European Food Safety Authority (EFSA)

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1 TECHNICAL REPORT APPROVED: 18 December 2017 doi: /sp.efsa.2018.en-1360 Outcome of the consultation with Member States, the applicant and EFSA on the pesticide risk assessment for spiroxamine 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 an active substance 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 spiroxamine are presented. The current report summarises the outcome of the consultation process organised by the rapporteur Member State Germany and presents EFSA s scientific views and conclusions on the individual comments received. European Food Safety Authority, 2018 Keywords: spiroxamine, peer review, confirmatory data, risk assessment, pesticide, fungicide Requestor: European Commission Question number: EFSA-Q Correspondence: pesticides.peerreview@efsa.europa.eu EFSA Supporting publication 2018:EN-1360

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 spiroxamine in light of confirmatory data. EFSA supporting publication 2018:EN pp. doi: /sp.efsa.2018.en-1360 ISSN: European Food Safety Authority, 2018 Reproduction is authorised provided the source is acknowledged. 2 EFSA Supporting publication 2018:EN-1360

3 Summary Spiroxamine has been approved under Regulation (EC) No 1107/2009 by Commission Implementing Regulation (EU) No 797/2011. It was a specific provision of the approval that the applicant was required to submit to the European Commission further studies on: a) the possible impact on the worker, the consumer and the environmental risk assessment of the potential stereo-selective degradation of each isomer in plant, animals and the environment; b) the toxicity of the plant metabolites formed in fruit crops and the potential hydrolysis of fruit crop residues in processed commodities; c) the groundwater exposure assessment for metabolite M03; d) the risk to aquatic organisms. The applicant was required to submit the information set out in point (a) by two years after the adoption of specific guidance and the information set out in points (b), (c) and (d) by 31 December In accordance with the specific provision, the applicant, Bayer CropScience, submitted an updated dossier in December 2013 that was completed in April 2014, which was evaluated by the designated rapporteur Member State (RMS), Germany, in the form of updates of the assessment report. In compliance with guidance document SANCO 5634/2009-rev.6.1, the RMS distributed the revised assessment report to Member States, the applicant and EFSA for comments on 10 April The RMS collated all comments in the format of a reporting table, which was submitted to EFSA on 22 November 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, Germany, and presents EFSA s scientific views and conclusions on the individual comments received. Spiroxamine is the ISO common name for 8-tert-butyl-1,4-dioxaspiro[4.5]decan-2- ylmethyl(ethyl)(propyl)amine (IUPAC). The representative formulated products for the evaluation were Spiroxamine EC 500 an emulsifiable concentrate (EC) containing 500 g/l spiroxamine and Prothioconazole + Spiroxamine EC 460 an EC formulation containing 300 g/l spiroxamine and 160 g/l prothioconazole. The representative uses evaluated comprised outdoor foliar spraying against fungal diseases in grapes with the Spiroxamine EC 500 formulation and in wheat, triticale, rye, barley and oats with the Prothioconazole + Spiroxamine EC 460 formulation. The residue definition for monitoring in poultry is currently defined as spiroxamine carboxylic acid (M06), expressed as spiroxamine (sum of isomers). The methods presented as monitoring methods are analysing only for spiroxamine carboxylic acid. This means that additional validation data covering also spiroxamine or new analytical methods are currently not required. In the mammalian toxicology area, it was noted that the genotoxic potential of PTBCOL (M13) had not been addressed according to the studies provided by the applicant. However, the data provided do not contradict the conclusion reported in a case study provided in the Guidance on the establishment of the residue definition for dietary risk assessment (EFSA PPR Panel, 2016), that metabolites M13, M14, M15 and M16 are predicted as negative by all (Q)SAR models and no new alerts are identified by read-across; hence, they are not of concern for genotoxicity. It is therefore concluded that the metabolite is unlikely to be genotoxic. Regarding the metabolite M28, its genotoxic potential was found of no concern based on a battery of negative genotoxicity tests in vitro. The RMS proposed toxicological reference values for both metabolites M13 and M28 using uncertainty factors of 5400 and 900 to derive the acceptable daily intake (ADI) and acute reference dose (ARfD) respectively. These values were not agreed during the commenting by other Member States and EFSA. To be consistent with other EFSA conclusions on metabolites and in general agreement with discussions held with Member States experts, uncertainty factors of 1000 and 300 were applied to 3 EFSA Supporting publication 2018:EN-1360

4 derive the ADI and ARfD respectively based on limited databases resulting in the following toxicological reference values for the two metabolites. For M13, the point of departure for both the ADI and ARfD is 31.5 mg/kg bw per day for the maternal NOAEL from the developmental toxicity study in rats with M13 acetate. The ADI is 0.03 mg/kg bw per day, applying and uncertainty factor (UF) of The ARfD is 0.1 mg/kg bw, based on the same NOAEL for clinical signs, noting that transient signs of neurotoxicity were also observed in the 28-day study in rats with M13 that are also relevant to acute exposures, and applying an UF of 300. With regards to M28, the ADI is 0.03 mg/kg bw per day based on the NOAEL of 30 mg/kg bw per day from the 28-day and developmental toxicity studies in rat, applying an UF of The ARfD is 0.5 mg/kg bw, based on the maternal NOAEL of 150 mg/kg bw per day for mortality and clinical observations in the developmental toxicity study in rats, and applying an UF of 300. It is noted that classification regarding developmental toxicity could be considered for the metabolite M28; however this classification would not have an impact on the risk assessment; it would be in line with the harmonised classification of the parent spiroxamine according to Regulation (EC) No 1272/2008. Data gaps remain regarding an assessment of read-across for the different metabolites to allow grouping. Upon toxicological evaluation of metabolites formed in fruit crops it is proposed to additionally consider metabolites containing the tert-butylcyclohexanone moiety and set the residue definition for dietary risk assessment (fruits) as sum of spiroxamine and metabolites containing the aminodiol (Nethyl-N-propyl-1,2-dihydroxy-3-amino-propane) and the tert-butylcyclohexanone moiety, expressed as spiroxamine. A new hydrolysis study simulating industrial and household food processing conditions indicated that spiroxamine may degrade ph dependently, although to a moderate extend. The main hydrolysis products (up to 23% AR) were M28 (spiroxamine-aminodiol) and M15 (tert-butylcyclohexanone) while significant proportions of unknown metabolites are not formed. The newly proposed residue definition for fruit crops covers these two compounds. Since a change of toxicological reference values for metabolites M13 and M28 was concluded as outcome of the confirmatory data review, and the grouping of metabolites has to be further substantiated, the consumer risk assessment provided by the RMS requires revision (data gap) and hence has to be considered provisional. This provisional consumer dietary risk assessment identified an acute dietary intake concern with regard to table grapes under worst case and refined conditions ( % of acute dietary reference values), confirmation pending availability of an updated consumer risk assessment. The information presented for environmental fate and behaviour satisfy the confirmatory data request to provide a groundwater exposure assessment for metabolite KWG4168-N-oxide (M03) using a better justified soil DT 50. Three laboratory soil degradation studies, evaluated during the peer review of spiroxamine, were kinetically assessed according to FOCUS (2006) in order to derive degradation endpoints for metabolite KWG4168-N-oxide (M03). The geometric mean normalised DT 50 of 21 days and the worst case formation fraction were used to calculate PECgw only with the model FOCUS PELMO (version 5.5.3). PECgw for metabolite KWG 4168-N-oxide (M03) were below µg/l for the uses of spiroxamine in cereals in all relevant FOCUS scenarios. PECgw calculations were not performed for the uses in vines. However, since during the peer review the DT 50 geometric mean value used was more conservative than the updated value and the PECgw were below µg/ L for the uses in vines, it is considered that an updated groundwater exposure assessment is not needed for metabolite KWG 4168-N-oxide (M03) for the uses of spiroxamine in vines. A peer review is proposed in the area of ecotoxicology for further discussing potential endocrine effects and risk assessment to fish. 4 EFSA Supporting publication 2018:EN-1360

5 Table of contents Abstract... 1 Summary Introduction Background and Terms of Reference as provided by the requestor Interpretation of the Terms of Reference Assessment... 7 Documentation provided to EFSA... 7 References... 7 Abbreviations... 8 Appendix A Collation of comments from Member States, applicant and EFSA on the pesticide risk assessment for the active substance spiroxamine in light of confirmatory data and the conclusions drawn by EFSA on the specific points raised Appendix B Used compound codes Appendix C Updated parts of list of endpoints EFSA Supporting publication 2018:EN-1360

6 1. Introduction 1.1. Background and Terms of Reference as provided by the requestor Spiroxamine has been approved under Regulation (EC) No 1107/ by Commission Implementing Regulation (EU) No 797/ EFSA previously finalised a Conclusion on this active substance on 1 September 2010 (EFSA, 2010). It was a specific provision of the approval that the applicant was required to submit to the European Commission further studies on: a) the possible impact on the worker, the consumer and the environmental risk assessment of the potential stereo-selective degradation of each isomer in plant, animals and the environment; b) the toxicity of the plant metabolites formed in fruit crops and the potential hydrolysis of fruit crop residues in processed commodities; c) the groundwater exposure assessment for metabolite M03; d) the risk to aquatic organisms. The applicant was required to submit the information set out in point (a) by two years after the adoption of specific guidance and the information set out in points (b), (c) and (d) by 31 December In accordance with the specific provision, the applicant, Bayer CropScience, submitted an updated dossier in December 2013 which was completed in April 2014, and evaluated by the designated rapporteur Member State (RMS), Germany, in the form of updates of the assessment report (Germany, 2017a). In compliance with guidance document SANCO 5634/2009-rev.6.1 (European Commission, 2013), the RMS distributed the revised assessment report to Member States, the applicant and EFSA for comments on 10 April The RMS collated all comments in the format of a reporting table, which was submitted to EFSA on 22 November 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 Germany, 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 spiroxamine 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 20 December 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 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 797/2011 of 9 August 2011 approving the active substance spiroxamine, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the market, and amending the Annex to Commission Implementing Regulation (EU) No 540/2011. OJ L 205, , p EFSA Supporting publication 2018:EN-1360

7 2. Assessment The comments received on the pesticide risk assessment for the active substance spiroxamine in light of confirmatory data and the conclusions drawn by the EFSA 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 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. Those parts of the list of endpoints that needed amendments following the assessment of the confirmatory data are presented in Appendix C. Documentation provided to EFSA 1. Germany 2017a. Revised assessment report on spiroxamine (Vol.1; Vol.2; Vol.3 B5, B6, B7, B8, B9; List of endpoints), confirmatory data, August Available online: 2. Germany, 2017b. Reporting table, comments on the pesticide risk assessment for spiroxamine in light of confirmatory data, November References EFSA, Conclusion on the peer review of the pesticide risk assessment of the active substance spiroxamine. EFSA Journal 2010;8(10)1719, 102 pp. doi: /j.efsa EFSA, 2015a. Reasoned opinion on the review of the existing maximum residue levels (MRLs) for spiroxamine according to Article 12 of Regulation (EC) No 396/2005. EFSA Journal 2015;13(1):3992. doi: /j.efsa EFSA, 2015b. Technical report on the outcome of the pesticides peer review meeting on general recurring issues in ecotoxicology. EFSA supporting publication 2015:EN pp. EFSA PPR Panel (EFSA Panel on Plant Protection Products and their Residues), Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters. EFSA Journal 2013;11(7):3290, 186 pp. doi: /j.efsa EFSA PPR Panel (EFSA Panel on Plant Protection Products and their Residues), Guidance on the establishment of the residue definition for dietary risk assessment. EFSA Journal 2016;14(12):4549, 129 pp. doi: /j.efsa 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. 6.1 FOCUS (Forum for the Co-ordination of Pesticide Fate Models and their Use), Guidance document on estimating persistence and degradation kinetics from environmental fate studies on pesticides in EU Registration Report of the FOCUS Work Group on Degradation Kinetics. EC Document Reference SANCO/10058/2005-v. 2.0, 434 pp. 7 EFSA Supporting publication 2018:EN-1360

8 Abbreviations a.s. ADI AR ARfD AUC BfR CF CLH GAP DT 50 ELS GC GI GIT HCD HR EC 10 EC ECHA- RAAF FBS FFLC FOCUS GAP HC HCD IESTI ILV LC-MS- MS LD 50 LOQ LoEP MLA MRL MS MSCA MSD active substance acceptable daily intake applied radioactivity acute reference dose area under the blood concentration/time curve Bundesinstitut für Risikobewertung conversion factor harmonised classification and labelling good agricultural practice period required for 50% dissipation (define method of estimation) early life stage gas chromatography gastrointestinal gastrointestinal tract historical control data highest residue effective concentration emulsifiable concentrate European Chemicals Agency Read-Across Assessment Framework fetal bovine serum fish full life cycle Forum for the Co-ordination of Pesticide Fate Models and their Use Good Agricultural Practice historical control historical control data international estimated short-term intake inter-laboratory validation liquid chromatography with tandem mass spectrometry lethal dose, median; dosis letalis media limit of quantification list of end points mouse lymphoma assay maximum residue level Member State Member State competent authority mass selective detector 8 EFSA Supporting publication 2018:EN-1360

9 MWHC NEU NOAEL NOEC NPD OECD PEC PECgw PHI (Q)SAR RAC RAR RMS SEU SF TG twa UF vtg maximum water-holding capacity Northern European Union no observed adverse effect level no observed effect concentration nitrogen phosphorus detector Organisation for Economic Co-operation and Development predicted environmental concentration predicted environmental concentration in groundwater pre-harvest interval (quantitative) structure activity relationship Risk Assessment Committee renewal assessment report rapporteur Member State Southern European Union safety factor test guideline time-weighted average uncertainty factor vitellogenin 9 EFSA Supporting publication 2018:EN-1360

10 Appendix A Collation of comments from Member States, applicant and EFSA on the pesticide risk assessment for the active substance spiroxamine 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 0(1) Vol 1 Level 4 4. Further information to permit a decision to be made. Column 2 Comments from Member States / applicant / EFSA ES: It is stated regarding Toxicology and metabolism: Part B of the Commission Implementing Regulation (EU) No 797/2011 mentions that the notifier shall submit confirmatory information to address the possible impact on the consumer risk assessment of the potential stereoselective degradation of each isomer in plant and animals. No such data were submitted by the applicant to address the requirements. However, no guidance is available on how to deal with isomers in consumer risk assessment. It is recommended that this issue is reconsidered once such guidance is available. A similar statement should be included regarding Environmental fate and behaviour Column 3 Evaluation by rapporteur Member State RMS: see comment 3(14) Noted. See 3(14) Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 10 EFSA Supporting publication 2018:EN-1360

11 Part B of the Commission Implementing Regulation (EU) No 797/2011 mentions that the notifier shall submit confirmatory information to address the possible impact on worker and the environmental risk assessment of the potential stereo-selective degradation of each isomer in the environment. No such data were submitted by the applicant to address the requirements. However, no guidance is available. It is recommended that this issue is reconsidered once such guidance is available. 1. Physical/Chemical Properties; Data on application and efficacy; Further Information; Methods of Analysis (B.1-B.5) Physical and chemical properties of the active substance No. Column 1 Reference to addendum to assessment report Column 2 Comments from Member States / applicant / EFSA Column 3 Evaluation by rapporteur Member State 1(1) AT: Not considered. 1(2) FR: No comment. Column 4 EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 11 EFSA Supporting publication 2018:EN-1360

12 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(3) Vol. 3, B Method Class, 2010, p.41 EFSA: The study ASB is considered an acceptable ILV of the method KIIA, 4.3 /10 Allmendinger H., The method is sufficiently validated for determination of spiroxamine carboxylic acid in bovine muscle, liver/kidney and milk. RMS: Agree. 1(4) Vol. 3, B Method Meyer, M., 2010, p.48 EFSA: The study ASB is considered an acceptable ILV of the method KIIA, Class, T.; Merdian, H., The method is sufficiently validated for determination of spiroxamine carboxylic acid in fat and eggs. However, as method Meyer, M., 2010 has the number of replicates 5 for all matrices, while method Class, T.; Merdian, H., 2009 has 4 replicates for fat, it can also be considered that this last one is the ILV of the first one as a primary method. RMS: Agree. Will be corrected in a revised version. Addressed: Method Class, T.; Merdian, H., 2009 (Germany, 2017a) can be considered a proper ILV of the method Meyer, M., 2010 (study ASB ) 1(5) Vol.1, Animal matrices, Residue definitions, p. 39 EFSA: Residue definition for monitoring in poultry is defined as sum of spiroxamine and spiroxamine RMS: Agree. Will be amended accordingly. Addressed: The legally established residue definition for animals is implemented 12 EFSA Supporting publication 2018:EN-1360

13 1(6) Vol.1, LoEP Residue definitions for monitoring, p (7) Vol.1, 3.1 Proposed decisions, Analytical methods, p (8) Vol.1, 3.2 Proposed decisions, Analytical methods, p (9) Vol.1, 4 Further information, Analytical methods, p. 206 carboxylic acid (M06), expressed as spiroxamine (sum of isomers). The methods presented as monitoring methods are analysing only for spiroxamine carboxylic acid. This means that additional validation data covering also spiroxamine or new analytical methods are required. EFSA: the residue definition for monitoring in poultry is not mentioned in the LoEP in the methods section, as a consequence nor the data requirement for such a method EFSA: Based on the changed residue definition for monitoring in poultry it seems that a proper method analysing for the compounds of the residue definition is missing. EFSA: based on the updated residue definition in animal matrices, additional data are required. EFSA: analytical method for the determination of the compounds of the residue definition for monitoring in poultry is needed. RMS: Agree. Will be amended accordingly. RMS: Agree. Will be amended accordingly. RMS: Agree. Will be amended accordingly. RMS: Agree. Will be amended accordingly. by Regulation (EU) 2016/452 3 : residue definition for animal origin commodities as spiroxamine carboxylic acid metabolite M06, expressed as spiroxamine (sum of isomers). The methods presented as monitoring methods are analysing only for spiroxamine carboxylic acid. This means that additional validation data covering also spiroxamine or new analytical methods are currently not required. Addressed: List of endpoints was updated. Addressed: See comment 1(5). Addressed: See comment 1(5). Addressed: See comment 1(5). 3 Commission Regulation (EU) 2016/452 of 29 March 2016 amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as regards maximum residue levels for captan, propiconazole and spiroxamine in or on certain products. OJ L 79, , p EFSA Supporting publication 2018:EN-1360

14 2. Effects on human and animal health (B.6) Acute 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(1) Vol. 3, B KWG4168-aminodiol, Study 3, V79/HPRT test, page 132 and Study 4, Micronucleus test in vitro, page 136 Under deviations in both studies it is mentioned that the procedure to set up the HCD (historical control data) is unclear. What information is needed in addition to the overview on HCD in the study reports to describe the setup of the HCD clearly? RMS: The necessary data are listed in the OECD TG and the literature referenced therein (e.g., Hayashi M., Dearfield K., Kasper P., Lovell D., Martus H.J., and Thybaud V. (2011). Compilation and Use of Genetic Toxicity Historical Control Data, Mutation,Res., 723, 87-90). 2(2) Vol. 3, B KWG4168-aminodiol, Study 4, Micronucleus test in vitro, page 138 Next to last paragraph: line 6 (Fehler! Verweisquelle konnte nicht gefunden werden.) should read: (see Table B below) RMS: Agree. Text revised accordingly. 2(3) Vol. 3, B , p- tert.-butyl cyclohexanol, Acute oral toxicity rat (rat LD50) - Dermal toxicity (rabbit), p. 158 EFSA: please correct the typo in table B , dermal LD 50 is > 5000 mg/kg bw RMS: Agree. Text revised accordingly EFSA Supporting publication 2018:EN-1360

15 Short term 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(4) Vol. 3, B KWG4168-aminodiol, Study 5, 28-day toxicity study in the rat, page 146 Regarding RMS criticism that the top dose was too low for an evaluation of the potential toxicological effects (hazard assessment) / top dose selection was not in line with OECD TG: The study design (testing equal mg/kg bw doses as used in the respective study on spiroxamine for comparison of toxicity) was agreed before study conduct in a meeting between BfR and Bayer. Toxicological effects at higher doses can be seen in females in the developmental toxicity study on KWG4168-aminodiol. RMS: It was Bayer s decision to conduct this study in the way it was conducted. According to the study report, the study was conducted according to OECD TG 407 (2008). Therefore, the basis for the assessment of the study s validity had to be TG 407. Indeed, the dose selection was not in line with the TG requirements. 2(5) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 205, last paragraph Regarding RMS criticism that the top dose was too low for an evaluation of the potential toxicological effects (hazard assessment) / top dose selection was not in line with OECD TG: The study design (testing equal mg/kg bw doses as used in the respective study on spiroxamine for comparison of toxicity) was agreed before study conduct in a meeting between BfR and Bayer. RMS: See 2(4) EFSA Supporting publication 2018:EN-1360

16 Toxicological effects at higher doses can be seen in females in the developmental toxicity study on KWG4168-aminodiol. 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) Vol. 3, B , p- tert.-butyl cyclohexanol (PTBCOL), gene mutation and chromosome aberration tests, p EFSA: the genotoxic, clastogenic and aneugenic potential of the metabolite PTBCOL has not been sufficiently addressed due to the short-comings of both studies (one is not acceptable and the other is supplementary which is insufficient evidence by itself). RMS: Noted. Addressed: The genotoxic potential of PTBCOL (M13) has not been addressed according to the studies provided by the applicant. However, the data provided do not contradict the conclusion reported in a case study provided in the Guidance on the establishment of the residue definition for dietary risk assessment (EFSA PPR Panel, 2016), that metabolites M13, M14, M15 and M16 are predicted as negative by all (Q)SAR models and no new alerts are identified by read-across; hence, they are not of concern for genotoxicity. It is therefore concluded that the metabolite is unlikely to be genotoxic. See also 2(2, 3, 4, 6, 8, 9) below 2(2) Vol. 3.B FR: RMS considers the MLA assay RMS: The study report did not contain See genotoxicity 2(1) 16 EFSA Supporting publication 2018:EN-1360

17 2(3) Vol. 3, B p-tertbutyl cyclohexanol, Study 3, Mouse lymphoma assay, page 163 performed with M13 as not acceptable due to the use of foetal bovine serum instead of horse serum. Could you please indicate which serum has been used in historical control tests? In case the assay is considered inacceptable, the uncertainty of M13 genotoxic profile should be further discussed. Study was not accepted due to use of fetal bovine serum (FBS) instead of horse serum in the medium: Based on recommendation of the producer of the cell line the assay was run since 2002 with the use of medium with inactivated fetal bovine serum (FBS); the historical control data are based on this protocol and the respective mutation rates fulfill the requirement of the former and current OECD GLs. Therefore, the study is valid and should be considered acceptable. details which serum was used in historical control tests. RMS: The information mentioned by the applicant was not submitted for this procedure. The L5178Y TK+/ C cell line was first described by Donald Clive. He used medium supplemented with heat inactivated horse serum (e.g., Clive & Spector (1975) Mutation Research 31, 17-29; Clive et al. (1979) Mutation Research 59, ; Clive et al. (1987) Mutation Research 189, ) to maintain the cells, which was confirmed later on by other authors (e.g., Clements (2000) Mutation Research 455, ) and in the OECD TG 490. It is noted that even the study plan for this study, required the use of horse serum. Hence the use of FBS in this study is extremely unusual. Therefore, the study is still considered non-acceptable. See genotoxicity 2(1) 2(4) Vol. 3, B p-tert- RMS: The numbers mentioned by See genotoxicity 2(1) 17 EFSA Supporting publication 2018:EN-1360

18 butyl cyclohexanol, Study 3, Mouse lymphoma assay, page 163 RMS comments that too few cells were exposed when compared to current TG (2015): OECD 490 (2016) states that the minimum number of cells used for each culture should be based on the spontaneous mutant frequency, i.e. sufficient cells have to be treated so as to maintain 10 (ideally) 100 spontaneous mutants / 10 6 cells in all test phases. According to LPT (the lab conducting the study) this can be achieved by treatment of at least 6 x 10 6 cells. In the study report it is mentioned that x 10 6 cells per culture were treated, so that the requirement is fulfilled. 2(5) Vol. 3.B FR: For better clarity, could you please indicate in Volume 3B6 which metabolites (codes and structures) are proposed to be in Group B and Group C respectively? Indeed, a list is given in Volume 1 Section Further Toxicological studies (page 31), but is different to the one mentioned in Volume 3B7. 2(6) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 207, third paragraph Study was not accepted due to use of fetal bovine serum (FBS) instead of horse serum in the medium: Based on recommendation of the producer of the cell line the assay was run since 2002 with the use of medium with inactivated fetal bovine serum (FBS); the historical Bayer are no accurate citations. The report reads as follows: The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures of x 106 cells/ml, respectively, and were seeded into a series of tubes, diluted to 5 x 105 cells/ml per tube. With a culture volume of 20 ml this would be 10 7 cells/culture. The respective comment will be deleted in a revised version. RMS: Unfortunately, this cannot be deduced unequivocally from the notifier s confirmatory data submission. Possibly, M13, M14, M15, M16, M22, M24, M32, M33, M34, M35 and M36 belong to group B and M28, M29, M30 and M31 belong to group C. Data gap: Grouping of metabolites regarding their toxicological profile should be further substantiated. RMS: See 2(8) See genotoxicity 2(1) 18 EFSA Supporting publication 2018:EN-1360

19 control data are based on this protocol and the respective mutation rates fulfill the requirement of the former and current OECD GLs. Therefore, the study is valid and should be considered acceptable. 2(7) Vol. 3.B FR: The RMS assessment of the grouping proposed by the applicant using the ECHA RAAF is welcomed. Nevertheless, could you please give more details on the in silico metabolism predictions used to group metabolites and to propose a representative metabolite for each group? Furthermore, the use of (Q)SAR and read-across is considered needed for each metabolite regarding genotoxicity and toxicity endpoints in order to substantiate grouping and selection of the representative metabolites. It is noted that spiroxamine is one of the case study presented in the new EFSA Guidance on the establishment of the residue definition for dietary risk assessment (2016), and as such, (Q)SAR and read-across data are available in this document. Overall, FR shares the doubts of the RMS concerning the uncertainties related to grouping and selection of representative metabolites from a RMS: All data and information received from the notifier were included and described in the assessment. Unfortunately, QSAR predictions on the metabolites toxicological properties were not among the submitted information. The toxicological assessment was prepared in April/July 2016; at that time the PPR panel opinion was not available. Besides to the information listed in the opinion, no details are available to RMS. It is noted that the appropriateness of the proposed grouping was not assessed in the opinion. Bayer conducted the in silico metabolism predictions were with Meteor Nexus: 1.5.1, Nexus: The reasons why M13 and M28 were selected as representative metabolites for the groups were not given. See 2(12) below 19 EFSA Supporting publication 2018:EN-1360

20 2(8) Vol3, B , Studies on metabolites, Gene mutation M13 2(9) Vol. 3, B , Toxicity of the plant metabolites formed in fruit crops, p (10) Vol3, B , Reference values for M13 and M28 (geno)toxicological point of view. AT: Could RMS please explain if gene mutation assay with M13 has been considered acceptable or not? Does RMS conclude that gene mutation endpoint for M13 is sufficiently covered? If not, could you please explain how reference values could be derived? EFSA: Regarding M13, considering that the genotoxicity potential of the metabolite has not been adequately addressed, it is considered that no toxicological reference values can be established. AT: Could RMS please explain in detail where the SF of 5400 (chronic) and 900 (acute) comes from? Although it might appear that these SFs might come from new GD on residue definition they are not in line with this document. Based on the information as presented we would not agree with reference values as currently set for M13 and M28 and consider them not in line with latest published documents and overly conservative. RMS: See 2(6, 14) See genotoxicity 2(1) RMS: Noted. See genotoxicity 2(1) RMS: See 2(14). The toxicological assessment was prepared in April/July 2016; at that time the PPR panel opinion was not available. The additional SFs were selected based on the precedents of previous peer-review decisions in similar cases and on the ECHA GD on information requirements/chemical safety assessments (chapter R.8). In the addendum following reasons are listed (section B ): When taking into account these elements of incomplete hazard assessment, time extrapolation and data from only one species, the uncertainty factor of usually 100 needs to be increased by 3 x See 2(12) below 20 EFSA Supporting publication 2018:EN-1360

21 2(11) LoEP EFSA: the agreed reference values for the metabolites should be included in the LoEP 2(12) Vol. 3.B FR: Generally, an uncertainty factor of 1000 (instead of 5400 as proposed by the RMS) is used to derive an ADI for a metabolite based on the NOAEL observed in a 28- or 90-day study. The additional UF of 10 to the standard UF of 100 takes into account the incomplete data package as well as the extrapolation from sub-acute/sub-chronic to longterm toxicity. On the same basis, an UF of 300 (instead of 900 as proposed by the RMS) could be used to determine the ARfD (additional UF of 3 due to the lack of a complete data package). The additional UF of 3 proposed by the RMS to take into account the lack of data on a second mammalian species is considered to be covered by the additional UF used to take 6 x 3 leading to 5400 for the reference dose for the chronic risk assessment. Similarly, the uncertainty factor of usually 100 needs to be increased by 3 x 3 (incomplete hazard assessment and data from only one species) leading to 900 for the reference dose for the acute risk assessment. RMS: Agree. Text revised accordingly. RMS: See 2(14,15) Addressed: To be consistent with other EFSA conclusions on metabolites and in general agreement with discussions held with MSs experts, an uncertainty factor of 1000 and 300 should be applied to derive the ADI and ARfD respectively based on limited databases resulting in the following toxicological reference values for M13 and M28: M13: The point of departure for both the ADI and ARfD is 31.5 mg/kg bw per day from the developmental toxicity study with M13 acetate (40 mg/kg bw per day *156 g/mol /198 g/mol to calculate the amount of M13 from the amount of M13 acetate). ADI = 0.03 mg/kg bw per day (dev tox study in rat performed with M13 acetate, UF 1000) 21 EFSA Supporting publication 2018:EN-1360

22 into account the lack of a complete data package. ARfD = 0.1 mg/kg bw (dev tox study in rat with M13 acetate, based on clinical signs, in addition, transient signs of neurotoxicity were observed in the 28-d study in rat with M13, UF 300) M28: ADI: 0.03 mg/kg bw per day (28-d rat, dev tox rat, UF 1000) ARfD: 0.5 mg/kg bw, maternal NOAEL from the dev tox study in rats, UF 300) 2(13) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 213, third paragraph For derivation of the reference doses RMS set in addition to the safety factor of 100 an uncertainty factor (UF) of 3 for incomplete hazard assessment / uncertainties and another 3 x for data only from one species. Also without the 3 x for data only in one species the remaining overall UFs of 1800 and 300 for the chronic and acute risk assessment would be very conservative, especially since both fruit metabolites based on the NOAELs are less toxic than parent. Moreover, a 10x for interspecies variation is already included in the standard safety factor of 100. Based on these considerations, Bayer considers that the extra 3 x for data only from one species should be removed. RMS: See 2(14, 15) See 2(12) See also 2(7, 10, 13, 14, 15) 22 EFSA Supporting publication 2018:EN-1360

23 2(14) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 213, third paragraph In case that a reduction of the overall safety factors will be needed: does the RMS agree that a 90-day mouse study would fulfil the requirement for data in a second species? RMS: From the RMS s point of view, currently no further animal studies should be conducted. See 2(12) Further toxicological studies No. Column 1 Column 2 Column 3 Column 4 Reference to addendum to assessment report Comments from Member States / applicant / EFSA Evaluation by rapporteur Member State EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 2(15 ) Vol. 3, B , KWG 4168-aminodiol, Oral (gavage) prenatal developmental toxicity study in the wistar rat, p EFSA: The incidence of gas filled parts of the GI tract of dams is treatment-related and may be considered as adverse. Regarding developmental toxicity, please list the effects considered adverse on ossification (some statistically significant results appear to be nonadverse). RMS: See 2(17). Regarding altered ossification, findings were increased above or decreased below the incidences in control group (and historical control range); no maternal toxicity was reported that would explain changes in ossification pattern in offspring. 2(16 ) Vol. 3, B KWG4168-aminodiol, Study 6, developmental toxicity study in rats, page 156 RMS comment reason for mortality was not elucidated : The animals sacrified in extremis / found dead showed gas-filled or distended regions of the gastrointestinal tract RMS: The relationship as proposed by Bayer between cabbage and the test material and the implications for the possible induction of adverse findings are unclear and remain 23 EFSA Supporting publication 2018:EN-1360

24 (GIT) and displayed gasping or rales before death. It is a well known clinical picture in rabbits, that eating too much cabbage can lead to excessive gas production in the GIT and, subsequently, to death by cardiovascular failure. It is very probable (but difficult to prove experimentally) that high doses of aminodiol lead to a similar picture in rats. speculative in the absence of data. 2(17 ) Vol. 3, B KWG4168-aminodiol, Study 6, developmental toxicity study in rats, page 156 RMS set the maternal NOAEL at 150 mg/kg bw/d based on the fact that the only finding at this dose were gaseous content of the intestines in one dam. Gas-filled and distended regions of the gastro-intestinal tract (stomach, cecum and colon) were seen frequently in this study as well as in the dose range finder > 500 mg/kg bw/d. Therefore, also the observation in one dam at 150 mg/kg bw/d clearly is a compound related effect and the maternal NOAEL should be set at 30 mg/kg bw/d. RMS: Gaseous content of intestines was reported in one of 24 animals at 150 mg/kg bw per day. Several parts of the GI tract had gaseous content in several animals in the top dose group. In summary, the incidence of one animal is considered not of sufficient severity to set the LOAEL at 150 mg/kg bw per day. No dose range-finding study was submitted. The summary in the report of the main study mentions The only finding at 480 mg/kg was a decrease in gestation body weight in comparison to controls (of up to 6.3%). Results of the animals treated with 30 or 120 mg/kg bw per day were not reported. This would support the NOAEL of 150 mg/kg bw per day 2(18 ) Vol. 3, B KWG4168-aminodiol, Study 6, developmental toxicity study in rats, page 156 RMS set the fetal NOAEL at 30 mg/kg bw/d, since some of the de- or increased incidences of several unossified or incompletely ossified RMS: See 2(20). No maternal toxicity was reported at 150 mg/kg bw per day that might explain delays in development. Classification regarding developmental toxicity could be considered for the metabolite M28; however this 24 EFSA Supporting publication 2018:EN-1360

25 bones were outside the historical controls (HCs). At 150 mg/kg bw/d only lumbar centra incompletely ossified (2 cases vs. of up to 1 case in HCD), at 30 & 150 mg/kg bw/d Sternebrae seg. 4 incompletely ossified (fetal incidence 96 or 100% vs. up to 92.3% in HCs) were slightly outside the HC range. These variations, which per se only indicate a slight delay in development of these skeletal structures, are not an adverse effect. Thus, the fetal NOAEL should be set at 150 mg/kg bw/d. classification would not have an impact on the risk assessment. 2(19 ) Vol. 3, B , Oral (gavage) developmental toxicity study of 4-tert butylcyclohexyl acetate (4-tBCHA) in rats, p EFSA: in the conclusion of the study, the opinion of the RMS regarding the maternal and developmental NOAELs is not clear (only the applicant s one). It should be clarified which clinical signs are the basis for the setting of the NOAELs (in both Vol. 3 and the LoEP). In addition, the toxicological equivalence between 4-tertbutylcyclohexanol and 4-tertbutylcyclohexyl acetate has not been demonstrated. RMS: The maternal NOAEL was 40 mg/kg bw per day based on clinical signs (mainly excessive salivation) at 160 mg/kg bw par day. The developmental NOAEL was 160 mg/kg bw per day based on lower foetal weights, increased incidences of dilatation of the renal pelvis and reduced ossification sites at 640 mg/kg bw per day EFSA Supporting publication 2018:EN-1360

26 Toxicological data on metabolites No. Column 1 Column 2 Column 3 Column 4 Reference to addendum to assessment report Comments from Member States / applicant / EFSA Evaluation by rapporteur Member State EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 2(1) Vol. 3, B , Possible impact on the risk assessment of the potential stereoselective degradation of isomers, p. 200 EFSA: the data gap regarding the possible impact on the worker, (resident), and consumer of the potential stereo-selective degradation of each isomer in plant and animals is noted. RMS: Noted. The data gap is acknowledged. 2(2) Vol. 3, B , Toxicity of the plant metabolites formed in fruit crops, p. 201 EFSA: the data gap regarding an assessment of read-across for the different metabolites within grouping is acknowledged. RMS: Noted. The data gap is acknowledged. 2(3) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 204, next to last paragraph RMS didn t understand why additional data on structurally related compounds pre-registered under REACH were not considered by the applicant. However, at the time of preparation of the confirmatory data, the additional compounds pre-registered under REACH were obviously not visible to the public on the ECHA website. RMS: RMS gave Bayer the information that structurally related compounds were pre-registered or registered during the mentioned presubmission meeting so that Bayer could make the case for readacross/grouping stronger. Bayer could have approached the data owners and negotiated access to the studies. However, it was Bayer s decision not to pursue this path EFSA Supporting publication 2018:EN-1360

27 Toxicological end points: ADI, ARfD, AOEL No. Column 1 Column 2 Column 3 Column 4 Reference to addendum to assessment report Comments from Member States / applicant / EFSA Evaluation by rapporteur Member State EFSA s scientific views on the specific points raised in the commenting phase conducted on the RMS s assessment of confirmatory data 2(1) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 206, first paragraph 2(2) Vol. 3, B Toxicity of the plant metabolites formed in fruit crops, page 206, first paragraph RMS set the maternal NOAEL at 150 mg/kg bw/d based on the fact that the only finding at this dose were gaseous content of the intestines in one dam. Gas-filled and distended regions of the gastro-intestinal tract (stomach, cecum and colon) were seen frequently in this study as well as in the dose range finder > 500 mg/kg bw. Therefore, also the observation in one dam at 150 mg/kg bw/d clearly is a compound related effect and the maternal NOAEL should be set at 30 mg/kg bw/d. RMS set the fetal NOAEL at 30 mg/kg bw/d, since some of the deor increased incidences of several unossified or incompletely ossified bones were outside the historical controls (HCs). At 150 mg/kg bw/d only lumbar centra incompletely RMS: See 2(22) See 2(15) RMS: See 2(20, 23) See 2(15) 27 EFSA Supporting publication 2018:EN-1360