An analysis of the 1987 list of IARC-identified human carcinogens and the correlated animal studies.

The thesis that the use of long-term, appropriately conducted and interpreted laboratory studies with rats and/or mice to test materials for potential carcinogenicity in humans is supported by the evidence on human carcinogens developed by Working Groups of the International Agency for Research on Cancer (IARC) is examined by a review of the Working Group reports on IARC-designated human carcinogens. The conclusion from that review is that the list of carcinogens does not constitute a sufficiently diverse group of agents to warrant a generalization; nor do the correlated animal studies justify the implied claim that most human carcinogens are demonstrated animal carcinogens, when the test for animal carcinogenicity is a properly conducted and interpreted study with rats and/or mice.

Rationales for the establishment of limits and regulations for mycotoxins.

Although 50 countries have enacted or proposed regulations for control of alfatoxins in food or feed, and 15 of these countries also have regulations for permitted levels of contamination by other mycotoxins, very few countries have formally presented the rationale for the need to regulate, or for the selection of a particular maximum tolerated level. After several successive inquiries, information concerning the rationale for regulation was obtained from 21 countries. Most of the responses concerned limits for aflatoxin in food, and most of these were based on a vague, unsupported statement of the carcinogenic risk for humans. There was a general consensus that exposure to a potential human carcinogen that could not be totally avoided should be limited to the lowest practical level; the definition of practicality depended on whether the country was an importer or producer of the potentially contaminated commodity. A claim to a hazard evaluation was made by six countries (Canada, Belgium, India, United Kingdom, United States, Switzerland) without providing specifics; and one country, South Africa, referred to a risk determination. The most comprehensive rationale for any mycotoxin regulation was provided by the United States in support of limits for aflatoxin in specific animal feedstuffs. The responses provided no rationale for setting limits for other mycotoxins; but scholarly risk assessments for zearalenone and ochratoxin A have been published by Canadian government scientists, and a symposium presentation provides the information that in Norway patulin is regulated for quality control purposes only. It is apparent that, in most countries, either the scientific basis for regulation of mycotoxins is nonexistent, or the science has not been fully utilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin is not a probably human carcinogen: the published evidence is sufficient.

Since the early 1960s, when aflatoxin, the mold-produced contaminant of a number of important food commodities, was found to be a potent hepatocarcinogen for laboratory rats, there has been a sustained search for evidence to support the regulatory presumption that aflatoxin is a probable human carcinogen. The developing laboratory evidence of differences between species in metabolism of aflatoxin and susceptibility to its oncogenic effects indicated that humans were probably refractory to aflatoxin carcinogenesis, but the early epidemiological evidence indicated otherwise. That epidemiological evidence, however, contained flaws so that Working Groups of the International Agency for Research on Cancer (IARC) meeting in 1970, 1976, and 1982, although ignoring the biochemical evidence, did consider the available epidemiological evidence insufficient for a conclusion of human carcinogenicity. During the 1970s and 1980s, studies on the connection between chronic infection with hepatitis B virus (HBV) and primary liver cell cancer (PLC), the expected lesion from aflatoxin exposure, had established a very strong etiological relationship between HBV and PLC. Since all the epidemiological studies of aflatoxin and PLC conducted prior to 1982 had been of populations with endemic HBV infection, and, in addition to other flaws, had not been controlled for this confounding factor, there was a solid basis for their rejection. Most epidemiological studies in the 1980s of aflatoxin and PLC were either in the United States, where HBV-infected groups could be excluded from the study, or, when in areas of chronic HBV infection, attempts were made to include that factor. The study of U.S. populations showed no difference in mortality rates from PLC that could be attributed to aflatoxin exposure. The studies of populations with endemic HBV infection produced no convincing evidence to support a primary role for aflatoxin in the induction of human PLC, although an accessory role to HBV infection for aflatoxin could not be ruled out. However, the epidemiological studies of the HBV/PLC relation indicate that an accessory factor is not an essential condition, a conclusion supported by animal models and a laboratory study that specifically found no interaction between aflatoxin and a hepatitis virus in the duck, a species in which liver cancer can be induced by either agent. It was surprising that an IARC Working Group meeting in 1987 concluded, on the basis of much of this evidence that was available at that time, and citing other studies that appear to be irrelevant to the issue, that there was sufficient evidence to consider aflatoxin a probable human carcinogen.

Aflatoxin control--how a regulatory agency managed risk from an unavoidable natural toxicant in food and feed.

The control by the Food and Drug Administration (FDA) of aflatoxin, a relatively recently discovered, unavoidable natural contaminant produced by specific molds that invade a number of basic food and feedstuffs, provides an example of the varying forces that affect risk assessment and management by a regulatory Agency. This is the story of how the FDA responded to the initial discovery of a potential carcinogenic hazard to humans in a domestic commodity, to the developing information concerning the nature of the hazard, to the economic and political pressures that are created by the impact of natural forces on regulatory controls, and to the restraints of laws within which the Agency must work. This story covers four periods: the years of discovery and action decisions on the basis of meager knowledge and the fear of cancer; the years of tinkering on paper with the regulatory process, the years of digestion of the accumulating knowledge, and the application of that knowledge to actions forced by natural events; and an audit of the current status of knowledge about the hazard from aflatoxin, and proposals for regulatory control based on that knowledge.

Determination of aflatoxins in peanut butter, using two liquid chromatographic methods: collaborative study.

Two methods for determining aflatoxins in peanut butter, one using normal phase and the other reverse phase liquid chromatography (LC), were studied by 8 and 10 collaborators, respectively. Fluorescence detection was used for the determinative step in both methods. For reverse phase LC, aflatoxins B1 and G1 were converted to B2a and G2a; for normal phase LC, a silica gel-packed flow cell was placed in the irradiating light path of the detector. The samples included spiked and naturally contaminated peanut butter with total aflatoxin levels from about 5 to 20 ng/g and controls in a balanced pair design. For the normal phase LC method, recoveries of B1, B2, G1, and G2 from spiked samples averaged 79, 92, 74, and 88%, respectively; for the reverse phase method, the recoveries were 103, 104, 89, and 163%. For the normal phase LC method, pooled repeatabilities were 20, 23, 28, and 17% for B1, B2, G1, and G2, respectively; for the reverse phase method, the repeatabilities were 19, 22, 38, and 31%. For the normal phase method, pooled reproducibilities were 34, 33, 39, and 34% for B1, B2, G1, and G2, respectively; for the reverse phase method, the reproducibilities were 32, 46, 51, and 52%. Both methods show an improved limit of detection and better within-laboratory precision over current AOAC methods; however, between-laboratory precision is no better, and the reverse phase method shows evidence of interferences being measured. For these reasons and because of no benefits of present value, neither method was submitted for adoption as official first action.

Determination of aflatoxicol and aflatoxins B1 and M1 in eggs.

Procedures from 2 methods, one for aflatoxins B1 and M1 in eggs and one for aflatoxicol in milk, blood, and liver, have been combined to determine the 3 toxins in eggs. The sample is blended with sodium chloride-saturated water and this mixture is then blended with acetone. After separation from the solid residue, the aqueous acetone extract is defatted with petroleum ether. The toxins are next partitioned into chloroform and separated from interferences on a silica gel column. Aflatoxicol is determined by fluorescence measurement after separation on a C18 reverse phase liquid chromatographic column, and aflatoxins B1 and M1 are determined by fluorescence densitometry after separation on a silica gel thin layer chromatographic plate. In a recovery study with eggs, mean recoveries of aflatoxicol added at levels of 0.1, 0.05, and 0.025 ng/g were 87, 77, and 78%, respectively. Mean recoveries of aflatoxins B1 and M1 added at a level of 0.1 ng/g were 75 and 87%, respectively, and at an added level of 0.05 ng/g were 86 and 75%. The within-laboratory precision (repeatability) ranged from 2 to 13%.

Aflatoxicol and aflatoxins B1 and M1 in eggs and tissues of laying hens consuming aflatoxin-contaminated feed.

This study was undertaken to relate quantitatively the aflatoxin residue found in eggs and tissues to the aflatoxin intake via feed. Eighteen hens were fed an aflatoxin B1 (B1)-contaminated feed (8 micrograms/g) for 7 days, after which half the group was sacrificed; the remainder were sacrificed after an additional 7 days on an aflatoxin-free diet. Eggs were collected over the entire 14-day period. Aflatoxicol (R0), B1, or both were found in eggs and tissues (kidneys, liver, muscle, blood, and ova). Aflatoxin M1 (M1) (.04 to .1 ng/g) was found only in the kidneys. Levels of R0 and B1 were approximately the same in eggs, ova, kidneys, and liver. In eggs, the levels of R0 and B1 (.02 to .2 ng/g) increased steadily for 4 or 5 days, after which time the levels plateaued and then decreased after B1 withdrawal at the same rate as they had increased. At 7 days after withdrawal, only trace amounts of R0 (.01 ng/g) remained in eggs. All tissues, except blood, from hens sacrificed immediately before aflatoxin withdrawal contained R0 (.04 to .4 ng/g) or R0 and B1 (.04 to .8 ng/g). The R0 (.03 to .11 ng/g) was the only aflatoxin detected in muscle, and B1 (.05 to .07 ng/g) was the only aflatoxin in blood. Seven days after aflatoxin withdrawal, B1 (.08 ng/g) was found in one of nine livers and R0 (.01 to .04 ng/g) in eight of nine muscles analyzed, but no aflatoxins were found in any other tissues.

Absorption and distribution patterns of aflatoxicol and aflatoxins B1 and M1 in blood and milk of cows given aflatoxin B1.

Two 600-kg lactating cows were each given a single oral dose (0.5 mg/kg of body weight) of aflatoxin B1 (B1). Samples were obtained at postdosing hours 0, 1, 2, 3, 4, 6, 8, 10, and 12 and thereafter every 12 hours for 10 days. Aflatoxicol (Ro), B1, and aflatoxin M1 (M1) were found in the milk, plasma, and RBC of both cows at postdosing hour 1. Maximum concentrations of the toxins were observed at 12 and 60 hours. The ratio of the concentrations for Ro, B1, and M1 was approximately 1:10:100. Both cows had clinical signs of distress at 24 hours; 1 cow died at 60 hours and the other cow recovered within 4 days. In the samples of liver, kidney, urine, bile, and rumen contents of the cow that died, the B1 concentrations were 5.1, 3.3, 4.1, 1.6, and 320 ng/g, respectively, and the M1 concentrations were 4.3, 20, 37, 16, and 8.6 ng/g. The Ro concentrations in the kidney were approximately equal to that of B1; however, liver, urine, bile, and rumen contents concentrations were 0.88, 0.10, 0.36, and 4.9 ng/g, respectively.

Aflatoxin as a cause of primary liver-cell cancer in the United States: a probability study.

Primary liver-cell cancer (PLC) mortality ratios, computed from death certificate records compiled by the National Center for Health Statistics, for the periods 1968-1971 and 1973-1976 were sorted by race, sex, urbanization, and region. From this sort, rural white males from the Southeast and the "North and West" regions were selected for comparison of mortality ratios and past dietary exposure to aflatoxin. Based on projections of recent aflatoxin contamination information back to the 1910-1960 period, and estimates of corn and peanut usage from household food consumption surveys relating to that period, the expected average daily ingestion of aflatoxin B1 for each group was calculated (Southeast, 13-197 ng/kg bw; North and West, 0.2-0.3 ng/kg bw). An age-adjusted excess PLC mortality ratio was observed for the Southeast population when compared with the "North and West"-10% excess PLC deaths at all ages, and 6% excess PLC deaths for the 30-49 year age-group-but although the difference was in the expected direction in relation to projected past exposure to aflatoxin, it was far from the manyfold difference that would have been anticipated from experiments with rats and from prior epidemiological studies in Africa and Asia. The remaining major portion of the PLC mortality in the Southeast may be attributed to many unidentified causes for which the two populations that were compared were not controlled, leaving in doubt the validity of any attribution of the excess PLC mortality to aflatoxin ingestion. A considerable excess over average US PLC mortality ratios was seen for all Orientals resident in the US and for urban black males. Occurrence of PLC in Orientals has been related to the presence of markers for hepatitis B virus in the blood serum of affected individuals.

Determination and thin layer chromatographic confirmation of identity of aflatoxins B1 and M1 in artificially contaminated beef livers: collaborative study.

An international collaborative study involving 13 laboratories was conducted to test methods for the determination and thin layer chromatographic (TLC) confirmation of identity of aflatoxins B1 and M1 in beef liver. For the determination, each collaborator furnished fresh or frozen beef liver. Samples were artificially contaminated by adding solutions containing various concentrations of aflatoxins B1 and M1 (0.032-0.69 ng/g). Two TLC confirmation methods were tested with extracts obtained from the determination. Two measurement methods using 2-dimensional TLC were evaluated. In the first, sample extracts were compared directly with B1 and M1 standards on TLC plates; in the second, internal standards plus sample extracts were compared with B1 and M1 standards on the plates. Average within-laboratory coefficients of variation (CV) for the direct method were 26% for B1 and 26% for M1 compared with 24 and 26%, respectively, for the internal standard method. The average between-laboratory CV values were 39% for B1 and 41% for M1 by the direct method and 36% for B1 and 39% for M1 by the internal method and 36% for B1 and 39% for M1 by the internal standard method. Recoveries ranged from 64 to 90% for B1 and from 72 to 86% for M1. These data indicate that the more convenient direct method was sufficient, and internal standards were unnecessary. An analysis of variance was calculated from combined sample data to determine components of variance. The within-laboratory CV values were 27.0 and 32.3% for B1 and M1, respectively, and the between-laboratory CV values were 47.1 and 53.2%, respectively. Both TLC confirmation methods gave satisfactory results and have been adopted official first action, along with the determination method.

Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin.

Aflatoxicol (AFL) and aflatoxins B1 and M1 were found in tissues (kidney, liver, and muscle) of feeder pigs given an estimated LD50 oral dose of B1 (1.0 mg/kg body weight) provided as a rice culture of Aspergillus flavus and of market-weight pigs fed a naturally contaminated feed, containing aflatoxin B1 at a level of 400 ng/g from corn, for 14 days. The residues in all tissues decreased with time after treatment in both groups, with no detectable residues (approximate detection limits, ng/g, B1 0.03, M1 0.05, AFL 0.01) in pig tissues from the feeding experiment 24 h after withdrawal of aflatoxin-contaminated feed. B1 and M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the dominant aflatoxin. The level of AFL, when detected, was about 10% of the B1 level.

Aflatoxin M1 in manufactured dairy products produced in the United States in 1979.

In a 1979 survey of manufactured dairy products (992 samples of nonfat dry milk, vanilla ice cream, yogurt, Cheddar cheese, and cottage cheese) for aflatoxin M1 contamination, one sample, a cottage cheese, had detectable aflatoxin equivalent to .08 ng/ml in the milk from which the product was made. Samples were taken by Food and Drug District inspectors from randomly selected establishments at three times throughout the year. The distribution of sample quotas to each District was weighted to double the representation of establishments in the southern tier of states. The conclusion from this survey is that in a "normal" year aflatoxin M1 should not be in a manufactured dairy product in the United States at a level in excess of that from milk with .1 ng aflatoxin M1/ml.

High performance liquid chromatographic determination of aflatoxicol in milk, blood, and liver.

Samples of milk were extracted with chloroform, the extract was transferred to methanol, and residual interferences were removed by liquid-liquid partition against hexane and by silica gel column chromatography. Aflatoxicol (AFL) in the purified extract was resolved on a mu Bondapak C18 column, using water - methanol - acetonitrile - tetrahydrofuran (70 + 15 + 20 + 3) as the mobile phase, and measured with a fluorescence detector (excitation 325-385 nm, emission greater than 408 nm). Recoveries of AFL added to samples of whole pasteurized milk at levels ranging from 0.025 to 0.10 ng/mL averaged 92% (range 78-100%). The method for AFL has also been applied to the analysis of raw milk, whole beef blood, and beef liver, with recoveries of 70-88%.

Effect of boiling, frying, and baking on recovery of aflatoxin from naturally contaminated corn grits or cornmeal.

Corn grits naturally contaminated with aflatoxins were used for making boiled grits, and portions of the boiled grits were used for making pan-fried grits; cornmeal naturally contaminated with aflatoxins was used for making corn muffins. Procedures and recipes were derived from cookbook and market package recommendations. From analyses of the products for aflatoxins before and after preparation of the table-ready products, it was determined that 72 +/- 9% (n = 15) of the aflatoxin found in the original grits could be recovered after the grits were boiled. The recovery of aflatoxin B1 after the grits were fried was either 66 +/- 10% (n = 6) or 47 +/- 8% (n = 9), depending on whether 3 cups of water or 4 cups of water per cup of grits, respectively, were used for preparing the boiled grits before frying. Similarly, it was determined that 87 +/- 4% (n = 9) of the aflatoxin B1 found in the original cornmeal could be recovered from the baked muffins. No detectable aflatoxin B2 a was present in the extracts from any of the table-ready products.

Comparison of three methods for determining aflatoxins in sunflower seed meals.

Methods developed for the determination of aflatoxins in peanut products, in cottonseed products, and in ginger were compared for their applicability to sunflower seed meal. The peanut products method was inapplicable. Separation from interferences adequate to obtain a limit of determination for aflatoxin B1 around 1 ng/g and recovery of total added aflatoxins of about 97% could be achieved with either the cottonseed or ginger methods. The cleanest extract and the most rapid operation were obtained with the ginger method.

Thin layer chromatographic determination of aflatoxins in dry ginger root and ginger oleoresin.

A method for determining aflatoxins in dry ginger root and ginger oleoresin, using 1-dimensional thin layer chromatography (TLC) for the determinative step, has been developed. The key cleanup steps that permit this change from the 2-dimensional TLC previously required are partitioning of the extract with carbon tetrachloride and use of an improved eluting system for the silica gel adsorption column. Recoveries of alfoxins B1, B2, G1, and G2 added to samples of ground ginger were 82, 101, 106, and 110%, respectively; recoveries of these aflatoxins added to ginger oleoresin were 75, 100, 93, and 125%, respectively. The method is applicable to fish meal and a number of mixed feeds.

Aflatoxin control: past and present.

Domestic commodities most susceptible to aflatoxin contamination are peanuts, corn, cottonseed, and tree nuts (almond, pecans, walnuts); the most susceptible imported commodities are Brazil nuts and pistachio nuts. The development, effectiveness, and shortcomings of the strategies used to limit consumer exposure to aflatoxin from these commodities are reviewed.

Survey for aflatoxins and Zearalenone in canned and frozen sweet corn.

Aflatoxins and zearalenone were determined in 263 samples of canned or frozen sweet corn, collected from packing plants during the 1976 and 1977 packing seasons. As anticipated from geographic, agronomic, and microbiological considerations, no aflatoxin or zearalenone was found. Based on this sampling, the highest incidence of detectable aflatoxin that could be statistically anticipated in the major packing areas is 1.5%.