SUMMARY Enramycin is a polypeptide antibiotic effective against pathogens of the gut flora. A fermentation product of Streptomyces fungidicus, enramycin, is a very large molecule that is not well absorbed from the intestinal lumen when consumed orally. Enramycin has 2 major homologs: enramycin A and enramycin B with enramycin A being the predominant homolog. The depletion of enramycin was determined in broiler chicken tissues (liver, kidney, muscle, and skin-with-attached-fat) when enramycin was administered orally to broiler chickens in the feed at the level of 23 ppm for 10 d prior to the typical slaughter age of 42 d. Enramycin A and enramycin B residues were quantified separately for each tissue sample. The muscle and skin-with-attached-fat did not contain any detectable levels of enramycin A or enramycin B. The liver samples had combined enramycin A and B concentration of 19.4 ppb, and the kidney samples had combined enramycin A and B concentrations of 14.1 ppb at 0-hour withdrawal. The Japanese MRL (maximum residue limit) for enramycin is 30 ppb for all edible chicken tissue. Enramycin administered in feed at 23 ppm, with 0-hour withdrawal, resulted in tissue levels well below the established Japanese MRL of 30 ppb. DESCRIPTION OF PROBLEM Enramycin is a large molecule polypeptide antibiotic effective against pathogens of the gut flora [1–3]. A fermentation product of Streptomyces fungidicus , enramycin, is composed of 2 homologs, enramycin A (molecular weight 2355.3 g/mol)  and enramycin B (molecular weight 2369.3 g/mol) , that are too large to be well absorbed from the intestinal lumen when consumed orally. It has strong inhibitory effect against Clostridium perfringens , the pathogen responsible for inducing necrotic enteritis in chickens [8, 9]. Enramycin has been used as a preventive feed medication at the level of 3 to 10 ppm in poultry feed  since its commercial introduction in Japan in 1974. Recent governmental and market pressures to remove preventive antibiotics from poultry feeds have led to an interest in the therapeutic use of enramycin for the treatment of necrotic enteritis caused by C. perfringens. The therapeutic level of enramycin is established at 20 ppm by MSD Animal Health (2 Giralda Farms, Madison, NJ). This study investigated the tissue concentration of enramycin residues following oral administration of 1.15 times the therapeutic dose level (23 ppm) of enramycin administered in feed. Additionally, in support of the safety of the therapeutic dose (20 ppm), a dose level up to 5X (100 ppm) the intended clinical dose was tested in a safety study conducted by Merck Animal Health and was found to have no adverse effect on the health or performance of the birds . MATERIALS AND METHODS The purpose of this study is to provide data supporting the registration of enramycin as a veterinary pharmaceutical product, and therefore, the study was conducted in accordance with the Guidance for Industry VICH GL 48(R) issued by the Center for Veterinary Medicine, U.S. Food and Drug Administration. The Guidance for Industry defines the requirements of such a study as having a minimum of 6 birds per test period and that the study should be run at the recommended dose and duration of treatment. This study was compliant with the Guidance. In addition, the guidelines presented in the Guide for the Care and Use of Agricultural Animals in Research and Teaching, 3rd Edition, 2010 were followed. The study was conducted in accordance with an IACUC-approved study protocol, the US Food and Drug Administration Good Laboratory Practice Regulations, and applicable Standard Operating Procedures at Sinclair Research Center and Primera Analytical Solutions Corp. Broiler chickens (Cobb 500 breed, 90 males and 90 females) were obtained as day-old chicks and raised in an open poultry colony in 2 pens separated by gender. They were reared on non-medicated starter feed until 14 d of age followed by non-medicated grower feed until 25 d of age. At 25 d old, the health records and gender were used to select 33 males and 33 females to undergo acclimation for 1 wk. During acclimation, 5 birds were housed in a pen by sex (12 pens total), and 2 pens had 3 birds each separated by sex. Each bird was tagged with an identification number and fed non-medicated finisher feed during acclimation until Study Day 1. On Study Day 1 (bird age 31 d), the 33 male and 33 female chickens were clinically evaluated and weighed prior to randomization for group assignment. Ten birds were randomly allocated to Group 1, the untreated control group, consisting of 2 pens of 5 male or 5 female chickens. Group 2, the enramycin treated group, consisted of 10 pens each containing 5 male or 5 female chickens. An additional reserve group for quality control (QC) chickens consisted of 2 pens, each housing 3 male or 3 female chickens. Group 1 and QC birds were housed in a separate independently ventilated room, while all Group 2 birds were housed in the same room. The target dose for the enramycin treatment group (10 pens of group 2 chickens) was 25 ppm of Enradin F-80 administered in finisher ration ad libitum. The analysis of medicated feed performed by the Eurofins Central Analytical Laboratory (CAL) measured 23.4 ppm enramycin; –6.4% vs. the targeted dose of 25 ppm. The feed analysis by the Eurofins CAL showed no measurable enramycin in non-medicated control feed. On Study Day 0 through Day 9, the chickens in Group 1 and Group QC were fed non-medicated pelleted finisher feed ad libitum while chickens in Group 2 (treated group) were offered enduracidin pelleted finisher feed ad libitum. On each morning during the treatment period, the appropriate feed (medicated or non-medicated feed) was added to the feeder. The feeder together with feed was weighed before and also after adding feed, and the feeder was replaced in the respective pen. The weights of the feeder plus feed were recorded and the remaining feed was recorded the next morning before starting the next treatment period. The feeder weights were used to calculate the amount of feed consumed per pen. The amount of feed added daily to each feeder (up to approximately 1000 g/d/pen), plus an amount in excess to ensure ad libitum feeding, was determined by the feeding rate measured during acclimation and feeding recommendations for broiler breed from the Cobb Broiler Broiler Performance and Nutrition Supplement . At the end of the treatment period, 10 treated birds (Group 2) and 10 control birds (Group 1) were humanely euthanized at time 0-h withdrawal, and the target tissues, liver, kidney, muscle, and skin-with-attached-fat, were harvested and frozen. The frozen tissues were later homogenized with dry ice and shipped frozen to Primera Analytical Solutions Corp for analysis of enramycin residues. The remaining birds were offered non-medicated feed, and euthanized at time points 0, 6, 12, and 24 h after withdrawal of medicated feed. The measured concentrations for enramycin residues for tissues collected at 6, 12, and 24 h post cessation of treatment will not be reported in this paper. Enramycin residues in tissue homogenates (liver, kidney, muscle, and skin/fat) were assayed by Primera Analytical Solutions Corp using validated procedures for each respective tissue. The extraction procedure was very similar for all 4 tissue types. Each tissue type was assayed in 2 sample analysis batches. Each sample batch included matrix calibration standards, control samples, double blank samples, QC samples, and unknown tissue samples from treated chickens. Tissue samples from control (untreated) chickens were used to prepare control samples, calibration standards, and QC standards. Duplicate control tissue fortified with the internal standard only (control blanks) and duplicate control samples without the internal standard or analyte (matrix blanks) were assayed with each sample analysis batch to monitor the background signal. Additionally, for the sample batches for each tissue type, duplicate QC samples fortified at 2 different concentrations (9.20 and 36.0 ppb enramycin A and 6.50 and 25.6 ppb enramycin B) were assayed. Matrix calibration standards and QC samples were prepared by adding an appropriate amount of fortification solution (containing enramycin and flunixin-d3 internal standard) to 1 g of homogenized tissue (liver, kidney, muscle, and skin/fat). To prepare a control blank and unknown samples, flunixin-d3 internal standard was added to 1 g of tissue homogenate. Double blank samples did not have enramycin or the internal standard. The samples were extracted with acidified acetonitrile and centrifuged to separate suspended precipitates from the extract. This process was repeated 3 times, and the supernatants were combined. An aliquot of the extract was partitioned with hexane and further cleaned on a solid phase extraction (SPE) cartridge. The SPE eluate was evaporated to dryness. The residue was dissolved in solution to yield the final extract and analyzed by liquid chromatography with mass spectrometry detection. All tissue samples were assayed by Primera Analytical Solutions Corp within 2 mo of collection. The limit of quantitation for enramycin A and B in all tissues is 4.60 and 3.30 ppb, respectively. The limit of detection for each tissue is listed below: Skin/Fat: 0.258 ppb enramycin A and 0.0980 ppb enramycin B Muscle: 0.264 ppb enramycin A and 1.47 ppb enramycin B Liver: 2.25 ppb enramycin A and 1.99 ppb enramycin B Kidney: 1.15 ppb enramycin A and 1.39 ppb enramycin B The withdrawal period for edible tissues (muscle, skin/fat, liver, kidney) was calculated according to CVMP Note for Guidance “Approach towards harmonization of withdrawal periods” (EMEA/CVMP/036/1995-FINAL), using the WT1.4 software (Withdrawal time calculation for tissues, EMA application software) for kidney tissue and SAS software (SAS Institute Inc, Cary, NC, release 9.2) for liver tissue. RESULTS AND DISCUSSION The residues in muscle and skin-with-attached fat were below the limit of quantification at 0-h withdrawal. The residues in liver and kidney were an average of 19.4 ppb (% CV = 18.8) and 14.1 ppb (% CV 25.5) respectively at 0-h withdrawal (Table 1). The amounts are below the maximum residue limit (MRL) of 30 ppb established by the Japanese Ministry of Health, Labour and Welfare (MRLs of Agriculture Chemicals in Foods, Enramycin). Table 1. Enramycin (A + B) Concentration in Tissue Samples at 0 Hours’ Withdrawal. Sample Kidney Muscle Liver Skin/Fat Sum A + B Sum A + B Sum A + B Sum A + B ppb ppb ppb ppb Negative Controls BLOQ1 BLOQ BLOQ BLOQ Average NA NA NA NA Enramycin 14.1 BLOQ 19.4 BLOQ Average NA NA % CV 25.5 NA 18.8 NA Sample Kidney Muscle Liver Skin/Fat Sum A + B Sum A + B Sum A + B Sum A + B ppb ppb ppb ppb Negative Controls BLOQ1 BLOQ BLOQ BLOQ Average NA NA NA NA Enramycin 14.1 BLOQ 19.4 BLOQ Average NA NA % CV 25.5 NA 18.8 NA 1 BLOQ = Below Limit of Qualification; for enramycin A = 4.60 ppb and enramycin B = 3.30 ppb. View Large Table 1. Enramycin (A + B) Concentration in Tissue Samples at 0 Hours’ Withdrawal. Sample Kidney Muscle Liver Skin/Fat Sum A + B Sum A + B Sum A + B Sum A + B ppb ppb ppb ppb Negative Controls BLOQ1 BLOQ BLOQ BLOQ Average NA NA NA NA Enramycin 14.1 BLOQ 19.4 BLOQ Average NA NA % CV 25.5 NA 18.8 NA Sample Kidney Muscle Liver Skin/Fat Sum A + B Sum A + B Sum A + B Sum A + B ppb ppb ppb ppb Negative Controls BLOQ1 BLOQ BLOQ BLOQ Average NA NA NA NA Enramycin 14.1 BLOQ 19.4 BLOQ Average NA NA % CV 25.5 NA 18.8 NA 1 BLOQ = Below Limit of Qualification; for enramycin A = 4.60 ppb and enramycin B = 3.30 ppb. View Large These results support the establishment of a withdrawal time of 0-d for poultry flocks treated with 20 ppm enramycin in the feed for 10 d. CONCLUSION AND APPLICATIONS Enramycin, when used at a therapeutic dose of up to 23 ppm, had residues below the limit of quantification in muscle and skin-with-attached fat at 0-d withdrawal time. Residues in liver and kidney were below the MRL established by the Japan Ministry of Health, Labour and Welfare at 0-d withdrawal time. Withdrawal time is established at 0-d for flocks treated with enramycin at a therapeutic dose up to 23 ppm. Notes Primary Audience: Broiler Producers, Veterinarians, Nutritionists, Meat Processors. REFERENCES AND NOTES 1. Asai M. , Muroi M. , Sugita N. , Kawashima H. , Mizuno K. , Miyake A. . 1968 . Enduracidin, a new antibiotic II. isolation and characterization . J. Antibiot. 21 : 138 – 146 . Google Scholar Crossref Search ADS PubMed 2. Tsuchiya K. , Kondo M. , Oishi T. , Yamazaki T. . 1968 . Enduracidin, a new antibiotic. III in vitro and in vivo antimicrobial activity . J. Antibiot. 21 : 147 – 153 . Google Scholar Crossref Search ADS PubMed 3. Kawakami M. , Nagai Y. , Fujii T. , Mitsuhashi S. . 1971 . Anti-microbial activities of enduracidin (enramycin) in vitro and in vivo . J. Antibiot. 24 : 583 – 586 . Google Scholar Crossref Search ADS PubMed 4. Higashide E. , Hatano K. , Shibata M. , Nakazawa K. . 1968 . Enduracidin, a new antibiotic I. Streptomyces fungicidicus no. B5477, an enduracidin producing organism . J. Antibiot. 21 : 126 – 137 . Google Scholar Crossref Search ADS PubMed 5. Enramycin A CID = 16132292. National Center for Biotechnology Information. PubChem Compound Database: PubChem Open Chemistry Database . 6. Enramycin CID = 56842192. National Center for Biotechnology Information: PubChem Compound Database . 7. Benno Y. , Endo K. , Mitsuoka T. . 1988 . Isolation of fecal Clostridium perfringens from broiler chickens and their susceptibility to eight antimicrobial agents for growth promotion . Japanese J. Vet. Sci. 50 : 832 – 834 . Google Scholar Crossref Search ADS 8. Songer J. G. 1996 . Clostridial enteric diseases of domestic animals . Clin. Microbiol. Rev. 9 : 216 – 234 . Google Scholar Crossref Search ADS PubMed 9. Opengart K. A , Songer J. G . 2013 . Necrotic enteritis . Pages. 949 – 953 in Diseases of Poultry . Glisson J. R. , Swayne D. E , McDougald L. R. , Nolan L. K. , Suarez D. L. , Nair V. , eds. Wiley-Blackwell Publishing : Ames, IA . 10. MSD . Enradin Product Details. Accessed 20 September 2017. http://www.msd-animal-health.co.in/products/enradin/020_product_details.aspx . 11. MSD . Enradin F80 (Enduracidin): 30-Day feed adminsitration target animal safety study in growing broilers . [Internal Report S14111-00; 9/14/2015] . 12. Cobb . 2015 . Broiler performance and nutrition supplement , in Cobb-Vantress : Cobb-Vantress.com . © 2018 Poultry Science Association Inc. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 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Journal of Applied Poultry Research – Oxford University Press
Published: Dec 1, 2018
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