Investigation of safety and efficacy of febantel and fenbendazole in fish and exposure assessment

Fish are susceptible to blood‑sucking parasite infections, which cause severe anemia, dyspnea, and ultimately death. However, veterinary drugs available for fish to treat such infectious diseases are lacking; thus, livestock drugs have been repurposed as aquatic animal drugs. Febantel (FBT) and fenbendazole (FBZ) are representative antiparasitic agents for livestock such as cattle, swine, and poultry, and are considered suitable as aquatic animal drugs. Therefore, we investigated the safety and efficacy of FBT and FBZ in fish and performed a risk assessment to determine the maxi‑ mum residue limit in fish. Most studies indicate that FBT is rapidly converted to FBZ, which is metabolized to oxfenda‑ zole and oxfendazole sulfone. FBZ was frequently detectable in the plasma and tissues (e.g., muscle, skin, and the liver) in significant quantities than other metabolites. We regarded the liver as the target organ because reversible hepatocytic changes were observed in fish after administration of 100 mg/kg FBT for 9 days. No toxicological effects, such as increased mortality or decreased appetite, were observed when the fish were administered 50 mg/kg FBT for 3 days. The efficacy of the drugs was verified in various parasites, including H. heterocerca, H. okamotoi or Z. japonica , and M. seriolae , as causative agents of beko disease through laboratory and field trials. Although toxicity studies on FBZ in fish are limited, its safety has been demonstrated from toxicity studies in a wide range of animal models. The risk from using FBT and FBZ was negligible for human health because the ratio of the estimates of dietary exposure and acceptable daily intake was 78.4%.


Introduction
Aquaculture has increasingly become the most important food-producing sector in terms of global food security and nutrients [1].Korean annual seafood consumption reached an average of 58.4 kg per capita during 2013-2015, indicating that Koreans were the top consumers of seafood among the countries investigated by the Food and Agriculture Organization [2].
To meet the growing consumption of aquatic foods, aquaculture is growing at high densities; however, intensive circumstances represent a risk of disease outbreaks [3].Hence, the use of effective aquatic animal drugs is inevitable in aquafarms to prevent and treat infectious diseases (i.e., parasitic diseases with high prevalence and intensity) [4].
Some studies indicate that fish farm workers use prohibited chemicals (e.g., formalin and malachite green) to control the prevalence of infectious diseases in fish [5,6].In line with American veterinary medical association, there are only a limited number of approved drugs for aquatic animals, so the demand for the development of new effective drugs against various parasites is high.To fulfill these demands, trials for repurposing livestock drugs as aquatic animal drugs have been reported.Oxytetracycline (OTC) is a useful example.OTC is a widely used antibacterial agent in various food-producing animals (e.g., mammals, birds, and fish) because of its pharmacological properties [7][8][9].It is actively used for farmed fish (e.g., salmonids, olive founders, and sea breams) worldwide [9].
Febantel (FBT) and fenbendazole (FBZ) are approved for use in multispecies animals, such as ruminants (cattle, sheep, and goats), nonruminants (swine and equine), and birds (growing turkeys), for controlling internal parasites, such as gastrointestinal roundworms or lungworms [10][11][12].FBT is a pro-benzimidazole anthelmintic with broad-spectrum activity against various nematode parasites in canine and feline species [13].FBT is metabolized to FBZ in vertebrates [14].FBZ is metabolized to oxfendazole (OXF) and further sulfoxidized to oxfendazole sulfone (OXFSO 2 ) [15].FBZ is interconvertible to OXF, which is considered the most toxic compound compared with FBT and FBZ [14].FBZ, the active metabolite of FBT, binds to microtubules in parasites and irreversibly blocks glucose uptake, leading to the depletion of energy stores and, ultimately, death [10].
Although the safety and efficacy of FBT and FBZ have been established in laboratory animals and livestock [11,12], research on FBT and FBZ in aquatic animals is lacking.Therefore, we investigated the efficacy and safety of FBT and FBZ in fish and performed exposure assessment to establish their maximum residue limits in fish.
The remainder of the study has been organized as follows.The materials and methods section provide details regarding searching materials and exposure assessment.The results section presents the analysis of toxicity studies, metabolism studies, residue depletion studies, pharmacological studies, and exposure assessment.Finally, the conclusion section summarizes the study and discusses future research directions.

Searching materials
Metabolism, safety pharmacology, and other related data of FBT and FBZ for fish were gathered from publications, such as the evaluation reports released by the Food and Drug Administration (FDA), Food Safety Commission of Japan (FSCJ), and European Medicinal Agency (EMA).
In addition, we investigated relevant studies by searching PubMed and Google databases.The searched keywords were the following: "febantel and fish, " "fenbendazole and fish(es), " "fenbendazole and aquatic animals, " "benzimidazoles and fish(es), " "febantel and efficacy" or "fenbendazole and efficacy."

Exposure assessment
To estimate the amount of FBT and FBZ exposure, the tentative maximum residue level was used, which is proposed by Ministry of Food and Drug Safety and Ministry of oceans and fisheries.An exposure model was applied to the global estimate of chronic dietary exposure, with a slight modification [16].This model was recommended by the Joint WHO/FAO Expert Committee on Food Additives (JECFA).The final calculation method is as follows, The minimum number of subjects for which percentiles could be used in dietary exposure assessment was obtained from the European Food Safety Agency guidance document [17].The acceptable daily intake (ADI) values by international bodies were investigated (see Additional file 1: Tables S1 and S2 for summarized version).In this study, the ADI was 0.007 mg/kg bw/day, as recommended by the JECFA and FSCJ (Additional file 1: Table S3).

Toxicity studies
Toxicity studies on FBT and FBZ were conducted in early life or at specific developmental stages in several species, including Japanese amberjack (Seriola quinqueradiata), great amberjack (Seriola dumerili), Japanese pufferfish (Takihugu rubripes), rainbow trout (Oncorhynchus mykiss), zebrafish (Danio rereio), silver perch (Bidyanus high dietary exposure for one food 90, 95 or 97.5 percentile consumption by consumers × tMRL + mean dietary exposure for all other foods(average consumption by generalpopulation × tMRL) body weight(kg) bidyanus), and sticklebacks (G.aculeatus).Table 1 presents additional toxicity data in detail.Mortality rates in amberjacks (S. quinqueradiata and S. dumerili) were dependent on the exposure concentration and duration.For example, the mortality rate reached above 90% when fish were exposed to more than 50 mg/ kg bw/day for 28 consecutive days, and the growth rate decreased by approximately 6.8% compared to that of the control.The mortality rate among fish was zero in exposure group with same dose for 14 consecutive days [18][19][20].According to Kimura et al. [35], no detrimental effects are observed when tiger puffers are administered up to 25 mg/kg bw/day FBT for five consecutive days.Fish consumed all feed within 5-10 min, and low daily mortality rates are observed at all concentrations.
An analysis using zebrafish embryos demonstrated the metabolism by embryos of FBT into FBZ and FBZ into OXF.Various malformations, such as underdeveloped eyes, head, and curved tail, were reported in embryos exposed to FBT at 0.043 or 2 mg/L levels.No observed effect concentration of FBZ and FBT were 0.02 and 0.2 mg/L at 144 h-post-fertilization, and 50 percent of effect concentration of FBZ and FBT corresponded to 0.024 and 0.34 mg/L, respectively.These findings suggest that FBZ may have harmful effects on the early life stages of fish exposed to FBT [21].

Metabolism studies
Metabolism studies of FBT and FBZ intended for various fish species, including Japanese amberjack (S. quinqueradiata), Japanese pufferfish (T.rubripes), channel catfish (Ictalurus punctatus), and rainbow trout (O.mykiss), were performed (see Table 1 for the detailed information).
In a persistence assessment using Japanese amberjack (S. quinqueradiata), FBT was not detected in any edible tissue (e.g., the liver, muscle, or kidney) at 1 day-postadministration (dpa); in contrast, FBZ was observed in the liver and kidney at 7 dpa.No compounds, including FBT, FBZ, OXF and OXFSO 2 were detected in any of the organs at 14 dpa.This study highlighted that the total degradation of FBT in S. quinqueradiata took more than a week; therefore, the appropriate withdrawal period from the last treatment was 21 dpa, which was comparable to that of T. rubripes [5,22].
In O. mykiss, depletion time differed between the two species.In a previous study, FBZ was detected up to 4 dpa in muscle exposed to FBZ after oral treatment, whereas the compound was detected in muscle until 1 dpa after bath treatment [23].Furthermore, another study revealed that FBZ remained until 20 dpa at a mean level of 0.13 mg/kg [24].
Research conducted on channel catfish (I.punctatus) showed that FBZ was detected in muscle at all time points when the fish were exposed to FBZ via intravenous injection at a concentration of 1 mg/kg bw or orally at 5 mg/kg bw.However, FBZ was observed in urine at insignificant levels, and OXFSO 2 was mainly detected in urine and bowel [25].
In an in vitro test, hepatocytes obtained from channel catfish were treated with FBZ, revealing that FBZ was primarily transformed into OXF, followed by p-hydroxyfenbendazole.Only small quantities of OXFSO 2 were detected, and fenbendazole amine was not present [26].

Residue depletion studies
Tissue depletion of FBZ was studied in a generally used laboratory model (rainbow trout) and in important fish species consumed in Japan (Japanese amberjacks and tiger puffers).Table 1 presents the study design and additional information.
A study conducted on rainbow trout via the gastrointestinal tract by a stomach tube at a single dose of 50 mg/ kg bw/day indicated that FBZ was observed in the liver and muscle 24 h after administration, and the levels in both tissues sharply decreased thereafter [24].Another study of rainbow trout by intubation or bathing revealed that the peak concentrations of both routes reached 24 h and then rapidly decreased.FBZ was detected in muscle at 4 days with a maximum concentration, whereas OXF was present in the skin until 4 days after administration [23] In a residue depletion trial using Japanese amberjack (S. quinqueradiata) administered via feed to a final dose of 10 mg/kg bw/day FBT for 5 consecutive days, compounds that could be transformed to OXFSO 2 were below the limit of quantification (LOQ) at all time points over 3 days after the last administration [27].However, the FBT, FBZ, and OXF levels were not reported in this study.The highest concentration of residues that could be metabolized into OXFSO 2 was found in the liver, followed by the kidneys and muscle.In particular, muscle was below the LOQ at all time points 3 days after the last treatment.After 14 days post the last treatment, levels above the LOQ were observed in both the liver and kidneys.The residues ranged from 0.0730 to 0.0842 μg/g in the liver and from 0.0578 to 0.0837 μg/g in the kidneys [27].
In a residual study in which greater amberjack (S. dumerili) was exposed to a dose of 10 mg/kg bw FBT via feed for 5 days, the levels of OXFSO 2 in muscle ranged from 0.0192 to 0.0582 μg/g 3 days after the last treatment.However, OXFSO 2 levels in muscle samples were below the LOQ at all time points after 5 days.Similar to the results for greater amberjack (Kanpachi), the highest concentration of OXFSO 2 was observed in the liver, followed by the kidneys.At day 3 from cessation, levels of OXFSO 2 ranged from 0.0192 to 0.0582, 2.14 to 3.42, and 0.775 to 1.50 μg/g in muscle, the liver, and kidneys, respectively.After 5 days from the last treatment, residues were not observed in muscle at all sampling points.On day 14 after suspension, the levels of OXFSO 2 in the liver and kidneys were detected at a range of 0.0924-0.135and 0.134-0.142μg/g, respectively [27].
Another residue depletion trial using Japanese pufferfish (T.rubripes) administered 25% formulation of FBT via feed at a dose of 50 mg/kg bw for 5 consecutive days showed that no compounds were detected in muscle without skin 7 days after the last feeding.In muscle without skin, FBT was absent in all fish 24 h after cessation, whereas OXFSO 2 was detectable at 0.51-1.39μg/g.At 24 h after cessation of treatment, the concentrations of OXFSO 2 in skin tissues of fish ranged from 1.00 to 2.64 μg/g [27].
In addition to the aforementioned residual trials, a similar study was conducted using pufferfish.The study indicated that at 1 day post-termination, residue concentration of FBT 0.27-0.41μg/g, FBZ, OXF and, OXFSO 2 as an equivalent to OXFSO 2 of 1.40-5.00μg/g in skin sample; however, in muscle sample, residue concentration of FBT 0.11-0.15μg/g, FBZ and its metabolites as an equivalent to OXFSO 2 of 0.80-2.80μg/g.None of the compounds were observed in both tissue samples (i.e., muscle and skin) after 14 days from the last dietary administration.
In the two residue trials conducted with pufferfish, the elimination of FBT and its metabolites in muscle tissue appeared to be faster than in the skin.This was observed because the concentration of FBT and its degradation products in muscle samples without skin was approximately twofold lower than in the skin samples.Additionally, OXFSO2 persisted longer than the parent compound in both tissues.

Pharmacological studies
Detailed pharmacological studies of FBT and FBZ in different fish species, including rainbow trout, pufferfish, and amberjacks, are outlined in Table 2.
In vivo and in vitro efficacy trials of FBT performed using rainbow trout infected with Gyrodactylus species showed that anthelmintic activity was not observed in vivo test and signs of toxicity were reported at a high concentration of 10 mg/L for 3 h.However, in vitro test showed that all parasites of Gyrodactylus species could be killed by treatment with FBT for a prolonged exposure time (i.e., 1 h) [28].
In vivo test on the efficacy of FBZ and OXF in rainbow trout demonstrated that FBZ treatment was uniquely effective to eliminate Gyrodactylus species at a dose ranging from 1.5 to 25 mg/L for 3 h.However, in vitro test of both compounds confirmed that they had no anthelmintic activity against Gyrodactylus species [29].
In a pharmacodynamic study in which Japanese pufferfish (T.rubripes) were administered 25% formulation of FBT via feed at a dose of 25 mg/kg bw consecutively for 5 days, the T max of FBT was 4 h with a C max of 0.58 μg/g.Plasma levels of FBZ were constant (approximately 4 or 5 μg/g over 24 h before cessation), although those of FBT, OXF, or OXFSO 2 were maintained at below 1, 0.5, or 0.5 μg/g, respectively [27].
When Japanese pufferfish (T.rubripes) were administered a single dose of 25 mg/kg bw/day FBT, FBT was only detectable at below 1.8 μg/g at 4 h after administration and was absent thereafter.The T max of the total residues (FBT, FBZ, OXF, and OXFSO 2 ) in the tissues (muscle, skin, and the kidneys) was 12 h, and C max ranged from 1.8 to 5.0 μg/g.The T max of the total residue in the liver was 4 h with a C max of 29.0 μg/g.The total residues, excluding FBT, were still detectable up to 24 h postadministration in various tissues and 72 h post-administration in the plasma [27].
An efficacy test using two amberjacks species, S. quinqueradiata and S. dumerili [30], demonstrated that FBT treatment was effective against infection by the gill flukes H. heterocerca and Z. japonica in two fish species, and the effective regimen was confirmed to be 10 mg/kg bw/ day for 5 days.Moreover, there were no obvious signs of toxicity, and all treated animals showed good feeding activity [20].Another study on the efficacy of FBT using the S. quinqueradiata and S. dumerili showed that infection with M. seriolae, a causative agent of beko disease, was prevented appropriately by oral FBT treatment at a dose of more than 3 mg/kg bw/day for 3-10 days [31].In field efficacy trials of FBT, amberjack-susceptible parasites such as H. heterocerca, Z. japonica, and M. seriolae were successfully removed through oral FBT administration.The frequency of infected fish decreased, and the progression of cyst formation was delayed [18,22].
An analysis regarding the antiparasitic effects of FBZ in stickleback fish that was naturally infected G. anomala highlighted that the number of infected fish was markedly reduced and mature parasites' polaroplast was completely damaged by multi or single applications through bath at a concentration up to 50 μg/mL for 6 h [32].
Research on the efficacy of FBZ in juvenile rohu fish infected with Dactylogyrus species, which are considered critical gill parasites causing severe economic damage to the aquaculture industry, showed that multiple applications of FBZ at a dose of 20 mg/kg bw/day for 7 days FBT Japanese Amberjack (S. quinqueradiata, S. dumerili); 0, 10 mg/kg bw/da for 5 day; feed (different four locations, total four trials) H. heterocerca and Z. japonica Decreased infection prevalence of both fish species against gill flukes Shirakashi et al. [30] FBT Tiger puffer (T.rubripes); 0, 12.5, or 25 mg/kg bw/day for 5 day; feed (total six trials)

M. seriolae
Concentration of FBT depends on cumulative mortality at the end of experiment; Significant decrease in cyst detection rate and progression of cyst formation after treatment; no adverse effects observed in both tests Kawakami et al. [18] could reduce the number of young fish infected with Dactylogyrus species [33].
Several studies aimed at evaluating the efficacy of FBT against H. okamotoi and the pharmacokinetics of FBT in tiger puffer fish demonstrated that FBZ was accepted as the main metabolite [34].The T max of FBT was approximately 4 h and gradually decreased after 8 h.In contrast, the T max of OXFSO 2 was three times higher than that of FBT, and OXFSO 2 was detectable until 3 days after suspension [5].Following FBT treatment at a dose of 25 mg/ kg bw/day for 5 days via feed, the number of H. okamotoi substantially reduced after 4-5 days [34].In addition, the efficacy did not differ with exposure concentration [35].
In an investigation of the pharmacological activities of baths and orally administered FBZ against the monogenean gill parasite L. bidyana of silver perch (Mitchell), the efficacies of bath and oral administration against mature L. bidyana were 91% and 95%, respectively, without any toxicological concerns, such as behavioral abnormalities or adverse physical signs and mortalities.The efficacies of both treatments against immature L. bidyana were substantially lower than those against mature ones (i.e., efficacy of 28%-76%) [36].In summary, previous pharmacological studies have shown that FBT treatment is remarkably effective against blood-sucking parasites responsible for gill fluke disease, such as H. okamotoi, M. sebastis, and Dactylogyrus sp.across various fish species.Specifically, M. sebastis is recognized as a prominent pathogen isolated from Korean farmed fishes like rockfish Sebastes schlegelil, blackhead seabream Acanthoparus schlegelii, and Red seabream Pagrus major [37].
Praziquantel is the only approved anthelmintic drug for aquatic animals in Korea, widely used in human medicine, veterinary practice, and aquaria.However, the development of praziquantel resistance has been documented in numerous studies [38].Significantly, a reduction in efficacy was noted when treating praziquantel to salmon infected with Eubothrium in Norway [39,40].
Considering the emergence of praziquantel resistance and the outstanding efficacy of FBT and its active metabolite FBZ, we anticipate that FBT and FBZ could serve as excellent alternatives to praziquantel in the Korean aquaculture industry.

Exposure assessment
Tables 3 and 4 show that the estimates of chronic dietary exposure are calculated as 0.3300 mg/person/day and the values are divided by 0.42 mg/person/day (60 kg of body weight).Hazard indices are shown up to 78.6%, suggesting that the residues from using FBT and FBZ will pose negligible consumer health risks.This study confirms that FBZ and its metabolites are more frequently detected in the skin than in muscle after FBT and FBZ treatment in fish.Furthermore, residues that can be metabolized into OXFSO 2 have been observed in the liver and kidneys in large quantities for a prolonged period compared with those in muscle.The beneficial effects of treating gill and skin diseases were demonstrated without any significant signs of toxicity in various fish species, including Japanese amberjacks, Indian carp (i.e., rohu), and pufferfish, through the application of FBT and FBZ.The effective dose and duration of FBT ranged from 10 to 50 mg/kg bw/day consecutively for 5 days without any signs of toxicity.Feeding or bath treatment was more effective than gavage or injection.
As a result of the exposure assessment, the primary source of FBT and FBZ exposure was milk, with 0.2766 mg capita per day, corresponding to 83.8 percent of the total exposure.The estimated exposure from fish was 0.0015 mg capita per day, accounting for 0.45 percent.The Hazard Index (HI) is calculated by dividing the estimated total exposure by the acceptable daily intake.Therefore, when the Hazard Index, as in the case of FBT and FBZ, is below 100 percent, it is considered that the human health risk from the consumption of food items, including veterinary drug residues, would be negligible.Consequently, it is concluded that the tentative Maximum Residue Limit (MRL) of FBT and FBZ for fish was acceptable.
Most research identifying residue levels in fish has focused on muscle.In contrast, residue investigations of potential edible tissues in fish, such as other visceral organs (i.e., the intestine, ovaries, or eggs), are limited.However, in several countries, these organs are generally consumed in salted form [41,42].
Additionally, an investigation in which aquatic animals were fed antimicrobials over a long period demonstrated that antimicrobial residues could negatively affect the intestine (i.e., altered intestinal enzyme activities and imbalanced relative abundance of gut intestinal microflora) [43][44][45].
Considering the great diversity of fish consumption patterns and the effects of veterinary drugs on the intestine, further depletion or metabolism studies on potential edible tissues in fish are warranted.

Table 1
Metabolism, toxicology, and residue depletion studies of febantel and fenbendazole

Table 1 (continued) Compound Study design (species, dosage, and admin. regime) Results References
[27]peaked at 4 h after admin.anddecreased to 30%of the peak at 8 h and gradually reduced thereafter in plasma; OXFSO 2 peaked at 12 h in plasma and was detected until 72 h after admin Kimura et al.[5]Metabolism FBZ Channel catfish (Ictalurus punctatus); intravenously 1 mg/kg bw or orally 5 mg/kg bw FBZ was detected in muscle at all sampling time; but it appeared in urine at a low concentration; OXFSO 2 was main metabolite in urine and intestinal contents Kiztman et al. [25] Comparative Metabolism FBZ Liver tissue from catfish (young nonbreeding) at 25 or 37 OXF was the majority; OXFSO 2 with small quantities; absence of FBZNH 2 ; no metabolic difference of temperature Short et al. [26] Residue depletion FBZ Rainbow trout (O.mykiss); 6 mg/kg bw/day (single) or 1.5 mg/L (12 h); intubation or bath FBZ was detected in muscle until 4d after last treatment per oral; Tmax (24 h) and sharply reduced thereafter in both routes; the concentration of skin was more than that of muscle; FBZ was detected until 1d after last treatment via bath exposure; OXF was observed in muscle up to 1d after cessation; OXF was detected in skin until 4d after cessation Iosifidou et al. [23] Residue depletion FBZ Rainbow trout (O.mykiss); 50 mg/kg bw/day (single); intubation FBZ was detected in liver and muscle at 24 h after treatment, and concentration of both organs sharply declined thereafter; appropriate withdrawal period as 497.6-degree days was confirmed Soukupova-markova et al. [24] Residue depletion FBT Japanese amberjack (S. quinqueradiata); 10 mg/kg bw/ day for 5 day; feed Residues were detectable until 14 days in liver and kidney, but not in muscle; highest concentration of residues were reported in liver followed by kidney and muscle FSCJ [27] Residue depletion FBT Greater amberjack (S. dumerili); 10 mg/kg bw/day for 5 day; feed In muscle, OXFSO 2 was detectable no longer than 5 day after cessation FSCJ [27] Residue depletion FBT Greater amberjack (S. dumerili); 10 mg/kg bw/day for 5 day; feed Residues were not detectable in muscle over 5d after last treatment; highest concentration: liver > kidney > muscle at 3 day after cessation; residues were detected until 14 day in liver and kidney FSCJ [27] Residue depletion FBT Japanese pufferfish (T.rubripes); 50 mg/kg bw/day for 5 day; feed Absence of FBT in muscle and skin after 24 h after cessation, and OXFSO 2 was detectable in both tissues ranging from 0.51 to 2.64 μg/g (skin > muscle) FSCJ [27] Residue depletion FBT Japanese pufferfish (T.rubripes); 50 mg/kg bw/day for 5 day; feed Absence of FBT in muscle and skin; OXFSO 2was detected in one muscle sample among five samples at a concentration of 0.08 μg/g at 7 day after cessation; none of residues were observed at 14 day after cessation FSCJ[27]

Table 2
Pharmacological studies of febantel and fenbendazole

Table 3
Results of febantel exposure assessment Ratios of marker to total radioactive residue (MR:TRR) were obtained from EMA (2013) *

Table 4
Results of fenbendazole exposure assessment Ratios of marker to total radioactive residue (MR:TRR) were obtained from EMA (2013) *