Reasoned opinion on the setting of a new MRL for isoprothiolane in rice 1

Transcription

1 EFSA Journal 2012;10(3):2607 Reasoned opinion on the setting of a new MRL for isoprothiolane in rice 1 European Food Safety Authority 2 European Food Safety Authority (EFSA), Parma, Italy SUMMARY According to Article 6 of the Regulation (EC) No 396/2005, the United Kingdom, herewith referred to as the rapportuer Member State (RMS), received an application from Exponent International Ltd. to set an import tolerance for isoprothiolane in rice. In order to accommodate the authorized use of isoprothiolane on rice in Japan, the RMS proposed to raise the existing MRL from 0.01* mg/kg to 5 mg/kg. The RMS United Kingdom 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 26 April On 12 May 2011 some data requirements were identified, which prevented EFSA to conclude on the consumer risk assessment. An updated evaluation report, addressing those data requirements, was submitted by the RMS on 23 August 2011 and taken into consideration by EFSA for finalization of this reasoned opinion. On 8 February 2012 a Member State consultation was launched on the draft of this reasoned opinion. By the end of the commenting period comments were received from the European Reference Laboratory for Pesticide Residues in Cereals and Feeding stuff (EURL CF), France, Germany, the United Kingdom and the Netherlands, and the comments were taken into consideration for finalizing this reasoned opinion. It is noted that isoprothiolane is an active substance not registered in Europe and the manufacturer does not intend to apply for an approval of isoprothiolane under Regulation (EC) No 1107/2009. Consequently, no EU assessment of the active substance has been performed. Therefore in the assessment of the current application EFSA relies solely on the evaluation report prepared by the RMS United Kingdom and the original studies provided by the manufacturer. The toxicological profile of isoprothiolane was assessed by the RMS and reviewed by EFSA. The data were sufficient to propose an ADI of 0.1 mg/kg bw/day and an ARfD of 0.12 mg/kg bw. The metabolism of isoprothiolane was investigated in rice after foliar application of isoprothiolane. The applied radioactivity generally stays stable along time and is mainly located in stem, leaves and rice hull, with lowest amounts recovered in rice grain. Large part of the radioactivity is incorporated in the natural constituents of rice plant. Isoprothiolane is the main residue in rice grain with all other metabolites being below the LOQ. EFSA identified some deficiencies in the metabolism study in rice, due to the low application rate which did not allow to identify traces of potentially formed 1 On request from the European Commission, Question No EFSA-Q , approved on 02 March Correspondence: pesticides.mrl@efsa.europa.eu Suggested citation: European Food Safety Authority; Reasoned opinion on the setting of a new MRL for isoprothiolane in rice. EFSA Journal 2012;10(3):2607. [29 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2012

2 metabolites. However, the potential contribution of isoprothiolane metabolites to the total consumer intake is covered by a wide safety margin of the calculated consumer exposure to isoprothiolane residues. EFSA concludes that the available metabolism study can be used to characterise the metabolism of isoprothiolane in rice when treated according to the authorised GAP. EFSA agrees with the proposal of the RMS to define parent isoprothiolane as a relevant residue for the risk assessment and enforcement purposes in rice. The submitted residue data are sufficient to derive an import tolerance of 5 mg/kg for the authorized use of isoprothiolane on rice in Japan. An adequate analytical method is available to enforce the proposed MRL of isoprothiolane in rice at the LOQ of 0.01 mg/kg. Studies investigating the effect of processing on the nature of isoprothiolane residues have not been submitted. Since rice is mainly consumed processed, studies investigating the nature of isoprothiolane under baking/boiling conditions are strongly recommended. Several studies investigating the effect of processing on the magnitude of isoprothiolane residues in processed rice were reported, but EFSA could not assess the validity of the submitted studies and therefore study results were not used to derive proposals for enforcement purposes. There are some indications that polishing reduces the concentration of isoprothiolane residues. For the consumer exposure the most conservative approach is used, assuming that no significant reduction of residues occur during processing. The residues of isoprothiolane in rotational crops are of no relevance for the import tolerance application. Rice is not considered as a livestock feed in Europe and therefore the nature and magnitude of isoprothiolane in commodities of animal origin was not investigated in the framework of the current application. The consumer risk assessment was performed with revision 2 of the EFSA PRIMo. For the calculation of the chronic exposure EFSA used the median residue value for rice as derived from the supervised field trials. For other food commodities of animal and plant origin the default MRL of 0.01 mg/kg was used as an input value. The acute exposure assessment was performed only with regard to rice. Rice is a bulked commodity and therefore the median residue value as derived from the supervised residue field trials was used as an 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 values ranged from 0.1 to 1.6% of the ADI (maximum for UK Infant diet). The contribution of residues in rice to the total consumer exposure accounted for a maximum of 1.2% of the ADI (PT general population diet). No acute consumer risk was identified in relation to the MRL proposal for rice. The calculated maximum exposure in percentage of the ARfD was 16.2% for rice. EFSA concludes that the import tolerance for rice supporting the authorized use of isoprothiolane in Japan will not result in a consumer exposure exceeding the toxicological reference values and therefore will not pose a public health concern. The recommendations of EFSA are compiled in the table below: EFSA Journal 2012;10(3):2607 2

3 Code Commodity Existing EU number a MRL (mg/kg) Proposed EU MRL (mg/kg) Justification for the proposal Proposed enforcement residue definition: Isoprothiolane Rice 0.01* 5 The import tolerance request is supported by residue data and no risk for consumers was identified. However, studies investigating the effect of processing on the nature of isoprothiolane residues are strongly recommended. The MRL proposal refers to brown rice (hulled rice). (*): Indicates that the MRL is set at the limit of analytical quantification. a according to Annex I of Regulation (EC) No 396/2005 KEY WORDS Isoprothiolane, import tolerance, rice, MRL application, Regulation (EC) No 396/2005, consumer risk assessment, fungicide and plant growth regulator EFSA Journal 2012;10(3):2607 3

4 TABLE OF CONTENTS Summary... 1 Table of contents... 4 Background... 5 Terms of reference... 5 The active substance and its use pattern... 6 Assessment Methods of analysis Methods for enforcement of residues in food of plant origin Methods for enforcement of residues in food of animal origin Mammalian toxicology 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 Consumer risk assessment Conclusions and recommendations References Appendix A. Good Agricultural Practices (GAPs) Appendix B. Pesticide Residues Intake Model (PRIMo) Abbreviations EFSA Journal 2012;10(3):2607 4

5 BACKGROUND Commission Regulation (EC) No 396/ establishes the rules governing the setting of pesticide MRLs at Community 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 1107/2009 5, 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 an application to set a new MRL for the active substance isoprothiolane in rice. This application was notified to the European Commission and EFSA and subsequently evaluated by the RMS in accordance with Article 8 of the Regulation. After completion, the evaluation report of the RMS was submitted to the European Commission who forwarded the application, the evaluation report and the supporting dossier to EFSA on 26 April The application was included in the EFSA Register of Questions with the reference number EFSA-Q and the following subject: Isoprothiolane - Application to set the MRL in rice. On 12 May 2011 some data requirements were identified, which prevented EFSA to conclude on the consumer risk assessment. An updated evaluation report, addressing those data requirements, was submitted by the RMS on 23 August 2011 and taken into consideration by EFSA for finalization of this reasoned opinion. On 8 February 2012 a Member State consultation was launched on the draft of this reasoned opinion. By the end of the commenting period on 29 February 2012, comments were received from European Reference Laboratory for Pesticide Residues in Cereals and Feeding stuff (EURL CF), France, Germany, the United Kingdom and the Netherlands and the comments were taken into consideration for finalizing this reasoned opinion. EFSA then proceeded with the assessment of the isoprothiolane residues in rice 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 regarding isoprothiolane residues in rice. 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 is 4 February Commission Regulation (EC) No 396/2005 of 23 February OJ L 70, , p Council Directive 91/414/EEC of 15 July 1991, OJ L 230, , p Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October OJ L 309, , p Exponent International Ltd., The Lenz, HG28RE, Harrogate, United Kingdom EFSA Journal 2012;10(3):2607 5

6 THE ACTIVE SUBSTANCE AND ITS USE PATTERN Isoprothiolane is the ISO common name for diisopropyl 1,3-dithiolan-2-ylidenemalonate (IUPAC). The chemical structure of the compound is herewith reported. Molecular weight: g/mol Isoprothiolane is a fungicide and a plant growth regulator. It is systemic with protective and curative action, absorbed by the leaves and roots, with translocation acropetally and basipetally. On rice plants it is used to control the following fungi: Pyricularia oryzae, Helminthosporium sigmoideum, Fusarium nivale. It is also used on rice to control Delphacidae as well as to accelerate the rooting. Isoprothiolane is an active substance which has not been approved in the EU and consequently no uses are registered in Europe. The manufacturer has not applied for the approval of isoprothiolane under Regulation (EC) No 1107/ Consequently, no EU assessment has been performed and no EFSA conclusion is available. Therefore in the assessment of the current application EFSA relies solely on the evaluation report prepared by the RMS United Kingdom and the original studies provided by the applicant Exponent International Ltd. According to Article 18(b) of Regulation (EC) No 396/2005 default MRLs at the LOQ of 0.01 mg/kg are currently applicable for isoprothiolane. Since JMPR has not been requested to evaluated isoprothiolane, no CXLs are set for this active substance. The authorised GAP of isoprothiolane on rice in Japan for which the import tolerance request of 5 mg/kg is made, is summarized in Appendix A. 7 Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October 2009, OJ L 309, , p.1-50 EFSA Journal 2012;10(3):2607 6

7 ASSESSMENT EFSA bases its assessment on the evaluation report submitted by the RMS and the dossier of the active substance as submitted by the applicant. The assessment is performed in accordance with the legal provisions of the Uniform Principles for the Evaluation of the Authorization of Plant Protection Products set out in 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, 2008, 2010, 2011; OECD, 2011a, 2011b). 1. Methods of analysis 1.1. Methods for enforcement of residues in food of plant origin Isoprothiolane residues in rice grain can be analysed with the QuEChERS method (Anastasiades et al, 2003). The validation of the method based on GC-MS/MS has been performed by the Danish National Food Institute (Denmark, 2010); the validation report is publicly available on the website of EU Reference Laboratory for Residues of pesticides The validation was performed as 5 replicates at three spiking levels: 0.01, 0.02 and 0.1 mg/kg. The mean recoveries for all spiking levels were within the acceptable range of % and the standard deviation was within the acceptable 20% level ( %). Thus, QuEChERS method is capable to determine isoprothiolane residues in rice at the lowest LOQ of 0.01 mg/kg. In the framework of the Member State consultation, the EURL for Pesticide Residues in Cereals and Feeding stuff submitted to EFSA an information that the EURL had organised a proficiency test where brown rice was spiked with isoprothiolane (assigned value 0.17 mg/kg). Most of the participants (38 out of 50 participating laboratories) used the QuEChERS method to analyse the samples. The overall results of the proficiency test demonstrated that the method is appropriate for enforcement practice. EFSA concludes that an adequate analytical method is available to determine isoprothiolane residues in rice with the LOQ of 0.01 mg/kg Methods for enforcement of residues in food of animal origin The analytical enforcement methods for the determination of isoprothiolane residues in commodities of animal origin were not assessed in the framework of the current application as MRLs for commodities of animal origin are not proposed. 2. Mammalian toxicology 2.1. Absorption, Distribution, Excretion and Metabolism (Toxicokinetics) Isoprothiolane is rapidly (peak blood levels at 1-2 hours post dosing) and extensively (about 80%) absorbed when orally administered to rats at a dose of 50 mg/kg bw. It is extensively distributed with highest levels of radioactivity in liver and kidney. Excretion is rapid and extensive, mainly in urine (50 70%), with ca % exhaled as CO 2. Isoprothiolane is extensively metabolised in rodents: in mice, a total of 20 metabolites were separated by TLC. Less than 5% of the radioactivity in urine and faeces was consistent with parent. Conjugated metabolites represented a small proportion of total 8 Commission Regulation (EU) No 546/2011 of 10 June 2011, OJ L 155, , p EFSA Journal 2012;10(3):2607 7

8 radioactivity. In vitro investigations with mouse liver slices showed that the initial stages of metabolism involved de-isopropylation and sulphoxidation. Subsequent reactions appear to include hydroxylation, removal of the carboxylate group and cleavage of the thiolane ring to give CO Acute toxicity Isoprothiolane database for assessing acute toxicity consists of one valid acute oral study and 2 further studies (oral and dermal) of a limited value. The LD50 was estimated to be between 300 and 2000 mg/kg; cut-off 1000 mg/kg bw, which classifies isoprothiolane in Category 4 in GHS. Neither inhalation studies nor studies to investigate the skin sensitisation potential of isoprothiolane were submitted. It did not show skin or eye irritation potential. Table 2-2. Summary of the acute toxicity studies Type of test/ Species Oral (acute toxic class) / Rat Test substance/ Purity of test substance Lot No ; 98.2% pure Results LD mg/kg bw (cut-off 1000 mg/kg bw) Acceptability of the study References Yes Oda S, 2010 Oral LD 50 / rat 98% LD mg/kg bw Supplementary Miyazaki T, 1970 Dermal LD 50 / rat 98% >10250 mg/kg bw Supplementary Miyazaki T, Short term toxicity Only one valid study was submitted to assess the short term toxicity potential of isoprothiolane: the 1- year toxicity study in dogs showed a NOAEL of 10 mg/kg bw/day, based on increased liver and thyroid weight and decreased body weight at 50 mg/kg bw/day. Table 2-3. Summary of the short term toxicity studies Type of test/ Species (purity of the test substance) Dose levels (mg/kg bw/day) NOAEL (mg/kg bw/day) Effects at LOAEL and higher doses (mg/kg bw/day) Acceptability of the study References 1 month Rat 97.1% Males: 0, 15, 38, 78, 225 & 357 Females 0, 16, 39, 77, 225 & 350 <15 increased liver & kidney wt (15 mg/kg bw/d) Supplementary (histopathology not performed) Yoshida, month Rat 97.1% Males: 0, 16, 75 & 350 Females: 0, 16, 79 & increased liver & kidney wt (75 mg/kg bw/d) Supplementary (histopathology not performed) Inui, week rat >99% Males: 0, 6, 17, 53 or 158 Females: 0, 7, 22, 62 or increased liver wt; decreased body wt (158 mg/kg bw/d) Supplementary (limited histopathology, no GLP) Miyazaki, 1972 EFSA Journal 2012;10(3):2607 8

9 Type of test/ Species (purity of the test substance) Dose levels (mg/kg bw/day) 90-day oral Rats Males: 0, 3.5, 20.5, Females: 0, 4, 23.4, (0, 50, 300, 3000 ppm) 4 week mouse 97.5% Males: 0, 13, 70, 322, 612 or 1079 Females: 0, 16, 74, 353, 688 or 1096 NOAEL (mg/kg bw/day) Effects at LOAEL and higher doses (mg/kg bw/day) 3.5 increase of relative weights of liver and kidneys of male rats, the increase of absolute weight of the liver of female rats, and the significantly high value of γ-gtp of male rats at 300 ppm 70 increased liver wt; decreased food consumption (322 mg/kg bw/d) Acceptability of the study References Yes Sunaga, 2006 * Supplementary (histopathology not performed) Yoshida, week mouse >99% 4 week dog Purity not given 1 year dog 97% 2 year dog 99.9% Males: 0, 15, 47, 132 or 472 Females: 0, 14, 47, 140 or 444 0, 10, 100, 1000 or ppm 47 decreased ovary wt (132mg/kg bw/d) 25 (1000 ppm) decreased body wt (250 mg/kg bw/d) 0, 2.0, 10, and increased liver & thyroid wt; decreased body wt (50 mg/kg bw/d) 0, 80, 400 or 2000 ppm 10 (400 ppm) *Study reported in the original dossier submitted by the applicant increased liver wt; decreased body wt (50 mg/kg bw/d) Supplementary (limited histopathology, no GLP) Supplementary (no guidelines, no GLP, 1 animal/sex tested) Acceptable Supplementary (due to lab facility) Miyazaki, 1972 Oshita, 1974 Osborne & Kalichman, 1989 Mastalski, Genotoxicity The RMS in detail summarized only the in vivo micronucleus assay (Kajowara (1986)), showing negative result. Isoprothiolane has been tested in a range of genotoxicity assays including bacterial gene mutation, chromosome aberrations and micronucleus formation: only the chromosome aberration study in Chinese hamster lung cells showed a slight increased chromatid exchanges and chromatid breaks at the highest concentration in the presence of metabolic activation. Overall, it can be concluded that isoprothiolane does not have genotoxic potential relevant to human exposure. Table 2-4. Summary of the genotoxicity studies Test substance (batch and purity) Test system Concentrations /dose Results Acceptability of the study References In vitro studies EFSA Journal 2012;10(3):2607 9

10 Test substance (batch and purity) Test system Concentrations /dose Results Acceptability of the study References Lot: DBBCFG; 97% pure Lot: unknown; 99% pure Lot: unknown; 99% pure Lot: ; 99.9% Lot: DBBCFG; 97% pure In vivo studies Lot: unknown; 99% pure Lot: DBADME; 97.5% pure S. typhimurium (5 strains); E.coli WP-2 S. typhimurium (5 strains); E.coli WP-2 B. subtilis (recassay) Chinese hamster lung cells (chromosomal aberrations) Human lymphocytes (chromosomal aberrations) S. typhimurium (G46), host mediated in mice Male ddy mice (bone marrow micronucleus) µg/plate +S µg/plate -S µg/plate +S µg/plate - S9 Negative Yes. Concentrations limited by cytotoxicity & precipitation. S9 only checked with 2-AA Asquith (1988) Negative Yes. Moriya & Shirasu (1977) mg/disc Negative Yes nonstandard test µg/ml µg/ml Equivocal increase at 134µg/ml Yes, but high purity noted. Concentrations limited by cytotxicity Moriya & Shirasu (1977) Moriya & Shirasu (1977) µg/ml Negative Yes Asoh (1985) 100 or 300 mg/kg bw (x2) 150, 300, 600 mg/kg bw; i.p. Negative Yes Asquith (1988) Negative Yes Kajowara (1986) 2.5. Long term toxicity Two valid studies allowed concluding that the relevant NOAELs of long term toxicity and carcinogenicity are 11 and 20 mg/kg bw/day, in rats and mice, respectively. In the rats keratoacanthoma occurred in male rats at the highest dose tested of 115 mg/kg bw/day. No mechanistic information was provided. Table 2-5. Summary of the long term toxicity studies Type of test/ Species (purity of the test substance) Dose levels (mg/kg bw/day) NOAEL (mg/kg bw/day) Effects at LOAEL and higher doses (mg/kg bw/day) Acceptability of the study References 2 year rat 97.1% Males: 1.8, 11, 115 Females: 2.1, 13, decreased body wt, liver toxicity; Keratoacanthoma (115 mg/kg bw/d) Acceptable Inui, month mouse 97.1% Males: 20, 104, 501 Females: 18, 96, decreased body wt (100 mg/kg bw/d) No increase of tumours Acceptable Inui, 1989 EFSA Journal 2012;10(3):

11 2.6. Reproductive toxicity Reasoned opinion on the setting of a new MRL for isoprothiolane in rice Isoprothiolane did not show evidence of reproductive toxicity based on a three-generation toxicity study in rats showing a parental and offspring NOAEL of 300 ppm (16 mg/kg bw/day) based on a suppression in body weight gain in male and female parent animals at 3000 ppm and low pup body weights rin F2 male and F3 males and females at 3000 ppm group during the lactation period. The reproductive NOAEL is 3000 ppm. To assess the teratogenicity potential, isoprothiolane was tested in rats and rabbits. The NOAEL of isoprothiolane in rats was 50 mg/kg bw/day for maternal rats and 12 mg/kg bw/day for foetuses, (incidence of unossified thoracic vertebral bodies and cervical and total vertebral bodies at 50 mg/kg bw/day in the developmental toxicity study in rats), whereas in rabbits the maternal NOAEL for isoprothiolane is 80 mg/kg bw/day, based on body weight and food consumption effects at 400 mg/kg bw/day; and the developmental NOAEL was 400 mg/kg bw/day, the highest dose tested. Table 2-6. Summary of the reproductive toxicity studies Type of test/ Species (purity of the test substance) Dose levels (mg/kg bw/day) NOAEL (mg/kg bw/day) Effects at LOAEL and higher doses (mg/kg bw/day) Acceptability of the study References Multigenerational Rat 3 generation >99.9% 0, 1.6, 16, Par.: 16 - Offsp.: 16 - Repro.:> 160 Decreased Body wt (160) Decreased Body wt (160) No effects Acceptable Ito, 1976 Developmental Rat 98.2% 0, 12, 50 and Mat.: 50 - Dev.: 12 Decreased Body wt (200) Skeletal anomalies (50) Not teratogenic (> 200) Acceptable Fujii, 2007 Rabbit 97.5% 0, 15, 80, and Mat.: 80 - Dev.:> 400 Decreased Body wt (400) No effects Acceptable Asoh, Neurotoxicity No neurotoxicity studies have been submitted; however, there are no indications from the available database that isoprothiolane is neurotoxic Further toxicological studies An acute oral toxicity of didehydroisoprothiolane was submitted, showing that the metabolite is of low acute toxicity to mice, hamsters guinea pigs and rats, whereas rabbits showed an oral LD50 of mg/kg bw. The genotoxicity of didehydroisoprothiolane was evaluated in a series of tests, which were negative. Available data showed 4-hydroxy-isoprothiolane to be of low acute oral toxicity in mice Medical data No data were submitted. EFSA Journal 2012;10(3):

12 2.10. Acceptable daily intake (ADI) and acute reference dose (ARfD) The toxicological profile of the active substance isoprothiolane was assessed by the RMS and reviewed by EFSA. The data were sufficient to derive toxicological reference values for isoprothiolane which are compiled in Table 2-9. The 2-year rat study (Inui, 1991) with the NOAEL of 11 mg/kg bw/day was considered relevant for setting the ADI, also supported by the NOAEL of the 1-year dog study, with the application of an uncertainty factor of 100, leading to an ADI of 0.1 mg/kg bw/day; this retains a margin of 1000 to the LOAEL for skin tumours seen in male rats. Reductions in food consumption and body weight / body weight gain occurred several times during the early phases of several studies, and they were considered potentially relevant for setting an ARfD. These effects could be related to irritation of high concentrations of the product, not relevant for exposures to crop residues, however, this was not evident from the available data. On the other side, the reductions in ossification seen in the rat developmental study could be the basis for setting an ARfD as well: they were considered by the EMS more likely to be related to repeated exposure than to a single exposure, and the consequence of maternal toxicity. However, clear proof of this thesis was not provided. Therefore, it is agreed to set an ARfD of 0.12 mg/kg bw, based on the NOAEL of the rat developmental study (12 mg/kg bw/day) and applying an UF of 100. Table 2-9: Overview of the toxicological reference values Isoprothiolane Source Year Value Study relied upon Uncertainty factor ADI EMS/EFSA mg/kg bw/d 1 year and 2 year dog studies; 2 year rat ARfD EMS/EFSA mg/kg bw Rat developmental toxicity EFSA Journal 2012;10(3):

13 3. Residues 3.1. Nature and magnitude of residues in plant Primary crops Nature of residues In studies submitted for the current application the metabolism of isoprothiolane was investigated in paddy rice by using [dithiolane-4,5-14 C]isoprothiolane. The overview of the metabolism study design is presented in the table below. Table 3-1: Summary of available metabolism studies in plants Group Crop Label position Cereals Rice [dithiolane- 4,5-14 C] isoprothiolane Cereals Rice [dithiolane- 4,5-14 C] isoprothiolane Method, F, G or P (a) Foliar (EC 40%); G In solution containing a.s. Rate Application details No/ Interval 0.6 kg a.s./ha 1/ before ear emergence (until BBCH 50) 1) 2 ppm (rice at BBCH 19 and rice at BBCH 60 were grown in a culture solution for 7 days) 2) 20 ppm (rice at BBCH 13 were grown in a culture solution) Sampling 7 DAT (immature) and 28 DAT 7 (experiment no 1), 14, 21, 35, 56 (mature) DAT (a): Outdoor/field use (F) or glasshouse/ /indoor application (G) or protected (P) * The study was not compliant with the currently applicable guidelines and serves as supplementary information only Study I Remarks Study I * Study II Grain, stem and leaves, and roots were harvested separately and grains after air-drying were separated into brown rice (unpolished) and hull. The TRR in brown rice, hull, stem and leaves and root 7 DAT accounted for 0.21, 5.38, 1.91 and 0.03 mg eq./kg and 28 DAT were 0.2, 4.05, 1.36 and 0.02 mg eq./kg, indicating no significant reduction of radioactivity along time. Good extractability of radioactivity was achieved 7 DAT and 28 DAT in hull (82-89%), stem and leaves (77-85%), and roots (45-48%). In grain the extractable radioactivity was slightly lower ranging from 33% 7 DAT to 42% 28 DAT. An overview of the study results is provided in Table 3-2 and Table 3-3. EFSA Journal 2012;10(3):

14 Table 3-2: Distribution of radioactivity in rice samples treated with [ 14 C] isoprothiolane %TRR (mg/kg parent equivalent) 7 DAT 28 DAT Grain Hulls Stems/ Stems/ Roots Grain Hulls Leaves Leaves MeOH rinse n/a n/a n/a n/a n/a (0.57) (0.27) MeOH extract (0.06) (3.97) (0.85) (0.02) (0.07) (2.57) (0.53) MeOH/water extract (0.01) (0.98) (0.21) (<0.01) (0.02) (1.04) (0.24) MeOH/HCl n/a n/a extract (<0.01) (0.04) (<0.01) (0.05) (0.05) MeOH/NaOH * n/a n/a n/a extract (<0.01) (0.08) (0.01) (0.17) Cellulase 46.2* 42.6* 1.5 n/a n/a n/a n/a treatment (0.10) (0.09) (0.02) 6N HCl treatment (0.01) (0.42) (0.16) (0.01) (<0.01) (0.28) (0.04) Post-extraction solids (PES) (0.01) (0.42) (0.16) (0.01) (<0.01) (0.28) (0.04) TRR (0.21) (5.38) (1.91) (0.03) (0.20) (4.06) (1.36) *No single metabolite accounting for greater than 10 %TRR Roots n/a 45.2 (<0.01) 9.3 (<0.01) n/a n/a n/a n/a 45.6 (<0.01) 100 (0.02) From the extractable residues, 7 DAT isoprothiolane accounted for 16.4% (0.03 mg/kg), 75.5% (4.06 mg/kg), 50.9% (0.97 mg/kg) and 42.4% (0.01 mg/kg) in rice grain, hulls, stems and root samples, respectively. The amount of parent isoprothiolane in brown rice 28 DAT had increased to 32.3% (0.06 mg/kg), whereas in other matrices it had slightly decreased (61.6% (2.5 mg/kg) in hull, 26.3% (0.36 mg/kg) in stem and leaves, and 34.3% (<0.01 mg/kg) in roots). In all rice matrices at both sampling intervals no other metabolites were identified individually accounting for more than 10% of the TRR. In brown rice 7 DAT and 28 DAT all identified metabolites - 4-hydroxy isoprothiolane 9, isoprothiolane monoester 10, isoprothiolane monosulfoxide 11 and didehydro isoprothiolane - were below 0.01 mg/kg (maximum of 0.3% TRR (<0.01 mg/kg) of isoprothiolane monosulfoxide 7 DAT and a maximum of 0.5% TRR (<0.01 mg/kg) of 4-hydroxy isoprothiolane 28 DAT). In rice hull, besides parent isoprothiolane (75.5% (7 DAT) and 61.6% TRR (28 DAT)) the most abundant metabolite was isoprothiolane monosulfoxide (8.5% (28 DAT) and 6.5% (7 DAT)). All other metabolites were below 2% of the TRR. In rice stem and leaves, besides parent isoprothiolane (50.9 % (7 DAT) and 26.3% (28 DAT)), monosulfoxide isoprothiolane was also present, but below 10% trigger value (8% (7DAT) and 7.7% (28 DAT)). All other metabolites individually accounted for <1.4% of the TRR. 28 DAT residue 9 Diisopropyl 4-hydroxy-1,3-dithiolan-2-ylidenemalonate 10 Isopropylhydrogen 1,3-dithiolan-2-ylidenemalonate 11 Diisopropyl 1-oxo-1,3-dithiolan-2-ylidenmalonate EFSA Journal 2012;10(3):

15 levels of all compounds, except didehydro isoprothiolane, had decreased in comparison to residue levels identified 7 DAT. Table 3-3: Metabolic profile of isoprothiolane in rice %TRR (mg/kg parent equivalent) 7 DAT 28 DAT Grain Hulls Stems/ Stems/ Roots Grain Hulls Leaves Leaves Total extract a (0.07) (4.96) (1.63) (0.02) (0.08) (3.61) (1.04) Isoprothiolane (0.03) (4.06) (0.97) (0.01) (0.06) (2.5) (0.36) 4-hydroxy-isoprothiolane n.d. (<0.01) (0.05) (0.03) (<0.01) (0.08) (0.02) Monoester < n.d. n.d. (<0.01) (0.01) (<0.01) (0.01) (<0.01) Monosulfoxide n.d. (<0.01) (0.35) (0.15) (<0.01) (0.35) (0.10) Didehydro n.d n.d. (<0.01) (0.01) (<0.01) (0.01) (<0.01) TLC-Origin 10.7 b b b 40.0 C (0.02) (0.34) (0.45) (<0.01) (0.01) (0.55) (0.54) Unidentified Metabolites (0.01) (0.14) (<0.01) (<0.01) (<0.01) (0.11) (<0.01) (a): Analyzed for methanol and methanol/water extract of brown rice and hull. For stem & leaves, sum of surface rinse, methanol and methanol/water extract were analyzed, for root, methanol extract was analysed (b): No single metabolite exceeded more than 10% TRR (c): No single metabolite exceeded more than 10% TRR except highly polar origin on TLC n.d. not detected Roots 45.2 (<0.01) 34.3 (<0.01) n.d. n.d. n.d. n.d. 9.8 (<0.01) 1.0 (<0.01) Study II The study II indicates that the incorporation of the radioactivity increases for 35 days and then becomes constant. As from 14th DAT the organosoluble radioactivity decreased while un-extractable radioactivity increased, indicating the incorporation of radioactivity in rice tissues. In polished rice 56 DAT 95% of the radioactivity was found in the un-extractable fraction. 14 DAT the residues in rice plant were identified as parent isoprothiolane, its monoester and sulfoxide, and 4-hydroxy isoprothiolane. From the study data EFSA could not retrieve adequate information on the quantitative distribution of residues in plant matrices. No differences were observed in the uptake of residues between vegetative and reproductive stage rice plants. The metabolism studies indicate that the applied radioactivity generally stays stable along time and is mainly located in stem, leaves and rice hull, with lowest amounts present in rice grain. Large part of the radioactivity is incorporated in the natural constituents of rice plant. Parent isoprothiolane is the main residue in rice grain. In general, the metabolism of isoprothiolane in rice proceeds via several oxidation and hydroxylation reactions at the side chain and at dithiolane ring, forming 4-hydroxyl form of isoprothiolane and sulfoxide isoprothiolane. The proposed metabolic pathway of isoprothiolane in rice is illustrated in Figure 3-1. EFSA Journal 2012;10(3):

16 Figure 3-1. Metabolic pathway of isoprothiolane in rice Reasoned opinion on the setting of a new MRL for isoprothiolane in rice The authorized use in Japan involves broadcast of a granular formulation to nursery box and two foliar treatments of rice with isoprothiolane. The available metabolism studies do not reflect metabolic pathway of isoprothiolane after granular treatment at the early growth stage. However, no qualitative differences in metabolism of isoprothiolane were observed between rice grown in a culture solution (this study would be representative for the granular treatment of nursery boxes) and foliar treatment of rice, therefore it can be concluded that early granular application will not result in a different metabolic pathway of isoprothiolane. It is noted that the submitted metabolism studies are performed with an application rate lower than the notified GAP (1*0.6 kg a.s./ha vs. 2*0.6 kg a.s./ha plus treatment of the nursery box before transplanting) and isoprothiolane residue concentrations found in the metabolism study (0.06 mg/kg 28 DAT) are significantly lower than the residue concentrations found in supervised field trials (0.7 mg/kg to 3.54 mg/kg measured 14 day after the application (see Table 3-4)). This difference can be partially attributed to a different time elapsed between the treatment and the sampling, but it may give also an indication that the metabolism study does not exactly reflect the situation expected in rice treated according to the GAP. This deficiency of the metabolism study is contributing to the uncertainty of the assessment. Although, according to the authorized use a more mature crop is treated, metabolism studies indicate that radioactivity in rice plant is stable along time with no changes in metabolic profile. It is therefore concluded that the available metabolism study can be used to characterise the metabolism of isoprothiolane in rice when treated according to the authorised GAP. EFSA agrees with the proposal of the RMS to define parent isoprothiolane as a relevant residue for the risk assessment and enforcement purposes in rice Magnitude of residues In support of the critical authorized use (combined nursery treatment (pre-transplanting) and two foliar applications close to harvest), the applicant submitted 6 residue trials on rice performed in EFSA Journal 2012;10(3):

17 Japan in 2007 and 2009 which reflect the GAP. Samples of hulled rice (brown rice) were analysed for isoprothiolane residues. It is noted that the study was not performed according to GLP standard, but the submitted studies were scientifically valid and were therefore considered as acceptable by EFSA and the EMS. According to the EU guidance document rice is a major crop and therefore at least 8 GAP compliant residue trials have to be provided (EC, 2011). To address this data gap the applicant submitted two additional residue trials where instead of a foliar spray a dust application at a significantly higher rate was used (1 kg a.s./ha vs. 0.6 kg a.s./ha). EFSA disregarded those trials as overdosed and as incompliant in terms of the type of application. In addition, the applicant submitted a summary reporting the results of 35 additional residue trials performed between 1971 and However, since none of the trials reflects exactly the authorized critical GAP in Japan, the trials are considered as supplementary information. These additional residue trials were performed with one to four applications, application rates ranging from 0.3 to 6 kg a.s./ha per application and PHI intervals from 10 to 84 days. Although the trials reflect more critical conditions (higher seasonal application rates, partially shorter PHI) than the notified critical GAP, residues in all these trials were generally lower than in trials submitted in support of the current application. From this database EFSA selected 3 residue trials which were performed with 2-3 foliar applications at the application rate comparable with the critical GAP (± 25% deviation) and sampling between day PHI intervals. According to the EMS, there are no indications that these trials are incompliant with general requirements on performance of residue trials. The results of the residue trials, the results of the supplementary trials (highlighted in grey), the related risk assessment input values (highest residue, median residue) and the MRL proposals are summarized in Table 3-4. The storage stability of isoprothiolane in rice was investigated in the framework of the current application concurrently with field residue trials. Homogenized samples were spiked with 0.5 mg/kg of isoprothiolane and stored at -20 C. After completion of analysis of field samples, spiked samples were analyzed and the stability of residues was examined. A sufficiently validated analytical method was used. Study results confirm the stability of isoprothiolane residues in rice for up to 195 days when stored deep frozen. As the supervised residue trial samples were stored under conditions for which integrity of samples was demonstrated (maximum of 195 days, -20ºC), it is concluded that the residue data are valid with regard to storage stability. According to the EMS, the analytical methods used to analyse supervised residue trial samples have been sufficiently validated and were proven to be fit for purpose. EFSA concludes that the MRL of 5 mg/kg as requested by the applicant would be appropriate for the authorized use of isoprothiolane on rice in Japan. EFSA Journal 2012;10(3):

18 Table 3-4: Overview of the available residues trials data Commodity Region (a) Outdoor /Indoor Individual trial results (mg/kg) Enforcement (Isoprothiolane) Risk assessment (Isoprothiolane) Median residue (mg/kg) (b) Highest residue (mg/kg) (c) MRL proposal (mg/kg) Median CF (d) Comments Rice Import (Japan) Outdoor 0.7 e ; 0.8 e ; 1.3 e ; 2 x 1.54; 1.54 e ; 1.68 e ; 1.88; 3.54 e 0.7 e ; 0.8 e ; 1.3 e ; 2 x 1.54; 1.54 e ; 1.68 e ; 1.88; 3.54 e The values highlighted in grey are considered as supplementary results which do not exactly reflect the critical GAP. R ber =3.56 R max =4.1 OECD MRL g =5 (a): NEU, SEU, EU or Import (country code). In the case of indoor uses there is no necessity to differentiate between NEU and SEU. (b): Median value of the individual trial results according to the enforcement residue definition. (c): Highest value of the individual trial results according to the enforcement residue definition. (d): The median conversion factor for enforcement to risk assessment is obtained by calculating the median of the individual conversion factors for each residues trial. (*): Indicates that the MRL is set at the limit of analytical quantification. (e): Higher residue within a trial observed at a longer PHI interval of days. (f): Higher residue within a trial observed at a longer PHI interval of 20 days (g) : The MRL calculated using the OECD MRL calculator (OECD, 2011b) EFSA Journal 2012;10(3):

19 Effect of industrial processing and/or household preparation The effect of processing on the nature of isoprothiolane has not been investigated in standard hydrolysis studies representing pasteurization, baking/brewing/boiling and sterilization. The applicant, instead, has performed studies investigating the stability of isoprothiolane in buffer solutions (ph 5, 7 and 9) incubated at 25ºC for 0, 1, 3, 7, 14, 21 and 28 days and at 50 ºC for 0, 3 and 7 days. The results indicate that isoprothiolane is hydrolytically stable at the investigated hydrolysis conditions. The study is not compliant with the EU guidance documents on how the effects of processing on the nature of the active substance should be investigated (EC, 1997) and therefore was considered as a supplementary information only. EFSA is of the opinion that the nature of isoprothiolane residues under baking/boiling conditions has to be further investigated in studies which are compliant with the currently applicable EU guidance documents. The applicant submitted a summary of a study which aimed to investigate the effect of cooking of rice on the magnitude of isoprothiolane residues. Brown rice (hulled rice) with field incurred residues of isoprothiolane was polished, by removing rice bran. No information is available on the residues in brown rice. Washing the polished grains was shown to reduce the isoprothiolane residues significantly (reduction by 50% in case rice is washed 3 times). The reduction is probably linked to the removal of adhering hulls. Immersion of rice in water overnight did not have an impact on the residue level. No study details are available regarding the exact time and temperature during this immersion. EFSA concludes that lacking a clear description of the study details, this information is considered as supplementary information only. According to the EMS, more information on the magnitude of residues in processed rice is available in a study sponsored by the cooperative of rice growers. The study had been performed for investigating the effect of cooking and polishing on the residue levels of isoprothiolane in rice. Study results indicate that the polishing of brown rice (hulled rice) reduced the residues from 0.2 mg/kg to 0.05 mg/kg. Then further washing and cooking (steaming) procedures reduces the residues from 0.05 mg/kg to 0.02 mg/kg. EFSA could not assess the validity of the submitted processing studies and therefore study results were not used to derive proposals for enforcement purposes. There are some indications that polishing reduces the concentration of isoprothiolane residues. For the consumer exposure the most conservative approach is used, assuming that no significant reduction of residues occur during processing Rotational crops The residues of isoprothiolane in rotational crops are of no relevance for the import tolerance application Nature and magnitude of residues in livestock Rice is not considered as a livestock feed in Europe (EC, 1996) and therefore the nature and magnitude of isoprothiolane residues in livestock was not assessed in the framework of this application. EFSA Journal 2012;10(3):

20 4. Consumer risk assessment Reasoned opinion on the setting of a new MRL for isoprothiolane in rice The consumer risk assessment was performed with revision 2 of the EFSA Pesticide Residues Intake Model (PRIMo). This exposure assessment model contains the relevant European food consumption data for different sub-groups of the EU population 12 (EFSA, 2007). For the calculation of the chronic exposure, EFSA used the median residue value as derived from the residue trials on rice (see Table 3-4). For the remaining commodities of plant and animal origin, the default MRL of 0.01 mg/kg was used as an input value. The acute exposure assessment was performed only with regard to rice assuming the consumption of a large portion of the food item as reported in the national food surveys containing residues at the median level as observed in supervised field trials. 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 Acute exposure assessment Input value (mg/kg) Risk assessment residue definition: Isoprothiolane Comment Input value (mg/kg) Comment Rice 1.54 Median residue 1.54 Median residue Other commodities of food and animal origin 0.01 Default MRL Acute risk assessment was undertaken only with regard to the crops under consideration. The estimated exposure was then compared with the toxicological reference values derived for isoprothiolane (see Table 2-9). The results of the intake calculation are presented in Appendix B to this reasoned opinion. No long-term consumer intake concerns were identified for any of the European diets incorporated in the EFSA PRIMo. The total calculated intake values ranged from 0.1 to 1.6% of the ADI (maximum for UK Infant diet). The contribution of residues in rice to the total consumer exposure accounted for a maximum of 1.2% of the ADI (PT general population diet). No acute consumer risk was identified in relation to the MRL proposal for rice. The calculated maximum exposure in percentage of the ARfD was 16.2% for rice. EFSA identified some deficiencies in the metabolism studies in rice, due to the low application rate which did not allow to identify traces of potentially formed metabolites. However, the potential contribution of isoprothiolane metabolites to the total consumer intake is covered by a wide safety margin of the calculated consumer exposure to isoprothiolane residues. 12 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 2012;10(3):

21 EFSA concludes that the import tolerance for rice supporting the authorized use of isoprothiolane in Japan will not result in a consumer exposure exceeding the toxicological reference values and therefore will not pose a public health concern. EFSA Journal 2012;10(3):

22 CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The toxicological profile of isoprothiolane was assessed by the RMS and reviewed by EFSA. The data were sufficient to propose an ADI of 0.1 mg/kg bw/day and an ARfD of 0.12 mg/kg bw. The metabolism of isoprothiolane was investigated in rice after foliar application of isoprothiolane. The applied radioactivity generally stays stable along time and is mainly located in stem, leaves and rice hull, with lowest amounts recovered in rice grain. Large part of the radioactivity is incorporated in the natural constituents of rice plant. Isoprothiolane is the main residue in rice grain with all other metabolites being below the LOQ. EFSA identified some deficiencies in the metabolism study in rice, due to the low application rate which did not allow to identify traces of potentially formed metabolites. However, the potential contribution of isoprothiolane metabolites to the total consumer intake is covered by a wide safety margin of the calculated consumer exposure to isoprothiolane residues. EFSA concludes that the available metabolism study can be used to characterise the metabolism of isoprothiolane in rice when treated according to the authorised GAP. EFSA agrees with the proposal of the RMS to define parent isoprothiolane as a relevant residue for the risk assessment and enforcement purposes in rice. The submitted residue data are sufficient to derive an import tolerance of 5 mg/kg for the authorized use of isoprothiolane on rice in Japan. An adequate analytical method is available to enforce the proposed MRL of isoprothiolane in rice at the LOQ of 0.01 mg/kg. Studies investigating the effect of processing on the nature of isoprothiolane residues have not been submitted. Since rice is mainly consumed processed, studies investigating the nature of isoprothiolane under baking/boiling conditions are strongly recommended. Several studies investigating the effect of processing on the magnitude of isoprothiolane residues in processed rice were reported, but EFSA could not assess the validity of the submitted processing studies and therefore study results were not used to derive proposals for enforcement purposes. There are some indications that polishing reduces the concentration of isoprothiolane residues. For the consumer exposure the most conservative approach is used, assuming that no significant reduction of residues occur during processing. The residues of isoprothiolane in rotational crops are of no relevance for the import tolerance application. Rice is not considered as a livestock feed in Europe and therefore the nature and magnitude of isoprothiolane in commodities of animal origin was not investigated in the framework of the current application. The consumer risk assessment was performed with revision 2 of the EFSA PRIMo. For the calculation of the chronic exposure EFSA used the median residue value for rice as derived from the supervised field trials. For other food commodities of animal and plant origin the default MRL of 0.01 mg/kg was used as an input value. The acute exposure assessment was performed only with regard to rice. Rice is a bulked commodity and therefore the median residue value as derived from the supervised residue field trials was used as an 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 values ranged from 0.1 to 1.6% of the ADI (maximum for UK Infant diet). The contribution of residues in rice to the total consumer exposure accounted for a maximum of 1.2% of the ADI (PT general population diet). No acute consumer risk was identified in relation to the MRL proposal for rice. The calculated maximum exposure in percentage of the ARfD was 16.2% for rice. EFSA Journal 2012;10(3):