TECHNICAL REPORT. European Food Safety Authority 2. European Food Safety Authority (EFSA), Parma, Italy

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1 supporting publication 2014:EN-680 TECHNICAL REPORT Outcome of the consultation with Member States, the applicant and on the pesticide risk assessment of for the active substance difenoconazole 1 European Food Safety Authority 2 European Food Safety Authority (), Parma, Italy ABSTRACT The European Food Safety Authority () was asked by the European Commission to provide scientific assistance with respect to the risk assessment of following approval of an active substance in accordance with Article 6(f) of Regulation (EC) No 1107/2009. In this context s scientific views on the specific points raised during the commenting phase conducted with Member States, the applicant and on the risk assessment of for difenoconazole are presented. The current report summarises the outcome of the consultation process organised by the rapporteur Member State Sweden and presents s scientific views on the individual comments received. The representative use of difenoconazole is as a fungicide on pome fruit, carrot, wheat, barley, triticale, rye and oats. European Food Safety Authority, 2014 KEY WORDS difenoconazole, peer review,, risk assessment, pesticide, fungicide 1 On request from the European Commission, Question No -Q , approved on 17 October Correspondence: pesticides.peerreview@efsa.europa.eu Suggested citation: (European Food Safety Authority), 2014; Outcome of the consultation with Member States, applicant and on the pesticide risk assessment of submitted for the active substance difenoconazole. supporting publication 2014:EN pp. Available online: European Food Safety Authority, 2014

2 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole SUMMARY Difenoconazole was included in Annex I to Directive 91/414/EEC on 1 January 2009 by Commission Directive 2008/69/EC, and has been deemed to be approved under Regulation (EC) No 1107/2009, in accordance with Commission Implementing Regulation (EU) No 540/2011, as amended by Commission Implementing Regulation (EU) No 541/2011 and Commission Implementing Regulation (EU) No 1100/2011 as regards the conditions of approval of difenoconazole. It was a specific provision of the approval that the applicant was required to submit to the European Commission further studies on the potential for endocrine disrupting effects on fish and the chronic risk to earthworms from the active substance and the metabolite CGA by 30 November In accordance with the specific provision, the applicant, Syngenta Ltd., submitted an updated dossier in November 2013, which was evaluated by the designated RMS, Sweden, in the form of an Addendum to the Draft Assessment Report. In compliance with the Guidance Document SANCO 5634/2009 rev.6.1, the RMS distributed the Addendum to Member States, the applicant and for comments on 7 May The RMS collated all comments in the format of a Reporting Table, which was submitted to on 25 September added its scientific views on the specific points raised during the commenting phase in column 4 of the Reporting Table. It can be concluded that the submitted fish study (fish full-life-cycle test) does not provide sufficient evidence to conclude that there are no endocrine disruptive effects on fish. Nevertheless, a long-term risk assessment was performed using a NOEC (no observed effect concentration) endpoint derived from this study. The risk assessment indicated a low risk (FOCUS step 2) for the seed treatment use on cereals. However a high risk was concluded for all the relevant scenarios for the uses on pome fruit and carrot. When appropriate risk mitigation measures (no-spray buffer zones) were considered in the exposure predictions, a low risk was concluded for the majority of the FOCUS step 4 scenarios for these representative uses. It is noted that the endpoint used in the risk assessment was considered by the RMS as potentially over conservative. It should also be noted that the exposure of the fish in the submitted study was longer than in the standard long-term studies. As regards the chronic risk to earthworms, a set of new laboratory chronic studies on soil organisms was available. Based on these data a low chronic risk was concluded for the pertinent metabolite for all the representative uses and for difenoconazole for the seed treatment use. The risk assessments that used the endpoint for difenoconazole and exposure estimations for the spray uses (pome fruit, carrot) resulted in a high chronic risk to earthworms (i.e. toxicity exposure ratios (TERs) < 5). When the endpoints from the studies of spray formulations were used, a low chronic risk was concluded for the pertinent representative uses. It is noted that there were some indications for increasing body weight of the earthworms with increasing concentration of difenoconazole in soil. This phenomenon was however not consistent among all the studies that were available. supporting publication 2014:EN-680 2

3 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Commission... 4 Terms of reference as provided by the European Commission... 5 Evaluation... 6 Documentation provided to... 6 References... 6 Appendix A... 7 Collation of comments from Member States, applicant and on the pesticide risk assessment of for the active substance difenoconazole and the conclusions drawn by on the specific points raised... 7 Appendix B Used compound code(s) Abbreviations supporting publication 2014:EN-680 3

4 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Difenoconazole was included in Annex I to Directive 91/414/EEC on 1 January 2009 by Commission Directive 2008/69/EC 3, and has been deemed to be approved under Regulation (EC) No 1107/2009 4, in accordance with Commission Implementing Regulation (EU) No 540/2011 5, as amended by Commission Implementing Regulation (EU) No 541/ and Commission Implementing Regulation (EU) No 1100/ as regards the conditions of approval of difenoconazole. previously finalised a Conclusion on this active substance on 17 December 2010 in the Journal 2011;9(1):1967 (, 2011). It was a specific provision of the approval that the applicant was required to submit to the European Commission further information as regards: a) further data on the specification of the technical material (to be submitted by 31 May 2012); b) residues of triazole derivative metabolites (TDMs) in primary crops, rotational crops, processed commodities and products of animal origin (to be submitted by 30 November 2013); c) the potential for endocrine disrupting effects on fish (fish full life cycle study) and the chronic risk to earthworms from the active substance and the metabolite CGA (to be submitted by 30 November 2013); d) the possible impact of the variable isomer-ratio in the technical material and of the preferential degradation and/or conversion of the mixture of isomers on the worker risk assessment, the consumer risk assessment and on the environment (to be submitted within 2 years from the adoption of specific guidance). The review of related to point a) concerning the specification of the technical material was already completed and finalised at the Standing Committee on the Food Chain and Animal Health in December The Review Report has been updated accordingly (European Commission, 2013a). Confirmatory data required related to point b) as regards residues for the triazole derivative metabolites were submitted by the applicant in November These data are evaluated by the United Kingdom for all TDMs therefore this assessment is not covered in the current report. As regards the requirement under point c) of the specific provision, the applicant, Syngenta Ltd., submitted an updated dossier in November 2013, which was evaluated by the designated rapporteur Member State (RMS), Sweden, in the form of an Addendum to the Draft Assessment Report (Sweden, 2014a). In compliance with the Guidance Document SANCO 5634/2009 rev. 6.1 (European Commission, 2013b), the RMS distributed the Addendum to Member States, the applicant and the for comments on 7 May The RMS collated all comments in the format of a Reporting Table and added its evaluation in column 3 of the Reporting Table (Sweden, 2014b), which was 3 Commission Directive 2008/69/EC of 1 July 2008 amending Council Directive 91/414/EEC to include clofentezine, dicamba, difenoconazole, diflubenzuron, imazaquin, lenacil, oxadiazon, picloram and pyriproxyfen as active substances. OJ L 172, , p Regulation (EC) No 1107/2009 of 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 Commission Implementing Regulation (EU) No 540/2011 of 25 May 2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 541/2011 of 1 June 2011 amending Implementing Regulation (EU) No 540/2011 implementing Regulation (EC) No 1107/2009 of the European Parliament and of the Council as regards the list of approved active substances. OJ L 153, , p Commission Implementing Regulation (EU) No 1100/2001 of 31 October 2011 amending Implementing Regulation (EU) No 540/2011 as regards the conditions of approval of the active substances dicamba, difenoconazole, and imazaquin. OJ L 285, , p supporting publication 2014:EN-680 4

5 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole submitted to on 25 September added its scientific views on the specific points raised during the commenting phase in column 4 of the Reporting Table. The current report summarises the outcome of the consultation process organised by the rapporteur Member State Sweden and presents s scientific views on the individual comments received. On the basis of the Reporting Table, the European Commission may decide to further consult to conduct a full or focussed peer review and to provide its conclusions on certain specific points. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The was asked by the European Commission to provide scientific assistance with respect to the risk assessment of following approval of an active substance in accordance with Article 6(f) of Regulation (EC) No 1107/2009. s scientific views on the specific points raised during the commenting phase conducted with Member States, the applicant and on the risk assessment of for difenoconazole are presented in compliance with the Guidance Document SANCO 5634/2009 rev To this end, a Technical Report containing the finalised Reporting Table is prepared by within 4 weeks after receipt of the Reporting Table. On the basis of the Reporting Table, the European Commission may decide to further consult to conduct a full or focussed peer review and to provide its conclusions on certain specific points. supporting publication 2014:EN-680 5

6 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole EVALUATION The comments received on the pesticide risk assessment of for the active substance difenoconazole and the conclusions drawn by the are presented in the format of a Reporting Table. The comments received are summarised in column 2 of the Reporting Table. The RMS considerations of the comments are provided in column 3, while s scientific views and conclusions are outlined in column 4 of the table. The finalised Reporting Table is provided in the Appendix A of this report. DOCUMENTATION PROVIDED TO 1. Sweden, 2014a. Addendum to the Draft Assessment Report on difenoconazole,, May 2014, revised in September Available online: 2. Sweden, 2014b. Reporting Table, comments on the pesticide risk assessment of for the active substance difenoconazole, September REFERENCES ACD/ChemSketch, Advanced Chemistry Development, Inc., ACD/Labs Release: Product version: (Build 29305, 25 Nov 2008 (European Food Safety Authority), Conclusion on the peer review of the pesticide risk assessment of the active substance difenoconazole. Journal 2011;9(1):1967, 71 pp. doi: /j.efsa European Commission, Guidance Document on Terrestrial Ecotoxicology Under Council Directive 91/414/EEC. SANCO/10329/2002 rev.2 final, 17 October 2002 European Commission, 2013a. Review Report for the active substance difenoconazole finalised in the Standing Committee on the Food Chain and Animal Health at its meeting on 14 March 2008 in view of the inclusion of difenoconazole in Annex I of Directive 91/414/EEC. SANCO/830/08 rev.3, 13 December 2013 European Commission, 2013b. Guidance document on the procedures for submission and assessment of following inclusion of an active substance in Annex I of Council Directive 91/414/EEC. SANCO 5634/2009 rev. 6.1 supporting publication 2014:EN-680 6

7 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole APPENDIX A COLLATION OF COMMENTS FROM MEMBER STATES, APPLICANT AND ON THE PESTICIDE RISK ASSESSMENT OF CONFIRMATORY DATA FOR THE ACTIVE SUBSTANCE DIFENOCONAZOLE AND THE CONCLUSIONS DRAWN BY ON THE SPECIFIC POINTS RAISED 5. Ecotoxicology Aquatic organisms (B.9.2) 5(1) Addendum for, B.9.2 Effects on AO : agrees with RMS s RMS: Agreed. conclusion that the submitted fish study does not provide sufficient evidence to conclude that there are no endocrine disruptive effects on fish. This conclusion seems to be in line with the opinion of DE who also evaluated the submitted study. However please consider s comments below. Further explanation: particularly agrees with the comments referring to the variable results and the questionable statistical power. Additionally notes that in F1 generation the average vitellogenin concentrations were clearly lower in all treated groups (except one female group) than in the control. Also, for some parameters (e.g. lengths, BW) some concentration related trends s scientific views on the specific There is an agreement among the RMS, and MSs (i.e. DE and DK, however see NL comment in 5(15)) that the submitted fish study does not provide sufficient evidence to conclude that there are no endocrine disruptive effects on fish. has noted some further potential effects on the F1 and F0 generations. Notes: Another study was available for the national product authorisation in Sweden. Although this study was not evaluated in detail, it was noted that there was a large variation of female vitellogenin levels among the tested fish and a low statistical power was anticipated. These findings are in line with the findings of the submitted study for the confirmatory dossier. supporting publication 2014:EN-680 7

8 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) s scientific views on the specific could also be observed. See also comments 5(3), 5(7) and 5(15), below. 5(2) Addendum for, B.9.2 Effects on AO : cannot see the figures and trends that are discussed in point b, on page 11. The only data on sex ratio are summarised in Table B where no large skewing to males are evident. Could you please clarify this? Also it was not clearly described that some groups were excluded as outliers for the analysis. It can only be worked out from indirect indications from points c., d. and e. and from the footnote for Table B RMS: The large skewing at individual time points mentioned in point b was cited from the German evaluation. We were not able to confirm the figure since only the combined values were presented in the original study submitted to Sweden. The applicant proposed that the combined figures from days 90, 143 and 178 post-hatch should be used for the analysis. This may need further discussion. Regarding exclusion/inclusion of outliers in Table B this has been clarified in the Addendum by moving the footnote text to the table heading. The applicant maintain that sex ratio data from individual time points should not be treated in isolation as these should be considered as biased samples. The whole population of fish is relevant for determining the sex ratio, no matter when they are removed for sampling (i.e. time is irrelevant). Therefore, they propose that only the data for the whole This point could not be fully clarified. However, the study evaluation made by DE clearly concluded a potential effect on fish skewing towards males (at least in the highest test concentration on day 143 post-hatch). The applicant argues that this phenomenon is an artefact (see comment 5(11)). Regarding the question whether the combined data (from days 90, 143 and 178 post-hatch) should be used for the analysis or these data sets should be handled separately, is of the opinion that the combined approach might be acceptable when the overall biological relevance is to be answered. When the potential ED properties are investigated then it would be better to analyse all available details separately (as apparently DE has done). If there are clearly biased (artefact) data sets, this should be noted. also agrees that an expert discussion would be valuable to see supporting publication 2014:EN-680 8

9 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(3) Addendum for, B.9.2 Effects on AO 5(4) Addendum for, B.9.2 Effects on AO : It is noted that a further study on zebrafish is available, but not yet evaluated. Ideally it should also be assessed as it could help to reduce the uncertainty from different aspects. In case if the peer-review result in disagreement between MSs, the detailed assessment of this study might become necessary. : An updated RA is available using the endpoint of NOEC 3.6 µg/l (median tested concentration from the submitted FLS). It is noted that this RA bears the uncertainties that were F0 generation should be used (i.e. taken at 90, 143 and 178 dph combined) to determine the sex ratio of the population. s scientific views on the specific more opinions on this issue, however as this biological indication is not the only one, further investigation of this issue is not necessarily essential (i.e. the final conclusion on potential ED properties will likely remain open). See also comments 5(6), 5(10), 5(11), 5(12) and 5(15), below. RMS: The study on zebrafish ( See notes in the column 4 entry at 2007) was not included in the comment 5(1), above. package but was available to the RMS from a product dossier and therefore mentioned in the Addendum. No indepth evaluation has been conducted. However, it was observed that there is a very large variation in the results on VTG levels indicating a low statistical power of the study. Hence, it is unlikely that the study would give sufficient evidence on lack of endocrine effects. RMS: Agree. The RMS proposed that the male body length effects observed at 7.8 ug/l was not biologically relevant but that the overall uncertainties related to Addressed. There is an agreement ( and RMS) that even if the precautionary endpoint of NOEC 3.6 µg/l is used in a risk supporting publication 2014:EN-680 9

10 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(5) B.9.2.3, pp. 10, Re-view / re-analysis of the study by 2009, point 1 discussed for the assessments for the submitted FLS study and extrapolation to field situations (i.e. to other species). DE: According to the RMS [...] the slight effect on male length should not be regarded as a biologically significant, and therefore we (RMS) agree with the NOAEC value of 7.8 µg/l proposed by the study author. No statement was included, why the effects are not relevant. Hence in our opinion the justification is not sufficient respectively not supported by DE. possible endocrine disruption justifies the use of a lower NOEC (3.6 ug/l). Two MS commented that the body length effect should be regarded as biologically significant and supported the NOEC of 3.6 ug/l for the risk assessment. It should be noted that if a NOEC of 7.8 ug/l would be concluded (as proposed by the applicant), the lower value from Daphnia will drive the risk assessment (see original ). s scientific views on the specific assessment, this risk assessment would bear some uncertainties (e.g. extrapolation to other species). See also column 4 entries at comment 5(5), below. RMS: The justification for disregarding The RMS has given further clarification the biological significance of the on the choice of their endpoint. The two male body length effect was: authorities, who have evaluated this 1) The effect was small, 4.1% study in detail, have different views on compared to the control. what should be the endpoint to be used 2) No significant effect was in a risk assessment (7.8 vs. 3.6 µg/l). observed on body weight of This fact indicates that even after a males of the same group. through evaluation of this study it is difficult to judge which water 3) The body length effect was concentration would be of no-concern limited to males in the first and an expert consultation would be generation out of two. ideal, however would not be essential as This justification was stated in the the follow up risk assessment performed Addendum. The applicant has added that by the RMS has used the precautionary the higher NOEC would be more supporting publication 2014:EN

11 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(6) B.9.2.3, pp. 10, Re-view / re-analysis of the study by 2009, point 2b DE: We would like to further confirm, that effects on the sex ratio (towards male) cannot be excluded. Referring to the argumentation by DE (included in the confimatory addendum), we would like to strengthen the NOEC of 3.6 µg/ll as relevant endpoint for risk assessment. 5(7) Volume 3, B DK: The issue of endocrine disruption in non-target species is still under scientific debate and currently there are no agreed criteria for assessment. DK agrees with RMS that there seems to be an effect on the sex ratio at the highest test concentration. DK agrees with RMS that there is not sufficient evidence to conclude that consistent with other available chronic fish data. s scientific views on the specific endpoint of 3.6 µg/l (thus covers DE s concerns). It should be noted that the risk See also comments 5(6) 5(8) and 5(9), assessment in the addendum is below. nevertheless based on the NOEC of 3.6 ug/l due to uncertainty related to possible endocrine effects. The applicant maintains that a NOEC of 7.8 µg/l should be used. RMS: Further clarification may be See column 4 entries at comments 5(2) needed from the German evaluation, and 5(5), above. since only the combined values were presented in the original study submitted to Sweden. RMS: Agree. However, the procedure See column 4 entry at comment 5(1), following this (lack of) conclusion above. is not clear to us at this stage. Further discussions may be needed. supporting publication 2014:EN

12 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(8) Volume 3, B.9.2.3, page 13 5(9) B Fish full life cycle study; study endpoint there are no endocrine disrupting effects. DK: In the last section on page 13 RMS proposes a NOEC of 7.8 µg/l. DK disagrees with the NOEC of 7.8 µg/l. Because of the statistical significant effect on male body length the NOEC is in our opinion 3.6 µg/l. DK agrees that the effect on body length is small and that it is not likely to have a severe biological effect. However as the effect on wet weight is in the same order of magnitude as the effect on length, and because we do not have other fixed criteria than the statistical significance to determine when an effect is acceptable, DK assess the NOEC to be 3.6 µg/l. Applicant: In response to the comment made during the UBA review of the study, the RMS has commented that minor body length effect on males can be considered as not adverse since no corresponding effect was observed on the body weight and they are in agreement with the study authors. Syngenta would echo these s scientific views on the specific RMS: See response to comment 5(5). See column 4 entry at comment 5(5), above. RMS: Noted. The argumentation See comment 5(5), above regarding inconsistent results compared to other available data has been included in the revised Addendum. The comments regarding lack of reproduction effect and on the unrealistic exposure are rejected since supporting publication 2014:EN

13 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(10) B Fish full life cycle study; sex ratio of F0 fish (combined fish from 90/143/178 dph) comments and add that the effect on male length at 95-dph is also not consistent with other available chronic fish studies for difenoconazole. In addition, the study demonstrates no reproductive effects up to and including 15 µg/l and therefore a statistically significantly difference in male length at one observation is not population relevant. Further, the exposure is unrealistic (continuous over 183 days). Therefore under field conditions of significantly lower exposure over significantly shorter durations it is highly unlikely to be relevant for the development of natural fish populations. Applicant: This represents an absolute difference in treatment means of only 5.2% and such a small difference is not statistically or biologically significant. Acceptable control variability far exceeds the small difference observed in this study. For example Fathead minnow controls in the early drafts of the FSDT test guideline were between 35 and 65% male. 1) The results on reproductive effects are considered inconclusive. 2) Possible long term effects due to short term exposure cannot be excluded based on the available data. s scientific views on the specific RMS: The relevance of the results on See comment 5(2), above. sex ratio may be disputable, however, this is not the only driving factor for the overall results/uncertainties of the study. supporting publication 2014:EN

14 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(11) B Fish full life cycle study; sex ratio of F0 fish thinned on 143 dph In addition, there is no concentration response or even trend in the data nor are there indications in the F1 generation. The samples size exceeds the requirement for studies with this species designed to detect differences in sex ratio (OECD 234). Applicant: Sex of fish thinned at 143 dph should not be treated as an endpoint as it is a biased sample; thinning was based on sex to achieve reproductive groups. More males would have been thinned as groups had a sex ratio of 2 males: 4 females (i.e. more males were removed). Across all treatment groups, an expected predominance of males were thinned (not following a concentration response). The best analysis of sex ratio is the combined F0 data as this is the largest sample size (greatest power to detect an effect), is an unbiased sample (the entire F0 population from dph) and the numbers (of individuals and replication) exceed those required for this endpoint as recommended by the OECD (OECD 234). No effect on sex ratio was observed at any of the s scientific views on the specific RMS: There was no explanation to the See comments 5(2), 5(6) and 5(15). high proportion of males before thinning at 143 days post-hatch. Therefore we agree with the conclusion from the German evaluation that there might be a possible effect on the sex ratio, although there were also a high proportion of males in the untreated control. This is stated in the Addendum. Further, the possible effect or lack of effect on sex ratio is not the only driving factor for the overall results/uncertainties of the study. supporting publication 2014:EN

15 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) unbiased (intended) sampling points. 5(12) B Fish full life Applicant: It is standard practise to cycle study; removal of remove replicates where reproductive outliers from groups are mis-sexed. If there are reproduction data fewer females, the number of (spawning frequency, potential spawns available are eggs per replicate, eggs different and females with a higher per female) ratio of males experience higher levels of aggression (which has consequences for reproductive performance). These effects are not treatment related but an artefact of unintended changes to test system. As previously demonstrated there is no effect on sex ratio; fish used in the reproduction trials contributed to the overall F0 sex ratio analysis. Syngenta has taken every measure possible to ensure the design was as powerful as possible for the determination of reproductive effects, using 8 independent replicate reproductive groups per test item treatment and 16 independent replicate reproductive groups for the control. Syngenta maintains that the design and approach to the statistical analysis is robust and concludes there is no evidence for a reproductive s scientific views on the specific RMS: Noted. From our point of view, all See comments 5(2), 5(6), 5(11) and replicates should be included in the 5(15). analysis, as was originally proposed by Germany. This may however need further discussion. supporting publication 2014:EN

16 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) 5(13) B Fish full life cycle study; General ED conclusion species selection effect up to and including the highest treatment of 15µg/L. Applicant: Syngenta is not aware of any compelling evidence for high compensatory competence in fathead minnow against disturbances of sexual development. With regard to fathead minnow showing reproductive variability, as already demonstrated, considerable effort was made to address this aspect of the study design. Syngenta believes it is pure speculation that other species such as zebrafish would be affected at 15µg/L. No endocrine disrupting diagnostic or apical effects have been seen in this fathead minnow full lifecycle and no endocrine disrupting diagnostic effects have been seen in the zebrafish screening assay ( 2007, Difenoconazole - Fish screening assay for endocrine activity in zebrafish (Danio rerio), even at 7 times 15 µg/l (104 µg/l high treatment level). Nor are there any indicators from the toxicology package. RMS: Noted. The available study on Danio rerio ( 2007) was not included in the package, and has not been fully evaluated by the RMS. However, it was noted that also this study suffers from large variability (eg. for the female VTG levels, standard deviations were 60-87% of the mean values), and hence a low statistical power is anticipated. s scientific views on the specific See notes in column 4 entry at comment 5(1), above. See also comments 5(3) and 5(4). 5(14) B Fish full life Applicant: To reiterate, there are no RMS: The applicant refers to the lack of See comment 5(1), above. supporting publication 2014:EN

17 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) cycle study; General ED conclusion 5(15) B Fish full life cycle study; sex ratio elimination of outliers overall conclusion on ED effects indicative of aromatase / sterol biosynthesis inhibition; - no effect on sex ratio; - no reduction of female vitellogenin - no histopathological findings Nor is there any convincing evidence of reproductive impairment up to and including 15 µg/l. In conclusion, there are no effects indicative of endocrine disruption and the study NOEAC is 7.8 µg/l. In response to comments made by the RMS on the variability of the VTG results, Syngenta provides the following additional statement: Attachment moved by to the Annex after the table. NL: Regarding the FFLC test, the NL is wary of placing too much emphasis on the culled group of fish from one treatment level as an indicator of sex ratio skewing particularly when at this cull in the FFLC test the sex ratio is purposefully skewed in the culling statistically significant effects on VTG levels and reproductive parameters. However, based on the statistical power analysis made by Germany, the minimum detectable effects from the study range between 16% (sex ratio for combined days post hatch) and 95% (fecundity). Hence, the results are considered to be inconclusive. It is acknowledged that the test design involved a sufficient number of replicates according to the relevant test guidelines. However, this is no guarantee that the results are acceptable. s scientific views on the specific Notes: It is agreed that there is no solid convincing evidence of reproductive impairment or effects indicative of endocrine disruption. However, equally there is no solid convincing evidence that the signs and indications from this study cannot be linked with relevant biological effects (which may include ED). It is also noted that potential ED properties should be particularly considered for this class of pesticides. It is acknowledged that the study was well conducted and used a long exposure period. RMS: It is acknowledged that there were See comments and column 4 entries in no statistically significant effects on 5(1), 5(2), 5(4), 5(6) and 5(7), above. reproductive parameters, and that the assessment of sex ratio is uncertain due to the purposely removed males. Regarding the VTG results, we do not supporting publication 2014:EN

18 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) in order to obtain the optimal numbers for the appropriate spawning groups (4 females, 2 males) and the percent males in the control culled group was also high. Since more females than males are required for the formation of reproductive groups, that more males were culled (based on secondary sex characteristics, when available) stands to reason. We note that in instances of aromatase inhibition (perchloraz, fadrozole, fenarimol) in FHM, changes in both sex ratio (male skewing) and female vitellogenin (lower levels) are normally seen, whereas no effects on vitellogenin were noted in this test. We also note that there was no effect on sex ratio in the randomly culled group at dph (when histological sex differentiation is certainly possible for FHM) or in the total dph group. Regarding the elimination of outliers from the statistical analysis of several reproduction-related endpoints, we note that the composition of the spawning group can greatly affect the agree that there were no effects, since due to the low statistical power no conclusion can be drawn. This is also the case for other reproductive parameters, which from the German analysis showed minimum detectable differences up to 95%. Therefore, from our point of view no conclusion can be drawn on possible endocrine disruption from the available data. The procedure following this (lack of) conclusion is not clear to us at this stage. Further discussions may be needed. s scientific views on the specific supporting publication 2014:EN

19 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Aquatic organisms (B.9.2) reproductive endpoints in this test. Additional males result in increased aggression/stress and lower spawning rates ( 1982), thus we do not consider effects in those groups as indicative, necessarily, of an endocrine-related effect on reproductive endpoints. s scientific views on the specific Considering that in any case a lower endpoint is used for the risk assessment, the discussion may be moot, since we also feel strongly that no one test can be used to eliminate or confirm the possibility of endocrine disruption, but that, for example, a WoE including all available data should be presented and considered in order to assist in making conclusions as regards endocrine disruption. Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) 5(16) Addendum for, : agrees with RMS s conclusion and the derived NOEC RMS: Agreed. s scientific views on the specific There is no conflict between views of the and the RMS. However see supporting publication 2014:EN

20 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) B Earthworms study by Friedrich, (17) Addendum for, B Earthworms study by Friedrich, 2006 on the metabolite 5(18) Addendum for, B Earthworms study by Friedrich, 2006 on the formulation (0.5 mg/kg). agrees that 15% or 21% changes from the control should be considered as biologically relevant even if statistically not significant (even the 7.6% change in the reproduction observed at 0.5 mg/kg level is not necessarily negligible; in this case however considered as acceptable). : also noted that the performance of the worms at the highest tested concentration was somewhat lower than in the control for both the biomass and the reproduction, but overall agrees with RMS s conclusion and the derived NOEC (9.6 mg/kg). Could you please confirm that the results from the reference treated groups were in line with the expected effects? : notes that the performance of the worms at the highest tested concentration was lower with clearly more than 10 % for both the biomass and the reproduction than in the control ( calculated 12.7% and 12.2% differences, respectively from the reported data in the table B RMS: Effects from the toxic reference Addressed. were 61 and 100% at the two tested concentrations respectively, confirming the validity of the test method. This information has been included in the revised Addendum. s scientific views on the specific FR comment in 5(20). is of the opinion that using the more precautionary endpoint of 0.5 mg/kg (as suggested by the RMS) is justified. See also comment in 5(22). Additional information was included in the updated addendum (Sweden, 2014a), which is helpful. RMS: Agree that the observed effect on Addressed. reproduction may be biologically significant. Hence the NOEC can be The endpoint has been changed to a set to 15.2 mg formulation/kg dw more conservative one, in line with the soil. This information has been original comment. Moreover, additional included in the revised Addendum. information was included in the updated Effects from the toxic reference were 61 addendum (Sweden, 2014a) in relation supporting publication 2014:EN

21 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) 5(19) Addendum for, B RA for soil macro-organisms and RMS evaluation on the biomass issue on page ). Therefore the NOEC of 22.8 mg/kg is questionable (although does not change the RA). Could you please confirm that the results from the reference treated groups were in line with the expected effects? : agrees with the risk assessment of the RMS on the active substance. Could the RMS confirm that the observed increase in biomass is probably linked to the formulation (or more than to the a.s.)? and 100% at the two tested concentrations respectively, confirming the validity of the test method. This information has been included in the revised Addendum. RMS: From our opinion there is not sufficient evidence that the biomass effects are more strongly linked to the formulation than to the active ingredient. Therefore, we would propose to use the lowest value (active ingredient or formulation for each representative use) for the risk assessment. Possibly, this would need further discussion (see also 5(21)). s scientific views on the specific to the reference controls. The RMS is of the opinion that there is insufficient evidence that the biomass effects are more strongly linked to the formulation than to the active ingredient. just notes that this phenomenon was more frequently observed in formulation studies. On the other hand, more formulation studies were available. Nevertheless, there is an agreement on the risk assessment provided by the RMS. It is noted that lowering of the endpoint for the formulation SCORE was agreed in comment 5(18). As a consequence, the related TER values should also be lowered (these were not corrected in the updated addendum (Sweden, 2014a)). However the conclusion on the risk would not change (i.e. TERs > 5). supporting publication 2014:EN

22 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) 5(20) Vol.3, B , p FR: No statistically significant effects on biomass and reproduction were observed at 1 mg/kg dw. Therefore, France is of the opinion to use the NOEC of 1 mg/kg dw. 5(21) Vol.3, B , p FR: France agrees with the higher tier refinement of the risk assessment using the sub-lethal study with the formulation SCORE 250EC. 5(22) Volume 3, B DK: Because of the 21% effect on reproduction at 1.0 mg/kg dw, DK agrees with RMS that the NOEC for reproduction is 0.5 mg/kg dw. DK also agrees with RMS that the NOEC must be divided by 2 for the risk assessment (NOEC/2 = 0.25 s scientific views on the specific See also comment in 5(21), below. RMS: Similar cases were discussed by supports the view of the RMS. the ecotox expert meeting in May See conclusion on this point in comment 2014, and it was concluded that 5(16). statistical significance is not the sole indication of a biologically See also comment in 5(22). significant effect. The risk assessment presented in the Addendum is consistent with these discussions. RMS: From our opinion there is not See column 4 entry at comment 5(19), sufficient evidence that the biomass above. effects are more strongly linked to the formulation than to the active ingredient. Therefore, we would propose to use the lowest value (active ingredient or formulation for each representative use) for the risk assessment. Possibly, this would need further discussion. RMS: Agreed. See column 4 entry at comment 5(16). See also comment in 5(20). supporting publication 2014:EN

23 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) 5(23) B Sublethal effects on earthworms 5(24) B Sublethal effects on earthworms mg/kg dw). Applicant: The RMS has proposed a NOEC (based on reproduction) of 0.5 mg/kg based on biologically significant (but not statistically significant) effects at 1 mg/kg (the NOEC reported in the study). Syngenta would propose that in this case, the EC10 value of 0.65 mg/kg is a more appropriate value to use in the risk assessment. The NOEC based on robust statistical analysis is 1.0 mg/kg, therefore using an EC10 value of 0.65 mg/kg (to account for the apparent biologically significant effects) can be seen as the more conservative option. Applicant: The RMS has proposed that since the Log Pow for difenoconazole is >2, the NOEC should be divided by 2, despite the test being conducted in 5% peat. Regarding the conduct of earthworm studies and the subsequent correction of the endpoint, Syngenta would like to reference the EPPO Standards: Environmental Risk Assessment Scheme for Plant Protection Products, where the relationship RMS: Similar cases were discussed by the ecotox expert meeting in May 2014, and it was concluded that statistical significance is not the sole indication of a biologically significant effect. The risk assessment presented in the Addendum is consistent with these discussions. Use of EC10 as an alternative was considered an option, but would in this case not chance the conclusion to a significant extent. RMS: The issue on use of correction factor has been discussed in previous expert meetings. It has been agreed that for compounds with log Pow>2, a correction factor of 2 should always be used even if the test was conducted with lower organic carbon content. The proposed risk assessment in the addendum is consistent with this approach. s scientific views on the specific agrees with the RMS column 3 entries. See conclusion on this point in comment 5(16), which considers also that the final conclusion would not be changed by using an EC 10 value for the representative uses. However this approach might be used as an alternative for other uses. See also comments in 5(20) and 5(22). agrees with the RMS that the correction factor should be used (until better guidance is available). The quoted expert discussion can be found in the general report of the Pesticides Peer Review Meeting 91. Notes: It should be noted that the quoted texts from do not suggest oneto-one linearity. The SANCO guidance (European supporting publication 2014:EN

24 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) between adsorption and soil organic matter content is discussed: For chemicals with log Pow > 2, sorption is expected to be linearly related to soil organic matter content. A factor of 2 (division of all toxicity end-points) is used to correct the toxicity data from earthworm studies conducted in artificial soil. This derives from the fact that the artificial soil of OECD guideline 207 has a peat content of 10%, whereas a typical agricultural soil has not more than 5% organic matter. The use of 5% organic matter soil is also supported under point 11 of OECD 222; Earthworm reproduction Test where it is noted: The organic carbon content of the artificial soil may be reduced, for example by lowering the peat content to 4-5% and increasing the sand content accordingly. By such a reduction in organic carbon content, the possibilities of adsorption of test chemical to the soil (organic carbon) may be decreased and the availability of the test chemical to the worms may increase. s scientific views on the specific Commission, 2002) does not describe any case for chemicals with logkow > 2 when the use of the correction factor might be skipped, except if it is demonstrated that the toxicity is independent of the OC content. Using natural soils is indeed an option. Please note that if tests on representative natural soils were available, the foc content of these soils were much less than 5 %. Also the nature of the OM (or OC) thus the adsorption and therefore the bioavailability was different. In this case would agree to skip the correction. supporting publication 2014:EN

25 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) The Guidance Document on Terrestrial Ecotoxicology (SANCO/10329/2002) also makes reference to dividing an endpoint by 2 where logkow is greater than 2 as it states: The artificial substrate of the earthworm laboratory tests has a higher foc than many natural soils, so it could be expected that the LC50 or NOEC would be lower if the test were conducted in natural soil. Again endorsing the opinion that conducting studies in soil with reduced organic matter, is a worst case option. Syngenta proposes that each of these guidance documents give a clear suggestion that organic matter content is an important factor in an earthworm test and that any reduction is expected to give a more conservative endpoint. To further support the exclusion of the correction factor when 5% peat has been utilised, the risk assessment scheme described in the Guidance Document on Terrestrial Ecotoxicology goes on to give the s scientific views on the specific supporting publication 2014:EN

26 Outcome of the consultation on the pesticide risk assessment of for the active substance difenoconazole Earthworms and other soil non-target organisms (macro and micro) (B.9.6, B.9.7 and B.9.8) 5(25) Addendum for, B Earthworms 5(26) Addendum for, B Collembolan study by Friedrich, 2006b on the metabolite option of refining the effects side of the risk assessment by conducting the earthworm test in natural soil (i.e. a soil with a lower organic matter content). Since this would likely give a more conservative endpoint, the implication is that the endpoint would not be divided by two in order to aid the refinement. Therefore, Syngenta would strongly suggest that the existing NOEC from the earthworm study is not corrected before being used in the risk assessment. NL: no comment chronic earthworm RMS: Thanks. Addressed. : does not necessarily disagree with the conclusion and the NOEC, just notes that the reproduction success was lower in all treated levels ( 10% from 0.3 mg/kg) than in the control in this study. RMS: Noted. s scientific views on the specific Addressed. There was no disagreement which would require further actions. supporting publication 2014:EN