identified for the use of imazapyr on imidazolinonetolerant oilseeds. The validity of the residue data on rape seed with regard to storage stability should be confirmed Soya bean 5 or 3 The import tolerance request is sufficiently supported by data and no consumer intake concerns were identified for the use of imazapyr on genetically modified soya bean BPS-CV Two MRL options are proposed to risk managers: 5 mg/kg derived with OECD methodology or 3 mg/kg as established in the country of origin. EFSA Journal 2014;12(6):3743 3

4 Code Commodity Existing EU number (a) MRL (mg/kg) Setting of MRLs for imazapyr in several oilseeds and in lentils Proposed EU MRL (mg/kg) Justification for the proposal Mustard seed 0.05* The import tolerance request, which is extrapolated from data on rape seed, is sufficiently supported by data and no consumer intake concerns were identified for the use of imazapyr on imidazolinonetolerant mustard seed plants Tissue 0.05* The reported uses of imazapyr on oilseeds Milk 0.01* lead to a dietary exposure exceeding the trigger value in all animal species Bird eggs 0.01* However, residues above the LOQ are not expected in products of animal origin. (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 2014;12(6):3743 4

5 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 5 Background... 6 Terms of reference... 6 The active substance and its use pattern... 7 Assessment Method of analysis Methods for enforcement of residues in food of plant origin Methods for enforcement of residues in food of animal origin Mammalian toxicology Absorption, Distribution, Excretion and Metabolism (Toxicokinetics) Acute toxicity Short term toxicity Genotoxicity Long term toxicity Reproductive toxicity Neurotoxicity Further toxicological studies Medical data Acceptable daily intake (ADI) and acute reference dose (ARfD) 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 Appendices Appendix A. Good Agricultural Practice (GAPs) Appendix B. Pesticide Residue Intake Model (PRIMo) Abbreviations EFSA Journal 2014;12(6):3743 5

6 BACKGROUND Regulation (EC) No 396/ establishes the rules governing the setting of pesticide MRLs at European Union level. Article 6 of that Regulation lays down that any party having a legitimate interest or requesting an authorisation for the use of a plant protection product in accordance with Council Directive 91/414/EEC 4, repealed by Regulation (EC) No 1107/2009 5, shall submit to a Member State, when appropriate, an application to set an import tolerance in accordance with the provisions of Article 7 of that Regulation. The United Kingdom, hereafter referred to as the evaluating Member State (EMS), received from the company Exponent International Ltd. 6, on behalf of BASF Corporation, an application to set import tolerances for the use of the active substance imazapyr in genetically modified soya bean, imidazolinone-tolerant lentils, rape and mustard plants grown in Canada and sunflowers in Argentina. This application was notified to the European Commission and EFSA and was subsequently evaluated by the EMS in accordance with Article 8 of the Regulation. After completion, the evaluation report was submitted to the European Commission who forwarded the application, the evaluation report and the supporting dossier to EFSA on 30 January The application was included in the EFSA Register of Questions with the reference number EFSA-Q and the following subject: Imazapyr - Application to set new MRLs in soya bean, lentil and oilseeds (except peanut and soya bean). The United Kingdom proposed to set MRLs of 5 mg/kg in soya bean, 0.08 mg/kg in sunflower seed, 0.05 mg/kg in rape seed and mustard seed and 0.2 mg/kg in lentils. On 4 April 2014 some data requirements were identified, which prevented EFSA to conclude on the consumer risk assessment. The EMS provided a response addressing those data gaps (United Kingdom, 2014), which was taken into consideration by EFSA on 6 May 2014 for the finalization of this reasoned opinion. On 28 May 2014 the draft of the reasoned opinion was circulated to the Member State experts for consultation; comments were received from Germany and were considered for the finalisation of this reasoned opinion. EFSA proceeded with the assessment of the application and the evaluation report as required by Article 10 of the Regulation. 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 3 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 Exponent International Ltd., The Lenz, Hornbeam Park, Harrogate, HG2 8RE, North Yorkshire, United Kingdom. EFSA Journal 2014;12(6):3743 6

7 requests supplementary information, the time limit laid down shall be suspended until that information has been provided. In this particular case the revised deadline for providing the reasoned opinion is 3 June THE ACTIVE SUBSTANCE AND ITS USE PATTERN Imazapyr is the ISO common name for 2-[(RS)-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl] nicotinic acid (IUPAC). The chemical structure of the compound is reported below. Molecular weight: g/mol Imazapyr (CL 243,997) is a systemic herbicide belonging to the imidazolinone chemical group. It is used pre- and post emergence against broadleaf weeds. Imazapyr inhibits the enzyme acetohydroxyacid synthase (AHAS) which is essential for the production of branched-chain amino acids. Imazapyr is an active substance for which no dossier has been submitted at EU level for approval under Regulation (EC) No 1107/ Hence, no peer review has been performed at EU level. No MRLs are established for imazapyr at EU level and according to Article 18(1)(b) of Regulation (EC) No 396/2005 the default LOQ of 0.01 mg/kg applies to all products. No CXLs are established yet for imazapyr. However, Codex Alimentarius has recently assessed the use of the active substance in few crops, including the crops under consideration, and animal origin commodities. The following Codex MRL were recommended: 0.05 mg/kg (LOQ) on rape seed, 0.08 mg/kg on sunflower seed, 0.3 mg/kg on lentils, 0.05 mg/kg (LOQ) on meat (from mammalians other than marine mammalians), mammalian fats (except milk fat) and edible offal (mammalian), 0.01 mg/kg (LOQ) on milks and meat, fats, edible offal and eggs of poultry. Since no GAP was available, no recommendation was given for soya bean (JMPR, 2013). The MRL/tolerance established in the countries of origin for imazapyr are 3 mg/kg in soya bean in Brazil 8, 0.05 mg/kg in sunflower seed in Argentina 9, 0.05 mg/kg in rape seed and mustard seed, 0.2 mg/kg in dry lentils in Canada 10. The details of the GAPs reported to be authorised in the countries of origin are given in Appendix A. The notified GAPs for imazapyr on soya bean refer to a genetically modified soya bean variety (code BPS-CV127-9) containing the csr1-2 gene which confers tolerance to imidazolinone herbicides. At EU level, EFSA GMO Panel has assessed the application regarding the authorisation of the genetically 7 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 Resolution RE No 1.315, of 16 April Federal Official Gazette of Brazil (DOU) No 73, 4th feira, 17 April 2013, p National Service for Agrifood Health and Quality of Argentina (SANASA) Direction of agrochemicals and biologicals. Maximum residue limit (MRL) database (March 2014). Imazapyr. Avaialble online: 10 Pest Management Regulatory Agency (PMRA) - Health Canada. Maximum residue limit (MRL) database for MRLs regulated under the Pest Control Products Act. Imazapyr. Avaialble online: EFSA Journal 2014;12(6):3743 7

8 modified soya bean BPS-CV127-9 for food and feed uses, import and processing in accordance with Regulation (EC) No 1829/ (EFSA Panel GMO, 2014). The notified GAPs for imazapyr on lentils and the oilseeds under consideration (except soya bean) refer to plant hybrids which have been developed through mutagenesis and conventional breeding to exhibit tolerance to the imidazolinone (IMI) family of herbicides. No novel DNA has been introduced to achieve the herbicide tolerance. Tolerance to imidazolinones has been shown to be the result of a point mutation in the acetohydroxyacid synthase (AHAS) large subunit gene (ahasl), which alters the binding of the IMI herbicides on the AHAS enzyme (United Kingdom, 2014). 11 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 EFSA Journal 2014;12(6):3743 8

9 ASSESSMENT EFSA bases its assessment on the evaluation report (United Kingdom, 2013) and the response to the EFSA request for additional information (United Kingdom, 2014) submitted by the EMS, the original data (dossier) submitted by the applicant and the JMPR evaluation report (FAO, 2013). The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation and the Authorisation of Plant Protection Products adopted by Commission Regulation (EU) No 546/ and the currently applicable guidance documents relevant for the consumer risk assessment of pesticide residues (EC, 1996, 1997a-g, 2000, 2010a,b, 2011; OECD, 2011). 1. Method of analysis 1.1. Methods for enforcement of residues in food of plant origin Two analytical methods for the determination of imazapyr residues in the crops under consideration were assessed by the EMS in the evaluation report (United Kingdom, 2013). The first method is based on extraction of imazapyr with a solution of methanol/water/hydrochloric acid and further dilution with a solution of water and methanol (50:50, containing 0.1 % of formic acid). The extract was further analysed by liquid chromatography with mass tandem spectrometry detection (LC-MS/MS). The method was successfully validated for the enforcement of imazapyr residues in soya bean seed (including its processed fractions oil, flaked soya bean, meal and toasted meal) and sunflower seed (high oil content matrices) at the LOQ of 0.01 mg/kg. An ILV conducted in soya bean matrices is available (United Kingdom, 2013). The second method is based on extraction of imazapyr with a solution of methanol/water/formic acid or water/formic acid and final determination by LC-MS/MS. The method was successfully validated in lentils (dry matrix) at the LOQ of 0.05 mg/kg. An ILV was not performed (United Kingdom, 2013). The multi-residue QuEChERS method in combination with HPLC-MS/MS as described by European Committee for Standardization (CEN, 2008) is applicable for the determination of imazapyr in dry commodities with an LOQ of 0.01 mg/kg (EURL, online). Since the commodities under consideration belong to the group of high oil content and dry commodities, EFSA concludes that sufficiently validated analytical methods for enforcing the proposed MRLs for imazapyr in the soya bean, sunflower seed, rape seed, mustard seed and lentils are available Methods for enforcement of residues in food of animal origin Analytical methods for the determination of imazapyr residues in commodities of animal origin were not assessed in the evaluation report as residues are not expected in animal origin commodities (Germany, 2013). According to JMPR analytical methods for the determination of imazapyr residues using capillary electrophoresis (CE) with UV absorption detection have been validated in bovine milk and tissues at the LOQ of 0.01 mg/kg for milk and milk fat and 0.05 mg/kg for muscle, liver, kidney and fat. No information on the determination of imazapyr residues in eggs was reported (FAO, 2013). Hence, there are indications that imazapyr can be enforced in commodities of animal origin with an LOQ of 0.05 mg/kg and 0.01 mg/kg in tissues and milk, respectively. However, a detailed evaluation of the analytical method (CE-UV) for the determination of the residues of imazapyr in animal origin commodities is desirable. An enforcement method for the egg matrix is missing and is required. 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 EFSA Journal 2014;12(6):3743 9

10 2. Mammalian toxicology Setting of MRLs for imazapyr in several oilseeds and in lentils EFSA bases it assessment on the evaluation report prepared by the EMS in the framework of the current application (United Kingdom, 2013) Absorption, Distribution, Excretion and Metabolism (Toxicokinetics) After oral administration to rats, imazapyr was rapidly and extensively absorbed, minimally metabolised and excreted mainly in urine. Less than 1 % of the administered dose was detected in the carcass, with no evidence of accumulation. Oxidative cleavage of the imidazolinone ring is the metabolic reaction that forms nicotinic acid derivatives, the minor metabolites identified in rat urine Acute toxicity Imazapyr was of low acute oral, dermal and inhalation toxicity. In rabbits, it was not a skin irritant but a severe eye irritant, producing persistent corneal opacity and slight/moderate corneal vascularisation. According to the Regulation (EC) No 1272/ , imazapyr is currently classified as Eye Irrit 2, H319 - Causes serious eye irritation. However, due to the irreversible nature of the effects, the classification Eye Dam.1 (H318 Causes serious eye damage) should be considered. No skin sensitisation study was provided in the dossier, and it is not considered necessary for the purpose of this evaluation. Table 2-1: Summary of the acute toxicity studies with imazapyr Type of test/ Species Purity of test substance Results Acceptability of the study Reference (ID No) Acute oral rat 99.5 % LD 50 >2500 mg/kg bw (both sexes) Yes IZ Acute oral rat 99.5% LD 50 >5000 mg/kg bw Yes IZ Acute dermal rat 95.9 % LD 50 >2000 mg/kg bw Yes IZ Acute dermal rabbit 99.5 % LD 50 >2000 mg/kg bw Yes IZ Acute inhalation rat 93 % LC 50 >1.3 mg/l Yes IZ Skin irritation rabbit 99.5 % Slight reversible irritation Yes IZ Eye irritation rabbit 99.5 % Severe eye irritant Yes IZ Short term toxicity The short term oral toxicity of imazapyr was investigated in rats and in dogs. In the first 90-day rat study, an increased relative kidney weight (111 % of control group) observed in females at the high dose (816 mg/kg bw per day) was not considered as toxicologically significant when considering the magnitude of the effect and the absence of histopathological correlates or effects on clinical chemistry parameters. It is noted that medullary cysts in the kidney were also observed in the same group (high dose females). Therefore, a lower NOAEL of 450 mg/kg bw per day is proposed by EFSA. In the second 90-day rat study with higher dose levels, the same effects were observed (increased relative kidney weight in females and medullary cysts in the kidneys in males) and no NOAEL can be derived (<1350 mg/kg bw per day). In the 90-day and 1-year dog studies, no adverse effects were observed up to the highest dose tested. The short term dermal toxicity was investigated in rabbits where an inflammatory cell infiltrate and changes in the haematological values were observed at the high dose only. 13 Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. OJ L 353, , p EFSA Journal 2014;12(6):

11 Table 2-2: Summary of the short term toxicity studies Setting of MRLs for imazapyr in several oilseeds and in lentils Type of test/ Species (purity of the test substance) Dose levels (ppm in the diet) NOAEL (mg/kg bw per day) Effects at LOAEL Acceptability of the study Reference (ID No) 90-day oral/ rat (97.2 %) (5000 ppm, f) Increased relative kidney weight and medullary cysts in the kidneys (f) Yes IZ day oral/ rat (99.3 %) <1350 (15000 ppm) Increased relative kidney weight (f) and medullary cysts in the kidneys (m) Yes IZ day oral/ dog (isopropylamine salt as 50 % aqueous solution) a (10000 ppm) No adverse effects Yes IZ year oral/ dog (99.5 %) (10000 ppm) No adverse effects Yes IZ day dermal/ rabbit (93 %) mg/kg bw per day (topical) 200 Decreased haematocrit and mean corpuscular volume (a) Dose of isopropylamine salt of imazapyr, corrected for water content. (f): female; (m): male. Yes IZ Genotoxicity Imazapyr has been investigated in a battery of in vitro and in vivo studies. Negative results were obtained in an Ames test. A mammalian cell mutation assay in vitro gave equivocal results due to high levels of variation but no dose response relationship was observed. An in vitro clastogenicity assay also provided equivocal results due to an increase in the proportion of aberrant cells at an intermediate concentration. The results of an unscheduled DNA synthesis (UDS) assay in hepatocytes in vitro are also considered inconclusive due to high variability. No evidence of a dominant lethal effect was seen in rats following administration of dose levels up to 1000 mg/kg bw per day; however the top dose was not considered to be sufficiently high. Even though the data in vitro show a general lack of consistency, as underlined by Germany during the Member State consultation, there is no evidence of clastogenicity or aneugenicity at a limit dose of 2000 mg/kg bw in an acceptable mouse micronucleus assay (even though evidence was missing that the test compound had reached the target organ). Supported by the absence of carcinogenic potential and limited evidence of reproductive toxicity, it can be concluded that impazapyr is not a genotoxic compound in vivo. Table 2-3: Summary of the genotoxicity studies Test substance (batch and purity) Test system Concentrations/ dose Results Acceptability of the study Reference (ID No) In vitro studies Imazapyr ( , 93 %) Ames µg/plate (+ and S9) Negative Yes IZ EFSA Journal 2014;12(6):

12 Test substance (batch and purity) Imazapyr ( , 93 %) Imazapyr ( , 93 %) Imazapyr ( , 93 %) In vivo studies Imazapyr (FH12861, 99.1 %) Test system Chinese hamster ovary (CHO) cells /HGPRT locus Chinese hamster ovary (CHO) cells /cytogenetics Rat hepatocyte /UDS Mouse bone marrow erythrocytes /micronucleus test Concentrations/ dose 125 to 2500 µg/ml (-S9) 250 to 5000 µg/ml (+S9) 50 to 5000 µg/ml (+ and S9) Results Equivocal (no concentrationresponse relationship) Equivocal (no concentrationresponse relationship) 50 to 5000 µg/ml Inconclusive (high variability within groups) mg/kg bw (oral) Acceptability of the study Yes Yes Yes Reference (ID No) IZ IZ IZ Negative Yes 2006/ Long term toxicity The chronic toxicity and carcinogenicity of imazapyr has been investigated in rats and mice. In the long term rat study, there is no clear evidence for a substance-related increase in adrenal tumours (adrenal medulla, in females) or for a substance-related increase in thyroid tumours (C-cell tumours, in males). No classification is proposed for carcinogenic properties. A decreased survival was observed in males at the high dose but the survival of females was not affected. In the long term mouse study, an increased incidence of renal cysts in males at the high dose, not accompanied by microscopic findings, was not considered treatment-related. Table 2-4: Summary of the long term toxicity studies Type of test/ Species (purity of the test substance) Dose levels (mg/kg bw per day) NOAEL (mg/kg bw per day) Effects at LOAEL Acceptability of the study Reference (ID No) 2-year diet/ rat (99.5 %) 19-month diet/ mouse (99. 5%) Reduced survival (males) Yes IZ No adverse effects Yes IZ Reproductive toxicity One two-generation study in rats and two developmental toxicity studies (in rats and rabbits) are available. In the multigenerational rat study, no adverse effects were observed upon the reproductive parameters or the offspring development. In the developmental toxicity study in rats, an increased post-implantation loss was observed but no foetal findings. No treatment-related effects were apparent in the rabbit developmental toxicity study. No classification is proposed for reproductive and developmental toxicity. EFSA Journal 2014;12(6):

13 Table 2-5: Summary of the reproductive toxicity studies Setting of MRLs for imazapyr in several oilseeds and in lentils Species (purity of the test substance) Multigenerational Rat (99.5 %) Developmental Rat (93 %) Rabbit (93 %) Dose levels (ppm in the diet) mg/kg bw per d (gavage) mg/kg bw per d (gavage) NOAEL (mg/kg bw per day) - Parental: Offspring: Reproductive: 738 (10000 ppm) - Maternal: Developmental: Maternal: Developmental: 400 Effects at LOAEL Acceptability of the study Reference No adverse effects Yes IZ Mat: salivation Dev: increased post-implantation loss Yes IZ No adverse effects Yes IZ Neurotoxicity No data or specific study were submitted or required. There were no indications of neurotoxicity in the available studies Further toxicological studies No further studies were submitted Medical data Clinical cases and incidents of poisoning have not been reported. Further medical data are not considered relevant for this assessment Acceptable daily intake (ADI) and acute reference dose (ARfD) The derivation of the ADI is based on the 1-year dog study (NOAEL of 250 mg/kg bw per day) supported by the 2-year rat study (NOAEL of 253 mg/kg bw per day), with the application of an uncertainty factor of 100. The clinical observations in the acute oral toxicity study with rats and in the developmental toxicity studies were attributed to an irritant response at high doses, and not considered relevant for a dietary ARfD. In the absence of relevant acute toxicity findings in the available studies, the derivation of an ARfD is not considered necessary for imazapyr. Table 2-6: Overview of the toxicological reference values Imazapyr Source Year Value Study relied upon Safety factor ADI UK mg/kg bw per day Dog, 1 yr study and rat, 2-yr study ARfD UK 2013 Not necessary. 100 EFSA Journal 2014;12(6):

14 The toxicological reference values proposed by the EMS have been accepted by EFSA and by Member States during the consultation process. It is noted that JMPR derived an ADI of 3 mg/kg bw per day; the setting of an ARfD was considered not necessary (FAO, 2013). 3. Residues 3.1. Nature and magnitude of residues in plant Primary crops Nature of residues The metabolism of imazapyr in primary crops was investigated in herbicide resistant soya bean and maize using 14 C-pyridine labelled imazapyr after foliar application. The studies were submitted under the current import tolerance application and assessed by the EMS (United Kingdom, 2013, 2014). These studies were also assessed by JMPR (FAO, 2013). The overview of the metabolism study designs is presented in the table below. Table 3-1: Summary of available metabolism studies in plants Group Crop Application details Pulses and oilseeds Cereals Soya bean (BPS-CV127-9) (b) Maize (pioneer hybrid 3245-IR) (b) Method, Rate No Sampling Remarks F or G (a) Spraying, G Spraying, F kg a.s./ha (at BBCH 65) kg a.s./ha (at BBCH 13) 0.08 kg a.s./ha (at BBCH 13) 1 1h (forage), 35 (hay), 98 (straw, pod, seed) DAT (a): Outdoor/field application (F) or glasshouse/protected crops/indoor application (G). (b): Crops which exhibit tolerance to imidazolinone herbicides. 1 0 and 14 (green plant), 30 and 62 (forage), 114 (grain, fodder) DAT Samples: forage, hay, straw, pod, seed. Samples: green plant, forage, stalk, husk cob with grain. In genetically modified soya bean BPS-CV127-9, the total radioactive residues (TRR) after foliar application of imazapyr (corresponding to 1.3 times the reported critical use on soya bean and sunflower seed and 12 times the reported use on rape seed, mustard seed and lentils) were 0.64, 0.25, 0.08, 0.15 and 0.07 mg eq./kg in forage, hay, straw, pod and seed, respectively. The extractability ranged from 99 to 78 % of the TRR in all matrices, except pods (73 % of the TRR). Imazapyr was metabolised to a number of components, however, the unchanged parent compound was detected in all matrices and was the most abundant component of the residue in soya been seeds (34 % of the TRR or 0.02 mg/kg), hay (37 % of the TRR or 0.09 mg/kg) and forage (93.6 % of the TRR or 0.6 mg/kg). A polar fraction M3 exceeded 10 % of the TRR in hay, straw, pods and seeds. It represented the most abundant component of the residues in pods (30 % of the TRR or 0.04 mg/kg) and straw (12 % of the TRR or 0.01 mg/kg). Further analysis on soya bean seeds showed that the polar peak M3 consisted of multiple components, each present at less than mg/kg. The metabolite M19 accounted for 18 % of the TRR in straw, and 9 % of the TRR in hay, but was not detected in seeds and forage. Given the very absolute levels (0.013 mg/kg and 0.02 mg/kg in soya straw and hay) observed in the soya bean metabolism study conducted at an higher application rate and the lack of potential exposure for humans, M19 was not further characterised/identified. All other identified metabolites were present in low proportions and concentrations. EFSA Journal 2014;12(6):

15 In the maize pioneer hybrid 3245-IR, the total radioactive residues (TRR) after foliar application of imazapyr were higher in the green plant (2.5 and 8.7 mg a.s. eq./kg, depending on the applied dose rate). TRR was significantly lower in grain (0.09 and 0.03 mg a.s. eq./kg, depending on the applied dose rate). The extractability ranged from 79 to 96 % of the TRR in all matrices, except fodder (70 % of the TRR). Parent imazapyr represented the major component of the extractable residues in green plant, forage, fodder (12 to 81 % of extracted TRR) and in grain (41 % of the extracted TRR or 0.01 mg/kg). A number of minor components was identified but not further characterised, since none of them was equal or higher than 0.01 mg/kg in the 30 DAT to the 114 DAT samples. A similar metabolic pathway was observed in the two tested crops with imazapyr largely degraded into several metabolites, none of them present in significant amounts in the crops for human consumption. The EMS proposed to establish the enforcement and risk assessment residue definition as parent imazapyr for pulses and oilseeds (United Kingdom, 2013). It is noted that the metabolism of imazapyr on GM soya bean was investigated only with parent compound labelled in the pyridine ring. No data are available for imidazole-ring labelled active substance. Based on the low residues observed in the metabolism study (using pyridine label) in soya bean seed (TRR: 0.07 mg a.s. eq./kg; imazapyr: 0.02 mg/kg), significant degradation products would not be expected in commodity for human consumption. Since the residues of imazapyr observed in soya bean seeds in the supervised residue trials conducted at a lower rate were significantly higher (<0.05 to 3 mg/kg), the presence of imidazole derivatives in relevant amounts in soya bean food and feed terminal residues cannot be excluded and further investigations would be required. Meanwhile, in absence of any significant degradation product from the metabolism study on soya bean with imazapyr labelled at the pyridine ring, EFSA agrees to apply the proposed residue definitions as parent imazapyr to the crops under consideration (group of pulses and oilseeds) Magnitude of residues The results of residue trials conducted on imidazolinone-tolerant crops according to the critical GAP (± 25 % deviation in application rate) were selected to support the import tolerance request. A part of the trials on soya beans and sunflowers was conducted with a water-dispersible granule (WG) formulation instead of the soluble concentrate (SL) formulation foreseen in the cgap. Since the different type of formulation is not expected to have an impact on the residue level in the treated crops, especially if application occurs early post-emergence and PHI is more than 7 days (EC, 2011), these trials were considered as GAP-compliant. a. Soya bean Eighteen GAP-compliant residue trials on genetically modified soya bean (BPS-CV127-9 and equivalent varieties 14 ) were submitted. The trials were carried out during more than two seasons in different regions of Brazil. In eleven trials an adjuvant was added to the spray formulation; imazapyr residues ranged from <0.05 to 3 mg/kg, whereas the residues of the trials conducted without adjuvant ranged from 0.07 to 1.27 mg/kg. The data are sufficient to derive a MRL proposal of 5 mg/kg for the reported uses on GM soya bean BPS-CV127-9 in Brazil. The MRL in Brazil for imazapyr in this crop is 3 mg/kg. b. Sunflower seed Fifteen GAP-compliant residue trials on IMI-tolerant hybrids of sunflowers were submitted. The trials were carried out during more than two seasons in different regions of Argentina and Uruguay. Samples were collected between 81 to 111 days. The number of trials is sufficient to derive a MRL proposal of 0.08 mg/kg (corresponding to the recommended Codex MRL; FAO, 2013) for the reported 14 The varieties used in the residue trials (Cultivance CV 127, Cultivance CV L603, Cultivance L08, Cultivance BRZ and Cultivance BRZ 53841) were developed from the same genetic background (EFSA, 2013). EFSA Journal 2014;12(6):

16 uses on imidazolinone-tolerant sunflowers in Argentina. The MRL in Argentina for imazapyr in this crop is 0.05 mg/kg. c. Rape seed, mustard seed Ten GAP-compliant independent residue trials on IMI-tolerant hybrids of rapes were submitted. The trials were carried out during a single season (2008) in different regions of Canada. The extrapolation from residues in rape seed to mustard seed is foreseen in the EU (EFSA, 2011). The number of trials is sufficient to derive a MRL proposal of 0.05*mg/kg for the reported use on imidazolinone-tolerant oilseed rape and mustard in Canada. d. Lentils Four GAP-compliant residue trials on IMI-tolerant hybrids of lentils were submitted. The trials were carried out during a single season (2008) in different regions of Canada. Since lentils are classified as minor crops (EC, 2011), the number of trials is sufficient to derive a MRL proposal of 0.3 mg/kg according to the OECD methodology (corresponding to the recommended Codex MRL; FAO, 2013) for the reported use on imidazolinone-tolerant lentils in Canada. The MRL in Canada for imazapyr in this crop is 0.02 mg/kg. The results of the residue trials, the related risk assessment input values (highest residue, median residue) and the MRL proposals are summarised in Table 3-2. The degradation of imazapyr during storage was assessed in soya bean (seeds and processed products) and in maize (grains) in the framework of the current import tolerance application (United Kingdom, 2013). Imazapyr showed to be stable in soya bean samples stored frozen at -20 ºC for 10 months (seeds) or 3 months (processed products) and in maize samples (grains) stored frozen at -5 to -26 ºC for up to 27 months 15. The supervised residue trial samples were stored for up to 272 days (soya bean), 746 days (rape seed), 339 days (sunflower seed) and 5.5 months (lentils). Apart the results on rape seeds, the validity of the residue data submitted is supported by the storage stability in high oil content commodities and dry commodities (United Kingdom, 2013). Supportive stability summary data in soya bean for up to 44 months of a structurally related compound, imazamox 16, give some evidence that the imidazolinone class of compounds is sufficiently stable in high oil content commodities (United Kingdom, 2013). This assumption should be confirmed by submitting storage stability data to cover the whole period of storage of the samples of sunflower seeds from the supervised residue trials. According to the EMS, the analytical methods used to analyse the supervised residue trial samples have been sufficiently validated and were proven to be fit for the purpose (United Kingdom, 2013). EFSA concludes that the data are sufficient to derive a MRL proposal 5 mg/kg on soya bean, 0.08 mg/kg on sunflower seed, the LOQ of 0.05 mg/kg on rape and mustard seed and 0.3 mg/kg on lentils. In the countries of origin the MRLs are set at 3 mg/kg on soya bean, 0.05 mg/kg on sunflower rape and mustard seed and 0.2 mg/kg on lentils, 15 Although the study on maize was not fully in compliance with the EU guidance document (huge range of storage conditions; single level tested; lack of analysis immediately after sampling and of repetitions at each sampling), these data give an indication that imazapyr residues are stable in dry matrices for more than 2 years. 16 Imazamox (BAS 720 H or CL ). EFSA Journal 2014;12(6):

17 Table 3-2: Overview of the available residues trials data Commodity Residue region (a) Outdoor /Indoor Individual trial results (mg/kg) Enforcement (imazapyr) Risk assessment (imazapyr) Median residue (mg/kg) (b) Highest residue (mg/kg) (c) MRL proposal (mg/kg) Median CF (d) Comments (e) Enforcement residue definition: imazapyr Soya bean (BPS-CV127-9 or equivalent IMI-tolerant GM varieties) Import (Brazil) Outdoor <0.05; 0.07; 0.11; 0.35; 0.48; 0.55; 0.83 (f) ; 0.92; 1.03 (f) ; 1.27; 2 x 1.3; 1.4; 1.5 (f) ; 1.9; 2 (f) ; 2; 3 <0.05; 0.07; 0.11; 0.35; 00.48; 0.55; 0.83 (f) ; 0.92; 1.03 (f) ; 1.27; 2 x 1.3; 1.4; 1.5 (f) ; 1.9; 2 (f) ; 2; (g) 1 R ber = 3.2 R max = 3.06 MRL OECD = 4.29/5 Sunflower seed (IMI-tolerant varieties) Rape seed mustard seed (IMI-tolerant varieties) Lentils (IMI-tolerant varieties) Import (Argentina) Import (Canada) Import (Canada) Outdoor 10 x <0.01; 0.01; 2 x 0.03; 2 x < x <0.01; 0.01; 2 x 0.03; 2 x < (g) 1 R ber = 0.06 R max = 0.06 MRL OECD =0.077/0.08 Outdoor 10 x < x < * 1-1 R = 0.16 Outdoor 2 x 0.06; 2 x x 0.06; 2 x or 0.2 (h) ber R max = 0.13 MRL OECD = 0.21/0.3 (a): NEU (Northern and Central Europe), SEU (Southern Europe and Mediterranean), EU (i.e. indoor use) or Import (country code) (EC, 2011). (b): Median value of the individual trial results according to the enforcement residue definition. (c): Highest value of the individual trial results according to the enforcement residue definition. (d): The median conversion factor for enforcement to risk assessment is obtained by calculating the median of the individual conversion factors for each residue trial. (e): Statistical estimation of MRLs according to the EU methodology (R ber, R max ; EC, 1997g) and unrounded/rounded values according to the OECD methodology (OECD, 2011). (f): Highest value measured at a longer PHI (80 days) than the PHI of the intended GAP (60 d). (g): The MRLs in the country of origin are of 3 mg/kg and 0.05 mg/kg for soya bean and sunflower seed, respectively. (h): The MRL of 0.2 mg/kg is based on the previous EU methodology (R ber /R max ) and corresponds to the MRL established in the country of origin (Canada). The MRL of 0.3 mg/kg is derived using the OECD methodology. (*): Indicates that the MRL is set at the limit of analytical quantification. EFSA Journal 2014;12(6):

18 Effect of industrial processing and/or household preparation Setting of MRLs for imazapyr in several oilseeds and in lentils The effect of processing on the nature of imazapyr was investigated in studies performed at three test conditions representing pasteurisation, baking/brewing/boiling and sterilisation (20 minutes at 90 C, ph 4; 60 minutes at 100 C ph 5; 20 minutes at 120 C, ph 6). The studies were reported under the framework of this application (United Kingdom, 2013). The compound showed to be hydrolytically stable under the representative processing conditions. Thus, for oilseed processed commodities the same residue definition as for raw agricultural commodities (RAC) is applicable. Specific studies investigating the magnitude of imazapyr residues in processed commodities are not required as the total theoretical maximum daily intake (TMDI) is below the trigger value of 10 % of the ADI (EC, 1997d). Nevertheless, processing studies investigating the effect of processing on the magnitude of imazapyr residues in the processed soya beans BPS-CV127-9, IMI-tolerant sunflower and rape seeds were provided (United Kingdom, 2013). The soya bean and rape seed samples from studies conducted at exaggerated rates were processed to flakes, meal, toasted meal, defatted meal, pressecake and oil, simulating commercial processing. In the processing studies with sunflowers the crops were treated at about half of the reported maximum application rate. Various processing factors (PFs) were derived which are compiled in Table 3-3. A significant reduction of residues was observed in oilseed oil as imazapyr was not found (<LOQ) in any individual sample of the processed product. A residue concentration was observed in oilseed meal, regardless of the processing (toasted, defatted) and in press cake, whereas imazapyr residues did not concentrated in soya bean flakes. The overview of the results of the processing studies is reported in Table 3.3. The samples of one processing study on soya bean were stored prior to analysis for a period (up to 31 months) exceeding the period for which storage integrity was demonstrated (see Section ). Since the seed samples stored for 31 months were re-analysed immediately before processing and considering that a structurally analogous compound (imazamox) was stable for period up to 44 months in high oil content commodities (see Section ), the results of this processing study are acceptable. The samples from the remaining study were valid as per the storage stability. The analytical methods used to analyse the samples have been sufficiently validated and were proven to be fit for the purpose (United Kingdom, 2013). Table 3-3: Overview of the available processing studies Processed commodity Number of studies Median PF (a) Median CF (b) Individual PFs Enforcement residue definition: Imazapyr soya bean, flakes ; 0.9; 0.93; 1.56 soya bean, meal (c) ; 1.03 (c) ; 1.18; 1.22; 1.5; 1.83 soya bean, toasted meal ; 1.09 (c) ; 1.26 (c) ; 1.37; 1.39; 1.43 soya bean, defatted meal (c) ; 1.34 (c) soya bean, oil 6 < x <0.01; 2 x<0.01 (c) ; <0.06; <0.07 Sunflower, presscake meal Sunflower, refined oil 1 < Rape seed, presscake meal Rape seed, refined oil 1 < EFSA Journal 2014;12(6):

19 (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. (c): PFs derived from a study where samples of the seeds were stored for 16 months (defatted and toasted meal, oil) and 31 months (meal). EFSA recommends the inclusion of the derived processing factors of <0.01 for soya bean oil and of 1.3 for soya bean meal in Annex VI of Regulation (EC) No 396/ Rotational crops Preliminary considerations The residues of imazapyr in rotational crops are of no relevance for the import tolerance application Nature and magnitude of residues in livestock Since oilseeds and their by-products are normally fed to livestock, the nature and magnitude of imazapyr residues in livestock was assessed in the framework of this application (EC, 1996) Dietary burden of livestock The median and maximum 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 intake of oilseeds assessed in this import tolerance application (see Table 3-2). To account for the concentration of residues in oilseed meal the more critical PF of 1.3 was used for soya bean meal (see Table 3-3) and as default value for the other oilseeds. The input values for the dietary burden calculation are summarised in Table 3-4. The default processing factors have been added to the table in brackets. Table 3-4: Input values for the dietary burden calculation Commodity Median dietary burden Maximum dietary burden Input value (mg/kg) Risk assessment residue definition: Imazapyr Comment Input value (mg/kg) Comment Soya bean, seed 1.15 Median residue 1.15 Median residue Soya bean, meal 1.5 (1.15*1.3) Sunflower seed, meal (0.01*1.3) Rape seed, meal (0.05*1.3) Median residue*pf 1.3 N/A Not relevant Median residue*pf (1.3) N/A Not relevant Median residue*pf (1.3) N/A Not relevant The results of the dietary burden calculation are summarised in the following table. EFSA Journal 2014;12(6):