Depletion of Intramuscularly and Subcutaneously InjectedProcaine Penicillin G from Tissues and Plasma of

Yearling Beef Steers

Gary 0. Korsrud, Joe 0. Boison, Mark G. Papich, William D.G. Yates, James D. MacNeil, Eugene D. Janzen,Roger D.H. Cohen, Donald A. Landry, Gerard Lambert, Man Sen Yong and Jacques R. Messier

ABSTRACT

Withdrawal periods requiredwhen doses of 24,000 IU and66,000 IU of procaine penicillinG/kg body weight were adminis-tered to yearling beef steers byintramuscular injection daily forfive consecutive days were investi-gated. These dosages are in excessof product label recommendations,but are in the range of procainepenicillin G dosages that have beenadministered for the treatment ofsome feedlot bacterial diseases. Theapproved dose in Canada is7,500 IU/kg body weight intramus-cularly, once daily, with a with-drawal period of five days. Based onthe tissue residue data from thisstudy, the appropriate withdrawalperiod is ten days for the24,000 IU/kg body weight dose and21 days for the 66,000 IU/kg bodyweight dose when administeredintramuscularly to yearling beefsteers.

In a related study, 18 yearlingbeef steers received 66,000 IU ofprocaine penicillin G/kg bodyweight administered by subcuta-neous injection, an extra-labeltreatment in terms of both dose androute of administration, typical ofcurrent practice in some circum-stances. Deposits of the drug werevisible at subcutaneous injectionsites up to ten days after injection,with more inflammation and hem-orrhage observed than for intra-muscular injections of the samedose. These results suggest that pro-

caine penicillin G should not beadministered subcutaneously athigh doses; and therefore a with-drawal period was not establishedfor subcutaneous injection.

RESUME

Les temps de retrait requislorsque des doses de 24 000 et66 000 UL/kg de penicilline G pro-caine (PGP) ont ete injectees parvoie intramusculaire (im) une foispar jour (SID) pendant cinq joursconsecutifs a des bouvillons ont etemesures. Ces doses de PGP,quoique plus hautes que les dosesrecommandees par le fabricant,sont quand meme administrees. Ladose approuvee au Canada estpresentement de 7 500 UI/kg, im,SID, et comporte une periode deretrait de cinq jours. Les resultatsde cette etude demontrent que si desdoses de 24 000 et 66 000 UT/kg sontutilisees en im, les periodes deretrait devraient etre respective-ment de 10 et 21 jours. Dans uneautre etude, 18 bouvillons ont requla dose de 66 000 UT/kg de PGP SIDpar voie sous-cutane'e (SC), ce quirepresente encore une voie et unedose d'administration du produitqui sont non approuvees auCanada. Jusqu'a 10 jours suivantl'injection SC, des depots aux sitesd'injection ont ete observes, avecdes lesions hemorragiques etinflammatoires plus severes quelors d'injection de la meme dose parvoie im. Ces re'sultats demontrent

que la PGP ne devrait pas etreadministree par voie sc a de fortesdoses et que le temps de retrait pourcette voie et dose d'injection n'a puetre etabli. (Traduit par Dr PascalDubreuil)

INTRODUCTION

Penicillin G (benzylpenicillin) isused frequently by injection in rela-tively large dosages for the treatmentof sick animals and in lower dosagesto prevent disease in susceptible ani-mals. Subtherapeutic levels of peni-cillin also may be added to animalfeeds to prevent or reduce disease andto improve feed efficiency. It hasbeen reported that only limited dataon benzylpenicillin residues in meatwere available and that no tissuedepletion studies were available forreview (1). In Canada, penicillin isthe antibiotic most frequently foundin samples subjected to confirmatorylaboratory testing (2). The approveddose in Canada is 7,500 IU/kg bodyweight intramuscularly once daily andthe recommended withdrawal periodis five days (3). A maximum residuelimit (MRL) of 50 pg penicillin G/kgwas established for edible tissues ofcattle by- Health and Welfare Canada(4).

Several explanations have beenproposed for the presence of peni-cillin G residues in animal tissues,including the use of extra-labeldosages (5) and failure to observeadequate withdrawal periods prior toslaughter (6,7). It is recognized that

Health of Animals Laboratory, Agriculture Canada, 116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3 (Korsrud, Boison, Yates, MacNeil),Western College of Veterinary Medicine (Papich, Janzen) and College of Agriculture (Cohen), University of Saskatchewan, Saskatoon, SaskatchewanS7N OWO and Bureau of Veterinary Drugs, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario K1A OL2 (Landry, Lambert, Yong,Messier). Present address of Dr. M.G. Papich: College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh,North Carolina 27606. Present address of Dr. W.D.G. Yates: Animal Diseases Research Institute, P.O. Box 640, Lethbridge, Alberta T1J 3Z4.

Funded by the Bureau of Veterinary Drugs, Health Protection Branch, Health and Welfare Canada.Submitted November 27, 1992.

Can J Vet Res 1993; 57: 223-230 223

extra-label dosages have been com-monly used in veterinary practice (8),yet there is little published informa-tion on the withdrawal periodsrequired for animals treated withextra-label dosages of penicillin (5).Penicillin G residues in meat are ofparticular concern because smallquantities that may be present in ani-mal-derived foods from treated ani-mals can pose a risk to penicillin-sensitized individuals.Mercer et al and English (9,10)

suggested that subcutaneous injec-tions of penicillin G in food-produc-ing animals might have advantagesover intramuscular injections to over-come the problem of drug residues inmuscle tissues at the injection site.The dosage per kg body weight thatMercer et al (9) injected was 4,400 IUprocaine penicillin G, 4,400 IU ben-zathine penicillin G and 7.3 mg dihy-drostreptomycin. Some practitionersprefer the subcutaneous route over theintramuscular route when giving largedoses because the injections are easierto give and cause less evidence of dis-comfort at and after the time of injec-tion. It has been reported that chang-ing the route of administration fromintramuscular to subcutaneous did notsignificantly change the concentrationof penicillin G found in the serum ofcalves (9), and the same was true forcats and dogs (1 1). However, Krainock(12) reported prolonged residues inmilk when two cows were injectedsubcutaneously with procaine peni-cillin G. Subcutaneous injection ofprocaine penicillin G in cattle is anextra-label use in Canada for whichwithdrawal times are not available.

In a preliminary study, the disposi-tion of penicillin G in plasma follow-ing intramuscular and subcutaneousinjection of doses of 24,000 and66,000 IU procaine penicillin G wasexamined (13). One objective of thecurrent study was to use a newly devel-oped liquid chromatographic assay(14) to investigate the persistence ofpenicillin G residues in yearling beefsteers after injection with extra-labeldosages of procaine penicillin G. Thesteers were injected with 24,000 or66,000 IU per kg body weight of pro-caine penicillin G intramuscularly inorder to generate tissue depletion datawhich would permit the determinationof appropriate withdrawal times for

these higher than label dosages. Otherobjectives were to determine if subcu-taneous would be preferred to intra-muscular injections at a dose of66,000 IU procaine penicillin G per kgbody weight, and to establish an appro-priate withdrawal time for that doseand route of administration.

MATERIALS AND METHODS

Experiments followed the guide-lines prepared by the CanadianCouncil on Animal Care (15).

EXPERIMENTAL ANIMALS

Sixty-five healthy crossbred year-ling beef steers weighing an averageof 485 kg (range 371 to 628 kg) wereobtained from the Animal and PoultryScience Department, University ofSaskatchewan, and were kept in pensat the University of SaskatchewanBeef Research Centre for the durationof the study. The steers were fedrations of silage, ground hay, barleygrain, mineral supplement and water.They had not been given an antimi-crobial agent for at least 60 days priorto use in this study. Each steer's bodytemperature was less than 40°C justprior to the first injection. The aver-age body temperature was 39.2°C(range 38.3 to 39.9°C).

DRUG ADMINISTRATION

Ethacilin, a sterile suspension ofprocaine penicillin G, 300,000 IU/mL(rogar/STB, Pointe Claire-Dorval,Quebec) was used at dosage rates of24,000 IU and 66,000 IU/kg bodyweight/day for five consecutive days(3.2 and 8.8 times the label recom-mended daily dosage rate). Each24,000 IU/kg body weight dosage wasadministered as a single intramuscularinjection of 30 to 48 mL with a16 gauge 3.8 cm needle, while due tothe large volume, the 66,000 IU/kgbody weight dosages each requiredthree injections of equal volumes (28to 46 mL) made into the same injectionarea at a given time in a triangular pat-tern with the injections 6-8 cm apart.Five consecutive daily intramuscularinjections were given: the injectionsites were the right lower biceps/semi-membranosus/semi-tendinosus (ham-string) area, left neck, right neck, rightupper biceps/semimembranosus/semi-

tendinosus (hamstring) area, and rightgluteal, with one day's injections beinggiven at each site. The subcutaneousinjection sites were the left neck, cau-dal to the left shoulder, caudal to theright shoulder, the right shoulder, andthe right neck.

EXPERIMENTAL DESIGN

Three steers served as untreatedcontrols for Experiments 1 to 4.

Experiment I - To determine theappropriate withdrawal period forintramuscular injections of 24,000 IUof procaine penicillin G suspensionper kg body weight, 18 steers weretreated with five consecutive dailyinjections and then divided into sixgroups of three animals each, whichwere slaughtered at each of days 1, 2,3, 4, 8 and 12, respectively, after thelast injection.

Experiment 2 - To determine theappropriate withdrawal period forintramuscular injections of 66,000 IUof procaine penicillin G suspensionper kg body weight, 26 steers weretreated with five consecutive dailyinjections and then divided into sixgroups of three animals each and twogroups of four animals each. One ofthe groups of three steers was slaugh-tered at each of days 1, 2, 3, 4, 8 and12 after the last injection. The twogroups of four steers were slaughtered10 and 16 days after the last injection.

Experiment 3 - To determine theappropriate withdrawal period forsubcutaneous injections of 66,000 IUof procaine penicillin G suspensionper kg body weight, 12 steers weregiven five consecutive daily injec-tions. Each day, three injections ofequal volume (32 to 38 mL) weremade into the same general injectionarea in a triangular pattern with theinjections 6-8 cm apart. The steerswere divided into four groups of threesteers each, which were slaughteredone, two, three and four days, respec-tively, after the last injection.

Experiment 4 - To determine howlong penicillin G residues remain atsubcutaneous injection sites, sixsteers, weighing an average of 490 kg(range 441 to 527 kg) were injectedsubcutaneously with 66,000 IU pro-caine penicillin G/kg body weight inthree injections of equal volume

224

(32 to 39 mL) made into the samegeneral injection area in a triangularpattern with the injections 6-8 cmapart. Three of the steers wereinjected in their left sides 15 daysprior to slaughter and in their rightsides 10 days (i.e. five days later)prior to slaughter. Each steer wasinjected in two areas on each side ateach day. The injection sites were theneck and caudal to the shoulder. Thesecond group of three steers wasinjected with procaine penicillin G intheir left sides 30 days before slaugh-ter and in their right sides 20 days(i.e. ten days later) before slaughter.

SAMPLES TAKEN FOR ANALYSIS

All steers were slaughtered at acommercial abattoir in Saskatoon,where samples of gluteal muscle,diaphragm, liver and kidney, eachweighing approximately 400 g, werecollected from each steer. Each samplewas divided into three equal portionswhich were stored at -20°C for up toseven days prior to analysis. The injec-tion sites (intramuscular weighed 1 to3 kg each and subcutaneous weighed0.5 to 2.0 kg each) were collected fromthe hanging carcasses after the hidehad been removed. Only the glutealintramuscular injection sites were ana-lyzed for penicillin G. The relativelylarge volume of tissue was taken toensure that the actual injection site wasnot missed, with the quantity of tissuecollected being dependent on the con-fidence with which the injection sitecould be identified at slaughter. Formore obvious sites, less tissue wasrequired to ensure that the completesite had been excised from the car-cass. Injection sites were ground in ameat grinder prior to sampling foranalysis. Twenty mL of blood werecollected into heparinized tubes atslaughter, and were centrifuged in thelaboratory at 4,500 x g for 10 min-utes to collect the plasma, which wasstored at -20°C up to seven daysprior to analysis. Penicillin residueshave previously been demonstrated tobe relatively stable in tissue andplasma at -20°C for such storagetimes (16).

ANALYSIS OF RESIDUES

High performance liquid chromato-graphic analyses were conducted foreach sample in triplicate using the

procedures described by Boison et al(14,17). Pure standards and fortifiedrecovery samples were included ineach analytical run.

EVALUATION OF LESIONS ATINJECTION SITES

Carcasses were examined at theabattoir at the time of slaughter.Injection sites, including overlyingsubcutaneous tissues, were removedfrom the carcasses and examined atthe laboratory for gross lesions,before being ground and analyzed forpenicillin residues.

RESIDUE DEPLETION ANALYSIS

The method of least squares wasused to estimate the linear regressionequation of concentrations of peni-cillin G residues (natural logarithm)versus time for the different tissues(18). The assumption of equal vari-ance over time was tested by using theBartlett's test at the 5% level. The lin-earity assumption was verified by astandard analysis of variance and thetest for departure from linearityshould be nonsignificant at the 5%level. The upper tolerance limit ofresidue concentration was calculatedfor each tissue using the 99th per-centile of the population and the 95%confidence level (19,20). The with-drawal period was determined whenthe upper tolerance limit was at orbelow the MRL of 50 jig penicillinG/kg of tissue, for the tissue with thelongest depletion time. Values thatwere below the detection limit wereexcluded from the calculationsbecause they would bias the estima-tion of error.

RESULTS

The coefficients of variation (theSD expressed as a percent of themean) ± SEM for triplicate analysesof kidney, liver, muscle and plasma,respectively, were 11.2 ± 2.6, 7.7 ±1.6, 7.5 ± 1.3 and 6.9 ± 1.6.

EXPERIMENT 1

Penicillin G was not detected in anyof the samples from the untreatedcontrol steers. The results fromtreated steers are presented in Tables Ito III. Table I shows the depletion ofpenicillin G residues in kidney, liver,

left gluteal muscle, diaphragm andplasma over a time period of 12 daysfollowing the intramuscular adminis-tration of 24,000 IU/kg body weight,once daily, for five consecutive days.Penicillin G was detected in muscleup to two days and in plasma for threedays after the last injection. Kidneyand liver had the highest concentra-tions of residues at days 1 and 2 ofwithdrawal, as compared to those ofleft gluteal muscle and diaphragm.The rate of residue depletion wasslowest in the kidney (days 1-3)although a wide variation among indi-vidual animals was noted. Individualvalues of penicillin G concentrationsfound in the kidney were used to cal-culate the upper tolerance limit for thedetermination of the withdrawalperiod (Fig. 1). Penicillin G was notdetected in one kidney after threedays of withdrawal. The time periodneeded for the residue of penicillin Gto be lower than the established maxi-mum residue limit of 50 jig/kg wasestimated to be 8.5 days in the kidney.The upper tolerance limits calculatedbased on these results reflect the largevariation in the residue concentra-tions. Analysis of the right glutealmuscle injection sites demonstratedthat at day 4 of withdrawal, penicillinG residues were below the detectionlimit in two of three animals while thethird animal had a residue level of62 jig/kg (Table III). The injectionsites for days 8 and 12 of withdrawalwere below the detection limit of5 jig/kg.

In the steers injected intramuscu-larly with 24,000 IU of procaine peni-cillin G suspension per kg bodyweight, hemorrhage, up to 3 x 6 cm,on the subcutaneous surface of themuscle was observed over all upperhamstring injection sites from thesteers slaughtered two or four daysafter the last injection, and over oneof three upper hamstring injectionsites from the steers slaughtered eightdays after the last injection. Drug wasobserved within the muscle of one ofthe upper hamstring injection sitesfrom a steer slaughtered two daysafter the last injection. Hemorrhagewithin the muscle at the needle depthwas observed at all upper hamstringinjection sites from the steers slaugh-tered four days after the last injection.Neck, gluteal and lower hamstring

225

TABLE I. Body weight and penicillin G levels in tissues and plasma from yearling steersinjected intramuscularly with 24,000 IU procaine penicillin G/kg body weight once daily forfive consecutive days (Experiment 1, three steers per group)

Days Body Penicillin G (pg/kg or L)aof weight Left

withdrawal (kg) Kidney Liver glutealb Diaphragm Plasma1 444 ± 8 1,100 ± 620 2,000 ± 280 32 ± 20 41 ± 2 430 ± 942 494±8 650±385 350±244 59±30 27± 17 210± 1243 582 ± 10 34 ± 24 21 ± 18 <5 <5 17 ± 84 421 ± 31 22 ± 3 21 ± 12 <5 <5 <58 472 ± 36 <5 <5 <5 <5 <512 439 ± 42 <5 <5 <5 <5 <5

aDetermined by high performance liquid chromatographic analysis. Detection limit = 5 pg/kg or L.The values represent the mean ± SEM. Values below the detection limit were considered to beequal to zero for the calculation of meansbNot injected

TABLE II. Body weight and penicillin G levels in tissues and plasma from yearling steersinjected intramuscularly with 66,000 IU procaine penicillin G/kg once daily for fiveconsecutive days (Experiment 2, three steers per group, except days 10 and 16, where n = 4)

Days Body Penicillin G (pg/kg or L)3

of weight Leftwithdrawal (kg) Kidney Liver glutealP Diaphragm Plasma

1 463±2 2,800±490 2,300±410 75± 19 150±20 1.500± 1202 465 ± 13 1,000 ± 340 1,600 ± 290 44 ± 16 48 ± 5 310 ± 1543 498±11 240±110 370±177 9±9 5±5 82±224 451 ± 37 13 ± 8 46 ± 23 <5 <5 <58 466± 17 8±4 69±37 <5 <5 <510 550±4 9±5 26±11 <512 404± 16 <5 <5 <5 <5 <516 607±7 14± 14 12± 12 <5 v v

aDetermined by high performance liquid chromatographic analysis. Detection limit = 5 pg/kgL. Thevalues represent the mean ± SEM. Values below the detection limit were considered to be equal tozero for the calculation of meansbNot injected,Not analyzed

TABLE III. Penicillin G levels (pg/kg)a in gluteal injection sites from individual yearlingsteers injected intramuscularly with procaine penicillin G

Dose (lU/kg body weight)Daysof

withdrawal4

24,000(Experiment I)

<5<562<5<5<5

8

10

12

16

66,000(Experiment 2)

190<583<5<5<5

1,200

<5<5<5

<5<5440

aDetermined by high performance liquid chromatographic analysis. Detection limit = S pg/kg. Eachvalue represents the mean of triplicate determinationsbNo sample

injection sites were not examined atthe laboratory.

EXPERIMENT 2

In the steers treated with five con-secutive daily intramuscular injectionsof 66,000 IU of procaine penicillin Gsuspension per kg body weight. peni-cillin G was undetectable in muscletissue, other than at injection sites, fourdays after the last injection (Tables IIand III). The final samples to beobtained were taken from the foursteers that were slaughtered 16 daysafter the last injection. Residues ofpenicillin G were present in one ofthese four steers, in the musculature ofthe injection site, liver, and kidney.

Individual values of penicillin Gconcentrations found in the liver sam-ples collected at 1, 2. 3, 4. 8 and10 days after final treatment wereused to calculate the upper tolerancelimit for the determination of thewithdrawal period (Fig. 2). Penicil-lin G was not detected in one liver ateach of days 4 and 8 of withdrawal.Residue data at day 12 of withdrawalwere below the sensitivity of the ana-lytical method and the group was notincluded in the calculation of theupper tolerance limit. Consequentlythe subsequent day 16 group was alsoexcluded from the calculation.A Q test for outliers indicated that theliver value of 49 ppb (near the MRL),the kidney value of 54 ppb and themuscle level of 7 ppb were all outlierswith respect to the other three steersin the group. The Q value was equalto one for each tissue. The time periodrequired for the residue of peni-cillin G to be less than the establishedMRL of 50 gg/kg was estimated to be18 days in the liver. When the residuedata for kidney were analyzed at thesame withdrawal intervals as liver thecriteria for linear depletion were notmet. The 16 day data were left inTable II to demonstrate what can hap-pen and why the withdrawal time wasincreased from 18 days to 21 days.

In the steers injected intramuscu-larly with 66,000 IU of procaine peni-cillin G suspension per kg bodyweight, and slaughtered two daysafter the last injection, hemorrhageand drug deposits were present withinthe muscle, but were most prominentin the fascial planes between muscles.In the upper hamstring injection sites

226

EXPERIMENT 3

10 '

1

0 1 2 3 4 5 6 7 8 9

Time (Day)

Fig. 1. The projected depletion time for penicillin G residues in kidney from yearling sinjected intramuscularly with 24,000 IU procaine penicillin G/kg body weight once daillive consecutive days (Experiment 1).

105 -

104 -

103 -

102

10 -2

I1.

Upper tolerance limit

^..m...muuu xX Maximum residue limit

*~~~~~mo w ..... '.'.-W -

w

X\ ,E~~~~~~~~~~~~~C

0 2 4 6 8I I 6I

10 12 14 16 18

Time (Day)Fig. 2. The projected depletion time for penicillin G residues in liver from yearling steersinjected intramuscularly with 66,000 IU procaine penicillin G/kg body weight once daily forrive consecutive days (Experiment 2).

from the steers slaughtered ten daysafter the last injection, there wasnecrosis in the fascial planes betweenmuscles. Hemorrhage, up to 5 x 7 cm,on the subcutaneous surface of themuscle was observed over most of theupper hamstring injection sites at tendays and was still observed in three offour steers slaughtered 16 days afterthe last injection. There was a palefocus within the upper hamstringmuscle at the injection site in one of

the four steers slaughtered 16 daysafter the last injection. At the glutealinjection sites, subcutaneous hemor-rhage on the surface of the musclewas observed in two of four steers,16 days after injection, and one

gluteal injection site was comprisedof a fluid-filled, walled-off area thatcontained the drug, also 16 days afterinjection. Neck and lower hamstringinjection sites were not examined atthe laboratory.

Table IV shows the depletion ofpenicillin G residues in kidney, liver,left gluteal muscle, diaphragm andplasma over a time period of fourdays following the subcutaneousadministration of 66,000 IU/kg bodyweight, once daily, for five consecu-tive days. After four days of with-drawal, penicillin G was still detectedin the kidney, liver, diaphragm andplasma from steers injected subcuta-neously with 66,000 IU procainepenicillin G/kg body weight, the peni-cillin G levels were higher than whenthe same dose was given intramuscu-larly (Experiment 2). Penicillin G

10 concentrations in left gluteal muscleand diaphragm were less than theMRL of 50 ,g/kg body weight after

steers three days of withdrawal, butly for remained greater than the MRL in

kidney, liver and plasma samples atfour days posttreatment. There wassevere congestion, edema and hemor-rhage at the subcutaneous injectionsites (Figs. 3 and 4), with deposits ofthe drug visible at most of these sites.Frequently, the hemorrhagic inflam-matory reaction extended into thesubjacent muscular tissue. All ofthese injection sites were visible atslaughter and would be trimmed fromthe carcasses under normal inspectionprocedures.

loom EXPERIMENT 4

One subcutaneous injection sitecontained 3,600 jig/kg penicillin G tendays after injection (Table V). Twentydays after injection, penicillin G lev-

20 els in one injection site were still justabove the MRL.At ten days postinjection, hemor-

rhagic lesions were present in five ofsix injection sites; the same was trueat 15 days postinjection, although theinjection sites having no visiblelesions (one at 10 days and one at15 days) were not in the same animal.The lesions consisted of hemorrhagicfoci with evidence of necrosis, andwere present subcutaneously and/or infascial planes between muscles. All ofthese injection sites that containedpenicillin G were visible at slaughterand would be trimmed from the car-

casses under normal circumstances.By 20 days postinjection, lesions

were found in only two of six injection

227

105

104

103

102

I-I

a.

._

106 -

x

0-.

0.0.

CL

A-

16

I

Fig. 3. Subcutaneous injection sites on days 1 (neck), 2 (shoulder) and 3 (behind shoulder)after injection. The dose was 66,000 IU procaine penicillin G/kg body weight per day(Experiment 3).

J-- -I I Ii. f:,-A t?, .r t!-' '. 3l .740 M .4_ '

Fig. 4. Subcutaneous injection site five days after injection. The dose was 66,000 IU procainepenicillin G per kg body weight (Experiment 3).

sites. These consisted of a subcuta-neous hemorrhagic focus in one, and afocus of hemorrhagic inflammation ina fascial plane between muscles in theother. At 30 days postinjection, oneinjection site was not sampled, onestill had notable focal hemorrhagiclesions, one had no lesions and threehad detectable lesions that were heal-ing. The 20 and 30 day injection siteswere not visible at slaughter andwould not have been trimmed from thecarcasses under normal circumstances.A deposit of the drug was visible atone subcutaneous injection site tendays postinjection.

DISCUSSION

The recommended dosage rate forEthacilin (rogar/STB) in Canada, asstated on the label, is 7,500 IU of pro-caine penicillin G suspension per kgbody weight to be administered intra-muscularly once daily until one to twodays following the disappearance ofclinical signs (3). The withdrawalperiod is five days. The two dosagerates chosen for this study fall withinthe range of penicillin G dosages thatsome veterinarians and livestock pro-ducers administer in an extra-labelmanner in attempts to deliver the bestpossible therapeutic results fromtreatment.

There is little information availableregarding the withdrawal time requiredfor extra-label doses of procaine peni-cillin G when given to cattle. In theonly report that we found in the scien-tific literature on this topic, the authorsreported that no penicillin G residueswere detectable in injection sites orkidneys, after 30 days of withdrawal,from steers injected intramuscularlywith 33,000 IU of procaine penicillinG/kg body weight using a commercialpreparation that contained 200,000 IUof procaine penicillin G and 250 mg ofdihydrostreptomycin/mL (21). Ourstudy was designed to establish thewithdrawal time for procaine penicil-lin G when administered intramuscu-larly at 24,000 and at 66,000 IU/kgbody weight to yearling beef steers.The human safety assessment of

veterinary drugs intended for use infood-producing animals entails a six-step procedure as outlined below(22,23):

228

TABLE IV. Body weight and penicillin G levels in tissues and plasma from yearling steersinjected subcutaneously with 66,000 IU procaine penicillin G/kg body weight once daily forfive consecutive days (Experiment 3, three steers per group)

Days Body Penicillin G (pg/kg or L)aof weight Left

withdrawal (kg) Kidney Liver gluteal Diaphragm Plasma1 469 ± 23 1,600 ± 200 4,700 ± 430 102 ± 5 290 ± 18 2,400 ± 2502 466 ± 6 830 ± 176 2,500 ± 710 54 ± 4 67 ± 24 1,300 ± 2103 463±9 500±241 880±336 14±4 7±4 150±284 472±9 390±98 480±95 <5 13±3 380± 160

aDetermined by high performance liquid chromatographic analysis. Detection limit = 5 ,ug/kgLor L. The values represent the mean ± SEM

1. Metabolism studies in the targetspecies.

2. Comparative metabolism studiesin the laboratory test species.

3. Toxicity/carcinogenicity testing todetermine a safe level or accept-able level of risk for residues.

4. Determination of the safe concen-tration level for total residuesresulting from administration ofthe drug, the target tissue for resi-due analysis, the marker residue(the parent drug or a metabolite)and the maximum residue limit forthe marker residue.

5. Development of an analyticalassay for the marker residue at therequired level of measurement.

6. Establishment of a withdrawalperiod required for the safe use ofthe drug.

Unlike a new chemical entity, pro-caine penicillin G has been used infood producing animals for a substan-tial period of time before the aboverequirements were instituted. Many ofthe older drugs, including penicil-lin G, have been regulated on thebasis of the sensitivity of the assaymethod. The present Canadian maxi-mum residue limit of 50 pg/kg forpenicillin G in edible tissue of cattlewas established according to this prin-ciple and was not lowered with theadvent of more sensitive assay meth-ods because this level is consideredwithout significant risk for individu-als sensitized to penicillin G (1).The most relevant criteria for

assessing the human safety of peni-cillin G residues in edible tissues is itspotential for causing allergic reac-tions. The overall prevalence ofallergy to penicillin G was estimatedto be 3% to 10% in human popula-tions (1). To date there is no evidenceto suggest that sensitization to peni-cillin G could be induced from

residues in food, but previously sensi-tized individuals should not ingestmore than 30 jig of penicillin G perday (1).

It is of interest to note that the JointFAO/WHO Expert Committee onFood Additives (JECFA), in its recentassessment of penicillin G, recom-mended an acceptable daily intake(ADI) per person of 30 jig for thisantibiotic (1). The Canadian maxi-mum residue limit represents anintake of 25 jg of penicillin G basedon a daily consumption of 0.5 kg edi-ble meat products derived from cattle,which is below the ADI recommendedby the JECFA.

In the study reported herein, deple-tion of penicillin G residues in muscle,liver, kidney, plasma and injectionsites was examined through use of ahigh performance liquid chroma-tographic method with a detectionlimit of 5 jig/kg. The residue depletiondata were used to calculate the uppertolerance limit for each tissue. The kid-ney gave the longest depletion time forthe 24,000 lU/kg intramuscular dosagewhile the liver showed the longestdepletion time for the 66,000 IU/kgintramuscular dosage.A source of concern, in terms of

residues being present in meat pur-chased by a consumer, is the possibil-ity of entrapment of drug at the site ofan injection. Examples of this wereobserved in one injection site from the66,000 IU/kg intramuscular treatmentgroup collected at ten days after finaltreatment, and another collected at16 days posttreatment. The steers inthe 10 and 16 day groups were larger,mean weights of 550 and 607 kg,compared to a mean weight of 404 kgfor the steers in the 12 day group. Thelarger steers required larger volumesfor injection, 3 x 42 mL for the 10 and16 day steers, compared to 3 x 30 mL

TABLE V. Penicillin G levels (tg/kg)a insubcutaneous injection sitesb after 66,000 IUprocaine penicillin G/kg body weight/injection site was given (Experiment 4,values for individual steers, three steers for10 and 15 days and three different steers for20 and 30 days)

Injection Days after injectionsite 10 15 20 30

Neck <5 <5 15 9<5 <5 10 18460 28 18 10

Behind <5 <5 26 9shoulder <5 <5 51

3,600 <5 10 10

-Determined by high performance liquid chro-matrographic analysis. Detection limit =5 ,ug/kg. Each value represents the mean fortriplicate determinationshSubcutaneous injections in the neck and caudalto the front shoulder were given on the sameday,Sample not analyzed

for the smaller 12 day steers. Thepenicillin G residues found in theinjection sites of three of the 10 dayand one of the 16 day steers, canlikely be attributed to the larger vol-umes injected. When the injectionvolume is larger (42 mL for the 10and 16 day steers), there is a greaterchance for some of the injected peni-cillin to be deposited between mus-cles and/or subcutaneously, but evensmall injections (2 and 5 mL) can beintermuscular as demonstrated bystudies in dogs (24).Two cases of drug entrapment were

encountered in the present study.Such injection sites, particularly whenthe residue of antibiotic is deep withinthe musculature, may be difficult todiscover on routine postmortemexamination. As well, prior to slaugh-ter, the animal may be free of observ-able clinical signs that would suggesta recent history of antibiotic adminis-tration. In this context, our opinion isthat volumes injected per site shouldnot exceed 30 mL, because highervolumes may result in drug retentionat the injection sites for longerperiods.Based on the tissue residue deple-

tion data reported herein and conser-vative measures to increase the safetymargin for consumers with regard tothe injection sites, it was establishedby Health and Welfare Canada thatthe appropriate withdrawal period forprocaine penicillin G is ten days forthe 24,000 IU/kg body weight dose

229

and 21 days for the 66,000 IU/kg bodyweight dose when administered intra-muscularly to yearling beef steers.

English (10) indicated that it mightbe preferable in beef cattle to adminis-ter benzathine penicillin G subcuta-neously rather than intramuscularly todecrease muscle damage caused by alocal reaction at the site of injection.Mercer et al (9) proposed but did notvalidate that subcutaneous administra-tion could be used in order to decreasethe retention of drug residue at theinjection site. On the contrary, ourresults with high dosage levels of pro-caine penicillin G suggest that subcu-taneous injections can cause more tis-sue damage at the injection site thanequivalent intramuscular injections.Penicillin G concentrations in kidneyand liver, after four days of with-drawal, were considerably higher aftersubcutaneous injections than afterintramuscular injections. The variabil-ity in penicillin G concentrationsobserved ten days after injection at thedifferent injection sites is attributed todifferences in retention, rather than tovariability in sampling. If procainepenicillin G is injected subcuta-neously, the probability that an injec-tion site will be overlooked by inspec-tors at slaughter, up to 15 days afterinjection, is reduced because of thevisible tissue reaction. However,based on our results, we recommendagainst the use of subcutaneous injec-tion of procaine penicillin G, particu-larly at high dosage levels such asthose used in this study; and thereforea withdrawal period has not beenestablished for subcutaneous injection.

ACKNOWLEDGMENTS

We thank Dr. F. Schumann for assis-tance with injections and sample col-lection. We gratefully acknowledge theskillful technical assistance of ShawnaGobeil, Lily Keng, Brad Petford, KeithPinsonneault and Dwayne Stonechild.

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The cooperation of Drs. G. Grahamand A. Klemmer, and other meatinspection staff at the packing plant, isgratefully acknowledged.

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