Concurrent use of two dual-combination drenches containing monepantel/abamectin and oxfendazole/levamisole in sheep: effect on marker residues 21 and 28 days after administration

ABSTRACT Aims: To determine the concentration, in comparison with the maximum residue limit (MRL), of anthelmintic marker residues in the target tissues (liver and fat) of sheep treated concurrently with two oral drenches, one containing monepantel and abamectin and the other oxfendazole and levamisole. Methods: On day 0 of the study, 12 sheep (six male and six female; 8–9-months old) were dosed according to individual body weight determined the day prior. Zolvix Plus (dual-active oral drench containing 25 g/L monepantel and 2 g/L abamectin) was administered to all animals prior to administration of Scanda (dual-active oral drench containing 80 g/L levamisole hydrochloride and 45.3 g/L oxfendazole). Six sheep (three male and three female) were slaughtered 21 and 28 days after treatment and renal fat and liver samples were collected. Using validated methods, analyses for monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone) were performed on liver samples while renal fat specimens were analysed for monepantel sulfone and abamectin residues only. Detected concentrations were compared to the established MRL in sheep for each analyte determined by the Ministry for Primary Industries. Results: All residues detected in samples of liver and fat collected 21 and 28 days after treatment were below the MRL for each analyte. All liver samples collected on day 21 had detectable monepantel sulfone (mean 232 (min 110, max 388) μg/kg) and oxfendazole (mean 98.7 (min 51.3, max 165) μg/kg) residues below the MRL (5,000 and 500 μg/kg, respectively). Monepantel sulfone (mean 644 (min 242, max 1,119) μg/kg; MRL 7,000 μg/kg) residues were detected in 6/6 renal fat samples. Levamisole residues were detected in 3/6 livers (mean 40.0 (min 14.3, max 78.3) μg/kg; MRL 100 μg/kg), and abamectin residues in 1/6 livers (0.795 μg/kg; MRL 25 μg/kg) and 2/6 fat samples, (mean 0.987 (min 0.514, max 1.46) μg/kg; MRL 50 μg/kg) 21 days after treatment. Conclusion and clinical relevance: These results suggest that concurrent administration of Zolvix Plus and Scanda to sheep is unlikely to result in an extended residue profile for any of the active ingredients, with all analytes measured being under the approved New Zealand MRL 21 days after treatment. This work was not completed in line with guidance for establishing official residue profiles, nor is it sufficient to propose a new withholding period.


Introduction
Anthelmintic resistance (AR) of gastrointestinal nematode parasites of ruminants is recognised as a significant challenge to the ongoing viability of pastoral farming in New Zealand.Following initial reports on the prevalence of AR on New Zealand sheep farms (Waghorn et al. 2006;McKenna 2010), subsequent non-peer-reviewed reports suggest that the prevalence of resistance in sheep roundworm parasites to the older benzimidazole, levamisole and macrocyclic lactone active ingredients in anthelmintic drenches is high and increasing across New Zealand sheep farms (McKenna 2016(McKenna , 2018;;Riddy 2022).More concerning are reports of resistance in sheep roundworms to combinations of the older anthelmintics, including the widely used triple combination drenches containing benzimidazole, levamisole and a macrocyclic lactone (Wrigley et al. 2006;Hodgson and Mulvaney 2017).In line with observations in the Americas (Torres-Acosta et al. 2012;Chaparro et al. 2017) and Europe (Rose et al. 2015;Ploeger and Everts 2018), the number of farms in New Zealand diagnosed with triple-resistant parasites is expected to increase.
Data collated by a veterinary pathology laboratory (Gribbles Veterinary, Auckland, NZ) indicates that the prevalence of triple-drench-resistant Trichostrongylus spp.diagnosed in faecal egg count reduction tests conducted on New Zealand sheep farms has increased from 3% of 93 tests in 2016-2017(McKenna 2018) ) to 33% of 165 tests in 2021 (Riddy 2022).Furthermore, resistance (defined as < 95% efficacy in faecal egg count reduction tests) to the newer novel active ingredients monepantel and derquantel has been reported in New Zealand (Scott et al. 2013;McKenna 2018) and Australia (Kaminsky et al. 2011;Sales and Love 2016).
To help manage the issue of anthelmintic resistance, a pan-industry New Zealand worm management strategy group, Wormwise, was established in 2007 (Wilson et al. 2015).Wormwise has developed a riskmanagement based approach to roundworm control (McAnulty and Cook 2019).This involves identifying activities on livestock farms that are considered a risk for developing resistance in parasites to anthelmintics, and devising strategies to reduce the risk of these activities.
A significant risk identified for all livestock farmers is the potential to acquire drench-resistant parasites from another property through introducing animals infected with these parasites (Leathwick and Besier 2014).This is of particular concern to lamb fattening and trading operations, but is relevant to all farming systems where animals are bought in.
Following the findings and recommendations published by Lawrence et al. (2006) and Leathwick et al. (2009), Wormwise encourages implementation of a comprehensive farm-level quarantine procedure to reduce the likelihood of introducing resistant parasites (McAnulty and Cook 2019).Optimally, treatment at the farm gate with four or more unrelated anthelmintics is recommended, including at least one of the newer anthelmintic molecules (monepantel or derquantel).Specific grazing management is also advised.
An example in the Wormwise Technical Manual (McAnulty and Cook 2019) describes how this may be achieved by treating animals using two dualactive combination drenches.In practice, this involves dosing animals in a drenching race with one combination drench containing either monepantel plus abamectin or derquantel plus abamectin, and then dosing again with a benzimidazole plus levamisole combination drench.
While this option might minimise the risk of introducing resistant worms, the residue profiles of the respective actives when used in these treatment regimes have not been measured.Drug-to-drug interactions might occur between the individual anthelmintics when they are administered concurrently, and this could alter the pharmacokinetics and pharmacodynamics of each constituent (Entrocasso et al. 2008), ultimately impacting the residue profile.Approved meat withholding periods (WHP) to prevent drug residues exceeding maximum residue limits (MRL) are based on specific registration trial data, and the New Zealand regulatory authority, the Agricultural Compounds and Veterinary Medicines (ACVM) group, recommend that the default meat WHP of 91 days is applied when no such data is available (MPI 2022).In contrast, the approved WHP in New Zealand for meat from sheep treated with anthelmintic drenches containing monepantel and abamectin is 14 days (MPI 2023a), while for levamisole and oxfendazole is 10 days (MPI 2017).
A meat WHP of 91 days is undesirable for lamb trading and fattening operations.As a result, farmers and their advisors are forced to rely on quarantine treatments with fewer active ingredients, which might not be effective, potentially exposing their operations to a greater risk of importing anthelmintic-resistant parasites (Kaplan and Vidyashankar 2012).As such, data to establish the risk of MRL being exceeded by concurrent administration are required.Similar to the process undertaken by the FAO/WHO Joint Expert Committee on Food Additives (JECFA) to establish the MRL for animal commodities, food safety authorities screen for marker residues (a substance or group of substances with a known quantitative relationship between the concentration detected and the total residue of concern) in the target tissue(s) where residues are expected to be highest (Sanders et al. 2016).
This study was undertaken in order to confirm marker residue concentrations in the target tissues of the various active ingredients, in comparison with the MRL, when a monepantel plus abamectin combination drench was administered concurrently to sheep with an oxfendazole plus levamisole hydrochloride combination formulation.For all actives concerned, the target tissue (i.e. the tissue with the highest expected residue) is liver; as abamectin and monepantel are lipophilic, fat is considered a secondary target tissue.Muscle and kidney are not considered target tissues for the actives of interest and as such were not tested in the current study.

Materials and methods
The animal phase of this study was conducted under Elanco Australasia Pty. Ltd.Animal Ethics Committee approval number ELA210636 at the Yarrandoo R&D Centre (Kemps Creek, NSW, Australia).The bioanalytical phase was conducted by Eurofins Agroscience Testing Pty. Ltd. (Girraween, NSW, Australia).
This was a randomised, un-blinded study to determine the tissue residue profiles of monepantel, abamectin, levamisole and oxfendazole in sheep following a single concurrent administration of Zolvix Plus (25 g/L monepantel, 2 g/L abamectin; Elanco New Zealand, Auckland, NZ) and Scanda (80 g/L levamisole hydrochloride 45.3 g/L oxfendazole; MSD Animal Health, Upper Hutt, NZ).
Sixteen commercially sourced, first-cross (Border Leicester x Merino) sheep weighing 37-42 kg (mean 39.8 kg) and aged 8-9 months, were acclimatised to indoor housing for 7 days prior to day 0. None of the sheep had been treated with any other product within 3 months prior to the start of the study.During acclimatisation all animals underwent a veterinary examination, and were weighed and the four animals with the lowest body weight were excluded.The remaining sheep were randomised to two groups on the basis of sex and body weight so that six animals (three male and three female) were allocated to be sacrificed on day 21 of the trial and the remaining six on day 28.Randomisation was performed using an internal, validated, Access-based (Microsoft, Redmond, WA, USA) program (Table 1).Throughout the study the sheep were fed an oaten hay-based roughage mix and commercial sheep pellets (Vella's Stock Feeds, Glendenning, NSW, Australia) once per day at a maintenance rate.
On day 0 of the study, all 12 animals (6 male and 6 female) were dosed according to individual body weight determined the day prior.All treatments were measured by volume and rounded to the nearest 0.1 mL and administered orally from separate 5-mL disposable syringes; a new syringe was used for each animal and drench.Both drenches were dosed at a volume of 1 mL per 10 kg bodyweight according to label directions.Actual doses administered were above the target dose (Table 1).
Zolvix Plus was administered to all animals in the working race prior to administration of Scanda to simulate normal farm practice.Animals were observed immediately prior to treatment and approximately 2 hours after treatment of the last animal.
Animals were slaughtered by captive bolt followed by exsanguination using a staggered approach of six sheep (three male and three female) on each of days 21 and 28 following treatment.Target tissue specimens consisting of renal fat and the entire liver were collected for residue analysis.Fresh scalpels were used for each specimen collection and disposable gloves were changed between animals.Gloved hands were also washed with hot water and detergent between specimen collections from the same animal.Tissues were stored on ice and transferred to the Yarrandoo Bioanalytical Laboratory within 1 hour of slaughter.
Following receipt in the laboratory information management system, all tissues were weighed, roughly chopped and stored frozen (-20°C) in the Yarrandoo Bioanalytical Laboratory.Following the final necropsy, liver and renal fat samples were transferred frozen to the Eurofins laboratory.

Analytical methods
Tissue specimens were analysed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) for residues of monepantel sulfone, abamectin, levamisole and oxfendazole (as total fenbendazole sulfone) using validated analytical methods.
Analyses for monepantel sulfone, abamectin, levamisole and oxfendazole were performed on liver Active ingredients: 25 g/L monepantel, 2 g/L abamectin equating to a dose of 2.5 mg/kg monepantel and 0.2 mg/kg abamectin when dosed at the label rate of 1 mL/kg.b Active ingredients: 80 g/L levamisole hydrochloride (equivalent to 69 g/L levamisole), 45.3 g/L oxfendazole equating to a dose of 6.9 mg/kg levamisole and 4.53 mg/kg oxfendazole when dosed at the label rate of 1 mL/kg.c Expressed as levamisole equivalent (not levamisole hydrochloride).F = female; ID = identification number; M = male.
specimens collected on days 21 and 28.Oxfendazole residues were expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone.Renal fat specimens collected on days 21 and 28 were analysed for monepantel sulfone and abamectin residues only.
Control and fortified quality control (QC) samples were included with every set of specimens analysed to determine losses incurred during extraction.The QC samples were fortified at 10-fold (monepantel, levamisole and oxfendazole) or 100-fold (abamectin) the limit of quantitation (LOQ), with an additional single QC sample at 150-fold LOQ for monepantel in liver (Supplementary Table 1).Control tissues were sourced from reputable suppliers.All samples were analysed within the validated stability period for each tissue.
As set by the analytical laboratory, the LOQ of the analytical method for monepantel sulfone, oxfendazole, fenbendazole, fenbendazole sulfone and levamisole was 10 μg/kg for each individual analyte in each matrix with a limit of detection (LOD) set at 3 μg/kg (30% of the LOQ).When expressed as total fenbendazole sulfone, oxfendazole, fenbendazole and fenbendazole sulfone had a combined LOQ of 32 μg/kg and a LOD of 9.5 μg/kg.The LOQ of the analytical method for abamectin was 0.5 μg/kg in each matrix with a LOD set at 0.25 μg/kg (50% of the LOQ).
All data were calculated by relevant instrument software.Linearity of calibration standards were calculated by a linear regression model as specified in the analytical method.Fortified QC samples were used to assess method performance.
Residues below the validated LOQ were reported as "< LOQ".Data was not corrected for recovery.If there was more than one fortified QC sample per analytical run, the mean was reported.
According to in-house methods (monepantel, abamectin and levamisole) or published methods (oxfendazole; Yamada et al. 2006) aliquots of 0.5, 0.5, 5 g or 10 g (for monepantel sulfone, abamectin, oxfendazole and levamisole analysis, respectively) of thawed liver and fat were homogenised with the appropriate extraction solvents, clean-up steps and dilution (in accordance with the relevant methods; full details of the analytical methods are provided in Supplementary Material 1).The final extract was then injected onto an ultra-performance liquid chromatography column, and the target compounds were quantified with tandem mass spectrometry according to the conditions described in Supplementary Table 2.
Residues had declined further in sheep slaughtered 28 days after treatment and remained below MRL (Table 3).Oxfendazole residues were above the LOQ for all liver samples (mean 39.1 (min 25.1, max 53.6) μg/kg) collected at this time point.Monepantel sulfone was detected in four liver (mean 90.2 (min 45.1, max 170) μg/kg) and five renal fat (mean 380 (min 14.9, max 868) μg/kg) samples, respectively.A single liver sample had levamisole residues > LOQ (10.0 μg/kg) and abamectin residues were < LOQ for all tissue samples.
No adverse events were observed during the study period indicating the treatments were well tolerated when administered concurrently.
In summary, residues of monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone) in liver were successfully determined and found to be less than MRL determined by MPI (2023b) by 21 days after administration.Residues of monepantel sulfone and abamectin in renal fat were similarly successfully determined and were below MRL by 21 days.

Discussion
This study successfully determined marker residues in target tissues of monepantel sulfone, abamectin, levamisole and oxfendazole in sheep following a single concurrent administration of Zolvix Plus (monepantel/abamectin) and Scanda (oxfendazole/levamisole) according to label directions.All residues measured in liver and fat collected 21 and 28 days after treatment were below the MRL set by MPI for sheep for each analyte.
It should be noted that the current study was not intended to provide data to establish a formal WHP for the concurrent use of monepantel sulfone, abamectin, levamisole and oxfendazole in sheep.Whilst the work was conducted in GLP (Good Laboratory Practice)-compliant facilities, the work was not conducted in compliance with GLP standards (OECD 1998).Several deficiencies according to the ACVM guidance for residue depletion (MPI 2017) also exist within the current work that would preclude establishment of a WHP based on these results (e.g.insufficient timepoints for a residue depletion curve and lack of formal statistical analysis of the data).Thus, the data set must be considered and interpreted with caution.
The assays used in the current study were not corrected for recovery as mean recovery for each analyte in each matrix, except monepantel sulfone in liver, was within the acceptable range for accuracy as set by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH 2015).In the case of monepantel sulfone the mean recovery was 111% (relative SD ± 9.6) in five replicates, marginally above the accepted upper limit of 110%.As the recovery for this analyte was high, any correction would have resulted in an over-estimate of residues, thus no corrections were applied.
Although the MRL for abamectin in New Zealand is defined as the sum of both avermectin B1a and B1b (MPI 2023b), the current study only measured avermectin B1a residues.Avermectin B1a is considered the marker residue for abamectin, as it is the major homologue of the mixture (≥ 80%; Danaher et al. 2006); thus the analysis of the current study is consistent with laboratories conducting food safety screening.
Residues of the analytes were below 50% of the relevant MRL in all cases except for a single animal with a levamisole residue concentration 78.3% of MRL.It is generally accepted that concentrations up to the defined MRL are safe for human consumption, with analytical methods for food residues only required to measure marker residues at half the defined MRL (Sanders et al. 2016).While peer-reviewed reports are readily available for pharmacokinetic profiles of monepantel, abamectin and oxfendazole (Marriner and Bogan 1981;Karadzovska et al. 2009;Singh et al. 2018), there is a dearth of residue depletion studies.
Our results for monepantel concentrations in liver (mean 90.2 (min 45.1, max 170) μg/kg) 28 days after treatment are similar to those previously reported by Boison and Sanders (2012), who reported concentrations of 51 (SD 40) μg/kg 29 days after treatment, while our results for residues in fat (mean 380 (min 14.9, max 868) μg/kg) are somewhat higher than those reported by that study (83 (SD 45) μg/kg).Similarly, at a dose of 5.0 or 5.9 mg/kg, oxfendazole residues were below LOQ in liver 21 and 16 days after treatment, respectively (FAO/WHO 1999), which is in contrast with the mean of < 98.7 μg/kg 21 days following a target dose of 4.75 mg/kg observed in this study.
It has been noted in the literature that abamectin residues in sheep were below MRL 10 days after treatment in all tissues except liver (< 10-31 μg/kg) with the highest residues noted at day 3 (226 and 307 μg/kg, respectively, for liver and fat) post-treatment (Danaher et al. 2006).These concentrations are substantially higher than the current study (< 0.5-0.795and < 0.5-1.46μg/kg, respectively, for liver and fat), which would be expected given the much earlier sampling points reported.
Following a dose of 7.5 mg/kg body weight, Ciucă et al. (2022) reported that levamisole residues in sheep livers were below LOQ, defined as 2 μg/kg, 7 days after treatment using LC-MS/MS, whilst Tyrpenou et al. (2017) reported 16.58 μg/kg remained 14 days after treatment using high performance liquid chromatography.While both reports indicate lower residue levels earlier than the current study (day 21: three animals > LOQ 14.3-78.3μg/kg, mean 40 μg/kg; and day 28: one animal = 10 μg/kg), differences in animal breed, formulation type, single drug administration and analytical methods may all have impacted on the final reported levels.
Similar to the individual analytes, there are no published reports of residues, or depletion in target tissues, of the commercial combinations used in this study.Perhaps, given the higher residues reported in the current study, some drug-drug interactions are occurring, albeit with minimal impact.However, it remains unclear whether the residues of the two commercial products would be comparable to the available reports of single analytes or the residues demonstrated in the current study.
It has been noted by several authors that the prevalence and distribution of AR, including multi-drug resistance, has been increasing at a rapid rate over recent decades (Rose et al. 2015;Gilleard et al. 2021;Charlier et al. 2022).With this increased prevalence, introduced stock are more likely to import AR nematodes onto farms, posing a risk to the existing livestock productivity (Leathwick et al. 2009;Kaplan and Vidyashankar 2012).Economic impacts of resistant nematodes on farm have been estimated between 10 and 33% overall loss in both wool and lamb production (Lawrence et al. 2006;Leathwick and Besier 2014).With this negative impact on profitability of individual farms threatening the longevity of pasturebased ruminant enterprises (Charlier et al. 2022), the importance of adequate quarantine procedures becomes even more imperative.Best practice for quarantine procedures relies on the use of effective anthelmintics (Prichard et al. 1980;Coles and Roush 1992) and ideally the development of a new anthelmintic class to prevent continued resistance development (Leathwick et al. 2009).Concurrent or sequential use of multiple anthelmintics has been suggested in the literature (McMahon et al. 2013;Kaplan 2020) and, when integrated with other best-practice management strategies on New Zealand sheep farms, increased anthelmintic efficacy and potentially longevity of individual anthelmintics (Leathwick et al. 2015).Thus, one could extrapolate that if farmers are using refugia practices effectively the concurrent administration of two dualactive combination products would achieve the greatest protection in quarantine drenching.
The results of the current study demonstrate that it is unlikely that there is any significant interaction between monepantel, abamectin, oxfendazole and levamisole hydrochloride when Zolvix Plus and Scanda are administered concurrently to sheep that would result in residues above MRL by 21 days after treatment.

Disclosure statement
No potential conflict of interest was reported by the authors.

Table 1 .
Dose (mg/kg) of monepantel, abamectin, levamisole and oxfendazole administered to individual sheep in a trial to determine the concentration of anthelmintic marker residues in target tissues following concurrent dosing with two dualactive oral drenches.

Table 2 .
Limits of detection (LOD) and quantitation (LOQ) and maximum residue limits (MRL) of actives in a trial to determine the concentration of anthelmintic marker residues in target tissues of sheep following concurrent dosing with two dual-active oral drenches.

Table 3 .
Mean residues (μg/kg) of monepantel sulfone, abamectin, levamisole and oxfendazole in liver and fat of sheep sacrificed 21 (n = 6) and 28 (n = 6) days after concurrent administration of two dual-active drenches.LOQ = 0.5 μg/kg for abamectin, 10 μg/kg for all other analytes.c Calculated on values > LOQ only.d Only calculated for analytes when n > LOQ was > 4. e Avermectin B1a residues only.f As calculated fenbendazole sulfone, converted using a factor of 1.05 from detected oxfendazole and 1.11 from detected fenbendazole. b