UNIVERSITY OF AGRICULTURAL SCIENCE AND VETERINARY MEDICINE CLUJ-NAPOCA

FACULTY OF AGRICULTUREMASTER COURSE: Food Quality Management

Chemical risk analysis for aflatoxins

Coropeţchi Cristina

Cluj-Napoca

2010

Table of content

I. Chemical risk assessment……………………………………………………………….…..11.What are aflatoxins?........................................................................................................ 2 2. Physical and chemical properties……………………………………………………... 33. Hazard identification…………………………………………………………………. .7 3.1. Factors favorizing aflatoxin production………………………………………….7 3.2. Occurrence……………………………………………………………………….74. Hazard characterization………………………………………………………………..9 4.1. Toxicity………………………………………………………………………… .9 4.2. Stability in foods………………………………………………………………. 10 4.3. Reccomended levels for aflatoxins presents in food……………………………115. Effects on health………………………………………………………………………12 5.1. Aflatoxins and animal health……………………………………………………12 5.2. Aflatoxins and human health……………………………………………………15

II. Risk management…………………………………………………………………………17

III. Risk communication……………………………………………………………………..19

IV. Chemical control…………………………………………………………………………201. Methods of analysis for aflatoxins in foods and feeds…………..…………………..202. Monitoring techniques for assessing human exposure to aflatoxins………………...213. Control and management of aflatoxins……………………………………………....21

V. Measures to prevent contamination with aflatoxins……………………..………………..22

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I. Chemical risk assessment

1. What are aflatoxins?

Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds. They are probably the best known and most intensively researched mycotoxins in the world. Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world. The occurence of aflatoxins is influenced by certain environmental factors: hence the extent of contamination will vary with geographic location, agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or processing periods. Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. As it is realized that absolute safety is never achieved, many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.

In the 1960 more than 100,000 young turkeys on poultry farms in England died in the course of a few months from an apparently new disease that was termed "Turkey X disease". It was soon found that the difficulty was not limited to turkeys. Ducklings and young pheasants were also affected and heavy mortality was experienced. A careful survey of the early outbreaks showed that they were all associated with feeds, namely Brazilian peanut meal. An intensive investigation of the suspect peanut meal was undertaken and it was quickly found that this peanut meal was highly toxic to poultry and ducklings with symptoms typical of Turkey X disease. Speculations made during 1960 regarding the nature of the toxin suggested that it might be of fungal origin. In fact, the toxin-producing fungus was identified as Aspergillus flavus (1961) and the toxin was given the name Aflatoxin by virtue of its origin (A.flavus--> Afla). Aflatoxins are produced primarily by some strains of A. Flavus and by most, if not all, strains of A. parasiticus , plus related species, A. nomius and A. niger

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Aspergillus flavus seen under an electron microscope.

This discovery has led to a growing awareness of the potential hazards of these substances as contaminants of food and feed causing illness and even death in humans and other mammals.

The aflatoxins consist of about 20 similar compounds belonging to a group called the difuranocoumarins, but only four are naturally found in foods. These are aflatoxins B1 , B2, G1

and G2 plus two additional metabolic products, M1 and M2, that are of significance as direct contaminants of foods and feeds. The aflatoxins M1 and M2 were first isolated from milk of lactating animals fed aflatoxin preparations; hence, the M designation. Whereas the B designation of aflatoxins B1 and B2 resulted from the exhibition of blue fluorescence under UV-light, while the G designation refers to the yellow-green fluorescence of the relevant structures under UV-light. These toxins have closely similar structures and form a unique group of highly oxygenated, naturally occuring heterocyclic compounds. Aflatoxins B2 and G2 were established as the dihydroxy derivatives of B1 and G1, respectively. Whereas, aflatoxin M1 is 4-hydroxy aflatoxin B1 and aflatoxin M2 is 4-dihydroxy aflatoxin B2.

2. Physical and chemical properties

Aflatoxins are potent toxic, carcinogenic, mutagenic, immunosuppressive agents, produced as secondary metabolites by the fungus Aspergillus flavus and A. parasiticus on variety of food products. Aflatoxin B1 (AFB1) is normally predominant in amount in cultures as well as in food products. Pure AFB1 is pale-white to yellow crystalline, odorless solid. Aflatoxins are soluble in methanol, chloroform, actone, acetonitrile. A.Flavus typically produces AFB1 and AFB2, where as A. parasiticus produce AFG1 and AFG2 as well as AFB1 and AFB2. Four other aflatoxins M1, M2, B2A, G2A which may be produced in minor amounts were subsequently isolated from cultures of A. flavus and A. parasiticus. A number of closely related compounds namely aflatoxin GM1, parasiticol and aflatoxicol are also produced by A. flavus. Aflatoxin M1and M2 are major metabolites of aflatoxin B1 and B2 respectively, found in milk of animals that have consumed feed contaminated with aflatoxins.

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Aflatoxins are normally refers to the group of difuranocoumarins and classified in two broad groups according to their chemical structure; the difurocoumarocyclopentenone series (AFB1, AFB2, AFB2A, AFM1, AFM2, AFM2A and aflatoxicol) and the difurocoumarolactone series (AFG1, AFG2, AFG2A, AFGM1, AFGM2, AFGM2A and AFB3). The aflatoxins display potency of toxicity, carcinogenicity, mutagenicity in the order of AFB1 > AFG1 > AFB2 > AFG2 as illustrated by their LD50 values for day-old ducklings. Structurally the dihydrofuran moiety, containing double bond, and the constituents liked to the coumarin moiety are of importance in producing biological effects. The aflatoxins fluoresce strongly in ultraviolet light (ca. 365 nm); B1 and B2 produce a blue fluorescence where as G1 and G2 produce green fluorescence.

Crystalline aflatoxins are extremely stable in the absence of light and particularly UV radiation, even at temperatures in excess of 100 ºC. A solution prepared in chloroform or benzene is stable for years if kept cold and in the dark. The lactone ring makes them susceptible to alkaline hydrolysis, and processes involving ammonia or hypochlorite have been investigated as means for their removal from food commodities, although questions concerning the toxicity of the breakdown products have restricted the use of this means of eradicating aflatoxins from food and animal feeds. If alkaline treatment is mild, acidification will reverse the reaction to reform the original aflatoxin. In acid, aflatoxins B1 and G1 are converted to aflatoxins B2a and G2a by acid catalytic addition of water across the double bond of the furan ring. Oxidising reagents react and the molecules lose their fluorescence properties.

Chemical structure of aflatoxins:

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Some important physical and chemical properties of the aflatoxins are given in the table

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Chemical reactions of aflatoxinsThe reaction of aflatoxins to various physical conditions and reagents have been

studied extensively because of the possible application of such reactions to the detoxification of aflatoxins contaminated material.

Heat: Aflatoxins in dry state are very stable to heat up to the melting point. However, in the

presence of moisture and at elevated temperatures there is destruction of aflatoxin over a period of time. Such destruction can occur either with aflatoxin in oilseed meals, aflatoxin in roasted peanuts or aflatoxin in aqueous solution at pH 7. Although the reaction products have not been examined in detail it seems likely that such treatment leads to opening of the lactone ring with the possibility of decarboxylation at elevated temperatures.

Alkalis: In alkali solution hydrolysis of the lactone moiety occurs. This hydrolysis appears to

be reversible, since it has been shown that recyclization occurs following acidification of a basic solution containing aflatoxin. At higher temperatures (ca. 100oC) ring opening followed by decarboxylation occurs and reaction may proceed further, leading to the loss of the methoxy group from the aromatic ring. Similar series of reactions also seems to occur with ammonia and various amines.

Acids: In the presence of mineral acids, aflatoxin B1 and G1 are converted in to aflatoxin

B2A and G2A due to acid-catalyzed addition of water across the double bond in the furan ring. In the presence of acetic anhydride and hydrochloric acid the reation proceeds further to give the acetoxy derivative. Similar adducts of aflatoxin B1 and G1 are formed with formic acid-thionyl chloride, acetic acid-thionyl chloride and trifluoroacetic acid.

Oxidizing agents: Many oxidizing agents, such as sodium hypochlorite, potassium permanganate,

chlorine, hydrogen peroxide, ozone and sodium perborate react with aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of fluorescence. The mechanisms of these reactions are uncertain and the reaction products remain unidentified in most cases. Reduction:

Hydrogenation of aflatoxin B1 and G1 yields aflatoxin B2 and G2 respectively. Further reduction of aflatoxin B1 by 3 moles of hydrogen yields tetrahydroxyaflatoxin. Reduction of aflatoxin B1 and B2 with sodium borohydride yielded aflatoxin RB1 and RB2 respectively. These arise as a result of opening of the lactone ring followed by reduction of the acid group and reduction of the keto group in the cyclopentene ring.

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3. Hazard identification

3.1. Factors favorizing aflatoxins production

Aflatoxin formation was considered to occur mainly in geographic regions with a tropical or subtropical climate. Conditions favorable for natural aflatoxin contamination of foods occur at latitudes between 40°N and 40°S of the equator. Production of aflatoxins is related to the highly variable relative humidity of the area, which influences moisture content of grains. Average relative humidity can be used to predict aflatoxin production. A fundamental distinction must be made between aflatoxin formation in crops before (or immediately after) harvest, and that occurring in stored commodities or foods. Peanuts, maize and cottonseed are associated with A. flavus, and in the case of peanuts, also with A. parasiticus, so that invasion of plants and developing seed or nut may occur before harvest. This close association results in the potential for high levels of aflatoxins in these commodities and is the reason for the continuing difficulty in eliminating aflatoxins from these products. In contrast, A. flavus lacks this affinity for other crops, so it is not normally present at harvest. Prevention of the formation of aflatoxins therefore relies mainly on avoidance of contamination after harvest, using rapid drying and good storage practice

Fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment. The appropriate combination of these factors determine the infestation and colonization of the substrate, and the type and amount of aflatoxin produced. However, a suitable substrate is required for fungal growth and subsequent toxin production, although the precise factor(s) that initiates toxin formation is not well understood. Water stress, high-temperature stress, and insect damage of the host plant are major determinig factors in mold infestation and toxin production . Similarly, specific crop growth stages, poor fertility, high crop densities, and weed competition have been associated with increased mold growth and toxin production. Aflatoxin formation is also affected by associated growth of other molds or microbes. For example, preharvest aflatoxin contamination of peanuts and corn is favored by high temperatures, prolonged drought conditions, and high insect activity; while postharvest production of aflatoxins on corn and peanuts is favored by warm temperatures and high humidity.

3.2. Occurence

In Raw Agricultural Products :Aflatoxins often occur in crops in the field prior to harvest. Postharvest contamination

can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mold growth. Insect or rodent infestations facilitate mold invasion of some stored commodities. Aflatoxins are detected occasionally in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, spices, and a variety of other foods and feeds . Milk, eggs, and meat products are sometimes contaminated because of the animal consumption of aflatoxin-contaminated feed . However, the commodities with the highest risk of aflatoxin contamination are corn, peanuts, and cottonseed.

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Food Group Range ( µg/kg) Mean Level

Peanuts & Peanut Products

1.6-26.0  1.45

Vegetable oil and fat 0.1-5.8  0.20

Cereals & Cereal Products 1.3-5.8  0.27

Aflatoxin detected in food groups

In Processed Foods :

Corn is probably the commodity of greatest worldwide concern, because it is grown in climates that are likely to have perennial contamination with aflatoxins and corn is the staple food of many countries. However, procedures used in the processing of corn help to reduce contamination of the resulting food product. This is because although aflatoxins are stable to moderately stable in most food processes, they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps. Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products, including non-fat dry milk, cheese, and yogurt.

 

 

 

 

 

Above: Observe the comparison between the same ear of corn before and after removing the husk from it: No major signs of infestation shows before, yet it is extensively damaged from

the inside.

 

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4. Hazard characterization

4.1. Toxicity

Aflatoxin really is one of the most toxic and carcinogenic substances known, as this label emphasizes.

The differences in suscepibility to aflatoxin across species and between persons depend largely on the fraction of the dose that is directed into the various possible pathways, with harmful "biological" exposure being the result of activation to the epoxide and the reaction of the epoxide with proteins and DNA. There is also evidence that the fractions thatfollow the different possible pathways are influenced by dosage, perhaps because of the saturation of chemically most competitive processes. Susceptibility to aflatoxin is greatest in the young, and there are very significant differences between species, individuals of the same species (according to their differing abilities to detoxify aflatoxin by biochemical processes), and the sexes (according to the concentrations of testosterone). Toxicity of aflatoxins also varies according to many nutritional factors as for instance recovery from protein malnutrition is delayed by exposure to aflatoxin. The dose and duration of exposure to aflatoxin clearly have a major effect on the toxicology and may cause a range of consequences such as:a. large doses may lead to acute illness and death, usually through liver cirrhosisb. chronic sublethal doses have nutritional and immunologic consequencesc. all doses have a cumulative effect on the risk of cancer.

Aflatoxins are both acutely and chronically toxic. Aflatoxin B1 is one of the most potent hepato-carcinogens known, and hence the long-term chronic exposure to extremely low levels of aflatoxins in the diet is an important consideration for human health. In the temperate, developed areas of the world, acute poisoning in animals is rare and in man is extremely unlikely.

Acute aflatoxin toxicity has been demonstrated in a wide range of mammals, fish, birds, rabbits, dogs and primates. Ducks, turkeys and trout are all highly susceptible. Age, sex and nutritional status all affect the degree of toxicity. Young animals are particularly

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susceptible and males more than females. For most species the LD50 (lethal dose) is between 0.5- and 10mg/kg body weight. The liver is the principal target organ, although the site of the hepatic effect varies with species. Effects on the lungs, myocardium and kidneys have also been observed and aflatoxin can accumulate in the brain. Teratogenic effects following administration of high doses of aflatoxin have been reported in some species. Acute poisoning of man by aflatoxins does occur occasionally in some areas of the world. Cases of human aflatoxicosis have been reported sporadically, mainly in Africa or Asia. The majority of reported cases involve consumption of contaminated cereals – most frequently maize, rice or cassava, or cereal products such as pasta or peanut meal. A classic case occurred in Malaysia in 1990 when approximately 40 persons were affected and 13 children died after eating noodles highly contaminated with aflatoxin and boric acid. High levels of aflatoxin were found on autopsy in liver, lung, kidney, heart, brain and spleen. Aflatoxin may not always be the primary cause of death in these acute cases. Autopsy brain (cerebrum) specimens from 18 kwashiorkor children and 19 other children who had died from a variety of other diseases in Nigeria showed aflatoxin present in 81% of the cases.

Aflatoxins have been implicated in sub-acute and chronic effects in humans. These effects include primary liver cancer, chronic hepatitis, jaundice, hepatomegaly and cirrhosis through repeated ingestion of low levels of aflatoxin. It is also considered that aflatoxins may place a role in a number of diseases, including Reye’s syndrome, kwashiorkor and hepatitis. Aflatoxins can also affect the immune system (Pier 1991). Aflatoxin B1 is a potent mutagen causing chromosomal aberrations in a variety of plant, animal and human cells. The carcinogenicity and mutagenicity of aflatoxins B1, G1 and M1 are considered to arise as the result of the formation of a reactive epoxide at the 8, 9 position of the terminal furan ring and its subsequent covalent binding to nucleic acid, and the carcinogenicity of aflatoxin B1 has been studied in at least 12 different species. Although aflatoxins G1 and M1 have been tested less extensively, they appear to be toxicologically similar to aflatoxin B1. They are slightly less potent liver carcinogens but slightly more potent kidney carcinogens.

4.2. Stability in foods

Aflatoxins are quite stable in many foods and are fairly resistant to degradation. Theeffectiveness of some processes in reducing concentrations of aflatoxins in food can beaffected by many factors, such as the presence of protein, pH, temperature and length oftreatment. Commercial processing of raw commodities using cleaning regimes includingthe removal of broken particles, milling and sorting can reduce aflatoxin concentration considerably.

Aflatoxins are quite stable compounds and survive relatively high temperatures with little degradation. Their heat stability is influenced by other factors, such as moisture level and pH, but heating or cooking processes cannot be relied upon to destroy aflatoxins. For example, roasting green coffee at 180oC for 10 minutes gave only a 50% reduction in aflatoxin B1 level. The stability of aflatoxin M1 in milk fermentation processes has also been studied and although appreciable losses do occur, significant quantities of the toxin were found to remain in both cheese and yoghurt. Aflatoxins can be destroyed by alkaline and acid hydrolysis and by the action of oxidising agents. However, in many cases, the resulting by-products also carry a risk of toxicity, or have not been identified

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4.3. Recommended levels for aflatoxins presents in food

Around 100 countries around the world have regulations governing aflatoxins in food and most include maximum permitted, or recommended levels for specific commodities. European Union

The EU sets limits for aflatoxin B1 and for total aflatoxins (B1, B2, G1 and G2) in nuts, dried fruits, cereals and spices. Limits vary according to the commodity, but range from 2-8 µg/kg for B1 and from 4-15 µg/kg for total aflatoxins. There is also a limit of 0.050 µg/kg for aflatoxin M1 in milk and milk products. Sampling and analytical methods are also specified. More recently limits of 0.10 µg/kg for B1 and 0.025 µg/kg for M1 have been set for infant foods. USA

US food safety regulations include a limit of 20 µg/kg for total aflatoxins (B1, B2, G1

and G2) in all foods except milk and a limit of 0.5 µg/kg for M1 in milk. Higher limits apply in animal feeds. Others

Both Australia and Canada set limits of 15 µg/kg for total aflatoxins (B1, B2, G1 and G2) in nuts. This is the same as the international limit recommended for raw peanuts by the Codex Alimentarius Commission.

Food and Drug Administration (FDA) has established action levels for aflatoxin present in food or feed to protect human and animal health.

Levels must not exceed: 20 ppb - For corn and other grains intended for immature animals (including immature

poultry) and for dairy animals, or when its destination is not known; 20 ppb - For animal feeds, other than corn or cottonseed meal; 100 ppb - For corn and other grains intended for breeding beef cattle, breeding swine,

or mature poultry; 200 ppb - For corn and other grains intended for finishing swine of 100 pounds or

greater; 300 ppb - For corn and other grains intended for finishing (i.e., feedlot) beef cattle and

for cottonseed meal intended for beef cattle, swine or poultry. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) concluded that

aflatoxins are amongst the most potent mutagenic and carcinogenic substances known. The JECFA estimated potency values for AFB1 from the epidemiological data. These corresponded to 0.3 cancers/year per 100,000 population per ng aflatoxin/kg b.w. per day (uncertainty range: 0.05-0.5) in hepatitis B virus antigen positive individuals and 0.01 cancers/year per 100,000 population per ng aflatoxin/kg b.w. per day (uncertainty range: 0.002-0.03) in hepatitis B virus antigen negative individuals.

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5. Effects on health

5.1. Aflatoxin and Animal Health

Aflatoxin B1 is a strong acute toxin in various animal species. The susceptibility of individual animals to aflatoxins varies considerably depending on species, age, sex, and nutrition. In fact, aflatoxins cause liver damage, decreased milk and egg production, recurrent infection as a result of immunity suppression (eg. salmonellosis), in addition to embryo toxicity in animals consuming low dietary concentrations. While the young of a species are most susceptible, all ages are affected but in different degrees for different species. Clinical signs of aflatoxicosis in animals include gastrointestinal dysfunction, reduced reproductivity, reduced feed utilization and efficiency, anemia, and jaundice. Nursing animals may be affected as a result of the conversion of aflatoxin B1 to the metabolite aflatoxin M1 excreted in milk of dairy cattle.

The induction of cancer by aflatoxins has been extensively studied. Aflatoxin B1, aflatoxin M1, and aflatoxin G1 have been shown to cause various types of cancer in different animal species. However, only aflatoxin B1 is considered by the International Agency for Research on Cancer (IARC) as having produced sufficient evidence of carcinogenicity in experimental animals to be identified as a carcinogen.

Aflatoxins are known to impair the cellular and humoral immune system, rendering animals more susceptible to bacterial, viral, fungal and parasitic infections. This immunosuppressive effect impairs also acquired resistance following vaccination, and may occur at a sub-clinical level of intoxication. Whereas acute clinical intoxications are rarely seen under the conditions of modern agricultural practice, sub-optimal weight gain, lower milk and egg production, as well as an increased susceptibility towards infectious diseases may lead to considerable economic losses in animal production due to aflatoxin exposure. The Panel concluded that for these effects, a no-effect level could not be defined from the available data. However, the Panel noted that the margin between toxic doses (> 1.5 mg/kg feed) and the statutory limit (0.020 mg/kg feed) of at least 75-fold would provide adequate protection from these effects.

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Above: Six rat livers injected with increasing doses of aflatoxin B1. The liver in the upper left corner received no aflatoxin (control), while the one at the lower right corner received the highest dose. Observe the color difference in these livers.

 

Above: A rat liver fed with high doses of aflatoxin B1. Notice the induced tumors in the liver.

 

 

A comparison between a control trout fish and another trout fed with high doses of aflatoxin B1. Observe the tumors (LCC) developed in the liver of the right side trout .

No animal species is resistant to the acute toxic effects of aflatoxins. A wide variation in LD50 values has been obtained in animal species tested with single doses of aflatoxins. For most species, the LD50 value ranges from 0.5 to 10-mg/kg body weight. Animal species respond differently in their susceptibility to the chronic and acute toxicity of aflatoxins. Environmental factors, exposure level, and duration of exposure beside age, health, and nutritional status of diet can influence the toxicity ( FAO web library 2000). Domestic

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animals (pets and agricultural), monkeys and laboratory rats and mice have been the subject of a large body of research on the adverse effects of aflatoxins (particularly B1). These effects include adducts and mutations, cancer, immunosuppression, lung injury and birth defects. Also, aflatoxins have been shown to interact with DNA (nuclear and mitochonndrial adducts) and polymerases responsible for DNA and RNA synthesis. Aflatoxins are immunosuppressive in a variety of animals making them more susceptible to infection by various microorganisms. The animals include sheep, cattle, mice, rats rabbits, pigs, poultry among others. Clinical signs in animals, associated with aflatoxin exposure consist of anorexia, icterus, depression, weight loss, nasal discharge, gastrointestinal affections, haemorrhages, ascitis and pulmonary oedema.

Acute toxicity of aflatoxin B1 expressed as a single oral dose LD50 (FAO web library)

Species LD50 mg kg -1 bodyweight

Rabbit 0.30

Duckling (11 day old) 0.43

Cat 0.55

Pig 0.60

Rainbow trout 0.80

Dog 0.50 - 1.00

Sheep 1.00 - 2.00

Guinea pig 1.40 - 2.00

Baboon 2.00

Chicken 6.30

Rat (male) 5.50 - 7.20

Rat (female) 17.90

Macaque (female) 7.80

Mouse 9.00

Hamster 10.20

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5.2. Aflatoxins and Human Health

Humans are exposed to aflatoxins by consuming foods contaminated with products of fungal growth. Such exposure is difficult to avoid because fungal growth in foods is not easy to prevent. Even though heavily contaminated food supplies are not permitted in the market place in developed countries, concern still remains for the possible adverse effects resulting from long-term exposure to low levels of aflatoxins in the food supply .

The incidence of chronic aflatoxicosis in humans is unknown and is almost impossible to estimate because the symptoms are so difficult to recognise. However, human liver cancer is quite common in parts of the world where aflatoxin contamination of food is likely and there may be a link, although this remains unproven. Other diseases possibly related to human exposure to aflatoxin B1 include toxic hepatitis and liver fibrosis, stunted growth in children,

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and Reye’s syndrome. Evidence of acute aflatoxicosis in humans has been reported from many parts of the world , namely countries, like Taiwan, Ouganda, India, and many others. The syndrome is characterized by vomiting, abdominal pain, pulmonary edema, convulsions, coma, and death with cerebral edema and fatty involvement of the liver, kidney, and heart. Conditions increasing the likelihood of acute aflatoxicosis in humans include limited availability of food, environmental conditions that favour fungal development in crops and commodities, and lack of regulatory systems for aflatoxin monitoring and control.

The expression of aflatoxin related diseases in humans may be influenced by factors such as age, sex, nutritional status, and/or concurrent exposure to other causative agents such as viral hepatitis (HBV) or parasite infestation. Ingestion of aflatoxin, viral diseases, and hereditary factors have been suggested as possible aetiological agents of childhood cirrhosis. There are evidences to indicate that children exposed to aflatoxin breast milk and dietary items such as unrefined groundnut oil, may develop cirrhosis. Malnourished children are also prone to childhood cirrhosis on consumption of contaminated food. Several investigators have suggested aflatoxin as an aetiological agent of Reye’s syndrome in children in Thailand, New Zealand etc. Though there is no conclusive evidence as yet. Epidemiological studies have shown the involvement of aflatoxins in Kwashiorkor mainly in malnourished children. The diagnostic features of Kwashiorkor are edema, damage to liver etc. These out breaks of aflatoxicosis in man have been attributed to ingestion of contaminated food such as maize, groundnut etc. Hence it is very important to reduce the dietary intake of aflatoxins by following the procedures for monitoring levels of aflatoxins in foodstuffs.

Foetal and childhood environment, including the nutritional status of the pregnant mother and the infant, are considered critical for growth and risk of disease in earlier life. Mal-nourishment is one of the common problems in developing countries. Apart from these, they are also exposed to high levels of mycotoxins. Aflatoxins are the major among these. It has been proved that these aflatoxins are immunogenic, teratogenic, and they retard the growth among experimental animals. In the developing countries like India and China, the environmental conditions favor their production. High exposure of these aflatoxins occurs through out these regions. A study in West Africa showed a significant correlation among the aflatoxin exposure and stunted growth in children who are exposed to aflatoxin right for neonatal stages. Apart from that due to the capacity of aflatoxins to cross the placental barrier, can cause genetic defects at foetal stages itself.

Because aflatoxins, especially aflatoxin B1, are potent carcinogens in some animals, there is interest in the effects of long-term exposure to low levels of these important mycotoxins on humans. In 1988, the IARC placed aflatoxin B1 on the list of human carcinogens. This is supported by a number of epidemiological studies done in Asia and Africa that have demonstrated a positive association between dietary aflatoxins and Liver Cell Cancer (LCC) . Additionally, the expression of aflatoxin-related diseases in humans may be influenced by factors such as age, sex, nutritional status, and/or concurrent exposure to other causative agents such as viral hepatitis (HBV) or parasite infestation.

Synergic relation of aflatoxin toxicity and epidemics of hepatitis viruses is of great concern to public health. In many developing countries, epidemics of hepatitis B virus (HBV) and hepatitis C virus (HCV) affect 20% of the population. A strong synergy is observed between aflatoxin and these biological agents for liver cancer. In hepatitis B surface antigen-positive subjects, aflatoxin is 30 times more potent than in persons without the virus, and the

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relative risk of cancer for HBV patients increases from 5 with only HBV infection to 60 when HBV infection is accompanied with aflatoxin exposure. The suggested mechanism for this synergy is that aflatoxin suppresses DNA repair mechanisms that help limit the development of cancer from HBV, and HBV prevents detoxification. It is also possible that the immunotoxicity of aflatoxin interferes with the suppression of cancer.

Human foods containing 4 to 30 ppb of aflatoxins are considered acceptable with differing specific ranges within the limit by are allowed, because this concentration not only provides protection against acute aflatoxicosis but also is low enough to allow most of the grain produced to be traded. Prolonged exposure to doses of <10 micrograms aflatoxin B1/kg/day causes no more than transient effects. Epidemiological data from human outbreaks suggest a minimum dose of 50 micrograms/kg/day for clinically significant effects. Wannemacher and others estimate the lethal aflatoxin B1 dose for 50% of the exposed population is one to four milligrams per kilogram of human body weight. In practical terms, a 175-pound person would have to breathe in or eat an acute dose of between 80 to 318 milligrams of pure aflatoxin B1 to cause death.

In well-developed countries, aflatoxin contamination rarely occurs in foodsat levels that cause acute aflatoxicosis in humans. In view of this, studieson human toxicity from ingestion of aflatoxins have focused on their carcinogenic potential. The relative susceptibility of humans to aflatoxins is notknown, even though epidemiological studies in Africa and Southeast Asia,where there is a high incidence of hepatoma, have revealed an associationbetween cancer incidence and the aflatoxin content of the diet. These studieshave not proved a cause-effect relationship, but the evidence suggests anassociation. One of the most important accounts of aflatoxicosis in humans occurred inmore than 150 villages in adjacent districts of two neighboring states innorthwest India in the fall of 1974. According to one report of this out-break, 397 persons were affected and 108 persons died. In this outbreak,contaminated corn was the major dietary constituent, and aflatoxin levels of0.25 to15 mg/kg were found. The daily aflatoxin B1 intake was estimated tohave been at least 55 ug/kg body weight for an undetermined number of days.The patients experienced high fever, rapid progressive jaundice, edema of thelimbs, pain, vomiting, and swollen livers. One investigator reported a pe-culiar and very notable feature of the outbreak: the appearance of signs ofdisease in one village population was preceded by a similar disease in domes-tic dogs, which was usually fatal. Histopathological examination of humansshowed extensive bile duct proliferation and periportal fibrosis of theliver together with gastrointestinal hemorrhages. A 10-year follow-up of theIndian outbreak found the survivors fully recovered with no ill effects fromthe experience. A second outbreak of aflatoxicosis was reported from Kenya in 1982. Therewere 20 hospital admissions with a 60% mortality; daily aflatoxin intake wasestimated to be at least 38 ug/kg bodyweight for an undetermined number ofdays. In a deliberate suicide attempt, a laboratory worker ingested 12 ug/kg bodyweight of aflatoxin B1 per day over a 2-day period and 6 months later, 11ug/kg body weight per day over a 14-day period. Except for transient rash,nausea and headache, there were no ill effects; hence, these levels may

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serve as possible control levels for aflatoxin B1 in humans. In a 14-yearfollow-up, a physical examination and blood chemistry, including tests forliver function, were normal.

II. Risk management

The economic impact of aflatoxins derive directly from crop and livestock losses as well as indirectly from the cost of regulatory programs designed to reduce risks to animal and human health. The Food and Agriculture Organization (FAO) estimates that 25% of the world's food crops are affected by mycotoxins, of which the most notorious are aflatoxins. Aflatoxin losses to livestock and poultry producers from aflatoxin-contaminated feeds include death and the more subtle effects of immune system suppression, reduced growth rates, and losses in feed efficiency. Other adverse economic effects of aflatoxins include lower yields for food and fiber crops . In addition, the abilitiy of aflatoxins to cause cancer and related diseases in humans given their seemingly unavoidable occurrence in foods and feeds make the prevention and detoxification of these mycotoxins one of the most challenging toxicology issues of present time.

A river in which huge batches of milk were dumped because their content of aflatoxin M1 exceeded the FDA action Level of 0.5 ppb for milk.

Mycotoxin regulatory programs are being introduced in some Asian countries. These regulatory programs are introduced primarily to protect the export market of agricultural commodities. These regulations are being strictly enforced or else the importing countries would reject the commodities, resulting in a loss of valuable foreign exchange earnings. On the other hand, domestic regulatory measures on aflatoxins received very little attention. In India, mycotoxin legislations have been introduced but the implementation was found to be inadequate. This might be due to the interference connected to businessmen dealing with aflatoxin products. Most farmers feel that it is another government ploy which calls for additional investment with no incentives given for the aflatoxin free produce .There has been no heavy penalty on the violators of aflatoxin regulations. Aflatoxin contaminated products are allowed to enter in the market. Governments should have a regulation to reject those food and feedstuffs which have an aflatoxin level above the acceptable limit. However, an

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equipped laboratory would be needed with adequate and well-trained staff to perform such analysis.

To minimize the risk of aflatoxin exposure, close tripartite cooperation among the trade, the public and the government is essential. The followings are some recommended risk reduction measure for the trade and the consumers.

Advice To Trade  The prime responsibility to ensure the wholesomeness of the foods lies with the

trade.  They are advised to adopt the Good Manufacturing Practice (GMP) and integrate it with HACCP based safety programme.  The following measures are useful:

a. Obtain raw materials from reliable and reputable suppliers b. Verify the specifications for quality product (e.g. decontamination process for

reduction of aflatoxin level, if indicated) c. Maintain good storage conditions

-dry and cool environment-stock rotation should be on a first-in first out basis

d. Keep documentation well in place Advice To Consumers

Consumers are advised to take the following measures to reduce the risk of aflatoxin exposure. Upon Purchase

a. Purchase from reliable and reputable retailers b. Observe whether foods are stored in cool condition. c. Reject any unclean, opened or damaged package

Storage a. Maintain at dry and cool environment (temperature preferably below 20¢XC and

relative humidity below 80%)b. Avoid direct sunlight c. Watch out the durability of the products d. Avoid stocking up excessive foods

Consumption a. Consume foods within the designated "best before date". b. Discard any foods that look mouldy, damped, shriveled and discoloured.

III. Risk communication

Generally, before people are receptive to risk information, they must belive that the source of that information is credible and fair. So, when designing the message to convey DDT risk, one must understand the communication difficulties that the audience may face. In risk communication, there are five common barriers listed in the mnemonic CAUSE:

- lack of Confidence- lack of Awareness- lack of Understanding- lack of Satisfaction

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- lack of EnactmentFirst, risk communicators often confront suspicion, so they need to employ strategies

that earn the confidence of their audience. Second, risk communication often is impeded by unfamiliarity with the subject, so the communicator must create awareness of the scope of information available. Third, because it involves concepts that may be difficult to grasp, communicators need to ensure that their audience is understanding the message. Fourth, satisfaction with solutions is critical: risk communicators must offer plausible precautionary approaches to risk management. Fifth, the adience must be stimulated toward enactment, so that it will embrace and implement the recommendations.

IV. Chemical control

1.Methods of Analysis for Aflatoxins in Foods and Feeds

For the monitoring of the presence of aflatoxins in food and feed materials, various validated methods of analysis exist.

Sampling and Sample Preparation :Sampling and sample preparation remain a considerable source of error in the

analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level. In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other commodities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample.

Solid-Phase Extraction :All analytical procedures include three steps: extraction, purification, and

determination. The most significant recent improvement in the purification step is the use of solid-phase extraction. Test extracts are cleaned up before instrumental analysis(thin layer or liquid chromatography) to remove coextracted materials that often interfere with the determination of target analytes.

Thin-Layer Chromatography :Thin layer chromatography (TLC), also known as flat bed chromatography or planar

chromatography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitate aflatoxins at levels as low as 1 ng/g. The TLC method is also used to verify findings by newer, more rapid techniques.

Liquid Chromatography :

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Liquid chromatography (LC) is similar to TLC in many respects, including analyte application, stationary phase, and mobile phase. Liguid chromatography and TLC complement each other. For an analyst to use TLC for preliminary work to optimize LC separation conditions is not unusual.

Liquid chromatography methods for the determination of aflatoxins in foods include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column derivatization (BCD), RPLC followed by postcolumn derivatization (PCD), and RPLC with electrochemical detection.

Immunochemical Methods :Thin layer chromatography and LC methods for determining aflatoxins in food are

laborious and time consuming. Often, these techniques require knowledge and experience of chromatographic techniques to solve sepatation and and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).

2. Monitoring Techniques for Assessing Human Exposure to Aflatoxins

In the last few years, new technologies have been developed that more accurately monitor individual exposures to aflatoxins. Particular attention has been paid to the analysis of aflatoxin DNA adducts and albumin adducts as surrogates for genotoxicity in people. Autrup et al.(1983) pioneered the use of synchronous fluorescence spectroscopy for the measurement of aflatoxin DNA adducts in urine. Urine samples collected after exposure to alfatoxins were found to contain 2,3-dihydroxy-2-(N7-guanyl)-3-hydroxyaflatoxin B1, trivially known as AFB-Gual . Wild et al.(1986) used highly sensitive immunoassays to quantitate aflatoxins in human body fluids. An enzyme linked immunosorbent assay (ELISA) was used to quantitate aflatoxin B1 over the range of 0.01 ng /ml to 10 ng/ml, and was validated in human urine samples. Using this method, aflatoxin-DNA adduct excretion into urine was found to be positively correlated with dietary intake, and the major aflatoxin B1-DNA adduct excreted in urine was shown to be an appropriate dosimeter for monitoring aflatoxin dietary exposure.

3. Control and Management of Aflatoxins

A- Regulatory Control : Aflatoxins are considered unavoidable contaminants of food and feed, even where

good manufacturing practices have been followed. The FDA has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of violative lots from commerce. The action level for human food is 20 ppb total aflatoxins, with the exception of milk which has an action level of 0.5 ppb for aflatoxin M1. The action level for most feeds is also 20 ppb. However, it is very difficult to accurately estimate aflatoxins concentration in a large quantity of material because

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of the variability associated with testing procedures; hence, the true aflatoxin concentration in a lot cannot be determined with 100% certainty.

B- Detoxification Strategies :Because aflatoxin contamination is unavoidable, numerous strategies for their

detoxification have been proposed. These include physical methods of separation, thermal inactivation, irradiation, solvent extraction, adsorption from solution, microbial inactivation, and fermentation. Chemical methods of detoxification are also practiced as a major strategy for effective detoxification : Structural Degradation Following Chemical Treatment :

A diverse group of chemicals has been tested for the ability to degrade and inactivate aflatoxins. A number of these chemicals can react to destroy (or degrade) aflatoxins effectively but most are impractical or potentially unsafe because of the formation of toxic residues or the perturbation of nutrient content and the organoleptic properties of the product. Two chemical approaches to the detoxification of aflatoxins that have received considerable attention are ammoniation and reaction with sodium bisulfite.

Many studies provide evidence that chemical treatment via ammoniation may provide an effective method to detoxify aflatoxin-contaminated corn and other commodities. The mechanism for this action appears to involve hydrolysis of the lactone ring and chemical conversion of the parent compound aflatoxin B1 to numerous products that exhibit greatly decreased toxicity. On the other hand, sodium bisulfite has been shown to react with aflatoxins (B1, G1 , and M1) under various conditions of temperature, concentration, and time to form water-soluble products.Modification of Toxicity by Dietary Chemicals : The toxicity of mycotoxins may be strongly influenced by dietary chemicals that alter the normal responses of mammalian systems to these substances. A variable array of chemical factors, including nutritional components (e.g. dietary protein and fat, vitamins, and trace elements), food and feed additives (e.g. antibiotics and preservatives), as well as other chemical factors may interact with the effects of aflatoxins in animals.Alteration of Bioavailability by Aflatoxin chemisorbents : A new approach to the detoxification of aflatoxins is the addition of inorganic sorbent materials, known as chemisorbents, such as hydrated sodium calcium aluminosilicate (HSCAS) to the diet of animals. HSCAS possesses the ability to tightly bind and immobilize aflatoxins in the gastrointestinal tract of animals, resulting in a major reduction in aflatoxin bioavailability.

V. Measures to prevent contamination with aflatoxin

Aflatoxin B1 contamination of animal feeding stuffs can be a very serious problem, occurring in part due to inadequate storage conditions. Contamination may also occur at the preharvest stage and be exacerbated by inadequate storage conditions. Good cropping practices, use of seed varieties bred for resistance to seed-infecting fungi and insect pests as well as the use of appropriate approved pesticides represent reasonable preventive measures to control contamination in the field. Even with application of these practices, conditions created by the environment and/or traditional agricultural procedures may defeat any

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preventative measures. Practices that reduce aflatoxin B1 contamination in the field and after harvest should be an integral part of animal feeding stuff production, particularly for the export market because of the additional handling and transport steps required to get the product to the final destination. The factors most amenable for prevention of fungal infection and aflatoxin B1 production involve proper drying and storage of the feeding stuff prior to transport. The problems created by too much moisture are magnified greatly by deficient post-harvest crop handling techniques. Investigations concerning the biological fate of aflatoxin B1 (AFB1) in lactating dairy cattle have demonstrated the transmission of residues into milk, occurring as the metabolite aflatoxin M1 (AFM1). Although AFM1 is considered to be less carcinogenic than AFB1 by at least an order of magnitude, its presence in dairy products should be limited to the lowest level practicable. The amount of daily ingested AFB1 which is transferred into milk is in the range of 0.17 to 3.3%. To ensure the lowest possible level of AFM1 in milk, attention should be given to residues of AFB1 in the lactating dairy animal's daily feed ration. To date there has been no widespread government acceptance of any decontamination treatment intended to reduce aflatoxin B1 levels in contaminated animal feeding stuffs. Ammoniation appears to have the most practical application for the decontamination of agricultural commodities, and has received limited regional (state, country) authorization for its use with animal feed under specified conditions (i.e. commodity type, quantity, animal). Also, research suggests that the addition of the anticaking/binding agent "hydrated sodium calcium aluminosilicate" to aflatoxin contaminated feeds may reduce AFM1 residues in milk, depending on the initial concentration of AFB1 in the feed.The Codex Code of Practice for the Reduction of Aflatoxin B1 in Raw Materials and Supplemental Feedingstuffs for Milk Producing Animals was adopted by the 22nd Session of the Codex Alimentarius Commission, 1997. The Code has been sent to all Member Nations and Associate Members of FAO and WHO.

RECOMMENDED PRACTICES

l. Crop production- Prepare seed bed for new crop by destroying or removing the seed heads or fruits (e.g. corn ears, peanuts, etc.) of aflatoxin susceptible crops.- Utilize soil tests if possible to determine fertilizer needs and apply fertilizer and soil conditioners to assure adequate soil pH and plant nutrition to avoid plant stress, especially during seed development.- When feasible, use seed varieties bred for fungal resistance and field tested for resistance toAspergillus flavus.- As far as practicable, sow and harvest crops at times which will avoid high temperature and drought stress during the period of seed development/maturation.- Minimize insect damage and fungal infection by the proper use of appropriate approved insecticides and fungicides and other appropriate practices within an integrated pest management program.- Use good agronomic practice, including measures which will reduce plant stress. Such measures may include: avoidance of overcrowding of plants by sowing at the recommended row and intra-plant spacings for the species/varieties grown; maintenance of a weed free environment in the growing crop by the use of appropriate approved herbicides and other

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suitable cultural practices; elimination of fungal vectors in the vicinity of the crop; and crop rotation.- Minimize mechanical damage to crops during cultivation.- Irrigation is a valuable method of reducing plant stress in some growing situations. If irrigation is used ensure that it is applied evenly and individual plants have an adequate supply of water.

2. Harvest- Harvest crops at full maturity unless allowing the crop to continue to full maturity would subject it to extreme heat, rainfall or drought conditions.- As much as possible avoid mechanical damage during harvest.- Where applicable dry crops to a minimum moisture content as quickly as possible.- If crops are harvested at high moisture levels dry immediately after harvest.- Avoid piling or heaping wet freshly harvested commodities for more than a few hours prior to drying or threshing to lessen the risk of fungal growth.- Ensure adequate protection from rain during sun drying.

3. Storage- Practice good sanitation for storage structures, wagons, elevators and other containers to ensure that stored crops will not be contaminated. Proper storage conditions include dry, well ventilated structures that provide protection from rain or seepage of ground water.- For bagged commodities, ensure that bags are clean and dry and stack on pallets or incorporate a water impermeable layer between the sacks and the floor.- Ensure that crops to be stored are free of mould and insects and are dried to safe moisture levels (ideally crops should be dried to a moisture content in equilibrium with a relative humidity of 70 %).- Prevent insect infestation by the use of appropriate approved insecticides.- Ensure that the storage facilities are free of insects and mould by good housekeeping and/or the use of appropriate approved fumigants.- Prevent access by rodents and birds.- Store at as low a temperature as possible. Where possible aerate commodities stored in bulk through continuous circulation of air through the storage vessel to maintain proper temperature and moisture.- Use of a suitable authorized preservative e.g. an organic acid such as proprionic acid, may be beneficial in that such acids are effective in killing moulds and fungi and preventing the production of mycotoxins. If organic acids are used, it is important that the amounts added are sufficient to prevent fungal growth and is consistent with the products end use.

4. Transport- Make sure that transport containers and vehicles are free of mould, insects and any contaminated material by thoroughly cleaning before use or re-use. Periodic disinfestation with appropriate approved fumigants or other pesticides may be useful.- Protect shipments from moisture by appropriate means such as airtight containers, covering with tarpaulins, etc. Care must be taken in the use of tarpaulins to avoid sweating of the

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commodity that could lead to local moisture and heat build up which are prime conditions for fungal growth.- Avoid insect and rodent infestation during transport by the use of insect resistant containers or insect and rodent repellent chemical treatments.

5. Feed production and disposition of AFB1 contaminated animal feeds- Ensure that milling equipment is kept clean, free of dust and feed accumulation.- Use an appropriate sampling and testing program to monitor outbound and inbound shipments for the presence of AFB1. Because AFB1 concentration in shipments may be extremely heterogeneous refer to FAO recommendations for sampling plans. Adjust frequency of sampling and testing to take into account conditions conducive to aflatoxin B1 formation, the regional source of the commodity and prior experience within the growing season.- If aflatoxin B1 is detected, consider one or more of the following options. In all cases ensure that the aflatoxin B1 level of the finished feed is appropriate for its intended use (i.e. maturity and species of animal being fed) and is consistent with national codes and guidelines or qualified veterinary advice.- Consider the restriction of AFB1 contaminated feed to a percentage of the daily ration such that the daily amount of AFB1 ingested would not result in significant residues of AFM1 in milk.- If feed restriction is not practical, divert the use of highly contaminated feedingstuffs to non-lactating animals only.

Decontamination Physical separation of contaminated material can be an effective means of reducing

aflatoxin levels in contaminated commodities. For example, colour sorting is often used to remove mouldy peanuts from bulk shipments. Density segregation, mechanical separation and the removal of fines and screenings from grain and nut shipments can also be effective measures. Chemical decontamination methods have been investigated, especially for material used in animal feed, but most of the methods investigated are impractical, or produce toxic by-products. So far, an ammoniation process has shown the most promise and has been successfully used to remove aflatoxins from feed in the USA. Biological decontamination has also been considered, and a single bacterial species, Flavobacterium aurantiacum, has been shown to remove aflatoxin B1 from peanuts and corn. Although decontamination methods for aflatoxin M1 in milk and dairy products have also been investigated, most of these are not practical for the dairy industry. The only really effective control is to minimise the contamination of materials used in animal feed for dairy cows.

In developed countries, where regulations allow higher aflatoxin concentrations in animals, the agricultural industries have developed alternative approaches [chemoprotection and enterosorption] to limit biologically effective exposure without the high cost of preventing contamination .. Chemoprotection is based on manipulating the biochemical processing of aflatoxin to ensure detoxification rather than preventing biological exposure. Enterosorption is based on the approach of adding a binding agent to food to prevent the absorption of the toxin while the food is in the digestive tract; the combined toxin-sorbent is then excreted in the feces. This approach has been used extensively and with great success in the animal feeding industry . The traditional approach to prevent exposure to aflatoxin has

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been to ensure that foods consumed have the lowest practical aflatoxin concentrations. In developed countries, this has been achieved for humans largely by regulations that have required low concentrations of the toxin in traded foods . Hydrated sodium calcium aluminosilicate (HSCAS) can prevent aflatoxicosis in chickens and swine. The basic mechanism for this action appears to involve sequestration of aflatoxin in the gastrointestinal tract and chemisorption (i.e., tight binding) to HSCAS, which results in a reduction in aflatoxin bioavailability . Garlic extract is found to possess an inhibitory effect on growth of Aspergillus flavus and its aflatoxin production

References

1. Chemical Hazards Evaluation, Aflatoxins in food, Food and Environmental Hygiene Department , 2001, Risk Assessment Studies

2.http://www.abc.cornell.edu/plants/toxicagents/aflatoxin/aflatoxin.html

3. http://www.fao.org/inpho/vlibrary/x0036e/x0036e04.htm

4. http://www.fao.org/docrep/x2100t/x2100t04.htm

5. http://www.cfs.gov.hk/english/programme/programme_rafs/files/report.pdf

6. http://www.foodsafetywatch.com/public/482.cfm

7. http://www.aflatoxin.info/health.asp

8.http://www.drthrasher.org/Aflatoxins_and_Aflatoxicosis.html

9. http://fsrio.nal.usda.gov/document_fsheet.php?product_id=48

10.http://www.micotoxinas.com.braflafacts.pdf.pdf .

11. http://www.worldscibooks.commedscietextbookp108p108_chap1.pdf

12. http://193.51.164.11/htdocs/Monographs/Vol56/09-AFL.htm

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13. Aflatoxin contaminated foods and health risk perspective for Pakistani population, Mycopath (2006), 4(2): 27-34

14.http://www. stason.org/.../13-1-6- Aflatoxin -What-Is-It.html

15. http://www.worldscibooks.com/medsci/etextbook/p108/p108_chap1.pdf

16. Code of practice for the reduction of aflatoxin B1 in raw materials and supplimental feedingstuffs for milk producing animals

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