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. 2024 Oct 26;48(3):163–169. doi: 10.1111/jvp.13488

The Pharmacokinetics of Subcutaneous Eprinomectin in Plasma and Milk in Dry Dairy Cattle

Ranee A Miller 1,✉, Tyana S McCluney 1, Jennifer L Halleran 1, Ronald E Baynes 1, Derek M Foster 1
PMCID: PMC12066900  PMID: 39460598

ABSTRACT

Parasitic infections in dairy cattle reduce herd immunity, milk production, and conception rates. This leads to higher production costs, compromised animal welfare, and increased interest in extralabel drug use. The extralabel use of anthelmintics poses food safety risks for consumers since appropriate withdrawal intervals in milk have yet to be established. Although topical eprinomectin has no milk withdrawal time, more research is needed to determine the residues present in milk after subcutaneous administration. This study aimed to characterize the pharmacokinetics of injectable eprinomectin in dry dairy cows. We hypothesized that, when given at the labeled dose, eprinomectin residues in dry dairy cattle would be below the FDA milk tolerance at the onset of lactation. Plasma was collected daily from 13 mature dairy cattle for 7 days postadministration, followed by periodic samples for 90 days. After calving, milk was collected daily until 90 days. Eprinomectin concentrations were measured using HPLC‐fluorescence detection. The maximum eprinomectin concentration in plasma and milk was approximately 36 ng/mL 43 h after administration and 3 ng/mL at the onset of lactation, respectively. The low eprinomectin levels in milk collected from these lactating dairy cattle suggest that administering eprinomectin at dry‐off is unlikely to result in violative residues. However, subcutaneous eprinomectin in lactating dairy cattle would be hard to justify unless there is evidence that the approved topical formulation is clinically ineffective.

Keywords: dairy cattle, drug residue, milk, pharmacokinetics, subcutaneous eprinomectin

1. Introduction

Parasitic infections in dairy cattle affect production performance by reducing milk yield, milk quality, and lowering contraception rates (Sanchez et al. 2004). Treating dairy cattle with anthelmintics can help mitigate these losses by increasing dairy production and average daily gain, improving animal welfare, and maintaining a healthy herd (Rashid et al. 2022). However, only a limited number of anthelmintic drugs are licensed for use in lactating animals (Imperiale and Lanusse 2021). This raises concerns about the development of anthelmintic resistance from the overuse of these products and the potential for increased extralabel drug use (ELDU). The ELDU of anthelmintics generates food safety risks for consumers as suitable milk withdrawal intervals have not yet been established (Canton, Lanusse, and Moreno 2021). There is growing pressure from consumers to grant production animals with pasture access due to perceived benefits in environmental conservation, animal welfare, and health (Stampa, Schipmann‐Schwarze, and Hamm 2020; Bousquet‐Mélou et al. 2021). However, with greater pasture access, dairy cattle will experience greater susceptibility to increased parasitic burden.

Currently, only two topical macrocyclic lactone products, eprinomectin and moxidectin, are approved for use in lactating dairy cattle. Topical eprinomectin is a broad‐spectrum anthelmintic used for the treatment and control of internal and external parasites (Eprinex, Boehringer Ingelheim Animal Health). This formulation has a zero‐day milk discard time, and the label claims up to 99.9% efficacy. However, topical eprinomectin has been shown to only provide 7–21 days of protection from reinfection depending upon the parasite species, and previous studies show a rapid decline in the efficacy of pour‐on macrocyclic lactones in comparison to injectable formulations (Gasbarre et al. 2015). Topical anthelmintics have also been shown to differ in absorption and efficacy depending on group housing conditions and licking behavior within a herd (Laffont et al. 2001). Although this product has a zero‐day milk discard time, the lack of residual efficacy is a potential concern for producers. Alternatively, an eprinomectin extended‐release injectable parasiticide is approved for use in beef or dairy cattle 20 months of age or younger (Longrange, Boehringer Ingelheim Animal Health). This formulation has provided up to 150 days of efficacy for parasitic control in beef cattle depending on the parasite species. The extent of efficacy is attributable to the polymer matrix that remains beneath the skin, allowing for a slow release of eprinomectin over time (Avgoustakis 2005). This long duration of action has been shown to provide considerable advantages for production parameters in heifers compared with the topical formulation with a much shorter duration of action (Dudley and Smith 2020). However, this long‐duration formulation has no published pharmacokinetic data and no label approval for use in mature, adult dairy cattle. FARAD has received several questions about the usage of this product in cattle, which is why its application was explored in this particular scenario.

This study aimed to provide new pharmacokinetic data utilizing the dry period in dairy cows with the goal of benefiting food safety, animal welfare, producers' economic interests, and educated treatment decisions for veterinarians. The objective of this study was to determine the elimination kinetics of eprinomectin when administered subcutaneously in dry dairy cattle. We hypothesized that a single subcutaneous, label dose of 1 mg/kg eprinomectin would result in drug concentrations falling below the FDA milk tolerance level by the time of postcalving lactation.

2. Materials and Methods

2.1. Animals and Housing

The study was approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC #21–172). Inclusion criteria for enrollment included cows with no history of anthelmintic administration in the previous 4 months, were multiparous, and were about to begin a 60‐day dry‐off period. Prior to enrollment, all cows were determined to be pregnant (by rectal palpation), healthy based on physical examination, and had a negative California Mastitis Test in all working quarters. Fourteen mature, adult dairy cows (Jersey, n = 5; Holstein, n = 9; weight range: 463–873 kg) were enrolled in this study. The cows were sourced and group‐housed at the North Carolina State University Dairy Educational Unit. After a final milking, the enrolled cows were administered dry‐cow therapy (Spectramast DC, Zoetis Animal Health) and a teat sealant (Lockout, Boehringer Ingelheim Animal Health) at one syringe/quarter. The cows were fed once daily, provided water ad libitum, and had pasture access for grazing throughout the duration of the study.

2.2. Dosing and Sample Collection

Following the last milking before dry‐off, each cow received a single dose of subcutaneous eprinomectin (5% sterile solution) at a dose of 1 mg/kg in the front of the right shoulder. Doses exceeding 10 mL were divided between two injection sites.

Blood sample collection time points were extrapolated from previous studies in dairy cattle (Soll et al. 2013). Heparinized blood samples (10 mL) were collected by tail venipuncture using a vacutainer needle prior to drug administration (0 h) and then at 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, 35, 49, 63, 77, and 91 days. Plasma was separated by centrifugation at 3500 × g for 10 min at 4°C and equally divided into two cryovials. Once the cows calved, milk samples were collected every 12 h for the first 7 days and then once daily until 91 days postdrug administration. Milk sample collection time points were taken based on the established milking frequency at the dairy unit. The cows were milked in a side‐by‐side parallel parlor by a milking claw. A milk meter (“Turn & Mix” Sampler Metatron; GEA Farm Technologies) collected a measured amount of milk throughout the entirety of milking, and a 15 mL aliquot was taken immediately from this collection to provide a representative sample of the total milking volume. Leftover milk was vacuumed out of the system, and a clean milk meter was used for every collection. If treated cows were milked in the same stall, there was a small chance for cross‐contamination from residual milk left in the milking claw or hoses, but careful attention was paid to avoid this from happening. Between milkings, the entire milking system was thoroughly cleaned during the rinsing cycle. Blank milk was taken from the bulk tank prior to the start of the study to be used for method development and spiking solutions during sample analysis. Eprinomectin was not used at this dairy, so there was no concern for contaminated blank milk from the bulk tank. Milk samples were divided into three cryovials. All samples were placed in a −20°C freezer until the study's completion and moved to a−80°C freezer until they could be analyzed.

2.3. Drug Concentration Analysis

Eprinomectin concentrations in all samples were determined by high‐performance liquid chromatography with fluorescence detection (HPLC‐FL) by modifying a method used previously in alpacas (Pollock et al. 2017). The analytical method for determining eprinomectin in plasma and milk was validated before use in this study. The HPLC‐FL system consisted of a Waters Alliance 2965 HPLC Separations Module System with a Waters 2475 HPLC Multi Fluorescence Detector set at an excitation wavelength of 355and emission wavelength of 465 nm. The LC column used was an XBridge C18 3.5 μm, 4.6 × 100 mm HPLC Column with an XBridge BEH C18 3.5 μm, 3.9 × 5 mm Vanguard precolumn (Waters Corp.) kept at a constant temperature of 30°C. The mobile phase consisted of 87% acetonitrile and 12.9% ultrapure water with the addition of 0.1% trifluoroacetic acid. The flow rate was 1 mL/min and was delivered in an isocratic mixture. The retention time for eprinomectin using this method and equipment was approximately 6 min. Eprinomectin USP reference standard (300 mg; MilliporeSigma) was dissolved in 100% HPLC‐grade acetonitrile to achieve a 1 mg/mL stock solution concentration. From this solution, further dilutions were made in acetonitrile to prepare spiking solutions for plasma and milk. The spiking solutions were made fresh weekly. The reference standard, stock solution, and spiking solutions were tightly sealed, protected from light, and stored at −20°C.

In glass tubes, 10 μL of the spiking solutions were added to 990 μL of blank bovine plasma to create a calibration curve consisting of one blank and seven calibration standards ranging from 0.625 to 100 ng/mL. To these tubes, 1000 μL of acetonitrile was added, vortexed briefly, and then centrifuged at 3500 × g for 10 min at 4°C. The supernatant was decanted, and 1000 μL was transferred to a clean glass tube and dried under 100% air at 55°C for 30 min. Plasma samples were reconstituted in 100 μL of a 1:1 acetonitrile:1‐methylimidazole solution and vortexed. Then, 100 μL of a 7:3 acetonitrile:trifluoroacetic anhydride solution was added to the samples and vortexed. The acetonitrile:1‐methylimidazole and acetonitrile:trifluoroacetic anhydride solutions were made fresh daily. The derivatized plasma samples were filtered with an EZFlow hydrophilic PVDF 13 mm 0.22 μm syringe filter (Foxx Life Sciences) into HPLC total recovery vials (Waters Corp.), and 50 μL was injected for analysis.

For milk sample analysis, the same method for plasma sample analysis was used except that the calibration curve had a different concentration range, a solid phase extraction technique was added for additional cleanup, and each milk sample was analyzed in triplicate. The milk calibration curve consisted of one blank and six calibration standards ranging from 1.25 to 50 ng/mL. After centrifugation, the milk supernatant was diluted with ultrapure water and loaded into Oasis PRiME HLB 3 cc (60 mg) Extraction Cartridges (Waters Corp.) onto a positive pressure manifold (Biotage). These samples were washed with 2000 μL of 95:5 water:methanol and eluted with 2000 μL of 90:10 acetonitrile:methanol into clean glass tubes. The samples were dried down, reconstituted with derivatizing reagents, syringe filtered, loaded into HPLC total recovery vials (Waters Corp.), and 50 μL was injected into the instrument for analysis.

The chromatograms were integrated with a computer program (Empower 3 Software; Waters Corp.) which quantified the unknown concentrations in both matrices.

Calibration, quality control, and blank samples were made fresh at the start of each run. The quality control samples were made by spiking blank plasma or milk with eprinomectin concentrations at 10 and 50 ng/mL. Quality control samples for plasma and milk were within 7.5% and 9.4% of the nominal value, respectively, and the relative standard deviation was ≤ 15.7% and ≤ 10.8%, respectively. All calibration curves were linear for both matrices with an average R 2 > 0.99. The limit of quantification (LOQ) and the limit of detection (LOD) for eprinomectin in both plasma and milk were 2.5 and 1.25 ng/mL, respectively.

Plasma and milk intraday precision and accuracy were obtained on the same day using four different concentrations repeated five times each at 0.625, 0.75, 1.25, and 25 ng/mL and 1.25, 2.5, 5, and 25 ng/mL, respectively. Plasma interday precision and accuracy were obtained by measuring five different concentrations on seven different days at 2.5, 10, 25, 50, and 100 ng/mL. Milk interday precision and accuracy were obtained by measuring six different concentrations on 22 different days at 1.25, 2.5, 5, 10, 25, and 50 ng/mL, respectively. Plasma intraday precision was 5.3%–15.9%, with an accuracy of 97.7%–125.3%. Milk intraday precision was 2.0%–13.1%, with an accuracy of 91.1%–102.6%. Plasma interday precision and accuracy for 10–100 ng/mL standards were 2.2%–15.6% and 91.3%–102.2%, respectively. Plasma interday precision and accuracy for the 2.5 ng/mL standard were 40.0% and 105.6%, respectively. Milk interday precision and accuracy for standards of 2.5–50 ng/mL were 1.4%–18.2% and 92.8%–101.2%, respectively. Milk interday precision and accuracy for the 1.25 ng/mL standard were 34.1% and 105.1%, respectively.

2.4. Pharmacokinetic and Statistical Analysis

A noncompartmental analysis of eprinomectin concentration versus time profiles was performed with analytical software (Phoenix Win‐Nonlin 8.0; Certara Inc.). The pharmacokinetic analysis of milk was performed on the average concentration value of the three replicates to account for any variability present in the raw milk samples. The noncompartmental analysis of plasma was conducted on points for the initial concentration peak only (approx. 49 days postdrug administration). All analyses were conducted on observed values that were above the LOQ (2.5 ng/mL) for plasma and above the LOD (1.25 ng/mL) in milk. The linear log trapezoid method calculated the area under the concentration–time curve from time zero to infinity (AUC0 → ∞; day × ng/mL). Other parameters determined were peak concentration (Cmax; ng/mL), time to reach peak concentration (Tmax; day), and the terminal half‐life (T1/2; day). This study could not calculate absolute bioavailability as an accompanying intravenous dose was not administered. Therefore, the apparent clearance and volume of distribution per fraction absorbed are not reported.

The mean pharmacokinetic parameters were compared between breeds using the Mann–Whitney U‐test based on the normality of data distribution. All statistical analyses were performed using R Statistical Software (v4.3.1; R Core Team 2023).

3. Results

No tissue site reactions were observed in the cows after subcutaneous eprinomectin administration. One Holstein cow was eliminated from the study due to spontaneous abortion shortly after dosing. Another Holstein cow was eliminated from the study due to insufficient quantities of plasma and milk samples for processing after they were spilled. A total of 12 cows (Jersey, n = 5; Holstein, n = 7) were used for plasma analysis. One Holstein cow's milk data was removed from analysis due to contracting metritis after calving, so a total of 11 cows (Jersey, n = 5; Holstein, n = 6) were used for milk analysis.

3.1. Plasma

The plasma concentration–time curves and pharmacokinetic parameters following a single subcutaneous dose of eprinomectin at 1 mg/kg are summarized in Figure 1 and Table 1. The observed values that fell below the LOQ (2.5 ng/mL) were removed before analysis. This study only reports pharmacokinetic parameters in plasma from the initial peak which lasted until approximately 49 days after dosing. The initial mean maximal plasma concentration (Cmax ± SD) for Holstein and Jersey cows in this study reached 37.52 ± 1.30 ng/mL at 1.74 ± 0.06 days (Tmax) and 33.34 ± 1.37 ng/mL at 1.89 ± 0.06 days, respectively. There may be apparent breed differences, but the analysis suggests there are no statistically significant differences in eprinomectin depletion in plasma (Figure 1). There were no significant differences in T1/2, Cmax, Tmax, AUCinf, AUClast, and MRT between the two groups (Table 1).

FIGURE 1.

FIGURE 1

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Plasma concentration versus time profile (linear scale axis, left; log scale axis, right) following a single 1 mg/kg dose of subcutaneous eprinomectin (geometric mean ± SD).

TABLE 1.

Plasma pharmacokinetic (PK) parameters* of eprinomectin based on noncompartmental PK analysis following a single 1 mg/kg dose of subcutaneous eprinomectin to dry dairy cattle (geometric mean (% CV)).

Subjects T1/2 (day) Tmax (day) Cmax (ng/mL) AUClast (day × ng/mL) AUCinf (day × ng/mL) AUCextrap (%) MRTinf (day)
Holstein (n = 7) 35.80 (45.88) 1.74 (42.31) 37.52 (26.41) 486.59 (21.87) 709.31 (25.83) 27.89 (48.35) 43.06 (42.93)
Jersey (n = 5) 22.05 (77.04) 1.89 (41.24) 33.34 (32.26) 335.61 (37.59) 466.20 (40.31) 26.18 (35.03) 29.08 (43.83)
All cows (n = 12) 26.90 (63.16) 1.80 (40.05) 35.72 (28.19) 416.81 (34.12) 583.46 (37.40) 25.49 (45.45) 35.54 (43.46)
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Abbreviations: AUCextrap, percentage of the area under the concentration–time curve extrapolated from the last observed time point; AUCinf, area under the concentration–time curve of total exposure of drug to the body; AUClast, area under the concentration–time curve from time zero to time of the last measurable concentration; Cmax, maximum plasma concentration; MRTinf, mean residence time of total exposure of drug to the body; T1/2, terminal half‐life of drug; Tmax, time to maximum concentration.

*

Values shown are for the initial plasma peak only (approx. 49 days postdrug administration).

3.2. Milk

Breed milk concentration–time curves following a single subcutaneous dose of eprinomectin at 1 mg/kg are shown in Figure 2. The median dry period for all cows was 65 days (Holstein, 66 days; Jersey, 64 days) with a range of 53–71 days. Eprinomectin concentrations were not present above the FDA milk tolerance for cattle of 12 ng/mL in any milk sample at the start of lactation or thereafter (Figure 2). Drug concentration analysis quantified most milk samples to be below the LOQ (2.5 ng/mL). Therefore, subsequent milk samples collected past 7 days in lactation were excluded from analysis. At the time of calving, 87% of milk samples fell below the LOQ in milk. Therefore, only Cmax and Tmax parameters are reported, and the observed values that fell below the LOD (1.25 ng/mL) were removed before analysis. The mean maximal milk concentration (Cmax ± SD) for Holstein and Jersey cows in this study reached 2.67 ± 1.27 ng/mL at 0.63 ± 0.06 days in lactation (Tmax) and 2.39 ± 1.33 ng/mL at 0.59 ± 0.06 days in lactation, respectively. The milk pharmacokinetic parameters and milk yield data are summarized in Table 2. The milk yield data are an average of all milkings for the first 7 days in lactation and does not accurately depict the increase in milk yield throughout the course of the study given these cows were in early lactation.

FIGURE 2.

FIGURE 2

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Milk concentration versus time profile (linear scale axis, left; log scale axis, right) following a single 1 mg/kg dose of subcutaneous eprinomectin (geometric mean ± SD). Negative values for the lower SD could not be plotted on the semilogarithmic scale. LOD, limit of detection (1.25 ng/mL); LOQ, limit of quantification (2.5 ng/mL).

TABLE 2.

Milk pharmacokinetic (PK) parameters of eprinomectin based on noncompartmental PK analysis following a single 1 mg/kg dose of subcutaneous eprinomectin to dry dairy cattle (geometric mean (% CV)).

Subjects Tmax (day) Cmax (ng/mL) Milk yield (kg)
Holstein (n = 6) 0.63 (36.97) 2.67 (23.93) 17.19 (± 4.08)
Jersey (n = 5) 0.59 (35.72) 2.39 (29.08) 13.02 (± 3.12)
All cows (n = 11) 0.62 (34.44) 2.55 (25.11) 15.42 (± 4.23)
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Note: Milk yield, average amount of milk collected per milking for the first 7 days in early lactation.

Abbreviations: Cmax, maximum plasma concentration; Tmax, time to maximum concentration.

The Holstein cow that contracted metritis after calving had detectable levels of eprinomectin above the LOQ for the first 7 days in lactation and reached a maximum concentration of approximately 6.90 ng/mL 144 h into lactation. Her observed values are excluded from our analyses because she was not a member of our target, healthy population.

4. Discussion

This study is the first to report on the pharmacokinetic parameters of subcutaneous eprinomectin in dry dairy cattle. Plasma and milk have a maximum eprinomectin concentration of approximately 36 ng/mL 43 h after administration and 3 ng/mL at the onset of lactation, respectively.

In this trial, the subcutaneous formulation of eprinomectin was not used per the label directions as it was administered to dry dairy cattle, a production class not listed on the label. On average, 90% of the milk samples in this study within 48 h were free of residues according to our LOQ of 2.5 ng/mL. If the Holstein cow with metritis whose milk sample concentrations remained above the LOQ for the first 7 days of lactation was removed from the dataset, 97% of the milk samples were free of residues by 48 h. The milk Cmax and Tmax parameters reported in this study are less accurate due to our decision to exclude only values that fell below our LOD instead of our LOQ. This decision was made to still report the milk concentration–time profile and emphasize the lack of residues present at the onset of lactation. The use of injectable eprinomectin in mature dairy cattle instead of the approved topical formulation would be considered prohibited ELDU under the Animal Medicinal Drug Use Clarification Act (AMDUCA) if used for convenience. FARAD does not encourage prohibited ELDU and conducted this research to provide veterinarians with pharmacokinetic data to make educated treatment decisions. We highly recommend veterinarians to request extralabel advice at https://www.farad.org/ prior to use in dairy cattle and abide by AMDUCA's requirement that there is evidence that shows the approved topical formulation is clinically ineffective for its intended use.

The mean plasma concentration profiles of eprinomectin in dairy cows measured in the current study are lower than those measured in lactating dairy cows, nonlactating dairy cows, and crossbred cows (Bos indicus × Bos taurus) (Baoliang et al. 2006; Aksit et al. 2016; do Nascimento et al. 2020) yet higher than those measured in beef cows (Soll et al. 2013). The differences among these studies may be explained by the various eprinomectin formulations administered or individual animal variability, such as body condition score and physiology across the classes of production animals. A study conducted in canines supported the conclusions that three macrocyclic lactones (ivermectin, moxidectin, and eprinomectin) show decreased clearance, increased volume of distribution, and longer terminal half‐lives in obese patients when compared to their lean counterparts (Bousquet‐Mélou et al. 2021). This is supported by the known pharmacokinetics of lipophilic drugs and their affinity for adipose tissue. This may explain the pharmacokinetic differences between dairy and beef cattle breeds as dairy breeds historically have less body conditioning and altered metabolism, but more research needs to be conducted to evaluate the effect of disease, breed, and production parameters on eprinomectin elimination kinetics.

Eprinomectin was chosen for evaluation in this study, because it has substantially reduced distribution in milk (Imperiale and Lanusse 2021) and offers a large margin of safety for human consumers due to its mechanism of action. Eprinomectin consists of a 90:10 mixture of two homologous components, B1a and B1b. The B1a component is the marker residue in all matrices. Previous studies have reported that the milk‐to‐plasma concentration ratio for eprinomectin in cattle ranges from 0.12 to 0.14 and remains constant for the duration of eprinomectin clearance (Alvinerie et al. 1999). This ratio is valuable as it estimates the amount of drug distributed into the milk after it is transferred out of systemic circulation. Applying the 0.14 partitioning ratio to the current study's plasma Cmax, Holstein and Jersey cows dosed during lactation instead of at dry‐off would have an estimated milk Cmax of 5.25 ± 1.36 and 4.67 ± 1.49 ng/mL, respectively. Anthelmintic treatment in heifers during the growing period has been shown to increase average daily gain, milk production, and reproduction parameters (DesCôteaux, Caldwell, and Doucet 1999; Volk et al. 2019; Dudley and Smith 2020). It is easy to assume that administering anthelmintics to prepartum dairy cows during a time of suppressed immunity would contribute the most to increasing production parameters during freshening. However, studies have shown that production parameters such as milk yield increase the most when dairy cows are treated in mid‐lactation (Gross, Ryan, and Ploeger 1999). Due to the concern of violative residues after ELDU of subcutaneous eprinomectin in lactating dairy cattle, mid‐lactation application is currently impractical for this formulation of eprinomectin.

There were multiple limitations in this study. The sampling duration, sample size, and lack of an efficacy component were the main limitations. Injectable eprinomectin exhibits a biphasic peak in plasma drug concentration, but this study did not ultimately capture the secondary plasma peak due to insufficient sampling duration. The secondary mean maximal plasma concentration has been shown to have reached approximately 30% of the initial mean maximal plasma concentration around 90 days in beef cattle when sampling continued 160 days after drug administration (Soll et al. 2013). It is unknown if there is also a biphasic peak in milk drug concentration and what the secondary mean maximal milk concentration value would be. It is recommended to have a minimum of 20 animal subjects for dry‐cow studies (FDA 2015). However, after evaluation to meet the study's inclusion criteria, only 14 subjects were available for enrollment at the dairy farm, 12 of which were used for plasma analysis and 11 for milk analysis. The cows were representative of commercial dairy practices but were not randomly selected as it was not appropriate for the pharmacokinetic assessment or practical for the extensive sampling with minimal animal availability. This study lacked an efficacy component however, previous publications indicate that plasma concentrations for avermectins ranging between 0.5 and 2 ng/mL would represent the minimal drug concentration required for optimal nematocidal activity (Lifschitz et al. 1999). Mean plasma concentrations for Holstein and Jersey cows remained above 1 ng/mL for the entire study duration. One exception was that Jersey cows had an average concentration that fell between our LOQ (2.5 ng/mL) and our LOD (1.25 ng/mL) on day 63 postadministration, which makes this quantity less accurate. Regarding an ectoparasite comparison, it is been documented that a plasma concentration for eprinomectin in cattle of at least 8 ng/mL is the necessary threshold for tick control (Lifschitz et al. 2016). More importantly, it was shown that this concentration was necessary for the length of tick development (21–27 days). Plasma concentrations for Holstein and Jersey cows remained above 8 ng/mL for at least 14 days, but concentrations dropped below the threshold level between days 14 and 21. This may contribute to larval reinfestation and concerns for resistance. This study did not assess parasitic resistance to eprinomectin, but there is growing concern that extended‐release formulations may expedite the development of resistance by increasing selection pressure for resistant parasites (EMA 2017).

In conclusion, low eprinomectin concentrations in milk collected from these dairy cattle in postcalving lactation could indicate a novel method for anthelmintic treatment without the concern for violative residue risks for consumers. Still, they will require a prolonged withdrawal interval to comply with a zero‐tolerance limit for ELDU. In addition, while this extended‐release product may seem advantageous to use due to its low concern for violative residues, we encourage veterinarians and producers to utilize labeled products first and look to extended‐release formulations as a last resort to minimize the formation of resistant parasites. FARAD does not encourage prohibited ELDU and conducted this research to provide veterinarians with pharmacokinetic data to make educated treatment decisions. Using subcutaneous eprinomectin in lactating dairy cattle would be hard to justify unless there is evidence that the approved topical formulation is clinically ineffective for its intended use.

Author Contributions

R.E.B. and D.M.F. conceived and designed the study. D.M.F., R.A.M., T.S.M., and J.L.H. collected samples and data. R.A.M. developed the analytical method and performed sample analysis. R.A.M. performed pharmacokinetic modeling. All authors were involved in the drafting and revising of the manuscript.

Ethics Statement

The study was approved by the North Carolina State University Institutional Animal Care and Use Committee (IACUC #21–172).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors thank the North Carolina State University Dairy Educational Unit for providing the facilities and animals used in this study. We also thank Lily Smith for sample collection assistance and Cassidy Konzelman for statistical consultation.

Funding: The USDA National Institute of Food and Agriculture (grant no. 2019–41480‐30292 and 2020–41480‐32520; Kansas City, MO) supported this research, which funds the Food Animal Residue Avoidance and Depletion Program (FARAD).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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