Scientific Opinion on safety and efficacy of Cycostat 66G (robenidine hydrochloride) for rabbits for breeding and fattening 1, 2

Transcription

1 EFSA Journal 2011;9(3):2102 SCIENTIFIC OPINION Scientific Opinion on safety and efficacy of Cycostat 66G (robenidine hydrochloride) for rabbits for breeding and fattening 1, 2 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 3,4 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy Cycostat 66G is a coccidiostat for rabbits for breeding and fattening, containing 66 g robenidine hydrochloride/kg feed. The FEEDAP Panel concludes that feed concentrations 66 mg robenidine hydrochloride from Cycostat 66G/kg complete feed are safe for breeding does. The margin of safety is about three. This conclusion can be extrapolated to rabbits for fattening. Robenidine hydrochloride is not genotoxic. Chronic studies in rats and dogs did not allow to determine a NOAEL but gave no indications of the tumorigenicity of robenidine hydrochloride. The lowest NOAEL of 11 mg/kg bw/day could be derived from the tolerance study in breeding does. An ADI of 0.11 mg robenidine hydrochloride/kg bw, equivalent to 6.6 mg for a 60 kg adult, is proposed, applying a safety factor of 100. Robenidine is the marker residue. No MRLs would normally be required. If MRLs should be set for control purposes they should concern only the liver and kidney (0.2 mg robenidine/kg tissue each). Robenidine hydrochloride is not a skin or eye irritant and not a skin sensitiser. The inhalation LC 50 (>5.2 mg/l) is higher than LC 50 values which require classification under Regulation (EC) No 1272/2008. The use of Cycostat 66G at the recommended dose range does not pose a risk for the environment. Robenidine hydrochloride at 66 mg/kg feed has a potential to control coccidiosis in breeding does and rabbits for fattening. 50 mg robenidine hydrochloride/kg has been demonstrated to be efficacious only in breeding does. Sensory quality of rabbit meat is ensured after a five-day withdrawal period. European Food Safety Authority, 2011 KEY WORDS Coccidiostats and histomonostats, robenidine hydrochloride, rabbits for breeding, rabbits for fattening, safety 1 On request from the European Commission, Question No EFSA-Q , adopted by written procedure on 7 March This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. The modified sections are indicated in the text. 3 Panel members: Gabriele Aquilina, Georges Bories, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Reinhard Kroker, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Derek Renshaw and Maria Saarela. Correspondence: FEEDAP@efsa.europa.eu 4 Acknowledgement: The Panel wishes to thank the members of the Working Group on Coccidostats and Histomonostats, including Werner Terhalle, for the preparatory work on this scientific opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on safety and efficacy of Cycostat 66G (robenidine hydrochloride) for rabbits for breeding and fattening. EFSA Journal 2011;9(3):2102. [32 pp.] doi: /j.efsa Available online: European Food Safety Authority, 2011

2 SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of Cycostat 66G as feed additive for rabbits for breeding and fattening purposes. Cycostat 66G, containing 66 g robenidine hydrochloride/kg as the active substance, is used to control coccidiosis caused by Eimeria intestinalis, Eimeria flavescens, Eimeria magna, Eimeria media, Eimeria perforans and Eimeria stiedai. The FEEDAP Panel concludes that feed concentrations 66 mg robenidine hydrochloride from Cycostat 66G/kg complete feed are safe for breeding does. The margin of safety is about three. This conclusion can be extrapolated to rabbits for fattening. The FEEDAP Panel reinforces its former statement that although robenidine hydrochloride is active against Gram-positive bacteria it has not induced resistance to robenidine hydrochloride in enterococci, Escherichia coli, Salmonella and Campylobacter in rabbits. Robenidine hydrochloride is not genotoxic. Chronic studies in rats and dogs did not allow to determine a NOAEL but gave no indications of the tumorigenicity of robenidine hydrochloride. The lowest NOAEL of 11 mg/kg bw/day could be derived from the tolerance study in breeding does. An ADI of 0.11 mg robenidine hydrochloride/kg bw, equivalent to 6.6 mg for a 60 kg adult, is proposed, based on the lowest NOAEL applying a safety factor of 100. Robenidine is the marker residue. No MRLs would normally be required. If MRLs should be set for control purposes they should concern only the liver and kidney (0.2 mg robenidine/kg tissue each). Robenidine hydrochloride is not a skin or eye irritant and not a skin sensitiser. The inhalation LC 50 (>5.2 mg/l) is higher than LC 50 values which require classification under Regulation (EC) No 1272/2008. The use of Cycostat 66G at the recommended dose range does not pose a risk for the environment. Robenidine hydrochloride at 66 mg/kg feed has a potential to control coccidiosis in breeding does and rabbits for fattening. 50 mg robenidine hydrochloride/kg has been demonstrated to be efficacious only in breeding does. Sensory quality of rabbit meat is ensured after a five-day withdrawal period. EFSA Journal 2011;9(3):2102 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 5 Terms of reference... 5 Assessment Introduction Characterisation Identity of the additive Characterisation of the active substance Manufacturing processes Stability and homogeneity Shelf-life of the additive Stability of the additive used in premixtures and feedingstuffs Homogeneity Conditions of use Evaluation of the analytical methods by the European Union Reference Laboratory (EURL)10 3. Safety Safety for the target animals Tolerance studies in rabbits for fattening Tolerance studies in breeding does Microbiological safety of the additive Interactions Conclusions Safety for the consumer Metabolic and residue studies Metabolism Residues Toxicological studies Acute toxicity Genotoxicity Sub-chronic oral toxicity Chronic oral toxicity/carcinogenicity Reproduction toxicity including developmental toxicity Determination of No Observed Adverse Effect Level (NOAEL) Assessment of consumer safety Proposal for the acceptable daily intake (ADI) Consumer exposure and proposal for maximum residue limits (MRLs) Proposal for a withdrawal period Safety for the user Safety for the environment Exposure assessment Fate and behaviour Predicted environmental concentrations (PEC) Conclusion Effect assessment Toxicity to soil organisms Toxicity to aquatic organisms Bioaccumulation Risk Characterisation Risk for soil organisms Risk for groundwater Risk for surface water EFSA Journal 2011;9(3):2102 3

4 Risk for secondary poisoning Conclusion Efficacy Efficacy for rabbits for fattening Battery cage studies simulating field conditions Field trials Efficacy studies for breeding does Battery cage studies simulating field conditions Field trials Studies on the quality of the animal products Conclusions on the efficacy for target species Post-market monitoring Conclusions and recommendations Documentation provided to EFSA References Appendix EFSA Journal 2011;9(3):2102 4

5 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC 6 for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC. 7 The European Commission received a request from the company Alpharma Belgium BVBA 8 for authorisation of the product Cycostat 66G, robenidine hydrochloride, to be used as a feed additive for rabbits for breeding and for fattening (category: coccidiostats and histomonostats) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 10(2)/(7) (re-evaluation of an authorised feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 9 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 10 March The additive Cycostat 66G is currently authorised for use in chickens for fattening, turkeys and rabbits for fattening until 29 October 2014, 10 and authorised for rabbits for breeding purposes until 30 September The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the use of robenidine in feedingstuffs for rabbits (EC, 1982) and on the extension of the use of robenidine in feedingstuffs for rabbits for breeding purposes (EC, 1995). In 2004 EFSA issued an opinion on the re-evaluation of Cycostat 66G in accordance with article 9G of Council Directive 70/524/EEC (EFSA, 2004a) and the update of the same opinion (EFSA, 2004b). In 2008 EFSA issued an opinion on MRLs and withdrawal period for Cycostat 66G for chickens and turkeys for fattening (EFSA, 2008). MRLs for chickens and turkeys for fattening entered into force with Regulation (EC) No 214/ TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animal(s), consumer, user and the environment and the efficacy of the product Cycostat 66G (robenidine hydrochloride) when used under the conditions described in Table 1. 5 Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. OJ L 268, , p Council Directive 70/524/EEC of 23 November 1970 concerning additives in feeding-stuffs. OJ L 270, , p Council Directive 82/471/EEC of 30 June 1982 concerning certain products used in animal nutrition. OJ L 213, , p.8. 8 Alpharma Belgium BVBA Laarstraat 16, B-2610 Antwerpen, Belgium. 9 EFSA Dossier reference: FAD Commission Regulation (EC) No 1800/2004 of 15 October 2004 concerning the authorisation for 10 years of the additive Cycostat 66 G In feedingstuffs, belonging to the group of coccidiostats and other medicinal substances. OJ L 317, , p Commission Regulation (EC) No 2430/1999 of 16 November 1999 linking the authorisation of certain additives belonging to the group of coccidiostats and other medicinal substances in feedingstuffs to persons responsible for putting them into circulation. OJ L 296, , p Commission Regulation (EC) No 214/2009 of 18 March 2009 amending Regulation 1800/2004 as regards the terms of authorisation of the feed additive Cycostat. OJ L 73, , p. 12. EFSA Journal 2011;9(3):2102 5

6 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(-ies) of additive Cycostat 66G E758 Coccidiostat Functional group(s) of additive / Composition, description Active substance: Robenidine hydrochloride Additive composition: Robenidine hydrochloride: 66 g/kg Calcium lignosulfonate: 40 g/kg Calcium sulphate dihydrate: 894 g/kg Chemical formula C 15 H 13 Cl 2 N 5.HCl 1,3-bis[(pchlorobenzyliden e)amino] guanidine hydrochloride, CAS Number: Description Purity criteria (if appropriate) Robenidine hydrochloride > 97% Related impurities: *TRIS=N,N,N - tris[(pchlorobenzylidene)ami no]guanidine: 0.5% *AZIN = Bis-(4- chlorobenzylidene)hyd razine: 0.5% * any unknown impurity: 0.2% Method of analysis (if appropriate) HPLC method Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Cycostat 66G Alpharma Belgium BVBA Conditions of use Species or category of animal Maximum Age Minimum content Maximum content Withdrawal period mg/kg of complete feedingstuffs (if appropriate) Rabbits for breeding Rabbits for fattening / / No withdrawal needed No withdrawal needed Other provisions and additional requirements for the labelling Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) / / Post marketing monitoring will be performed in accordance with the available scientific techniques / EFSA Journal 2011;9(3):2102 6

7 Marker residue Robenidine hydrochloride Maximum Residue Limit (MRL) Species or category of animal Rabbits Target tissue(s) or food products Liver, muscle, kidney, fat Maximum content in tissues Not required EFSA Journal 2011;9(3):2102 7

8 ASSESSMENT 1. Introduction Cycostat 66G is a feed additive containing 66 g robenidine hydrochloride/kg, used in rabbits for the prevention of coccidiosis caused by Eimeria intestinalis, Eimeria flavescens, Eimeria magna, Eimeria media, Eimeria perforans and Eimeria stiedae. The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the use of robenidine in feedingstuffs for rabbits (EC, 1982) and on the extension of the use of robenidine in feedingstuffs for rabbits for breeding purposes (EC, 1995). In 2004, EFSA issued an opinion on the re-evaluation of the safety and efficacy of Cycostat 66G used for chickens, turkeys and rabbits for fattening, in accordance with article 9G of Council Directive 70/524/EEC (EFSA, 2004a). Additional data on the safety of Cycostat 66G were assessed and adopted in October 2004 (EFSA, 2004b). EFSA issued an opinion on MRLs and withdrawal period for Cycostat 66G for chickens and turkeys for fattening (EFSA, 2008). MRLs for chickens and turkeys for fattening entered into force with Regulation (EC) No 214/ The additive Cycostat 66G is currently authorised for use in rabbits for fattening, chickens for fattening and turkeys until 29 October and for rabbits for breeding purposes until 30 September Characterisation 2.1. Identity of the additive Cycostat 66G is composed of the active substance robenidine hydrochloride (66 g/kg), calcium sulphate dihydrate (894 g/kg) as a carrier and calcium lignosulphonate (40 g/kg) as binder. Analysis of eight batches of Cycostat 66G indicated product consistency, mean robenidine hydrochloride content was 68.6 g (range: g/kg). The additive is a greyish coloured free-flowing powder. A study 16 of five batches showed that 0.6 % of the particles were below 150 μm, and 2.2 % of the particles were above 850 μm. Tapped density of the product was 1.13 kg/l, and the bulk density 0.87 kg/l. Dustiness test showed that 14 mg of the product were retained on the filter per 25 g equivalent to 0.14g/m 3 (another Heubach s test, showed 0.13 g/m 3 ). 17 Five recent batches of Cycostat 66G were analyzed for heavy metals contents. 18 The maximum values measured were 1.88 mg/kg arsenic, 1.17 mg/kg lead, <0.50 mg/kg cadmium and 0.06 mg/kg mercury, which comply with Directive 2002/32/EC 19 on undesirable substances. The levels of dioxin/dioxin-like compounds (PCDD and PCDF s) and dioxin-like PCB s (constantly monitored) were measured 20 in one batch of Cycostat 66G and resulted in compliance with the requirements of 13 OJ L 73, , p OJ L 317, , p OJ L 296, , p Technical dossier/section II/ Annex II Technical dossier/section II/Annex II Supplementary information/july Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in feed. OJ L 140, , p Certificate/November EFSA Journal 2011;9(3):2102 8

9 Directive 2002/32/EC 21 and related amendments (<0.17 WHO-TEQ ng/kg and <0.32 WHO-TEQ ng/kg, respectively). Non-routinely performed tests on microbial purity show that Enterobacteria, fungi and yeasts are below 100 CFU/g of finished product. Moreover, Salmonella is absent in 10 g of finished product, whereas E. Coli and S. aureus are absent in 1 g of the final additive. 22, Characterisation of the active substance 24 Robenidine hydrochloride [N1,N3-bis[(p-chlorobenzilidene)amino] guanidine hydrochloride; CAS number : ] is a non-chiral chemically synthesized substance. A minimal purity of 97 % is given by the applicant. Its molecular formula is C 15 H 13 Cl 2 N 5 HCl; the molecular weight is Its structural formula is given in Figure 1. Figure 1 Structural formula of robenidine hydrochloride Two major impurities associated to the synthetic process have been identified as: N,N,N -tris[(pchlorobenzylidene)amino]guanidine (TRIS), Bis-(4-chlorobenzylidene)hydrazine (AZIN). The analysis of three batches of robenidine hydrochloride 25 using an HPLC method and chemical standards, indicated maximum values of 0.02 % for TRIS, 0.01 % for AZIN. Considering a water content <0.5 %, this corresponds to a robenidine hydrochloride purity > 99 % These data comply with the specifications given by the applicant, i.e. >97 % global purity, 0.5 % TRIS, 0.5 % AZIN and 0.2 %, total unknown impurities. In the former assessment of Cycostat 66G (EFSA, 2004a), three main impurities were identified in robenidine hydrochloride: the two mentioned above plus N1-[p-chlorobenzilidene)-1,2- hydrazinedicarboxamide (resulting also from the oxidative degradation of robenidine hydrochloride). The applicant proposed a specification of 0.5 % for each of these impurities, an HPLC method being validated for that purpose. The FEEDAP Panel considers that the present specification of <0.2 % covers the third impurity. The relevant physico-chemical properties of robenidine hydrochloride are summarized on Table 2. Table 2: Physicochemical properties of robenidine hydrochloride 26 Dissociation constant (pka) 3.5 Melting Point C Solubility in water at 20 C LogK ow * * Measurement performed at unknown ph g/l 3.3 (HPLC method) 21 OJ L 140, , p Technical Dossier/Section II. 23 Technical dossier/section II/Annex II This section has been edited following the provisions of Article 8(6) and 18 of Regulation (EC) No 1831/ Technical dossier/section II/Annex II Technical dossier/section II/Annex II.2.1. EFSA Journal 2011;9(3):2102 9

10 2.3. Manufacturing processes 27 The manufacturing process of the active substance and additve is fully described in the dossier Stability and homogeneity Shelf-life of the additive Shelf-life of the additive (three batches) was evaluated when kept under two environmental conditions: C/75 % relative humidity (RH) for six months and 25 C/60 % RH for up to 36 months. No significant loss of robenidine hydrochloride (and no concomitant appearance of degradation products TRIS and AZIN) was observed after 36 months under normal conditions. Recovery of robenidine hydrochloride under accelerated conditions was >97 % after three months and 93 % after six months Stability of the additive used in premixtures and feedingstuffs The stability in vitamin/mineral premixtures was determined 29 in three different premixtures (for broilers, turkeys (~ 8 g robenidine hydrochloride/kg) and rabbits (15 g robenidine hydrochloride/kg)). Two batches of the additive were used, and the premixtures were kept at 40 C/75 % RH for three months and 25 C/60 % RH for up to 12 months. Recovery of robenidine hydrochloride after 12 months at 25 C was > 96 %; recovery of robenidine hydrochloride in broilers, turkeys and rabbit premixtures after 3 months at 40 C was 90, 91 and 84 %, respectively. The stability during preparation (conditioning/pelleting) and storage of feedingstuffs was examined. 30 Pelleting of turkey complete feed (33 mg robenidine hydrochloride /kg) did not influence the robenidine hydrochloride content but that of rabbits diets (66 mg robenidine hydrochloride/kg) recovery was only 92 %. The pelleted feeds were kept at 25 C for three months, the recoveries were 85 % for the turkey feed, and 87 % in the rabbit feed Homogeneity The coefficient of variation of seven samples each from the premixture already described above was 6.8 %, 4.7 % and 10 % for broiler, turkey and rabbit premixtures, respectively. The coefficient of variation for broiler, turkey and rabbit complete feed ranged from 10.9 % to 14.9 % for mash feed, and 3.3 to 6.0 % for pelleted feed. Rabbit premixture and feedingstuffs were packed in 25 kg packs and transported for 1200 km. Homogeneity of the mixtures was not influenced by segregation during transport Conditions of use Cycostat 66G is a feed additive for the treatment and prevention of coccidiosis in rabbits for fattening and breeding. The recommended feed inclusion level is 50 to 66 mg robenidine hydrochloride/kg complete feed. The additive is administered during the complete life cycle of the animals. No withdrawal period before slaughter is foreseen Evaluation of the analytical methods by the European Union Reference Laboratory (EURL) EFSA has verified the EURL report as it relates to the methods used for the control of the active substance in animal feed. The Executive Summary of the EURL report can be found in the Appendix. 27 This section has been edited following the provisions of Article 8(6) and 18 of Regulation (EC) No 1831/ Technical Dossier/Section II/Annex II Technical Dossier/Section II/Annex II Technical Dossier/Section II/Annex II.4.4. EFSA Journal 2011;9(3):

11 3. Safety 3.1. Safety for the target animals Tolerance studies in rabbits for fattening No new studies have been provided. The FEEDAP Panel assessed in June 2004 (EFSA, 2004a) a tolerance study in rabbits for fattening, where doses of 0, 60, 100, 100/60, 200 and 500 mg robenidine hydrochloride/kg feed have been administered for the fattening period of 48 days, and concluded: Robenidine at 500 mg/kg feed, corresponding to a safety factor of about seven with respect to the maximum dose proposed for use, is not associated with adverse effects in rabbits for fattening during the fattening period. FEEDAP Panel notes (i) that the above conclusion is based only on zootechnical parameters, and (ii) that following the rules established by Regulation (EC) No. 429/ , the study would not have been considered because of the lack of haematological and biochemistry data as well as of gross necropsy Tolerance studies in breeding does In a preliminary study, female New Zealand White received a basal diet (n=32) or a diet supplemented with 660 mg robenidine hydrochloride/kg (n=33). The does were fed the experimental diets from six weeks before insemination until the end of lactation. The progeny was observed until weaning (28 days). Cycostat 66G at tenfold the maximum supplementation rate did not influence the reproduction capacity nor viability and growth of the kits during lactation. In a second study, 33 robenidine hydrochloride was administered daily by gavage to four groups of rabbits (24 per group) at 0, 20, 50 and 125 mg/kg body weight on days 6-28 of gestation. The trial is considered a developmental study (described more fully in Section ) and not acceptable for assessing the safety of a long term use of robenidine hydrochloride in breeding does. Upon request of the FEEDAP Panel, the applicant provided a third study 34 conducted according to Regulation (EC) No. 429/2008. A total of 80 breeding does (New Zealand rabbits, body weight about 3 kg) was divided into four groups, an un-supplemented control group, and three groups receiving feed supplemented with 66 (use level), 198 (intermediate dose = 3-fold overdose) and 330 mg robenidine hydrochloride/kg (high dose = 5-fold overdose). The intended robenidine hydrochloride contents were analytically confirmed. The diets were fed from three weeks prior to insemination until weaning (84 days). Fourteen days after insemination, the group size was randomly reduced to 15 animals. Different endpoints were recorded: zootechnical (body weight, feed intake), clinical (physical examinations, daily observations, mortality), reproductive (fertility, pregnancy, litter size, litter weight and pup mortality), on eight randomly selected does/group haematology and blood biochemistry (at three different occasions) as well as necropsy and histopathology (at the end of the study). Statistical evaluation war performed using ANOVA. The use level and the intermediate dose were well tolerated during the study. The results of necropsy, histopathology, haematology and blood biochemistry were comparable between all groups, no treatment related effects were found including the high dose group. Adverse effects were found in the high dose group concerning lower fertility rate (not significant to the control group), higher incidence of stillbirth rate (significant to the control group), lower kit body weight and average daily growth rate 31 Commission Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC) No 429/2008 of the European Parliament and of the Council as regards the preparation and presentation of applications and the assessment and authorisation of feed additives. OJ L 133, , p Technical Dossier/Section III/Annex Technical dossier/section III/Annex Supplementary information/september 2010/Annex 1. EFSA Journal 2011;9(3):

12 of kits (significant to the control group). The intermediate dose of 198 mg/kg (equal to 11 mg/kg bw/day) was taken as the NOAEL for this study Microbiological safety of the additive No new data were provided. The FEEDAP Panel concluded in June 2004 (EFSA, 2004a): Robenidine is active against Gram-positive bacteria and Gram-positive bacteria are susceptible at concentrations relevant for the in vivo situation, considering the proposed dose range. The FEEDAP Panel also concluded when assessing a study on the in vivo development of resistance to robenidine hydrochloride in rabbits that: Robenidine feed supplementation at recommended doses to rabbits does not cause in vivo resistance to robenidine in the investigated bacterial groups (Enterococcus, Escherichia coli, Salmonella and Campylobacter) Interactions No interactions or incompatibilities with feed materials, carriers, other approved additives or veterinary drugs have been recorded or reported Conclusions The conclusion drawn on the safety of robenidine hydrochloride (66 mg/kg feed) for rabbits for fattening by the FEEDAP Panel in 2004 was not longer maintained because essential end points, mandatory for substances with a margin of safety 10, have not been studied. Among the trials submitted as tolerance studies (including the preliminary study), only one could be assessed as a tolerance study. Feeding the 5-fold overdose to breeding does resulted in an impairment of reproductive parameters; the 3-fold overdose was tolerated. The FEEDAP Panel concludes that feed concentrations 66 mg robenidine hydrochloride from Cycostat 66G/kg complete feed are safe for breeding does. The margin of safety is about three. This study is also considered sufficient to allow conclusions on the safety of the additive for rabbits for fattening. Although exposure of suckling rabbits to feed was rather short (about three weeks), the kids have been already intrauterinally exposed and received for four weeks milk from exposed does. Consequently, feed concentrations 66 mg robenidine hydrochloride from Cycostat 66G/kg complete feed are safe for rabbits for fattening. The FEEDAP Panel reiterates its former statement that feed supplementation with robenidine hydrochloride, active against Gram-positive bacteria and Gram-positive bacteria at concentrations relevant for the in vivo situation, does not cause in vivo resistance to robenidine hydrochloride in Enterococci, Escherichia coli, Salmonella and Campylobacter in rabbits at use dose level Safety for the consumer Metabolic and residue studies No new studies on the metabolism and residues of robenidine hydrochloride in rabbit have been submitted since the last assessment (EFSA, 2004a) Metabolism The main conclusions of the former assessment (EFSA, 2004a) where the following: EFSA Journal 2011;9(3):

13 i) Robenidine hydrochloride (radiolabelled) is absorbed and processed rapidly in the rabbit, 80 % of the administered dose being recovered in the faeces and 20 % in the urine. The metabolic equilibrium in plasma is reached after one day. ii) The metabolic pathway of robenidine hydrochloride in the rabbit involves the hydrolysis of the semi-carbazide bonds of the molecule, followed by the oxidation of the resulting p- chlorobenzaldehyde to p-chlorobenzoic acid which is in turn conjugated to glycine giving rise to p-chlorohippuric acid. No significant qualitative gender difference is observed. iii) In the faeces, unchanged robenidine hydrochloride is the major compound (70 to 80 %) excreted, three minor metabolites representing each less than 10 %. In the urine, no robenidine hydrochloride is detected and, depending on the study, p-chlorobenzoic acid or p- chlorohippuric acid are the major metabolites (more than 50 %), with a further 11 metabolites representing each less than 10 %. iv) In tissues and at zero-day (the only withdrawal time investigated), about 50 %, 60 %, 75 % and 65% of total residues in liver, kidney, abdominal fat and muscle, respectively, are extractable. Unchanged robenidine hydrochloride represents a very limited fraction of total residues in the liver (12 and 2 % for male and female, respectively) and kidney (3 and 3 %) and is absent from the muscle and abdominal fat. The major metabolite in liver is p- chlorobenzoic acid (6 % and 12 %), six unidentified metabolites representing each less than 10 %. The major metabolite in muscle is p-chlorobenzoic acid (18-22 %), three unidentified metabolites representing 4 % to 13 %; very similar figures are found in the kidney. In the abdominal fat, a major unidentified metabolite represents 60 % and 36 % (male and female) of total metabolites, three other metabolites representing each less than 10 %. The acid hydrolysis of the non-extractable fraction of liver (protein-linked) generates p-chlorobenzoic acid. v) The metabolic fate of robenidine hydrochloride in the rat is similar qualitatively and quantitatively to that in the rabbit. The re-assessment of residue data in fat from chicken, turkey and rabbit (EFSA, 2004a) indicate that, in similar experimental conditions, the major unidentified metabolite found in rabbit abdominal fat (60 % in males) has the same chromatographic behaviour as the major metabolite found in chicken s abdominal fat (60 %) and skin/fat (3 %), but also in the turkey at much lower levels (3 and 1 %, respectively). No such metabolite was observed in rat excreta and tissues, even if its presence cannot be excluded considering the limitation of the analytical tools at the time the study was performed (1968) which did not allow to separate and identify minor metabolites in fat besides unchanged robenidine hydrochloride and p-chlorobenzoic acid. These new considerations challenge to a certain extent the claimed similarity of the metabolic fate in the rat and rabbit, and the validity of the toxicological data from rodents to derive human safety Residues Robenidine total residue data for rabbits for fattening administered the maximum dose proposed for use (66 mg/kg feed) have been taken from the former assessment (EFSA, 2004a) and completed with the standard deviation values (Table 3). EFSA Journal 2011;9(3):

14 Table 3: Kinetics of robenidine-derived total residues in tissues (mg equivalent robenidine/kg) of rabbits administered 66 mg [ 14 C]-robenidine/kg feed for 7 consecutive days and following a withdrawal period. Withdrawal (day) Liver Kidney Muscle Fat (abdominal) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± It appears that all along the withdrawal period the highest residues were found in the liver, which appeared as the target tissue, and by decreasing order in the kidney, abdominal fat and muscle. Robenidine was measured simultaneously in the same tissue samples using a validated (internally) analytical method with a LOQ of 0.1 mg/kg; all values were below the LOQ in all tissues at zero-day withdrawal time and beyond. The FEEDAP Panel considered p-chlorobenzoic acid as a more relevant marker residue than robenidine and asked the applicant to supply an analytical method for its determination in tissues. The applicant gathered data showing that p-chlorobenzoic acid is a degradation compound of polychlorobiphenyls, but also the degradation compound/metabolite of certain drugs, and therefore not robenidine-specific. 35 Taking these data into consideration, the FEEDAP Panel concludes that p- chlorobenzoic acid is not an appropriate marker residue and that robenidine should be retained by default Toxicological studies The main conclusions of the former assessment (EFSA, 2004a, 2004b) and the results of a new 90-day rat study are reported below Acute toxicity The acute oral toxicity of robenidine hydrochloride was investigated in both sexes of rats giving a LD 50 of just over 3000 mg/kg bw. A study in male mice (1968) indicated a LD 50 of 150 mg/kg bw while in rabbits the value was 1500 and 2900 mg/kg bw/day for males and females, respectively (EFSA, 2004a) Genotoxicity For testing genotoxicity two in vitro and one in vivo test have been conducted (EFSA, 2004a). Robenidine hydrochloride was tested for mutagenic activity in Salmonella typhimurium, (TA1535, TA1537, TA98 and TA100) and in Escherichia coli (WP2uvrA) in the presence and in the absence of metabolic activation (S9-mix prepared from liver of rats treated with Aroclor 1254), in compliance with OECD guideline 471 (1997). The test material did not induce gene mutations in any bacterial strain up to a concentration of 100 μg/plate, while cytotoxicity, detected as reduction of the background bacterial growth, was observed at 33 and 100 μg/plate, with and without S9-mix, respectively. The positive controls gave the expected response. The potential for inducing chromosomal aberrations was studied in CHO cells, with and without S9- mix prepared from liver of rats treated with Aroclor 1254 (OECD guideline 473). Robenidine hydrochloride was tested within a concentration range from 1.25 to 20 μg/ml. No clastogenic effect was observed, while the positive controls resulted significantly effective. A weak increase in 35 Supplemetary information/july EFSA Journal 2011;9(3):

15 polyploidy was reported only in the presence of S9-mix and only at cytotoxic concentrations (cell count below 40 %). An in vivo micronucleus test was conducted in bone marrow erythrocytes of CD-1 mice. Five animals per sex per dosage group were treated orally twice, at 0 h and 24 h, at three exposure levels in corn oil, up to the maximum tolerated dose of 100 mg/kg bw, and bone marrow samples were taken 48 h after the initial 0 h administration. Although no local bone marrow toxicity was reported, the treatment caused evident systemic toxicity and one animal death occurred in the high dose group. No micronucleus induction was detected in animals treated with robenidine hydrochloride, while the positive control group, dosed orally with cyclophosphamide, responded with a substantial increase in the number of micronuclei. Conclusion Robenidine hydrochloride resulted negative in bacterial reverse mutation assay, not clastogenic in cultured mammalian cells and did not induce micronuclei in the bone marrow of treated mice. The incidental observation of polyploidy in the in vitro chromosomal aberration test is interpreted as the consequence of cytoxicity. Therefore robenidine hydrochloride can be considered not genotoxic Sub-chronic oral toxicity Sub-chronic studies (90-days) have been performed in rats, mice and dogs. In the rat study (performed in 1968) no substance-related changes have been observed and a NOAEL of 13.5 mg/kg bw/day based on the highest dose in males has been identified. A NOAEL of 14 mg/kg bw/day in mice (study performed in 1968) due to renal changes (focal nephritis) was proposed. A re-evaluation of the 90-day dog study (performed in 1968) showed that the original robenidine hydrochloride concentrations were given in mg/kg feed. Growing dogs were used and a constant daily amount of feed was given. Calculating from these data (375, 750 and 1500 mg robenidine hydrochloride/kg feed) and the daily feed of 325 g/dog, 19, 34 and 66 mg/kg bw/day were used. The lowest NOEL in this study was therefore 19 mg/kg bw/day derived from the data on the increase in relative liver weight. It should be noted that this effect has not been observed in a two-year dog study. The NOAEL is about 2.5 times higher than the value the FEEDAP Panel calculated in Because of the low quality of the data and the uncertainties to establish a robust NOAEL, the Benchmark Dose approach (BMD) was applied to re-analyse some of the data from the 90-day dog and rat studies (organ weights, haematological and biochemical parameters). A precondition for applying the BMD approach is the existence of a dose dependency of the different experimental parameters. The approach determines the dose (Benchmark Dose, BMD) that most likely leads to a Benchmark response (BMR) together with its one-sided confidence interval (here, a confidence level of 90 % BMD has been chosen). Then, with a probability of 90 %, the minimum dosage leading to a critical response is higher than the lower confidence limit (Benchmark Dose Lower Limit, BMDL). The re-analyses and model fittings (linear, polynominal and power models) were performed using BMDS Version 1.4.1c (National Center For Environmental Assessment). In the dog study only two parameters, white blood cell (WBC) increase and relative liver weight, showed a dose dependency and both parameters were assessed by the BMD approach. For the parameter WBC change, the polynomial model was the only one that fitted the data adequately; the resulting BMDL was 190 mg/kg (9.6 mg robenidine hydrochloride/kg bw). However, the toxicological relevance of these findings remains unclear. For the parameter relative liver weight, the power model was the only one that fitted the data adequately but was not appropriate as a large ratio BMD/BMDL indicated a large uncertainty in the modelling. Because modelling of the limited data set could lead to over- or underestimation of the BMDL, the approach has not been further taken into consideration, and a new sub-chronic toxicity study was considered necessary. In the rat study, no reliable dose-response curves could be estimated and the BMD approach could not be applied. EFSA Journal 2011;9(3):

16 A new 90-day GLP-compliant rat study has been provided. 36 The study was performed according to OECD guideline 408. Based on the results of a dose-finding study 37 doses of 0, 15, 37 und 95 mg robenidine hydrochloride/kg bw/day were chosen. For the main study ten female and ten male rats were used for each dose group and ten of each gender served as controls. Clinical signs, functional observations, body weight, dietary intake and water consumption were monitored during the study. Hematology, blood chemistry and urine analysis were evaluated for all animals during week six and at the end of the treatment period (week 13). Ophthalmoscopic examination was also performed on all animals prior to start of treatment and all control group and high dose animals during week 12 of the study. All animals were subjected to gross necropsy examination, and histopathological evaluation of tissues was undertaken for all animals. Rats treated with the highest dose showed reduced feed intake and body weight gain. In the high dose group small changes of haematological parameters could be detected (mean corpuscolar hemoglobin, mean corpuscolar haemoglobin concentration). At the study mid-point and terminal examinations in the high dose group an increase of serum bile acids, creatinine and urea concentrations were observed in both genders together with lower serum concentrations of bilirubin and cholesterol values and decreased triglyceride concentrations. A significant increase in glucose levels was seen in all male treatment groups in comparison to the controls. However this increase was minimal and not dose related. No other treatment-related changes were detected in animals treated with 15 and 37 mg robenidine hydrochloride/kg bw/day. The histopathological investigation demonstrated in the kidney an increased incidence and severity of tubular basophilia along with tubular-epithelial cell necrosis, granular casts, tubular dilatation and tubulo-interstitial inflammation in males and females of the high dose. In the mesenteric lymph nodes aggregation of histiocytes could be observed and in the liver an increase in weight and of inflammatory foci were seen at 95 mg/kg bw/day. A significant decrease in absolute and relative thymus weight in male rats (p<0.05) was observed in all dose groups, but without histopathological changes in the low and intermediate dose group. The differences between the treatment groups were small and within the expected historical range. Consequently, this finding was considered not to be of toxicological significance. Males in the mid-dose but not in the high dose group showed a reduction in the testes weight. As no treatment related changes could be observed at the mid dose of 37 mg/kg bw/day, this dose is considered as the NOAEL of the study Chronic oral toxicity/carcinogenicity The two available chronic studies, an 84-week rat study and a two-year dog study, were performed in 1970 and 1969, respectively. In the 84-week rat study, groups of 60 (control) or 35 (all other groups) Wistar rats of each sex received via feed either 0 (control), 4, 12 and 24 mg robenidine hydrochloride/kg bw/day for 26 weeks after which time 10 (control) or 5 (all other groups) rats per sex per group were killed. After 64 weeks the low dose group received 80 mg robenidine hydrochloride/kg bw. Histopathology examination was limited to 10 controls and 5 animals from each treated group per sex surviving at the termination of the study. There is lack of data on interim post mortems and interim deaths. No tumour developments or pre-neoplastic lesions were observed. In the dog study groups of five (high dose) or six (all other doses) beagle dogs of each sex were fed diets containing either 0, 200, 400 or 1000 mg robenidine hydrochloride/kg feed (approximately 0, 36 Supplementary information/september 2010/Annex Supplementary information/september 2010/Annex 2. EFSA Journal 2011;9(3):

17 6.5, 13 and 32.5 mg/kg bw/day). Histopathological examination was performed mainly on the control and high dose animals. Tumour developments were not recorded. Neither study cannot be considered as carcinogenicity studies mainly due to the short duration and the insufficient number of animals examined. However, it can be noted that no treatment related tumors or neoplastic lesions were reported. The lack of genotoxic potential considered alongside the absence of any findings of pre-carcinogenic lesions in toxicological studies supports the conclusion that robenidine hydrochloride is not carcinogenic. Both studies are also considered inadequate in design, conduct and reporting by present standards as chronic toxicity studies. Consequently, no NOAELs could be derived Reproduction toxicity including developmental toxicity A reproduction study in rabbits (52 controls animals, 53 treated) was provided. 38 The purpose of this study was to evaluate the effects of a feed supplemented with the maximum authorised concentration of Cycostat 66G (66 mg robenidine/kg feed) during three consecutive pregnancies and lactations from six weeks before insemination until the end of the study. The endpoints selected were: i) evolution of the body weight of the females, ii) losses of females (morbidity, mortality, culling), iii) frequency of the females in oestrus and fertility and iv) viability of the litter during the period of lactations. No negative maternal effects or effects on litters by the treatment were seen at any stage of the study. A two generation study in rats was assessed in 2004 (EFSA, 2004a). Groups of 10 male and 20 female rats were fed diets containing either 0 (control) 10, 50 or 500 mg robenidine hydrochloride/kg for nine weeks (corresponding to 0; 0.07; 3.2; 33 mg robenidine hydrochloride/kg bw/day (P1 females before mating)). Each male was then caged with two females from the same treatment group. The males were transferred to different pairs of females from the same treatment group each week for three weeks. Females were allowed to give birth to their litters (F1a) that were culled to give standard maximum litter size of eight pups. Approximately one third of the pups from the first generation were necropsied. The parents were then rebred, as before, to produce a second set of litters (F1b). Ten male and 20 females were selected at random from each treatment group of the F1b pups to provide the second parental generation (P2). The P2 parents were bred as previously for the P1 but the time of mating was determined for each female and for approximately half the P2 females caesarean sections were performed on day 19 of gestation. Foetuses were preserved for alizarin staining (two thirds) or serial sectioning (one third) to investigate possible skeletal or visceral abnormalities, respectively. Remaining P2 females were allowed to produce their litters and rear to weaning, when they were brought to necropsy. A full range of observations was made at each stage of the study. No teratogenic effects or influences on reproduction by the treatments were observed at any stage of the study on females, litters and foetuses. In mated female New Zealand White rabbits a developmental toxicity study was provided. The animals were randomised into three test groups and one vehicle control group, each containing approximately 24 animals. The animals were treated once daily by gavage over day 6-28 of gestation. The dose levels were 0, 20, 50 and 125 mg robenidine hydrochloride/kg bw/day. Animals were killed on day 29. Maternal effects of robenidine hydrochloride were indicated by convulsions in one animal at 50 mg/kg bw/day, and one animal at 125 mg/kg bw/day. Maternal toxicity at 125 mg robenidine hydrochloride/kg bw/day was also indicated by reduced body weight gain and food consumption. Mean foetal weights at 50 and 125 mg robenidine hydrochloride/kg bw/day were lower than control. The incidence of foetuses with the costal cartilage of Rib 7 not attached to the sternum at 20 mg/kg/day (11 %) was similar to control in contrast to higher doses. In summary, the maternal and foetal NOAEL in this study was 20 mg robenidine hydrochloride/kg bw/day. 38 Technical dossier/section III/Annex 5. EFSA Journal 2011;9(3):

18 Determination of No Observed Adverse Effect Level (NOAEL) The NOAELs of the previous 90-day studies are in a narrow range: 13.5 mg robenidine hydrochloride/kg bw/day in the rat, 14 mg/kg bw/day in mice and 19 mg/kg bw/day in dogs. The recent 90-day rat study revealed a NOAEL of 37 mg/kg bw/day. The FEEDAP Panel also evaluated a tolerance study in rabbits 39 for its potential to derive a NOAEL. Study duration was comparable with the 90 days applied in rodents and the dog. Based on accurate measurements of the weekly feed intake and of body, a NOAEL of 11 mg robenidine hydrochloride/kg bw/day could be derived, based on reproductive effects at the higher dose. The NOAEL derived from the rabbit tolerance study is lower than that derived from the other sub-chronic studies with rodents and dogs and it cannot be ignored. As the similarity of the metabolism of robenidine hydrochloride in the rat and the rabbit was not established unambiguously a necessary condition to derive an ADI the FEEDAP Panel decided not to consider further this uncertainty and to use the conservative approach offered by the NOAEL from the rabbit tolerance study Assessment of consumer safety Proposal for the acceptable daily intake (ADI) The FEEDAP Panel proposes an ADI of 0.11 mg robenidine hydrochloride/kg bw, equivalent to 6.6 mg for a 60 kg adult, based on the lowest NOAEL of 11 mg robenidine hydrochloride/kg bw/day derived from the rabbit tolerance study, applying a safety factor of Consumer exposure and proposal for maximum residue limits (MRLs) Considering the re-assessment of metabolism data in mammals and birds and the toxicological data on rabbit, the FEEDAP Panel concludes that: (i) the toxicological evaluation of robenidine hydrochloride and robenidine-derived residues in the rabbit is thoroughly investigated through the toxicological package submitted, (ii) the similarity of the metabolic fate of robenidine hydrochloride in the rat and the target animal (reflecting consumer exposure) is no longer a critical point. The theoretical exposure of the consumer has been calculated according to daily human food consumption values set by Regulation (EC) No 429/ and the highest robenidine-derived total residue levels (average values plus 2SD, 95 % confidence limit) measured at steady state (0-day withdrawal (Table 4). Table 4: Human consumer daily exposure to whole robenidine-derived residues from relevant rabbit tissues taken at 0-day withdrawal and corresponding percentage of the acceptable daily intake (ADI). Liver Kidney Muscle Fat Sum Total residues* Daily intake (mg) % ADI (0.96 mg/person/day) *Total residues calculated as average + 2 SD (mg equivalent robenidine/kg tissue) The results indicate that consumer exposure to the whole robenidine residues present in rabbit tissues at zero-day withdrawal complies with the ADI and consequently do not pose a risk. Consequently, no MRL would normally be required. If MRLs should be set for control purposes they should concern only the liver and kidney, no significant marker residue (robenidine) concentrations being found in the muscle and fat. The most conservative ratios robenidine to total residues retained for the liver and kidney were 0.07 and 0.03, respectively (see ). MRLs were chosen as twice the LOQ of the 39 Supplementary information/september 2010/Annex OJ L 133, , p.1. EFSA Journal 2011;9(3):