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Chapter 11 FURAZOLIDONE O2N<\c>N\N~o H \~/ Furazolidone occurs as an odorless, yellow crystals that melt at 275°C. The crystals will darken under strong light and are decomposed by alkali. Furazolidone ' s volubility in water (pH 6 ~ is approx ima te ly 4 0 mg/1 . I t is a lso known by the following synonyms and trade names: 3-[ [~5-nitro-2-furanyl~methylenelamino]- 2-oxazolidinone, 3-~5-nitrofurfurylideneamino)-2-oxazolidinone, _-(S-nitro-2-furfurylidene)-3-amino-2-oxazolidone, NF 180, Furovag, Furoxane, Furoxone, Giarlam, Giardil, Medaron, Neftin, Nicolen, Nifulidone, Ortazol, Roptazol, Tikofuran, and Topazone. Furazolidone is produced synthetically from furfural, hydroxyethylhydrazine, and d iethyl carbonate . PRODUCTI ON The sole U. S . producer of furazol idone is the Norwich Pharmaceutical Company in Norwich, N.Y. (Stanford Researab Institute International 1979 ~ . The compound is prepared according to procedures described in U.S. patents 2,759,931 and 2,927,110 to Norwich Pharmaceutical Co. It was first produced in 1953 as a veterinary medicinal and feed additive, and in 1957 as a human 288
systemic medicine! for U.S. and worldwide use (Federal Register, 1976a,b; Bryan, 19783. The U.S. International Trade Commission (1976-1978) reports furazolidone data obtained on group of 20 pher~ceutical chemicals whose combined production totals approximately 3.2 million kg/year. The fact that production is reported to and by the commission indicates that annual production is 450 kg or ore. USES Furazol idone is one of f ive S-nitrofurans currently approved for use as systemic veterinary medicines in the United States (Federal Register, 1976a,b; Bryan, 1978) . Its use was approved in 1953 to treat turkeys and chickens for fowl typhoid, paratyphoid, and pullorum; blackhead (histomoniasis); nonspecific enteritis (blue comb, mud fearers , ulcerative enteritis (quail disease), and synovitis (ar thr itis due to f Alterable virus); and paracolon infection (Paracolobactrum). Furazolidone use is permitted in chickens for infectious hepatitis and coccidiosis, in turkeys for hexamitiasis, and in swine for bacterial enteritis (necrotic enteritis, black scours) or vibrionic (bloody) dysentery (Federal Register, 1976a) . Furazol idone is one of two 5-nitrofurans that have been approved in the United States as veterinary feed additives (Federal Register, 1976a,b), and it accounts for approximately 97% of the 5-nitrofurans administered to food-producing animals (Federal Register, 1976b) . As a feed additive, the compound has been approved 289
for use in chickens and turkeys to prevent fowl typhoid, paratyphoid, pullorum, air-sac infection and paracolon infection, and to enhance growth and feed ef f iciency. In swine, it is approved for the prevention of bacterial enteritis and vibrionic dysentery, and for growth promotion and enhanced feed eff iciency (Federal Register, 1976a) . In 1976, the Food and Drug Administration (FDA} pub' ished proposals to withdraw approval of furazolidone for the Deter inary purposes for which it is now used (Federal Register, 1976a ,b) . To date, no f inal action on these proposals has been taken. Furazol idone has been used in humans to treat bacillary dysentery, typhoid and paratyphoid fevers, giardiasis, brucellosis, and intestinal infections of undetermined etiology {Bryan, 1978; Miura and Reckendorf, 1967: Paul and Paul, 1964, 1966~. EXPOSURE Furazolidone has been the subject of controversy in recent years. The chemical has been determined to cause cancer when ingested by rats and mice, although the producer has challenged this finding. In 1976, the FDA proposed to withdraw approval for use of the drug in food-producing animals (Federal Register, 1976a} . At that time, the agency ruled that data were not adequate to determine the total drug-related residues that can occur, that the analytic techniques for measuring the drug were not reliable for the lower concentrations found in food, that the drug was present in edible tissue following medication when no withdrawal period was observed, 290
and that a reliable withdrawal period could not be established from the information ava ilable. The results of testing for furazolidone in controlled exper iments a re shown in Table 11-1 . As ind icated, each study concluded the t residues were undetectable (usually with a detection limit of 1 to 5 mg/g) after a 2- to 19-day withdrawal period. The 1976 proposal to ban the use of furazolidone in food-product ng an imals was withdrawn because of problems in obta in ing supportive data, but another proposal is being issued in 1980 (Moy, Food An ima 1 Add i t ive Bra nch, FDA, per sona 1 conm~un ice t ion, 1980 ~ . Although FDA requ ires that food products con ta in no res idue, compliance is handled by the U. S. Department of Agriculture. Because analytic techniques for sampling at low concentrations are unreliable, the existing regulations have not been enforced. It is not possible to estimate the degree to which humans are exposed to this drug. The largest potential for exposure occurs from its presence in food. However, the residue disappears or, at the very least, becomes undetectable, within 20 days after the withdrawal of medication . Thus, the concentrations expected in furazolidone-treated food products cannot be estimated. Moreover, there is no information concerning the percentage of the total poultry and swine treated with the drug. Minute quantities may be released to the air, water, and solid waste in the vicinity of the plant. 291
Table 11-1 Results of Testing for Furazol idone in Food Food Hype Dose Res idue Reference Chicken ~Excessive. None after 2 days Krieg and Loeliger, 1973 Eggs 100 mg/100 ml water 8 mg/g on 3rd day; Krieg, 1972 for 3 days, and none after 10 S mg/100 mg for 2 days days Eggs 40 mg/100 g water 23 micrograms/9 Krieg, 1972 for 15 days on 6th day; none after 19th day Vea 1 Unknown None found Nouws, 1973 292
ANALYTIC METHODS Most of the analytic methods for furazolidone are based on thin-layer chromatography (TLC), high-pressure liquid chromatography IMPLY), W absorption spectrophoto~netry, or comb ina t ion s of the se techn ique s . A general procedure using TLC to identify 18 drugs, including furazolidone, in animal feed was reported by Williams (1978~. The sample was extracted with acetonitrile-chloroform (4:1), and an aliquot of the extract was cleaned on a column of aluminum oxide. The concentrated eluate was then subjected to TLC on silica gel G by using chloroform-methanol (9:1) to develop the plate. Spots were made visible on the plates by spraying with 1,2-diaminoethane or with Dragendorff's reagent. Cieri (1978) also reported a method for determining furazolidone in animal feed. The sample was extracted with acetone and cleaned on TLC plates of silica gel H by using chloroform-methanol (9 :1) to develop the plates. The appropriate zone was scraped from the plate, eluted with ethyl alcohol, and quantitated at its absorption maximum near 360 nm. The method was reported not to be applicable at levels below 0.005%. Moreta in et al . (1979 ~ described a procedure for determining furs zol idone and furaltadone in admixture at the levels expected in animal feed ~ i.e., SO to 200 ppm) . The sample was extracted 293
with dimethylformamide (DMF) and subjected to TLC on silica gel plates developed with chloroform-acetone (7:31. The appropriate zones were then scraped from the plates, extracted with DMF, and determined spectrophotometr ically at 370 nm. Methods reported recently have all been based on HPLC. Lefeb~re (1979 ~ demonstrated that furazolidone could be assayed by HPLC, using a glassy carbon or carbon paste electrode, coupled with a voltametr ic-amperometric detector. Detection limits were reported to be in the low nanogram range; however, no actual residue assays were performed. Using HPLC Jones et al. (1978) determined furazolidone levels in swine and poultry feed at levels as low as 5ppm, with a silica column and a mobile phase of water-saturated dichloromethone. The W-visible detector was set at 360 nm. The sample preparation consisted only of extracting the feed with methanol and 2 N hydrochloric acid (1:1), partitioning the residue into dichloromethane, and concentrating the solvent for a 20 pi injection into the HPLC system. Again using HPLC, Cieri (1979) determined residues of furazolidone and nitrofurazone in feeds at levels as low as 0.5 ppm. The sample was extracted with DMF-acetone (1:1), cleaned on a column of silica gel, and analyzed on a reverse-phase column with 30% acetonitrile as the mobile phase. The detector was set at 365 nm. Hoener et al. (1979) recently reported an HPLC procedure for determining residues of furazolidone in turkey tissue at levels as low as 2 ppb. The tissue was ground with methanol and centrifuged. 294
The extract then was either injected directly or concentrated be fore be ing in jec ted into the HPLC system. A Bondapak Cue column was used with a mobile phase consisting of methanol and 0.01 mol sodium acetate (1:47. We W absorption detector was set at 365 nm. Recoveries from muscle and liver spiked at the 2 ppb level were 103 + 19% and 112 + 12%, respectively. Bagon { 1979 ~ used a Spher isorb S5-ODS column for HPLC separations of several antibiotics and nitrofurans in pharmaceutical preparations. Furazolidone was separated from nitrofurazone by using a mobile phase of water-methanol (11:9) with the absorption detector set at 375 nm. Although no sables other than pharmaceutical preparations were analyzed, the procedure is sa id to be generally capable of distinguishing the parent compounds from decomposition products and likely congeners. Problems associated with the analysis of furazolidone in edible tissues of at levels of 0.5 to 4.0 ppb with methods ava liable prior to 1976 are well documented (Federal Register, 1976~. Various spectrophotometric and TLC procedures failed to yield satisfactory and reproducible recoveries at these levels. 295
HEALTH EFFECTS Metabol Sam Furazolidone is biotransformed In Vito in mammals to a variable but major extent (Swaminathan and Lower, 1978~. Two to five metabolites have been suggested from In vitro or in viva analyses (Federal Register, 1976a; Tatsumi et al., 1978~. One biotransformation product, 3-~4-cyano-2-oxobutylideneamino)-2- oxazolidone, was characterized by mass-, ultraviolet-, and nuclear-magnetic-resonance-spectroscopic methods following furazol idone incubation In vitro with milk xanthine oxidase or administration in viva to rabbits, when it was identif fed in urine. In contrast to furazolidone, this metabolite was not an active mutagen in Salmonella typhimurium TA 100 (Tatsumi et al., 1978) . Acute Toxicity Humans. Reported symptons of acute toxicity of furazolidone in humans include nausea, emesis, occasional diarrhea, abdominal pain, and bleeding (Cohen, 1978~. Infrequently, there have been reports of an Antabuse-like reaction to alcohol (Cohen, 1978) or, rarely, idiosyncratic or hypersensitivity reactions such as pneumonitis (Cohen, 1978; Collins and Thomas, 1973; Cortez and Pankey, 1972; Jira sek and Kalensky, 1975 ~ . Hemolytic anemia due to glucose-6-phosphate- dehydrogenase def iciency has been reported with furazo' idone (Cohen, 1978) . Furazolidone demonstrated monoamine-oxidase inhibition (Pettinger and Gates, 1968 ~ and 296
required precautions when coadministered with other moneamine-oxidase inhibitors, sympathomimetic amines, or tyramine-containing foods. No data have been reported concerning chronic toxic effects of f u ra zol idone in humans . Animals. Furazolidone induced emesis and necrologic changes in dogs (Miura and Rekkendorf, 1967; Rogers et al., 1956) . No published oral LD50 data exist for manunals. The drug also induced cardiomyopathy in turkeys (Czarnecki et al. , 1978; Staley et al.. 1978 ) . Ch ron ic Tox ic i ty Care inogenic ity Humans. No furazolidone-associated carcinogenicity in humans teas been repor ted . An ima Is . Fura zol idone wa s eve lusted for ca rc inogen ic activity in six studies in rats and one study in mice (Federal Register, 1976a,b; Cohen, 1978), but no review of these studies teas yet appeared. However, the FDA has summarized the statistically signif icant ef fects resulting from its analyses of the data submitted (Federal Register, 1976a,b) and has used these evaluations as the basis for proposed regulatory action (Federal Register, 1976a,b). Statistical comparisons of significant effects in rats and mice of both sexes exposed to varying dose levels of furazolidone administered orally for 18 or more months are presented in Tables 11-2 through 11-5. 297
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Furazolidone was fed to 35 female Sprague-Dawley (Holtzman strain) rats for 45 weeks followed by a drug-free diet for 8 additional weeks before the rats were sacrificed. A control group of 35 rats was fed a drug-free diet for 53 weeks. Rats fed furazolidone had a signif icantly higher incidence. of mammary tumors than did control rats (Federal Register, 1976a). In second study, 20 male and 20 female rats (CFE strain) were fed furazolidone for 45 weeks. The animals then received a drug-free diet for 7 more weeks. Control groups of both sexes were fed a drug-f ree diet for the 52 weeks . The female rats fed furazol idone had a higher inc idence of mammary tumors than did the female controls. No signif leant effects on tutor development were noted in male rats (Federal Register, 1976a) A third 2-year evaluation of furazolidone toxicity was also reported (Federal Register, 1976a) for 60 rats divided into three groups, each consisting of 10 males and 10 females. One group of each sex was fed a furazolidone-free control diet. The diets for the other groups contained two levels of furazolidone. There were three times as many tumors in rats fed furazolidone at a level of 0.01% in the diet as compared to the number of tumors in the control group Federal Register, 1976a) . A four th study involved 2-month-old ma le and fema le Sprague-Dawley (Charles River stra in) rats fed furazolidone (Table 11-2~. The number of female rats with malignant mammary tumors 302
increased as the dosage level increased, and the dose-response relationship was reported as linear and significant {P <0.01~. At the highest dose level (1 , 000 ppm), the proportion of female rats with malignant tumors was significantly higher (P <0.05) than that of the control rats fed the furazolidone-free diet. Furazolidone also concurrently induced benign mammary tumors in female rats. Furazolidone did not induce mammary tumors in male rats; however, it did randomly induce tumors in other body tissues. The proportion of wale rats that developed tumors of other tissues at the 1,000 ppm dose level was significantly higher (P <0.05) than that of controls. Finally, furazolidone-treated male and female rats exhibited a drug-related early mortality (Federal Register, 1976a) . A fifth study in which furazolidone was fed to 2-month-old male and female Fischer 344 rats is summarized in Table 11-3. The proportion of female rats with malignant mammary tumors at the 1,000-ppm level was significantly higher (P <0.05) than that of control rats. Malignant mammary tumors were diagnosed only in fema le ra ts fed the highest dosage of furazolidone. At all dosage levels, the proportion of female rats with mammary tumors was signif icantly higher (P <O. 01} than that of control rats. A signif icant relationship between the dosage of furazolidone and the proportion of female rats with mammary tumors was reported (Federal Reg i s te c, 19 7 6a ~ . The proper t ion of fema le ra ts w i th mu 1 t iple mammary tumors was signif icantly higher (P <0. 01) for all treatment groups than in controls. Other tumors that occurred more signif icantly (P <0 . 05) in male and female rats fed furazolidone 303
include thyroid and sebaceous adenomas (both sexes), testicular masotheliomas, and basal cell epitheliomas in male rats (Table 11-3~. A dose-response (early mortality) relationship was signif leant for both male and female rats (Federal Register, 1976a) . A sixth study, in which furazolidone was fed to 2-month-old male and female Sprague-Dawley (Charles River strain) rats, is summarized in Table 11-4 . There was a signif icant (P <0 .05 ~ linear dose-response relationsh ip for the number of female rats with tumors, and a significant increase {P <0.05) in the proportion of female rats at the 375-ppm dose level with tumors, as conga red to females fed a furazolidone-free diet. The types of tumors observed are shown in Table 11-4 . A signif icant increase (P <0 .05 ~ of the inc. idence of multiple mammary tumors for both the 125- and 375-ppm dose groups (as compared to controls) was noted, with a significant (P ~ O .05 ~ linear dose-response relationship. Furazolidone resulted in signif icant and early onset and development of mammary tumors in female rats at the 375-ppm dose level as compared with that of controls. There was a significant linear dose response for mammary tumor development in female rats dur ing the f irst 16 months of the study. Early significant mortality (P <0.05l, which was dose related, was reported for female rats. No significant tumor or mortality rate differences were found for male rats (federal Reg ister, 1976a ~ . A study of furazolidone fed to 2-month-old male and female Swiss MBR/ICR mice is summarized in Table 11-S. Bronchial adenocarcinomas were signif icantly higher (P <0 .05 ~ in both male and female mice 304
than in controls fed a furazolidone-free diet. Incidence of this growth in both sexes showed a significant (P <0.01) and linear dose response (Federal Register, 1976a). me dose-response relationships for malignancies of all tissue types as well as for the incidence of all tumors were significant (P <0.05) and linear for both sexes. There was a s ignif icant increase (P <0. 05) both in the development of malignancies and in the development of benign and malignant tumors combined in ma le mice at the lSO and 300 ppm levels, and in female mice at the 300 ppm level, as compared with those of control mice. Male mice that received the 300 ppm dose tad significantly more (_ <0 .05 ~ mult iple tumors than did control mice. mere was also a significant linear dose-response relationship for tumor multiplicities for female mice fed furazolidone. Male and female mice fed furazolidone both exhibited significant {P <0.05) early mortality which was linear in dose response (Federal Register, 1976a) . Mutagen icity The data from mutagenicity tests of furazolidone are summarized in Table 11-6. Furazolidone was found to be mutagenic in Escherichia cold (Lu et al., 1979; McCalla and Voutsinos, 1974) and Drosoph i la me lanoga s ter ~ B1 i j leveh _ a 1 ., 19 77 ~ caused ch romosoma l damage in human lymphocytes (Cohen and Sag i , 1979 ), and cross-linking of DNA in Vibrio cholerae (Chatter jee et al., 1977) . 305
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Bacterial Tests. Furazolidone was among 22 nitrofurans that McCalla and Voutsinos (1914 ~ found to be mutagenic in E. cold WP2 and its u~rrA-der ivative by reversion from trp~ to trp+. ExrA~or recA tester strains were not induced to mutate by the nitrofurans, indicating that mutants ar ise when the lesions induced by these compounds are repaired by the ~error-prone. repa ir system, components of which are coded for by rec and exr genes. Results obta ined with the nfr-stra ins, which lack n itrofuran reductase I, suggest that the nitro group is ~ key structural component, and that only when the nitro group is reduced and the drug converted into a more reactive compound are mutants induced. In this respect, mutagenesis of nitrofurans is similar to the fornication of alkali-labile DNA lesions by these compounds {McCalla et al., 1971) . The mutagenic activity of the nitrofurans so far tested covers a wide range (Lu _ al., 1979, McCann et al., 19751. Lu et al. {1979) reported an approximately 10,000-fold range in mutagenicity of eight nitrofurans. including furazolidone. The most active compound was 2- (2-furyl) -3- (5-nitro-2-furyl} acrylamide, (AF-2) which was approximately 6 times as mutagenic as furazolidone. Lu et al. (1979) ranked these compounds from most to least toxic to E. cold AS follows: AF-2 > N- ~ (5-n itro-2-furyl ~ th is zolyl ~ formamide (FANPT} ~ 2-amino-4- (5-nitro-2-furyl) thiazole (ANFT} ? furazolidone 1- ~ ~ 3 - (5 -n itro-2-fura nyl ~ -2-propenyl id ine] amino] -2, 4-imidazol idinedione (furag in) ~ nitrofurazone > methylnitrofuroste > nitrofuroate. In general, mutagen ic activity was found to parallel toxicity. Insect Tests. The genetic ef feats of furazolidone were determined in _. melanogaster by the induction of sex-linked recessive lethals, 307
which is the most sensitive mutation test in this organism (Blijle~reh _ al. 19771. Furazolidone was fed to adult males, which were then mated with fresh virgins for three consecutive periods of 3, 2, and 2 days. A consistent increase in the frequency of sex-linked recessive lethals was observed for furazolidone under these Conditions, indicating that the spermatids are sensitive to this compound, as they were to AF-2 {Blijleven et al., 1977) . Although the mutation frequency for furazolidone is low in this system (as in AF-2), and a demonstration of dose-response effect is lacking, the present data indicate that furazolidone and AF-2 are mutagen ic in Drosophila . Chron~osoma l Damage Mammalian Cells in Culture. The capacity of furazolidone to induce chromosomal damage (chromosomal breaks, sister-chromatic exchange, DNA repa ir synthesis, and inhibition of mitosis) in cultured human per ipheral lymphocytes was examined by Tonomura and Sasa k i ( 1973 ~ and Cohen and Sag i ( 1979 ~ . Tomomura and Sasak i ( 1973 ~ did not report either a signif leant amount of chromosomal abberation or unscheduled DNA synthesis for furazolidone over an 0.5 to 100 x 10 6 M dose range. In contrast, Cohen and Sagi (1979) found that furazol idone produced dose-dependent mitotic suppression, chromosomal breakage, and sister-chromatic exchanges (SCE 's) . file different results are surprising and difficult to explain, in that the experimental design of the two studies (e.g., range of drug concentration used, exposure times, solvents, methods of cytologic scoring) were similar. However, the fact that furazolidone induced 308
dose-dependent SCE in the Cohen and Sagi study (1979 ~ and that this type of chromosomal damage was not reported by ]'omomura and Sasaki (1973 ~ suggests that this compound is capable of inducing chromosomal damage in human cells. Further studies are needed to clarify this point. Bacterial DNA. Furazolidone inhibits DNA synthesis in V. cholerae cells while stimulating RNA and protein synthesis and causing filamentation of these cells (Chatterjee and Haiti, 19737. Chatterjee et al. (1977) reported that interstrand cross-linking in DNA takes place with in the furazol idone-treated V. cholerae cells, which therefore might explain the actual mechanism of inhibition of DNA biosynthesis by this drug. The In vivo action of furazolidone has considerable similar ity to that of miton~cin C (Iyer and Szybalsk i, 1964 ~ . Both agents induce interstrand cross-linking in DNA, inhibit DNA synthesis, and cause filamentation of the cells at the appropr late dose level by inhibiting cell division. Te ratogen ic ity No data were ava liable to evaluate the teratogenicity or embryotoxicity of furazolidone. CONCLUS IONS Furazol idone has exhibited carcinogenic effects in male and female rats and mice in a variety of tissues. Susceptible tissues are dif ferent for the two species. Furazolidone, in common with 309
most 5-nitrofurans studied, is a significant carcinogen in rodents (Cohen, 1978~. Furazolidone is highly mutagenic in both microbial (E. coli) and insect (D. melanogaster) test systems, produces chromosomal damage (breakage, SCE, mitotic suppression) in human lymphocytes, and forms interstrand cross-linking in bacterial (V. cholere) DNA. It is for the above reasons that the use of furazolidone is now being reviewed by the FDA. Resolution of this matter awaits the development of a sufficiently sensitive and reliable analytic method. If risk to human health does exist, it most certainly would be associated with the use of furazolidone for veterinary purposes. A ~solution" may involve the substitution of an efficacious product that could be demonstrated not to have the mutagenic and carcinogenic potential of furazolidone. 310
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