Assessment of the feed additive consisting of robenidine hydrochloride (Cycostat® 66G) for rabbits for breeding and rabbits for fattening for the renewal of its authorisation (Zoetis)

Abstract

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 feed additive consisting of robenidine hydrochloride (Cycostat® 66G) for rabbits for breeding and rabbits for fattening for the renewal of its authorisation. Since the potential aneugenic activity of robenidine hydrochloride cannot be excluded, the FEEDAP Panel is not in the position to conclude on the safety of the additive for the target species and the consumer. In the absence of data, the FEEDAP Panel cannot conclude on the safety of robenidine hydrochloride for the environment. Robenidine hydrochloride has potential for bioaccumulation; however, a risk for secondary poisoning for worm/fish‐eating birds and mammals is not likely to occur. No concern for groundwater is expected. Robenidine hydrochloride is not a skin or eye irritant and not a skin sensitiser. Due to the lack of data on the genotoxicity (aneugenicity) of the substance, the exposure to the additive of the unprotected users should be minimised. Owing to the lack of sufficient data, the FEEDAP Panel cannot conclude that robenidine hydrochloride from Cycostat® 66G is still efficacious against recent Eimeria spp. strains in rabbits.

1. Introduction

1.1. Background and Terms of Reference

Regulation (EC) No 1831/2003 ¹ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 14(1) of that Regulation lays down that an application for renewal shall be sent to the Commission at the latest 1 year before the expiry date of the authorisation.

The European Commission received a request from Zoetis Belgium SA ² for the renewal of the authorisation of the additive consisting of robenidine hydrochloride (Cycostat® 66G), when used as a feed additive for rabbits for breeding and rabbits for fattening (category: coccidiostats and histomonostats).

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 14(1) (renewal of the authorisation). EFSA received directly from the applicant the technical dossier in support of this application. The particulars and documents in support of the application were considered valid by EFSA as of 9 October 2020.

According to Article 8 of Regulation (EC) No 1831/2003, 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. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and on the efficacy of the product robenidine hydrochloride (Cycostat® 66G), when used under the proposed conditions of use (see Section 3.1.3).

1.2. Additional information

The additive is currently authorised for rabbits for breeding and for fattening with end of period of authorisation of 21 June 2021. ³ Maximum residue limits (MRLs) are in force pursuant of the same regulation: 200 μg robenidine hydrochloride/kg wet weight for liver and kidney and 100 μg robenidine hydrochloride/kg wet weight for all other tissues. The same additive (under the name Robenz® 66G) is also authorised in chickens for fattening until 25 February 2030 ⁴ while the same additive for use in turkeys has been withdrawn from the European market in 2021. ⁵

In 2011, the EFSA FEEDAP Panel re‐evaluated the additive in accordance with Regulation (EC) No 1831/2003 for its use in rabbits for breeding and fattening (EFSA FEEDAP Panel, 2011) and in 2019, adopted the opinion on the re‐evaluation for chickens and turkeys for fattening (EFSA FEEDAP Panel, 2019a).

2. Data and methodologies

2.1. Data

The present assessment is based on data submitted by the applicant in the form of a technical dossier ⁶ in support of the authorisation request for the use of robenidine hydrochloride (Cycostat® 66G) as a feed additive.

The FEEDAP Panel used the data provided by the applicant together with data from other sources, such as previous risk assessments by EFSA or other expert bodies, peer‐reviewed scientific papers and other scientific reports, to deliver the present output.

The European Union Reference Laboratory (EURL) considered that the conclusions and recommendations reached in the previous assessment are valid and applicable for the current application. ⁷

2.2. Methodologies

The approach followed by the FEEDAP Panel to assess the safety and the efficacy of robenidine hydrochloride (Cycostat® 66G) is in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents: Guidance on the renewal of the authorisation of feed additives (EFSA FEEDAP Panel, 2013), Guidance on the identity, characterisation and conditions of use of feed additives (EFSA FEEDAP Panel, 2017a), Guidance on the assessment of the safety of feed additives for the target species (EFSA FEEDAP Panel, 2017b), Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c). Guidance on the assessment of the efficacy of feed additives (EFSA FEEDAP Panel, 2018), Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019b) and Guidance on studies concerning the safety of use of the additive for users/workers (EFSA FEEDAP Panel, 2012).

3. Assessment

The additive under assessment robenidine hydrochloride (Cycostat® 66G) is authorised as coccidiostat for use in rabbits for breeding and fattening in the prevention of coccidiosis with a minimum content of 50 and a maximum content of 66 mg robenidine HCl/kg complete feed.

The applicant has applied for the renewal of this authorisation under the same conditions of use as currently authorised.

3.1. Characterisation

Robenidine hydrochloride is produced via chemical synthesis. The final product Cycostat® 66G is manufactured by blending the active substance with calcium lignosulfonate and calcium sulfate dihydrate and subsequent granulation. The manufacturing process has been assessed in former opinions and no changes have been introduced since then (EFSA FEEDAP Panel, 2011, 2019b).

3.1.1. Characterisation of the additive

The composition of Cycostat® 66G is the same as described in the previous opinion (EFSA FEEDAP Panel, 2011); it contains 6.6% robenidine HCl, 4% calcium lignosulfonate and 89.4% calcium sulfate dihydrate. Analysis of six recent batches of the additive indicated product consistency: robenidine HCl content was in the range of 6.5–6.8% (specification: 6.1–7.1%). ⁸

Three batches were tested for impurities. ⁹ Cadmium, mercury and fluorine were all below the limit of quantification (LOQ). ¹⁰ Lead content was in the range of 0.71–0.73 mg/kg and arsenic in the range of 0.80–0.87 mg/kg. The content of Aflatoxin B1 was below the LOQ. ¹¹

The levels of polychlorinated dibenzo‐p‐dioxins and dibenzofurans (PCDD/F) and the sum of PCDD/F and dioxin‐like polychlorinated biphenyls (PCBs) were 0.16 ng WHO‐PCDD/F‐TEQ/kg and 0.30 ng WHO‐PCDD/F‐PCB‐TEQ/kg, respectively, in all three batches tested. Non‐dioxin‐like PCB content was 1.7 μg/kg. The impurities do not represent a concern.

The same three batches of the additive were analysed for microbiological contamination. Salmonella spp. was not detected in 25 g, Enterobacteriaceae were < 10 CFU/g and the total plate count of aerobic mesophilic bacteria was < 100 CFU/g. Moulds and yeasts were both < 100 CFU/g in all three batches. No concern has been identified.

Cycostat® 66G is a greyish coloured free‐flowing granular preparation with particles between 150 and 850 μm. Since the manufacturing process did not change, no change is expected in the technological properties and physical characteristics of the product which has been recently reviewed by the FEEDAP Panel (EFSA FEEDAP Panel, 2019a). The applicant provided new data by sieve analysis on the particle size distribution; results were in line with the specifications (≤ 5% of particles below 150 μm; ≤ 5% above 850 μm). ¹² The dusting potential reported in the FEEDAP opinion in 2019 was measured in three batches (Stauber–Heubach) and was 0.14 g/m³ (EFSA FEEDAP Panel, 2019a); no recent data on dusting potential were submitted. ¹³ A new stability study has been also provided which confirmed the shelf life of 36 months for the product under assessment. ¹²

It is noted that the particle size data available do not allow the risk assessors to exclude the presence of small/nano particles as foreseen in the Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles (EFSA SC, 2021a). Therefore, the applicant was requested to provide information by choosing any of the appraisal routes as indicated by the aforementioned guidance document. The applicant made reference to the existing safety studies (appraisal route described in Section 4 of the Guidance on technical requirements) including tolerance studies, toxicokinetic and residue studies performed with the additive under assessment (see Section 3.2) to conclude that the safety of robenidine hydrochloride from Cycostat® 66G can be adequately covered by the conventional risk assessment. During the risk assessment, the FEEDAP Panel reviewed the available studies and concluded that any risks from particles that are potentially below 500 nm in size have already been covered by the existing data.

3.1.2. Characterisation of the active substance

For robenidine hydrochloride (C₁₅H₁₃Cl₂N₅·HCl; N1,N3‐bis[(p‐chlorobenzilidene)amino] guanidine hydrochloride; CAS number 25875‐50‐7; EC number 247‐307‐8) a minimum purity of 97% is specified by the authorising regulation. In addition, two related impurities are specified as N,N′,N″‐tris[(p‐Cl‐benzylidene)amino]guanidine (TRIS) and bis‐4[4‐Cl‐benzylidene]hydrazine (AZIN) with a maximum content of 0.5%, each. Analytical data ¹⁴ from six recent batches showed compliance with these specifications: average robenidine HCl concentration ranged between 99.0–100.3%, TRIS and AZIN < 0.05%. ¹⁵ The analysis of six recent batches of the robenidine HCl concentrations of the solvents used in the manufacturing process (■■■■■) below the company specification (3,000 and 5,000 mg/kg, respectively) and raised no concern. ¹⁴

3.1.3. Conditions of use

Cycostat® 66G is currently authorised as a feed additive for the prevention of coccidiosis in rabbits for breeding and fattening with a minimum content of 50 mg and a maximum content of 66 mg robenidine HCl/kg complete feed. The authorisation under other provisions foresees: (i) the additive shall be incorporated in compound feed in the form of a premixture, (ii) robenidine hydrochloride shall not be mixed with other coccidiostats, (iii) for safety: breathing protection, glasses and gloves shall be used during handling, (iv) a post‐market monitoring programme on the resistance to Eimeria spp. shall be planned and executed by the holder of authorisation and (v) use is prohibited at least 5 days before slaughter. The applicant proposes to keep the same conditions of use as authorised.

3.2. Safety

The safety of the additive and its active substance for the target species, consumer, user and environment was re‐evaluated in previous FEEDAP opinions (EFSA FEEDAP Panel, 2011, 2019a).

The applicant carried out a literature search providing information on the safety of robenidine HCl using three different databases (PubMed, Cab Abstracts, FSTA) covering the period 2010–2020. ¹⁶ The search terms used were related to the active substances or the product name. The inclusion criteria were related to species (rabbits, human, animal target species including dogs and cats) reporting safety, adverse events and toxicity. In vitro studies were also included. The FEEDAP Panel reviewed the papers retrieved and quoted them in the below chapters, if considered relevant.

3.2.1. Absorption, distribution, metabolism, excretion and residues

The data on the absorption, distribution, metabolism, excretion (ADME) of robenidine HCl in rabbits and laboratory animals, as well as residue data in the target species, were evaluated in 2011 and 2019 in the context of the re‐evaluation of the additive (EFSA FEEDAP Panel, 2011, 2019a). The main conclusions are summarised below:

• –Robenidine HCl (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 1 day.
• –The metabolic pathways of robenidine HCl 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.
• –In the faeces, unchanged robenidine HCl is the major compound excreted (70–80%), along with three minor metabolites representing each less than 10%. In the urine, no robenidine HCl 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%.
• –In tissues and at zero‐day (the only withdrawal time investigated), about 50%, 60%, 75% and 65% of total residues in the liver, kidney, abdominal fat and muscle, respectively, are extractable. Unchanged robenidine HCl represents a very limited fraction of total residues in the liver (12 and 2% for male and female, respectively) and kidney (3% for both) and is absent from the muscle and abdominal fat. The major metabolite in liver is p‐chlorobenzoic acid (6% and 12% for male and female, respectively), with six unidentified metabolites representing each less than 10%. The major metabolite in muscle is p‐chlorobenzoic acid (18–22%), followed by three unidentified metabolites representing 4–13%; very similar figures are found in the kidney. In the abdominal fat, a major unidentified metabolite represents 60% and 36% (male and female, respectively) 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.
• –The similarity of the metabolic fate of robenidine HCl in the rat and in rabbits could not be unequivocally confirmed due to the following considerations: in similar experimental conditions, the major unidentified metabolite found not only in rabbits' 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 rats' excreta and tissues, even if its presence cannot be excluded considering the limitation of the analytical tools at the time the study was performed (year 1968), which did not allow to separate and identify minor metabolites in fat besides unchanged robenidine HCl and p‐chlorobenzoic acid. As a consequence, the validity of the toxicological data from rodents to derive human safety was put into question.
• –From total residue studies, it appeared 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 should be retained as the marker residue.

For the current assessment, the applicant did not submit new studies but made reference to the former evaluations and submitted a literature review.

The FEEDAP Panel reviewed the outcome of the literature search and reviewed the ADME and residue studies on robenidine HCl already assessed in previous FEEDAP opinions (EFSA FEEDAP Panel, 2011, 2019a), considering the most recent FEEDAP guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c).

As a result of the literature review, the applicant identified two relevant full publications in relation to the ADME and residues of robenidine HCl. The first one is a pharmacokinetic and bioavailability study in rabbit performed with 100 mg/kg bw, i.e. 25 times the oral dosage via feed (Li et al., 2017); the second one is a study of the excretion of robenidine in rabbit urine and faeces performed with the non‐radioactive item (Tian et al., 2015). The FEEDAP Panel noted that these publications do not add new information on the ADME of robenidine.

The Panel noted that, in order to compare the toxicological profile of robenidine HCl in laboratory animals and in the target species, additional information (structural identification and toxicological profile) would be required on the major unidentified metabolite found in the abdominal fat of rabbit representing 60% of the whole activity. Since such information was not submitted, ¹³ the Panel cannot conclude on the similarity of metabolic pathways of the additive between the laboratory animals and the target species.

3.2.2. Toxicological studies

The toxicological profile of robenidine HCl was last assessed by the FEEDAP Panel in 2019 (EFSA FEEDAP Panel, 2019a). For the current assessment, the applicant made reference to the toxicological studies previously assessed and performed a literature review.

As a result of the literature review, the applicant did not identify relevant papers in relation to the toxicology of robenidine HCl.

General toxicology

The overview on the toxicological profile of robenidine HCl, as already reported in the Panel opinion adopted in 2019 (EFSA FEEDAP Panel, 2019a), is reiterated for the current evaluation as follows:

‘Subchronic studies (90 days) were performed in rats, mice and dogs. In the rat study (performed in 1968), no substance‐related changes were observed and a no observed adverse effect level (NOAEL) of 13.5 mg/kg body weight (bw) per day, based on the highest dose in males, has been identified. A NOAEL of 14 mg/kg bw per day in mice was proposed (study performed in 1968) based on renal changes (focal nephritis) seen at the dose of 28 mg/kg bw per day […]. A re‐evaluation of the 90‐day dog study (performed in 1968) showed that the original robenidine HCl concentrations were given in mg/kg feed. The lowest no observed effect level (NOEL) in this study was recalculated to be 19 mg/kg bw per day derived from the data on the increase in relative liver weight observed at the dose of 34 mg/kg bw per day […]. The recalculated NOAEL resulted to be about 2.5 times higher than the value the FEEDAP Panel calculated in 2004 […]. In a 90‐day Good Laboratory Practice (GLP)‐compliant rat study performed according to OECD 408, no treatment‐related changes were observed at the mid‐dose of 37 mg/kg bw per day and this dose was considered as the NOAEL of the study […].

Two chronic studies were conducted in rats and dogs. In the rat study, no tumour developments or preneoplastic lesions were observed; in the dog study, tumour developments were not recorded. The Panel noted that the studies had some limitations in their design. However, the lack of genotoxic potential [see below] considered alongside the absence of any findings of pre‐carcinogenic lesions in toxicological studies supports the conclusion that robenidine HCl is not carcinogenic […].

No negative maternal effects or effects on litters by the treatment were seen at any stage of a reproduction study in rabbits; no treatment‐related teratogenic effects or influence on reproduction were observed at any stage of a two‐generation study in rats on females, or on litters and fetuses; the maternal and fetal NOAEL in a developmental toxicity study in rabbits was 20 mg robenidine HCl/kg bw per day.

In its opinion in 2011, the FEEDAP Panel noted that the NOAELs of the available 90‐day studies are in a narrow range: 13.5 and 37 mg robenidine HCl/kg bw per day in the rat, 14 mg/kg bw per day in mice and 19 mg/kg bw per day in dogs. In the same opinion, the Panel evaluated a tolerance study in rabbits performed in line with the requirements of Regulation (EC) No 429/20087 and concluded that the study was adequate to derive a NOAEL, considering also that study duration (84 days) was comparable with the 90‐day studies in rodents and the dog. The NOAEL of 11 mg robenidine HCl/kg bw per day was taken from the study based on the absence of reproductive effects.

Considering that the literature review performed by the applicant covering the last 10 years (until 2015) did not identify any new data that would require modification of the previous assessment […], the lowest NOAEL of 11 mg robenidine HCl/kg bw per day from the tolerance study in rabbits is still considered adequate for the establishment of a health‐based guidance value for the assessment of consumer safety’.

Genotoxicity

In previous Panel evaluations (EFSA FEEDAP Panel, 2011, 2019a), it was concluded that ‘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’.

The available data set was reconsidered in light of the most recent Guidance on the assessment of the safety of feed additives for the consumer (EFSA FEEDAP Panel, 2017c) and EFSA Guidance on aneugenicity assessment (EFSA SC, 2021b). Based on the available in vitro chromosomal aberration test and the results of the in vivo micronucleus test, the potential aneugenic activity of robenidine HCl could not be excluded. No information on the potential aneugenicity of robenidine HCl was made available in the current application. ¹³ In the absence of such data, the Panel is not in the position to exclude the potential aneugenic activity of robenidine HCl.

Conclusions on toxicology

The lowest NOAEL from the toxicity data available is the one from the tolerance study in rabbits (11 mg robenidine HCl/kg bw per day). In previous FEEDAP evaluations, it was concluded that robenidine HCl is not carcinogenic based on the lack of genotoxic potential considered alongside the absence of any findings of pre‐carcinogenic lesions in toxicological studies. No information on the potential aneugenicity of robenine HCl were made available in the current application. Therefore, the Panel cannot conclude on the genotoxicity (aneugenicity) of the substance.

3.2.3. Safety for the target species

In the opinion on the safety and efficacy of Cycostat® 66G (robenidine HCl) for rabbits for breeding and fattening (EFSA FEEDAP Panel, 2011), the FEEDAP Panel concluded that the additive was safe at the proposed maximum concentration of 66 mg robenidine HCl/kg complete feed for rabbits for breeding with a margin of safety of about three based on a tolerance study conducted in breeding does following the applicable European guidelines. This conclusion was extrapolated to rabbits for fattening. Robenidine is active against Gram‐positive bacteria. In 2011, the FEEDAP Panel reiterated its former statement that feed supplementation with robenidine HCl, at concentrations relevant for the in vivo situation, did not cause in vivo resistance to robenidine HCl in enterococci, Escherichia coli, Salmonella and Campylobacter in rabbits at use level.

As a result of the literature review, the applicant identified six publications in relation to the safety for the target species. The FEEDAP Panel noted that the papers dealt with the efficacy and antimicrobial properties of robenidine and were not directly relevant to the safety of the additive in rabbits.

In addition, the applicant indicated that no serious pharmacovigilance cases have been reported for the product under assessment in the authorisation period. ¹⁷

Considering that (i) the literature review did not indicate safety concerns for the target animals, (ii) the post‐marketing report did not indicate any serious event, (iii) the composition and the production process have not been modified, and (iv) the proposed conditions of use of the additive have not been modified, the FEEDAP Panel would consider that the use up to 66 mg of robenidine HCl from Cycostat® 66G per kg complete feed remains safe for rabbits for breeding and fattening.

However, the Panel notes that since the potential aneugenic activity of robenidine HCl cannot be excluded based on the available data, the safety for the target species cannot be confirmed.

3.2.4. Safety for the consumer

Since the potential aneugenic activity of robenidine HCl cannot be excluded based on the available data, the FEEDAP Panel is not in the position to confirm the acceptable daily intake (ADI) of 0.055 mg robenidine HCl/kg bw (EFSA FEEDAP Panel, 2019a) and cannot conclude on the safety of the additive for the consumer.

3.2.5. Safety for user

No new studies were submitted and the outcome of the literature search ¹⁶ did not identify papers relevant for the assessment of user safety in the present evaluation.

Based on the information provided by the applicant and the fact that the manufacturing and composition of the additive have not been modified, the FEEDAP Panel considers that there is no evidence to reconsider the conclusions reached in the previous opinion (EFSA FEEDAP Panel, 2019a): ‘robenidine HCl is not a skin or eye irritant and not a skin sensitiser. Based on the low acute inhalation toxicity and low exposure, the risk via inhalation is considered negligible’.

Due to the lack of data, the FEEDAP Panel cannot conclude on the genotoxicity (aneugenicity) of the substance. Therefore, the exposure to the additive of the unprotected users should be minimised.

3.2.6. Safety for the environment

For the current assessment, the applicant submitted an environmental risk assessment following the requirements of the updated FEEDAP guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019b). ¹⁸ The assessment is based on (i) studies already evaluated in previous Panel evaluations, (ii) new studies on the octanol/water partition coefficient ¹⁹ and stability and solubility in aqueous media and octanol, ²⁰ and (iii) a literature search (3.2) ¹⁶ ; the outcome of the literature search did not identify papers relevant for the current evaluation.

3.2.6.1. Phase I

The physico‐chemical properties used for the environmental risk assessment of robenidine HCl are summarised in Table 1. ²¹

Table 1.

Physico‐chemical properties of robenidine HCl used for the assessment

Property	Value	Unit
Molecular weight	370.7	g/mol
Octanol/water partition coefficient (log K ow) by shake flask method (pH = 7) (OECD 107)	4.7
Solubility at 20°C (pH around 3.5)	4.9	mg/L
Dissociation constant pKa	3.4	–
Vapour pressure	Not provided	Pa

In a previous FEEDAP opinion, the octanol/water partition coefficient (log K_ow) for robenidine HCl was determined according to both OECD guideline 117 (HPLC method) and 107 (shake flask method) (EFSA FEEDAP Panel, 2019b). A new study (shake flask method) was submitted for the current evaluation.

The FEEDAP Panel reviewed the data available for the log K _ow of robenidine HCl and confirmed that the log K _ow value of 4.7, derived from the study performed according to OECD 107 (shake flask method), has to be used for the present evaluation (EFSA FEEDAP Panel, 2019b). The log K _ow of 3.3 derived from the study performed according to OECD guideline 117 (HPLC method), and already evaluated by the Panel in 2019, cannot be used for the assessment since the measurement were performed at an unknown pH. Finally, the measurement for the determination of log K _ow obtained in the newly submitted study with the shake flask method (OECD guideline 107) ²² was performed in unbuffered water and the resultant pH of the aqueous phase was not reported (although presumably acidic) and, therefore, it is considered not relevant for the environmental risk assessment.

As already noted by the Panel in 2019 (EFSA FEEDAP Panel, 2019a), robenidine HCl is protonated at acidic pH and not charged at pH 7. The physico‐chemical properties of the neutral form (robenidine) are relevant for the environmental risk assessment. The FEEDAP Panel noted that the solubility of the neutral molecule was not provided. The solubility of the neutral form of robenidine was estimated to be 4.9 mg/L using EPI Suite and the FEEDAP Panel accepted its use for further calculations. The vapour pressure was not provided but estimated to be 1.22 × 10⁻⁶ Pa. This value was used for the calculation of the predicted environmental concentrations (PECs).

Fate and behaviour

Fate in soil and fate in water

The degradation of robenidine HCl and its adsorption in soil were already assessed by the FEEDAP Panel in 2019 (EFSA FEEDAP Panel, 2019a). For the current evaluation, the same values are used. In particular, a half life (DT₅₀) of 118 days at 20°C (corresponding to 251 days when it is adjusted to an incubation temperature of 12°C using the Arrhenius equation (DT₉₀ > 1 year)) ²³ and an adsorption coefficient (K_oc) value of 426,580 L/kg.

The FEEDAP Panel reiterates its previous conclusions that robenidine HCl is hydrolytically stable at pH 7 and 9 but hydrolysis occurs at more acidic conditions (pH 4).

Predicted environmental concentrations

PECs were calculated according to the Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019b) and are given in Table 2.

Table 2.

Initial predicted environmental concentrations (PECs) of robenidine in soil and groundwater

Input	Value
Dose (mg/kg feed)	66
Molecular weight (g/mol)	370.7
Vapour Pressure (Pa)	1.22 × 10−6
Solubility (mg/L)	4.9
K oc (L/kg)	426,580
Output
PECsoil (μg/kg)	898
PECgroundwater (μg/L)	0.03

The Phase I PEC trigger value is exceeded for soil. Therefore, a Phase II assessment is necessary.

3.2.6.2. Phase II

Exposure assessment

PECs calculation refined in Phase II

PEC soil refinement based on metabolism

Based on a metabolism study in rabbit, already evaluated by the FEEDAP Panel in 2011 (EFSA, FEEDAP Panel, 2011), the excretion of unchanged robenidine in the faeces of rabbits administered 66 mg [¹⁴C]‐robenidine hydrochloride/kg feed amounted to 77% (72% in males and 82% in females), no excretion occurring in the urine. Considering that 80% of the radioactivity administered was excreted in the faeces, the robenidine whole excretion represents 62% of the total radioactivity administered. Therefore, it was considered appropriate to refine the PEC_soil calculation taking into account 62% of the initial dose administered.

PEC soil refinement based on accumulation

Since the additive is not expected to degrade within a year (DT₅₀ at 12°C of 251), the refined PEC_soil plateau has been calculated in line with Guidance on the assessment of the safety of feed additives for the environment (EFSA FEEDAP Panel, 2019b).

The re‐calculated PEC values, considering both metabolism and accumulation, are given in Table 3.

Table 3.

Predicted environmental concentrations of robenidine in soil, groundwater, surface water and sediment) refined for persistent compounds and for metabolism

Compartment	Value
PECsoil (μg/kg)	876
PECgroundwater (μg/L)	0.03
PECsurfacewater (μg/L)	0.009
PECsediment (μg/kg dry weight)	365

Conclusions on PECs used for assessment

The following values are used for the assessment in rabbits for fattening: a PEC_soil of 876 μg/kg, a PEC_{surface water} of 0.009 μg/L and a PEC_sediment of 365 μg/kg dry weight. No concern is expected for groundwater.

Ecotoxicity studies

No new studies were submitted. The applicant made reference to the ecotoxicity studies evaluated in previous FEEDAP opinions (EFSA FEEDAP Panel, 2011, 2019a).

In 2019, the FEEDAP Panel concluded the following for the ecotoxicological effect of robenidine on soil, water and sediment: ‘For the terrestrial compartment, data are available for plants, earthworms and microorganisms. Acute toxic concentrations resulted in 1,000 and 100 mg/kg of dry soil for earthworms and plants, respectively. For the aquatic compartment, data are available for algae, aquatic invertebrates and fish. The lowest toxicity value of 96‐h EC₅₀ of 36 μg/L for the aquatic compartment was found in a study on the effect on fish. Nevertheless, considering the very low solubility of the substance at neutral pH, the values obtained are uncertain. Ecotoxicological data for sediment‐dwelling invertebrate Chironomus riparius is provided for the sediment compartment resulting in NOEC of 50 mg/kg’.

For the current assessment, the available data set was reconsidered in light of the most recent FEEDAP guidance on the safety of the additive for the environment (EFSA FEEDAP Panel, 2019b) and it was noted that for some of the tests the requirements were not fulfilled. In particular, in the study on plant performed according to OECD guideline 208, three plant species (wheat (Triticum aestivum), radish (Raphanus sativus) and mung bean (Vigna radiata)) were used and this is not in compliance with the provisions set in the abovementioned guidance which requires one growth test in six different terrestrial plant species (at least two monocotyledonous and two dicotyledonous species). Also, the Nitrogen Transformation Test submitted is not in line with the current requirements of the abovementioned guidance, since the test was not performed with an exposure of 10× PEC_soil.

No new data which could address such limitations were made available, ¹³ and therefore, it is not possible to update the conclusions on the ecotoxicological effects of robenidine on soil, water and sediment and the risk ratios for the terrestrial and aquatic compartments cannot be assessed.

3.2.6.3. Bioaccumulation and risk for secondary poisoning

To assess potential for bioaccumulation and risk for secondary poisoning, the FEEDAP Panel made an assessment using the method proposed in the relevant Guidance from the European Medicines Agency (EMA, 2016) as indicated in the FEEDAP Guidance to evaluate the risk of feed additives for the environment (EFSA FEEDAP Panel, 2019b). The log K _ow of robenidine HCl is 4.7 thus it has a potential for bioaccumulation. The calculated bioconcentration factor (BCF) values for fish and earthworm are 1,970 L/kg and 602 L/kg, respectively. Since both values are above the threshold for bioaccumulation potential used as trigger value when assessing risk for secondary poisoning (≥ 100 L/kg) (ECHA, 2017), the FEEDAP Panel proceeded with the assessment.

Using the lowest NOAEL for rabbits of 11 mg/kg bw per day, which corresponds to a no observed effect concentration (NOEC) of 366 mg/kg feed, the predicted no effect concentration (PNEC_oral) resulted to be 1.22 mg/kg. This value is higher than the estimated concentration of 0.11 and 0.036 mg/kg in the worms and fish, respectively, which are based on the PECs reported in Table 3. The PEC/PNEC ratios for the assessment of secondary poisoning via the aquatic and terrestrial food chain are given in Table 4. Based on the calculated ratios, a risk for secondary poisoning for worm/fish‐eating birds and mammals is not likely to occur.

Table 4.

The assessment of secondary poisoning for robenidine via the aquatic and terrestrial food chain

	PECfish (mg/kg)	PECworm (mg/kg)	PNECoral (mg/kg)	PECfish/PNECoral	PECworm/PNECoral
Robenidine	0.036	0.11	1.22	0.03	0.09

Conclusions on the safety for the environment

In the absence of adequate information, the FEEDAP Panel cannot conclude on the safety for the environment of robenidine HCl from Cycostat® in feed for rabbits for breeding and rabbits for fattening up to 66 mg/kg complete feed. Robenidine HCl has potential for bioaccumulation; however, a risk for secondary poisoning for worm/fish‐eating birds and mammals is not likely to occur. No concern for groundwater is expected.

3.3. Efficacy

3.3.1. Efficacy for rabbits for fattening and breeding

Two anticoccidial sensitivity tests (AST) in rabbits for fattening (AST‐1 and AST‐2) and one in rabbits for breeding (AST‐3) were submitted using robenidine HCl from Cycostat®. The ASTs followed a similar design (Table 5). ²⁴ In each AST, three treatment groups were included: an uninfected untreated control (UUC), an infected untreated control (IUC) and an infected treated (IT) group. The treatments were replicated 15 times with three rabbits in each cage. The experimental diets were offered to the rabbits from the day of allocation to treatment groups, corresponding to day 1 (study start), until study completion on day 31. Rabbits in IUC and IT were orally challenged on day 3 with a recently collected Eimeria field isolate. ²⁵ Body weight gain and feed intake were monitored on weekly basis, feed to gain ratios were calculated. Faecal samples were collected on days 10, 14, 17, 24 and 31 for counting of oocysts. Clinical observations were performed at the cage and animal level, and a score was recorded for each day for faecal consistency ²⁶ and depression/apathy. ²⁷ Mortality was monitored; the body weight and cause of death (intestinal and/or hepatic coccidiosis related or not) was determined. In AST‐3, livers were assessed for the presence of lesions during necropsy at the end of the study on all animals.

Table 5.

Experimental design of ASTs with rabbits fed robenidine HCl from Cycostat®

Trial No (Date of conduct)	Inoculum characteristics			Age and sex of animals at study start	Robenidine HCl analysed in feed of IT (mg/kg)
	Country, date of isolation	Dose (number of oocysts) and strain per animal
1 (9/2019)	The Netherlands 2/2019	E. magna/irresidua	1,200	4 to 8 weeks old ♂	43.7
		E. vejdovskyi/coecicola/media/stedai	50,700
		E. piriformis/intestinalis/flavescens	30
		E. perforans	900
2 (12/2019)	Belgium 11/2019	E. magna/irresidua	50,000	4 to 8 weeks old ♂	41.1
		E. vejdovskyi/coecicola/media/stedai	75,000
		E. perforans	6,000
3 (1/2020)	France 2/2019	E. magna/irresidua	59,000	16 weeks old ♀ (not pregnant)	50.1
		E. vejdovskyi/coecicola/media/stedai	72,000
		E. perforans	27,000

The data were analysed using a general linear mixed model considering the treatment as the fixed effect and the cage was the statistical unit. All hypothesis tests were conducted at the 0.10 level of significance using two‐sided tests.

In AST‐1 and AST‐3, only one coccidiosis‐related mortality was recorded in the UUC and in the IUC group. In AST‐2, a high mortality rate was observed in all treatment groups reaching almost 50% in both IUC and IT groups (26% in UUC, 46% in IUC and 47% in IT). This was potentially attributed to a combination of intestinal coccidiosis and a concurrent Clostridium infection. The Panel agreed with the observation of the pathologist report that a possible Clostridium infection in the rabbits could have made the rabbits more susceptible to coccidiosis, and concluded that this test cannot be further considered to assess efficacy of robenidine HCl in the prevention of coccidiosis.

There were no significant differences between oocyst counts per gram of excreta (OPG) in AST‐1 and AST‐3 of the IUC and IT groups at any of the timepoints.

In AST‐1, no alteration was observed in faecal consistency in any of the experimental groups. In AST‐3, the challenge applied induced clinical coccidiosis characterised by diarrhoea (scored at 1 or 2) in all the experimental groups. In the overall period (days 3–31), the challenge increased significantly (P < 0.10) the days of diarrhoea (expressed as percentage of days with diarrhoea over the total days of the observation period) from 6.4% of the UUC to 12.3% in the IUC. Treatment with robenidine HCl reduced significantly (P < 0.10) this percentage to 3.2%.

The number of other clinical observations (due to depression/apathy) attributed to coccidiosis was very low both in AST‐1 and AST‐3. Livers were assessed for lesions at study end in AST‐3; no lesions were detected. No significant difference in the performance parameters (weight gain, feed intake and feed to gain ratio) was observed between the IUC and IT groups in any of the time intervals in AST‐1 and AST‐3.

Conclusions on the efficacy for the target species

Only one anticoccidial sensitivity test indicated the potential of the additive to be efficacious in rabbits for breeding based on reduced diarrhoea incidence. Owing to the lack of sufficient data, the FEEDAP Panel cannot conclude that robenidine HCl from Cycostat® 66G is still efficacious against recent Eimeria spp. strains in rabbits.

3.4. Post‐market monitoring

The FEEDAP Panel considers that there is no need for specific requirements for a post‐market monitoring plan other than those established in the Feed Hygiene Regulation ²⁸ and Good Manufacturing Practice.

4. Conclusions

Since the potential aneugenic activity of robenidine hydrochloride cannot be excluded due to lack of data, the FEEDAP Panel is not in the position to conclude on the safety of the additive for the target species and the consumer.

In the absence of data, the FEEDAP Panel cannot conclude on the safety of robenidine hydrochloride for the environment when used in feed for rabbits for breeding and rabbits for fattening up to 66 mg/kg complete feed. Robenidine hydrochloride has a potential for bioaccumulation, however a risk for secondary poisoning for worm/fish‐eating birds and mammals is not likely to occur. No concern for groundwater is expected.

Robenidine hydrochloride is not a skin or eye irritant and not a skin sensitiser. Due to the lack of data on the genotoxicity (aneugenicity) of the substance, the exposure to the additive of the unprotected users should be minimised.

Owing to the lack of sufficient data, the FEEDAP Panel cannot conclude that robenidine hydrochloride from Cycostat® 66G is still efficacious against recent Eimeria spp. strains in rabbits.

5. Documentation as provided to EFSA/Chronology

Date	Event
22/06/2020	Dossier received by EFSA. Assessment of the feed additive consisting of robenidine hydrochloride (Cycostat® 66G) for rabbits for breeding and rabbits for fattening for the renewal of its authorisation. Submitted by Zoetis Belgium SA.
31/07/2020	Reception mandate from the European Commission
09/10/2020	Application validated by EFSA – Start of the scientific assessment
04/02/2022	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: characterization, safety for the consumer, safety for the environment.
15/07/2022	Request of supplementary information to the applicant in line with Article 8(1)(2) of Regulation (EC) No 1831/2003 – Scientific assessment suspended. Issues: Safety evaluation strategy and corresponding testing strategy.
18/07/2022	Reception of supplementary information from the applicant (reply to questions sent on 04/02/2022)
03/08/2022	Reception of supplementary information from the applicant (reply to questions sent on 15/07/2022) ‐ Scientific assessment re‐started
31/01/2023	Opinion adopted by the FEEDAP Panel. End of the Scientific assessment

Abbreviations

ADI

acceptable daily intake

ADME

absorption, distribution, metabolism, excretion

AST

anticoccidial sensitivity test

AZIN

bis‐4[4‐Cl‐benzylidene]hydrazine

BCF

bioconcentration factor

bw

body weight

CAS

Chemical Abstracts Service

DT₅₀

time to degradation of 50% of original concentration of the compound in the testedsoils

DT₉₀

time to degradation of 90% of original concentration of the compound in the testedsoils

EC₅₀

the concentration of a test substance which results in 50% of the test organismsbeing adversely affected, i.e. both mortality and sublethal effects

EMA

European Medicines Agency

IT

infected treated group

IUC

infected untreated group

KOC

adsorption or desorption coefficient corrected for soil organic carbon content

LOQ

limit of quantification

Log Kow

logarithm of octanol–water partition coefficient

MRL

maximum residue limit

NOAEL

no observed adverse effect level

NOEC

no observed effect concentration

OPG

oocyst counts per gram of excreta

PEC

predicted environmental concentration

PNEC

predicted no effect concentration

TRIS

N,N′,N″‐tris[(p‐Cl‐benzylidene)amino]guanidine

UUC

uninfected untreated group

Suggested citation: EFSA FEEDAP Panel (EFSA Panel on Additives and Products or Substances used in Animal Feed) , Bampidis V, Azimonti G, Bastos ML, Christensen H, Dusemund B, Durjava M, Kouba M, López‐Alonso M, López Puente S, Marcon F, Mayo B, Pechová A, Petkova M, Ramos F, Sanz Y, Villa RE, Woutersen R, Gropp J, Rychen G, Holczknecht O, Rossi B and Vettori MV. Scientific Opinion on the assessment of the feed additive consisting of robenidine hydrochloride (Cycostat® 66G) for rabbits for breeding and rabbits for fattening for the renewal of its authorisation (Zoetis). EFSA Journal 2023;21(3):7863, 16 pp. 10.2903/j.efsa.2023.7863