European Food Safety Authority (EFSA)

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1 TECHNICAL REPORT APPROVED: 10 August 2016 Outcome of the consultation with Member States, the applicant and EFSA on the pesticide risk assessment for triazole derivative metabolites in light of confirmatory data Abstract European Food Safety Authority (EFSA) The European Food Safety Authority (EFSA) was asked by the European Commission to provide scientific assistance with respect to the risk assessment for triazole derivative metabolites in light of confirmatory data requested following approval in accordance with Article 6(1) of Directive 91/414/EEC and Article 6(f) of Regulation (EC) No 1107/2009. In this context EFSA s scientific views on the specific points raised during the commenting phase conducted with Member States, the applicant and EFSA on the confirmatory data and their use in the risk assessment for triazole derivative metabolites are presented. The current report summarises the outcome of the consultation process organised by the rapporteur Member State the United Kingdom and presents EFSA s scientific views and conclusions on the individual comments received. European Food Safety Authority, 2016 Keywords: triazole derivative metabolites, peer review, confirmatory data, risk assessment, pesticide, fungicide Requestor: European Commission Question number: EFSA-Q Correspondence: pesticides.peerreview@efsa.europa.eu EFSA Supporting publication 2016:EN-1080

2 Suggested citation: EFSA (European Food Safety Authority), Technical report on the outcome of the consultation with Member States, the applicant and EFSA on the pesticide risk assessment for triazole derivative metabolites in light of confirmatory data. EFSA supporting publication 2016:EN pp. European Food Safety Authority, 2016 Reproduction is authorised provided the source is acknowledged. 2 EFSA Supporting publication 2016:EN-1080

3 Summary During the second and third stages of the European s pesticides review programme [referred to in Article 8(2) of Council Directive 91/414/EEC], data gaps were identified, and confirmatory data requirements set, for a number of triazole fungicides. A group of manufactures of the triazolecontaining fungicides (BASF SE, Bayer CropScience AG, BASF, DOW Agrosciences LLC, Isagro S.p.A., and Syngenta Crop Protection AG) formed an industry taskforce, known as the Triazole Derivative Metabolite Group (TDMG) which made a joint submission of new toxicological, metabolism and residues data to meet these data requirements. The new data submitted by the TDMG were intended to support the derivation of revised reference values for each of the triazole derivative metabolites (TDMs) in order to prevent the need for the application of the additional assessment factors as concluded at Pesticides experts meeting (PRAPeR 14, 2007), and to also set reference values for the metabolite triazole lactic acid (TLA). These new reference values could then be used in consumer risk assessments to address the confirmatory data requirements set for the various triazole fungicides. The TDMG submitted data, which were evaluated by the designated rapporteur Member State (RMS), the United Kingdom, in the form of an addendum to the draft assessment reports on various triazole containing pesticides. In compliance with guidance document SANCO 5634/2009-rev.6.1, the RMS distributed the addendum to the Member States, the applicant and EFSA for comments on 1 December The RMS collated all comments in the format of a reporting table, which was submitted to EFSA on 24 May EFSA 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 RMS, the United Kingdom, and presents EFSA s scientific views and conclusions on the individual comments received. Several issues were identified which would need further consideration and Member States experts consultation in the areas of mammalian toxicology and residues. In particular, in regards to mammalian toxicology, issues in reproductive toxicity and other toxicological studies were highlighted and need to be further discussed. The overall consumer exposure assessment with regards to the TDMs cannot be concluded on considering that the nature and the magnitude of the TDMs residues in primary crops, rotational crops and in products of animal origin need to be further discussed in an experts consultation. 3 EFSA Supporting publication 2016:EN-1080

4 Table of contents Abstract... 1 Summary Introduction Background and Terms of Reference as provided by the requestor Interpretation of the Terms of Reference Assessment... 6 Documentation provided to EFSA... 6 References... 6 Abbreviations... 7 Appendix A Collation of comments from Member States, applicant and EFSA on the pesticide risk assessment for the triazole derivative metabolites in light of confirmatory data and the conclusions drawn by EFSA on the specific points raised... 9 Appendix B Used compound codes EFSA Supporting publication 2016:EN-1080

5 1. Introduction 1.1. Background and Terms of Reference as provided by the requestor During the second and third stages of the European s pesticides review programme [referred to in Article 8(2) of Council Directive 91/414/EEC 1 ], data gaps were identified, and confirmatory data requirements set, for a number of triazole fungicides. A group of manufactures of the triazolecontaining fungicides (BASF SE, Bayer CropScience AG, BASF, DOW Agrosciences LLC, Isagro S.p.A., and Syngenta Crop Protection AG) formed an industry taskforce, known as the Triazole Derivative Metabolite Group (TDMG) which made a joint submission of new toxicological, metabolism and residues data to meet these data requirements. The new data submitted by the TDMG were intended to support the derivation of revised reference values for each of the triazole derivative metabolites (TDMs) in order to prevent the need for the application of the additional assessment factors as concluded at Pesticides experts meeting (PRAPeR 14, 2007), and to also set reference values for the metabolite triazole lactic acid (TLA). These new reference values could then be used in consumer risk assessments to address the confirmatory data requirements set for the various triazole fungicides. Indeed, experts evaluated the existing toxicological data for the TDMs and set reference values for T and TA. For TAA only very limited data existed and experts proposed to use the lower endpoints of T for TAA. Finally, TLA was not considered. The TDMG submitted data, which were evaluated by the designated rapporteur Member State (RMS), the United Kingdom, in the form of an addendum to the draft assessment reports on various triazole containing pesticides. In compliance with guidance document SANCO 5634/2009-rev.6.1, the RMS distributed the addendum to Member States, the applicant and EFSA for comments on 01 December The RMS collated all comments in the format of a reporting table, which was submitted to EFSA on 24 May EFSA 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 RMS, the United Kingdom, and presents EFSA s scientific views and conclusions on the individual comments received Interpretation of the Terms of Reference On 22 December 2014 the European Commission requested EFSA to provide scientific assistance with respect to the risk assessment of confirmatory data following approval of an active substance in accordance with Article 6(1) of Directive 91/414/EEC and Article 6(f) of Regulation (EC) No 1107/2009. EFSA s scientific views on the specific points raised during the commenting phase conducted with Member States, the applicant and EFSA on the risk assessment of confirmatory data for triazole derivative metabolites are presented. To this end, a technical report containing the finalised reporting table is being prepared by EFSA. The deadline for providing the finalised report is 10 August On the basis of the reporting table, the European Commission may decide to further consult EFSA to conduct a full or focused peer review and to provide its conclusions on certain specific points. 1 Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market. OJ L 230, , p EFSA Supporting publication 2016:EN-1080

6 2. Assessment The comments received on the pesticide risk assessment for triazole derivative metabolites in light of confirmatory data and the conclusions drawn by the EFSA are presented in the format of a reporting table. They are summarised in column 2 of the reporting table. The RMS considerations of the comments are provided in column 3, while EFSA s scientific views and conclusions are outlined in column 4 of the table. The finalised reporting table is provided in Appendix A of this report. Documentation provided to EFSA 1. United Kingdom, Addendum to the assessment reports on various triazole containing pesticides, confirmatory data, November 2015 updated in May Available online: 2. United Kingdom, Reporting table, comments on the pesticide risk assessment for triazole derivative metabolites in light of confirmatory data, May References European Commission, Residues: guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (Part A, Section 4) and Annex III (Part A, Section 5) of Directive 91/414. SANCO/3029/99-rev. 4, 11 July 2000 European Commission, Guidance Document on Assessment of the Relevance of Metabolites in Groundwater of Substances Regulated under Council Directive 91/414/EEC. SANCO/221/2000-rev. 10 final, 25 February 2003 European Commission, Guidance document on the procedures for submission and assessment of confirmatory information following approval of an active substance in accordance with Regulation (EC) No 1107/2009. SANCO 5634/2009-rev EFSA Supporting publication 2016:EN-1080

7 Abbreviations a.s. ADI AF AOEL ARfD bw CAS CNS CPK DAR FOB GAP DG SANCO EU GAP GLP HPLC HR LC- MS/MS LDH LH MCH MCV MoA MRL MS NEU NOAEL OECD OSR PEC gw PRAPeR PRIMo RD RMS SEU active substance acceptable daily intake assessment factor acceptable operator exposure level acute reference dose body weight Chemical Abstracts Service central nervous system creatine phosphokinase draft assessment report functional observation battery good agricultural practice European Commission Directorate General Health and Consumers European Union good agricultural practice good laboratory practices high-pressure liquid chromatography or high-performance liquid chromatography hazard rate liquid chromatography with tandem mass spectrometry lactate dehydrogenase luteinizing hormone mean corpuscular hemoglobin mean corpuscular volume mode of action maximum residue level Member State northern Europe no observed adverse effect level Organisation for Economic Co-operation and Development oilseed rape predicted environmental concentration in groundwater Pesticides Risk Assessment Peer Review Pesticide Residue Intake Model residue definition rapporteur Member State southern Europe 7 EFSA Supporting publication 2016:EN-1080

8 SF STMR TDM TRR safety factor supervised trials median residue triazole derivative metabolites total radioactive residue 8 EFSA Supporting publication 2016:EN-1080

9 Appendix A Collation of comments from Member States, applicant and EFSA on the pesticide risk assessment for the triazole derivative metabolites in light of confirmatory data and the conclusions drawn by EFSA on the specific points raised 0. General General No. Column 1 Reference to addendum to assessment report Column 2 Comments from Member States / applicant / EFSA Column 3 Evaluation by rapporteur Member State Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 0(1) Addendum Nov Table of content BE: [editorial comment] Considering the length of the addendum and the fact that this addendum will have to be consulted regularly at MS level (for comparison of GAPs/results), an automatically generated Table of contents (with integrated hyperlinks) would be highly appreciated. RMS (UK): Thank you for your comment, but whilst integrated hyperlinking would be nice we do not consider it essential at this stage (and, given it is such a large document, it would involve a lot of resource). 0(2) Addendum Nov Introduction and background (3 rd paragraph & Table 1) BE: It should be noted that also for the triazole fungicide Bromuconazole, confirmatory data requirements were set in the framework of the EU review under Dir. 91/414/EEC. Table 1 is thus incomplete. Sumitomo Chemical Agro Europe S.A.S. (SCAE), who supported approval of bromuconazole, is not a member of the TDMG (according to SCAE due to the prohibitive fee that was required) and confirmatory data submitted by SCAE were already assessed by RMS BE (see DAR addendum Oct. 2013), RMS (UK): Table 1 only outlines the triazole pesticides covered by the TDMG and hence bromuconazole is not included. The UK was only in a position to evaluate data submitted by the TDMG. Data requirement: Applicant to provide data available in bromuconazole dossier and assessed by BE. 9 EFSA Supporting publication 2016:EN-1080

10 leading to the identification of several data gaps. In this context, it also remains to be clarified whether studies on toxicology (and residues) owned by the TDMG can be relied upon by other (generic) authorisation holders (cf. data protection/access). 0(3) Addendum Nov Introduction and background (Table 1) BE: Concerning the a.s. myclobutanil, it should be noted that confirmatory data related to i.a. the question of TDM occurrence in following crops and/or growing seasons (CRC study and) was assessed by RMS BE (cf. myclobutanil DAR addendum July 2013). It may be useful to mention this and clarify possible interference between the UK assessment and the previous assessment. However, we note that a field rotational crop study for myclobutanil was not included in the TDMG data package assessed by UK. RMS (UK): The UK has only considered the data provided in the submission to the UK. It is not possible in this assessment to start including/referencing assessments conducted by other MS, particularly as at the time of the UK assessment a final decision on the confirmatory data assessment of other triazole pesticides had not been reached. However, the EU Commission may need to mandate EFSA to make a full consideration of all the available assessments on the triazole pesticides. Data requirement: Applicant to provide the confirmatory data related to the occurrence of the TDMs in rotational crops for myclobutanil and that were assessed by BE. Open point: RMS to report the assessment of confirmatory data for TDMs for myclobutanil in a revised Addendum. 0(4) Page 9: Methods of Analysis NOT (TDMG): The TDMG believe that sufficient data have been provided to fully support the yield and precision of the derivatisation step in methods ARAM 217, 2280 and RMS (UK): The information/data provided were fully considered and as outlined in the assessment were deemed not to be sufficient. Further data are required. The yield and precision of the derivatisation step in methods 2280 and 2281 were not demonstrated, however the validation data of the whole method show that its validation can be considered acceptable. The yield and precision of the derivatisation step in method ARAM 217 was not demonstrated. The number of recovery 10 EFSA Supporting publication 2016:EN-1080

11 determinations at each fortification level was not in accordance with EU guidance SANCO 3029/99 rev.4, however, when considering all the validation data and the procedural recovery data available as a whole it can be concluded that the method allows accurate determination of TAA. 0(5) Page 9/10: Application of several triazole fungicides within one season NOT (TDMG): Although the considerable set of supervised field trials performed (>1200 trials) does not include trials in which several triazole fungicides were deliberately applied within one season in a sequential manner, the TDMG considers that the residue levels which result from this scenario are covered by the submitted trials and the dietary exposure assessment. RMS (UK): The UK presented the argumentation from the TDMG and as outlined in the assessment the UK does not agree with the position taken by the TDMG. A full consideration of the potential residue levels arising from the use of several different triazole pesticides in the same season is still required. See experts consultation 3(30) 0(6) Page 9/10: Livestock feeding study for TLA NOT (TDMG): According to CRD s evaluation, the approach to assess residue levels arising in food of animal origin from T and TLA requires further consideration. The TDMG does not agree there is a need to conduct additional feeding studies with TLA as they will not significantly change the conclusions of the consumer safety assessment (max 2.6% ADI) and that reasoned arguments have been provided as to why the existing data can be used to evaluate levels of TLA in animal commodities. RMS (UK): The full details of the approach taken by the task force have been outlined in a transparent way for MS and EFSA to consider. The approach taken is unusual and it is therefore appropriate that it is fully considered by MS and EFSA. The overall livestock exposure assessment should be discussed in an experts meeting. See data requirement 3(20), experts consultations 3(23), 3(24), 3(28) EFSA Supporting publication 2016:EN-1080

12 0(7) Page 9/10: Livestock feeding study for parent triazoles NOT (BASF): In Appendix E, it is stated that livestock information on epoxiconazole is not available to CRD. The relevant data for ruminants and poultry have been submitted to Germany. The studies provided are summarized in Addendum 8 to the Draft Assessment Report (18 April 2015). The relevant EFSA Peer review report was published in June 2015 (doi: /j.efsa ). RMS (UK): The details were not available to the UK at the time the assessment was conducted. The evaluation cannot be updated with this information at this stage in the process. Data requirement: Applicant to provide the epoxiconazole metabolism data addressing the fate of TDMs in livestock matrices. Open point: RMS to report and assess the epoxiconazole metabolism data addressing the fate of TDMs in livestock matrices. 0(8) Page 9/10: GAP compliance of residue trials NOT (TDMG): We agree that GAP compliance of residue trials is important, especially when they are used for deriving MRLs. However in the case of the TDMs, the situation is different. In many cases the magnitude of TDM residues was not found to be strongly related to the GAP and was also linked to triazole fungicide uses in previous seasons and subsequent uptake from soil. Based on this the TDMG considers GAP compliance of lower importance for the assessment of TDM residues. RMS (UK): The UK agrees that a clear link between residue levels of the TDM and a specific GAP could not be established. In addition, residue levels in untreated control samples were often higher. However, it is important to recognise that specific confirmatory data requirements were set for the triazole pesticides and these related to a specific representative GAP. Where specific residue trials data for the TDM are not available to cover a representative GAP considered for an approval then for completeness and transparency this must be highlighted. See experts consultation 3(30). This issue needs to be fully considered by MS and EFSA. 0(9) Page 9/10: Agreed approach to future NOT (TDMG): We appreciate to have clearly defined data requirements for RMS (UK): The UK assessment does not exclude the possibility of using See experts consultation 3(30) EFSA Supporting publication 2016:EN-1080

13 data requirements the submission of TDM data, but from our point of view an approach based on the intended GAP for a specific product in each crop is unnecessary. Because of this a broad extrapolation approach was suggested by TDMG and also followed by CRD in their assessment. Any process for requiring more residue data (if needed at all) should be more based on active substance and crop groups rather than on individual products within residue zones. Such a procedure would also considerably reduce workload for evaluation at individual member state level. The exposure estimates performed in context of this evaluation clearly indicate the low possibility of having any consumer risk concerns which also reduce the need for additional residue data. the broader extrapolations as used in the assessment. It is important that at the product authorisation stage that it is clearly demonstrated that the intended uses are fully covered with respect to TDM and if not then further trials data will be required. It is important that MS and EFSA fully consider the available data and an EU agreed position is reached on how all registered uses are supported in terms of the TDM. It might be feasible to consider the data required to support GAPs as part of any future MRL reviews, and this would reduce the work load at a MS level. However, such an approach would not be possible where the article 12 MRL review has already been conducted. This evaluation is specifically to address the confirmatory data requirements set for the approval of various triazole pesticides. In this context it is not feasible for the UK to make an assessment of the acceptability of the data for all authorised uses in the EU as well as import tolerances. The article 12 MRL review is a significant amount of work that involves the input from the RMS, other MS, IND and EFSA to collect all the info on GAPs. Such an approach 13 EFSA Supporting publication 2016:EN-1080

14 can be taken to ensure all registered uses are supported but it will need to be fully considered as the resource for one MS will be significant. 0(10) Addendum Nov A provisional outline of the use of this assessment in the future (p. 498 ff.) BE: The proposal for a harmonised approach to deal with the authorisations of triazole plant protection products at national level is appreciated. However, in this context, it should also be clarified how authorisations of authorisation holders that have no access to the data used in this assessment should be dealt with (e.g. companies not included in Task force, generic applicants). This is important, because the proposed stepwise decision scheme is based on comparison of GAPs and simple reference to this TDM assessment in case the GAP is covered (cf. step 1). RMS (UK): The data protection and data access to confirmatory data is a generic issue and not specific to this assessment. 0(11) Addendum Nov A provisional outline of the use of this assessment in the future (Figure 3, p. 502) BE: To be consistent with the step 1 and step 2 and the explanation of the approach, we presume the flow diagram outlining step 3 (Figure 3) should be amended accordingly, as follows (entry in central box): Are the total TDM residue levels in the crop, in the processed commodities and in products of animal origin below those considered in the TDM assessment? (See Appendix E, Table RMS (UK): No changes are required at this time and it is made clear in the UK assessment this is only a proposal and an agreed EU approach is required. See experts consultation 3(30) EFSA Supporting publication 2016:EN-1080

15 7.7-1) Cross-references to the appropriate tables for comparison of results for processed commodities and products of animal origin still need to be added. 0(12) Addendum Nov Reference lists (p. 503) BE: The list of References relied on for the confirmatory data submission needs to be completed for section B.5 (methods of analysis) and B.7 (Residues). Currently, no references are reported in the list for these sections. RMS (UK): The data list is in preparation and will be uploaded in due course. Open point: RMS to complete the list of References relied on for sections B.5 (methods of analysis) and B.7 (Residues). Addendum to be revised accordingly. 1. Methods of analysis Methods of analysis No. Column 1 Reference to addendum to assessment report Column 2 Comments from Member States / applicant / EFSA Column 3 Evaluation by rapporteur Member State Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 1(1) References relied on for the confirmatoy data submission DE: References for section B.5 are missing. RMS, please add. RMS (UK): The data list is in preparation and will be uploaded in due course. 1(2) B , analytical method to generate residue data in plants, specificity of the methods DE: The correction of blank values for calculation of recoveries during method validation is an acceptable approach. Nevertheless, the range of detectable residues in untreated controls should be reported to estimate the lowest detectable residues above the blank values. RMS, RMS (UK): The UK has made a full and robust assessment of the methods and where relevant outlined shortcomings in the methods and clear data gaps identified. Adding the requested information would not impact on the outcome of the assessment and is not warranted EFSA Supporting publication 2016:EN-1080

16 please add. 1(3) B , Methods for risk assessment in plants and B Products of animal origin DE: A critical assessment of the methods would be useful. Critical aspects influencing the performance of the methods, i.e. matrix effects in MS, false-positive signals and interfering signals in the chromatograms should be discussed. RMS, please add. RMS (UK): The UK has made a full and robust assessment of all the methods used in accordance with the appropriate guidance. Further details are not required in the assessment and would add no value to the evaluation. 1(4) B method 01062; B method 01062/M001; B method 01062/M002, B method GRM053.01A, B method DE: Which HPLC columns were used for the determination of 1,2,4-triazole, triazole alanine and triazole acetic acid? RMS, please add the information. RMS (UK): The specific column used is not a data requirement and will not impact on the outcome of the risk assessment. MS are reminded that comments should be restricted to major issues. Addressed: The requirements under 1.5 of SANCO/3029/99 rev.4 relating to instrumentation might include also the column, however there is no agreed procedure how to report the methods in the evaluations done by MS. 1(5) B method 536/0 DE: Is the inital extract injected in the LC-MS/MS? Which column is used? RMS, please add the information. RMS (UK): Such minor details will not impact on the outcome of the validity of the method or risk assessment. MS are reminded that comments should be restricted to major issues. 1(6) B method D0604 and B method D0508 DE: An acid hydrolysis step was performed for the analysis of triazole alanine in fatty matrices. What is the reason for the hydrolysis step? RMS, please add. RMS (UK): Acid hydrolysis of fatty matrices is a standard approach to facilitate complete extraction and for these analytes such an approach was necessary. Details of the method development procedures undertaken prior to a method being validated are not data requirements so further 16 EFSA Supporting publication 2016:EN-1080

17 1(7) B Page 23/24 B Page 24/25 B Page 33 Conclusion on the methods used for the generation of preauthorisation data NOT (TDMG): The TDMG believe that sufficient data have been provided to fully support the yield and precision of the derivatisation step in methods ARAM 217, 2280 and information is not necessary. RMS (UK): The information/data provided were fully considered and as outlined in the assessment were deemed not to be sufficient. Further data are required. Addressed: The yield and precision of the derivatisation step in methods 2280 and 2281 were not demonstrated, however the validation data of the whole method shows that its validation can be considered acceptable. The yield and precision of the derivatisation step in method ARAM 217 was not demonstrated. The number of recovery determinations at each fortification level was not in accordance with EU guidance SANCO 3029/99 rev.4, however, when considering all the validation data and the procedural recovery data available as a whole it can be concluded that the method allows accurate determination of TAA. 1(8) Addendum, B Method 2280and 2281, p. 23 EFSA: The validation for the determination of residues of triazolyl alanine and triazolyl acetic acid in/on plant commodities is not in accordance with EU guidance SANCO 3029/99 rev.4, in support of pre-registration studies. As the method used a derivatisation step in which standards of the analytes were also derivatised, the efficiency of the derivatisation should be assessed. RMS (UK): The UK notes that EFSA agree with the UK assessment that the method has not been fully validated and hence a data gap has been identified. Addressed: The yield and precision of the derivatisation step in methods 2280 and 2281 were not demonstrated, however the validation data of the whole method shows that its validation can be considered acceptable. 1(9) Addendum, B Method 217, p. 25 EFSA: The validation for the determination of residues of triazolyl RMS (UK): The UK notes that EFSA agree with the UK assessment Addressed: The yield and precision of the 17 EFSA Supporting publication 2016:EN-1080

18 alanine and triazolyl acetic acid in/on plant commodities is not in accordance with EU guidance SANCO 3029/99 rev.4, in support of pre-registration studies. As the method used a derivatisation step in which standards of the analytes were also derivatised, the efficiency of the derivatisation should be assessed. that the method has not been fully validated and hence a data gap has been identified. derivatisation step in method ARAM 217 was not demonstrated. The number of recovery determinations at each fortification level was not in accordance with EU guidance SANCO 3029/99 rev.4, however, when considering all the validation data and the procedural recovery data available as a whole it can be concluded that the method allows accurate determination of TAA. 1(10) Addendum, Toxicological studies, B , B , B , B , B , B , EFSA: there is no information about the methods used for the determination of the achieved concentrations of triazole acetic acid in analysed samples of test diet used on the studies. RMS (UK): It must be taken into consideration that the confirmatory data are being assessed in accordance with Regulation (EC) 544/2011 and not Regulation (EC) 283/2013. Based on this then it would be appropriate to only consider requiring validation data for studies critical to an endpoint rather than all toxicological studies. Addressed The reference values used for the risk assessment for TAA originate from investigations with 1,2,4-triazole. So currently these studies are not critical to an endpoint supporting the reference value. Therefore currently high confidence in analyses results of the diet of the investigations is not essential. 1(11) Addendum, Toxicological studies, B , p.63 EFSA: there is no information about the methods used for the determination of the achieved concentrations of triazole alanine in analysed samples of test diet used on the studies and no information about the LC-MS/MS method used for the analysis of the urine and faeces. RMS (UK): It must be taken into consideration that the confirmatory data are being assessed in accordance with Regulation (EC) 544/2011 and not Regulation (EC) 283/2013. Based on this then it would be appropriate to only consider requiring validation data for studies critical to an endpoint 18 EFSA Supporting publication 2016:EN-1080

19 rather than all toxicological studies. 1(12) Addendum, Toxicological studies, B , B EFSA: there is no information about the methods used for the determination of the achieved concentrations of triazole alanine in analysed samples of test diet used on the studies. RMS (UK): It must be taken into consideration that the confirmatory data are being assessed in accordance with Regulation (EC) 544/2011 and not Regulation (EC) 283/2013. Based on this then it would be appropriate to only consider requiring validation data for studies critical to an endpoint rather than all toxicological studies. 1(13) Addendum, Toxicological studies, B EFSA: there is no information about the methods used for the determination of the achieved concentrations of triazole in analysed samples of test diet used on the studies. RMS (UK): It must be taken into consideration that the confirmatory data are being assessed in accordance with Regulation (EC) 544/2011 and not Regulation (EC) 283/2013. Based on this then it would be appropriate to only consider requiring validation data for studies critical to an endpoint rather than all toxicological studies EFSA Supporting publication 2016:EN-1080

20 2. Mammalian toxicology Genotoxicity No. Column 1 Reference to addendum to assessment report Column 2 Comments from Member States / applicant / EFSA Column 3 Evaluation by rapporteur Member State Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 2(1) p p SE: For an independent assessment of the new genotoxicity studies, please include results (tabulated data) - currently, the reviewer must rely on the RMS opinion. RMS (UK): The idea of the RMS is to summarise the data on behalf of others. Where there are equivocal findings we have presented data in the DAR. We can confirm that the studies are negative. Open point: In studies B (in vitro chromosome aberration assay) and B (in vitro mammalian cell gene mutation assay) for 1,2,4-triazole and in studies B (bacterial mutation assay-ames), B (in vitro mammalian cell gene mutation assay) and B (in vitro chromosome aberration mammalian assay) for triazole lactic acid, RMS to include the tabled results in a revised RAR. Reproductive toxicity No. Column 1 Reference to addendum to assessment report Column 2 Comments from Member States / applicant / EFSA Column 3 Evaluation by rapporteur Member State Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 2(2) B month study in rats, IIA 5.8/09 A chronic (12 month) toxicity testing study in BE: taking into account the slight increase of mineralisation of the GIT, and also the optic nerve degeneration at the top-dose, toxicity NOAEL is RMS (UK): The optic nerve findings are not present in the neurotoxicity group and therefore are considered not to be treatment related. We note that this The increased mineralisation of the GIT, the optic nerve degeneration and the NOAEL of the 12-month study in rats with triazole alanine should be 20 EFSA Supporting publication 2016:EN-1080

21 the Wistar rat with triazole alanine precautionously set at 300 mg/kg b.w./d. It is observed that, while all groups experienced the same retro-orbital bleeding, the effects of it are only seen at top-dose, perhaps indicating a weakening of this group. proposal will not alter the derived reference doses. discussed and agreed in an experts meeting (see also 2(20)). 2(3) B Developmental toxicity study in rabbits, IIA 5.8/10 Triazole alanine oral (stomach tube) developmental toxicity study in rabbits. BE: proposal: Maternal NOAEL = 100 mg/kg b.w./d, based upon soft/liquid faeces, b.w. effects at 250 mg/kg/day. Developmental NOAEL = 30 mg/kg b.w./d, based upon no. foetuses showing hyoid alae angulated at 100 mkd and above. The meaningful increase of this variation is dose-dependent, and cannot be disregarded because of the high spontaneous incidence. Taking into account the type of anomaly (common variation rather than a malformation), there is no proposal for classification. This proposal, both for setting developmental NOAEL and nonclassification is in line with previous conclusions on a.s. RMS (UK): The hyoid variation is dose related with a clear increase at 250 mg/kg bw/day. The finding at 100 mg/kg bw/d is not statistically significant (5 versus 2 fetuses; 4/25 versus 2/23 litters) and is within the cited historic control range of 0 50% of litters. We do not consider this to be adverse and consider 100 mg/kg bw/d to be a NOAEL. The developmental NOAEL in the developmental toxicity study in rabbit should be discussed and agreed in an experts meeting. 2(4) B month study in rats, IIA 5.8/13 A chronic (12 month) toxicity testing study in the Wistar rat with 1,2,4-triazole BE: For the setting of the toxicity NOAEL, the decreased MCV and MCH at 375 ppm and above in males and at 1000 ppm and above in females, at the 3, 6 and 12 month sampling intervals should not be ignored. Other RBC parameters are also affected, albeit in a less consistent way. The RMS (UK): There is no dose response and the magnitude of the change is <10%. Therefore the haematology changes are considered not adverse. We agree with the concern for the neurological changes at the top dose level. However, there is a Haematological changes in the 12 month study in rats should be discussed and agreed in an experts meeting (see also 2(14)) EFSA Supporting publication 2016:EN-1080

22 neurohistopathological top-dose findings are worrying, and no explanation is found for this severe effect, although involvement of RBC deficit (possibly indicative of some ischemia) cannot be excluded. The proposed NOAEL is supported. clear threshold. Your support for the proposed NOAEL is noted thank you. Other toxicological studies & Medical data No. Column 1 Reference to addendum to assessment report 2(5) B.6.8.1, TAA, 28-days rat study 2(6) B.6.8.1, TAA, 28-days mouse study Column 2 Comments from Member States / applicant / EFSA AT: AT agees with the conclusion of the RMS that the NOAEL of the study is the highest tested dose (13000 ppm; 993 mg/kg bw/d in males and 940 mg/kg bw/d in females). It would be appreciated to include in the table (please not that the numbering of the table should be ) the percentage of the kidney weight increase compared to the control (males ppm statistically significantly increased relative kidney weight but only +9.3% of the control males value, no histopatho findings). AT: AT is not convinced that the increase in LH is not a treatment related effect. RMS stated that the increase of LH in females of 7000 ppm group (+29.5% of control) was due to Column 3 Evaluation by rapporteur Member State RMS (UK): Thank you for identifying the error in the table number the DAR has been revised. We considered that enough information to calculate percentages was in the table, however values for the top dose group, have been added. RMS (UK): The clinical chemistry data do not present a consistent pattern. Both CPK and LDH are increased in males, but CPK is decreased in females while LDH is Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data LDH and CPK changes in the 28-day mouse study should be discussed and agreed in an experts meeting (see also 2(16)) EFSA Supporting publication 2016:EN-1080

23 one animal, however, the same was observed in high dose males (+36.7%). Please note that increased LDH usually indicates cell damage. Damaged cells release LDH which is than accumulated in blood. The same is true for CPK, which indicated a muscle damage. This was increased in high dose males (+67% of the control males), although it is also obvious that there was a high variability between animals. It would be appreciated to include in the table the percentage of the increase of LDH and CPK compared to the control animals. increased, indicating the changes are not due to the same mode of action. Ranges from the males are in the table below:- Control CPK LDH ppm ppm * * *Highest values both in the same animal. Although no histopathological findings were observed we would propose not to disregard these findings and set the NOAEL for the study at 3000 ppm (483 mg/kg bw/d in males and 542 mg/kg bw/d in females). Irrespective of the relevance of this finding, the reference doses will not be altered. 2(7) B.6.8.1, TAA, onegenaration reproduction study in rats AT: RMS derived the NOAEL for general parental toxicity at 100 mg/kg bw/d. However, at 300 mg/kg bw/d no significant effects on body weight or body weight gain above 10% during premating (week 0-16) were observed. Also no effects on terminal body weight was observed. Therefore, we would propose to set the NOAEL for general parental toxicity at 300 mg/kg bw/d, based on statistically significantly decreased body weights in males of 1000 mg/kg bw/d (weeks 6, 10 and 16) and significantly decreased body weight gain in males of 10% RMS (UK): We agree this appears to be a conservative approach. This is the NOAEL proposed in the study report and the company submission. Our general policy is not to propose higher NOAELs as the study report authors are the ones who have the greatest understanding of the test system and what constitutes a response outside the norm. The NOAEL for general parental toxicity of the one-generation reproduction study in rats should be discussed and agreed in an experts meeting (see also 2(21)) EFSA Supporting publication 2016:EN-1080

24 (weeks 0 to 16). The slight increase in relative liver weight in males of 1000 mg/kg bw/d and in relative kidney weight at 300 and 1000 mg/kg bw/d can be contributed to slightly lower terminal body weight (males 300 mg/kg bw/d: - 3.9% body weight, males 1000 mg/kg bw/d: - 6.3% body weight compared to the control males). AT agrees with the developmental NOAEL of 1000 mg/kg bw/d. 2(8) B.6.8.1, TAA, developmental range finding study in rats and main developmental study in rats 2(9) B.6.8.1, TAA, developmental range finding study in rats AT: While in the range finding developmental study in rats 1000mg/kg bw/d was well tolerated in the main developmental rat study 1000 mg/kg bw/d was poorly tolerated (3 dams were killed prematurely due to poor clinical condition). Could RMS please clarify if there is any explanation available for the different tolerability of 1000 mg/kg bw/d? The same rat strain, the same laboratory, the same study author are stated and the studies have been conducted almost simultanously. In the dose finding study the same low ph must habe been achieved but no comparable findings (local intestinal irritation) attributed to this have been observed. AT: RMS stated that maternal body weight gain was slightly reduced at 1000 mg/kg bw/d. AT is of the opinion that decrease of 26.6% body weight gain at 1000 mg/kg bw/d RMS (UK): We agree with the point made. There is no indication why there was greater toxicity in the main test. Additional reassurance regarding TAA developmental toxicity can be taken from the absence of fetal abnormalities in the rabbit study at 750 mg/kg bw/day and the fact that the typical triazole effect of cleft palate would have been evident in the range-finding study which included an external examination. RMS (UK): Agree. This was taken into account in proposing the NOAEL for the study. The DAR has been revised accordingly EFSA Supporting publication 2016:EN-1080

25 2(10) B.6.8.1, TAA, Table , Summary 2(11) B , 28-day study in rats, selection of dose levels, page 38 comparing to control from day 6-20, corrected for uterine weight, even if not statistically significant, is rather remarkable. AT: In the table it is written that in the main rat developmental study at LOAEL (1000 mg/kg bw/d) the observed maternal effects were decline in clinical conditions and slight decrease in body weight gain. AT is of the opinion that beside decline in clinical conditions it should be added followed by sacrifice and that the decrease in body weight gain was rather remerkable than slight (-26% of control). EFSA: it is reported that the dose levels of the current study were selected on the basis of a 14-day preliminary study which was not submitted. The submission of such study would be welcome. RMS (UK): Agree. The effects resulted in premature sacrifice and while this is mentioned in a separate sentence it would be clearer to include this in the sentence on clinical condition. The DAR has been revised RMS (UK): It is not clear what would be gained by reviewing the 14 day study, as the 28 day study used dose levels of over 900 mg/kg bw/day. The limit dose for these studies is 1000 mg/kg bw/day. Open point: RMS to provide the assessment of the 14-day study in the rat. 2(12) B , 28-day study in mice, selection of dose levels, page 41 EFSA: it is reported that the dose levels of the current study were selected on the basis of a 14-day preliminary study which was not submitted. The submission of such study would be welcome. RMS (UK): It is not clear what would be gained by reviewing the 14 day study, as the 28 day study used dose levels of over 1000 mg/kg bw/day. The limit dose for these studies is 1000 mg/kg bw/day. Open point: RMS to provide the assessment of the 14-day study in the mouse. 2(13) B.6.8.2, TA, developmental study in rabbits AT: In the rabbit developmental study with TA, slight effects on maternal body weight at 250 mg/kg bw/d could be observed (corrected for gravid uterine weight: -1.6% of control), RMS (UK): It is unclear what is meant by dam-fetus-direct comparison. The body weight effects in dams are relatively small in terms of absolute weight. However there is 25 EFSA Supporting publication 2016:EN-1080

26 however, the effects on foetuses at this dose were stronger (mean fetal body weight 10.3% of control foetuses, high percentage of litters effected by skeletal variations). Could RMS please add the information of dam-foetus-direct comparison? It is questionable if rather mild observed toxicity in dams could explain the effects observed on foetuses. a clear effect on food consumption (10% ) and body weight change corrected for uterine weight over the dosing period of GD6 29:- Controls minus-70g versus minus-130g in the top dose group. We consider the mild developmental effects (reduced fetal weight and skeletal anomalies) could be related to the maternal toxicity. Both maternal and fetal effects were taken into account in determining the NOAEL. 2(14) B.6.8.3, 1,2,4-triazole, 12 months study in rats, toxicology subgroup AT: RMS considered the effects observed on blood parameters (MCH, MCV) from 375 ppm onwards in males from 3 months to the end of the study and from 1000 ppm in femals at 6 and 12 months as not treatment related since there were no consistent dose related changes apparent in the haematology parameters from which MCV and MCH are calculated namely RBC, Hb and Hct. AT is of the opinion that only Hb is directly related to MCH, but not RBC and Hct. MCV is the mean size of the erytrocytes and not directly connected to their number or to Hb. Therefore, the connection should be established first with Hb. And Hb was also decreased, although not statistically significantly, but at least in males in a dosed-related manner. RMS (UK): See response to point 2(4) from BE. We agree the text on calculation of MCV and MCH was unclear and the DAR has been revised accordingly. The changes in haematology values were all <10%. A consistent reduction in Hb is seen in both sexes at 2000 ppm but the magnitude is <6% and this is considered not adverse. The body weights in the main group were decreased at 375 ppm, but were increased in the neurotoxicity group between days 0 and 343 (Table B ); therefore the findings in the main group are considered to be within normal variability. See experts Consultation 2(4) EFSA Supporting publication 2016:EN-1080

27 2(15) B.6.8.3, 1,2,4-triazole, 12 months study in rats, Conclusion 2(16) B.6.8.3, 28-day study in mice, CPK and LDH values, page 42 Therefore, at is of the opinion that effects observed on blood parameters (MCH, MCV) should not be disregarded from 375 ppm onwards. Additionally the body weight gain in females (0-343) at 375 ppm was % of the control females. Therefore, AT is rather of the opinion that the NOAEL of the study (toxicology sub-group) should be set at 125 ppm (6.9 mg/kg bw/d in males and 8.3 mg/kg bw/d in females). AT: In the Conclusion of the study the RMS derived the NOAEL of the study at 375 ppm, based on the results from the toxicology group. Could RMS please include the NOAEL observed for neurotoxic effects (1000 ppm) since also a neurotoxicity sub-group was involved. AT is of the opinion that the observed neurotoxic effects on Purkinje cells should be clearly stated as such and 1,2,4 triazole considered to produce neurotoxic effects. The absence of findings in the FOB can not overrule the histopathological findings in nervous system since FOB are frequently difficult to interpret. EFSA: it is acknowledged that the changes in CPK levels observed at high dose in males are of doubtful toxicological effect, also considering that such changes were not observed at the same dose level in females. However, for LDH it seems that the effect is consistent in high dose RMS (UK): Agree. Clear neuropathic findings are seen. The DAR has been revised accordingly. RMS (UK): See response to point 2(6) from AT. The absence of an impact on the overall conclusions is agreed. See experts Consultation 2(6) EFSA Supporting publication 2016:EN-1080