Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Relay Toxicity J. Boisseau To cite this article: J. Boisseau (1990) Relay Toxicity, Drug Metabolism Reviews, 22:6-8, 685-697, DOI: 10.3109/03602539008991463 To link to this article: http://dx.doi.org/10.3109/03602539008991463
Published online: 22 Sep 2008.
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DRUG METABOLISM REVIEWS, 22(6-8), 685-697 (1990)
RELAY TOXICITY* J. BOISSEAU Luboratoire des Medicaments Vkte'rinaires Centre National d'Etudes Vkterinaires et Alimentaires Ministere de I'Agriculture JAVENE-35133 FOUGERES-France
I
I.
INTRODUCTION ..........................................................
686
11.
ANABOLIC AGENTS ....................................................
687
111.
p-TOLUOYL CHLORIDE PHENYLHYDRAZONE ................688
IV.
CARBADOX ................................................................
688
V.
AFLATOXIN ................................................................
689
VI
CAMBENDAZOLE ........................................................
690
VII. ALBENDAZOLE ...........................................................
690
VIII. DISCUSSION ...............................................................
692
References .................... ........ .... .................. ..................695 *This paper was refereed by Thomas Sulkowski, Ph.D., Division of Toxicology, HFV- 150, Center for Veterinary Medicine, 5600 Fishers Lane, Rackville, MD 20857. 685 Copyright 0 1991 by Marcel Dekker, Inc.
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I. INTRODUCTION The main objective of veterinary pharmaceutical legislation is the protection of public health from the toxic effects of drug residues likely to be present in foods from treated animals. Usually, the toxicity of veterinary drug residues is assessed through experiments performed on animals since, given the present state of the art, human epidemiology is unable to supply the relevant data on this topic. It is well known that a veterinary drug may be more or less intensely metabolized in the organism of the treated animal, and it is thus very difficult to perform a qualitative comprehensive and qualitative study of all the drug residues present in the muscles or offal of this animal. Consequently, the research for possible toxic effects of a given veterinary drug residue depends mainly on the toxicological study of the parent drug through a series of toxicological trials on laboratory animals. However it is technically and financially impossible to contemplate repeat ing these toxicological tests for al I the compounds produced by drug metabolism. At best, when the parent product is shown to be toxic, a few metabolites may be tested in order to check whether they show the same potentialities. In that case, the metabolites tested are always the same: the ones most easily identified because they are stable. Now, it is well known that metabolites, whether quantitatively important or not, may be responsible for toxic effects. On the other hand, as toxic compounds possess a strong reactivity, they are often unstable and consequently their biological half-lives are very short. The most favorable conditions for conducting experiments are met when the mechanism of toxic action is understood. In these conditions, which unfortunately are rarely encountered, it is possible to test only the metabolites that may be involved in the toxic effects caused by the parent drug, if the financial cost of these studies is not prohibitive. In practice, the safety evaluation of veterinary drug residues relies on the observation of toxic effects in laboratory animals, usually rodents. But there is no absolute guarantee that the rat and the target animals, such as cattle, will metabolize a veterinary drug in the same way. So we are not sure that the laboratory animal will be exposed, because of its own metabolic capacities, to the same residues as those present in the tissues of the target animal treated with veterinary drug. All these problems facing the usual procedure of residue safety assessment led Professors Truhaut and Ferrando to devise a methodology for an-
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imal feed additives, which is better adapted to this problem. They named it relay toxicity [I-31. Its principle is simple. The laboratory animal is considered as a consumer surrogate which, throughout the test, will have to ingest feed (meat, offal, milk, eggs) derived from animals treated by the additive under consideration. Thus, the laboratory animal is exposed to all the additive residues which contaminate a food after the target animal has been treated. This methodology is very attractive since it allows a global approach to the difficult safety assessment of all the residues likely to be present in a food from a treated animal. Several experiments have been performed based on this methodology. Some of them are briefly presented as examples in the first part of this paper: anabolic agents p-toluoyl chloride phenylhydrazone carbadox aflatoxin cambendazole albendazole These examples provide information on the toxicological evaluation of all the residues existing in a food and not only of the bound residues, which are the subject of our meeting.
11. ANABOLIC AGENTS [4-71 Male and female rats and mice were given a feed containing either 6% liver or 20% meat from calves to which different anabolic substances have been administered. Whereas the administration to these laboratory animals of liver and muscle from calves treated with estradiol, testosterone, or progesterone does not affect their physiology, in any way, the reproductive functions of rats were impaired when the rats were fed tissues from calves implanted with D.E.S. These results confirm the observations made after administration to animals of a feed spiked with 60-120 ppb D.E.S. It is difficult to draw conclusions from this experiment. As no information regarding levels of D.E.S. in calf tissues has been provided, it is not
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possible to compare the effects observed on the reproductive function based on the doses used in the two methodologies.
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111. /I-TOLUOYL CHLORIDE PHENYLHYDRAZONE
Jaglan et al. have examined by the relay toxicity method TCPH, an antiparasitic drug active against a wide variety of sheep’s cestodes and nematodes [8]. This product was chosen because its residues remain for a long time in the tissues of treated sheep, especially in the blood. For 90 days, rats were given feed containing 8.6% of heat-dried sheep blood containing 39 ppm of phenyl groups. The tests usually performed in such studies (hematology, biochemistry, histology) did not reveal any toxic effect in the rats used in the experiment.
IV. CARBADOX Carbadox is a product used as a growth-promoter in the pig. Its safety for the consumer was questioned when long-term toxicity studies in the rat revealed the incidence of liver lesions. Thus, while 10% of control rats had hepatic lesions, the rates were 26% when the dose was 2.5 mg/kg/d, 35% with 5 mg/kg/d dose, 71% with 10 mg/kg/d, and 78% with 25 mg/kg/d, with development towards neoplasia and metastasis in the last case. Moreover, carbadox is rapidly metabolized in the organism of the treated animal into a major metabolite in the liver, quinoxaline carboxylic acid 2, the level of which is I ppm 24 h after administration of the authorized dose of 50 mg/kg. Three relay toxicity studies were carried out to assess the toxicity of all the residues due to carbadox administration to pigs [9-1 I]. Pigs were given feed containing 0, 20, or 200 mg/kg carbadox. These two doses represent, respectively, 0.4 and 4 times the authorized dose. Neither of the two long-term studies carried out in the rat revealed any toxic effect. The group of rats fed with the highest dose of carbadox received:
9 pglg x 56 b/kg/d = 504 pg/kg/d 9 pg/g = level of carbadox in pig liver 56 glkgld = mean daily consumption of pig liver by rats
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As the 0.5 mg/kg/d carbadox dose is much lower than the lowest dose, 2.5 mg/kg/d, used in the usual toxicological protocol, it is not surprising that it was not possible to detect hepatic lesions nor, of course, neoplasic lesions. The long-term study on dogs that ingested meat of pigs given 20 mg/kg/d carbadox did not reveal any toxic effects. But it is not possible to compare these results with those of a similar conventional toxicological study.
V. AFLATOXIN Aflatoxins have been recognized as carcinogenic compounds and as potential contaminants of food, particularly milk. Thus, they require specific care. For this reason, different relay toxicity experiments aimed at assessing the toxicity of aflatoxins present in milk were carried out on I-day-old ducklings, which are particularly sensitive to the toxic effect of aflatoxins. For 23 days, the ducklings were given feed containing 20% of heat-dried milk from goats which had ingested cakes containing 0, 54, 64,or 1136 pg/kg of aflatoxin B 1 . The milk produced by goats fed with the most contaminated cake contained 0.5 pg aflatoxin B I , traces of aflatoxin B2, and 16.2 pg/kg of aflatoxin M I . No mycotoxins were detected in the milk of goats that received the other cakes [12, 131. Examination of the livers of the ducklings given such feeds did not reveal lesions that could be definitely attributed to aflatoxin. Other groups of ducklings also received feed containing contaminated cakes. Only the ducklings given the most contaminated cake developed typical hepatic lesions. Since ducklings fed with cakes containing 54 and 64 pg/kg aflatoxin BI did not develop decisive hepatic lesions, it is not surprising that no lesions were observed in ducklings fed with a milk containing no more than 0.5 pg/kg aflatoxin BI and 16.2 pg/kg aflatoxin M I . A similar experiment was conducted with I-day-old ducklings given, for 69 days, feed containing 30% of heat-dried milk contaminated by 7.5 pg/kg of aflatoxin M I from cows fed a ration containing contaminated peanutcakes [ 141. Other ducklings received feed containing cakes contaminated by 80, 240, and 288 pg/kg of aflatoxin B1. As in the preceding experiment the ducklings fed with contaminated milk did not develop aflatoxicosic lesions.
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VI. CAMBENDAZOLE Cambendazole belongs to the benzimidazole family, which is known for its embryotoxicity and the persistence of its residues in liver. Baer et al., in a paper devoted to the study of bound cambendazole residues, give the results of a study by relay embryotoxicity performed on female rats fed with livers of cattle treated with cambendazole IIS]. The data they provide, which are rather scant, indicate that the quantity of cambendazole given to the animals through their feed amounted to 10 mglkgld and that no teratogenic effect was observed. When cambendazole was administered directly to the female rats, according to the usual embryotoxicity study, the dose of 60 mg/kg/d through the feed was embryotoxic. It is thus not surprising that this relay toxicity study did not reveal embryotoxic effects since the dose ingested by this method represents I/a of the toxic dose of cambendazole administered through the feed according to the standard methodology.
VII. ALBENDAZOLE Albendozole embryotoxicity in the rat is well established. It is embryolethal, teratogenic, and fetotoxic. By systematically studying the embryotoxicity of 10 metabolites of albendazole, it was possible to prove the responsibility of two of them: albendazole itself and its sulfoxide metabolite [ 16, 171. The “no-effect’’ doses are the same for the two products:
6 mg/kg/d by gavage 12 mg/kg/d when administered through the feed The total inhibition of albendazole toxic effects when administered in association with an inhibitor of microsomial oxidation, P-diethylaminoethyl2.2-diphenylpentanoate (SKF 525A), shows that, most probably, albendazole itself is devoid of toxic effect and that it causes toxic effects only after transformation into its sulfoxide. Indeed, SKF 525A. when given to a rat: suppresses the embryotoxicity of albendazole decreases the plasma concentration of sulfoxide albendazole has no influence on the level of radioactivity linked to the fetal microsomial proteins
69 1
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These results show that albendazole induces toxic effects through its free sulfoxide metabolite and not through metabolites covalently bound to macromolecules. It is known that, after administration to an animal, albendazole is metabolized to such an extent that i t cannot be detected in cattle liver 24 h after administration of 20 mg/kg. It was thus profitable to use the relay toxicity method to ascertain the results obtained from the direct assessment of the embryotoxicity of albendazole metabolites [ 18, 191. The livers of cattle which had received 20 mg/kg albendazole were heat dried and incorporated at a mean level of 38.7% in the feed given to pregnant rats between the 8th and 15th day of gestation. Four batches of female rats were given feed to which was added heat-dried liver of cattle slaughtered 1, 2, or 3 days after they had been administered 20 mg/kg albendazole. No embryotoxic effect was noted for any batch of animals, even for those fed with liver of cattle slaughtered I day after the administration of albendazole. In this case, where the rats had to ingest the greatest quantity of albendazole residues, the mean daily intake of sulfoxide albendazole was 1.3 mg/kg/d.
I I pg/g x
I00
x
38.7
1~x
92 g/kg/d = 1.3 mg/kg/d
I I pg/g is the sulfoxide albendazole level in the liver I day after treatment 29.9% is the ratio of heat dried liver to fresh liver 38.7% is the % of heat dried meat incorporated into the feed of the rats 92 g/kg/d is the mean daily consumption of animals
This dose, 1.3 mg/kg, is far lower than the “no embryotoxic effect” of albendazole sulfoxide, which is 12 mg/kg/d when given through the feed. The relay methodology did not reveal the embryotoxic effects of albendazole residues present in cattle liver. However, The dose administered was 2.7 times higher than the approved therapeutic dosage. The withdrawal time observed was very short ( I day) so as to obtain a maximum level of residues in the liver. The rate of incorporation of heat dried liver into the feed was high (40%). much higher than the dose recommended by Truhaut and Ferrando. This experiment is particularly valuable as it enables comparison, in appropriate conditions, of the results of the safety assessment of albendazole
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residues by both methods: the conventional one and relay toxicity (the residue levels in cattle liver were known). Thus, the protocol followed to study relay toxicity was not appropriate, as it did not permit the administration to the laboratory animals of a sufficient amount of metabolites, particularly toxic ones, to detect an embryotoxic effect.
VIII. DISCUSSION From these examples, it is possible to conclude that the rationales of safety assessment of veterinary drug residues through conventional procedure and of animal feed additives through relay toxicity are quite different. The conventional assessment of residue safety relies on experimental procedures aimed at identifying the nature of the possible toxic properties of the compound under consideration. This goal is usually reached through the administration to the laboratory animals of doses of veterinary drugs which are much higher than the levels of residue likely to be found in the food from a treated animal. Then, by decreasing the amount of drug administered, a “no observed toxic effect” dose is determined. After adjustment by a safety factor, the importance of which depends on the nature of the observed toxic effect, it is possible to derive an A.D.I. from this “no observed toxic effect” dose. This A.D.I. in turn provides the basis of the calculation of maximum residue limits (M.R.L.), which have to be considered when determining an appropriate withdrawal time. Moreover, these M.R.L. are necessary as key points for the monitoring of food hygienic quality. Therefore, a withdrawal time is a parameter which is the endpoint of the rationale of the residue safety assessment. The relay toxicity procedure used for the safety evaluation of animal feed additive is quite different. The above-mentioned examples mainly show that this procedure leads to the conclusion that food derived from treated animals does not cause any toxic effect in laboratory animals. Therefore, it may be assumed that the withdrawal time adopted during this relay toxicity experiment ensures that the residues likely to be present in the food fed to these animals cannot endanger public health. As the authors pointed out, however, this methodology was devised and developed to assess the safety of animal feed additives: The problem is to know to what extent this method can be used for the toxicological evaluation of veterinary drug residuies. One way to answer this question is to compare the results obtained with both methods: the conventional method
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of direct administration to laboratory animals of the compound to be studied and relay toxicity. As already stated, it is not possible to obtain relevant information from the studies performed on anabolic agents and p-toluoyl chloride phenylhydrazone. The other studies regarding aflatoxin, carbadox, cambendazole, and albendazole show that all the doses administered during relay toxicity experiments are lower than the lowest doses which caused a toxic effect in laboratory animals in a conventional toxicity study. This means that, in these four examples, relay toxicology would not have revealed a toxic effect, particularly the carcinogenic potency of aflatoxin B 1 and carbadox, nor the embryotoxic potentialities of cambendazole and albendazole. From the results, it would have been inferred that the quantities of residues administered to the animals are safe for the consumer. However, the conclusions drawn from conventional assessment of residue safety are different. In the case of embryotoxic potentialities of albendazole, the loo0 safety factor usually taken to calculate an A.D.I. from the “no observed toxic effect” dose in animals gives a lower figure. The “no effect dose” in animals for albendazole being 12 mg/kg/d, the calculated A.D.I. is 12 p.g/kg/d, a figure much lower than the 1.3 mg/kg/d dose which appears to be safe according to the relay toxicity experiment. The same demonstration may be applied to compounds with carcinogenic potency. The problem of the level of doses to be administered to animals for the toxicological evaluation of veterinary drug residues is, clearly, a difficult one. As the quantities of products administered are far higher than the levels of residues, it is possible to fear metabolic and toxicologic artifacts. But, on the other hand, the probability calculations show that such high doses of product have to be used to detect weak or infrequent toxic effects to the extent that, for economic and ethical reasons, the experiments have to be performed on only a limited number of animals. Thus, this question remains open. The issue regarding the quantity of compound likely to be administered to the consumer surrogate animal in relay toxicity is the crucial point of this procedure. To try to get round this problem, Truhaut and Ferrando used a set of three coefficients, which are presented below with the carbadox relay toxicity study in the dog as an example. Coefficient of rhe compared consumption. This is determined by the ratio of the daily quantity of tissue ingested by the laboratory animal and man. In this example, it has been estimated at 18. Coefficient of the dose used. This is calculated as the ratio between the dosage of the product administered to the target animal in the experiment and the recommended dosage. In this example, this coefficient is 4.
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BOISSEAU Coefficienr of withdrawal rime. This is determined by the ratio of residue level in food from animals slaughtered without withdrawal time to the residue level in the same tissue after observing the official withdrawal time. This coefficient thus varies according to the withdrawal time observed. It is 2 with a 4-day withdrawal time and 128 with a 4-week withdrawal time. So the final and total safety factor for residues of carbadox in pork, according to this relay toxicity study, is 144 with a 4-day withdrawal time and 9216 for a 4-week withdrawal time. It is worth discussing these three safety factors to see what they may represent for the toxicological evaluation of the veterinary drug residues. Coefficient of rhe dose used: The value of this coefficient will also decrease when relay toxicity is applied to veterinary drugs. The reason is that the dosages usually prescribed for veterinary drugs are higher than for animal feed additives. As it is not possible, for animal safety purposes, to increase too greatly the amount of veterinary drugs administered to the target animal in a relay toxicity experiment, the value of this coefficient will be reduced. In the case of albendazole, this coefficient was 2.7. Another problem to be solved concerns the different dosages prescribed for the different animal species for which a veterinary drug is intended. Coefficient of wirhdrawal rime: In this case, there is also an important difference between the approval of animal feed additives and veterinary drugs. The approval of animal feed additives is granted to one compound and stipulates the dosage through the feed and the withdrawal time for the animal species considered. Thus, the conditions of approval of an animal feed additive are both standardized and limited. The situation is different for veterinary drugs. In this case, the approval is granted for a finished product taking into account different parameters such as the formulation, dosage, route of administration. animal species, and so on. Consequently, it is not possible to prescribe only one withdrawal time for an active ingredient but. on the contrary, one withdrawal time or even more has to be established for each veterinary drug, regarded as a finished product. As already mentioned, for veterinary drugs, the withdrawal time is an endpoint in the procedure of residue safety evaluation. Therefore, it is difficult to consider withdrawal times, which may vary with each veterinary drug, as an appropriate parameter for assessing the safety of veterinary drug residues and stable maximum residue limits necessary for monitoring the hygienic quality of food. Let us take the example of the different formulations, oral suspension, bolus, pour-on of an anthelmintic drug intended for cattle. It is normal to expect different residue kinetics for each of these different formulations and dosages. Nevertheless, it is also quite possible that the relay toxicity studies
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carried out with these different formulations may demonstrate that these different veterinary drugs are safe even if the level of residues in meat, offal, and milk are different. Therefore, what approach should be adopted to assess maximum residue level and monitor the hygienic quality of food derived from the animal treated by these different veterinary drugs? In conclusion, it would not appear that relay toxicity study represents for the time being an acceptable alternative to the currently used rationale for the safety evaluation of veterinary drug residues. This procedure cannot meet the requirement of stable maximum residue limits likely to be applied to an active ingredient, whatever the veterinary drugs involved. In the case where benefits can be derived from relay toxicity, this would be only an additional test to obtain a better understanding of the results provided by the usual toxicological studies. But as a first step, it is absolutely essential to establish the validity of the relay toxicity procedure. This will have to rely on the comparison of the effects observed in laboratory animals of the same dosages administered according to both methodologies: relay toxicity and classical toxicological studies. As a first step, this should concern mainly free metabolites, which are easier to handle. If positive results make it possible to go further, it will be possible, in a second step, to try to use relay toxicity for the safety evaluation of bound residues. However, bearing in mind the low levels of bound residues in tissue, it is possible to expect that relay toxicity will probably be useful only in some very favorable situations.
REFERENCES [ I ] Truhaut, R., FerraGdo, R. La toxicite de relais-I. Principes gtntraux d’une approche mithodologique nouvelle pour I’Cvaluation toxicologique des additifs aux aliments des animaux d’ilevage, Toxicology, 3, 361-368 (1975). [2] Truhaut, R., Ferrando, R. RCsultats de 8 ans de recherches sur l’evaluation toxicologique des additifs a I’alimentation animale par la methode dite toxicite de relais, European Journal of Toxicology (supplement), 9 (7), 413-422 (1976). [3] Ferrando, R., Truhaut, R. Aspects gtneraux de la toxicite de relais; ses applications, Toxicological European Research, 4 ( 5 ) . (1982). [4] Ferrando, R. Toxicitt de relais de la viande et du foie de veaux recevant des implants d’hormones: premiers resultats, C.R. Acad. Sc. Paris, t. 273 (4 October, 1971).
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[5] Ferrando, R., Henry, N., Klur, M., Valette, J. P. Influence de la restriction de consommation d’aliment due a la viande de veaux implant& au diethylstilboestrol (DES) sur la fecondite des rats consommant cette viande, C.R. Acad. Sc. Paris, t. 275 (24 July, 1972). [6] Ferrando, R., Valette, J. F?, Henry, N., Boivin, R., Parodi, A. Toxicite de relais des viandes et foies provenant de veaux traites avec diverses hormones. Resultats globaux, C.R. Acad. sc. Paris, t. 278 (17 April, 1974). [7] Ferrando, R., Truhaut, R. V1/2 Considerations toxicologiques spkciales sur les anabolisants; toxicite de relais, Environmental Qualify and Safety, 5 , 219-226 (1976). [8] Jaglan, F? S.. Weldon Glenn, M., William Neff, A. Experiences in dealing with drug-related bound residues. Journal of Toxicology and Environmental Health, 2 , 815-826, (1977). 191 Ferrando, R., Truhaut, R., Raynaud, J. P., Spanoghe, J. P. La toxicite de relais - 11. Application de la methodologie “toxicite de relais” a I’kvaluationde la securitt d’emploi pour les consommateurs humains du carbadox, facteur de croissance, ajoute a la ration du porc charcutier, Toxicology, 3, 369-398 (1975). [lo] Ferrando, R., Truhaut, R., Raynaud, J. P. Principles of a full “relay toxicity” experiment and results conducted with carbadox a feed additive used as growth promoter for growing swine. Folia Ver. Latinu, 7 (4), 333 (1977). [ I l l Ferrando, R., Truhaut, R., Raynaud, J. P., Spanoghe, J. P. La toxicite de relais-Ill. SCcuritt d’emploi pour le consommateur humain du carbadox, facteur de croissance pour le porc, estimee par toxicite de relais d’une duree de 7 ans chez le chien beagle, Toxicology, I / , 167183 (1978). [I21 Ferrando, R., Parodi, A., Henry, N., Delort-Laval, J., Lamine, A. “Milk aflatoxine” et toxicite de relais, C.R. Acad. Sc. Paris, t. 284 (7 March, 1977). [13] Ndiaye, Ah.L. La toxicite de relais dans ses rapports avec les substances naturellement presentes dans les aliments. Toxicological European Research, 4 (3,243-255 (1982). [I41 Ferrando, R., Palisse-Roussel, M., Jacquot, L. Toxicite de relais de I’aflatoxine MI de la poudre de lait. Etude a moyen terme sur le caneton, C.R. Acad. Sc. Paris, t. 298, sdrie 111, no 13 (1984). [15] Baer, J. E., Jacob, T. A., Wolf, F. J. Cambendazole and nondrug macromolecules in tissue residues, Journal of Toxicology and Environmental Health, 2 , 895-903 (1977). [I61 Martin, I? Albendazole These E.N.V., Lyon, 1980. [ 171 Delhoste, C. Albendazole: Essai de corrdlation entre embryotoxicite, metabolites libres, mktabolites lids, Thtse E.N.V., Lyon, 1983.
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[ 181 Grannec, C. Albendozole: Embryotoxicit6 de relais, T h b e E.N.V.,
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Lyon, 1980. [I91 Delatour, P, Parish, R. C . , Gyurik, R. J. Albendazole: a comparison of relay embryotoxicity with embryotoxicity of individual metabolites, Ann. Rech. Vet., 12 (21, 159-167 (1981).
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Relay Toxicity J. Boisseau To cite this article: J. Boisseau (1990) Relay Toxicity, Drug Metabolism Reviews, 22:6-8, 685-697, DOI: 10.3109/03602539008991463 To link to this article: http://dx.doi.org/10.3109/03602539008991463
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 7
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of California, San Diego]
Date: 07 November 2015, At: 13:23
Downloaded by [University of California, San Diego] at 13:23 07 November 2015
DRUG METABOLISM REVIEWS, 22(6-8), 685-697 (1990)
RELAY TOXICITY* J. BOISSEAU Luboratoire des Medicaments Vkte'rinaires Centre National d'Etudes Vkterinaires et Alimentaires Ministere de I'Agriculture JAVENE-35133 FOUGERES-France
I
I.
INTRODUCTION ..........................................................
686
11.
ANABOLIC AGENTS ....................................................
687
111.
p-TOLUOYL CHLORIDE PHENYLHYDRAZONE ................688
IV.
CARBADOX ................................................................
688
V.
AFLATOXIN ................................................................
689
VI
CAMBENDAZOLE ........................................................
690
VII. ALBENDAZOLE ...........................................................
690
VIII. DISCUSSION ...............................................................
692
References .................... ........ .... .................. ..................695 *This paper was refereed by Thomas Sulkowski, Ph.D., Division of Toxicology, HFV- 150, Center for Veterinary Medicine, 5600 Fishers Lane, Rackville, MD 20857. 685 Copyright 0 1991 by Marcel Dekker, Inc.
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BOISSEAU
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I. INTRODUCTION The main objective of veterinary pharmaceutical legislation is the protection of public health from the toxic effects of drug residues likely to be present in foods from treated animals. Usually, the toxicity of veterinary drug residues is assessed through experiments performed on animals since, given the present state of the art, human epidemiology is unable to supply the relevant data on this topic. It is well known that a veterinary drug may be more or less intensely metabolized in the organism of the treated animal, and it is thus very difficult to perform a qualitative comprehensive and qualitative study of all the drug residues present in the muscles or offal of this animal. Consequently, the research for possible toxic effects of a given veterinary drug residue depends mainly on the toxicological study of the parent drug through a series of toxicological trials on laboratory animals. However it is technically and financially impossible to contemplate repeat ing these toxicological tests for al I the compounds produced by drug metabolism. At best, when the parent product is shown to be toxic, a few metabolites may be tested in order to check whether they show the same potentialities. In that case, the metabolites tested are always the same: the ones most easily identified because they are stable. Now, it is well known that metabolites, whether quantitatively important or not, may be responsible for toxic effects. On the other hand, as toxic compounds possess a strong reactivity, they are often unstable and consequently their biological half-lives are very short. The most favorable conditions for conducting experiments are met when the mechanism of toxic action is understood. In these conditions, which unfortunately are rarely encountered, it is possible to test only the metabolites that may be involved in the toxic effects caused by the parent drug, if the financial cost of these studies is not prohibitive. In practice, the safety evaluation of veterinary drug residues relies on the observation of toxic effects in laboratory animals, usually rodents. But there is no absolute guarantee that the rat and the target animals, such as cattle, will metabolize a veterinary drug in the same way. So we are not sure that the laboratory animal will be exposed, because of its own metabolic capacities, to the same residues as those present in the tissues of the target animal treated with veterinary drug. All these problems facing the usual procedure of residue safety assessment led Professors Truhaut and Ferrando to devise a methodology for an-
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imal feed additives, which is better adapted to this problem. They named it relay toxicity [I-31. Its principle is simple. The laboratory animal is considered as a consumer surrogate which, throughout the test, will have to ingest feed (meat, offal, milk, eggs) derived from animals treated by the additive under consideration. Thus, the laboratory animal is exposed to all the additive residues which contaminate a food after the target animal has been treated. This methodology is very attractive since it allows a global approach to the difficult safety assessment of all the residues likely to be present in a food from a treated animal. Several experiments have been performed based on this methodology. Some of them are briefly presented as examples in the first part of this paper: anabolic agents p-toluoyl chloride phenylhydrazone carbadox aflatoxin cambendazole albendazole These examples provide information on the toxicological evaluation of all the residues existing in a food and not only of the bound residues, which are the subject of our meeting.
11. ANABOLIC AGENTS [4-71 Male and female rats and mice were given a feed containing either 6% liver or 20% meat from calves to which different anabolic substances have been administered. Whereas the administration to these laboratory animals of liver and muscle from calves treated with estradiol, testosterone, or progesterone does not affect their physiology, in any way, the reproductive functions of rats were impaired when the rats were fed tissues from calves implanted with D.E.S. These results confirm the observations made after administration to animals of a feed spiked with 60-120 ppb D.E.S. It is difficult to draw conclusions from this experiment. As no information regarding levels of D.E.S. in calf tissues has been provided, it is not
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possible to compare the effects observed on the reproductive function based on the doses used in the two methodologies.
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111. /I-TOLUOYL CHLORIDE PHENYLHYDRAZONE
Jaglan et al. have examined by the relay toxicity method TCPH, an antiparasitic drug active against a wide variety of sheep’s cestodes and nematodes [8]. This product was chosen because its residues remain for a long time in the tissues of treated sheep, especially in the blood. For 90 days, rats were given feed containing 8.6% of heat-dried sheep blood containing 39 ppm of phenyl groups. The tests usually performed in such studies (hematology, biochemistry, histology) did not reveal any toxic effect in the rats used in the experiment.
IV. CARBADOX Carbadox is a product used as a growth-promoter in the pig. Its safety for the consumer was questioned when long-term toxicity studies in the rat revealed the incidence of liver lesions. Thus, while 10% of control rats had hepatic lesions, the rates were 26% when the dose was 2.5 mg/kg/d, 35% with 5 mg/kg/d dose, 71% with 10 mg/kg/d, and 78% with 25 mg/kg/d, with development towards neoplasia and metastasis in the last case. Moreover, carbadox is rapidly metabolized in the organism of the treated animal into a major metabolite in the liver, quinoxaline carboxylic acid 2, the level of which is I ppm 24 h after administration of the authorized dose of 50 mg/kg. Three relay toxicity studies were carried out to assess the toxicity of all the residues due to carbadox administration to pigs [9-1 I]. Pigs were given feed containing 0, 20, or 200 mg/kg carbadox. These two doses represent, respectively, 0.4 and 4 times the authorized dose. Neither of the two long-term studies carried out in the rat revealed any toxic effect. The group of rats fed with the highest dose of carbadox received:
9 pglg x 56 b/kg/d = 504 pg/kg/d 9 pg/g = level of carbadox in pig liver 56 glkgld = mean daily consumption of pig liver by rats
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As the 0.5 mg/kg/d carbadox dose is much lower than the lowest dose, 2.5 mg/kg/d, used in the usual toxicological protocol, it is not surprising that it was not possible to detect hepatic lesions nor, of course, neoplasic lesions. The long-term study on dogs that ingested meat of pigs given 20 mg/kg/d carbadox did not reveal any toxic effects. But it is not possible to compare these results with those of a similar conventional toxicological study.
V. AFLATOXIN Aflatoxins have been recognized as carcinogenic compounds and as potential contaminants of food, particularly milk. Thus, they require specific care. For this reason, different relay toxicity experiments aimed at assessing the toxicity of aflatoxins present in milk were carried out on I-day-old ducklings, which are particularly sensitive to the toxic effect of aflatoxins. For 23 days, the ducklings were given feed containing 20% of heat-dried milk from goats which had ingested cakes containing 0, 54, 64,or 1136 pg/kg of aflatoxin B 1 . The milk produced by goats fed with the most contaminated cake contained 0.5 pg aflatoxin B I , traces of aflatoxin B2, and 16.2 pg/kg of aflatoxin M I . No mycotoxins were detected in the milk of goats that received the other cakes [12, 131. Examination of the livers of the ducklings given such feeds did not reveal lesions that could be definitely attributed to aflatoxin. Other groups of ducklings also received feed containing contaminated cakes. Only the ducklings given the most contaminated cake developed typical hepatic lesions. Since ducklings fed with cakes containing 54 and 64 pg/kg aflatoxin BI did not develop decisive hepatic lesions, it is not surprising that no lesions were observed in ducklings fed with a milk containing no more than 0.5 pg/kg aflatoxin BI and 16.2 pg/kg aflatoxin M I . A similar experiment was conducted with I-day-old ducklings given, for 69 days, feed containing 30% of heat-dried milk contaminated by 7.5 pg/kg of aflatoxin M I from cows fed a ration containing contaminated peanutcakes [ 141. Other ducklings received feed containing cakes contaminated by 80, 240, and 288 pg/kg of aflatoxin B1. As in the preceding experiment the ducklings fed with contaminated milk did not develop aflatoxicosic lesions.
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VI. CAMBENDAZOLE Cambendazole belongs to the benzimidazole family, which is known for its embryotoxicity and the persistence of its residues in liver. Baer et al., in a paper devoted to the study of bound cambendazole residues, give the results of a study by relay embryotoxicity performed on female rats fed with livers of cattle treated with cambendazole IIS]. The data they provide, which are rather scant, indicate that the quantity of cambendazole given to the animals through their feed amounted to 10 mglkgld and that no teratogenic effect was observed. When cambendazole was administered directly to the female rats, according to the usual embryotoxicity study, the dose of 60 mg/kg/d through the feed was embryotoxic. It is thus not surprising that this relay toxicity study did not reveal embryotoxic effects since the dose ingested by this method represents I/a of the toxic dose of cambendazole administered through the feed according to the standard methodology.
VII. ALBENDAZOLE Albendozole embryotoxicity in the rat is well established. It is embryolethal, teratogenic, and fetotoxic. By systematically studying the embryotoxicity of 10 metabolites of albendazole, it was possible to prove the responsibility of two of them: albendazole itself and its sulfoxide metabolite [ 16, 171. The “no-effect’’ doses are the same for the two products:
6 mg/kg/d by gavage 12 mg/kg/d when administered through the feed The total inhibition of albendazole toxic effects when administered in association with an inhibitor of microsomial oxidation, P-diethylaminoethyl2.2-diphenylpentanoate (SKF 525A), shows that, most probably, albendazole itself is devoid of toxic effect and that it causes toxic effects only after transformation into its sulfoxide. Indeed, SKF 525A. when given to a rat: suppresses the embryotoxicity of albendazole decreases the plasma concentration of sulfoxide albendazole has no influence on the level of radioactivity linked to the fetal microsomial proteins
69 1
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These results show that albendazole induces toxic effects through its free sulfoxide metabolite and not through metabolites covalently bound to macromolecules. It is known that, after administration to an animal, albendazole is metabolized to such an extent that i t cannot be detected in cattle liver 24 h after administration of 20 mg/kg. It was thus profitable to use the relay toxicity method to ascertain the results obtained from the direct assessment of the embryotoxicity of albendazole metabolites [ 18, 191. The livers of cattle which had received 20 mg/kg albendazole were heat dried and incorporated at a mean level of 38.7% in the feed given to pregnant rats between the 8th and 15th day of gestation. Four batches of female rats were given feed to which was added heat-dried liver of cattle slaughtered 1, 2, or 3 days after they had been administered 20 mg/kg albendazole. No embryotoxic effect was noted for any batch of animals, even for those fed with liver of cattle slaughtered I day after the administration of albendazole. In this case, where the rats had to ingest the greatest quantity of albendazole residues, the mean daily intake of sulfoxide albendazole was 1.3 mg/kg/d.
I I pg/g x
I00
x
38.7
1~x
92 g/kg/d = 1.3 mg/kg/d
I I pg/g is the sulfoxide albendazole level in the liver I day after treatment 29.9% is the ratio of heat dried liver to fresh liver 38.7% is the % of heat dried meat incorporated into the feed of the rats 92 g/kg/d is the mean daily consumption of animals
This dose, 1.3 mg/kg, is far lower than the “no embryotoxic effect” of albendazole sulfoxide, which is 12 mg/kg/d when given through the feed. The relay methodology did not reveal the embryotoxic effects of albendazole residues present in cattle liver. However, The dose administered was 2.7 times higher than the approved therapeutic dosage. The withdrawal time observed was very short ( I day) so as to obtain a maximum level of residues in the liver. The rate of incorporation of heat dried liver into the feed was high (40%). much higher than the dose recommended by Truhaut and Ferrando. This experiment is particularly valuable as it enables comparison, in appropriate conditions, of the results of the safety assessment of albendazole
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residues by both methods: the conventional one and relay toxicity (the residue levels in cattle liver were known). Thus, the protocol followed to study relay toxicity was not appropriate, as it did not permit the administration to the laboratory animals of a sufficient amount of metabolites, particularly toxic ones, to detect an embryotoxic effect.
VIII. DISCUSSION From these examples, it is possible to conclude that the rationales of safety assessment of veterinary drug residues through conventional procedure and of animal feed additives through relay toxicity are quite different. The conventional assessment of residue safety relies on experimental procedures aimed at identifying the nature of the possible toxic properties of the compound under consideration. This goal is usually reached through the administration to the laboratory animals of doses of veterinary drugs which are much higher than the levels of residue likely to be found in the food from a treated animal. Then, by decreasing the amount of drug administered, a “no observed toxic effect” dose is determined. After adjustment by a safety factor, the importance of which depends on the nature of the observed toxic effect, it is possible to derive an A.D.I. from this “no observed toxic effect” dose. This A.D.I. in turn provides the basis of the calculation of maximum residue limits (M.R.L.), which have to be considered when determining an appropriate withdrawal time. Moreover, these M.R.L. are necessary as key points for the monitoring of food hygienic quality. Therefore, a withdrawal time is a parameter which is the endpoint of the rationale of the residue safety assessment. The relay toxicity procedure used for the safety evaluation of animal feed additive is quite different. The above-mentioned examples mainly show that this procedure leads to the conclusion that food derived from treated animals does not cause any toxic effect in laboratory animals. Therefore, it may be assumed that the withdrawal time adopted during this relay toxicity experiment ensures that the residues likely to be present in the food fed to these animals cannot endanger public health. As the authors pointed out, however, this methodology was devised and developed to assess the safety of animal feed additives: The problem is to know to what extent this method can be used for the toxicological evaluation of veterinary drug residuies. One way to answer this question is to compare the results obtained with both methods: the conventional method
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of direct administration to laboratory animals of the compound to be studied and relay toxicity. As already stated, it is not possible to obtain relevant information from the studies performed on anabolic agents and p-toluoyl chloride phenylhydrazone. The other studies regarding aflatoxin, carbadox, cambendazole, and albendazole show that all the doses administered during relay toxicity experiments are lower than the lowest doses which caused a toxic effect in laboratory animals in a conventional toxicity study. This means that, in these four examples, relay toxicology would not have revealed a toxic effect, particularly the carcinogenic potency of aflatoxin B 1 and carbadox, nor the embryotoxic potentialities of cambendazole and albendazole. From the results, it would have been inferred that the quantities of residues administered to the animals are safe for the consumer. However, the conclusions drawn from conventional assessment of residue safety are different. In the case of embryotoxic potentialities of albendazole, the loo0 safety factor usually taken to calculate an A.D.I. from the “no observed toxic effect” dose in animals gives a lower figure. The “no effect dose” in animals for albendazole being 12 mg/kg/d, the calculated A.D.I. is 12 p.g/kg/d, a figure much lower than the 1.3 mg/kg/d dose which appears to be safe according to the relay toxicity experiment. The same demonstration may be applied to compounds with carcinogenic potency. The problem of the level of doses to be administered to animals for the toxicological evaluation of veterinary drug residues is, clearly, a difficult one. As the quantities of products administered are far higher than the levels of residues, it is possible to fear metabolic and toxicologic artifacts. But, on the other hand, the probability calculations show that such high doses of product have to be used to detect weak or infrequent toxic effects to the extent that, for economic and ethical reasons, the experiments have to be performed on only a limited number of animals. Thus, this question remains open. The issue regarding the quantity of compound likely to be administered to the consumer surrogate animal in relay toxicity is the crucial point of this procedure. To try to get round this problem, Truhaut and Ferrando used a set of three coefficients, which are presented below with the carbadox relay toxicity study in the dog as an example. Coefficient of rhe compared consumption. This is determined by the ratio of the daily quantity of tissue ingested by the laboratory animal and man. In this example, it has been estimated at 18. Coefficient of the dose used. This is calculated as the ratio between the dosage of the product administered to the target animal in the experiment and the recommended dosage. In this example, this coefficient is 4.
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BOISSEAU Coefficienr of withdrawal rime. This is determined by the ratio of residue level in food from animals slaughtered without withdrawal time to the residue level in the same tissue after observing the official withdrawal time. This coefficient thus varies according to the withdrawal time observed. It is 2 with a 4-day withdrawal time and 128 with a 4-week withdrawal time. So the final and total safety factor for residues of carbadox in pork, according to this relay toxicity study, is 144 with a 4-day withdrawal time and 9216 for a 4-week withdrawal time. It is worth discussing these three safety factors to see what they may represent for the toxicological evaluation of the veterinary drug residues. Coefficient of rhe dose used: The value of this coefficient will also decrease when relay toxicity is applied to veterinary drugs. The reason is that the dosages usually prescribed for veterinary drugs are higher than for animal feed additives. As it is not possible, for animal safety purposes, to increase too greatly the amount of veterinary drugs administered to the target animal in a relay toxicity experiment, the value of this coefficient will be reduced. In the case of albendazole, this coefficient was 2.7. Another problem to be solved concerns the different dosages prescribed for the different animal species for which a veterinary drug is intended. Coefficient of wirhdrawal rime: In this case, there is also an important difference between the approval of animal feed additives and veterinary drugs. The approval of animal feed additives is granted to one compound and stipulates the dosage through the feed and the withdrawal time for the animal species considered. Thus, the conditions of approval of an animal feed additive are both standardized and limited. The situation is different for veterinary drugs. In this case, the approval is granted for a finished product taking into account different parameters such as the formulation, dosage, route of administration. animal species, and so on. Consequently, it is not possible to prescribe only one withdrawal time for an active ingredient but. on the contrary, one withdrawal time or even more has to be established for each veterinary drug, regarded as a finished product. As already mentioned, for veterinary drugs, the withdrawal time is an endpoint in the procedure of residue safety evaluation. Therefore, it is difficult to consider withdrawal times, which may vary with each veterinary drug, as an appropriate parameter for assessing the safety of veterinary drug residues and stable maximum residue limits necessary for monitoring the hygienic quality of food. Let us take the example of the different formulations, oral suspension, bolus, pour-on of an anthelmintic drug intended for cattle. It is normal to expect different residue kinetics for each of these different formulations and dosages. Nevertheless, it is also quite possible that the relay toxicity studies
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carried out with these different formulations may demonstrate that these different veterinary drugs are safe even if the level of residues in meat, offal, and milk are different. Therefore, what approach should be adopted to assess maximum residue level and monitor the hygienic quality of food derived from the animal treated by these different veterinary drugs? In conclusion, it would not appear that relay toxicity study represents for the time being an acceptable alternative to the currently used rationale for the safety evaluation of veterinary drug residues. This procedure cannot meet the requirement of stable maximum residue limits likely to be applied to an active ingredient, whatever the veterinary drugs involved. In the case where benefits can be derived from relay toxicity, this would be only an additional test to obtain a better understanding of the results provided by the usual toxicological studies. But as a first step, it is absolutely essential to establish the validity of the relay toxicity procedure. This will have to rely on the comparison of the effects observed in laboratory animals of the same dosages administered according to both methodologies: relay toxicity and classical toxicological studies. As a first step, this should concern mainly free metabolites, which are easier to handle. If positive results make it possible to go further, it will be possible, in a second step, to try to use relay toxicity for the safety evaluation of bound residues. However, bearing in mind the low levels of bound residues in tissue, it is possible to expect that relay toxicity will probably be useful only in some very favorable situations.
REFERENCES [ I ] Truhaut, R., FerraGdo, R. La toxicite de relais-I. Principes gtntraux d’une approche mithodologique nouvelle pour I’Cvaluation toxicologique des additifs aux aliments des animaux d’ilevage, Toxicology, 3, 361-368 (1975). [2] Truhaut, R., Ferrando, R. RCsultats de 8 ans de recherches sur l’evaluation toxicologique des additifs a I’alimentation animale par la methode dite toxicite de relais, European Journal of Toxicology (supplement), 9 (7), 413-422 (1976). [3] Ferrando, R., Truhaut, R. Aspects gtneraux de la toxicite de relais; ses applications, Toxicological European Research, 4 ( 5 ) . (1982). [4] Ferrando, R. Toxicitt de relais de la viande et du foie de veaux recevant des implants d’hormones: premiers resultats, C.R. Acad. Sc. Paris, t. 273 (4 October, 1971).
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[5] Ferrando, R., Henry, N., Klur, M., Valette, J. P. Influence de la restriction de consommation d’aliment due a la viande de veaux implant& au diethylstilboestrol (DES) sur la fecondite des rats consommant cette viande, C.R. Acad. Sc. Paris, t. 275 (24 July, 1972). [6] Ferrando, R., Valette, J. F?, Henry, N., Boivin, R., Parodi, A. Toxicite de relais des viandes et foies provenant de veaux traites avec diverses hormones. Resultats globaux, C.R. Acad. sc. Paris, t. 278 (17 April, 1974). [7] Ferrando, R., Truhaut, R. V1/2 Considerations toxicologiques spkciales sur les anabolisants; toxicite de relais, Environmental Qualify and Safety, 5 , 219-226 (1976). [8] Jaglan, F? S.. Weldon Glenn, M., William Neff, A. Experiences in dealing with drug-related bound residues. Journal of Toxicology and Environmental Health, 2 , 815-826, (1977). 191 Ferrando, R., Truhaut, R., Raynaud, J. P., Spanoghe, J. P. La toxicite de relais - 11. Application de la methodologie “toxicite de relais” a I’kvaluationde la securitt d’emploi pour les consommateurs humains du carbadox, facteur de croissance, ajoute a la ration du porc charcutier, Toxicology, 3, 369-398 (1975). [lo] Ferrando, R., Truhaut, R., Raynaud, J. P. Principles of a full “relay toxicity” experiment and results conducted with carbadox a feed additive used as growth promoter for growing swine. Folia Ver. Latinu, 7 (4), 333 (1977). [ I l l Ferrando, R., Truhaut, R., Raynaud, J. P., Spanoghe, J. P. La toxicite de relais-Ill. SCcuritt d’emploi pour le consommateur humain du carbadox, facteur de croissance pour le porc, estimee par toxicite de relais d’une duree de 7 ans chez le chien beagle, Toxicology, I / , 167183 (1978). [I21 Ferrando, R., Parodi, A., Henry, N., Delort-Laval, J., Lamine, A. “Milk aflatoxine” et toxicite de relais, C.R. Acad. Sc. Paris, t. 284 (7 March, 1977). [13] Ndiaye, Ah.L. La toxicite de relais dans ses rapports avec les substances naturellement presentes dans les aliments. Toxicological European Research, 4 (3,243-255 (1982). [I41 Ferrando, R., Palisse-Roussel, M., Jacquot, L. Toxicite de relais de I’aflatoxine MI de la poudre de lait. Etude a moyen terme sur le caneton, C.R. Acad. Sc. Paris, t. 298, sdrie 111, no 13 (1984). [15] Baer, J. E., Jacob, T. A., Wolf, F. J. Cambendazole and nondrug macromolecules in tissue residues, Journal of Toxicology and Environmental Health, 2 , 895-903 (1977). [I61 Martin, I? Albendazole These E.N.V., Lyon, 1980. [ 171 Delhoste, C. Albendazole: Essai de corrdlation entre embryotoxicite, metabolites libres, mktabolites lids, Thtse E.N.V., Lyon, 1983.
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[ 181 Grannec, C. Albendozole: Embryotoxicit6 de relais, T h b e E.N.V.,
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Lyon, 1980. [I91 Delatour, P, Parish, R. C . , Gyurik, R. J. Albendazole: a comparison of relay embryotoxicity with embryotoxicity of individual metabolites, Ann. Rech. Vet., 12 (21, 159-167 (1981).
