Residue depletion study and withdrawal period for flunixin-N-methyl glucamine in bovine milk following intravenous administration

The objective of this study was to establish a withdrawal period for flunixin in milk by quantifying 5-hydroxyflunixin, the marker residue, in bovine milk as a function of time, following intravenous treatment of lactating dairy cows with flunixin-N-methyl glucamine (Banamine or Finadyne). Lactating dairy cows were dosed on three consecutive days at 2.2 mg of flunixin free acid/kg of body weight/day. Milk was collected twice daily and assayed using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) procedure. The method was validated at concentrations in the range 0.5-250 ppb. The concentrations for 5-hydroxyflunixin measured 12 h after the last administration of drug ranged from 1.56 to 40.6 ppb for all cows. Milk concentrations for 5-hydroxyflunixin were used to establish withdrawal periods of 36 h using guidelines established by the U.S. Food and Drug Administration/Center for Veterinary Medicine and 24 h using guidelines established by the European Medicinal Evaluation Agency/Committee on Veterinary Medicinal Products.     

Determination of total 14C residues of sarafloxacin in eggs of laying hens

[14C]sarafloxacin was orally administered to six laying hens for five consecutive days. Eggs were collected for 15 days after the initial drug treatment. Egg yolk and egg albumen were separated and assayed for total radioactive residues (TRR) using a combustion oxidizer and scintillation counting techniques. Radioactivity was detected in egg yolk and egg albumen on the second day of dosing and reached a maximum at 24 h after drug withdrawal. Thereafter, the sarafloxacin TRR levels in egg albumen declined rapidly and were undetectable 2 days after the last dose, whereas the levels in egg yolk declined at a much slower rate and were undetectable 7 days after drug withdrawal. In both the egg albumen and yolk, HPLC analysis indicated that the parent sarafloxacin was the major component.     

Tissue distribution, elimination, and metabolism of sodium [36Cl]perchlorate in lactating goats

Perchlorate has contaminated water sources throughout the United States but particularly in the arid Southwest, an area containing large numbers of people and few water sources. Recent studies have demonstrated that perchlorate is present in alfalfa and that perchlorate is secreted into the milk of cows. Studies in lactating cows have indicated that only a small portion of a perchlorate dose could be accounted for by elimination in milk, feces, or urine. It was hypothesized that the remainder of the perchlorate dose was excreted as chloride ion. The purpose of this study was to determine the fate and disposition of (36)Cl-perchlorate in lactating dairy goats. Two goats (60 kg) were each orally administered 3.5 mg (16.5 muCi) of (36)Cl-perchlorate, a dose selected to approximate environmental perchlorate exposure but that would allow for adequate detection of radioactive residues after a 72 h withdrawal period. Blood, milk, urine, and feces were collected incrementally until slaughter at 72 h. Total radioactive residue (TRR) and perchlorate concentrations were measured using radiochemical techniques and liquid chromatography mass spectrometry (LC-MS-MS). Peak blood levels of TRR occurred at 12 h ( approximately 195 ppb) postdose; peak levels of parent perchlorate, however, occurred after only 2 h, suggesting that perchlorate metabolism occurred rapidly in the rumen. The serum half-life of perchlorate was estimated to be 2.3 h. After 24 h, perchlorate was not detectable in blood serum but TRR remained elevated (160 ppb) through 72 h. Milk perchlorate levels peaked at 12 h (155 ppb) and were no longer detectable by 36 h, even though TRRs were readily detected through 72 h. Perchlorate was not detectable in skeletal muscle or liver at slaughter (72 h). Chlorite and chlorate were not detected in any matrix. The only radioactive residues observed were perchlorate and chloride ion. Bioavailability of perchlorate was poor in lactating goats, but the perchlorate that was absorbed intact was rapidly eliminated in milk and urine.     

Tissue distribution, metabolism, and residue depletion study in Atlantic salmon following oral administration of [3H]emamectin benzoate

Atlantic salmon (approximately 1.3 kg) maintained in tanks of seawater at 5 +/- 1 degrees C were dosed with [3H]emamectin B1 benzoate in feed at a nominal rate of 50 microg of emamectin benzoate/kg/day for 7 consecutive days. Tissues, blood, and bile were collected from 10 fish each at 3 and 12 h and at 1, 3, 7, 15, 30, 45, 60, and 90 days post final dose. Feces were collected daily from the tanks beginning just prior to dosing to 90 days post final dose. The total radioactive residues (TRR) of the daily feces samples during dosing were 0.25 ppm maximal, and >97% of the TRR in pooled feces covering the dosing period was emamectin B1a. Feces TRR then rapidly declined to approximately 0.05 ppm by 1 day post final dose. The ranges of mean TRR for tissues over the 90 days post dose period were as follows: kidney, 1.4-3 ppm; liver, 1.0-2.3 ppm; skin, 0.04-0.09 ppm; muscle, 0.02-0.06 ppm; and bone, <0.01 ppm. The residue components of liver, kidney, muscle, and skin samples pooled by post dose interval were emamectin B1a (81-100% TRR) and desmethylemamectin B1a (0-17% TRR) with N-formylemamectin B1a seen in trace amounts (<2%) in some muscle samples. The marker residue selected for regulatory surveillance of emamectin residues was emamectin B1a. The emamectin B1a level was quantified in individual samples of skin and muscle using HPLC-fluorometry and was below 85 ppb in all samples analyzed (3 h to 30 days post dose).     

Analysis of veterinary drugs and metabolites in milk using quadrupole time-of-flight liquid chromatography-mass spectrometry

A quadrupole time-of-flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze veterinary drug residues in milk. Milk samples were extracted with acetonitrile. A molecular weight cutoff filter was the only cleanup step in the procedure. Initially, a set of target compounds (including representative sulfonamides, tetracyclines, β-lactams, and macrolides) was used for validation. Screening of residues was accomplished by collecting TOF (MS(1)) data and comparing the accurate mass and retention times of found compounds to a database containing information for veterinary drugs. The residues included in the study could be detected in samples fortified at the levels of concern with this procedure 97% of the time. Although the method was intended to be qualitative, an evaluation of the MS data indicated a linear response and acceptable recoveries for a majority of target compounds. In addition, MS/MS data were also generated for the [M + H](+) ions. Product ions for each compound were identified, and their mass accuracy was compared to theoretical values. Finally, incurred milk samples from cows dosed with veterinary drugs, including sulfamethazine, flunixin, cephapirin, or enrofloxacin, were analyzed with Q-TOF LC-MS. In addition to monitoring for the parent residues, several metabolites were detected in these samples by TOF. Proposed identification of these residues could be made by evaluating the MS and MS/MS data. For example, several plausible metabolites of enrofloxacin, some not previously observed in milk, are reported in this study.     

Metabolism and distribution of [2,3-(14)C]acrolein in lactating goats

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.     

Liquid chromatographic determination of depletion of bithionol sulfoxide and its two major metabolites in bovine milk

A liquid chromatographic method is described for determining bithionol sulfoxide and its metabolites, bithionol and bithionol sulfone, in milk. Samples are treated with HCl to precipitate proteins and to permit extraction of bithionol sulfoxide in nonionized form. Tetrahydrofuran is added to the organic phase to facilitate extraction in diethyl ether; the dried residue is dissolved in chloroform, hexane, and sodium hydroxide and subjected to LC analysis. Residues of bithionol sulfoxide and its 2 metabolites were determined in milk of lactating cows. Holstein-Friesian dairy cows were administered a single oral dose of bithionol sulfoxide (50 mg/kg). Milk samples were analyzed with a reliable detection level of 0.025 microgram/mL for each compound. Residues of bithionol sulfoxide and bithionol were detected during 30 and 16 milkings, respectively; bithionol sulfone was never present at detectable levels.     

Nature of the residue of [(14)C]Cloransulam-methyl in lactating goats

Two lactating goats were given a daily oral dose of either [UL-aniline-(14)C; AN] or [triazolopyrimidine-7,9-(14)C; TP]cloransulam-methyl for 5 consecutive days. Each animal received a dietary equivalent of approximately 10 mg/kg of test material, approximately 2225 times the realistic maximum dietary exposure for a dairy animal. Milk, urine, and feces samples were collected in the morning and afternoon for each animal. Each goat was sacrificed within 23 h of receiving the last dose, and the liver, kidneys, samples of blood, fat, muscle, and gastrointestinal tract contents, and urine from the bladder were collected. All of these samples were analyzed for (14)C content. Cloransulam-methyl (CM) was rapidly excreted by the animals, with 99.9% of the recovered radioactivity appearing in the urine and feces. Radiochemical analysis showed very low residues, with the highest being in the kidneys at 0.122 and 0. 128 mg equiv of CM/kg (AN and TP labeled compounds, respectively). Radioactive residues were extracted and fractionated from kidney, liver, and milk. Analysis showed approximately 0.066 mg/kg CM in the kidney but <0.003 mg/kg in the liver. Only one metabolite, cloransulam, was identified (in liver, 9.5% of total radioactive residue; 0.005 mg/kg). All other metabolites were present at lower levels. Sulfonanilide bridge cleavage was not a significant degradation route for cloransulam-methyl in ruminants. These data indicated a very low bioaccumulation potential for cloransulam-methyl and its metabolites in ruminants. For a ruminant exposed to anticipated levels of cloransulam-methyl in its diet, parent and metabolites, in total, would not be expected to exceed 50 ng/kg in the kidney and liver.     

Marker residue determination of tritium-labeled ivermectin in the muscle of aquacultured largemouth bass, hybrid striped bass, and yellow perch following oral treatment

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered 3H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 μCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of 3H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.     

An LC/MS/MS method for improved quantitation of the bound residues in the tissues of animals orally dosed with [(14)C]Benomyl

Livers of goats orally dosed with [phenyl(U)-(14)C]benomyl contained radioactive residues which were not extractable using conventional, solvent-based extraction methods. We report a new residue method capable of enhanced extraction of benomyl-derived residues with selective and sensitive quantitation capability for methyl 4-hydroxybenzimidazol-2-ylcarbamate (4-HBC), methyl 5-hydroxybenzimidazol-2-ylcarbamate (5-HBC), and methyl benzimidazol-2-ylcarbamate (MBC). This method involves rigorous Raney-nickel reduction of hypothesized thioether bonds between benomyl residues and polar cellular components. Following acidic dehydration (desulfurization), the polar benomyl-derived residues are extracted into ethyl acetate and analyzed by LC/MS/MS. We have shown this method to be superior to alternative extraction approaches. When applied to goat liver tissue containing [phenyl(U)-(14)C]benomyl-bound residues, the extraction efficiency of total radioactive residues was approximately 30%, and the major benomyl-derived residue was 5-HBC (91-95% of extractable residue) with minor levels of carbendazim (MBC) (5-9%). HPLC/LSC data were consistent with the LC/MS/MS data. The overall method satisfies U.S. regulatory requirements in extraction efficiency, selectivity in detection, and limits of quantitation for benomyl-bound residues.     

Uniformly (14)C-ring-labeled 2,4-dichlorophenoxyacetic acid: a metabolism study in bluegill sunfish, Lepomis macrochirus.

The fate of 2,4-dichlorophenoxyacetic acid (2,4-D), a mixture of [phenyl(U)-(14)C]-2,4-D and unlabeled 2,4-D, in bluegill sunfish was investigated after exposure to approximately 11 ppm under static conditions for 4 days. Total radioactive residues (TRR) in whole fish increased from 0.41 ppm on day 1 to 0.60 ppm on day 3. TRR levels in fillet (edible) and viscera (nonedible) of treated fish on day 4 were 0.41 and 1.9 ppm, respectively. Most residues in both matrices were acetonitrile soluble; small amounts were hexane soluble or unextractable with solvents. Acid and base hydrolyses with ethyl acetate partitioning were used to release the fillet unextractable residues. The identification of 2,4-D and 2,4-dichlorophenol (2,4-DCP) in the fillet was conclusively confirmed by GC-MS analysis. On the basis of the experimental data from this study, a metabolic pathway for 2,4-D in bluegill sunfish in which the 2,4-D is metabolized to 2,4-DCP and conjugates of 2,4-D and 2,4-DCP is proposed.     

Metabolism of [(14)c]chlorantraniliprole in the lactating goat

Metabolism of [(14)C]chlorantraniliprole {3-bromo-N-[4-chloro-2-methyl-6-[(methylamino)carbonyl]phenyl]-1- (3-chloro-2-pyridinyl)-1H-pyrazole-5-carboxamide} was investigated in a lactating goat following seven consecutive daily single oral doses. Each dose was equivalent to 10.4 mg/kg of feed. There was no significant transfer of residues of either chlorantraniliprole or its metabolites into fat, meat, or milk. Chlorantraniliprole and its metabolites accounted for 93.57% of the administered dose and were eliminated primarily in the excreta. Residues in meat, milk, liver, and kidney together accounted for ca. 1.5% of the administered radioactivity. A total of 19 metabolites including 3 glucuronide conjugates and intact chlorantraniliprole were identified in the feces, urine, or tissues by comparison of their HPLC retention times, mass spectral fragments (LC-MS/MS), or multiple reaction monitoring (MRM) transitions to authentic synthesized standards. The major metabolic pathways of [(14)C]chlorantraniliprole in the goat were N-demethylation, methylphenyl hydroxylation, and further oxidation to the carboxylic acid; loss of water from the N-hydroxymethyl group to yield various cyclic metabolites; and hydrolysis of N-methyl amides to form benzoic acid derivatives. Minor metabolic reactions involved cleavage of the amide bridge between the phenyl and heterocyclic rings of chlorantraniliprole.     

Determination of depletion and statistical distribution of morantel-related residues in bovine milk following administration of morantel tartrate to dairy cows

Residue depletion studies were conducted in dairy cattle to monitor morantel-related residues in milk following oral administration of morantel tartrate (Rumate. Eleven lactating cows of various ages, periods of lactation, and known milk production were orally dosed with the bolus formulation of morantel tartrate with an actual dose range of 8.4-9.8 mg/kg body weight. Representative samples of milk were collected at 10-14 h intervals post-dose, and subsamples were assayed for the major and minor hydrolysis products of morantel-related residues, MAPA and CP-20,107. Residues assayed as precursors of MAPA peaked at the second milking (24 h post-dose) and were below 25 ppb (range: less than 12-24 ppb). Precursors of CP-20,107, which confirm the identity of morantel, also peaked at 24 h post-dose (range: 2.1-3.3 ppb) and declined rapidly thereafter. A statistical model was used to project the level of residues at the upper limit of 99% of the total target animal (i.e., dairy cattle) population with 95% confidence. The calculated peak levels from this model were 50 and 5.0 ppb for morantel-related residues convertible to MAPA and CP-20,107, respectively.     

Naturally occurring estrogens in processed milk and in raw milk (from gestated cows)

The occurrence of the steroid hormones estrone (E1), 17alpha-estradiol (alphaE2), 17beta-estradiol (betaE2), and estriol (E3) in processed bovine milk with different fat contents and in raw milk from (non)gestated cows was investigated. Following liquid extraction, optional enzymatical deconjugation, C18 solid-phase extraction, and derivatization, estrogens were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free and deconjugated E1 (6.2-1266 ng/L) was the major estrogen followed by alphaE2 (7.2-322 ng/L) and betaE2 (5.6-51 ng/L), whereas E3 was detected regularly at the detection limit of 10 ng/L. The lowest and highest concentrations were determined in raw milk from nonpregnant and from cows in the third trimester of gestation, respectively. The estrogen concentration in processed milk coincides with that of raw milk between first and second trimesters, reflecting the contribution of lactating pregnant cows to the final consumable product. The daily intake of total investigated estrogens through milk is 372 ng, which is dramatically more than currently recognized.     

Effect of sodium [36Cl]chlorate dose on total radioactive residues and residues of parent chlorate in beef cattle

The objectives of this study were to determine total radioactive residues and chlorate residues in edible tissues of cattle administered at three levels of sodium [36Cl]chlorate over a 24-h period and slaughtered after a 24-h withdrawal period. Three sets of cattle, each consisting of a heifer and a steer, were intraruminally dosed with a total of 21, 42, or 63 mg of sodium [36Cl]chlorate/kg of body weight. To simulate a 24-h exposure, equal aliquots of the respective doses were administered to each animal at 0, 8, 16, and 24 h. Urine and feces were collected in 12-h increments for the duration of the 48-h study. At 24 h after the last chlorate exposure, cattle were slaughtered and edible tissues were collected. Urine and tissue samples were analyzed for total radioactive residues and for metabolites. Elimination of radioactivity in urine and feces equaled 20, 33, and 48% of the total dose for the low, medium, and high doses, respectively. Chlorate and chloride were the only radioactive chlorine species present in urine; the fraction of chlorate present as a percentage of the total urine radioactivity decreased with time regardless of the dose. Chloride was the major radioactive residue present in edible tissues, comprising over 98% of the tissue radioactivity for all animals. Chlorate concentrations in edible tissues ranged from nondetectable to an average of 0.41 ppm in skeletal muscle of the high-dosed animals. No evidence for the presence of chlorite was observed in any tissue. Results of this study suggest that further development of chlorate as a preharvest food safety tool merits consideration.     

Metabolic fate of 2,4-dichlorophenol and related plant residues in rats

This study compared the metabolic fate of [(14)C]-DCP, [(14)C]-residues from radish plants, and purified [(14)C]-DCP-(acetyl)glucose following oral administration in rats. A rapid excretion of radioactivity in urine occurred for [(14)C]-DCP, [(14)C]-DCP-(acetyl)glucose, and soluble residues, 69, 85, and 69% within 48 h, respectively. Radio-HPLC profiles of 0-24 h urine from rats fed [(14)C]-DCP and [(14)C]-DCP-(acetyl)glucose were close and qualitatively similar to those obtained from plant residues. No trace of native plant residues was detected under the study conditions. The structures of the two major peaks were identified by MS as the glucuronide and the sulfate conjugates of DCP. The characterization of a dehydrated glucuronide conjugate by MS and NMR of DCP was unusual. In contrast to soluble residues, bound residues were mainly excreted in feces, 90% within 48 h, whereas total residues were eliminated in both urine and feces. For total residues, the radioactivity in feces was higher than expected from the percentage of soluble and bound residues in radish plants. This result highlighted that less absorption took place when residues were present in the plant matrix as compared to plant-free residues and DCP.     

Plasma and tissue depletion of florfenicol and florfenicol-amine in chickens

Chickens were used to investigate plasma disposition of florfenicol after single intravenous (i.v.) and oral dose (20 mg kg-1 body weight) and to study residue depletion of florfenicol and its major metabolite florfenicol-amine after multiple oral doses (40 mg kg-1 body weight, daily for 3 days). Plasma and tissue samples were analyzed using a high-performance liquid chromatography (HPLC) method. After i.v. and oral administration, plasma concentration-time curves were best described by a two-compartment open model. The mean [ +/- standard deviation (SD)] elimination half-life (t1/2beta) of florfenicol in plasma was 7.90 +/- 0.48 and 8.34 +/- 0.64 h after i.v. and oral administration, respectively. The maximum plasma concentration was 10.23 +/- 1.67 microg mL-1, and the interval from oral administration until maximal concentration was 0.63 +/- 0.07 h. Oral bioavailability was found to be 87 +/- 16%. Florfenicol was converted to florfenicol-amine. After multiple oral dose (40 mg kg-1 body weight, daily for 3 days), in kidney and liver, concentrations of florfenicol (119.34 +/- 31.81 and 817.34 +/- 91.65 microg kg-1, respectively) and florfenicol-amine (60.67 +/- 13.05 and 48.50 +/- 13.07 microg kg-1, respectively) persisted for 7 days. The prolonged presence of residues of florfenicol and florfenicol-amine in edible tissues can play an important role in human food safety, because the compounds could give rise to a possible health risk. A withdrawal time of 6 days was necessary to ensure that the residues of florfenicol were less than the maximal residue limits or tolerance established by the European Union.     

Liquid chromatographic analysis of incurred amoxicillin residues in catfish muscle following oral administration of the drug

Improper application of antibiotic chemicals to livestock and aquaculture species may lead to the occurrence of residues in food supplies. An appropriate depletion period is needed after the administration of drugs to animals for ensuring that residues in edible tissues are below established tolerance levels. This study was conducted to determine incurred amoxicillin residues in catfish muscle following oral administration. Dosed fish were harvested after four depletion periods, and muscle fillets were analyzed for amoxicillin residues using an HPLC method with precolumn derivatization and fluorescence detection. The residue levels in fish after a 6-h depletion ranged from 40 to 64 ng/g with one exception at 297 ng/g. Average residue levels decreased to 5.4 and 2. 8 ng/g after 24- and 48-h depletions, respectively. Residue levels after a 72-h depletion decreased to below the method's limit of quantitation (1.2 ng/g). An LC-MS/MS confirmatory method was developed. Confirmation of the presence of amoxicillin was demonstrated in incurred fish samples containing residues at approximately 50-300 ng/g.     

Neutral cleanup procedure for 2,3,7,8-tetrachlorodibenzo-p-dioxin residues in bovine fat and milk.

A neutral cleanup method for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in milk and animal tissue was developed involving solvent extraction and liquid adsorption chromatography on magnesia-Celite 545, alumina, and Florisil. Cleaned up extracts were subjected to dual-ion analysis in a direct probe high resolution mass spectrometer, interfaced to a multi-channel analyzer for signal averaging. Calibration experiments were carried out with bovine milk and beef fat samples containing added TCDD. The 37CI isotopic isomer of TCDD was added as an internal standard. The response was linear for concentrations in the ppt range, with recoveries about 80%. Milk from a cow fed TCDD was cleaned up by the neutral procedure or, alternatively, a base-acid extraction procedure. The TCDD recoveries for both procedures were essentially the same. Recoveries of TCDD from liver samples of a rat given 14C-TCDD intraperitoneally, subjected to neutral cleanup and radioactive counting, were about 70%.     

Effect of pressure cooking on aflatoxin B1 in rice

The effect of pressure cooking on aflatoxin residues in polished rice was conducted to determine reduction of aflatoxin and mutagenic potentials. Three rice lots consisting of naturally contaminated, A. parasiticus-infested, and aflatoxin-spiked rice were steamed by ordinary and pressure cookers after they were washed with water. They were chemically analyzed for aflatoxins using a silica solid phase extraction tube and high-performance liquid chromatography (HPLC)-fluorescence detection (FD), and the presence of aflatoxin residues was confirmed using HPLC-electrospray ionization (ESI)-mass spectrometry (MS). An in vitro mutagenicity test with Salmonella typhimurium TA100 was employed to verify the results based on chemical analyses. The aflatoxin loss (78-88%) was notable after pressure cooking, and the reduction of aflatoxin-induced mutagenic potential (68-78%) was in good agreement with the HPLC results. It can be concluded that Koreans are safe from the aflatoxin-related risk if a pressure cooker is employed for cooking rice. The average Korean daily intake of aflatoxin through the consumption of staple rice would fall to 0.15 ng/kg bw/day, which would not exceed the established tolerable daily intake (0.40 ng/kg bw/day).