HAL Id: hal-00901335https://hal.archives-ouvertes.fr/hal-00901335

Submitted on 1 Jan 1981

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

BOUND RESIDUES OF VETERINARY DRUGS :BIOAVAILABILITY AND TOXICOLOGICAL

IMPLICATIONSViviane Burgat-Sacaze, P. Delatour, A. Rico

To cite this version:Viviane Burgat-Sacaze, P. Delatour, A. Rico. BOUND RESIDUES OF VETERINARY DRUGS :BIOAVAILABILITY AND TOXICOLOGICAL IMPLICATIONS. Annales de Recherches Vétéri-naires, INRA Editions, 1981, 12 (3), pp.277-289. �hal-00901335�

BOUND RESIDUES OF VETERINARY DRUGS :BIOAVAILABILITY AND TOXICOLOGICAL IMPLICATIONS

Viviane BURGAT-SACAZE1 P. DELATOUR2 A. RICO

1 Laboratoire de Pharmacie- Toxicologie, Eco% Nationale Vétérinaire, 31076 Toulouse Cedex, France.2 Laboratoire de Biochimie, Eco% Nationale Vétérinaire de Lyon, 69260 Charbonnières-les-Bains, France.

3 Laboratoire de Radioéléments et d’Etudes Métaboliques, Eco% Nationale Vétérinaire,31076 Toulouse Cedex, France.

Résumé

RÉSIDUS LIÉS DES MÉDICAMENTS VÉTÉRINAIRES : BIODISPONIBILITÉ ET SIGNIFICATIONTOXICOLOGIQUE. ― Les auteurs envisagent la nature et la toxicité des résidus de médicamentsvétérinaires. Le problème des résidus liés par covalence est particulièrement étudié sous troisaspects concernant : 1) Les problèmes analytiques, qui opposent les résidus solubles, rapidementéliminés des tissus, aux résidus liés nettement plus rémanents ; 2) leur biodisponibilité, qui est diffé-rente ; 3) leur signification toxicologique et les conséquences de ces faits sur l’évaluation des nor-mes de sécurité des denrées alimentaires.

One of the main objects of the French law ofMay 29 th, 1975 on veterinary pharmacy is toprotect the consumers against possible harm-ful residues of veterinary drugs in food pro-ducts of animal origin. To this end, the lawprovides for a « withdrawal period », or inter-val, to be observed between administration ofdrugs and « consumption » of food productsfrom treated animals. The « withdrawalperiod » has been discussed recently in litera-ture (Milhaud, 1978 ; Burgat-Sacaze and Rico,1980) and is schematized in figure 1 ; it is cal-culated from metabolic (nature, pharmacoki-netics and bioavailability of residues) and toxi-cological data. In practice, one of two lines ofaction may be possible :

- to set a « withdrawal period » of suchduration that residues are no longer measura-ble,- to demonstrate that existing residues arenot harmful ; this is justifiable on scientific,toxicological, public health and economic

grounds, but calls for evaluation of the natureof residues and of their toxicity.The problem is beset with some difficulties

which we intend to tackle. Far from using thisas an excuse to discard the protocol nowadopted by many countries, we make it a pre-text for studying the problem and for workingtowards its solution, as do other organisationsabroad, such as FDA (Cordle, 1978 ; Weber,1978).

Nature of residues

1 - The problem

1.1. - Definition of residues

« The term residues is used to describe allactive principles and their metabolites whichpersist in meats or other food products fromanimals which have been treated with the drugin question » (Arrete July 4th, 1977, JournalOfficiel of July 20th, 1977).The term metabolite has not been defined. It

is generally accepted that it applies to « anyby-product of biotransformation of the initialactive principle ».

However, it is difficult to assess the « fate »

of the parent compound within a living orga-nism. In spite of progress achieved, it must be

admitted that most present rules have been

drawn without prior knowledge of the« exact » nature of residues. Let us considerthe example of antibiotics which are detectedand measured by bacteriological techniques.Thus, for benzyl penicillin, we know only thefate of the initial antimicrobial compound, andvery few authors have shown interest in per-sistence of truly toxic, allergenic residues,which are in fact metabolites. This problemhas very deservedly been raised by Wal (1979).- Should we therefore give priority to

determining exact nature of residues ? In

many cases this appears to be both impossibleand unnecessary.- The problem is well known since regula-

tions require that the « principal metabolites »should be identified. But quantitative selectionof « major » over « minor » metabolites is nowdevoid of any rational biologic foundation.(Weiner and Newberne, 1977). In fact, many

results show that the metabolite responsiblefor a toxic effect is often present in very smallquantities. (This will be discussed in detail inthe second part of this paper).- Therefore, attention has been paid to

« relay toxicity » studies (Ferrando and Tru-haut, 1972), but limitations of the method,fundamental as well as practical, will causenew protocols to prevail (Cordle, 19781.

1.2. - Residual fractions

In this view, the first purpose of the studyshould be to determine residual fractions. In

fact, detailed experiments on metabolism ofcompounds has shown that three types ofresidues rapidly appear :a) Totalresidues, determined by overall quan-titative assessment of residual radioactivityafter administration of the labelled compound.This value is often used, but has the majordisadvantage of expressing a non-specificradioactivity as « equivalents » of the onlyknown form, i.e. the parent molecule. In thisway, residues, the nature of which is unknownor different from that of the initial molecule,are sometimes given an ADI (acceptable dailyintake) which is relative to the initial com-

pound. This practice is very often disadvanta-geous. In fact, total residues can easily be splitinto :b) Extractable residues, namely a fraction

which, as the name implies, can be extractedfrom biological tissues or fluids using varioussolvents (water at varying pH, organic sol-vents) before and after denaturation of macro-molecules.

This fraction includes all compounds, i.e.the parent compound and the metabolites, infree form or loosely bound to tissues.c) Non-extractable or bound residues, namelythe fraction of radioactivity which persists in

tissue extracts after the above-mentionedtreatments.

The nature of these residues can be determi-ned only after almost complete breakage, par-ticularly of proteins (enzymatic or acid

hydrolysis, for instance) ; it may, or may not,be related to that of the initial molecule.

In fact, this fraction is found to contain :

1 - « endogenous compounds », natural,resulting from incorporation of the degrada-tion products of the parent compound at theintermediate stage of metabolism (aminoacids, for instance).2 ― « new compounds » resulting fromcovalent binding of the parent drug or meta-bolites to endogenous macromolecules.

This distinction is all the more valuablesince :- Extractable residues are early residues.This fraction is the largest in the first days afteradministration of the drug, and nature of these

residues is very often closely related to that ofthe initial compound.- Bound residues, on the other hand, makeup the larger fraction of late residues, as

shown by a radioactivity which lasts for seve-ral weeks and even two months. Their bioavai-lability and toxicity often differ from those ofthe parent compound.These general notions are corroborated by

results from several experiments.

2 ― Concrete examples

2.1. ― Mebendazole

Our study of the metabolism of 14C-mebendazole after oral administration in lambsshowed poor absorption and low tissue con-centrations, except in the liver. Tissue totalresidue concentrations fell rapidly. At day 15and after, radioactivity persisted only in theliver ; expressed as mebendazole equivalent, it

amounted to 1.07 ppm, i.e. 0.13 % of thedose (table 11.

Residue levels were determined at varioustimes after dosing by measuring total radioac-tivity and radioactivity of each fraction (liquidscintillation). Mebendazole and four of its

major urinary metabolites (fig. 2) were estima-ted by thin-layer chromatography (and autora-diography) and high performance liquid chro-matography.Treatment of liver homogenates (fig. 3),

separated three different residual fractions :- fraction A : which contained mebendazoleand its metabolites (known and unknown)soluble in acetonitrile.- fraction B : contained radioactive polarconstituents, soluble in neutral, acid, or alka-line water.- fraction C : non-extractable residue,bound to proteins, even denaturated (aftertreatment with urea or by heat).

Mebendazole and its main metabolites wereidentified only in fraction A which accountedfor only 13 % of total radioactivity at 48 h afterdosing. At day 10, 98 % of this radioactivitywas no longer directly due to mebendazole(table 2).More detailed studies of the exact nature of

free residues B and bound residues C are in

progress, but we already know that acid

hydrolysis of fraction C does not liberatemebendazole-related compounds. At the pre-sent experimental stage, it is pertinent to askwhether we are justified in drawing up analyti-cal protocols which may later on prove toocomplicated. In fact, a number of research

teams carried out such studies, which wereprotracted and have yielded doubtful results,e.g. with cambendazole.

2.2. ― Cambendazole

Baer et a/. (1977) studied residues of 14C-cambendazole in calves, in which residual

radioactivity can be detected 70 days after sin-gle injection. At day 30, liver residual activityamounted to 0.9 ppm cambendazole equiva-lent ; hydrolysis with ficine released 54 % ofbutanol-extractable material.

Exact nature of bound residues could not be

determined ; 83 % were recovered in the pro-tein fraction. Benzimidazole nucleus is presentin tissues of drug-treated animals ; the princi-pal binding site is almost surely through inter-action of thiazole moiety with endogenouscompounds. Mechanism and nature of thebound remains unknown, as well as toxicolo-gical assessment of this non-extractable frac-tion.

The problem is complicated further by thefact that, as a result of changes in the labellingof the benzene ring, residual radioactivity isdetected at day 30 in glycogen, nucleic acids,lipids and all protein fractions (as 14C-glutamicacid) (Rosenblum et al., 1971). ).

3 ― Nature of bound residues

This shows the complexity of the exactnature of residues, particularly that of boundresidues. General theories cannot yet be basedupon the few available results but they pointout the important problem of non-drug relatedresidue.

3.1. ― Incorporation into endogenous meta-bolism

3.1.1. ― DichlorvosA simple model is provided by dichlorvos

(fig. 4). When (14 C-vinyl)-dichlorvos is orallyadministered to rats, the compound is rapidlymetabolised. At day 4, 40 % is eliminated in

expired air (C02) and 12 % in urine (hippuricacid and urea). At day 5, 75 % of liver radioac-tivity is non extractable material ; 14C is incor-porated in endogenous compounds such asglycocol, serine and cysteine (derived fromC02) and thus in proteins (Hutson et al.,1971 ).Such models can be very complex, e.g.,

nihydrazone (fig. 5).The same type of incorporation has been

demonstrated in the case of other veterinarydrugs.3.1.2. - ParbendazoleBound residues found in liver of sheep at

day 15 are due to the incorporation of &dquo;C intofive amino acids (Di Cuollo et aL, 1974).3.1.3. ― Thiabendazole

Late radioactivity present in the liver is alsofound in proteins (20 to 60 %) lipids (12 to16 %), nucleic acids (1 %) and glycogen(10 %) (Rosenblum, 1965 ; Rosenblum et al.,19711. ).

3.1.4. ― RonidazoleWhich breaks down giving acetate and very

persistent late residues (Rosenblum et al.,1972).

In fact, study of the metabolism of 14C-sodium acetate in mice (Skipper et al., 1949),rats (Rosenblum etal., 1971) and pigs (Rosen-blum, 1977) showed that at 60 days withdraw-al the residual radioactivity was equivalent to17 ppm acetate, and had a very long half-life(about 9-days), almost identical in all species.A similar half-life was recorded in calves afteradministration of thiabendazole.

In practice, the above instances demons-trate only the slow turnover of endogenousmacromolecules.

3.2 ― Formation of « non-natural » com-

poundsLess metabolized than in the previous ins-

tances, drugs or their metabolites can reactwith macromolecules through covalent boundformation.

This seems to happen partly for cambenda-zole and has been clearly demonstrated for theanthelmintic p-toluoyl-chlorophenyl hydra-zone (TCPH) (fig. 6). After oral treatment ofsheep, persistent blood residues (5-6 ppm)were observed for at least 21 days. The 14C

residues were largely localized in erythrocytesand covalently bound to both heme and glo-bin. Only the phenyl group of the phenylhy-drazine part of TCPH was present as 14Cbound residues (Jaglan et al., 1976, 1977).What is the toxicity of this phenyl-

haemoglobin for the consumer ?These few examples give some indication of

difficulties encountered in the study of theseproblems. Nature of residues quickly appearsto be heterogenous and complex ; that ofbound residues is also dependent on variousfactors associated with method used by inves-

tigator. For instance, it may vary accordingto :

- the way in which compounds are labelled.For TCPH methods I and II do not provide thesame kinetics of tissue depletion (table 31.- the way in which covalent complexes arebroken down, since a residue not extracted byone process may be unmarked or liberated byanother. As a general rule, the term a boundresidue » applies to a covalently bound conju-gate, which can be liberated by breakdown ofthe primary structure of proteins. But there isa need to bring general protocols into line and

to design suitable models which closely repro-duce biological conditions under which thosecomplexes are broken down (heat, digestivemedium, etc.)

In fact, the root of the problem is to eva-luate when to stop analysing. One can, ofcourse, always strive after better understan-ding of the nature of residues ; but one alsohas to consider relevance of this informationto the assessment of biological and toxicologi-cal importance of residues.

Bioavailability and toxicological impor-tance of residues

In the last few years, a new approach to theproblem of residues has been discussed in lite-rature.

The term « bioavailability » has appearedand studies are carried out to determine intes-tinal absorption of residues. Obviously,absorption varies from residue to residue. Thevalue of such studies, very rarely undertakenat present, is that they enable to show easilythat a non-absorbed residue is virtually nolonger a toxic residue.

Furthermore our knowledge of the intrinsicmechanism of action of toxic substances

increases rapidly, e.g. xenobiotic biotransfor-mations. Various metabolic pathways can leadto both toxic and non toxic compounds. The-refore, residues have a toxicological signifi-cance which varies widely.

1 - Bioavailability of residues

The veterinarian must look at bioavailabilityin two ways :- Bioavailability of the drug in animals,

which we will call primary. It complies with thefollowing general definition (FDA, 1973 ;OMS, 1974).« The bioavailability of a pharmaceutical formis the quantity of drug which reaches generalblood circulation and the speed at which it isreached ».

Any other interpretation, which mightinclude, for instance, the fate of a substanceafter this first pharmacokinetic stage, leads toerrors which regrettably are still to be found.- Bioavailability of residues, or secondary

bioavailability, is concerned with gastro-intestinal absorption of drug residues contai-ned in food products of animal origin. Cordle(1978) proposed this definition :« Bioavailable residues are parent drug or

metabolites which are absorbed by the gastro-intestinal tract and which may be found in thecells of the gastro-intestinal tract, tissues,fluids or expired CO, of the ingestingspecies ».

In fact, bioavailability of the components ofa residue varies from component to compo-nent. More particularly, there are marked dif-ferences between the free and the bound frac-tion. In vitro, residual radioactivity of thebound fraction can be eliminated only by dras-tic procedures ; it is almost obvious that its

bioavailability may be low, as it has beendemonstrated by few relay bioavailability stu-dies.

The study of cambendazole by Baer et al.(1977) included determination of the bioavaila-bility : -

- of the parent drug, alone or mixed with calfliver (condition similar to that of the consu-mer). In rat, about 50 % of the product wasabsorbed.- of total residues, by giving calf liver treatedwith cambendazole to rats. Only 15 % of thetotal radioactivity was absorbed. Thus, on thesame withdrawal day, the amount of residuesfound in the liver was one-thirtieth of thatfound in the previous case (table 41.

Studies by relay toxicity have confirmed

that, at « apparently » equal doses, calf liver isdevoid of the embryotoxic properties of cam-bendazole. Various residues are found and

only one fraction is toxic. In addition, theiroverall bioavailability is less than that of the

parent compound.These results do not preclude the possibility

of the in vivo formation of metabolites moretoxic than the parent compound e.g., alben-dazole, as will be discussed further on.

2. - Toxicological significance of residues.

All constituents of a residue are not equallytoxic. In fact, certain metabolites may bedevoid of the toxicity of the parent compoundwhereas others are in fact responsible for itstoxicity.

Undoubtedly, one has to consider andunderstand the part played by the animal

relay. There are several approaches and solu-tions to this problem, such as the coefficient ofsafety applied to the maximum tolerated dose,the study of the toxicity of the principal meta-bolites, relay toxicity studies, etc. At present,some of these are being called into questionagain, and it appears that these problemsshould be tackled differently : toxicologicalsignificance of biotransformations and bioa-vailability are too often neglected.

In this respect, it seems that free and earlyresidues have a toxicological importancewhich is quite different from that of bound andlate residues.

In fact, residues of the first group include

the initial compound and its « primary » meta-bolites ; toxicity of the initial compound is wellknown and certain metabolites may be more

dangerous than the parent compound. Apriori, this residual fraction is suspect, andcaution regarding early and free residues is

justified.On the other hand, late residues may occa-

sionally be considered as non-toxic. This is

normally the case when residual radioactivityis incorporated into normal endogenous pro-ducts. But what does happen when non-

natural macromolecular compounds are for-med ? This is shown by the two models givenhereafter as examples.

alThe covalent, so-called « irreversible »,

binding of toxic substances to endogenousproducts is currently much studied. It

accounts for many deleterious effects fromnecrosis to chemical carcinogenesis. Thesemechanisms are not easily demonstrated

since, in most cases, the initial compound

must be converted in the body to chemicallyreactive metabolite. The ultimate toxic meta-bolite is hardly ever isolated ; but it is indirectlysuspected by the demonstration mainly of acovalent binding to endogenous components,i.e. of bound residues.

Bromobenzene, for example, is an extensi-vely studied model hepatotoxin. Metabolicpathways now suggested are shown in figure 7.This compound can be converted, by the hepa-tic mixed function cytochrome P-450 oxyge-nase system, to epoxides. The 2,3-epoxide mayrearrange non enzymatically to form o-

bromophenol, the major urinary metabolites,which is not hepatotoxic. On the contrary, the3,4-epoxide is probably the ultimate toxicmetabolite that covalently binds to liver cells,leading to hepatic necrosis ; it is also conver-ted to non-toxic compounds : mercapturicacid (70 % ), 3,4-dihydrodiol, p.bromophenol.Bromobenzene is said to be prenecrogenic

and the 3,4-epoxide is the ultimate necrogenicagent. The other metabolites are devoid of

hepatotoxicity when they are administered

directly to animals.The ultimate necrogenic agent is an inter-

mediate which is highly reactive and hardlyever isolated from tissues. On the other hand,the covalent complex macromolecule is easilyquantified and its role can also be demonstra-ted directly by modifying biotransformations ;the higher the detoxication processes thelower the toxicity (e.g. by increase gluta-thione) ; on the contrary, their inhibitionincreases necrosis.

This example shows that the existence of amajor metabolic route does not justify itsimplication a priori in the causation of harmfuleffects ; on the contrary these effects are veryoften attributed to a « minor » route.

b) The anthelmintic Albendazo% (Martin,1980 ; Grannec, 1980 ; Gyurik et al., 19811, isembryotoxic in rats when the dosage exceeds6.00 mg/kg. Its bioavailability is 31 %.Among urinary metabolites identified in

mice, rats, sheep and cattle, only the sul-phoxide metabolite exhibits the same embryo-toxicity as its bioavailability is 73 %. In theory,two toxic compounds can be found in edibletissues. On the other hand, bioavailability ofbound residues is only 3 % (kidney) or 4.4 %(beef liver).

By relay toxicity, total residues are not

embryotoxic and a simple a posteriori calcula-tion shows that female rats have ingested adaily dose of potentially toxic metabolitesequal to one tenth the maximal non-effectivedose.

c) How can these models contribute to thetoxicological assessment of residues ?

1 - Toxicity studies of urinary metabolitescan lead to the discovery of activation anddetoxication routes but are beset with difficul-ties. All the metabolites, at least the urinaryones, must be identified and tested, and thereis no guarantee that they will include the reallytoxic compounds which are often present ear-lier. These tests appear to be clumsy by com-parison with other methods of investigation.

2 ― Bound residues show that the parentdrug and/or a few of its metabolites are poten-tially dangerous. Their identification is essen-tial from a toxicological point of view. A risk isassociated with any residue preceeding the

necrogenic agent (in the case of bromoben-zene).On the other hand, bound residues them-

selves no longer have the same toxicity. Thesecovalent complex macromolecules can, of

course, be broken down and, for bromoben-zene for instance, no longer yield a toxic

epoxide. Similar examples are numerous, evenin the case of carcinogens. Thus, dimethylni-trosamine is rapidly degraded in vivo to yield amethonium CH3 ion which becomes bound tonucleic acids, and the resulting 7-(N)-methylguanine and 6-(O)-methyl guanine are not car-cinogenic (Kruger and Schmahl, 19711, theydo not yield the dangerous carbocation.Bound residues can therefore be harmless

for different reasons ; but one cannot assumethis a priori and always has to prove it.

3 ― The above statements do not apply toall types of toxicity. For instance, when therisk involves an allergen, bound residues can-not be considered as harmless, and their abi-lity to produce or trigger off hypersensitivyreaction cannot be dismissed.

However, all covalent complex compoundsdo not possess the same immunological pro-perties. Thus anaphylactic reactions are

exceptional for monovalent haptens (De Wecket al., 1973). Moreover, ingestion of a haptencan produce immunological tolerance (Chase,1946).

The problem is therefore complex, and ourknowledge of residues of veterinary drugs,particularly bound residues, is very scanty.

Conclusion

Use of veterinary drugs must comply withpublic health requirements, and two points areworthy of stress :The imperfect but convenient method by

which all residual radioactivity is transformedinto equivalent of the initial compound allowsdetermination by excess of the toxic substan-ces which are present. Caution justifies thisexcess when the nature of the residues is notknown, but it should be understood that, in sodoing, we indirectly increase the coefficient ofsafety and that this coefficient should not beincreased further arbitrarily.

If this line of action cannot be adopted, e.g.when the withdrawal period is too long, new

approaches to the problem should be lookedfor : determination of residual fractions, bioa-vailability of these fractions, toxicologicalsignificance on the biotransformation routesand, possibly, further studies to assess toxicityof bound residues.

We have tried to show that solutions to the

problem include overall assessment of various

results. Determination of the withdrawal

period always amounts to assessing a risk, andwe must look for and develop methods whichwill allow us to assess this risk in order to ans-wer the questions asked.

Accepted for publication, September l8th,1981.

Summary

Basic considerations dealing with the biological significance of drug residues and safety evalua-tion are studied. The specific problem of covalently bound residues is considered from three dif-ferent points of view. (1) Analytical characteristics clearly distinguish between soluble residues,which deplete relatively fast, and bound material which is slowly eliminated from edible tissues oftarget animals. (2) These two residual fractions have not the same bioavailability. (3) Finally, toxi-city assessment of bound residues is considered.

References

BAER J.E., JACOB T.A., WOLF F.J., 1977. Cambendazole and non-drug macromolecules in tissue residues.J. Toxicol. Environ. Health, 2, 895-903.

BURGAT-SACAZE V., RICO A.G., 1980. Thérapeutique et hygiene publique : le temps d’attente desmedicaments veterinaires. Bull. Group. Tech. V6t., 3, 65-73.

CHASE M.W., 1946. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc.Soc. Exp. Biol. Med., 61, 257-259.

CORDLE M.K., 1978. When is a residue a residue. Symposium on relay toxicity and residue bioavailability.Paris, France, October 5th, 1978.

DI CUOLLO C.J., MILLER J.A., MENDELSON W.L., PAGANO J.F., 1974. Metabolic and tissue residuestudies on Parbendazole in sheep. J. Agric. Food Chem., 22, 948-953.

FERRANDO R., TRUHAUT R., 1972. La toxicité de relais. Nouvelle approche pour la méthodologie d’éva-cuation toxicologique des additifs aux aliments des animaux d’61evage. C. R. Acad. Sci., 275D, 279-283.

FOOD AND DRUG ADMINISTRATION, 1973. Proposed bioavailability requirements. Federalregister USA,38, 885-887.

GRANNEC C., 1980. Albendazole : embryotoxicité de relais. Thèse Doctorat Vétérinaire, Lyon.GYURIK R.J., CHOW A.W., ZABER B., BRUNNER E., MILLER J.A., VILLANI A.J., PETKA L.A.,

PARISH C., 1981. Metabolism of Albendazole in cattle, sheep, rats and mice. Drug Metab. Disposition,(accepted for publication).

HERRETT A.L., WILLIAMS C.W., HEOTIS J.P., BUZARD J.A., 1967. Metabolism of 5-nitro,2-furaldehyde acetylhydrazone-C&dquo; (Nihydrazone) in the chick. J. Agric. Food Chem., 15, 433-438.

HUTSON D.H., HOADLEY E.C., PICKERING B.A., 1971. The metabolic fate of (vinyl-1-&dquo;C)dichlorvos in the rat after oral and inhalation exposure. Xenobiotica, 1, 593-611.

JAGLAN P.S., GOSLINE R.E., NEFF A.W., 1976. Metabolic fate of p-toluoyl chloride phenyl-hydrazone(TCPH) in sheep. The nature of bound residues in erythrocytes. J. Agric. Food Chem., 24, 659-664.

JAGLAN P.S., GLENN M.W., NEFF A.W., 1977. Experiences in dealing with drug-related bound residues.J. Toxicol. Environ. Nealth, 2, 815-826.

KLEIN G.M., HEOTIS J.P., BUZARD J.A., 1963. Alkaline hydrolysis of ethyl 5-amino-2-furoate to

a-ketoglutaramic acid. J. Biol. Chem., 238, 1625-1627.KRUGER F.W., SCHMAHL D., 1971. Fehlen einer carcinogen Wirkung von 7-methyl-guanin bei Ratten.

Z. Krebsforsch., 75, 253-254.MARTIN D., 1980. Albendazole : !tude embryotoxique de dix métabolites. These Doctorat Vétérinaire,

Lyon.MILHAUD G., 1978. L’utilisation des medicaments veterinaires et le temps d’attente. Recl. Med. V!L Ec.

Alfort, 154, 177-185.

OLIVARD J., VALENTI S., BUZARD J.A., 1962. M6tabolisme de I’acdtylhydrazone du nitro-5furaid6hyde-2. J. Med. Pharm. Chem., 5, 524-531.

ORGANISATION MONDIALE DE LA SANTE, 1974. Biodisponibilit6 des medicaments : principes et

problbmes. Séries de rapports techniques, Gen6ve, 536, 1-20.ROSENBLUM C., 1965. Non-metabolite residues in radioactive tracer studies. /n : ROTH L.J., Isotopes in

experimental Pharmacology, Univ. Chicago Press, pp. 353-360.ROSENBLUM C., 1977. Non-drug-related residues in tracer studies. J. Toxicol. Environ. Health, 2, 803-814.ROSENBLUM C. TRENNERP N.R., WOLF D.E., 1971. The non-metabolite residue. A limitation to the tracer

method. J. Labelled Compd, 3, 225-232.ROSENBLUM C., TRENNERP N.R., BUHS R.P., HIREMATH C.B., KONINSZY F.R., WOLF D.E., 1972.

Metabolism of Ronidazole (1-methyl-5-nitroimidazole-2 yl, methyl carbamate). J. Agric. Food

Chem., 20, 360-371.SKIPPER H.E., WHITE L., BRYAN C.E., 1949. Body retention of carbon-14 from labeled sodium

bicarbonate. Science, 110, 306-307.WAL J.M., 1979. Evolution du concept de résidu dans les produits animaux des antibiotiques

utilises en 6levage. Ann. Nutr. Aliment., 33, 325-341.WEBER N.E., 1978. Bioavailability of bound residues. Symposium on relay toxicity and residue

bioavailability, Paris, France, October 5th, 1978.DE WECK A.L., SCHNEIDER C.H., SPENGLER H., TOFFLER 0., LAZARY S., 1973. Inhibition of allergic

reactions by monovalent haptens. /n : SEHON A., Mechanisms of Reaginic Allergy, pp 323-337,Dekker, New York.

WEINER M., NEWBERNE J.W., 1977. Drug metabolites in the toxicologic evaluation of drug safety.Toxicol. Appl. Pharmacol., 41, 231-233.